Fahim Halim Khan

The Elements of
Immunology
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The Elements of
Immunology
FAHIM HALIM KHAN

A L I G A R H M U S L I M U N I V E R S I T Y
Delhi • Chennai • Chandigarh

Upper Saddle River, NJ • Boston • London
Sydney • Singapore • Hong Kong • Toronto • Tokyo
The publishers are grateful to the organizations and individuals who have
allowed the use of their copyrighted material. Each source is acknowledged in the
appropriate place in the text. While every effort has been made to trace the
owners, the publishers apologize for any inadvertent errors or omissions and
would welcome corrections to be incorporated into the next edition or reprint of
the book.
About the Cover

Phagocytosis of Foreign Debris by Monocyte White Blood Cell.
Blood monocyte and phagocytosis of foreign debris on a blood
The Elements of
Immunology
vessel. Monocytes are derived from bone marrow monoblasts and
Fahim Halim Khan
promonocytes. They are delivered to the blood and can enter the
tissues to develop into macrophages. Magnification of 1,400. Cover
courtesy of Visuals Unlimited/Corbis.
Library of Congress Cataloging-in-Publication Data
Khan, Fahim Halim.

The elements of immunology/Fahim Halim Khan.
p. cm.
Includes bibliographical references and index.
ISBN 978-8131711583 (pbk.)
1. Immunology—Textbooks. I. Title.
QR181.K43 2009
616.07'9—dc22
2008050350
Copyright © 2009 Dorling Kindersley (India) Pvt. Ltd.

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Dedicated to
my mother
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BRIEF CONTENTS
Preface xxix
1
An Introduction to the Immune System 2
2
Cells and Organs of the Immune System 24
3
Antigens 58
4
Antibodies 70
5
Generation of Antibody Diversity 92
6
Major Histocompatibility Complex 118
7
T-cell Receptors 136
8
T-cell Development and Activation 160
9
B-cell Development and Activation 184
10
The Complement System 208
11
Antigen Presentation and Processing 228
12
Cell-mediated Immunity 246
13
Hypersensitivity 266
14
Cell Migration and Inflammatory Response 294
15
Immune Response to Infectious Agents 314
16
Vaccines 342
17
Transplantation Immunology 360
18
Cancer and the Immune System 380
19
Primary and Secondary Immunodeficiencies 404
20
Autoimmunity and Autoimmune Diseases 428
Glossary 449
Index 461
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CONTENTS
PREFACE xxix
1 An Introduction to the Immune System

1.1 INTRODUCTION
2
3
1.2 THE COMPONENTS OF IMMUNITY 4

1.2.1 INNATE IMMUNITY 4
Mechanical Barriers 4
Chemical Barriers 5
Phagocytosis 6
Fever 7
Inflammation 7
Acute-phase Proteins 9
1.2.2 ADAPTIVE IMMUNITY 10
1.2.3 CELLS OF THE IMMUNE SYSTEM 10
B Lymphocytes 11
T Lymphocytes 11
Antigen-presenting Cells 12
Natural Killer Cells 12
Eosinophils 12
Basophils and Mast Cells 13
Mononuclear Phagocytes 13
Neutrophils 13
1.2.4 ANTIGENS AND ANTIGEN RECOGNITION 13
1.2.5 MHC AND ANTIGEN PRESENTATION 13
Processing of Endogenous Antigens 14
Processing of Exogenous Antigens 14
1.3 TYPES OF IMMUNE RESPONSE 15

1.3.1 ANTIBODY-MEDIATED IMMUNITY 16
1.3.2 CELL-MEDIATED IMMUNITY 17
1.4 ACTIVATION OF THE IMMUNE RESPONSE 17

1.4.1 HUMORAL RESPONSE 17
1.4.2 SELECTIVE ACTIVATION OF B CELLS AND GENERATION OF HUMORAL RESPONSE 18
1.4.3 CELL-MEDIATED RESPONSE 18
1.5 IMMUNE DISORDERS 19

1.5.1 AUTOIMMUNITY 19
1.5.2 IMMUNODEFICIENCY 20
1.5.3 HYPERSENSITIVITY 20
1.6 EVOLUTION OF IMMUNITY 20

x CONTENTS
SUMMARY 21
KEY WORDS 21
REVIEW QUESTIONS 22
QUIZ YOURSELF 22
FURTHER READING 23
2 Cells and Organs of the Immune System

2.1 HAEMATOPOIESIS
24
25
2.2 REGULATION OF HAEMATOPOIESIS 29

2.2.1 APOPTOSIS 29
2.3 CELLS OF THE IMMUNE SYSTEM 31

2.3.1 LYMPHOCYTES 32
Lymphocyte Development 32
B Lymphocytes 34
T Lymphocytes 34
Natural Killer Cells/Null Cells 36
2.3.2 MONONUCLEAR PHAGOCYTES 38
2.3.3 GRANULOCYTES 39
Neutrophils 39
Eosinophils 40
Basophils and Mast Cells 41
2.3.4 DENDRITIC CELLS 41
2.3.5 PLATELETS 42
2.4 PHAGOCYTOSIS 42
2.4.1 BACTERIAL KILLING MECHANISM 43
Oxygen-dependent Killing Mechanism 43
Oxygen-independent Killing Mechanism 43
2.4.2 OVERCOMING PHAGOCYTIC DEFENCES 44
2.5 LYMPHOID TISSUES 44

2.5.1 PRIMARY LYMPHOID ORGANS 46
Bone Marrow 46
Thymus 47
Thymic Education 47
2.5.2 SECONDARY LYMPHOID ORGANS AND TISSUES 48
Lymph Nodes 48
Spleen 49
Mucosa-associated Lymphoid Tissue 51
Nasal-associated Lymphoid Tissue 51
Gut-associated Lymphoid Tissue 51
Bronchus-associated Lymphoid Tissue 52
Cutaneous-associated Lymphoid Tissue 52
2.6 LYMPHATIC SYSTEM 53

‡ F LO W C Y TO M E T R Y 54
SUMMARY 55
KEY WORDS 56
CONTENTS xi
REVIEW QUESTIONS 56
QUIZ YOURSELF 56
FURTHER READING 57
3 Antigens
3.1 INTRODUCTION
58
59
3.2 GENERAL PROPERTIES OF ANTIGENS 62

3.2.1 MOLECULAR SIZE 62
3.2.2 SELF OR FOREIGN 63
3.2.3 CHEMICAL COMPLEXITY 63
3.2.4 ROUTE OF ENTRY 63
3.3 B-CELL AND T-CELL EPITOPES 63

3.3.1 PROPERTIES OF B-CELL EPITOPES 64
3.3.2 PROPERTIES OF T-CELL EPITOPES 64
3.4 SUPERANTIGENS 64
3.5 HAPTENS 65
3.6 ADJUVANTS 65
‡ G E L F I LT R AT I O N C H R O M ATO G R A P H Y 67
SUMMARY 68
KEY WORDS 68
REVIEW QUESTIONS 68
QUIZ YOURSELF 69
FURTHER READING 69
4 Antibodies
4.1 INTRODUCTION
70
71
4.2 LANDMARKS IN THE ELUCIDATION OF ANTIBODY STRUCTURE 71
4.3 ANTIBODY STRUCTURE 74

4.3.1 HINGE REGION 76
4.3.2 J CHAIN 77
4.3.3 DISULPHIDE BONDS 77
4.4 CLASSES OF IMMUNOGLOBULIN 78

4.4.1 IMMUNOGLOBULIN G 78
4.4.2 IMMUNOGLOBULIN A 80
4.4.3 IMMUNOGLOBULIN M 80
4.4.4 IMMUNOGLOBULIN D 81
4.4.5 IMMUNOGLOBULIN E 82
xii CONTENTS
4.5 ANTIBODY-MEDIATED EFFECTOR FUNCTIONS 84

4.5.1 ACTIVATION OF COMPLEMENT SYSTEM BY IgG AND IgM 84
4.5.2 CELL-MEDIATED CYTOTOXICITY TARGETED BY IMMUNOGLOBULINS 85
4.5.3 OPSONIZATION 85
4.6 MUCOSAL IMMUNITY 85
4.7 NEONATAL IMMUNITY 85
4.8 ANTIBODIES CAN BE ANTIGENS TOO 86

4.8.1 ISOTYPE 86
4.8.2 ALLOTYPE 86
4.8.3 IDIOTYPE 86
4.9 IMMUNOGLOBULIN SUPERFAMILY 87

‡ A N T I G E N – A N T I B O DY I N T E R AC T I O N S : I M M U N O P R E C I P I TAT I O N 89
SUMMARY 89
KEY WORDS 90
REVIEW QUESTIONS 90
QUIZ YOURSELF 90
FURTHER READING 91
5 Generation of Antibody Diversity

5.1 INTRODUCTION
92
93
5.2 GENETIC ORGANIZATION OF IMMUNOGLOBULIN GENES 96

5.2.1 LIGHT-CHAIN LOCI 97
l-chain Family 97
k-chain Family 97
5.2.2 HEAVY CHAIN LOCI 97
5.3 REARRANGEMENT OF GENES 98

5.3.1 HEAVY-CHAIN GENE REARRANGEMENT 98
Rearrangement of Genes at DNA Level 98
Rearrangement of Genes at RNA Level 99
5.3.2 LIGHT-CHAIN GENE REARRANGEMENT 100
Rearrangement of Genes at RNA Level 100
5.3.3 REARRANGEMENT OF V, (D), J GENE SEGMENTS 101
12/23 Rule 102
Recombination Sequence-directed Joining of Gene Segments 102
Mechanism of V, (D), J Gene-segment Rearrangement 103
Defects in V, (D), J Recombination 104
5.4 ALLELIC EXCLUSION 105

5.4.1 ALLELIC EXCLUSION OF HEAVY CHAINS 106
5.4.2 ALLELIC EXCLUSION OF LIGHT CHAINS 106
5.5 THE GENERATION OF DIVERSITY IN IMMUNOGLOBULINS 106

5.5.1 MULTIPLE GERM-LINE GENE SEGMENTS 107
5.5.2 V–J AND V–D–J RECOMBINATION 107
CONTENTS xiii
5.5.3 JUNCTIONAL DIVERSITY 107

5.5.4 GENE CONVERSION 108
5.5.5 SOMATIC HYPERMUTATION 109
5.5.6 ASSOCIATION OF VARIED LIGHT AND HEAVY CHAINS 110
5.6 MEMBRANE-BOUND AND SECRETED IMMUNOGLOBULINS 110

5.6.1 GENERATION OF MEMBRANE-BOUND OR SECRETED IMMUNOGLOBULINS 110
5.6.2 CO-EXPRESSION OF MEMBRANE-BOUND IgM AND IgD 111
5.7 ASSEMBLY AND SECRETION OF IMMUNOGLOBULINS 112
5.8 CLASS SWITCHING 112
5.9 REGULATION OF IMMUNOGLOBULIN GENE TRANSCRIPTION 114

‡SINGLE RADIAL IMMUNODIFFUSION 115
SUMMARY 115
KEY WORDS 116
REVIEW QUESTIONS 116
QUIZ YOURSELF 116
FURTHER READING 117
6 Major Histocompatibility Complex

6.1 INTRODUCTION
118
119
6.2 CLASS I MHC MOLECULES 120
6.3 CLASS II MHC MOLECULES 121
6.4 CLASS III MHC MOLECULES 122
6.5 STRUCTURE OF PEPTIDE-BINDING CLEFT 123
6.6 PEPTIDE–MHC INTERACTION 124
6.7 GENE MAP OF THE MAJOR HISTOCOMPABILITY COMPLEX 125

6.7.1 MURINE MHC LOCI 126
Classical Murine Class I Loci 126
Non-classical Murine Class I Loci 127
6.7.2 MURINE CLASS II MHC LOCI 127
6.7.3 I-GENE IN H-2 LOCUS 128
6.8 HUMAN MHC LOCI 128

6.8.1 HUMAN CLASS I MHC LOCI 128
6.8.2 HUMAN CLASS II MHC LOCI 129
6.8.3 HUMAN CLASS III LOCI 130
6.8.4 WHY THE NAME HLA? 130
6.9 MHC POLYMORPHISM 130

xiv CONTENTS
6.10 DISTRIBUTION OF CLASS I AND CLASS II MHC MOLECULES 130
6.11 TRANSCRIPTIONAL REGULATION OF MHC MOLECULES 132

6.11.1 CLASS I MHC: CONSTITUTIVE EXPRESSION 132
6.11.2 CLASS I MHC: CYTOKINE-INDUCED EXPRESSION 132
6.11.3 CLASS II MHC EXPRESSION 132
‡ A F F I N I T Y C H R O M ATO G R A P H Y 133
SUMMARY 134
KEY WORDS 134
QUIZ YOURSELF 135
FURTHER READING 135
7
7.1
T-cell Receptors
INTRODUCTION
136
137
7.2 STRUCTURE OF T-CELL RECEPTOR 139

7.2.1 T-CELL RECEPTORS ARE RELATED TO IMMUNOGLOBULINS 139
7.2.2 GLYCOSYLATION OF TCR CHAINS 141
7.3 ORGANIZATION OF T-CELL RECEPTOR GENES IN THE GERM LINE 141

7.3.1 MOUSE TCR α-GENE LOCUS 141
7.3.2 MOUSE TCR β-GENE LOCUS 141
7.3.3 HUMAN TCR α-GENE LOCUS 142
7.3.4 HUMAN TCR β-GENE LOCUS 142
7.3.5 MOUSE TCR γ-GENE LOCUS 142
7.3.6 MOUSE TCR δ-GENE LOCUS 143
7.3.7 HUMAN TCR γ-GENE LOCUS 143
7.3.8 HUMAN TCR δ-GENE LOCUS 143
7.4 REARRANGEMENT OF GENES TO FORM MATURE AND β GENES 144

7.4.1 GENE REARRANGEMENT TO FORM MATURE TCR β GENE 144
Splicing and Maturation of Primary Transcript 144
7.4.2 REARRANGEMENT OF α-CHAIN GENES 145
7.5 REARRANGEMENT OF AND δ GENES 146
7.6 ALLELIC EXCLUSION OF TCR GENES 146
7.7 INHIBITION OF IMMUNOGLOBULIN GENE REARRANGEMENT IN T CELLS 147
7.8 GENERATION OF STRUCTURAL T-CELL RECEPTOR DIVERSITY 147

7.8.1 PRESENCE OF MULTIPLE GERM-LINE V, D, AND J SEGMENTS 147
7.8.2 JUNCTIONAL DIVERSITY 147
7.8.3 IMPRECISE RECOMBINATION 147
7.8.4 P- AND N-NUCLEOTIDE ADDITION 148
7.8.5 PAIRING OF AND CHAINS 148
7.9 COMPLEMENTARITY DETERMINING REGIONS (CDR) OF T-CELL RECEPTOR 149

CONTENTS xv
7.10 TCR GENES DO NOT UNDERGO SOMATIC HYPERMUTATION 149
7.11 PROMOTERS, ENHANCERS AND SILENCERS OF T-CELL RECEPTORS 149

7.11.1 a CHAIN 149
7.11.2 b CHAIN 149
7.11.3 g AND δ CHAINS 150
7.12 T-CELL RECEPTOR COMPLEX 150

7.12.1 CD3 PROTEINS 150
7.12.2 CD3 COMPLEX 150
g Chain of CD3 Complex 151
d Chain of CD3 Complex 151
e Chain of CD3 Complex 152
z Chain of CD3 Complex 152
h Chain of CD3 Complex 152
7.12.3 SURFACE EXPRESSION OF TCR COMPLEX 152
7.12.4 FUNCTION OF CD3 COMPLEX 152
7.13 ACCESSORY MOLECULES ON T CELLS 153

7.13.1 CD4 ACCESSORY MOLECULES 153
7.13.2 CD8 ACCESSORY MOLECULES 154
7.14 ANTIGEN–MHC–T-CELL RECEPTOR COMPLEX 154
7.15 CROSS-REACTIVITY OF T CELL WITH ALLOGENEIC MHC 155

‡ T R A N S M I S S I O N E L E C T R O N M I C R O S CO PY 157
SUMMARY 158
KEY WORDS 158
QUIZ YOURSELF 159
FURTHER READING 159
8 T-cell Development and Activation

8.1 INTRODUCTION
160
161
8.2 T-CELL DEVELOPMENT 162
8.3 POSITIVE AND NEGATIVE SELECTION 165

8.3.1 POSITIVE SELECTION 165
Experimental Evidence of Positive Selection 166
8.3.2 NEGATIVE SELECTION 168
Experimental Evidence of Negative Selection 168
8.3.3 MECHANISMS OF POSITIVE AND NEGATIVE SELECTION 169
8.4 ACTIVATION OF T LYMPHOCYTES 171

8.4.1 RECOGNITION OF ANTIGEN–MHC COMPLEX AND SIGNAL TRANSDUCTION BY TCR 171
8.4.2 COSTIMULATORS AND T-CELL ACTIVATION 175
8.4.3 THE FALL OF T-CELL RESPONSE 176
8.5 SUPERANTIGEN-INDUCED T-CELL ACTIVATION 176

xvi CONTENTS
8.6 T LYMPHOCYTES 178

8.6.1 SPECIFICITY OF gδ T CELLS 178
8.6.2 FUNCTIONS OF gδ T CELLS 178
8.7 NKT CELLS 179

‡ S C A N N I N G E L E C T R O N M I C R O S CO PY 180
SUMMARY 181
KEY WORDS 181
QUIZ YOURSELF 182
FURTHER READING 183
9 B-cell Development and Activation

9.1 INTRODUCTION
184
185
9.2 B-CELL DEVELOPMENT 185

9.2.1 STAGES OF B-LYMPHOCYTE DEVELOPMENT 186
9.2.2 NEGATIVE SELECTION OF B CELLS 187
9.2.3 B1 SUBSET OF B CELLS 188
9.3 ACTIVATION OF B CELLS 189

9.3.1 ANTIGEN RECOGNITION 189
9.3.2 SIGNALLING THROUGH B-CELL CO-RECEPTOR COMPLEX 190
9.3.3 PROLIFERATION PHASE 191
9.4 THYMUS-DEPENDENT AND THYMUS-INDEPENDENT ANTIGEN 192
9.5 ROLE OF TH CELLS IN B-CELL ACTIVATION 193

9.5.1 ANTIGEN PRESENTATION BY B CELLS TO TH CELLS 193
CD40–CD40 Ligand Interaction 194
TH cell’s Cytokine in B-cell Proliferation and Differentiation 194
9.5.2 B-CELL DIFFERENTIATION INTO EFFECTOR PLASMA CELLS 195
9.5.3 B-CELL DIFFERENTIATION INTO MEMORY B CELLS 197
9.6 PRIMARY AND SECONDARY HUMORAL IMMUNE RESPONSE 198
9.7 ROLE OF TH CELLS IN HUMORAL RESPONSE 199
9.8 SITES FOR INDUCTION OF HUMORAL RESPONSE 199
9.9 GERMINAL-CENTRE REACTIONS 200

9.9.1 AFFINITY MATURATION OF B CELLS 200
9.9.2 SOMATIC HYPERMUTATION 200
9.9.3 SELECTION OF HIGH-AFFINITY B CELLS 203
9.10 REGULATION OF IMMUNE RESPONSE 203

9.10.1 REGULATION BY ANTIGEN 204
9.10.2 ANTIBODY-MEDIATED REGULATION 204
9.10.3 REGULATION BY LYMPHOCYTES 205
9.10.4 REGULATION BY IDIOTYPIC–ANTI-IDIOTYPIC NETWORK 205
‡ D O U B L E D I F F U S I O N AG A R A S S AY 205
CONTENTS xvii
SUMMARY 206
KEY WORDS 206
QUIZ YOURSELF 207
FURTHER READING 207
10 The Complement System

10.1 INTRODUCTION
208
209
10.2 CLASSICAL PATHWAY 210
10.3 ALTERNATIVE PATHWAY 213
10.4 THE MANNAN-BINDING LECTIN PATHWAY 214
10.5 THE FORMATION OF MEMBRANE-ATTACK COMPLEX 215
10.6 BIOLOGICAL FUNCTIONS OF COMPLEMENT PROTEINS 216

10.6.1 CYTOLYSIS 216
10.6.2 ANAPHYLATOXINS AND INFLAMMATION 217
10.6.3 C3b GENERATION AND PROMOTION OF PHAGOCYTOSIS 218
10.6.4 SOLUBILIZATION OF IMMUNE COMPLEXES OR IMMUNE COMPLEX CLEARANCE 219
10.6.5 NEUTRATIZATION OF VIRAL INFECTION 220
10.6.6 INDUCTION OF IMMUNE RESPONSE 220
10.7 REGULATION OF COMPLEMENT CASCADE 220

10.7.1 REGULATION OF C1 220
10.7.2 REGULATION OF C3 CONVERTASES 220
10.7.3 REGULATION OF MEMBRANE-ATTACK COMPLEX 222
10.8 COMPLEMENT DEFICIENCIES 222

‡ CO M P L E M E N T F I X AT I O N T E S T 224
SUMMARY 225
KEY WORDS 226
QUIZ YOUSELF 226
FURTHER READING 227
11 Antigen Presentation and Processing

11.1 INTRODUCTION
228
229
11.2 ANTIGEN-PRESENTING CELLS 230

11.2.1 DENDRITIC CELLS 231
11.2.2 MONONUCLEAR PHAGOCYTIC CELLS 231
11.2.3 B LYMPHOCYTES 231
11.2.4 NON-PROFESSIONAL ANTIGEN-PRESENTING CELLS 232
xviii CONTENTS
11.3 TWO PROCESSING AND PRESENTATION PATHWAYS 232

11.3.1 PRESENTATION OF ENDOGENOUS PATHOGENS TO CLASS I MHC MOLECULES 232
Proteasome Complex 233
Ubiquitination of Proteins 233
Peptide Transport from Cytoplasm to ER 234
Presentation of Peptides Derived from Membrane-bound and Secreted Proteins 235
Evidence for Cytosolic Degradation of Membrane-bound and Secreted Proteins 235
Binding of Class I MHC Molecules and Peptides 235
Blockage of Endogenous Pathway by Viruses 236
11.3.2 PRESENTATION OF EXOGENOUS ANTIGEN TO CLASS II MHC MOLECULES 237
Endocytosis of Proteins 237
Binding of Processed Peptides to Class II MHC Molecules 238
Role of Specialized Class II MHC-like Molecules — HLA-DM 240
11.4 PRESENTATION OF NON-PEPTIDE BACTERIAL ANTIGENS 240

‡GEL ELEC TROPHORESIS 242
SUMMARY 243
KEY WORDS 243
QUIZ YOURSELF 244
FURTHER READING 245
12 Cell-mediated Immunity
12.1 INTRODUCTION
246
247
12.2 LYMPHOCYTES AND CELL-MEDIATED RESPONSE 248

12.2.1 CYTOTOXIC T LYMPHOCYTES 248
Mechanism of Cytotoxicity 250
12.2.2 NATURAL KILLER CELLS 252
Surface Markers on NK Cells 252
Mechanism of NK-cell Cytotoxicity 254
12.3 ANTIBODY-DEPENDENT CELL-MEDIATED CYTOTOXICITY 256
12.4 DELAYED-TYPE HYPERSENSITIVITY 257

12.4.1 SENSITIZATION AND EFFECTOR PHASES IN DTH RESPONSE 257
Sensitization or Activation Phase 257
Effector Phase of DTH 258
12.5 CYTOKINES AND DTH REACTION 260

12.5.1 GROWTH FACTORS/CYTOKINES SECRETED BY MACROPHAGES 261
12.6 DETECTION OF DTH REACTION 261
12.7 SIGNIFICANCE OF DTH REACTION 261

‡ AG G LU T I N AT I O N 262
SUMMARY 263
KEY WORDS 263
QUIZ YOURSELF 264
FURTHER READING 265
CONTENTS xix
13 Hypersensitivity
13.1 INTRODUCTION
266
267
13.2 GELL AND COOMBS CLASSIFICATION 268

13.2.1 TYPE I HYPERSENSITIVITY REACTION 268
What Is An Allergen? 269
IgE 269
13.2.2 BASOPHILS, MAST CELLS AND EOSINOPHILS 270
Mast Cells 270
Eosinophils 271
13.2.3 RECEPTORS FOR IgE 272
High-affinity Receptor (FcεRI) 272
Low-affinity Receptor (FcεRII) 272
13.2.4 ACTIVATION OF MAST CELLS AND BASOPHILS 273
Events Leading to Mast-cell Activation 273
13.2.5 BIOLOGICAL MEDIATORS OF TYPE I REACTIONS 273
Primary Mediators 275
Secondary Mediators 275
Cytokines 276
13.2.6 CLINICAL CONSEQUENCES OF TYPE I HYPERSENSITIVITY 276
Features of Anaphylaxis 276
Features of Atopy 277
Allergic Rhinitis (Hay Fever) 277
Asthma 277
Food Allergies 278
Atopic Dermatitis (Eczema) 278
13.2.7 LATE-PHASE REACTION 278
13.2.8 TESTS FOR DIAGNOSIS OF TYPE I HYPERSENSITIVITY 278
Skin Test 278
Patch Test 280
Radioimmunosorbent Test (RIST) 280
Radioallergosorbent Test (RAST) 280
13.2.9 THERAPEUTIC MEASURES FOR TYPE I HYPERSENSITIVITY 280
13.3 TYPE II HYPERSENSITIVITY: ANTIBODY-DEPENDENT

CYTOTOXIC HYPERSENSITIVITY 281
13.3.1 DRUG-INDUCED HYPERSENSITIVITY REACTION 282
13.3.2 TRANSFUSION REACTIONS 282
13.3.3 RHESUS ANTIGEN INCOMPATIBILITY 283
13.4 TYPE III HYPERSENSITIVITY: IMMUNE COMPLEX– MEDIATED

HYPERSENSITIVITY 284
13.4.1 HOW ARE IMMUNE COMPLEXES REMOVED IN NORMAL INDIVIDUALS? 284
What Goes Wrong in Type III Hypersensitivity Reactions? 286
13.4.2 MECHANISM OF TYPE III HYPERSENSITIVITY REACTIONS 286
13.4.3 LOCALIZED TYPE III REACTIONS 287
13.4.4 GENERALIZED TYPE III REACTIONS 288
13.5 TYPE IV HYPERSENSITIVITY REACTIONS: DELAYED-TYPE

HYPERSENSITIVITY REACTIONS 289
13.5.1 CONTACT HYPERSENSTIVITY 289
13.5.2 TUBERCULIN REACTION 290
xx CONTENTS
13.5.3 GRANULOMATOUS HYPERSENSTIVITY 290

‡IMMUNOELEC TROPHORESIS 291
SUMMARY 291
KEY WORDS 292
QUIZ YOURSELF 292
FURTHER READING 293
14 Cell Migration and Inflammatory Response

14.1 INTRODUCTION
294
295
14.2 CELL-SURFACE ADHESION MOLECULES 296

14.2.1 IMMUNOGLOBULIN SUPERFAMILY CAMs 297
14.2.2 SELECTINS 297
14.2.3 INTEGRINS 297
14.2.4 MUCIN FAMILY 297
14.2.5 CADHERINS 298
14.3 LEUKOCYTE MIGRATION 298

14.3.1 CHEMOTACTIC MOLECULES 299
14.3.2 CHEMOKINES 300
14.3.3 CHEMOTACTIC MOLECULES 302
14.4 MEDIATORS OF INFLAMMATION 302

14.4.1 KININ SYSTEM 303
14.4.2 CLOTTING SYSTEM 303
14.4.3 FIBRINOLYTIC SYSTEM 303
14.4.4 COMPLEMENT SYSTEM 303
14.4.5 INFLAMMATORY CYTOKINES 303
14.4.6 INFLAMMATORY LIPID MEDIATORS 303
14.5 THE PROCESS OF INFLAMMATION 304

14.5.1 ACUTE INFLAMMATORY RESPONSE 304
Localized Inflammation 304
Systemic Acute-phase Response 305
14.5.2 CHRONIC INFLAMMATORY RESPONSE 306
14.6 ANTI-INFLAMMATORY AGENTS 308

14.6.1 ANTI-CAM ANTIBODIES 309
14.6.2 CORTICOSTEROIDS 309
14.6.3 NON-STEROIDAL DRUGS 309
‡ E L I S A — I N D I R E C T A S S AY 310
SUMMARY 311
KEY WORDS 311
QUIZ YOURSELF 312
FURTHER READING 313
CONTENTS xxi
15 Immune Response to Infectious Agents

15.1 INTRODUCTION
314
315
15.2 IMMUNITY TO VIRUSES 316

15.2.1 INNATE IMMUNE RESPONSE TO VIRUSES 316
15.2.2 VIRAL NEUTRALIZATION BY ANTIBODY AND COMPLEMENT 317
15.2.3 T-CELL-MEDIATED ANTIVIRAL MECHANISM 318
15.3 VIRUS STRATEGIES FOR THE EVASION OF HOST IMMUNE RESPONSE 319
15.3.1 VIRAL AVOIDANCE OF IMMUNE RESPONSE 320
Downregulating Cellular Protein 320
Evasion of Antibody Detection 320
Evasion of T-cell Response 320
Hiding in Immune-privileged Sites 320
Evasion by Inhibiting Host Immunity 320
15.4 VIRAL INFECTIONS: INFLUENZA 322

15.4.1 THE INFLUENZA VIRUS 322
15.4.2 ANTIGENIC DRIFT AND ANTIGENIC SHIFT 323
15.4.3 IMMUNE RESPONSE TO INFLUENZA INFECTION 323
15.5 IMMUNITY TO BACTERIAL INFECTIONS 324

15.5.1 FIRST LINE OF DEFENCE 324
15.5.2 IMMUNE RESPONSE TO EXTRACELLULAR BACTERIA 324
Innate Defence 324
Adaptive Immune Response 325
15.5.3 IMMUNE RESPONSE TO INTRACELLULAR BACTERIA 325
Innate Immune Response 325
Adaptive Immune Response 326
15.5.4 EVASION OF HOST DEFENCES BY BACTERIA 326
Antiphagocytic Mechanism 327
Feigning of Antigen (Antigenic Disguise) 328
Neutralization of Antibody 328
Changing Antigens on Bacterial Surface 329
15.6 BACTERIAL INFECTIONS 329

15.6.1 CORNYEBACTERIUM DIPHTHERIAE 329
15.6.2 MYCOBACTERIUM TUBERCULOSIS 330
15.6.3 LYME DISEASE: BORRELIA BURGDORFERI 331
15.7 PROTOZOAN DISEASES 332

15.7.1 MALARIA 332
Pathogenesis of Malaria 333
Host Immune Response to Plasmodium 334
Antimalarial Drugs and Vaccines 334
15.7.2 AFRICAN SLEEPING SICKNESS 335
Pathogenesis of Trypanosomiasis 335
Host Immune Response 335
15.7.3 LEISHMANIASIS 335
Host Response to Leishmania spp. 336
xxii CONTENTS
15.8 DISEASES CAUSED BY PARASITIC WORMS 337

15.8.1 HOST IMMUNE RESPONSE 337
15.8.2 EVASION OF IMMUNE MECHANISM BY HELMINTHS 338
‡ E L I S A — A N T I B O DY S A N D W I C H A S S AY 339
SUMMARY 340
KEY WORDS 340
QUIZ YOURSELF 340
FURTHER READING 341
16 Vaccines
16.1 INTRODUCTION
342
343
16.2 TYPES OF VACCINES 344

16.2.1 NATURAL LIVE VACCINES 345
16.2.2 LIVE ATTENUATED VACCINES 345
Advantages of Attenuated Vaccines 346
Disadvantages of Attenuated Vaccines 346
16.2.3 INACTIVATED VACCINES 346
16.2.4 TOXOID VACCINES 347
16.2.5 POLYSACCHARIDE VACCINES 347
16.2.6 RECOMBINANT ANTIGEN VACCINES 348
16.2.7 LIVE VECTOR VACCINES 348
Viral Vector Vaccine 349
Bacterial Vector Vaccine 350
16.2.8 DNA VACCINES 351
16.3 NEW VACCINE STRATEGIES 353

16.3.1 SYNTHETIC VACCINES 354
16.3.2 MICROENCAPSULATION DELIVERY SYSTEM 354
16.3.3 LIPOSOMES AND MICELLES 354
16.3.4 ISCOMS—IMMUNE-STIMULATING COMPLEXES 355
16.3.5 SOLID MATRIX-ANTIBODY-ANTIGEN COMPLEXES (SMAA) 355
16.3.6 ANTI-IDIOTYPE VACCINES 355
16.3.7 ANTIGEN-COCHELATE 355
16.4 WHAT SHOULD AN IDEAL VACCINE HAVE? 355

‡ I O N E XC H A N G E C H R O M ATO G R A P H Y 357
SUMMARY 357
KEY WORDS 358
QUIZ YOURSELF 358
FURTHER READING 359
17 Transplantation Immunology
17.1 INTRODUCTION
360
361
17.1.1 TRANSPLANTATION ANTIGEN 362
CONTENTS xxiii
17.1.2 IMMUNOLOGY OF ALLOGENEIC TRANSPLANTATION 363

Presentation of Allogeneic MHC to T Cells 363
T-cell Response to Alloantigens in vitro: Mixed Leukocyte Reaction 364
Cell-mediated Response to Allografts in vivo 365
17.2 TYPES OF GRAFT REJECTION 367

17.2.1 HYPERACUTE REJECTION 367
17.2.2 ACUTE REJECTION 368
17.2.3 CHRONIC REJECTION 368
17.3 IMMUNOSUPPRESSIVE THERAPY OF ALLOGRAFT REJECTION 369

17.3.1 ANTIGEN-NON-SPECIFIC IMMUNOSUPPRESSIVE AGENTS 369
Cyclosporin A 369
FK-506 (Tacrolimus) 370
Rapamycin 370
Mycophenolate Mofetil (MMF) 370
Corticosteroids 370
Azathioprine/Cyclophosphamide 370
17.3.2 SPECIFIC IMMUNOSUPPRESSIVE AGENTS 371
Monoclonal Antibodies to T-cell Antigens 371
Blocking Costimulatory Signals 371
17.4 IMMUNOLOGY OF XENOGENEIC TRANSPLANTATION 372

17.4.1 HYPERACUTE XENOGRAFT REJECTION 372
17.4.2 DELAYED XENOGRAFT REJECTION 373
17.4.3 T-CELL-MEDIATED XENOGRAFT REJECTION 373
17.5 TRANSPLANTS TO PRIVILEGED SITES 373
17.6 ORGAN TRANSPLANTATION 373

17.6.1 KIDNEY TRANSPLANTATION 373
17.6.2 LUNG TRANSPLANTATION 374
17.6.3 HEART TRANSPLANTATION 374
17.6.4 LIVER TRANSPLANTATION 375
17.6.5 PANCREAS TRANSPLANTATION 375
17.6.6 SKIN TRANSPLANTATION 375
17.6.7 BONE MARROW TRANSPLANTATION 375
17.7 GRAFT VERSUS HOST DISEASE (GVHD) 376

‡ I M M U N O F LU O R E S C E N C E 377
SUMMARY 378
KEY WORDS 378
QUIZ YOURSELF 379
FURTHER READING 379
18 Cancer and the Immune System

18.1 INTRODUCTION
380
381
18.1.1 MALIGNANT TRANSFORMATION OF CELLS 382
18.1.2 ONCOGENES AND CANCER INDUCTION 383
xxiv CONTENTS
Induction of Cellular Proliferation 384

Activation of Proto-oncogene to Oncogene 385
Tumour Suppressor Genes 385
Immortalization and Transformation 386
18.2 TUMOURS OF THE IMMUNE SYSTEM 387

18.2.1 TUMOUR ANTIGENS 387
18.2.2 TUMOUR-SPECIFIC ANTIGENS 387
Expression of Antigen in the Wrong Place 387
Virus-induced Tumour-specific Antigens 387
Mutated Cellular Gene Product as Tumour-specific Antigen 388
18.2.3 TUMOUR-ASSOCIATED ANTIGENS 388
Oncofoetal Tumour Antigen 388
Oncogenic Product as Tumour Antigen 389
Aberrantly Expressed Normal Cell Protein 389
Altered Glycolipid and/or Glycoprotein Antigens 389
18.3 IMMUNE RESPONSE TO TUMOURS 390

18.3.1 T-CELL-MEDIATED IMMUNITY 390
18.3.2 NK-CELL- AND MACROPHAGE-MEDIATED IMMUNITY 390
18.4 EVASION OF IMMUNE RESPONSE BY TUMOURS 392

18.4.1 DOWNREGULATION OF CLASS I MHC MOLECULES 392
18.4.2 BLOCKING OF TCYT RESPONSE BY ANTIBODIES 393
18.4.3 MODULATION OF TUMOUR ANTIGENS 393
18.4.4 LACK OF COSTIMULATORS 394
18.4.5 SUPPRESSION OF ANTI-TUMOUR IMMUNE RESPONSE 394
18.4.6 ANTIGEN MASKING 394
18.4.7 PREVENTING INFLAMMATORY RESPONSE 394
18.5 IMMUNOTHERAPY FOR CANCER 394

18.5.1 STIMULATION OF ACTIVE IMMUNITY AGAINST TUMOUR 394
Augmentation of Immune Response by Costimulators 395
Stimulation by Cytokines 395
Augmentation of Antigen Presentation 396
18.5.2 NON-SPECIFIC STIMULATION OF THE IMMUNE SYSTEM 396
18.5.3 PASSIVE IMMUNOTHERAPY FOR TUMOURS 397
18.5.4 ADOPTIVE CELLULAR IMMUNOTHERAPY 398
18.5.5 HUMORAL IMMUNOTHERAPY 398
‡ I M M U N O B LOT T I N G 400
SUMMARY 401
KEY WORDS 401
QUIZ YOURSELF 402
FURTHER READING 403
19 Primary and Secondary

Immunodeficiencies 404
19.1 INTRODUCTION 405
19.1.1 PRIMARY IMMUNODEFICIENCY 405
19.1.2 SECONDARY OR ACQUIRED IMMUNODEFICIENCY 405
CONTENTS xxv
19.2 PRIMARY IMMUNODEFICIENCIES 406

19.2.1 LYMPHOID CELL DISORDER 406
Severe Combined Immunodeficiency (SCID) 406
Defects in B-cell Maturation 409
Defects in T-cell Development 412
Combined B-cell and T-cell Disorders 413
19.2.2 DEFECTS IN MYELOID LINEAGE 414
Chronic Granulomatous Disease (CGD) 414
Leukocyte Adhesion Deficiency-1 (LAD-1) 414
Chediak–Higashi Syndrome 415
Neutropenia or Granulocytopenia 415
19.3 DEFECTS IN THE COMPLEMENT SYSTEM 416
19.4 TREATMENT APPROACHES FOR IMMUNODEFICIENCY 416
19.5 ANIMAL MODELS OF PRIMARY IMMUNODEFICIENCY 416

19.5.1 NUDE MOUSE 416
19.5.2 SCID MOUSE 417
19.5.3 CBA/N MOUSE 417
19.5.4 BEIGE MOUSE 417
19.6 SECONDARY IMMUNODEFICIENCY AND AIDS 417

19.6.1 THE AIDS EPIDEMIC 417
19.6.2 THE HIV VIRUS 418
The Genetic Composition of HIV 418
The Life Cycle of HIV 419
19.6.3 HIV’S MECHANISM OF IMMUNOSUPPRESSION 420
19.6.4 MECHANISM OF EVASION USED BY HIV 422
19.6.5 THE COURSE OF HIV INFECTION AND AIDS 422
19.6.6 TREATMENT AND PREVENTION OF AIDS 423
Development of Effective Drugs 424
Development of an Effective Vaccine 424
‡ R A D I O I M M U N O A S S AY 425
SUMMARY 426
KEY WORDS 426
QUIZ YOURSELF 427
FURTHER READING 427
20 Autoimmunity and Autoimmune

Diseases 428
20.1 INTRODUCTION 429
20.2 SINGLE-ORGAN AUTOIMMUNE DISEASE 430

20.2.1 AUTOIMMUNE DISEASES DUE TO TISSUE DESTRUCTION OF ORGANS 430
Chronic Thyroiditis (Hashimoto’s Thyroiditis) 430
Pernicious Anaemia 431
Autoimmune Haemolytic Anaemia 431
Drug-induced Haemolytic Anaemia 431
Thrombocytopenic Purpura 433
xxvi CONTENTS
Goodpasture Syndrome 433

Insulin-dependent Diabetes Mellitus (Type I Diabetes) 433
20.2.2 AUTOIMMUNE DISEASES INDUCED BY ANTIBODY BINDING 433
Graves’ Disease (Hyperthyroidism) 433
Myasthenia Gravis 434
20.3 SYSTEMIC AUTOIMMUNE DISEASES 435

20.3.1 SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) 435
20.3.2 RHEUMATOID ARTHRITIS 435
20.3.3 MULTIPLE SCLEROSIS 437
20.3.4 SCLERODERMA 437
20.3.5 GUILLIAN–BARRÉ SYNDROME 437
20.4 ANIMAL MODELS OF AUTOIMMUNE DISEASES 437

20.4.1 MODELS OF SPONTANEOUS AUTOIMMUNITY 437
20.4.2 MECHANISM FOR INDUCTION OF AUTOIMMUNITY 438
Failure of B-cell Tolerance 438
Molecular Mimicry by Cross-reactive Microbial Antigens 438
Availability of Sequestered Self-antigens 440
Aberrant Expression of Class II MHC Molecules 440
20.5 THERAPEUTIC APPROACHES TO AUTOIMMUNE DISEASES 442
20.6 OTHER STRATEGIES 442

20.6.1 TOLERANCE INDUCTION 443
20.6.2 MONOCLONAL ANTIBODY AGAINST AUTOANTIGENS 443
20.6.3 BLOCKAGE OF MHC MOLECULES 444
20.6.4 INDUCTION OF T-CELL SUPPRESSION 444
20.7 ROLE OF MHC, TH CELLS AND TCR IN AUTOIMMUNITY 444

20.7.1 ROLE OF MHC 444
20.7.2 TH CELLS IN AUTOIMMUNITY 444
20.7.3 ROLE OF T-CELL RECEPTORS IN AUTOIMMUNITY 445
‡ E L I S P OT 445
SUMMARY 446
KEY WORDS 446
QUIZ YOURSELF 446
FURTHER READING 447
GLOSSARY 449
INDEX 461
A B B R E V I AT I O N S
ABC ATP-binding cassette proteins IgSF Immunoglobulin superfamily

ADCC Antibody-dependent cell-mediated Ii Invariant protein
cytotoxicity
IL Interleukin
AID Activation-induced cytosine
deaminase KAR Killer activatory receptor
APC Antigen-presenting cells KIR Killer inhibitory receptor
BALT Bronchus-associated lymphoid tissue MAC Membrane-attack complex
CD Cluster of differentiation MALT Mucosa-associated lymphoid tissue
CDR Complementarity determining region MBP Mannose-binding proteins
CLIP Class-II-associated invariant chain MHC Major histocompatibility complex

peptide MIIC Class II MHC compartment
CRP C-reactive complement protein MMC Mucosal mast cells
CSF Colony-stimulating factor NALT Nasal-associated lymphoid tissue
CTMC Connective tissue mast cells NK cells Natural killer cells
DTH Delayed-type of hypersensitivity PALS Periarteriolar lymphatic sheath
ER Endoplasmic reticulum PAMP Pathogen-associated molecular
ES Embryonic stem cells patterns
FcR Fc receptor PRR Pattern-recognition receptor
FR Framework region TAP Transporter of antigenic peptides
GALT Gut-associated lymphoid tissue TCR T-cell receptor
HLA Human leukocyte antigen Tcyt Cytotoxic T cells
HSC Haematopoietic stem cells TdT Terminal deoxynucleotidyl transferase
IFN Interferon TH Helper T cells
Ig Immunoglobulin TNF Tumour necrosis factor

THE AUTHOR
Fahim Halim Khan is a gold medallist in biochemistry from Aligarh Muslim University and a re-
cipient of the UGC–CSIR fellowship. His doctoral work was directed at studying the mechanism of
the key plasma proteinase inhibitor, α-2 macroglobulin, with a focus on its purification and charac-
terization. The methods and protocols devised by him are in use till date. Following the acceptance
of his doctoral thesis, he was offered a position at the Aligarh Muslim University.
Currently, Dr Khan is an assistant professor of biochemistry at the Aligarh Muslim University
where he works on identifying and characterizing novel mutations that cause glaucoma in the Indi-
an population. The central theme of his research continues to be the exploration of free-radical-in-
duced damage to the antiproteinase barrier that occurs in inflammatory diseases. He has published
numerous peer-reviewed papers in national and international journals. He was also the principal
investigator for the CSIR-funded study on the effect of free radicals on the defence mechanisms in
humans.
Dr Khan, with over thirteen years of experience, has taught varied courses in basic and ad-
vanced immunology at both the undergraduate and postgraduate levels. Extremely popular among
his students for his well-structured, lucid and stimulating classroom sessions, Dr Khan is known
for his ability to deconstruct complex observations into a series of simple steps—a style that is re-
flected in his treatment of the complex concepts and mechanisms of immunology in the book.
P R E FAC E
The science of immunology is primarily directed towards understanding the immune system—
how it develops, functions and sometimes malfunctions, thereby causing disease. It is no surprise
then that immunology is more closely related to human health and well being than any other
discipline of biological science. That we can now manipulate the human immune system to fight
a number of diseases including cancer has propelled research in potential treatments of these
diseases—from drug, cytokine, cell and antibody-based treatments to gene therapy and cancer
vaccine—as is evident from the volume of publications in this area in recent times.
The rise of immunology to this position of prominence has been accompanied by an increas-
ingly pressing need for texts that are appropriate for different levels of courses. Most existing texts
are written at the “comprehensive” level despite the fact that most students and beginners would
benefit from a text that explains the basic concepts of immunology without going into overwhelm-
ing details.
As a student, I dreamt of such a textbook. In my years as a teacher, I have observed this need
in my students as well. From experience, I have learnt that a text on immunology is best utilized
when accompanied by colourful artwork. This, in combination with a concise and structured style
of presentation, ensures that the students are not intimidated by the details of the subject matter.
Finally, a student can never learn from a text that they cannot comprehend. So when I decided to
write a book on immunology, I knew what I had to do.
My intention was to write a “student-friendly” textbook of immunology that introduces the
intellectual framework of the discipline. True to its title and in keeping with my intention, this
book emphasizes the fundamentals of immunology and immunological processes. This book edu-
cates, not intimidates or overwhelms students. Though this text carries encyclopaedic content, it is
concise in presentation. Several pedagogical features have been incorporated into this text to help
students master its content. The comprehension of core concepts is facilitated as there is minimal
use of complex jargon.
SEE THE BIG PICTURE

Immunology is a broad discipline that encompasses specialities as diverse as biochemistry, clinical
biology, medicine and molecular genetics. This book provides a balanced introduction to this dis-
cipline. It considers each concept in context with etymological roots and historical perspective and
then takes the reader through the more detailed current and updated research in the field. Care has
been taken to ensure that students are not distracted by details till they understand the concept. A
deliberate effort has been made to avoid any complex topics at initial levels, which usually distract
students and hinder the formation of the big picture in their minds. The comprehensive treatment
of a topic is broken down into simple and easy-to-remember steps that facilitate comprehension.
VISUALIZE THE CONCEPT

The text of this book is tied to a consistent art programme that includes more than 300 colour-
ful conceptual diagrams apart from photographs and micrographs. This book “shows” you the
concepts described in the text. Even within the figures representing dynamic processes, complex
molecular mechanisms have been deconstructed into a number of simple steps to facilitate com-
prehension. These illustrations are integrated with the text that helps in mastering complex topics
with ease.
RICH PEDAGOGY ENHANCES LEARNING

The opening vignette talks about real people, contributors and landmark experiments that bring
immunology alive to the reader. To enrich the content of various topics without disrupting the
flow of information, hundreds of interesting facts are presented in the form of margin snippets
and margin definitions. Margin snippets present lesser-known interesting trivia in an attractive,
xxx P R E FA C E
easy-to-remember format. Margin definitions, presented in similar format, impart added value by
highlighting definitions of terms encountered within a particular topic. Further, focused learning
objectives and a detailed summary, along with numerous end-of-chapter questions make learning
easier.
Each chapter incorporates a pertinent laboratory technique in a nutshell. This section
describes the principle, methodology and application of a particular technique in a concise and
easy-to-understand way. These techniques provide the student with a sense of both the experiment
and its application. These can be read in isolation to gain useful insights.
THE TEACHING AND LEARNING PACK AGE

This textbook is packed with supplementary and media resources including lecture slides, clinical
case studies and hundreds of study questions on the companion Web site, which takes the learning
experience to a whole new level. There are more than 30 animations linked closely to the text that
will help students explore various key concepts. These animations provide a vibrant and clear de-
piction of various fundamental concepts and processes, which helps to visualize molecular mecha-
nisms, thereby enhancing the learning experience.
INSTRUCTORS’ RESOURCES
Instructors’ resources include a question bank (a database of 400 multiple choice questions from
all the twenty chapters of the book) and PowerPoint presentations for each chapter. These presen-
tations, which include both text and artwork, cover each of the 20 chapters. All the figures have
been optimized for projection with enhanced colours, higher resolution and enlarged fonts for easy
reading in the lecture hall.
ACKNOWLEDGEMENTS
It is a great pleasure to acknowledge the contribution and support of people who helped to mould
this book in its present form.
It is a great pleasure to acknowledge the late Mr Saleemuddin Ahmad, an illustrator par excel-
lence, who deciphered and transformed my tiny scribbles into visually appealing diagrams. The
efforts of Modabir Azam and his team, particularly computer whiz kid Goldy, who transformed
these diagrams into a piece of art, is gratefully acknowledged.
I also gratefully acknowledge the invaluable assistance of the editorial team at Pearson
Education.
I would like to thank family members and friends, especially Ahmad Waseem, Saima Waseem,
Naila Waseem, Hina Rafiq, Amir Rafiq and Nishi Rafiq, for their forbearance and support. I would
specially like to thank my two sisters, Naushin and Sumbul, for their encouragement and support,
and for not letting me quit the Herculean task of writing this textbook. To them, I owe a lot.
I would also like to acknowledge immense support and affection of all of my students, from
whom I continue to learn. I look forward to more cooperation with my students, some of whom
have mental antennae with a keen reception that anybody would aspire to.
Finally, and most importantly, I wish to thank my best friend and lifeline, my mom, who over
the years has smoothed the course of my life, but passed away suddenly before this book could see
the light of the day.
A Student’s Guide to Using This Text
The following pages walk you through some of the main features of this text.
The learning framework in this book will help you develop the essential knowledge
and skills you need to succeed in and enjoy immunology.
The opening vignette brings

alive the world of immunology
by describing landmark
experiments and significant
contributors.
Focused learning objectives

present a convenient overview
of the key concepts covered in
the chapter.
xxxii A STUDENT’S GUIDE TO USING THIS TEXT
Whenever you see this icon, log on to the Margin snippets highlight thought-provoking,
companion Web site and view the animation new, and lesser-known facts to enrich the
to understand the concept better. learning experience.
8 THE ELEMENTS OF IMMUNOLOGY AN INTRODUCTION TO THE IMMUNE SYSTEM 9
Tissue injury region which results in the deposition of insoluble strands of fibrin. This seals off the injured area
from the rest of the body, thereby preventing the spread of infection. This pus, enclosed in a wall of
fibrin, usually forms an opening on the surface of the body, from where it empties out. The fibrin
clot dissolves, tissue repair occurs and scar tissue is formed.
ACUTE PHASE PROTEINS

Stimulate Acute-phase proteins are a group of heterogeneous plasma proteins that are important in the innate
Phagocytic assault Antibody complement
on microbes pathway defence against microorganisms (mostly bacteria and protozoa) and in limiting the damage caused
by infection, trauma, malignancy and other diseases to tissues. In response to tissue insult, cells « Following tissue injury the con
Microbes
centration of acute phase proteins
circulating in the blood, such as macrophages and neutrophils, secrete a variety of cytokines that n blood can increase (or decrease)
stimulate the liver to produce acute-phase proteins. Some important acute-phase proteins include by 25 per cent or more
C-reactive complement protein (CRP) components, mannose-binding metal protein (MBP), bind-
ing proteins and protease inhibitors Acute-phase proteins function by stimulating phagocytosis
Mast cells and Basophils Opsonins
(acting as opsonins), activating the complement system and clumping the invading microbes. All The molecules that bind to the
these aid in the non-specific removal of pathogens. CRPs have an aesthetically designed pentagonal surface of pathogens and increase
the r susceptibility to phagocytosis
Vasoreactive and
structure to clump bacteria (Pneumococci) that bear C proteins on their surface. CRPs are present
are cal ed opsonins These nclude
chemotactic factors in primitive invertebrates. MBP binds to the mannose residue of glycolipids present on the surface antibodies and comp ement
Diapedesis
of protozoan and microbial cells and activates the complement system. Complement components mo ecules that bind pathogens
on the one hand and phagocytes
that act as acute-phase proteins, act as opsonins as well. These complement proteins coat the in-
on the other thereby enhancing
vading microbes, making them susceptible to phagocytosis. The functions of some acute-phase phagocytos s
proteins are listed in Table 1.1.
Margination Neutrophils
In addition to the soluble molecules of the innate immune system, an increasing number
of cell-surface receptors present on a variety of cells in the human body also provides a major
defence against invading pathogens. The recep-
Figure 1.5 tors are called pattern-recognition receptors
The major events of inflammat on (PRR) (see Figure 1.6). PRRs comprise a group Lipopolysaccharide receptor
Inflammat on is a non specifc immune
of proteins that are used by cells of the immune
response that s st mu ated by a variety
of actors system to identify conserved molecules common
to pathogens. Though they do not have the abso- Scavenger Toll
lute specificity of lymphocytes, they have evolved receptor receptor
microglia (in brain). However, tissue macrophages are present in small numbers initially. In the to recognize molecular patterns associated with
case of inflammation, these macrophages immediately proceed towards the injury site to begin different types of pathogens. These structures on
their phagocytic actions. They serve as the first line of defence against infection. microbes are called pathogen-associated molec-
The next step in inflammation is the redirection of blood phagocytes towards the lesion site. ular patterns (PAMP). Examples of PAMPs in-
This is facilitated by vasodilation of blood vessels and capillaries at the site of injury. The increase clude bacterial molecules such as peptidoglycans, Macrophage
in the diameter of blood vessels is brought about by chemical mediators like histamine and brady- teichoic acid, lipopolysaccharide, flagellins and
Figure 1.6
kinins. These mediators bind to the receptors on nearby capillaries and vessels, causing vasodi- viral double-stranded RNA. PRR include Toll- Mannose Pattern recogn tion receptors involved in
lation. The engorged capillaries are responsible for increased blood accumulation and the redness like receptors (which bind lipopolysaccharide and receptor the nnate immune system
of the inflamed tissue (erythema).
The increased permeability of the capillaries permits the influx of fluid from the engorged
Protein Function
capillaries into the site of irritation or injury. The accumulation of fluid at the site of irritation
C react ve protein Binds C polysaccharide of S pnemoniac; b nds phosphatidylcho
results in tissue swelling (oedema).
l ne of microbes damaged t ssue and activates complement (C1)
pathway/cascade
MARGINATION AND DIAPEDESIS The products released from the inflamed area also cause
phagocytes (now mainly neutrophils) to move towards the inflamed area, a process facilitated Serum amyloid protein Binds DNA activates complement (C1) pathway/cascade
by vasodilation and increased capillary permeability. The emigration of phagocytes involves the Complement components C2 C3 C4 C5 C9 Chemotax s and cell lysis
adherence of the cells to the capillary walls, a process called margination. The adhered phagocytes Factor B
then pass from the blood vessels into tissue spaces through the spaces between capillary endothelial Haptoglob n/ Haemopexin Binds iron and makes it unavailable for bacterial growth
Necrosis cells. This process is termed diapedesis. Once in the tissue spaces, the phagocytes migrate towards
Necrosis is the death of one or more Ceruloplasmin Binds copper and renders it unavailable for bacteria
cel s due to injury to the ce l. the injured tissue. Mannose binding prote n; Lipopolysaccharide Binds mannose and l popolysaccharide on bacterial surface
Once phagocytes engulf the invading bacteria and the necrotic tissue, many phagocytes even- bind ng protein (respectively) acts as opsonin act vates complement pathway/
tually die. However, some enzymes may leak out into the extracellular environment from the neu- cascade
Apoptosis trophils before the phagosome closes. This process, termed sloppy eating, damages healthy cells. Fibr nogen Blood coagulation
Apoptosis s a type of cell death After a few days, a cavity containing necrotic tissue, dead bacteria and dead phagocytes is formed
through which unwanted ce ls at the site of inflammation. This fluid mixture is often called pus. Ordinarily, pus formation con- Von Willebrand factor
commit su c de Table 1.1
tinues until all infection is suppressed. The blood-clotting system is also activated in the inflamed Antitrypsin Antichymotrypsin Proteinase nhibitor
Acute phase prote ns and their functions
Margin definitions highlight definitions

of terms that appear within the text. Set
in boxes, these highly visible definitions
ease recapitulation of key terminology.
A consistent art programme helps

visualize concepts and facilitates
comprehension.
A STUDENT’S GUIDE TO USING THIS TEXT xxxiii
EXPERIMENTAL INSIGHT
Flow Cytometry/Fluorescence Activated Cell Sorter
Flow cytometry is a technology that measures and then analyses Cells in buffer
various components or structural parameters of cells primarily by
optical means. Cells from solid tissue is first disaggregated before
they are subjected to flow cytometry. The tissue sample is broken
up into single cells. Any cell which is 0.5–150 μm in size is suitable
for analysis. These cells are then hydrodynamically focused to form a
thin stream of cells which is pumped into a flow chamber. Cells flow Experimental Insight presents a
through the flow chamber one at a time at a speed of about 500
cells per second. The cells may be alive or fixed (dead) at the time of pertinent immunological technique
measurement. A small laser beam hits the cells as they pass through
the flow chamber. Every cell that is hit by laser scatters some of the
in a nutshell to familiarize students
laser light, and also emits a fluorescent light after getting hit by the with various prevalent methods.
laser. The way the light scatters off each cell gives information about Side-scatter
the cell’s physical characteristics. Light can be bounced off at low detector
angles, which is called forward scatter. Low angle forward scatter is
directly proportional to cell diameter. Light scattered off in other di-
rections is called side scatter. The side scatter which is collected Forward-scatter
detector Incident laser beam
at 90˚ to the incident laser beam is directly proportional to the
quantity of granular structures or complexity in the cell. Scattered
and emitted light from cells are converted to electrical pulses by op-
tical detectors (see Figure 2.23). The detectors produce electronic
signals which are proportional to the optical signals striking them.
The fluorescent light, which is usually emitted by fluorescent probes, Laser
report specific components of cells such as cell surface markers. In
most of the applications, after a cell exits the laser beam, it is sent to
The Summary presents a compre-
waste. Cell sorting, takes this to the next level by allowing scientists hensive review of the concepts
to identify cells as they pass through the laser and then mark them
for sorting.For such sorting,cells are first labelled with a fluorescent discussed in the chapter.
dye that binds specific target molecule on the cell surface. A few
micro-seconds later, this marked cell is deflected away from the rest
S U M M A R Y
of the sample and collected in a separate tube. This fluorescence-
activated cell sorting allows the capture and collection of cells of
• The body s resistance to disease-causing pathogens can be non- • Tcyt cells are responsible for destruction of tumour cells/virus-
interest for further analysis. The collected cells can be further charac- speci? c (innate immunity) or specific (adaptive immunity). infected cells/cells harbouring intracellular pathogens.
terized biochemically, functionally or microscopically.
• Innate immunity comprises (a) mechanical and chemical barriers, • Major histocompatibility complex (MHC) comprises membrane
Figure 2.23 (b) phagocytosis, (c) fever, (d) inflammation and (e) acute-phase proteins that are present on the cell surface and are involved in
Flow cytometry and, more specifically, fluorescence-activated
Flowcytometry proteins. antigen presentation.
cell sorting can be used to distinguish subpopulations of different
cell types, such as Tcyt from B cell or TH cells. It can also be used to detect and sep • Adaptive immunity has two exquisite qualities (a) specificity and • MHC proteins are of two main types—class I MHC and class II
(b) memory. MHC proteins.
measure total cellular DNA or newly synthesized DNA. It can also available.
• The two arms of adaptive immunity include humoral immunity (com- • Class I MHC proteins display antigenic peptides that originate
prising B cells) and cell-mediated immunity (comprising T cells). endogenously, that is, within the cytosol of the cell. Class I MHC
molecules present antigenic peptides to Tcyt cells.
• When a B cell encounters antigen via its membrane-bound antibody,
it multiplies and differentiates into plasma cells and memory cells. • Class II MHC proteins display exogenous antigens that enter the
cells via phagocytosis or endocytosis. TH cells recognize antigen
• Plasma cells secrete antibodies that neutralize and clear pathogens. only when they is associated with class II MHC proteins.
• T cells are comprised of two groups of cells—T helper (TH) cells • The immune system may sometimes misdirect itself or may break
and cytotoxic T (Tcyt) cell. down causing autoimmune diseases, hypersensitivity diseases or
• TH cells secrete cytokines that help B cells to divide, differentiate immunode? ciency diseases.
and secrete antibodies.
Key Words highlight the important terms K E Y W O R D S

discussed in the chapter.
• acute-phase protein • humoral immune response • primary immune response
• adaptive immunity • immune disorder • secondary immune
• antigen-presenting cell • inflammation response
• B cell • innate immunity • T cell
• cell-mediated immunity • mechanical barrier
• chemical barrier • MHC proteins
• fever • phagocytosis
R E V I E W Q U E S T I O N S
Review Questions have been designed 1. What is the need for an innate immune response when there is a 4. Why are those B and T cell eliminated or suppressed that are reac-
more specific adaptive immune defence mechanism? tive to self-antigens? What do you think happens in autoimmunity
to be used for reviewing the chapter H I N T —Innate immune response is immediate but non-specific adaptive when body mounts an attack on self-antigens?
defence is specific but requires time. H I N T —The body’s defence is made tolerant to self-antigens. This tolerance
contents. 2. Why is an individual passively immunized, when he or she, given
breaks down in autoimmunity.
time, can develop active immunity? 5. What is the difference between primary and secondary immune
H I N T —Given time! Sometimes pathogen action is so fast and lethal that response?
they do not give time to the person to react. So it is better to inject preformed H I N T —Time and type of antibody formed.
antibodies into infected individual.
3. Most virus and bacteria enter cells via specific receptors. Why
would a cell have receptors that allow pathogens to enter?
H I N T —You don’t build windows or doors so that burglars can enter; you
need them for your own convenience and need.
Quiz Yourself comprises multiple choice Q U I Z YO U R S E L F

questions designed to assess the reten-
Choose the Appropriate Option
tion of factual information from the
1. Which one of the following is NOT a chemical barrier? 6. The term vaccine was first used for:
chapter. (a) Stomach acid
(b) Interferon
(a) Fowl cholera bacilli
(b) Fluid from cowpox pustule
(c) Lysozyme (c) Crusts from smallpox vesicle
(d) Mucous (d) None of the above.
xxxiv A S T U D E N T ’S G U I D E T O USI N G T H IS T E X T
The companion Web site—www.pearsoned.co.in/fahimhalimkhan—features

a variety of resources that enable both students and instructors
to use the book to the maximum.
Learn includes chapter summaries Teach, meant exclusively for instruc- Practice features an
and a glossary. It features several tors, provides relevant material— exhaustive question bank
clinical case studies that help students PowerPoint slides, transparencies, an comprising questions both
identify symptoms as manifestations image bank and a solutions manual— for beginners and advanced
of immunological malfunctions. that can be downloaded and custom- learners.
ized for use in the classroom.
Animations help visualize complex molecular

mechanisms, and identify the participants and steps
in the process. These enhance comprehension An exhaustive question bank, with over 400
and retention of critical details. questions, covers every major topic in the book.
REVIEWERS
C O N S U LT A N T B O A R D
The consultant board provided us with a detailed and critical analysis of each chapter and worked
with us throughout the development of the book. We would like to thank the following for their
time and commitment:
Vivien Amonkar Ashok Munjal

St. Xavier’s College, Mumbai Banasthali Vidyapeeth, Banasthali
Milind M. Gore S. Subramanian
National Institute of Virology, Pune University of Madras, Chennai
Naveen Kaushal
Pennsylvania State University, U. S. A.
REVIEWERS
The guidance and thoughtful recommendations of many helped us improve this book. We are
grateful for the comments and helpful suggestions received from the following reviewers:
Sibaprasad Adhikary S. Mohan Karuppayil

Utkal University, Bhubaneswar Swami Ramanand Theerth Marathwada University,
Nanded
Aradhna
Sardar Bhagwan Singh Post Graduate Institute of Samir Mukherjee
Biomedical Sciences and Research, Dehradun Kalyani University
Mamatha Ballal
K. M. C. International Centre, Manipal H. K. Nayak
S .C. S. College, Bhubaneswar
Sheila Bedi
Patna Women’s College Kusum Paul
The Oxford College of Engineering, Bangalore
N. Behera
Sambalpur University Birendra Prasad
Patna University
Basanti Biswal
Sambalpur University A. N. Rafique
M. G. Science College, Ahmedabad
G. B. Chand
Patna University T. Raghava Rao
Andhra University, Visakhapatnam
A. K. Dalai
Ravenshaw University, Cuttack B. Sashidhar Rao
R. L. Deopurkar Osmania University, Hyderabad
University of Pune Biswajit Rath
Ravi Dhar North Orissa University, Bhubaneswar
National Institute of Immunology, Delhi
Gourab Roy
Smriti Dhawan Institute of Management and Computer Science,
G. G. D. S. D. College, Chandigarh Burdwan
M. Anwar Mallick Partha Roy
St. Columba’s College, Hazaribag Indian Institute of Technology Roorkee
xxxvi REVIEWERS
Mukesh Lal Shah Vivek N. Upasani

Kumaun University, Nanital M. G. Science College, Ahmedabad
S. K. Sinha Viveka Vardhini Vadlamudi
B. N. College, Patna Nagarjuna University, Guntur
A. K. Srivastava Jugsharan Singh Virdi
Marwari College, Bhagalpur University of Delhi, South Campus
Akshay Tikoo Shahla Yasmin
Sri Mata Vaishno Devi University, Udhampur Patna Women’s College
Devyani Tipre
Gujarat University, Ahmedabad
The Elements of
Immunology
All studies tracing the development of immunology begin by describing “This is the
variolation and Jenner’s vaccinations against smallpox. This implies short and long
that theories of acquired immunity had to wait till the 18th century of it.”
when Pasteur’s germ theory of disease was advanced. This is not true. —WILLIAM SHAKESPEARE,
The Merry Wives of Windsor, II, ii, 60
The earliest description of immunity came from Thucydides who gave

a contemporary description of the Plague of Athens of 430 BC. He noted
that plague never attacked the same man twice.
It is believed that Mithridates VI, King of Pontus, took an increasing

daily dose of poisons to render himself safe from attempts on his
life by poisoning. He believed that he would develop resistance to
these poisons on repeated exposure. Throughout the Middle Ages, a
specially concocted mixture of poisons, known as mithridaticum, was After studying this chapter,
you should be able to:
universally used by the members of the royal family to develop resistance
• Explain immunity and barriers
against poisoning. Around 1714, two Italian physicians, Emanuele against infection
• Describe phagocytosis and
Timoni and Jacob Pylarini, drew the attention of Western medicine to inflammation
the Eastern practice of variolation. • Describe acute-phase proteins
and pattern-recognition
receptors
This procedure was popularized by Mary Wortley Montagu, the wife • Explain active and passive
immunity
of the British ambassador to Constantinople, who had this technique • Distinguish between
B lymphocytes, T lymphocytes
applied to her own children for protection against small pox and antigen-presenting cells
(see Figure 1.1). • Differentiate between humoral
and cell-mediated immune
response
In 1798, an English physician, Edward Jenner, used the fluid from a • Describe primary and
secondary immune response
cowpox pustule and inoculated an eight-year-old boy. This boy was • Differentiate between class I
MHC and class II MHC proteins
later intentionally injected with smallpox. As predicted, the child did and their related processing
pathways
not develop smallpox. Though Jenner had no clear idea of how the
technique worked, the success of this bold step set the stage for
further progress.
An Introduction to
the Immune System 1
1.1 INTRODUCTION
The word immunology owes its origin to the Latin words immunitas and immunis, which are not « Arrhenius, the noted chemist and
remotely related to present day immunology. Initially, in Rome, these words implied “exemption of Nobel laureate, coined the term
immunochemistry.
an individual from service or duty”. Later, during the Middle Ages, it came to mean “the exemption
of the Church and its properties and personnel from civil control”. A. M. Silverstein, in his book The
History of Immunology, states that it was the Roman poet Marcus Annaeus Lucanus (ad 39–65) who
first applied the word immunes in the present-day biological context. Lucanus described the resist-
ance to snakebite of the Psylli tribe of North Africa in his poem Pharsalia. Later, in the 14th century,
Vaccination
Colle used the word to describe his escape from a plague epidemic when he wrote Equibus Dei gratia The deliberate introduction of
ego immunis evasi. Although these terms (immunis, immunitas, immunes) were used intermittently antigen into the host body with the
intention of eliciting a protective
thereafter, the word immunology attained currency only in the 19th century, following the rapid
immune response. Of more than
spread of Edward Jenner’s historic technique for small pox vaccination. Since then, immunity came 1,400 known viral, bacterial and
to be known as the ability of an individual to resist diseases. The study of the immune reaction was parasitic pathogens that infect
human beings, we have eradicated
initially referred to as immunochemistry. Gradually, the meaning of the word immunology evolved
only smallpox.
to mean an experimental discipline that manipulates the function of the immune system.
Figure 1.1
Lady Mary Wortley Montagu with her
son Edward Wortley Montagu, and
attendants. The wife of the British
ambassador to Constantinople, Lady
Montagu was one of the first to
popularize the practice of variolation in
the early 18th Century. (National Potrait
Gallery, London.)
4 THE ELEMENTS OF IMMUNOLOGY
By the start of the 19th century, society as a whole was better prepared to accept, and grow with,
the advancement of science in general, and immunology in particular.
1.2 THE COMPONENTS OF IMMUNITY

The human body provides two levels of protection from infectious diseases:
(a) non-specific resistance or innate immunity; and
(b) specific resistance or adaptive immunity.
1.2.1 I N N AT E I M M U N I T Y
The non-specific component of immunity, also known as innate immunity (Latin: innasci—to
be born in) is not directed against any particular pathogen but is a general defence mechanism as
the human body must constantly defend itself from microbial invasion. This defence mechanism
is active right from the time a child is born (hence innate). The specificity of innate immunity is
relatively low as it lacks the ability to distinguish one microbe from another.
Innate immunity provides the early lines of defence against microbes while adaptive immun-
ity against an antigen occurs after five to six days of antigen exposure. In general, most pathogens
encountered by a healthy individual are rapidly cleared within a few days of their entry into the
host body by the non-specific defences of the host, much before the full force of the adaptive
immune response is unleashed.
The principal components of innate immunity are (a) mechanical and chemical barriers, (b) phagocytosis,
(c) fever, (d) inflammation and (e) acute-phase proteins.
MECHANICAL BARRIERS
A mechanical or physical barrier refers to the various physical hindrances blocking the entry of
the microbes into the host body. Mechanical barriers include the skin and the mucous membrane.
These barriers act as the first line of defence against infection.
» The skin is the largest organ in the
human body.
SKIN. The skin consists of two distinct layers—the thinner outer epidermis and the thicker inner
dermis. The epidermis consists of several layers of tightly packed epithelial cells. The outer layer
of epidermis is coated with a tough protein called keratin that does not support viral replication
or penetration by bacteria. The inner layer of skin, the dermis, contains most of the skin’s living
Sebaceous glands structures such as blood vessels, nerve endings, elastic fibres, sweat glands and sebaceous glands.
Small glands attached to hair The intact surface of the healthy epidermis provides an excellent defence against penetration by
follicles, which secrete sebum onto
the body surface.
bacteria or viruses. Moreover, the epidermis of the skin is constantly shed off, resulting in the
continual removal of any clinging pathogens. Any break in the integrity of the skin facilitates
the entry of pathogens. The bacteria that are most likely to cause dermal infections under such
conditions are those that infect hair follicles, for example Staphylococci. Some viruses such as
Papillomoviruses (which produce human warts) enter the skin at sites where cuts and abrasions
have resulted in the loss of epithelial integrity.
The skin is also penetrated by insect bites (for example mosquitoes, ticks, fleas and flies). Pathogen-
infected arthropods introduce the pathogen into the host. Plasmodium, the causative agent of malaria,
is introduced into the human body by mosquito bites. Similarly, viruses such as human Papillomaviruses,
Myxomavirus and Flaviviruses are introduced into the host by arthropod vectors during feeding.
MUCOUS MEMBRANE. The regions of the body that are not protected by intact skin are lined by the
mucous membrane. The gastrointestinal tract, respiratory tract, urogenital tract and conjunctiva
all are lined by the mucous membrane.
The mucous membrane protects the human body in several ways. In the respiratory tract,
goblet cells secrete mucous that entraps dust and microbes, and is propelled by the action of cili-
» Sneezing generates an enor- ated epithelial cells, thereby clearing foreign material from the respiratory tract. The mucous mem-
mous number of small aerosolized brane of the gastrointestinal tract offers the same protection. Some (mechanical) factors that assist
particles that travel up to 5 feet
at speed of 100 feet per second, in protecting the mucous membrane include:
while coughing generates higher
aerosol speeds of up to 850 feet (a) the lavaging action of physiological fluids, for example tears and saliva, that assist in flush-
per second! ing microbes from the body;
AN INTRODUCTION TO THE IMMUNE SYSTEM 5
(b) the trapping action of mucous-coated

hair in the anterior chambers of the
nose;
(c) the expulsive effects of coughing and Lungs Skin
sneezing, which protect the respiratory (Low pH)
and gastrointestinal tracts; and
(d) the cool temperature of the upper respira-
tory tract which inhibits replication by
many viruses.
Stomach
However, the protection provided by the mucous
membrane against pathogenic invasion is not
enough. The mere ingestion of typhoid bacilli
Acid
or tubercle bacilli or Picornaviruses in sufficient
numbers will lead to penetration of the gastro-
intestinal mucosa. A similar penetration of the Enzymes
mucosal barrier of the conjunctiva by Leptospira
or adenoviruses, and of the respiratory tract by
Pneumococci or Rhinoviruses, can occur follow-
ing a heavy exposure to these pathogens.
CHEMICAL BARRIERS
Mechanical barriers alone do not account for
the remarkable resistance provided by the in-
nate defence mechanism to pathogenic invasion.
The host body has several chemical/physiological Figure 1.2
barriers that contribute to innate immunity. Antibodies Complement The various chemical and mechanical
Protein barriers to microbial attack.
These include acidic gastric secretions, low pH of
the skin, gastric and duodenal enzymes, antibod-
« pH — The term pH is derived from
ies and inhibitors, interferons, complement proteins and anti-microbial peptides (see Figure 1.2). the original French word puissance
de Hydrogen, meaning the power of
ACIDIC GASTRIC SECRETIONS. The physiochemical environment in the stomach appears to Hydrogen. Hence the small p and
the capital H!
be extremely inhospitable to invading pathogens. The secretion of hydrochloric acid by gastric
parietal cells maintains the pH of the stomach at 2.0, which kills most microorganisms (except
some resistant ones such as Hepatitis A virus, Picornaviruses and typhoid bacilli). « The gastrointestinal tract in
humans is 30 feet long!
LOW pH OF THE SKIN. Sebum, secreted by the sebaceous glands present in the dermis, contains
organic acids.These organic acids maintain the pH of the skin in the range of 3–5. This low pH
inhibits or retards the growth of most microorganisms present on the surface of the skin.
LYSOZYME. Lysozyme is a hydrolytic enzyme present in all mucous secretions, including tears,
saliva and nasal secretion. It can lyse Gram-positive bacteria by cleaving the peptidoglycan layer
found in the bacterial cell wall.
GASTRIC AND DUODENAL ENZYMES. A large array of enzymes, including proteases and lipases,
digests a variety of structural and metabolic chemical components of pathogens. Rhinoviruses, for Rhinovirus
example, are easily inactivated by gastric acids. In rare cases, however, the infectivity of the pathogen is Rhino is from the Greek word
rhin, indicating nose or nasal. The
increased by acidity: for example the infectivity of Coronavirus is enhanced by an acidic environment. common cold virus is an example of
rhinovirus.
ANTIBODIES AND INHIBITORS. The mucous secreted by gastric and intestinal cells usually contains
IgA molecules as well as non-specific inhibitors of viral infections(for example, sialic acid found in
mucous inhibits the attachment of influenza virus particles to cells). Antibodies (IgA) are also found
in tears and saliva. Secretory IgA protects the body surface against invading pathogenic microbes.
INTERFERONS. The name interferons refers to a group of proteins produced by virus-infected

cells that induce a generalized antiviral state in neighbouring un-infected cells. These proteins also
augment innate immunity.
COMPLEMENT PROTEINS. Complement proteins are a group of serum proteins (discussed in Chapter
10) that circulate in an inactive state in the plasma. A variety of specific and non-specific mechanisms
activate these proteins. The activated forms of these proteins damage the invading pathogen.
ANTIMICROBIAL PEPTIDES. All insects and mammals, including humans, secrete a number of
antimicrobial peptides, such as defensins, for their protection. The human body is protected by 1 μm
thick biofilm of defensins, that protects the external surface of the body from microbial assault.
PHAGOCYTOSIS
Phagosome
A membrane-bound intracellular Phagocytosis of invading microorganisms is another important innate defence mechanism. Phago-
vesicle formed by membrane infold- cytosis can be defined as ligand-induced uptake of particulate material of 150–200 nm diameter or
ing of the trapped phagocytosed more. This basically includes large particles such as cell debris and microbial cells.
material. A phagosome is also
referred to as a parasitophorous When bacteria or other invad-
vacuole. ing parasites penetrate the skin or
Bacteria the mucous membrane, phago-
Membrane infolding
cytes, such as neutrophils, blood
monocytes and tissue macro-
phages, surge towards the site
Phagosome of infection. These phagocytes
Lysosome engulf the bacteria to form a
large intracellular vesicle, called
Figure 1.3
phagosome, containing the bac-
Phagocytosis is a non-specific immune Phagolysosome teria. Then, the involuntary guest
response that destroys invading Golgi apparatus trapped within the phagosome
microorganisms. When a phagocyte
comes into contact with a microorganism,
is destroyed by fusing it with
it engulfs the organism to form a “granules” (lysosomes) found in
membrane-bound structure called the Absorption of digested the cytoplasm of the phagocytes.
phagosome. This fuses with lysosmes substance These granules discharge their
to form a phagolysosome. The release Egestion of debris
of lysosomal enzymes degrades the contents (enzymes and reactive
bacteria. The useful products are oxygen species) inside the phago-
absorbed back into the cell while the
waste is egested out from the cell.
cytic vacuole, thereby degrading
the bacteria. As shown in Figure
1.3, the insoluble remnants of
degradation in the phagocytic vacuole are egested from the phagocyte.
It should be clarified that phagocytosis, which is non-specific, is different from specific receptor-
mediated endocytosis wherein extracellular molecules are ingested after they bind to specific
cellular receptors. Phagocytosis is also different from pinocytosis—the mechanism by which cells
take up fluids (and dissolved solutes) from the surrounding medium.
Obviously, phagocytes must be selective of cells and material they phagocytose, otherwise
normal cellular structures would be ingested. The occurrence of phagocytosis depends on the
following factors.
PRESENCE OF STRONG ELECTRIC CHARGE. Dead tissue or foreign particles (such as bacteria)
that have a strong electric charge on their surface are ideal for phagocytosis.
PRESENCE OF ANTIBODIES AND COMPLEMENT COMPONENTS ON THE CELL SURFACE. The

immune system develops antibodies against invading pathogens (such as bacteria) that adhere to
the bacterial membrane. These antibodies are recognized by the receptors present on phagocytes,
which bind them, making the bacteria susceptible to phagocytosis. A similar effect is mediated by
some complement components (such as C3b) that coat the bacterial surface.
SURFACE OF THE PARTICLE. The surface of the particle should be rough. This is best exemplified
by the fact that phagocytosis works more efficiently for non-encapsulated bacteria, but less so
for encapsulated bacteria which have a relatively smooth surface. To phagocytose such resistant
encapsulated bacteria, the immune system uses antibodies that bind the capsule on the bacteria,
enabling the phagocytic cells to ingest such microbes using their Fc receptors.
FEVER
Fever is the condition of an Infection
abnormally high body tempera-
ture, accompanied by increased
Lipopolysaccharide
pulse rate and dry skin. It pro-
vides a non-specific defence
against disease. Fever is a physi-
ological response to infection.
As depicted in Figure 1.4,
many proteins, and breakdown Monocyte
activation
products of proteins, toxins and
lipopolysaccharides (particularly
from Gram-negative bacteria),
released by microbes can affect
the endothelial cells of the hy- Pyrogens
pothalamus to raise the body (cytokines)
temperature from its “set point”
of 36.5°C. Such substances that
can increase the body tempera-
ture are called pyrogens.
Gram-negative bacteria PGE2
release a class of very potent
Hypothalmus
endotoxins called endogenous Thermoregulatory
pyrogens. Just a few nanograms centre
Endotoxin
of endogenous pyrogens can Endotoxin is a metabolic poison
cause very high fever. This class produced by Gram-negative
bacteria. This microbial poison is
of molecules is released when
released only upon lysis of bacteria.
either the bacteria or its break-
down products bind to macro-
phages and neutrophils. These Figure 1.4
cells then release several cyto- The scheme of fever response. The
kines like IL-1β, TNF-α and IL-6 hypothalamus is the thermostat of the
body that maintains “normal” body
that act on the hypothalamus to 102˚Celsius temperature. Substances that act upon
produce fever. the hypothalamus to increase the body
Fever is beneficial to the temperature are known as pyrogens.
host because it inhibits the

growth of temperature-sensitive
pathogens. Also, increased cell metabolism encourages rapid tissue repair and phagocytosis.
I N F L A M M AT I O N
Inflammation is the reaction of living tissue to either an injury or an infection. Inflammation is
characterized by heat (calor), redness (rubor), swelling (tumor) and pain (dolor). It is a non-specific
response of the body to injury. The tissue injury could be caused by either mechanical agents (such
as cut or pinprick) or chemical agents (such as bee venom, acid or alkali). Physical agents (heat or
ultraviolet radiation) and infectious agents (such as bacteria or other pathogens) can also induce
inflammation.
The process of inflammation may be initiated by a variety of tissue products such as histamine,
bradykinin, serotonin and prostaglandins released by a number of cells (such as mast cells and
basophils, found in most tissues). The mediators released by damaged cells, chemicals released by
invading microorganisms, products of the complement system and reaction products of the blood-
clotting system also trigger the process of inflammation. Many of these substances strongly acti-
vate macrophages and other cells of the phagocytic system. However, the inflammatory response
occurs in several different stages as depicted in Figure 1.5.
ATTACK BY TISSUE MACROPHAGES. Macrophages are already present in the tissues—the

alveolar macrophages (in lungs), histocytes (in subcutaneous tissue), Kupffer cells (in liver),
Tissue injury
Stimulate
Phagocytic assault Antibody complement
on microbes pathway
Microbes
Mast cells and Basophils
Vasoreactive and
chemotactic factors
Diapedesis
Margination Neutrophils
Figure 1.5
The major events of inflammation.
Inflammation is a non-specific immune
response that is stimulated by a variety
of factors.
microglia (in brain). However, tissue macrophages are present in small numbers initially. In the
case of inflammation, these macrophages immediately proceed towards the injury site to begin
their phagocytic actions. They serve as the first line of defence against infection.
The next step in inflammation is the redirection of blood phagocytes towards the lesion site.
This is facilitated by vasodilation of blood vessels and capillaries at the site of injury. The increase
in the diameter of blood vessels is brought about by chemical mediators like histamine and brady-
kinins. These mediators bind to the receptors on nearby capillaries and vessels, causing vasodi-
lation. The engorged capillaries are responsible for increased blood accumulation and the redness
of the inflamed tissue (erythema).
The increased permeability of the capillaries permits the influx of fluid from the engorged
capillaries into the site of irritation or injury. The accumulation of fluid at the site of irritation
results in tissue swelling (oedema).
MARGINATION AND DIAPEDESIS. The products released from the inflamed area also cause
phagocytes (now mainly neutrophils) to move towards the inflamed area, a process facilitated
by vasodilation and increased capillary permeability. The emigration of phagocytes involves the
adherence of the cells to the capillary walls, a process called margination. The adhered phagocytes
then pass from the blood vessels into tissue spaces through the spaces between capillary endothelial
Necrosis cells. This process is termed diapedesis. Once in the tissue spaces, the phagocytes migrate towards
Necrosis is the death of one or more
cells due to injury to the cell. the injured tissue.
Once phagocytes engulf the invading bacteria and the necrotic tissue, many phagocytes even-
tually die. However, some enzymes may leak out into the extracellular environment from the neu-
Apoptosis trophils before the phagosome closes. This process, termed sloppy eating, damages healthy cells.
Apoptosis is a type of cell death After a few days, a cavity containing necrotic tissue, dead bacteria and dead phagocytes is formed
through which unwanted cells at the site of inflammation. This fluid mixture is often called pus. Ordinarily, pus formation con-
commit suicide.
tinues until all infection is suppressed. The blood-clotting system is also activated in the inflamed
region which results in the deposition of insoluble strands of fibrin. This seals off the injured area
from the rest of the body, thereby preventing the spread of infection. This pus, enclosed in a wall of
fibrin, usually forms an opening on the surface of the body, from where it empties out. The fibrin
clot dissolves, tissue repair occurs and scar tissue is formed.
ACUTE-PHASE PROTEINS
Acute-phase proteins are a group of heterogeneous plasma proteins that are important in the innate
defence against microorganisms (mostly bacteria and protozoa) and in limiting the damage caused
by infection, trauma, malignancy and other diseases to tissues. In response to tissue insult, cells « Following tissue injury, the con-
centration of acute-phase proteins
circulating in the blood, such as macrophages and neutrophils, secrete a variety of cytokines that in blood can increase (or decrease)
stimulate the liver to produce acute-phase proteins. Some important acute-phase proteins include by 25 per cent or more.
C-reactive complement protein (CRP) components, mannose-binding metal protein (MBP), bind-
ing proteins and protease inhibitors Acute-phase proteins function by stimulating phagocytosis
Opsonins
(acting as opsonins), activating the complement system and clumping the invading microbes. All The molecules that bind to the
these aid in the non-specific removal of pathogens. CRPs have an aesthetically designed pentagonal surface of pathogens and increase
their susceptibility to phagocytosis
structure to clump bacteria (Pneumococci) that bear C proteins on their surface. CRPs are present
are called opsonins. These include
in primitive invertebrates. MBP binds to the mannose residue of glycolipids present on the surface antibodies and complement
of protozoan and microbial cells and activates the complement system. Complement components molecules that bind pathogens
on the one hand and phagocytes
that act as acute-phase proteins, act as opsonins as well. These complement proteins coat the in-
on the other, thereby enhancing
vading microbes, making them susceptible to phagocytosis. The functions of some acute-phase phagocytosis.
proteins are listed in Table 1.1.
In addition to the soluble molecules of the innate immune system, an increasing number
of cell-surface receptors present on a variety of cells in the human body also provides a major
defence against invading pathogens. The recep-
tors are called pattern-recognition receptors
(PRR) (see Figure 1.6). PRRs comprise a group Lipopolysaccharide receptor
of proteins that are used by cells of the immune
system to identify conserved molecules common
to pathogens. Though they do not have the abso- Scavenger Toll
lute specificity of lymphocytes, they have evolved receptor receptor
to recognize molecular patterns associated with
different types of pathogens. These structures on
microbes are called pathogen-associated molec-
ular patterns (PAMP). Examples of PAMPs in-
clude bacterial molecules such as peptidoglycans, Macrophage
teichoic acid, lipopolysaccharide, flagellins and
Figure 1.6
viral double-stranded RNA. PRR include Toll- Mannose Pattern recognition receptors involved in
like receptors (which bind lipopolysaccharide and receptor the innate immune system.
Protein Function
C-reactive protein Binds C polysaccharide of S. pneumoniae; binds phosphatidyl-
choline of microbes, damaged tissue and activates complement
(C1) pathway/cascade
Serum amyloid protein Binds DNA, activates complement (C1) pathway/cascade
Complement components C2,C3, C4,C5,C9, Chemotaxis and cell lysis
Factor B
Haptoglobin/ Haemopexin Binds iron and makes it unavailable for bacterial growth
Ceruloplasmin Binds copper and renders it unavailable for bacteria
Mannose-binding protein; Lipopolysaccharide- Binds mannose and lipopolysaccharide on bacterial surface
binding protein (respectively), acts as opsonin, activates complement pathway/
cascade
Fibrinogen Blood coagulation
von Willebrand factor
Table 1.1
Antitrypsin, Antichymotrypsin Proteinase inhibitor
Acute-phase proteins and their functions.
» The innate immune system recog- bacterial lipoprotein among others), CD14 (which binds lipopolysaccharide), mannose receptors
nizes approximately 103 pathogen-
associated molecular patterns.
(mannose- and fucose-specific) and scavenger receptors (bind lipids/carbohydrate, lipopolysac-
charide and lipoteichoic acid). These receptors have been identified on macrophages, B cells and
dendritic cells (see Table 1.2).
1.2.2 ADAPTIVE IMMUNITY

In contrast to innate immunity, adaptive immunity is a more evolved and specific defence mecha-
nism. The characteristics of adaptive immunity are exquisite antigenic specificity and the ability
to “remember” different types of antigens. Adaptive immunity is activated only against invading
foreign material and never against its own molecules (except in autoimmune diseases). Thus, it has
the ability to distinguish between self and non-self. Since it can differentiate between a variety of
different antigens, invading pathogens and self-antigens, and induce different levels/types of im-
mune response, adaptive immunity is often referred to as specific immunity.
However, adaptive immunity is not independent of innate immunity. Adaptive and innate
immunity cooperate to produce a more effective, evolved and vast defence mechanism against
infectious agents. For example, phagocytes, a crucial part of innate defence, generate “signals”
that stimulate specific immune response. This, in turn, facilitates the participation of the specific
immune system in the destruction and elimination of pathogens. The adaptive immune system
produces various soluble factors that stimulate and increase the effectiveness of the innate immune
response. For example, some T cells synthesize and secrete soluble factors that increase the ability
of macrophages to kill the microbes they have engulfed. Thus, both the adaptive and innate
immune responses make up an integrated and interactive system of host defence that erects an
effective and formidable barrier to infection. Some important characteristics of the innate and
adaptive immune systems are listed in Table 1.3.
1.2.3 CELLS OF THE IMMUNE SYSTEM

An effective immune response is mediated by a variety of cells including neutrophils, lympho-
cytes, natural killer (NK) cells, eosinophils, basophils and antigen-presenting cells. The two
main groups of cells, lymphocytes and antigen-presenting cells, are briefly discussed here. (For
Lymphocyte details on all cell types, see Chapter 2.) Lymphocytes are mainly responsible for initiating adaptive
A type of white blood cell formed in immune response in the human body. All lymphocytes are produced in bone marrow stem cells by
the lymphoid tissue.
a process known as haematopoeisis. The two major populations of lymphocytes—T lymphocytes
(which develop in the thymus) and B lymphocytes (which develop in bone marrow)—are briefly
described here.
Micro-
organism
Cellular that Express
Name Structure Specificity Location Ligands
Mannose 180 kDa transmembrane Mannosyl/Fucosyl Macrophages, Pseudomonas
receptor receptor structures endothelial aeruginosa,
cells, dendritic Mycobacterium
cells tuberculosis
CD14 Phosphoinositolglycan- Lipopolysaccharides Macrophages E. coli,
linked cell- surface Neisseria spp.,
receptor
Salmonella spp.
Toll Transmembrane recep- Lipopolysaccharides, Macrophages, B Some Gram-
receptor tor having extracel- peptidoglycans, cells, antigen- positive and
lular leucine-rich repeat glucans, teichoic acid, presenting Gram-negative
domain arabinomannans cells bacteria
Scavenger Dimer or trimer, with Carbohydrates/lipids Macrophages Bacterial spp.,
receptor transmembrane, coiled Yeast spp.
Table 1.2 coil and collagen–like
Cell-surface receptors that recognize
domain.
pathogens.
Characteristics Innate Immunity Adaptive Immunity

Recognition Broad specificity—recognition of Highly specific—recognition of
conserved molecular patterns specific antigenic determinants
Diversity Limited Large
Immunogenic memory None Present
Self–Foreign discrimination Present Present
Genes of receptor No rearrangement required Rearrangement required
Response Rapid (minutes) Delayed (usually days)
Components Mechanical and chemical bar- Antibodies , T and B lymphocytes,
riers, phagocytes, natural killer antigen-presenting cells
cells, complement, acute-phase
Table 1.3
proteins,
Innate and adaptive immunity.
B LY M P H O C Y T E S
B lymphocytes originate in the bone marrow where they continue to differentiate and mature. Each B cell
is genetically programmed to encode a unique antigen-binding receptor on its membrane. This B-cell
receptor is actually a membrane-bound antibody molecule (see Figure 1.7). When a naïve B cell (which
has not previously encountered antigen) comes in contact with an antigen via its membrane-bound
antibody, it multiplies and differentiates into two types of cells—plasma cells and memory cells.
Figure 1.7
Surface antibody
molecules T-cell receptor Lymphocytes in the immune system.
Also known as white blood cells (WBC),
lymphocytes include B cells, helper T
cells and cytotoxic T cells. These cell
types are collectively responsible for
B cell T cell intiating adaptive immune response.
Plasma cells lack B-cell receptors. However, plasma cells produce large numbers of soluble
molecules called antibodies. B-cell receptors and antibodies differ only at their c terminal. B-cell
receptors have an additional transmembrane segment at their c terminal that anchors them to the
cell membrane. The antibody molecule is a large, polyfunctional glycoprotein found in blood and
other tissue fluids. One antibody molecule consists of two identical heavy chains and two identical
light chains. Each light chain is linked to one heavy chain to form a heterodimer. Two such het-
erodimers are linked by disulphide bonds to form a tetrameric Y-shaped antibody molecule. The
tips of the two arms of the Y-shaped molecule form the antigen-binding site. Thus, both the heavy
chain and the light chain form a part of the antigen-binding site.
Unlike plasma cells which last for a few days, memory B cells have a longer life (sometimes
up to 20 years) than naïve B cells. In humans, B cells last for several months. Memory cells express
some membrane-bound antibody molecules like their parent naïve cells. Memory cells are func-
tionally inactive unless they are stimulated by the same antigen again.
T LY M P H O C Y T E S
Like B lymphocytes, T lymphocytes too are produced in the bone marrow but they migrate to the
thymus to mature. During maturation, each T cell acquires a specific receptor on its membrane
termed as the T-cell receptor. A T-cell receptor does not recognize soluble antigens. It recognizes
antigens only when they are associated with a protein complex called the major histocompatibility
complex (MHC).
MHCs are diverse transmembrane glycoproteins present on a variety of cells. These molecules
present (display) the antigens to the cells of the immune system. MHC molecules are so named
because they were first identified as “antigens” responsible for the acceptance or rejection of tissue
grafts (histocompatibility). There are three classes of MHC proteins. Class I MHC molecules are
expressed on nearly all nucleated cells. They present antigenic determinants to a specific class of
effector T cells. Class II MHC molecules are expressed only by antigen-presenting cells. They pres-
ent antigens to the other class of effector T cells. Class III MHC proteins are of diverse types and
are indirectly involved in the immune response.
There are several different groups of T cells. One group interacts with antigen–class II MHC
molecule complex, gets activated and starts secreting cytokines. This group of T cells is called T help-
er (TH) cells. TH cells activate B cells via cytokines and help them to divide, differentiate and secrete
antibody molecules. Another group of T cells is responsible for the destruction of virus-infected host
cells, intracellular pathogens harbouring host cells, tumour cells and cells of a foreign tissue graft.
These T cells are called cytotoxic T cells (Tcyt). They recognize an antigen when it is associated with
class I MHC molecules. It should be emphasized here that T cells recognize antigens only when they
are presented in association with MHC molecules. The receptor that recognizes an antigen–MHC
complex is present on the cell membrane of T cells and is termed T-cell receptor (TCR).
ANTIGEN-PRESENTING CELLS
Any cell that is capable of processing and presenting antigens to the cells of the immune system
may be termed an antigen-presenting cell. However, this term is reserved for only those cells that
display antigens associated with class II MHC molecules. These antigen-presenting cells present
antigens to TH cells. Antigen-presenting cells include dendritic cells (interdigitating), macrophages,
and B lymphocytes (see Figure 1.8).
Figure 1.8
Antigen-presenting cells. These cells
present the antigens to phagocytes, NK
cells and lymphocytes. The antigens are
displayed on the surface of these cells by Class II MHC molecule
class II MHC molecules. and processed peptide
Antigen-presenting cells take up antigens either by endocytosis or phagocytosis. The intern-

alized antigen is then degraded and processed. Small peptides derived from antigen processing
are then displayed on the surface of the antigen-presenting cells in association with class II MHC
molecules. This antigen–class II MHC complex is recognized by TH cells. This binding with TH cell
also activates antigen-presenting cells, which produces signal molecules (cytokines) that leads to
the activation of the TH cell which further augments the immune response.
N AT U R A L K I L L E R C E L L S
Natural killer (NK) cells play an important role in immune surveillance and innate immunity.
These large granular cells, that have a natural “instinct” to kill tumour or virus-infected cells with-
out prior immunization, are called natural killer cells. It is believed that NK cells recognize cells
that have lost class I MHC molecules from their surface. Many surface changes occur on tumour
cells and some virus-infected cells, including loss of class I MHC molecules. NK cells recognize and
damage these class-I-MHC-negative cells very effectively.
EOSINOPHILS
Eosinophils are a specialized group of leukocytes that are weak phagocytes exhibiting chemotaxis.
Eosinophils are produced in large numbers in individuals with parasitic infections. Sometimes
when a parasite is too big to be phagocytosed, (such as schistosomes or flatworms), they attach
themselves to these parasites and release their intracellular granular contents that contain several
toxic proteins to kill the parasite.
BASOPHILS AND MAST CELLS

Basophils circulating in the blood are very similar, though not identical, to the large mast cells lo-
cated outside many blood capillaries (mainly in tissues). These cells (basophils and mast cells) have
stored intracellular granules that contain a variety of molecules capable of triggering inflammation
upon their release. These mediators are released when the cells are activated. Basophils and mast
cells have bound antibody (IgE) molecules on their surface. The binding and cross-linking of these
antibody molecules by an allergen stimulates the release of pharmacologically active granules that
are stored in basophils/mast cells.These granules, when released inside the host body, elicit symp-
toms of an allergic reaction.
MONONUCLEAR PHAGOCYTES
The mononuclear phagocyte system comprises blood monocytes and tissue macrophages (histo-
cytes). Monocytes are incompletely differentiated cells that circulate in blood. These monocytes
are referred to as macrophages when they migrate through blood vessels and are sequestered into
the surrounding organs and connective tissues. Monocytes are derived from the myeloid lineage of
bone marrow stem cells. Their major functions involve phagocytosis and destruction of invading
pathogens. Moreover, these cells are also very effective at presenting antigens to T lymphocytes.
NEUTROPHILS
A neutrophil is another important phagocyte that has a multilobed nucleus, and is often referred to
as polymorphonuclear neutrophil (PMN). Neutrophils express Fc receptors for antibodies (recep-
tors that bind the Fc region of the antibody) and receptors for complement proteins that help to Fc region of antibody
phagocytose the opsonized bacteria. This region is the constant region
of the antibody molecule and
These cells are also involved in inflammation, and migrate from blood vessels into inflamed corresponds to stem (I) of the
tissue in response to chemical signals received from the lesion site. However, after phagocytosing Y-shaped antibody molecule. This
5–20 bacteria, neutrophils themselves die, probably by the action of a variety of lytic enzymes re- region has the ability to bind a group
of receptors called Fc receptors
leased near the neutrophils during phagocytosis. Thus, neutrophils are short-lived cells. present on a variety of cells such as
neutrophils and macrophages.
1.2.4 ANTIGENS AND ANTIGEN RECOGNITION
An antigen can be defined as any macromolecule that is capable of inducing B cells to produce
Antigen
specific antibody molecules. Now its usage has been broadened to include any molecule that can be Classically, an antigen was defined
specifically recognized by either B cells or T cells, or both. as a molecule that can elicit the
Antigen, a macromolecule, is not recognized as a whole by either B or T lymphocytes. Instead, generation of antibodies. We know
now that by and large any molecule
each lymphocyte receptor/antibody binds to a small or restricted part of the antigen called anti- that elicits the generation of
genic determinant (a shorter version of “the region that determines antigenicity”) or epitope. A antibodies also stimulates T cells. So
particular antigen can have several different antigenic determinants on its surface or the same an- a more apt term, immunogen, was
introduced later that includes those
tigenic determinant may be repeated several times. B-cell receptors, antibody molecules and T-cell molecules that elicit the generation
receptors are specific for a particular antigenic determinant rather than the whole antigen. of an immune response. In other
B lymphocytes express on their surface, antibody molecules that can recognize only a specific words, they stimulate both the arms
of the immune system.
antigen (more specifically, antigenic determinant). T lymphocytes, responsible for cell-mediated
immunity, express receptors that recognize short peptide sequences only when these sequences are
associated with MHC molecules present on the surface of the cell. Thus, humoral immunity rec- Antigenic determinant
A small or restricted part of antigen
ognizes various foreign proteins, polysaccharides, lipids, toxins and lipopolysaccharides released
(immunogen) that binds antibody
by invading pathogens into the blood stream In contrast, cell-mediated immunity recognizes an- or is displayed on the surface of the
tigenic determinants displayed on self-cells and altered self-cells (such as tumour cells or virus- cell by MHC is termed as antigenic
determinant.
infected cells) by MHC molecules.
1.2.5 M H C A N D A N T I G E N P R E S E N TAT I O N
The major histocompatibility complex (MHC) consists of approximately 15 different genes, most of
them highly polymorphic (over 500 different alleles of MHC have been identified). Initially, these
genes were identified as their gene products (that is, proteins) and were responsible for the acceptance
or rejection of tissue grafts (hence, the name histocompatible). If two mice had the same MHC al-
leles (like identical twins), the tissue graft was accepted. However, the same graft was rapidly rejected
if the two mice were of different strains. Though that was how MHC was discovered, it was obvious Antigen presentation
that MHC did not evolve to prevent indiscriminate organ transplantation. Long after their discovery The display of antigenic peptides on
the “plates” of MHC molecules.
as transplantation antigens, MHC proteins have been shown to be involved in antigen presentation.
MHC proteins are membrane-bound gly-

coproteins and can be grouped into two major
A2 S α1 classes. Class I and class II MHC molecules
S
have somewhat different structures (see Fig-
ure 1.9) and functions. (There are three class-
β 2 -microglobulin es of MHC molecules but we will focus on two
S S
important ones.) Class I MHC molecules con-
α3 sists of one transmembrane polypeptide chain
S S
of variable sequence known as the heavy chain,
associated non-covalently with an invariant
(non-polymorphic) small peptide known as
β2-microglobulin. Alterations in the variable
region of the heavy chain are responsible for
the dramatic changes in the peptide-binding
groove of the MHC molecule. The peptide-
Class I MHC
binding groove forms a cleft within which
the antigenic peptide sits and is presented to
T lymphocytes. Tcyt recognizes antigens com-
S
α chain α1 β1 β chain bined with class I MHC molecules. The class
S
II MHC molecule is a heterodimer of two sub-
units (α and β), each having a variable amino
acid sequence at its distal region (the region
S S farthest from the membrane). The variable
α2 S β
S 2
Figure 1.9 regions of both the subunits form the peptide-
Class I and class II MHC proteins. binding groove in class II MHC molecule. As
These molecules are a part of the noted previously, TH cells generally recognize
immunoglobulin superfamily and play a
vital role in antigen presentation. antigens combined with class II molecules.
Both classes of MHC molecules as well as
β2-microglobulin contain an Ig-like domain
Class II MHC and, thus, are members of the immunoglobu-
lin superfamily.
MHC molecules have a peptide-binding
groove that displays antigenic peptides that are generated within the cell. Class I MHC molecules are
predominantly responsible for displaying antigens that originate within the cytosol of a cell, that is,
endogenous proteins. In contrast, class II MHC molecules primarily display peptides of exogenous
antigens that are taken into the cell by endocytosis. Antigenic proteins are first processed and partially
digested inside the cell to generate antigenic peptides, capable of binding to MHC molecules.
PROCESSING OF ENDOGENOUS ANTIGENS

Antigens located in the cytosol of cells are degraded by proteases of the proteasome complex. Two
common examples of endogenous antigens are viral proteins synthesized inside virus-infected cells
and unique proteins synthesized by cancerous cells. These proteins are degraded into 8–15 residue long
fragments that are suitable for binding to the groove of class I MHC molecule. These peptides that are
generated in the cytosol, are transported into the lumen of the endoplasmic reticulum (ER) by a pep-
tide transporter known as transporter of antigenic peptide (TAP) located in the membrane of the ER.
Once in the lumen of ER, the peptide binds to class I MHC molecule and this peptide–class I MHC
complex is then transported to the cell membrane where the peptide is displayed (see Figure 1.10).
Tcyt recognizes antigen only when it is associated with class I MHC molecule and hence is said to be
class-I-MHC-restricted.
PROCESSING OF EXOGENOUS ANTIGENS

Exogenous antigens are produced/present outside the host cell and enter the cells via phagocytosis
or endocytosis. Antigen-presenting cells such as macrophages ingest and degrade antigens in the
endosomal or lysosomal compartment by acid proteases. The peptides generated within the lyso-
some are 13–18 residues long, slightly longer than those generated by proteasomes. These 13–18
residue peptides then fuse with the endocytic vesicle containing class II MHC molecules. The an-
tigenic peptide binds to the cleft within the class II MHC molecule and this complex then moves
Class I MHC
Exogenous antigen
Class II MHC
Enzyme
action
Endocytic
Endosome
vesicle
Digested
invariant chain
Golgi
complex
Processed
peptide Invariant chain
Class II MHC
molecules
Proteasome
complex
Lumen of
endoplasmic
reticulum
Class I MHC Class II MHC molecules

Protein molecules Figure 1.10
endogenous The processing of endogenous and
antigen exogenous antigens. Note that the two
pathways involve different MHC proteins.
to the cell membrane where it is displayed. Since only antigen-presenting cells predominantly dis-
play class II MHC molecules, the presentation of exogenous antigens is limited to these cells. TH
cells recognize antigen only when it is associated with class II MHC molecules and hence are said
to be class-II-MHC-restricted. Thus, TH cells are activated primarily by exogenous (extracellular)
antigens such as bacterial toxins or those that are part of bacterial cell walls.
1.3 TYPES OF IMMUNE RESPONSE

The specific immune response is normally stimulated when the host is exposed to invading microbes
over a period of time. This form of immunity, in which the immune system of the host plays an active
role in responding to the foreign antigen, is called active immunity. However, specific immunity can also
be conferred upon an individual by transferring cells or serum from specially immunized individuals
(or animals). This form of immunity, called passive immunity, makes the recipient individual immune
to a particular antigen or pathogen without even being exposed to it. Passive immunization is usually
used against those pathogens or toxins which act so rapidly and (in most cases) lethally, that the affected
individual is unable to mount an active and effective immune response.
Immune response can also classified as antibody-mediated or cell-mediated, depending on
which effector arm of the immune system is stimulated.
1.3.1 A N T I B O D Y - M E D I AT E D I M M U N I T Y
Antibody-mediated immunity is mediated by B cells and antibodies. These antibodies react
with pathogens (bacteria and viruses), soluble antigens, toxins and foreign cells within the body.
Antibodies function by binding to pathogens or toxins, neutralizing them and facilitating their
elimination. Pathogens and foreign cells coated with antibodies can be killed by proteins of the
complement system or can be readily ingested by phagocyte (see Figure 1.11). Toxins are neutral-
ized by antitoxin antibodies.
Antibody-mediated immunity is commonly referred to as humoral immunity as it is centred
around antibodies which are found primarily in the blood. (Blood was one of the four humors
formerly thought to constitute the body.)
Humoral response Antigen

Cell-mediated response
MHC
Antigen-presenting
cell
Antigen
B cells processing
Activation and Display of antigenic

proliferation determinants on MHC
Antibody synthesis
and binding
Tcyt cell
Fc receptor
Binding of
Figure 1.11 antibodies
The same antigen can elicit both
by phagocytes
humoral and cell-mediated immune
response. The activation of both the arms and its
of adaptive immunity helps the immune clearance Pores
system to deal with the antigen more
Perforin
effectively and rapidly.
1.3.2 C E L L - M E D I AT E D I M M U N I T Y
Cells of the immune system are directly involved in conferring cell-mediated immunity. The effector T
lymphocytes that are generated during cell-mediated immunity are TH cells and Tcyt. Tcyt lymphocytes
play a key role in killing virus-infected cells, transplanted cells and tumour cells, while TH cells can ac-
tivate various phagocytic cells enabling them to phagocytose and kill microbes effectively. TH cells also
activate humoral response simultaneously. Thus, while antibody-mediated immunity is the principal
defence mechanism against toxins, bacteria, viruses or foreign cells in the blood stream, cell-mediated
immunity is primarily directed against intracellular microbes such as viruses and some bacteria that
survive and proliferate inside host cells where they are inaccessible to circulating antibodies.
1.4 A C T I VAT I O N O F T H E I M M U N E
RESPONSE
There are two arms of the immune response—humoral response mediated by B cells and antibodies,
and cell-mediated response effected by T cells.
1.4.1 HUMORAL RESPONSE

Antigen-specific recognition by lymphocytes induces two major changes in lymphocytes—
proliferation and differentiation. The initial development of antigen-specific clones of lymphocytes
Clones
occurs before a lymphocyte ever encounters an antigen. As B cells mature in the bone marrow, Cells that are genetically and
random gene shuffling occurs in the gene segments that code for antibody molecules. Ultimately, phenotypically identical to each
each mature B cell possesses a single active gene for each light and heavy chain. The B cells therefore other and are derived from same
progenitor cell.
produce and display one type of antibody (that is, antibody of single specificity) on its membrane.
But different cells in a population of B lymphocytes undergo different genome rearrangements,
leading to the production of different antibody molecules by B cells.
There are about 105 antibody molecules displayed on a given B cell, all having the same speci-
ficity. Each B lymphocyte and its clone show a distinct specificity for a particular antigenic deter-
minant. Although it is difficult to place an upper limit on the number of antigenic determinants
that can be recognized by the human B-cell repertoire, it is estimated to be of the order of 109 to
1011. This huge diversity in the population of mature B cells is subsequently reduced by a screening
process that eliminates any B cell that recognizes self-antigens. This selection process ensures that
antibodies that react with self-antigens are not produced.
T-cell maturation involves rearrangements of a series of gene segments that encode T-cell re-
ceptors. Each T lymphocyte expresses about 105. receptors on its surface and the number of antigenic
specificities recognized by T-cell receptors of the entire human immune system is of the order of
1015. This enormous repertoire of T cells is diminished, as we have seen, through a selection process
in the thymus that ensures that any T cell capable of reacting with self-antigen is eliminated. The an-
tigen selects a specific pre-existing clone by binding to the membrane-bound receptor (mature B cell
or T cell) leading to a proliferation of cells with a given antigenic specificity. In this process, which is
referred to as clonal selection, an antigen binds to a particular T or B cell and stimulates it to divide
repeatedly into a clone of cells, each with antigenic specificity identical to that of the original parent
cell. These antigen-stimulated clones start differentiating into effector plasma cells and memory
cells. Memory B cells appear to have a longer lifespan than the naïve lymphocytes from which they Memory cells
Memory cells are long-lived cells
arise. Though antigens bind and stimulate membrane receptors of B cells very rapidly, antibody that retain “antigenic memory”.
levels in plasma start to increase after about a week of antigen exposure. This could be due to (a) time These cells when re-stimulated by
the antigen can again transform
required for expansion of clone of B lymphocytes into plasma cells, (b) time required for TH cell-
themselves into antibody-secreting
induced B-cell proliferation and differentiation. This initial and slightly delayed immune response plasma cells and memory cells
that occurs after the initial antigen exposure is known as primary response. It peaks in approximately within a short span of time.
14 days, when it shows the highest antibody concentration and then slowly starts to decline, as
plasma cells produced after initial antigen exposure begins to die. Subsequent contact with the same
antigen, elicits a faster, stronger and sustained immune response with a shorter lag period of one to
two days. This is termed as the secondary immune response and results in much higher antibody
levels. This is due to the presence of a large number of memory cells generated after the first antigenic
exposure. The increased antibody levels in secondary immune response is primarily due to the
fact that there are more memory B cells than naïve B cells that can be stimulated. The shorter lag
period is due to the fact that memory B cells re-

Membrane-bound antibody that spond to antigen more quickly as compared to
binds antigen naïve B cells. As memory cells are stimulated,
they again divide to produce a progeny of mem-
ory cells and antibody-secreting plasma cells and
consequently antibody levels rise between 100
Ag internalized,
B cell degraded and
and 1,000 fold. The memory cells continue to live
displayed for weeks, months or even years while plasma
MHC
cells die after few days.
Antigenic determinant
Cytokines T-cell receptor

1.4.2 S E L E C T IV E AC T I VAT I O N
O F B CE L L S A N D
GE N E R AT IO N O F
H UM O R AL RE SP O N SE
TH cell
Typically, B cells make antibodies in two forms—
secretory and membrane-bound. A membrane-
bound antibody, called a B-cell receptor, is attached
to the cell surface by a transmembrane tail present
T-cell receptor
at the c terminal of its Fc region. This leaves the com-
bining site on Fab region free to bind antigens. The
binding of antigens to these antibody molecules
on the surface of B cell triggers its activation and
Antigen ingested
proliferation (see Figure 1.12). Antigen binding
by macrophage, cross-links membrane-bound antibody molecules
Figure 1.12 digested and displayed resulting in their internalization by receptor-
The binding of antigen-presenting cell on antigen-presenting cell mediated endocytosis. After endocytosis, the
to TH cell. antigen is partially digested or processed. The re-
sulting peptides are combined with class II MHC (or class I MHC) molecules on their membrane.
The antigen–class II MHC complex is recognized by specific TH cells. The stimulated TH cells secrete a
variety of cytokines that simulate various stages of B-cell division and differentiation. These cytokines
expand the population of the B cells specific for the invading antigen. This increased population of
B cells then differentiates into antibody-secreting plasma cells and memory cells.
1.4.3 C E L L - M E D I AT E D R E S P O N S E
Cell-mediated immunity, which is centred on T lymphocytes, responds to cells infected with patho-
gens such as viruses and bacteria. As mentioned earlier, T lymphocytes recognize short peptide se-
quences only when these peptide sequences are associated with MHC molecules and displayed on
the cell surface. This peptide–MHC complex acts as a signpost that is recognized by T lymphocytes.
The recognition of an antigen–MHC complex by a mature, specific T lymphocyte activates these
lymphocytes. Activated T lymphocytes undergo proliferation and differentiation into various types
of effector (TH and Tcyt) and memory T cells. Memory T cells have a longer life than naïve T cells
and are more easily activated than naïve cells. Short-lived effector cells perform either helper or
cytotoxic functions. For example, Tcyt cell binds to antigenic peptides that are associated with class
I MHC molecules on the membrane of the altered self-cell. The self-cell can be altered by a viral or
bacterial infection, or by a tumour. Tcyt cell mediates the specific killing of these altered cells (see
Figure 1.13). The cytokines secreted by specific TH cell stimulate proliferation and differentiation of
T cells forming memory Tcyt cells.
TH cells are stimulated upon binding to antigen–class II MHC complex present on antigen-
presenting cells. This activation of TH cells causes rapid proliferation and differentiation into ef-
fector TH cells and memory TH cells. This clonally expanded population of antigen-specific TH
cells is actively engaged in stimulating B and T lymphocytes, thereby generating effective humoral
and cell-mediated responses. Apart from stimulating B and T lymphocytes, the cytokines secret-
ed by TH cells also activates non-specific effector cells that play different roles in a cell-mediated
response. These cells, which includes macrophages,

neutrophils and NK cells, play a significant role Tcyt cell
in phagocytosing pathogens or lysing virus-in-
fected cells or tumour cells non-specifically. These
phagocytes and NK cells serve as the non-specific
effector arm of cell-mediated immunity.
A specific class of TH cells is the delayed-type
hypersensitivity cells (TDTH) which are usually T-cell
helper T cells of TH1 type. These cells are involved receptor
in a specific type of hypersensitivity reaction called
delayed-type hypersensitivity. Delayed-type hyper-
sensitivity is a form of cell-mediated immune reac- MHC
tion and is designed as a primary defence against
intracellular bacteria that may survive within pha- Processed
antigen
golysosomes or in the cytosol of the host cell (such
as monocyte). However, if the antigen is not easily
cleared from the system, prolonged delayed-type Target cell
hypersensitivity response can become destructive
to the host as the intense inflammatory response Release of
perforin
that ensues can damage blood vessels and cause ex-
tensive tissue necrosis.
1.5 IMMUNE DISORDERS Tcyt cell
The immune system, complete with its innate and

specific immunity, is almost a flawless asset, pro-
tecting the body against pathogenic invasion and
cancer. However, there are several occasions when
the immune system misdirects itself, causing a
disease or other undesirable consequences.
The immune system can be compromised in
one of the following three ways: autoimmunity,
immunodeficiency and hypersensitivity.
Figure 1.13
The binding of Tcyt to target cell and its
1.5.1 AUTOIMMUNITY Target cell lysis killing by perforin.
Normally the immune system recognizes all for-
eign antigens and reacts against them. The immune system does not attack its own cells and mole-
cules. That is, the immune system is tolerant towards the self. Sometimes this tolerance mechanism
breaks down, leading to the production of autoantibodies and autoreactive T cells that cause tissue Tolerance
The inability to elicit an adaptive
damage and induce pathogenesis. Consequently, the body mounts an immune response against immune response despite the
itself. This is when an autoimmune disease occurs. presence of antigen is termed as
tolerance.
Multiple sclerosis is an autoimmune disease in which antibodies/T cells are formed against
components of the myelin sheath of the nerve cells. This immunological attack interferes with the
conduction of nerve impulse, causing severe and progressive neurological damage. Rheumatoid
arthritis is characterized by the attack of B and T lymphocytes on the joints due to a series of in-
flammatory responses. The destruction of cartilage and bone occurs, which causes pain and finally
results in the immobilization of joints. Another autoimmune disease, Graves’ disease, is character-
ized by an immune response against the thyroid-stimulating hormone (TSH) receptors present
on thyroid cells. The binding of antibodies to TSH receptors causes continual stimulation of these
receptors, resulting in hyperthyroidism. Thyroiditis develops from an immune attack against one
or more proteins of the thyroid cells. The destruction of the thyroid gland leads to hypothyroidism.
Other autoimmune diseases include insulin-dependent diabetes mellitus, systemic lupus erythe-
matosus (SLE) and pernicious anaemia.
Retroviruses 1.5.2 IMMUNODEFICIENCY

Retroviruses encompass a family of There are four elements of the immune system—T cells, B cells, phagocytes and complements,
animal viruses containing single- that function together to protect the host body. If any of these elements is defective since birth
stranded RNA. The viral genome of
retroviruses is replicated through a
(primary immunodeficiency) or becomes defective subsequently (secondary or acquired immuno-
double-stranded DNA intermediate deficiency), the individual may not be able to fi ght infections properly. Examples of primary
that is integrated in the host immunodeficiency include complement deficiencies, DiGeorge syndrome (defective T-cell develop-
genome. These viruses have been
known to infect a large number of
ment) and neutropenia (few neutrophils). The commonest example of acquired immunodeficiency
vertebrates including reptiles, birds is acquired immuno deficiency syndrome (AIDS) caused by the human immunodeficiency virus
and mammals. (popularly known as HIV), which is a retrovirus.
1.5.3 HYPERSENSITIVITY
Hypersensitivity Hypersensitivity is an abnormal state of heightened immune response that damages the normal
Hypersensitivity refers to the state
tissues of the host body. In other words, hypersensitivity is the outcome of the overreaction of the
of heightened immune response
that causes inconvenience to the immune system. The antigen (referred to as allergen) could be anything—from pathogens to pollen
individual. grains to innocuous food molecules. Hypersensitivity reactions cause substantial damage to unsus-
pecting individuals. Examples of hypersensitivity reactions include atopy (allergy to environmental
allergen), anaphylaxis and serum sickness.
At times, the immune system acts normally yet inconveniences the individual. The most im-
portant examples of this are blood transfusion and graft rejection. In case of blood transfusion and
organ grafting from incompatible or mismatched donors, the ordinary immune reaction that ensues
can lead to life-threatening consequences. So it is important to carefully match donor and recipient
blood (in blood transfusion) or tissue (in tissue grafting), to avoid rejection. These problems, however,
are a small price to pay for the constant and vast protection provided by the immune system.
1.6 EVOLUTION OF IMMUNITY

The vertebrate immune system probably evolved from the primitive cell-surface molecules that
were involved in cell–cell interaction. The duplication and divergence of this primordial recogni-
tion system led to the development of an immune system that resembled the T-lymphocyte system.
It seems plausible that the B-lymphocyte system and antibodies arose from this recognition system,
liberated from the constraint of interaction with the MHC proteins.
It is wrongly assumed that the presence of a defence mechanism against pathogen invasion is
limited only to vertebrates. Though the occurrence of antibodies, B cells and T cells are limited to
vertebrates alone, it would be a mistake to assume that even a non-adaptive immunity (innate im-
munity) does not occur in invertebrates. A rich variety of mechanisms that can distinguish self from
non-self (foreign) antigens and provide a non-specific barrier to the entry of pathogens has evolved.
Table 1.4 compares some important features of immune response in invertebrates and vertebrates.
The non-specific barrier of invertebrates includes mucous that surrounds the body of coelen-
trates and annelids. The presence of a tough exoskeleton, such as shells, forms a mechanical barrier
in the case of arthropods, echinoderms and molluscs. Other barriers include plasma gelation in ar-
thropods (akin to vertebrate blood coagulation) at the site of injury to prevent the fatal loss of body
fluids and phagocytosis by leukocytes (different from mammalian leukocytes) facilitated by lectins
Reactive oxygen species (akin to antibodies) that coat the foreign invader. If the pathogens to be phagocytosed are too large
This chemical species is derived from
or too many in number, they are encapsulated (as in an arthropod) in a multicellular aggregate.
oxygen and is known for its highly
reactive nature. Reactive oxygen Sequestered organisms are killed by lysosomal enzymes, lysozymes, reactive oxygen species and
species include free radicals such as nitrogen species released by surrounding invertebrate leukocytes.
hydroxyl radical, superoxide radical,
The body fluids of invertebrates contain various factors that have strong antibacterial and anti-
singlet oxygen and non-radical
oxidants such as hypochlorous microbial activity. These include agglutinins, lysozymes, non-lysozyme bactericidins, lysosomal en-
acid and hydrogen peroxide. These zymes and antimicrobial proteins. Antibacterial/antimicrobial proteins, found in insects such as
reactive species, if produced in
moths, flies and bees, can be induced within a few hours of an antigen injection. One such factor,
excess or in an inappropriate place,
can cause substantial damage to Cecropin A, found in silk moths shows approximately 40 per cent homology with immunoglobulin
DNA, RNA, proteins, lipids and domains and could represent a primitive form of immunoglobulin. Surprisingly, plants, which di-
carbohydrates that constitute the
verged from vertebrates at least a billion years ago, also respond to invading pathogens by producing
framework of cells.
a wide variety of antimicrobial peptides and non-peptide organic molecules that kill the pathogen.
This chapter provides a brief overview of the immune system comprising a diverse range of
cells and processes providing innate and adaptive immune responses. It is designed to provide a
bird’s-eye view of important components of the immune system. In the chapters to come, we will
learn more about cells, organs and different immunological processes that make up the delicately
entwined and perfectly regulated immune system. The subsequent chapters will delve into a vari-
ety of topics such as humoral immunity, cell-mediated immunity, generation of antibodies, T-cell
diversity and recent updates on vaccines. We will learn not only about the concepts but also about
scientists who helped to shape the discipline of immunology. The final chapters will deal with
the malfunctions of the immune system. These include misdirected immune response (auto-
immunity), exaggerated immune response(hypersensitivity) and diminished or absence of immune
response (immunodeficiency).
Pattern- Natural
recognition Killer B and Graft
Receptor Phagocytes Cells T cells Antibodies Rejection
Invertebrates + (usually) + – – – +**
Vertebrates + + + + + +
* Phagocytes are common to both invertebrates and vertebrates and represent the most primitive defence system. The emergence of
the first immune system having lymphocytes occurred around 500 million years ago in jawless fishes. This primitive immune system
Table 1.4
then evolved into two discernible population of B and T lymphocytes in all higher vertebrates.
Generalized representation of evolution
** Yet to be established in some invertebrates. of immune response.*
S U M M A R Y
• The body’s resistance to disease-causing pathogens can be non- • Tcyt cells are responsible for destruction of tumour cells/virus-
specific (innate immunity) or specific (adaptive immunity). infected cells/cells harbouring intracellular pathogens.
• Innate immunity comprises (a) mechanical and chemical barriers, • Major histocompatibility complex (MHC) comprises membrane
(b) phagocytosis, (c) fever, (d) inflammation and (e) acute-phase proteins that are present on the cell surface and are involved in
proteins. antigen presentation.
• Adaptive immunity has two exquisite qualities: (a) specificity and • MHC proteins are of two main types—class I MHC and class II
(b) memory. MHC proteins.
• The two arms of adaptive immunity include humoral immunity (com- • Class I MHC proteins display antigenic peptides that originate
prising B cells) and cell-mediated immunity (comprising T cells). endogenously, that is, within the cytosol of the cell. Class I MHC
molecules present antigenic peptides to Tcyt cells.
• When a B cell encounters antigen via its membrane-bound antibody,
it multiplies and differentiates into plasma cells and memory cells. • Class II MHC proteins display exogenous antigens that enter the
cells via phagocytosis or endocytosis. TH cells recognize antigen
• Plasma cells secrete antibodies that neutralize and clear pathogens. only when they are associated with class II MHC proteins.
• T cells include two groups of cells—T helper (TH) cells and cyto- • The immune system may sometimes misdirect itself or may break
toxic T (Tcyt) cell. down causing autoimmune diseases, hypersensitivity diseases or
• TH cells secrete cytokines that help B cells to divide, differentiate immunodeficiency diseases.
and secrete antibodies.
K E Y W O R D S
• acute-phase protein 9 • humoral immune response 17 • primary immune response 17

• adaptive immunity 10 • immune disorder 19 • secondary immune
• antigen-presenting cell 12 • inflammation 7 response 17
• B cell 11 • innate immunity 4 • T cell 11
• cell-mediated immunity 17 • mechanical barrier 4
• chemical barrier 5 • MHC proteins 13
• fever 7 • phagocytosis 6
1. What is the need for an innate immune response when there is a H INT —You don’t build windows or doors so that burglars can enter; you
more specific adaptive immune defence mechanism? need them for your own convenience and need.
H I N T —Innate immune response is immediate but non-specific adaptive
defence is specific but requires time.
4. Why are those B and T cell eliminated or suppressed that are reac-
tive to self-antigens? What do you think happens in autoimmunity
2. Why is an individual passively immunized, when he or she, given when body mounts an attack on self-antigens?
time, can develop active immunity? H INT —The body’s defence is made tolerant to self-antigens. This tolerance
H I N T —Given time! Sometimes pathogen action is so fast and lethal that breaks down in autoimmunity.
they do not give time to the person to react. So it is better to inject preformed
antibodies into infected individual. 5. What is the difference between primary and secondary immune
response?
3. Most virus and bacteria enter cells via specific receptors. Why
would a cell have receptors that allow pathogens to enter? H INT —Time and type of antibody formed.
Q U I Z YO U R S E L F

1. Which one of the following is not a chemical barrier? 6. The term vaccine was first used for:
(a) Stomach acid (a) Fowl cholera bacilli
(b) Interferon (b) Fluid from cowpox pustule
(c) Lysozyme (c) Crusts from smallpox vesicle
(d) Mucous (d) None of the above.
2. The process of inflammation is not initiated by: 7. Memory cells are formed in all, except:
(a) Histamine
(a) Primary immune response
(b) Bradykinin
(b) Active immunization
(c) Epinephrine
(c) Passive immunization
(d) Prostaglandins
(d) Secondary immune response
3. An example of acute-phase protein is: 8. Delayed type of hypersensitivity (TPTH) cells are primarily due
(a) Fibrinogen
to:
(b) Hemoglobin
(a) TH cells
(c) Haptoglobin
(b) Tcyt cells
(d) Prothrombin
(c) B cells
(d) Large granular cells
4. Which one of the following is not a phagocyte?
(a) Eosinophils
9. Cells least effective in controlling tumour cells are:
(b) Neutrophils
(a) NK cells
(c) NK cell
(b) Tcyt -cells
(d) Macrophages
(c) Neutrophils
(d) Basophils
5. The form of defence least effective against extracellular
pathogens: 10. Cells that are involved in inflammation include all, except:
(a) Macrophages
(a) Basophils
(b) TH cells
(b) Neutrophils
(c) B cells
(c) NK cells
(d) Tcyt cell
(d) Mast cells
Fill in the Blanks with Appropriate Terms

1. The ancient practice of introducing pathogenic smallpox crusts 4. The adherence of immune cells to capillary walls during the
into the host body is called_________________________. process of inflammation is called___________________.
2. The naturally occurring non-specific defence mechanism against 5. Class I MHC are expressed on ________________ cell while
invading pathogen is called_______________________. class II MHC are restricted to ____________ cells.
3. Two chemical barriers that are also proteins are ___________
and _____________.
F U R T H E R R E A D I N G
Autran, B., G. Carcelain, B. Combadiere, and P. Debre (2004). Morens, D. M., G. K. Folkers, and A. S. Fauci (2004). “The
“Therapeutic Vaccine for Chronic Infections”, Science, Challenge of Emerging and Re-emerging Infectious Diseases”,
305: 205–208. Nature, 430: 242–49.
Boes, M., and H. L. Ploegh (2004). “Translating Cell Biology in
vitro to Immunity in vivo”, Nature, 430: 264–71. Orkin, S. H., and S. J. Morrison (2002). “Stem Cell Competition”,
Nature, 418: 25–27.
Enver, T. (1999). “B-cell commitment: Pax-5 Is the Deciding
Factor”, Current Biology, 9: R933–35. Rhazes (1848). A Treatise on the Small Pox and Measles
Holmdahl, R. (1999). “Autoimmunity: Another Pathway Towards (translated by William A. Greenhill). London: Sydenham
Arthritis”, Current Biology, 9: R528–30. Society.
Karttunen, J., T. J. Trowsdale, and P. J. Lehner (1999). “Antigen Silverstein, A. M. (1992). The History of Immunology. San Diego,
Presentation: TAP Dances with ATP”, Current Biology, 9: R820–24. CA: Is Academic Press.
Le Bon, A., and D. F. Tough (2002). “Links Between Innate and
Adaptive Immunity via Type I Interferon.” Current Opinion in Stevenson, L. G. (1959). The Meaning of Poison. Lawrence,
Immunology, 14: 432–36. KS: University of Kansas Press.
In ancient days, disease was considered to be a maladjustment of the
four vital humors or body fluids. This belief, which continued for several
centuries, was challenged by the German anatomist Rudolf Virchow
in 1858. He suggested that disease arose because of a malfunction of
body cells, not due to maladjustment of body humor. It was “Millions of …
Virchow’s cellular concept that shifted the focus away from “humoralism”. creatures walk
Twenty-five years later, a Russian professor of zoology, Elie Metchnikoff, the earth
put forward his iconoclastic view in the form of the phagocytic theory. unseen, both
He suggested that phagocytic cells in the human body form the first when we sleep
line of defence by virtue of their ability to digest the invading patho- and when we
gens. The concept of cells and cellular immunity had come to stay. wake.”
– MILTON
The term stem cell, like the term immunity, has been used in more than
one context. This term initially appeared in E. B. Wilson’s great treatise on
cell biology in 1896 to describe the ancestral cell of a nematode
(Ascaris megalocephela). Pluripotential haematopoietic stem cells were
first described and studied by J. E. Till, E. A. McCulloch and their
colleagues in 1967. They showed that a lethally irradiated mouse could After studying this chapter,
survive if the bone marrow (which contains haematopoietic stem cells) from
• Differentiate between
another mouse of the same inbred strain is transplanted into it. Embryonic embryonic stem cells, adult
stem cells and progenitor cells
stem cells were isolated much later in 1981 by M. J. Evan and
• Briefly describe the various
M. H. Kauffman. stages of lymphocyte
development
• Describe B and T lymphocytes,
The role of lymphoid organs in immune response was brought to light NK cells and monocytes/
macrophages
by McMaster and Hudack in 1935. They injected antigen into the ear of
• Describe granulocytes,
rats and recovered antibodies from the lymph nodes before they could dendritic cells and mast cells/
basophils
be detected elsewhere. This confirmed for the first time that lymph • Explain the process of
nodes produced antibodies. The central role of the thymus in immunity phagocytosis
• Distinguish between primary
was first established by Jacques F. A. Miller in 1961. He demonstrated and secondary lymphoid
organs
the occurrence of two subsets of lymphocytes—B and T cells. He pointed
• Explain the structure and
out that B cells produced antibodies, and T cells, which did not produce function of the bone marrow,
lymph nodes and the spleen
antibodies, stimulated B cells to differentiate into antibody-secreting • Explain how an antigen
stimulates the formation
cells. Antigen-presenting cells constitute another subset of cells that of secondary follicles and
germinal centres, and elicits
play a key role in immune response.These cells include B cells, macro- antibody formation in lymph
nodes and the spleen
phages and dendritic cells. Dendritic cells (see Figure 2.1) of the epider-
mis were first described by Paul Langerhans in 1868 in Berlin, and hence
were named after him as Langerhans cells. The name dendritic cell was
coined much later, in the 1970s, by Ralph Steinman and Zanvil Cohn.
Cells and Organs of
the Immune System 2
2.1 H A E M AT O P O I E S I S
All cells and organ systems in the human body are derivatives of stem cells. The simplest definition Stem cells
of a stem cell is that it is a cell that can reproduce and generate differentiated progeny. There is no universally acceptable
definition of stem cells. They are
The capacity to remain in an undifferentiated state and have the ability, upon division, to gen- probably best defined by Marshek et
erate one or more differentiated cell types is at the heart of a stem cell’s unique role. Stem cells can al. in their book Stem Cell Biology as
be of two types: embryonic stem (ES) cell and “adult” tissue-specific (TS) stem cells. Embryonic “those cells that retain the capacity
to self-renew as well as to produce
stem cells are pluripotential (or pluripotent) cells derived from very early human (or mouse) em- progeny that is more restricted
bryo. These cells proliferate indefinitely in culture in an undifferentiated state. ES cells differentiate in both the mitotic potential and
into all cell lineages in vivo (and, many types, in vitro) and have the capacity to generate cells from the range of distinct types of
differentiated cells they can give
all three germ layers (see Figure 2.2). After birth, animals use stem cells to build and replenish rise to”.
particular organ systems such as haematopoietic, neural, hepatic and epidermal systems. Com-
pared with ES cells, TS cells are undifferentiated cells found among the differentiated cells in some
organs of the body. TS cells have “less” self-renewal ability and although they differentiate into
multiple lineages, their pluripotential nature is still debated. Some the properties of embryonic and « Anna Wobus and Tom
adult stem cells are given in Table 2.1. Duetschman in 1984 demonstrated,
for the first time, that ES cells can
Haematopoiesis is the formation and development of blood cells. All blood cells including differentiate into multiple cell
erythrocytes, monocytes, granulocytes, lymphocytes and megakaryocytes (platelets) are derived types.
Figure 2.1
An artist’s impression of dendritic cells.
from a type of cell called haematopoietic stem cell (HSC). In humans, the site of haematopoiesis—
formation and development of blood cells—changes during embryonic development. Initially, this
occurs inside the yolk sac. In the third month of gestation, haematopoietic stem cells become es-
tablished in the foetal liver. Around the fifth month, haematopoiesis starts occurring in the spleen
Keratinocyte
Liver cell Pluripotent

embryonic stem cell
Nerve cell Muscle cell Red blood cells
a)
Fibroblast
Osteoclast
Langerhans cell
Multipotent
adult stem cell
Granulocytes
Figure 2.2
A schematic diagram of pluripotent B lymphocyte Red blood cells
embryonic stem cells and multipotent T lymphocyte
adult stem cells showing that pluripotent
cells can diverge into a wide variety of
cells while multipotent cells differentiate
b)
into only a few specific cell types.
CELLS AND ORGANS OF THE IMMUNE SYSTEM 27
Property Embryonic Stem Cells Adult Stem Cells

Origin Embryo Adult bone marrow
Growth potential Indefinite Indefinite
Differentiate into all cell types Yes Yes* Table 2.1
Comparison of embryonic and adult
Expression of marker protein Oct-4 High Very low stem cells. Adapted from Orkin,
Stuart H. and Sean J. Morrison
Produce blood cells on transplantation No Yes
“Stem-cell Competition“, Nature,
* This thought is still debatable, awaiting conclusive evidence. 418: 25–27.
as well as the liver. By the fourth to sixth month haematopoiesis starts in the bone marrow, which « Haematopoietic stem cells
produce blood cells during the
becomes the major organ of HSC differentiation from the sixth month onwards. By the time of lifetime of an individual. They are
birth, all vestiges of haematopoiesis mainly are lost from the spleen and liver. Shortly after puberty, described by Weissman as “those
haematopoiesis mainly occurs in the axial skeleton (marrow of sternum, ribs and vertebrae). Blood cells, which upon division, produce
haematopoietic stem cells and a
cells are also formed, though to a limited extent, in the marrow of the pelvis, femur and tibia. population of progenitor cells that
As pointed out previously, HSC can differentiate to generate erythrocytes, monocytes, granu- becomes committed to different
locytes, lymphocytes and megakaryocytes, as is shown in Figure 2.3. The early events in haemato- haematopoietic lineages”. Orkin
defines haematopoietic stem cells
poiesis include the division of a stem cell into two, of which one is destined to differentiate into a as “those cells that are capable of
specific cell type. This new cell will give rise to either a lymphoid stem cell or a myeloid stem cell. reconstituting the haematopoietic
The lineage of cells differentiating into either lymphoid or myeloid stem cells is determined by the system of a recipient”.
type and amount of cytokines. These lymphoid or myeloid derivatives of stem cells have lost their
capacity of self-renewal and are committed to a particular cell lineage. These derivatives of stem Cytokine
cells are now referred to as progenitor cells. Cytokine is a generic term reserved
Lymphoid progenitor cells give rise to T, B and natural killer (NK) cells while myeloid for secretory (glyco)proteins that
typically act on neighbouring cells.
progenitor cells generate red blood cells, white blood cells (neutrophils, basophils, eosinophils, However, a cytokine may act on
monocytes, mast cells) and platelets. The commitment of progenitor cells to a cell-specific lineage distant cells if it is secreted into the
(for example, whether myeloid progenitor cells will differentiate into red blood cells, neutrophils, circulatory system. Cytokines are
mainly synthesized by leukocytes,
basophils or any other cell types) will depend on both cytokines and stromal cells. Stromal cells are although some stationary cells such
non-haematopoietic cells that support the growth and differentiation of haematopoietic cells (see as endothelial cells can also secrete
Figure 2.4). Stromal cells include epithelial cells, macrophages, fibroblasts, fat cells and endothelial cytokines.
cells. Within the foetal liver or adult bone marrow, haematopoietic cells grow and mature on a
meshwork of stromal cells. These stromal cells provide haematopoietic-inducing microenviron-
ment (HIM) involving both cytokines and adhesion molecules. The progenitor cells then differ- Progenitor cells
Progenitor cells are a subset of
entiate into a particular cell lineage. The red and white blood cells that are formed, pass into the cells between haematopoietic
sinusoids of the bone marrow from where they enter into circulation. stem cells and fully differentiated
The commitment of a progenitor cell to a particular differentiation pathway is associated with cells. These cells are derived from
(haematopoietic) stem cells and
the expression of specific receptors for particular cytokines on the cell membrane. The binding of become committed to various
specific cytokines on the progenitor cell surface, as well as the direct contact of the former with (haematopoietic) lineages.They do
stromal cells, switches on the specific genes coding for molecules required for the function of dif- not have the capacity of self-renewal.
For example, haematopoietic stem
ferent cell lineages: for example, those used for phagocytosis in macrophages and neutrophils, and cells give rise to lymphoid progenitor
for receptors on lymphocytes (antibodies) which determine specificity for antigens. cells (which are precursors of T, B and
Various cytokines (Greek: cyto—cell and kinesis—movement) are required for proliferation, NK cells) and myeloid progenitor
cells (which give rise to red blood cells,
differentiation and maturation of different blood cell types as well as renewal of HSC. Among the neutrophils and several other cells).
cytokines known to be involved in haematopoiesis are Flt-3 ligand, four colony-stimulating fac-
tors (CSFs), stem-cell factors (SCF) and erythropoietin (EPO). The four colony-stimulating fac-
tors include multi-lineage-CSF (multi-CSF or IL-3); macrophage-CSF (M-CSF); granulocyte-CSF
(G-CSF) and granulocyte–macrophage-CSF (GM-CSF). Though an oversimplification, it can be
loosely said that HSC-regeneration depends on SCF, IL-1 and IL-3, whereas the development of
monocytes and granulocytes involves the production of, among others, M-CSF and G-CSF, secreted
by the stromal cells. Erythropoietin (EPO), another cytokine produced by the kidneys, induces the
development and production of red blood cells. Erythropoietin
In general, stem-cell differentiation results in two types of changes: the expression of special- A cytokine that coaxes a progenitor
ized, lineage-specific and lineage-determining gene products, and a partial or complete restriction cell towards the erythroid cell
lineage.
of the capacity of cells to divide. The mechanism that regulates the expression of differentiation-
specific genes is incompletely understood. The expression of specific gene products marks both the
cell lineage and the stage of differentiation. The expression of differentiation-specific genes is gen-
erally under the control of a small number of master regulator genes known as homeotic genes.
Pluripotent stem cell
Myeloid progenitor Lymphoid progenitor
IL-3 EPO IL-2,IL-3,IL-4,IL-1,IL-4,IL-2,

IL-5,IL-6
IL-3,GM-CSF
IL-11 EPO
B cell T cell
IL-4
Erythroid Granulocyte-monocyte
progenitor progenitor
Megakaryocyte
EPO IL-6 IL-5
EPO
G-CSF M-CSF
Erythrocyte Basophil Platelet Eosinophil Neutrophil Monocyte Dendritic cell
Macrophage
Figure 2.3
The origin of cells involved in the immune system. All the cells of the immune system arise from haematopoietic stem cells under the influence of varying levels of cytokines and other factors.
Homeotic genes were first identified in the fruit Haematopoietic-inducing

fly, Drosophila. Similar genes have been identi- microenvironment
Pluripotent
fied in the genomes of higher eukaryotes, includ-
stem cell or
ing humans. Haematopoietic
In humans, about 40 homeotic genes are found cell
to be expressed not only in embryonic tissues but Cytokines
also in the adult tissue of haematopoietic lineage
The gene products of homeotic genes are transcrip-
tion factors. The target genes of these homeotic
transcription factor are largely unknown. Figure 2.4
Stromal cell Stromal cells and haematopoietic-
Transcription factors may either affect only
(Epithelial cell, macrophages, etc.) inducing environment.
a single lineage or influence many different hae-
matopoietic lineages. MyoP and c/EBP are nu-
clear factors that activate the transcription of muscle- and adipocyte-specific genes respectively.
GATA-1 gene-coded transcription factor is essential for the development of the erythroid lineage
while GATA-2 transcription factor is essential for the development of the lymphoid, myeloid and
erythroid lineages.
2.2 R E G U L AT I O N O F H A E M AT O P O I E S I S
Haematopoiesis is regulated by several complex mechanisms. These regulatory mechanisms main-
tain steady-state levels of various blood cells, by adding almost the same number of cells as are
removed from the system (due to natural death or aging). Adult tissue generally expresses a variety
of factors that act to maintain both the proliferation and differentiation of various cells lineages.
These include soluble factors and their receptors, transcription factors and processes such as
DNA methylation.
Soluble factors that regulate differentiation include those molecules that bind to cells surface
receptors, such as fibroblast growth factors, granulocyte colony-stimulating factors, stem cell fac-
tors, interleukins and several others. Some soluble factors can freely cross the plasma membrane
and bind to cytoplasmic or nuclear receptors. These include retinoic acid and its derivatives. These
soluble factors have a multitude of effects on the target cell undergoing differentiation, both during
embryogenesis and in adult tissue. Abnormalities in the expression of cytokines or their receptors
could lead to unregulated cellular proliferation and, hence, to the development of cancer. Basic
fibroblast growth factor can confer neoplastic properties when expressed in an inappropriate cell
type (for example, in fibroblasts).
DNA methylation in eukaryotes involves the addition of a methyl group to the 5-carbon posi-
tion of the cytosine ring. Presumably, DNA methylation affects gene expression because transcription
regulatory proteins that bind to methylated DNA differ from those that bind to unmethylated DNA.
DNA methylation is probably not a universal mechanism for regulating the expression of genes, but it is
believed to play an important role in the expression of some (not all) genes during cell differentiation.
Finally, the number of cells formed or differentiated is balanced by the controlled removal of
cells by programmed cell death. Failure in one, or a combination, of these regulatory processes can
produce cells with many properties of malignant tumour cells.
Apoptosis
Cell death resulting from an orga-
2.2.1 APOPTOSIS nized sequence of events that occur
There are two main ways by which cells die. They could be killed by mechanical or chemical injury, in a regulated manner and includes
the characteristic fragmentation of
toxic substances such as chemotherapeutic agents, or infection (necrosis), or cells may be induced nuclear genome. It can be induced
to die in a defined and orderly manner (apoptosis) Cells undergoing necrosis swell and their by physical agents (such as heat
intracellular contents leak out. The disintegration of cells and the release of their contents, includ- or UV radiation), chemical agents
(reactive oxygen species or, gluco-
ing lytic enzymes and proteins, usually the cause inflammation of surrounding tissues. corticoids) or biological (bacteria or
During apoptosis (from Greek: falling away) several different structural and functional changes virus). It involves the activation of
occur in a cell (see Figure 2.5). The cell shrinks, and the cytoskeleton modifies to develop bubble-like caspases that stimulate caspase-
activated DNase leading to fragmen-
blebs on its surface, the chromatin in the nucleus is degraded and the mitochondria breaks down with tation of the genome and character-
a release of cytochrome-c. The whole cell then breaks down into small, membrane-wrapped fragments istic DNA laddering that irreversibly
that contain intact organelles. These membrane-bound vesicles, or apoptotic bodies, have phosphati- causes cell death.
dylserine (which is normally present on the inner surface of the membrane in a normal cell) exposed
» Lockshin and William introduced on the surface. Macrophages and dendritic cells quickly phagocytose these apoptotic bodies ensur-
the term programmed cell death in
1964 to describe a predetermined
ing that the cells’ intracellular contents, which are now enclosed in apoptotic vesicles, are not released
pattern by which cell death occurs into the surrounding tissue. Thus, apoptosis does not induce local inflammatory reactions. Moreover,
in insects. The term apoptosis was macrophages and dendritic cells secrete cytokines that inhibit inflammation. The events occur during
introduced by John Kerr and his
colleagues in 1972.
apoptosis in a sequence, and in such an orderly fashion that this process is often called programmed
cell death.
Blood cells, like other cells of the body, have a characteristic lifespan and finally die by pro-
grammed cell death. If apoptosis fails to occur in leukocytes, a leukodermic state may develop.
Apart from its role in haematopoiesis, apoptosis is also needed for destroying cells infected with
viruses (Tcyt lymphocyte-mediated killing of virus-infected cells).
The signals that trigger the cell to take the road of self-destruction could be generated within
the cell or come from outside (see Figure 2.6). External signals (death activators) may bind to the
receptor on the surface of a cell that is going to die. The internal signals that trigger cell death in-
volve damage to the mitochondria of the cell. Damaged mitochondria releases cytochrome-c into
the cytosol. The released cytochrome-c binds Apaf-1(apoptotic protease-activating factor-1) pres-
ent in the cytosol and oligomerizes using ATP to form an apoptosome. This apoptosome activates
a set of cysteine proteases called caspases within the cell. The activation of caspase (specifically
caspase-9) results in the digestion of structural proteins, lamins of nuclear envelope, the activation
of endonuclease and chromatin degradation, and the inactivation of molecules such as focal adhe-
sin kinase, leading to cell detachment and, ultimately, cell death.
Apoptosis involving external signals is also brought about by caspases (in this case caspase-8),
which are activated by a different mechanism. This involves the activation of Fas and tumour
Perforin
Perforin is a 70 kDa protein necrosis receptors on the surface of target cells. The binding of death activators (Fas ligand and
secreted by Tcyt and NK cells, that tumour necrosis factors) transmits the signal to the cytoplasm that activates caspase-8, which
forms pores in the target cell. results in the final destruction of the cell.
Perforin also controls lymphocyte
proliferation. Mice that lack perforin The induction of apoptosis in the target cell by Tcyt may involve the interaction of Fas ligand
suffer from lymphohistiocytosis, a present on T cells, with Fas (CD95) on the target cell. Alternatively, T cells may release pore-forming
lymphoproliferative disorder. lethal proteins, perforin and enzymes—granzymes, which activate the caspases of the target cell.
Normal cell Normal cell
Chromatin degradation
Mechanical or
Blebbing (formation of
chemical injury
membrane bulb like structure)
Cell shrinks
Organelles swell Fragmentation of nucleus
Formation of apoptolic bodies
Release of
intracellular
Figure 2.5
The two common modes of cell death— contents
Released
(a) necrosis and (b) apoptosis. Necrosis
lytic enzyme causes
is the result of an injury to the cell. Apoptotic bodies caten up
Apoptosis, or programmed cell death, inflammation
by phagocytes
follows a sequence of events. Note the of surrounding cells.
formation of bulb-like membranous
structures. This is known as blebbing and
is characteristic of apoptosis. a) Necrosis b) Apoptosis
Fas ligand
(or tumour necrosis factor α /β )
Reactive oxygen species

UV/X-ray
Receptor
Chemotherapeutic drugs
Intrinsic pathway Extrinsic pathway

Activates
Mitochondrial Caspase 8
lysis
Cyt-C
DNA
fragmentation Figure 2.6
+ + Activates An overview of the two pathways that
ATP Proteolysis
Apaf-1 elicit apoptosis. Note that the caspase
Caspase 9 ATP Caspase 3 Membrane family of enzymes plays a critical role
Apoptosome blebbing in both the pathways. (Adapted from
Katoch et al. (2002).“ Programmed Cell
Death and Its clinical Implications,”
Indian Journal of Experimental Biology,
40: 513–24.)
« Parapoptosis is another form of

cell death, apart from necrosis and
2.3 CELLS OF THE IMMUNE SYSTEM apoptosis. Commonly found in
lower organisms, it is characterized
The immune system is composed of many interdependent cell types that collectively protect the by empty spaces within the cytosol.
body from microbial infections and the growth of tumour cells. The cells of the immune system can
engulf bacteria, kill parasites or tumour cells, and destroy viral-infected cells. The cellular composi-
« The human immunological
tion of adult human blood is depicted in Table 2.2. system, complete with cells and
The only cells that are capable of specifically recognizing and eliciting specific immune organs, weighs approximately 1 kg.
responses are lymphocytes. The other types of white blood cells play an important role in non-
specific immune response, antigen presentation and cytokine production.
Cell Type Number of Cells Per μl Approximate Percentage

Red blood cells 4200,000–6500,000
White blood cells
Agranulocytes
Lymphocytes 1500–4000 20–30
Monocytes 200–950 2–7
Granulocytes
Neutrophils 2000–7000 50–70

Basophils 50–100 <1
Eosinophils 40–500 2–5 Table 2.2

The cellular composition of adult human
Platelets 150,000–500,000 blood.
2.3.1 LY M P H O C Y T E S
Lymphocytes (Latin: Lympha—water and cyte—container) are the main cells of the immune sys-
tem. They are responsible for the specificity and memory in the adaptive immune response. Lym-
phocytes constitute about 20 per cent of the white blood cell population in the circulation. There
are three main subpopulations of lymphocytes—B cells, T cells and NK cells, that are quite different
in their functions, even though they all appear to be morphologically similar. B cells, are so called
Bursa of Fabricus because in birds they were first shown to mature in an organ called the bursa of Fabricus. In mam-
A cloacal organ in birds first mals, there is no anatomical equivalent of the bursa. B cells mature in the bone marrow. The fact
described by the 17th century
anatomist Hieronymus Fabricus.
that the place of maturation of B cells is the bone marrow, a tissue starting with the letter “b” is
mere coincidence. B lymphocytes are the only cells capable of producing antibodies. The antigen
receptors of B lymphocytes are membrane-bound form of antibodies. There are about 105 antibody
molecules on a single B cell.
The class of lymphocytes called T lymphocytes develop from their precursor in the thymus.
Immature T cells, also known as prothymocytes, leave the bone marrow and migrate into the
thymus. Through a maturation process known as thymic education, mature T cells are formed
and then released into the blood stream. Like B cells, T lymphocytes have receptors for antigen
binding. Unlike the receptors present on B cells, the T-cell receptors are not antibody molecules.
T-cell receptors have unusual specificity for antigens: they recognize antigen only when they are
presented on self-MHC molecules localized on the surface of the cell. T-cell receptors do not rec-
ognize free antigens.
The third subpopulation of lymphocytes does not express antigen receptors of either T or B
cells. These cells are called natural killer (NK) cells because they can lyse a variety of virus-infected
and tumour cells without any overt antigen stimulation that is, they are “born” (natural) killers of
these cell types. They are also referred to as null cells because they do not express the membrane
molecules and receptors that distinguish T- and B-cell lineages.
LY M P H O C Y T E D E V E L O P M E N T
» In the human body, about 106 new Like all blood cells, lymphocytes originate daily in the primary or central lymphoid organs (thymus
lymphocytes are produced every and the bone marrow). In the initial stages of development, lymphocytes do not produce surface
minute.
receptors for antigens. Hence, resting B or T lymphocytes cannot be distinguished morphologi-
cally. Such small, non-motile lymphocytes that have not interacted with antigens are referred to as
naïve cells or small lymphocytes. These naïve cells are 6–10 μm in diameter with a large nucleus.
The barely discernible rim of cytoplasm contains a few mitochondria and lysosomes, poorly devel-
oped endoplasmic reticulum and Golgi apparatus. These cells are in the Go state of the cell cycle. It
is believed that naïve lymphocytes eventually die after a short lifespan, which is not established and
may be just months.
In response to antigenic stimulation, resting small lymphocytes enter the G1 stage of the cell
Lymphoblast cycle. They become larger (approximately 15 μm in diametre) and are called lymphoblasts. Lym-
Rapidly dividing cells of the phoblasts have a wider cytoplasm rim, well-developed organelles and increased amount of cyto-
lymphocyte lineage. Such rapid
division usually occurs after
plasmic ribonucleic acid compared with small lymphocytes. The cells then progress to the S phase
antigenic stimulation. and finally the activated lymphocytes divide. This complex series of events is referred to as blast
transformation. The mature lymphoblasts divide and are differentiated into antigen-responsive
effector cells and memory cells. The sequence of events occurring in B and T lymphocytes is sum-
marized in Figure 2.7.
The effector cells of B-cell lineage include plasma cells which are antibody-producing B cells.
They are found at the site of immune response or are localized in lymphoid organs, and normally
do not circulate in the blood or lymph. The morphology of plasma cells is characterized by the
presence of abundant cytoplasm, acentric nucleus, abundant and prominent endoplasmic reticu-
lum and Golgi apparatus. Plasma cells have little capacity to undergo mitosis and are believed to be
terminally differentiated. These cells churn out large numbers of antibodies into the blood stream.
The effector cells of T-cell lineage include two major subsets which are functionally and phe-
notypically different—TH cells and Tcyt cells or lymphocytes. The cytokine-secreting TH cells have
the same morphological appearance as small or large lymphocytes. Their main function is to aug-
ment immune responses by the secretion of cytokines that activate the other cells of the immune
system to fight off infection. The Tcyt cells have an increased number of cytoplasmic granules whose
contents include the protein perforin that lyses the target cells. Tcyt cells are important in directly
Bone marrow
Multipotent stem cell
Lymphoid stem cell
Progenitor B cell Progenitor T cell
Bursa
Bone marrow Thymus
Peyers’ patches
Naive B cell Naive T cell
Antigenic stimulation Antigenic stimulation
B lymphoblast T lymphoblast
Figure 2.7
The maturation of B and T lymphocytes.
Descendents of the lymphoid progenitor
cell, B and T lymphocytes mature in
Memory B cell Effector B cell Memory T cell Effector T cell
different lymphoid organs in the human
(Plasma cell) (TH or Tcyt cell ) body.
killing certain tumour cells, viral-infected cells and sometimes parasites. Both types of T cells can
be found throughout the body. The activation of T cells often occurs in secondary lymphoid organs
(lymph nodes and the spleen).
Some of the progeny of B and T lymphoblasts become memory lymphocytes. These memory
cells are capable of surviving for long periods of time (months or years, sometimes even up-to
20 years). It is not clear whether memory cells survive without any stimulation or are maintained by
low levels of antigenic stimulation by the same or some other cross-reacting antigen. Memory cells
resemble small lymphocytes but they have virtually no common phenotypic markers to distinguish
them from naïve cells. In humans, memory T cells express the CD45RO marker while memory
B cells usually express the CD27 marker on their cell surface. Memory cells, whether of T- or B-cell
origin, are defined by their long survival time as compared with the other cells of the immune system.
As mentioned previously, T cells and B cells mature in the thymus and bone marrow respec-
tively. In the resting state, both T and B lymphocytes have similar morphologies. They have,
however, unique and different antigen receptors and other cell surface molecules. These molecules
are needed for lymphocyte activation and for movement into and out of the tissues.
B LY M P H O C Y T E S
B cells constitute about 10 per cent of human peripheral blood lymphocyte population. These cells
constitutively express surface immunoglobulin molecules (that is, antibodies localized on the cell
surface), where they act as specific antigen receptors. B cells are produced in the bone marrow and
migrate to secondary lymphoid organs and tissues for maturation.
+ ⫺ ⫺
B cells can be divided into two subsets—B1 (MacI , CD23 ) and B2 (MacI , CD23⫹):
B1 cells—B1 cells arise early in ontogeny and express mainly IgM antibodies. B1 cells spontaneous-
ly produce antibodies against multimeric sugar/lipid antigens of microbes, and sometimes against
autoantigens such as DNA, phospholipids and cytoskeletal components. They mature independ-
ently of the bone marrow and their response against antigens is independent of T cells.
B2 cells (conventional B cells)—B2 cells are primarily responsible for humoral-mediated immunity.
They are involved in the synthesis of IgG, IgA and IgE antibody molecules, which are T-cell-dependent.
A mature B cell expresses membrane-bound immunoglobulins (mIgM and mIgD) with a sin-
gle antigenic specificity. Less than 10 per cent of the B cells express IgG, IgA or IgE. Surface immu-
noglobulins are associated with Igα (CD79a) and Igβ (CD79b) to form a B-cell–antigen-receptor
complex, which constitutes the distinguishing feature of B cells. Other markers expressed on the
membrane of mature B cells include:
• CD19, CD20 and CD24—These markers are present on all B cells and their precursors are
currently used to identify human B cells. Plasma cells lack these markers.
• CD72—This is found on all B cells and on macrophages. Similarly, B220 is a frequently
used marker of B cells even though it is not found uniquely on B-lineage cells.
• Fc receptors (FcγRII CD32)—These are also present on the surface of B cells and mediate
negative signalling to the B cells.
• CD80 and CD86—These markers signal B-cell activation after being engaged by CD154
on activated T cells.
Other molecules that are involved in the more general functions of adhesion (ICAM-1) and antigen
presentation (class II MHC molecules), are also found on the surface of B cells. Some prominent
B cell markers are shown in Figure 2.8.
Once mature naïve B cells are appropriately stimulated, they divide and differentiate, form-
ing clones which progress to form a population of plasma cells and memory cells. Plasma cells
which lack surface immunoglobulins synthesize and secrete antibodies of a single specificity and
immunoglobulin (Ig) class. These plasma cells are infrequently present in blood and are normally
restricted to the secondary lymphoid organs and tissues. Plasma cells have a short life (one to two
weeks) and eventually die by apoptosis.
» In humans, the average popula- T LY M P H O C Y T E S

tion of lymphocytes at any given T lymphocytes constitute approximately 80 per cent of human peripheral blood lymphocytes.
time is 1012, of which approximately
one-fifth constitutes B cells and the
T-cell precursors migrate from the bone marrow to the thymus where they mature into antigen-
remaining four-fifth, T cells. specific T cells. These cells then migrate to the secondary lymphoid organs and tissues of the body.
CD20 CD21(Binds iC3b)

(Calcium
channel) IgM/IgD
(Binds antigen)
CD19
(Co-receptor ICAM-1
subunit) (Adhesion molecule)
(Mediates Ig B
B-cell
activation) IgA
CD40
Class II MHC (Triggers B-cell

(Antigen activation)
presentation)
CD35
(Complement
receptor)
CD72
CD32 CD80/86 Figure 2.8
(Binds IgG) (Binds CD154/CD28) Surface markers present on B cells.
A definitive T-cell marker is the T-cell receptor. A T-cell receptor is structurally and func-
tionally distinct from a B-cell receptor. B-cell receptors are membrane-bound antibodies
which recognize soluble antigens. On the other hand, a T-cell receptor is a heterodimer of two
disulphide-linked polypeptides, recognizing antigen only when it is presented/displayed on self-
molecules [major histocompatibility complex (MHC)]. Thus, to be recognized by most T cells,
antigen must be displayed together with MHC molecules on the surface of antigen-presenting
cells or on virus-infected cells, tumour cells and grafts.
There are two different types of T-cell receptors: one is a heterodimer of two disulphide-linked
polypeptides (α and β) and the other consists of γ and β polypeptides. Both receptors are associ-
ated with the CD3 complex (comprising five polypeptides) and this is known as the T-cell receptor
(TCR–CD3) complex. About 95 per cent of blood T cells express αβ TCR and the remaining 5 per
cent express γδ-receptors.
Apart from TCR and CD3 complexes, T cells also express some membrane markers, as follows:
• CD7 is expressed by all T cells.
• CD2 is also expressed by all T cells, though NK cells also express CD2 antigen.
• CD28, a receptor for B7 molecules, is present on B cells and antigen-presenting cells.
• CD45RA is present on naïve T cells and CD45RO on memory T cells. Figure 2.9 shows
some important surface molecules of T cells.
The αβ T cells can be further distinguished into two distinct populations: a subset of T cells that
expresses the CD4 marker (a surface glycoprotein) and “helps” or induces immune response (helper
T cells) and a subset which expresses the CD8 marker on its surface and is involved in killing
altered or infected cells (cytotoxic T cells). CD4⫹T cells, recognize specific antigens in association
with class II MHC molecules whereas CD8⫹T cells recognize antigens in association with class I
MHC molecules. It means that the presence of CD4 or CD8 limits (restricts) the type of cells, that
T cells can interact with. In general, CD4⫹T cells generally function as helper T (TH) cells and are
class-II-MHC-restricted and CD8⫹T cells are class-I-MHC-restricted and function as cytolytic or
cytotoxic T (Tcyt) cells. It should noted that most γδ T cells in a tissue express the CD8 marker.
The TH cells are an absolute requirement for immune responses to protein antigens in general,
and for helping B cells to make the different classes of antibodies. However, TH cells show func-
tional diversity. CD4⫹ T cell can be divided into two groups, depending on the pattern of cytokine
secretion. The TH1 subset secretes pro-inflammatory cytokines, such as IL-2, TNF-α and IFN-γ,
and is important for the killing of intracellular microbes and generation of Tcyt cells. Consequently,
these TH1 cells are important for combating intracellular pathogens such as viruses, bacteria and
parasites. TH2 cells secrete anti-inflammatory cytokines such as IL-4, IL-10 and IL-13, among
CD3 (a signaling complex associated with

the TCR, mediates T-cell activation)
T-cell receptor (A /B dimer or G /D dimer)
CD8 (on Tcyt

binds class I MHC molecule)
Memory T cell
CD71 (Binds transferrin)

CD45 RO
CD45 RA
LFA-1 Binds ICAM-1,
(Facilitates B-/T cell
interaction)
Naive T cell
(Signal CD7
transduction) CD28 (Binds CD
80/86 cell,triggers
CD27 T-cell activation)
CD4 (Only TH cells, binds

class II MHC molecules)
Figure 2.9
Surface markers present on T cells. CD2 (Adhesion molecule, binds LFA-3)
others, which are important for B-cell proliferation, and for differentiation and immunoglobulin
class switching to IgA and IgE. TH2 cytokines are also important in eradicating parasitic infections
as they lead to the production of IgE. TH2 cytokines also aid in the recruitment of eosinophils
» TH1 cells induce phagocyte-depen- (which have powerful antiparasitic functions).
dent immune responses to combat
bacterial and viral pathogens. TH2 Tcyt cells are activated when they interact with antigen presented on class I MHC complex on
cells are important in B-cell prolif- the surface of an altered self-cell. This activation results in the proliferation of Tcyt cells. Tcyt cells bind
eration and humoral response. TH2 and effectively interact and eliminate these altered self-cells (virus-infected cells or tumour cells).
cells also promote-IgE production ⫹
and eosinophil function, both of Recent evidences have suggested that the classification of CD4 T cells as cytokine-secreting,
⫹
which influence the pathogenesis and TH cells and CD8 T cells as Tcyt cells, is not absolute. Some CD4⫹T cells can have cytotoxic
of allergic reactions. functions and can kill cells. On the other hand, some CD8⫹T cells have been shown to secrete a
variety of cytokines. Thus, the distinction between TH and Tcyt cells is not absolute and there may
be ambiguous functional activities.
Recent research has suggested clear functional evidences for the existence of another sub-
population of T cells—T-suppressor cells (Ts) which are now called regulatory Treg cells. These
CD25⫹CD4⫹Treg cells are involved in maintaining immune unresponsiveness to self-molecules,
that is in maintaining tolerance. However, the exact mechanism by which Treg cells exert their im-
munosuppressive effect remains largely unknown.
N AT U R A L K I L L E R C E L L S / N U L L C E L L S
Natural killer (NK) cells account for approximately 10 per cent of blood lymphocytes. These periph-
eral blood cells do not express the antigen receptors that distinguish T and B cells and, hence, are
called null cells. They originate in the bone marrow from CD34⫹ haematopoietic progenitor cells.
Most NK cells are large lymphocytes with numerous cytoplasmic granules and, hence, some-
times referred to as large granular lymphocytes. They are produced in the bone marrow and are
found throughout the body, but mainly in the blood. These cells are not antigen-specific and there-
fore do not exhibit immunological memory. Although these cells lack antigen-binding receptors, that
is membrane-bound antibodies or heterodimeric T-cell receptors, they do express various surface
antigens. Some of these NK cell markers, however, are shared by other blood cells. These include:
• CD16 (FcγRIII), also expressed by neutrophils, macrophages, some T cells.
• CD56, an adhesion molecule also found on some T cells. Other markers include killer
activation receptors (KARs) and killer inhibitory receptor (KIRs), and C-type lectin
receptors composed of CD94 and NKG2 proteins. The absence of CD3, but the presence
of either CD16 or CD56 or both, is currently the most reliable marker for NK cells.
• Surface molecules of NK cells are shown in Figure 2.10.
CD16 (Fc γ RIII Binds IgG)

CD2 (Binds LFA-3)
CD18
CD56
CD94 (C-type
(Adhesion
NKG2 lectin receptor)
molecule)
CD38 Killer
Activatory Receptor
(on activation, cell releases
perforin )
Killer Inhibitory Receptor (binds Figure 2.10
class I MHC, prevents killing) Surface molecules of NK cells.
The function of NK cells is to recognize and kill self-cells which contain viruses, as well as
tumour cells, prior to the appearance of specific Tcyt lymphocytes. NK cells differentiate between
tumour, or virus-infected cells, and normal cells in two main ways. One involves KIR and the
other involves Fc receptors (CD16) present on the cell surface. When NK cells bind to uninfected
self-cells, the NK receptor KIR binds to the conserved region of class I MHC molecules associated
with the self-peptide. This provides a negative signal to the NK cells and prevents the killing of
the self-cell. However, the infection of cells by some viruses (such as Herpesvirus) or tumour cells
reduces the expression of class I MHC molecules on the cell surface. Now the KIR receptor can-
not bind class I MHC molecules. Moreover, the absence of class I molecules activates the receptor
NKG2D to induce the killing of the infected cells by the NK cells. This is known as the missing self
hypothesis, proposed by Ljunggren and Kare in 1990 (see Figure 2.11). They proposed that the
missing self-class-I-MHC molecules are detected by NK cells, and these cells are then lysed. This
is an important mechanism by which NK cells recognize and differentiate normal self-cells and
infected or aberrant self-cells. When an NK cell is triggered to kill a target cell, cytotoxic granules
are released. The granule contents can induce either perforin-mediated osmotic lysis or suicide
(apoptosis) of the target cell.
NK cells are also able to kill target cells coated with lgG antibodies via their receptors for IgG
(FcγRIII; CD16). NK cells attach to the Fc region of antibodies bound on the virus-infected or tumour
cells and subsequently lyse the target cells. This property is referred to as antibody-dependent cellular
Virus-infected or tumour cell

MHC,Class I MHC molecule
Normal self-cell downregulated
Missing
class I MHC
KAR Perforin-
mediated
KIR Empty KIR
Activation NK Cell
NK Cell
No activation Figure 2.11
Missing self hypothesis for NK-cell action.
KIR activated KAR activated
No target cell lysis Target cell lysis
cytotoxicity (ADCC). Although, ADCC can commonly be identified in vitro, its contribution in vivo
is still not known. NK cells release IFN-γ and other cytokines (IL-1) when activated, by recog-nizing
virus-infected cells. This helps in protecting the surrounding cells from virus infection. In addition,
IFN-γ plays an important role in the regulation of haematopoiesis and immune response.
2.3.2 MONONUCLEAR PHAGOCYTES

The mononuclear phagocyte system represents the second major immune cell population that has
a common lineage and shares an important common function called phagocytosis.
Cells of the mononuclear phagocyte system include monocytes and macrophages, which originate
in the bone marrow. Myeloid progenitor cells in the bone marrow differentiate into promonocytes and
finally into blood monocytes. The human blood monocyte (Greek: monos—single and cytos—cell) is
large (10–18 μm in diameter), has a bean-shaped nucleus and granular cytoplasm containing lysosomes,
phagocytic vacoules and cytoskeletal filaments. Cells from this circulating pool of monocytes migrate
through blood vessel walls into the various organs and tissues. Once they settle into different tissues,
these cells mature and become tissue-specific macrophages (Greek: macros—large and cytos—cell).
The differentiation of blood monocytes into tissue macrophages involves a number of changes.
They swell to much larger sizes, synthesize higher levels of hydrolytic enzymes and acquire in-
creased phagocytic ability. Tissue macrophages can live for months or sometimes even years unless
destroyed by performing phagocytic functions. These macrophages form the basis of the tissue
macrophage system that defends the tissue against infection. Macrophages are dispersed through-
out the body, taking up residence in different organs and tissues for which they have been given
special names. For instance, they are called Kupffer cells in the liver, mesangial cells in the kidneys,
alveolar macrophages in the lungs, sinus macrophages in the spleen and lymph nodes, microglial
cells in the brain, serosal macrophages in the peritoneal cavity and osteoblasts in the bone. Some
mononuclear cells differentiate into another cell type called dendritic cells (discussed later).
Like other human blood cells, monocytes/macrophages have monocyte-/macrophage-based
surface markers. These include:
• Mannosyl–fucosyl receptors on monocyte/macrophage.
• CD14—lipopolysaccharide receptors present on monocytes/macrophages, B cells and
some granulocytes.
• CD64—high affinity Fc receptors (FcγRI) present on monocytes/macrophages and some
neutrophils.
• CD32—medium affinity Fc receptor.
Other molecules found on human macrophages include CD15, CD13 and CD68, among others. It
should be clarified that none of the markers is lineage-specific, though the most practical monocyte/
macrophage marker is CD64. Monocytes and macrophages also have receptors for cytokines such
as IL-2, IL-4 and IFN. Some important cell surface molecules are shown in Figure 2.12.
Mononuclear phagocytes serve as an important link between innate immunity and specific
immune response. On the one hand, they phagocytose the invading microbes as a reaction of in-
nate immunity, and, on other hand, they act as antigen-presenting cells, displaying foreign antigen
on their surface that can be recognized by antigen-specific T lymphocytes. Activated macrophages
are 10 to 100 times more efficient than antigen-presenting B cells, though they are less efficient
than major antigen-presenting cells-dendritic cells. Macrophages produce cytokines that stimulate
T-cell proliferation and differentiation, recruit other inflammatory cells, especially neutrophils,
and are responsible for many of the systemic effects of inflammation.
Macrophages function as “scavenger cells” of the body, phagocytosing invading microbes,
antigens and even injured or dead self-tissue and cells. The scavenger activity of macrophages is
further enhanced by the cytokines secreted by the TH cells. Since macrophages express surface re-
ceptors for antibodies, certain complement proteins, and receptors for the components of bacterial
cell wall, they eliminate a broad range of pathogens ranging from bacteria to tumour cells.
Thus, the major function of macrophages include their participation in the elimination of in-
vading pathogen as well as the stimulation and amplification of specific immunity by acting as an-
tigen-presenting cells. Interestingly, macrophages also sometimes become a reservoir of pathogens
that persist for a long time inside the cell; for example, the measles virus, lentiviruses and
Cytomegaloviruses are transported by macrophages to distant parts of the body.
CD14 CD18/CD11b
(Lipopolysaccharide (C3b receptor)
receptor)
CD35
(C3b receptor)
CD16 CD25
(Antibody (Interleukin
receptor) receptor)
Mannosyl-fucosyl
CD32
receptor
(Antibody
CD64
receptor) Figure 2.12
(Fc H RI, antibody receptor)
Surface molecules on a macrophage.
2.3.3 GRANULOCYTES
Granulocytes are a class of white blood cells of the myeloid lineage, which typically contain
abundant cytoplasmic granules (and hence the name). These include eosinophils, basophils and
neutrophils (see Figure 2.13), distinguished histologically by the staining properties of these
granules. These leucocytes act as effector cells in inflammatory reactions as well as innate immu-
nity, and are also stimulated by T-cell derived cytokines. Granulocytes (mainly neutrophils and,
to some extent, eosinophils) have the ability to phagocytose the invading microbes or opsonized
particles.
NEUTROPHILS
Neutrophils (Latin: neuter—neither and philien—to love) are so called because their granu- « 1011 neutrophils are generated in a
lar cytoplasm stain readily at neutral pH. They are also called polymorpho-nuclear leukocytes day in a normal adult.
because of the presence of a multi-lobed nucleus that has three to five lobes connected by
thread-like chromatin.
Neutrophils comprise over 95 per cent of the circulating granulocytes. They are released into
the peripheral blood and circulate for about eight to ten hours before settling into the tissue, where
they stay for a few days. They respond rapidly to chemotactic stimuli, phagocytose and destroy in- Chemotactic stimuli
vading pathogens. The chemotactic agents include complement components (for example, C5a), Chemical stimuli that summon cells,
products of certain bacteria, products derived from the fibrinolytic and kinin systems, products usually phagocytes, to a particular
site.
of other leukocytes and platelets, and cytokines secreted by activated TH cells and macrophages.
Chemotactic stimuli result in a transient increase in the number of circulating neutrophils at the site
of inflammation. This phenomenon is called neutrophilia (or, loosely, leukocytosis). The movement
of neutrophils from the blood vessels into the tissues involves, first, the adhesion to endothelial
cells of the blood vessels, followed by margination (in which neutrophils change their shape and
show increased binding to endothelial cells) and, finally, the movement of neutrophils through
the gap between adjacent endothelial cells lining the blood vessels (diapedesis) into the tissue
spaces.
Neutrophils have a large arsenal of lytic enzymes and antibiotic proteins stored within
the granules. The granules are of two main types. The primary granules are large lysosomes
containing acid hydrolases, myeloperoxidase, lysozyme and other acid hydrolases. The smaller
secondary granules contain lysozyme, lactoferrin and collagenase, high concentrations of bac-
teriocidal proteins such as defensins, sperocidins and cathelicidins, and bacterial permeability
protein. The ingested organism is enclosed in the phagosome, which then fuses with the pri-
mary and secondary granules (lysosomes) to form a phagolysosome, whose contents are then
digested.
Neutrophil Eosinophil
Lymphocyte
Figure 2.13
False colour representation of
eosinophils, basophils, lymphocytes, and
monocytes (Adapted from An Atlas of
Drawings of Whole-Cell Structures.
W. B. Saunders, Philadelphia.) Basophil Monocyte
Neutrophils also possess receptors for immunoglobulin G (IgG). The binding of IgG mole-
cules (via their Fc region) results in the cross-linking of these Fc receptors, causing endocytosis of
the pathogen. Thus, they function as effector cells of humoral immunity. Sometimes, this complex
is so large that it cannot be taken inside neutrophils. This results in the release of cytotoxic sub-
stances stored in the granule, into the tissue. This can result in pathological conditions, as in im-
mune complex hypersensitivity.
EOSINOPHILS
» The normal concentration of Eosinophils (Greek: eos—dawn and philein—to love) comprise about 5 per cent of the total leu-
eosinophils is 350 cells per μl. Any kocyte population. Eosinophils have bilobed nuclei and many cytoplasmic granules that stain red
increase in the eosinophils beyond
500 cells per μl of blood is called with acid dye. Eosinophils are motile, though weak, phagocytes that migrate from the blood into
eosinophilia. the tissue spaces. Their phagocytic role is less important than that of neutrophils, though eosi-
nophils are capable of phagocytosing and killing ingested microorganisms.
Eosinophils are often produced in very large numbers in persons with parasitic infections
such as schistosomiasis and trichinosis.
Since most parasites (even juvenile forms) are too large to be phagocytosed by eosinophils
or any other phagocytes, eosinophils bind/attach to parasites coated with IgE (or IgG) via their Fc
receptors and degranulate, killing many of them.
The granular contents of eosinophils contain toxins known as “major basic protein” and
“eosinophils cationic proteins”. The major basic protein is toxic for helminths. These parasites are
relatively resistant to the lysosomal enzymes of neutrophils and macrophages, but are often killed
by the specialized granule proteins of eosinophils.
Eosinophils are also abundant at sites of immediate hypersensitivity reactions, such as in the
skin in allergic skin reactions or the bronchial tissue of the lungs in asthmatic persons. The growth
and differentiation of eosinophils is stimulated by TH-cell-derived cytokines, which contribute to
eosinophil accumulation at the site of allergic reaction or parasitic infections.
BASOPHILS AND MAST CELLS

Basophils (Greek: basis—base and philien—to love) are non-phagocytic, circulating granulocytes.
They have ill-defined lobed nuclei often obscured by granules that stain bluish-black with the ba-
sic dye methylene blue. Basophils are found in very small numbers in the circulation—less than
0.5 per cent of the total leukocytes.
Basophils originate from those haematopoietic cells in the bone marrow that are committed
progenitor basophil cells. Basophils migrate from the bone marrow to the peripheral blood pool.
They remain in the blood for five to eight hours and then migrate to tissues, where they can remain
for several days. Mature granulocytes in the peripheral blood are in transit to their potential sites
of action in the tissue.
Basophils express high affinity receptors for IgE antibodies and function by releasing phar-
macologically active substances from their cytoplasmic granules. These substances play a principal
role in certain allergic reactions and some delayed hypersensitivity states.
Mast cells, which are normally not found in the circulation, are similar to basophils in a number
of ways. They arise from bone marrow haematopoietic stem cells. They do not mature in the bone
marrow, or circulate in a recognized mature form. Unrecognized mast cell precursors enter the
tissue where they undergo proliferation, differentiation and maturation. Mast cells can be found
in a wide variety of tissues, such as, among others, cutaneous and mucosal surfaces. They can be
broadly divided into mucosal mast cells (MMC) found in the mucosae of digestive, respiratory or
genitourinary tracts, and connective tissue mast cells (CTMCs) found in the connective tissues of
various organs. Mucosal mast cells depend on T cells for proliferation whereas connective tissue
mast cells do not.
Mast cells are bigger than basophils, and each has a round or oval nucleus. These cells have
granules which have a crystalline structure. They also have receptors for IgE. Mast cells play the
main role in IgE-mediated type-I hypersensitivity reactions. As both basophils and mast cells ex-
press high-affinity receptors for IgE, they bind IgE antibodies via their Fc receptors (FcεRI). The
cross-linking of these receptors, which occurs because of the binding of an allergen to IgE antibod-
ies, induces these cells to degranulate. The release of granule contents, which contains heparin,
SRS-A, histamine and ECF-A, subsequently mediates adverse symptoms of IgE-mediated imme-
diate hypersensitivity. Basophils express additional surface receptors for activated complement
components C3a and C5a through which mediator release can be effected.
2.3.4 DENDRITIC CELLS

Dendritic cells (DC) are so named because their surface has many folds or spike-like projections « It is now believed that there are
that resemble dendrites of the nervous system (see Figure 2.1). These membrane extensions allow three different subsets of DC, one of
lymphoid origin and two of myeloid
maximum interaction with other cells of the immune system. These cells are derived from the bone origin.
marrow and are located in the blood and the lymph and most other tissues.
There are several different types of dendritic cells that have different properties, functions
and locations:
• Langerhans cells are present in the skin and mucous membrane.
• Tissue-resident dendritic cells (also known as interstitial dendritic cells) are found in
most tissues and organs (for example, liver, lungs, heart).
• Interdigitating dendritic cells are present in the thymic medulla, splenic white pulp and
lymph nodes.
• Dermal dendritic cells are found in the dermis of the skin.
• Circulating dendritic cells (also known as veiled cells) are found in the blood and lymph.
Irrespective of their location and properties, all dendritic cells process and present antigens to
CD4⫹TH cells. Activated dendritic cells express high levels of MHC molecules (class I and class II)
CD antigen together with accessory (costimulatory) molecules such as CD80, CD86, CDIA, and CD83. They
A cluster differentiation or cluster also produce a variety of chemokines that attract naïve T cells.
designation system of nomenclature
used to standardize the naming For this reason, dendritic cells are 10 to 100 times more potent antigen-presenting cells
of leukocyte differentiation than macrophages. An infection of the tissue by bacteria or virus, or even simple inflamma-
antigen recognized by monoclonal tion, leads to the maturation of immature dendritic cells: for example, after a respiratory virus
antibodies.
infection, immature dendritic cells are infected. These immature cells process the viral antigens,
become activated, circulate and carry viral antigens to lymphoid organs. These cells now appear
as interdigitating dendritic cells and efficiently stimulate naïve T cells, producing an antigen-
specific response.
» In vitro studies have shown that Another type of dendritic cells is called follicular dendritic cells. Follicular dendritic
one dendritic cell can stimulate 102 cells are not derived from a precursor in the bone marrow and are unrelated to interdigitating
to 103 cells in a mixed lymphocyte
reaction.
dendritic cells. These dendritic cells are so named because they are present in the germinal
centres of lymphoid follicles in the lymph nodes, spleen and mucosa-associated lymphoid
tissues. Follicular dendritic cells do not express class II MHC molecules and, therefore, are
unable to present antigen to TH cells. These cells, however, express high levels of receptors for
complement components and antibodies. They bind the immune complexes very effectively
through either of these receptors, and are very effective in presenting antigens to B cells.
These immune complexes have intact antigens with which B cells can interact. It is believed
that antigen–antibody complexes remains bound on the surface of follicular dendritic cells for
long periods of time (weeks, or even months). It is thought that the interaction between im-
mune complexes, bound to follicular dendritic cells and B cells (in B-cell follicles of lymphoid
tissue) is important in B-cell survival and development of memory B cells (within the primary
follicles).
As dendritic cells play an important role in the initiation of antigen presentation leading to
immunity, a number of viruses have evolved mechanisms to use these cells for their benefit. The
best example is that of human immunodeficiency virus (HIV). It is believed that dendritic cells in
the mucosal membrane are infected initially by HIV. These antigen-presenting cells then migrate
to lymphoid organs for effective presentation of HIV to both CD8⫹Tcyt and CD4⫹TH cells. In doing
so, HIV is inadvertently transported to the liver, and lymph nodes where it targets CD4⫹TH cells
found in abundance. This contributes to the spread of the virus to TH cells. The virus finally over-
whelms the immune system by weakening CD4⫹TH cells.
2.3.5 P L AT E L E T S
Blood platelets, in addition to their major role in blood clotting, are also involved in immune res-
ponses. The cells are derived from megakaryocytes in the bone marrow and contain granules. At
the site of injury of endothelial cells, platelets adhere at the damaged surface and degranulate.
The released substances which include serotonin and fibrinogen activate, the complement system,
increase vascular permeability and attract white blood cells to the site of tissue damage. Platelets
express class I MHC molecules as well as receptors for IgG and IgE. Parasite coated with IgG and/or
IgE can also activate (and hence degranulate) platelets through surface Fc receptors.
2.4 PHAGOCYTOSIS
Phagocytosis (Greek: phagein—to consume) refers to the ingestion of particulate matter (with a
diameter of more than 150 nm) such as whole microbes, cellular debris, activated clotting fac-
tors and so on. The first step in phagocytosis is the adherence of the particles to receptors on the
cell membrane of the phagocyte (macrophage, neutrophils and, to some extent, eosinophils).
The phagocyte then extend pseudopodia to encircle the particle. The pseudopodia on the op-
posite ends fuse to create a membrane-bound chamber which breaks away to form free-floating
phagocytic vesicles, or phagosomes. Phagosomes, once formed, fuse with lysosomes to form
phagolysosomes. A lysosome is a single-membrane-bound organelle that contains more than
20 different types of acid hydrolases (ranging from proteinases and lipases to sulphatases). Lyso-
somal enzymes digest the ingested materials. The undigested remnants of the ingested matter is
egested out by exocytosis.
A neutrophil can usually phagocytose five to twenty bacteria before the neutrophil itself be-
comes inactivated and dies. Macrophages are more powerful phagocytes than neutrophils. They
have the ability to engulf much larger particles and often five or more times as many bacteria than
neutrophils. Macrophages also have the ability to phagocytose necrotic tissue, even dead neutro-
phils, and continue to perform their function for months, sometimes even years.
Both macrophages and neutrohils have receptors for certain classes of IgG molecules and certain
complement components (C3b). When an antigen is coated with an appropriate antibody, a process
called opsonization, the Fc region of bound antibody binds to Fc receptors present on these phagocytes,
resulting in enhanced phagocytosis. IgG 1 and IgG 3 are most efficient in promoting phagocytosis. In
the same way the complement fragment C3b can also opsonize bacteria or other antigen by coating the
bacteria on one end and binding its receptors on phagocyte at the other. Opsonization either by com-
plement component or by an antibody leads to more than a thousand-fold increase in phagocytosis.
2.4.1 BACTERIAL KILLING MECHANISM

Studies in the biochemistry of phagocytosis carried out on neutrophils and macrophages have shown
that a number of cytotoxic substances are produced by phagocytes that can destroy the ingested
pathogen. There are two main but interlinked ways which mediate the killing of the pathogen.
OX YGEN-DEPENDENT KILLING MECHANISM

The opsonized pathogen/particle binds to the surface of phagocyte. The binding of the particle Respiratory burst
The increased O2 uptake is called
causes membrane infolding and capture of the particle by the phagocytes. At the onset of phagocy-
respiratory burst and is unrelated to
tosis, both cell types show increased O2 uptake, known as respiratory burst. mitochondrial electron transport.
A respiratory burst involves the activation of the membrane-bound enzyme NADPH oxidase Neutrophils as well as macrophages
consume very little oxygen in the
complex located on the cell surface of phagocytes. The activation of the NADPH oxidase, with the
resting state.
resultant production of superoxide radical (O2⫺) on the phagocyte (neutrophil) surface, occurs
within 30–60 seconds of phagocyte stimulation. Since the bacteria or other engulfed pathogen are
“enveloped” in a plasma membrane vesicle, the phagosome, they are exposed to high flux of O2⫺. NADPH oxidase
The superoxide anion also generates other powerful oxidizing agents such as hydrogen perox- NADPH oxidase is a membrane-
bound superoxide-radical-producing
ide (generated from O2⫺ by dismutation reaction) or hydroxyl radical (generated from hydrogen enzyme that is present on the
peroxide by iron-catalalyzed Haber–Weiss reaction). Phagosomes contain pathogens which have phagocyte surface. NADPH oxidase
been assaulted by reactive oxygen species such as superoxide radical, hydroxyl radical and hydro- contains membrane-bound
flavocytochrome b, three cytosolic
gen peroxide. The fusion of phagosomes with other granules (lysosomes) in neutrophil cytoplasm proteins p40phox, p47 phox and
empties, among other enzyme, the enzyme myeloperoxidase, over the engulfed particles. (Myelo- p67 phox, and GTP-binding protein
peroxidase is not normally present in macrophages.) The myeloperoxidase enzyme reacts with hy- p21rac.
drogen peroxide and Cl⫺ ion to form hypochlorite. Hydrogen peroxide (H2O2), hydroxyl radicals
(OH), hypochlorite (HOCl) formed are powerful and highly reactive oxidizing agents that are able
to oxidize many biological molecules and kill a number of pathogens. The stepwise generation of
oxidants generated in neutrophils is given below
NADPH oxidase O2˚
Dismutation Myeloperoxidase
O2˚ + O2˚ H2O2 HOCl
Cl˚
Fe
ions
OH˚
Macrophages, in addition to the above oxidative system, can express the enzyme nitric oxide
synthase that generates another powerful free radical, nitric oxide. Nitric oxide, like the hydroxyl
radical, has potent antimicrobial activity. Moreover, it can combine with a superoxide anion to
form peroxynitrite, an antimicrobial substance more toxic than nitric oxide. It is believed that
most of antimicrobial activity of macrophages is due to the ability to produce and use nitric oxide
and peroxynitrite against various pathogens.
OX YGEN-INDEPENDENT KILLING MECHANISM

As we have seen in previous sections, phagocytes possess higher levels of various hydrolytic enzymes,
whose activities do not require oxygen. The enzymes include lipase, protease and glycosidase, as also
Bacteria lysozyme which cracks open ingested microbial

invader. In addition, activated phagocytes pro-
duces a group of cytotoxic and microbial peptides
that help kill a variety of microbes. These include
CRI (CD35) defensins, lactoferrins, sperocidins and cathelici-
dins. Defensin is a bacterial permeability protein.
It is a circular peptide that forms ion-permeable
C3b Fc receptor
CD64/CD16/CD32
channels in bacterial cell membranes. Defensin
can kill organisms as diverse as Pseudomonas
a) aeruginosa, Escherichia coli, Streptococcus pneu-
moniae and the enveloped virus, Herpes simplex.
Lactoferrin, produced by neutrophils, binds iron
and renders it unavailable to bacteria even at an
Living cell
acid pH. These mechanisms of bacterial killing
require the fusion of lysosomes (where these pro-
teins are found) with phagosomes.
Ligand CD31 Repulsion
No Phagocytosis
It is interesting to note that phagocytes can
also recognize microbial invaders by recognizing
CD31
a limited number of conserved structures (pathogen-
Phagocyte associated molecular patterns—PAMPs) present
on microorganisms and not on multicellular host
cells. They can also differentiate between living
cells of the host which have to be ignored, and
dying or dead cells which are to be eaten up by
No signal Dead cell the phagocytes. All cells of the host express the
adhesion molecule CD31. The “handshake”
No repulsion binding between the living host cell CD31 ligand
Phagocytosis and phagocytes CD31 create repulsive impulses
occurs from healthy cells that actively discourage preda-
tory phagocytes. These impulses are disabled dur-
Phagocyte
Figure 2.14 ing cell death, allowing prolonged contact with
Schematic diagram showing the prey and predator (phagocytes) which even-
(a) phagocytosis of opsonized bacteria;
tually leads to “eating up” of the dead host cells
and (b) the role of CD31 the process of b)
phagocytosis. (see Figure 2.14).
2.4.2 OVERCOMING PHAGOCYTIC DEFENCES

Most of the phagocytosed microorganisms are killed by oxygen-dependent or oxygen-independent
mechanisms of phagocytes. Some microorganisms have successfully devised strategies to overcome
phagocytic defences. These includes the masking of bacterial antigens; for example, Staphytococcus
aureus produces cell-bound coagulase which clots fibrin on the bacterial surface, inhibiting phago-
cyte absorption or engulfment. Bacteria such as Treponema pallidium, Salmonella typhi, E.coli, Group
A Streptococci have antigens (K antigen in E.coli, M-protein and fimbriae in Streptococci) that inhibit
phagocytic engulfment. Inhibiting chemotaxis, for example, streptococcal streptolysin, suppresses
neutrophil chemotaxis. Some bacteria have devised novel ways to survive even inside the host phago-
cytes. These include strategies that prevent the fusion of phagosomes with lysosomes (for example, in
Rickettsia M.tuberculosis), escaping from phagosomes into the cytoplasm (for example, Rickettsia). Some bacte-
Rickettsia are small, rod-shaped ria such as Bacillus anthracis and Staphylococcus aureus have an unknown tenacious resistant mecha-
intracellular bacteria, usually
transmitted by arthropods. nism that enables them to survive inside phagolysosomes. Most of such intracellular pathogens are
combated by Tcyt cells or γδ T cells, or by a delayed type of hypersensitivity (discussed later). Some of
the mechanisms used by pathogen to overcome phagocytic defences are shown in Figure 2.15.
2.5 LY M P H O I D T I S S U E S
The cells involved in the immune response are concentrated in anatomically defined tissues and
organs where cellular interactions necessary for the recognition and effective activation of specific
immune responses can occur. These structures are collectively called lymphoid tissues or organs.
Fibrin clot
Fimbriae
K antigen
Bacteria
Hiding from immune cells
Inhibition of phagocyte binding
Lysosome Phagosome
Phagocyte
Phagosome
Inhibition of Escape from Survival inside

phagosome and phagosome phagolysosome Figure 2.15
lysosome fusion Overcoming phagocytic defences.
The major lymphoid organs are classified as primary (central, generative) organs and secondary
(peripheral) organs (see Figure 2.16). Included in the primary organs are the thymus and bone
marrow where lymphocytes
first express antigen recep-
tors and undergo maturation.
In birds, another primary Primary lymphoid Secondary lymphoid
organs organs
lymphoid organs is bursa of
Fabricus, where B cells un-
Thymus Lymph nodes
dergo maturation. Secondary
lymphoid tissues are those
where antigen is concen- Spleen
trated and allowed to interact
with immune cells for proper Lamina propria
immune response. These in- Peyers’ patch in
clude spleen, lymph node and small intestine
mucosa-associated lymphoid
Bone
tissue (MALT), that include Lymph node
marrow
lymphoid tissue associated
with the nasal (NALT), gut
(GALT), bronchus (BALT)
and genitourinary systems.
In addition, there are terti-
ary lymphoid tissues that Spleen Thymus
consist of loose aggregates of
lymphocytes often found in
Bursa of
connective tissue. The most
Fabricus
important tertiary tissues
Bone marrow
are the cutaneous-associated
Figure 2.16
lymphoid tissues. Schematic diagram showing various
lymphoid organs in the human body.
2.5.1 P R I M A R Y LY M P H O I D O R G A N S
Primary lymphoid organ Undifferentiated and immature lymphocytes formed in the bone marrow migrate to primary lym-
These are organs such as the phoid organs to become mature and committed to one antigenic specificity. The thymus and bone
thymus and bone marrow where
lymphocytes mature. marrow are the primary lymphoid organs in mammals. T cells mature in the thymus and B lym-
phocytes in the foetal liver and bone marrow. It is in primary lymphoid organs that lymphocytes
acquire their specific antigen receptors. This is followed by a selection procedure that selects only a
small percentage of cells that are capable of recognizing non-self antigens.
BONE MARROW
All cells of the immune system are derived from the bone marrow. The bone marrow is a richly
cellular connective tissue within the bone that produces blood cells and delivers them to the circu-
lation. During early foetal life, blood cells are produced in the mesenchyme of the yolk sac. As the
development of the foetus progresses, the spleen and liver take over this function. From the fifth
month of foetal life and postnatally, the bone marrow becomes a major haematopoietic tissue. Bone
marrow can be red or yellow. Red marrow, which is red because of the presence of red blood cell
and its precursors, is indicative of active haematopoiesis. Yellow marrow, which is yellow owing to
the presence of fat cells, is indicative of reduced haematopoiesis. Red and yellow marrow, however,
are inter-convertible as demands change.
The internal surface of the bone has several ridges with shelves and spicules of bone, the tra-
beculae that form sponge-like reticular framework of bony tissue. The spaces in this hollow re-
ticulum are filled with fats cells, collagen fibre, fibroblasts, osteoblasts, dendritic cells and reticular
cells, which lie associated with blood cell precursors of various lineages and maturity. The bone
marrow is richly supplied by venous and arterial vessels, however, it lacks lymphatic vessels. The
schematic representation of bone marrow is depicted in Figure 2.17.
The proliferation as well as the differentiation of these cells are stimulated by a number of
cytokines.These cytokines are produced by macrophages and stroma cells present in the bone mar-
row. Different cytokines promote the proliferation and maturation of different lineages of cells.
T-cell differentiation is initiated in the marrow, then the thymocytes migrate to the thymus where
differentiation is completed. B cells originate and mature in the bone marrow. They also undergo a
selection process that deletes those B cells that are self-reactive (that is, capable of producing anti-
bodies against autoantigens). Mature B cells exit via vascular sinuses and enter the blood stream.
In birds, B cells differentiate in the bursa of Fabricus. It receives stem cells from bone marrow
and differentiates and matures them into B cells.
Fat cell
Developing blood
cells
Megakaryocyte-
releasing platelets
Arteriole
Vein
Figure 2.17
Schematic representation of bone
marrow. (Adapted from Cell and Tissue
Blood cells releasing
Biology—A Textbook of Histology, Urban
and Schwarzenberg, Germany).
in sinusoids
THYMUS
The thymus is a flat bilobed encapsulated organ located behind the sternum, above and in front of the
heart. Each lobe of the thymus is divided into multiple lobules separated from each other by strands of « The thymus is at its largest at
connective tissue called septae. Within each lobule (see Figure 2.18), thymocytes (as the developing puberty (50 g), after which it starts
T cells are called) are arranged into an outer cortex and an inner medulla. There is a dense collection to shrink.
of thymocytes in the cortex, while the lighter staining inner medulla is sparsely populated with thy-
mocytes. The cortex contains most of the lymphocytes and the medulla contains all of the hassal’s cor-
puscles. Hassall’s corpuscles or thymic corpuscles are composed of tightly packed whorls of epithelial Hassall’s corpuscles
cells that may be remnants of degenerating cells, as well as lymphocytes, eosinophils and macrophages. Concentrically arranged whorls of
epithelial cells found in the medulla.
Both the cortex and medulla have a large number of non-lymphoid stromal cells. These in-
clude epithelial-reticular cells, interdigitating dendritic cells and macrophages. Some epithelial
cells, called nurse cells, have abundant cytoplasm and long membranous extensions. These mem-
branous extensions of the nurse cells as well as of interdigitating dendritic cells interact simultane-
ously with many thymocytes, forming large multi-cellular complexes. These “accessory” cells are
important in the development and differentiation of thymocytes and their “education” (positive
and negative selection) prior to their migration into the secondary lymphoid tissue.
The sequence of T-cell maturation is not completely known. The sub-capsular region of the
thymus which is the most exterior, is the arrival point of bone-marrow-derived thymocytes cells.
These precursor cells enter the thymus via the high endothelial venules in this region. Thymocytes High endothelial venules
These are specialized venules from
enter the cortex where they undergo rapid proliferation. This rapid cell division is coupled with a
where lymphocytes pass from the
high rate of cell death so that finally a very small subset of thymocytes migrate from the cortex to the blood to the lymph
medulla. En route to the medulla, thymocytes begins to express receptors for antigens and surface
markers that are present on mature T lymphocytes. It is believed that the cortico-medullary junction
is the site where the selection of “right” T cells occurs. Other T cells that are either self-reactive or do
not recognize foreign antigen displayed on self-MHC are deleted. Only those T cells whose receptor
recognizes a foreign antigen plus self-MHC molecule are allowed to mature and enter the medulla.
Thus the medulla contains T lymphocytes committed to T-helper or cytolytic function (that is, CD4⫹
or CD8⫹), which finally exit from the thymus via the high endothelial venules. The T cells leaving the
thymus are mature but innocent or naïve. They go to the spleen guided by cell surface molecules. It is
here that they finally mature and join the long-lived group of recirculating lymphocytes.
T H Y M I C E D U C AT I O N
The thymocytes maturing in the thymus start expressing their antigen-binding receptors (T-cell
receptors (TCR)). These T cells are then positively selected (chosen) for self-MHC recognition and
negatively selected (deleted) for receptors that are reactive to other self-antigens. This paradoxical
requirement, some-
times called thymic
education, ensures Capsule
that the thymocytes
that are unable to
recognize self MHC
molecules or that Epithelial reticular cell
do have a high affin-
ity for self-antigen
bound on self-MHC Thymocytes
are eliminated by ap-
optosis. So only those
Septae artery
T cells which recog-
nize foreign antigen Macrophage
on self-MHC are al-
lowed to mature. A Septae
detailed account of
thymic education is
Hassall’s corpuscles
given in Chapter 14. Figure 2.18
The activity of the Schematic diagram of the thymus
showing lobes, lobules and the dividing
thymus is maximal in Eosinophils septae.
the foetus and in early childhood, and then undergoes slow atrophy or age involution from pu-
berty, although it never totally disappears. The involution of the thymus begins within the cortex
which might completely disappear, to be replaced by fibrous and fatty tissues. The medullary part
usually persists though lobules atrophy and septae widen. Involution can also be induced by stress,
illness, pregnancy or even an accident, or can be mediated by cortisols and related steroids.
2.5.2 S E C O N D A R Y LY M P H O I D O R G A N S A N D T I S S U E S
Secondary lymphoid tissues The secondary lymphoid tissues comprise well organized encapsulated organs (spleen and lymph
Sites where antigen meet and nodes) and non-encapsulated lymphoid tissues. In these tissues, antigens are first trapped and then
interact with lymphocytes, for
example, lymph nodes and the exposed to mature lymphocytes. Hence, secondary lymphoid organs are the major sites of pro-
spleen. Secondary lymphoid tissues duction of effector T cells and antibody-secreting B cells. The non-encapsulated lymphoid tissues
are also involved in rejection of include Peyers’ patches in the lamina propria of the small intestine, tonsils in the pharynx and
transplanted tissue.
submucosal lymphoid follicles in the appendix and throughout the upper airways. These lymphoid
tissues that are found in association with mucosal surfaces are collectively called mucosa-associated
lymphoid tissue. In addition, there is cutaneous-associated lymphoid tissue that is specially adapt-
ed to combat pathogens that enter through the skin.
LY M P H N O D E S
Human lymph nodes are encapsulated bean-shaped or ovoid structures ranging from a few mil-
limetres to more than a centimetre in their largest dimension. Strategic sites rich in nodes are in the
areas such as neck, retroperitoneum, mediasternum, axillae, groin and abdominal cavity.
The lymph node is surrounded by a capsule composed of dense collagenous tissue. Its outer
surface is convex but contains an indentation, the hilus, through which efferent lymphatic vessels
leave the node and blood vessels enter. Afferent lymphatic vessels pierce the convex surface of the
capsule and empty into the node. Radial trabeculae, together with reticular fibres and reticular cells,
provide structural support to the various components of the node. Beneath the capsule is the sub-
capsular sinus followed by three areas: the outer region rich in B cells (cortex), a middle T-cell area
(paracortex) and the medulla (see Figure 2.19). The cortex contains B cells organized into primary
or, more commonly, secondary follicles. A group of lymphoid and non-lymphoid cells is usually
organized in a spherical or ovoid structure termed lymphoid follicle (sometimes termed as nod-
ule). The lymphoid follicles that consist of uniform tightly packed small naïve lymphocytes, as well
as follicular dendritic cells are termed primary follicles. Primary follicle lymphocytes have not yet
Afferent vessel
Primary follicle
Cortex (B-cell rich)
Medulla(B,T,plasma cells)
Secondary follicle
Germinal centre
Paracortex (T-cell rich)

Efferent vessel
Capsule
Figure 2.19
Schematic diagram of lymph nodes
showing cortex, paracortex and medulla.
encountered antigen. After antigenic exposure, a primary follicle becomes a larger secondary follicle.
The secondary follicle contains a central, pale staining area called germinal centre. The zone of small
lymphocytes surrounding the germinal centre of the secondary follicle constitutes the mantle zone.
The germinal centre consists of a central zone of large, concentrically arranged lymphocytes,
some TH cells, macrophages and follicular dendritic cells. Follicular dendritic cells which reside in
the germinal centre display antigens on their surface and selectively activate B cells. The forma-
tion of plasma cells and memory B cells is initiated here, but few plasma cells are present in the
germinal centres. This is as B cells differentiate into plasma cells they leave the germinal centres
and move towards the medulla. Germinal centres are not present in primary follicles. Beneath the
cortex is the paracortex, which is populated largely by TH cells and relatively sparsely by cytotoxic
Tcyt cells. It also contains many antigen presenting cells, which express high levels of class II MHC
molecules. These include antigen-presenting cells such as Langerhans cells (which have migrated
from the skin), follicular dendritic cells and interdigitating dendritic cells. These antigen-present-
ing cells transport antigens from the external and internal surfaces of the body to the lymph nodes
where they encounter naïve T-lymphocytes. The naïve T lymphocytes enter the lymph nodes ei-
ther with the lymph or via specialized venules (high endothelial venules) that are abundant in
paracortex region. It is this site where antigen-presenting cells present antigen to naïve T lympho-
cytes, particularly TH cells. Thus, the paracortex region of the lymphnode is the site where T-cell
responses to lymph-borne antigen are initiated.
The medulla lies central to the cortex and extends to the hilus. It contains both T and B cells in
addition to macrophages, plasma cells and some granulocytes.
The mechanism that sequesters lymphocytes into two distinct zones (B-cell rich and T-cell
rich) is still unclear. This compartmentalization might involve binding of T/B cells to different adhe-
sion proteins or stromal cells which are themselves localized in different regions. This anatomic
organization of the lymph nodes provides multiple sites for interaction between antigen-presenting
cells and lymphocytes, and between different classes of lymphocytes. Lymph arriving from tis-
sues or from a proceeding lymph node in the chain, passes via the afferent lymphatics into the
sub-capsular sinus, then into the cortex, around the follicles into the paracortex and then into the
medulla. Lymph is drained from medullary sinuses into efferent lymphatics and through the larger
lymphatic vessels back into the blood stream.
Lymph nodes constitute extensive filtration beds. As lymph-carrying antigens percolate
through the lymph nodes, they encounter macrophages, interdigitating dendritic cells which read-
ily phagocytose the antigens. These antigens are then processed, and presented together with class
II MHC molecules by these antigen-presenting cells to TH cells in the paracortex. Follicular den-
dritic cells in the germinal centres present antigens to activated B cells. The initial activation of B
cells is also believed to occur in the T-cell rich paracortex. Once activated, TH and B cells form small
ovoid structures (secondary follicles) constituted mainly by rapidly dividing B cells at the edge
of the paracortex. These B cells differentiate into IgM- and IgG-secreting B cells and memory B
cells. A few B cells and TH cells migrate to the primary follicles where cellular interaction be-tween
follicular dendritic cells, B cells and TH cells results in the formation of secondary follicles with a
central germinal centre. Some of the plasma cells generated in the germinal centre move to the
medulla and leave the node by efferent lymphatics.
The lymphocytes, whether T or B, remain in their characteristic sites for several hours, and if they
do not engage in antibody production they migrate to the medulla and exit via efferent lymphatics.
The lymphocytes then recirculate and may also enter a lymph node again through afferent lymphat-
ics. If T and B cells become engaged in antibody formation/induction in a node, the lymph leaving a
node is enriched with newly synthesized antibodies and/or increased concentration of lymphocytes.
The increase in lymphocytes in the lymph leaving the node is due to increased production of plasma
cells and memory cells (of T or B cells), that augments the recirculating lymphocytes pool.
Lymph nodes appear in the human foetus in the third month along the course of lymphatic
vessels. In humans, most nodes survive for 60 years or more with only slight atrophy and retain the
capacity to enlarge throughout life.
SPLEEN
The spleen is a large, encapsulated, bean-shaped organ with a spongy interior (splenic pulp) and is
situated on the left side of the body below the diaphragm. Like lymph nodes, the spleen is encapsulated
by white connective tissue, a few millimetres thick. A rich network of trabeculae extends into the inte-
rior, subdividing the organ into several communicating compartments. When the spleen is cut across
and viewed grossly, two major divisions of the pulp are evident: red and white. The white pulp forms
the “island” within a background of red pulp (see Figure 2.20). The capsule of the spleen is indented
medially at the hilus through which a large splenic artery enters. (Blood is carried out by splenic vein.)
Unlike the lymph nodes, the spleen is not supplied by lymphatic vessels. The splenic artery divides and
subdivides into smaller branches inside the spleen. These arterioles are surrounded by the periarteri-
PALS olar lymphatic sheath (PALS), composed of B- and T-cell-rich areas.
PALS refer to aggregates of Enclosed in the sheath are T cells (composed of mainly TH cells and partly Tcyt cells) around
lymphocytes around the splenic
artery arterioles, interdigitating dendritic cells, primary lymphoid follicles containing follicular dendritic
cells and naïve B cells. During an immune response, these primary follicles develop into germinal
centres and become secondary follicles. The whole splenic material enclosed within PALS consti-
tutes the white pulp. Several white pulp structures are scattered in the spleen.
The splenic red pulp consists of a reticular network of splenic cords and long anastomosing
channels (vascular sinuses), containing macrophages, platelets, granulocytes, lymphocytes, plasma
Haemoclasia cells and, most importantly, red blood cells, which imparts the red colour to splenic pulp. This is
The destruction of old and damaged the site where old or damaged blood cells are destroyed.
red blood cells is called haemoclasia.
The spleen efficiently traps lymphocytes, monocytes macrophages, antigen-presenting cells,
immune complexes and, more importantly, antigens from the blood and permits them to interact
and produce antibody. The splenic artery empties into vascular sinuses which finally reaches the
marginal zone. The marginal zone is the region between red pulp and white pulp that contains
B cells, TH cells and macrophages. The marginal zone constitutes the major receiving depot of the
spleen, receiving large quantities of blood from the arterial terminal. Here antigen is trapped by
macrophages and interdigitating dendritic cells, which carries it to the PALS. The interdigitating
dendritic cells and macrophages present the antigen-bound on class II MHC to TH cells. Once
» Follicular dendritic cells which activated, these TH cells activate B cells together with follicular dendritic cells. These activated
reside in the primary and secondary B cells and TH cells then migrate to primary follicles in the marginal zones. Upon antigenic exposure,
follicles trap, and retain extracellu-
larly in a non-processed form, some the primary follicles, develop into secondary follicles containing germinal centres where rapidly di-
of the pathogens that the body viding B cells and plasma cells migrate to the periphery of the sheaths and start secreting antibodies.
encounters. Thus, the function of the spleen is similar to lymph nodes, the essential difference being that
spleen is the major site of immune response to blood-borne antigens while lymph nodes are special-
ized for responses to antigens in the lymph, which trickle from the tissues. Though the major func-
tion of the spleen is the control of infectious disease, it has several non-immunological functions.
The human spleen serves as a reservoir of platelets and stores one-third of the platelets of the body.
Capsule
Trabeculae
Primary follicle
Marginal zone
PALS (Containing T cells and
dendritic cells) White pulp
Secondary follicle showing
germinal centre.
Figure 2.20
Schematic cross-section of the spleen Red pulp Vein Artery
showing red and white pulp.
The spleen receives T and B cells of the recirculating lymphocyte-pool from the blood, sorts
them into different compartments wherein they interact with one another and mount an effective
immune response.
The spleen originates in the dorsal mesogastrum in the human embryo. In the first trimester,
the human foetal spleen is erythropoietic and myelopoietic. These activities fade after the fifth pre-
natal month, pre-empted successively by the liver and marrow.
M U C O S A - A S S O C I AT E D LY M P H O I D T I S S U E
The mucous membranes lining the digestive, respiratory and urogenital systems represent the main
sites for the entry of microbes into the body. It is for this reason that almost 50 per cent of the lym-
phoid tissue in the human body is located within the lining of the major tracts, defending these
venerable membrane surfaces against microbial invasion. The lymphoid tissues are collectively
called mucosa-associated lymphoid tissue and include nasal-associated lymphoid tissues, gut-associated
lymphoid tissues, bronchus-associated lymphoid tissues and lymphoid tissue associated with the
genitourinary system. Lymphatic tissues such as Peyers’ patches in the small intestine, adenoids,
appendix and tonsils are well demarcated. In addition to standard sites, lymphocytes and their
accessory cells, being migratory cells may, set up lymphatic loci at virtually any place in the body.
N A S A L - A S S O C I AT E D LY M P H O I D T I S S U E
The nasal-associated lymphoid tissues are found in three locations: pharyngeal tonsils at the back of
the nose, palative tonsils at the sides of the back of the mouth and lingual tonsils at the base of the
tongue. The three main kinds of tonsils (palantine, lingual and pharyngeal) constitute an anatomi-
cal structure called Waldeyer’s ring. Because of their strategic location in the nose and back of the
mouth, these lymphoid tissues are directly involved in handling airborne pathogens.
In humans, the tonsils contain a meshwork of reticular fibres and cells, co-mingled with
lymphocytes, granulocytes, macrophages and mast cells. The B cells are organized in large secondary
follicles with germinal centres and the intervening regions show T-cell activity and high endothelial
venules. Airborne antigens and foreign particles are trapped within the deep crypts of the lympho-
epithelium from where they reach the lymphoid follicles for appropriate immune response.
G U T - A S S O C I AT E D LY M P H O I D T I S S U E
The gastrointestinal tract is exposed to an ever-changing mixture of antigens, microbes and other
potentially harmful foreign substances that are separated from the mucosal blood vessels only by a
single columnar epithelium and variable layer of mucous. It is not surprising, then, that the mucosa
is heavily populated with cells of the immune system. In the normal adult, about one-quarter of
the mucosa consists of lymphoid tissue, which includes organized cellular aggregates such as lam-
ina propria, Peyers’ patches in the submucosa, as well as lymphoid cells or intermingled between
epithelial cells or intraepithelial lymphocytes (IEL).
Before understanding how the immune system in gastrointestinal tract works, let us under-
stand a few strategic details of these lymphoid tissue. The outer mucosal epithelial layer contains
the IEL. Most of these lymphocytes are T cells but B cells are also present. Most of these T cells
express unusual γδ T-cell receptors which exhibit limited diversity. The actual function of these
cells is still largely unknown as many of them express CD8 antigen on their surface. IEL have been
found to release cytokines such as IL-5 and γ-IFN and may be involved in immune surveillance.
The accumulation of lymphoid tissues is seen in the lamina propria of the gastrointestinal wall as
well as in the appendix. The lamina propria contains loose clusters of B cells, plasma cells, activated
TH cells and macrophages. This region, which lies under the epithelial layer, harbours more than
10,000 diffuse lymphoid follicle. The submucosal layer beneath the lamina propria contains Peyers’
patches, nodules of 30–40 lymphoid follicles. Peyers’ patches are found in the lower ileum and, like
the lymphoid follicles of the spleen and nodes, can develop into secondary follicles upon antigen
exposure (see Figure 2.21).
However, epithelial cells of the mucous membrane that overlie Peyers’ patches contain a pe-
culiar type of flattened epithelial cells whose apical (luminal) surface has numerous microfolds.
These specialized cells are called as M cells (M for microfolds). These sites, where M cells are lo-
cated, are called inductive sites. In addition, M cells contain deep invaginations in the basolateral
plasma membrane which forms pockets filled with B and T cells, macrophages and dendritic cells.
Columnar
mucosal
M cell epitheluin
B
Submucosa
Plasma cells Germinal centre

Figure 2.21
Schematic representation of Peyers’ Peyers’ patches
patches.
It should be noted that M cells are not specific for Peyers’ patches but can also be found associated
with lymphoid cells at other mucosal sites.
In order to distinguish between harmless food and harmful pathogens, an efficient sampling
mechanism has been evolved. The specialized epithelial cells (M cells) endocytose and transport
antigen in endocytic vesicles to the underlying basolateral (pocket) membrane. This process is
called transcytosis. The vesicles fuse with the pocket membrane, delivering antigens to the clus-
ters of lymphocytes, macrophages and dendritic cells. Hence, antigens are transported across the
mucous membrane to the underlying B cells aggregated in lymphoid follicles in the lamina propria
(or Peyers’ patches) (see Figure 2.22). The activated B cell mature (with help of TH cells) into
antibody-producing plasma cells. These plasma cells leave the follicles, pass into mesenteric lymph
nodes and enter the lymphatic and blood circulation.
B cells proliferate within lymphatic nodules of Peyers’ patches, and differentiate into seden-
tary, local plasma cells. These cells secrete antibodies that are transported across the epithelium
into the lumen as secretory immunoglobulins (IgA or IgM) to protect the individual from ingest-
ed pathogens. Moreover, lymphocytes stimulated in the GALT can migrate to other MALT sites
(such as salivary glands, mammary glands, respiratory passages and other parts of the gastrointes-
tinal tract) and protect these surfaces from invasion by pathogens. However, these lymphocytes
never “home” in one lymph nodes because they lack “homing” molecules (such ICAM-1, ICAM-
2,VCAM) to which lymphocyte attach. Thus, antigen stimulation at one of the sites in the mucosa
elicits immune responses that are restricted to MALT.
B R O N C H U S - A S S O C I AT E D LY M P H O I D T I S S U E
These lymphoid tissues are found in all the lobes of the lungs and are situated along the bron-
chi. BALT is composed of aggregates of lymphoid cells mainly B cells, organized into follicles
situated under the epithelium. Antigens are, captured by the M cells. Antigen-presenting cells are
transported to underlying lymphoid tissues which give an appropriate immune response to the
invading pathogens.
C U TA N E O U S - A S S O C I AT E D LY M P H O I D T I S S U E
The skin serves as a protective barrier and sensing buffer between an organism and the environment.
The outer region of the skin, the epidermis, is composed of tightly bound stratified squamous epithe-
lium covered by keratin. Keratin is a physically tough, insoluble protein product of epidermal cells.
Bacteria
M cell
Columnar
mucosal epithelium
B cell
T cell
Intra-epithelial lymphocyte
Macrophage Dendritic cell
B-and T-cell activation and migration to

germinal centre which produces antibodies
Figure 2.22
The role of M cells in immunity.
The epidermis of the skin is made up of cells that produces keratin and is, therefore, called keratino-
cyte. Keratinocyte play a role in immune response by producing number of cytokines such as IL-1, IL-3,
IL-6, tumour necrosis factor and, after activation by T-cell derived cytokines, IFN-γ. In addition to
serving as sources of cytokines, keratinocytes can express class II MHC molecule when stimulated
by IFN-γ. This suggests that keratinocytes may augment local inflammation and lymphocyte activa-
« Langerhans cells, named after
tion. The other cells of epidermis include melanocyte, Langerhans cell and Merket cell. their discoverer Paul Langerhans in
Langerhans cells constitute only about 1 per cent of the epidermal cells and they cover almost 1868, are a type of dendritic cells
25–30 per cent of the surface because of their long membranous dendritic processors. These cells found in the skin.
form almost a continuous meshwork that enables them to pick up any antigen that enter through
the skin.
These cells ingest antigen by phagocytosis or endocytosis. Then they migrate from the
epidermis to the regional lymph nodes where they differentiate into interdigitating cells of the
immunological system. These cells express high levels of class II MHC molecules and function as
antigen-presenting cells to activate TH cells.
The epidermis also contains intraepidermal lymphocytes. These cells are similar to intraepi-
thelial lymphocytes found in the mucosa of MALT. These intraepidermal cells are CD8⫹ Tcyt cells
and bear γδT-cell receptors which have limited diversity as compared to αβT cells. The underlying
dermal layer of the skin contains scattered CD4⫹ TH and CD8⫹ Tcyt cells and a few macrophages.
It is believed that intraepidermal T cells, Langerhans cells as well as CD4⫹ and CD8⫹ T cells are the
cells that combat antigens that enter through the skin and generate effective immune responses.
2.6 LY M P H AT I C S Y S T E M
The walls of blood capillaries and venules act as a “semiperimeable membrane”. As blood circulates
through these capillaries large volumes of fluid together with some low molecular weight materi-
als escapes through endothelial lining of the capillaries into the surrounding tissue. This bathing of
the cells with this transduate constitutes an essential physiological process by which hormones,
Flow Cytometry
Flow cytometry is a technology that measures and then analyses Cells in buffer
various components or structural parameters of cells primarily by
optical means. Cells from solid tissue is first disaggregated before
they are subjected to flow cytometry. The tissue sample is broken
up into single cells. Any cell which is 0.5–150 μm in size is suit-
able for analysis. These cells are then hydrodynamically focused to
form a thin stream of cells which is pumped into a flow chamber.
Cells flow through the flow chamber one at a time at a speed of
about 500 cells per second. The cells may be alive or fixed (dead)
at the time of measurement. A small laser beam hits the cells as
they pass through the flow chamber. Every cell that is hit by la-
ser scatters some of the laser light, and also emits a fluorescent
light after getting hit by the laser. The way the light scatters off Side-scatter
detector
each cell gives information about the cell’s physical characteristics.
Light can be bounced off at low angles, which is called forward
scatter. Low-angle forward scatter is directly proportional to cell
diameter. Light scattered off in other directions is called side scat- Forward-scatter
detector Incident laser beam
ter. The side scatter which is collected at 90˚ to the incident laser
beam is directly proportional to the quantity of granular struc-
tures or complexity in the cell. Scattered and emitted light from
cells are converted to electrical pulses by optical detectors (see
Figure 2.23). The detectors produce electronic signals which are
Laser
proportional to the optical signals striking them. The fluorescent
light, which is usually emitted by fluorescent probes, report spe-
cific components of cells such as cell surface markers. In most of
the applications, after a cell exits the laser beam, it is sent to waste.
Cell sorting, takes this to the next level by allowing scientists to
identify cells as they pass through the laser and then mark them
for sorting.For such sorting,cells are first labelled with a fluorescent
dye that binds specific target molecule on the cell surface. A few
microseconds later, this marked cell is deflected away from the rest
of the sample and collected in a separate tube. This fluorescence- Waste
activated cell sorting allows the capture and collection of cells of
Figure 2.23
interest for further analysis. The collected cells can be further char- Flow Cytometry.
acterized biochemically, functionally or microscopically.
Flow cytometry and, more specifically, fluorescence-activated measure total cellular DNA or newly synthesized DNA. It can also
cell sorting can be used to distinguish subpopulations of different detect and separate any cellular structures for which antibodies are
cell types, such as Tcyt from B cell or TH cells. It can also be used to available.
antibodies, enzymes and mediators reach the cells and the intercellular matrix of the body. Most of
this fluid (interstitial fluid) returns to blood capillaries. A part of this protein containing cell-free fluid
(lymph) flows from connective tissue spaces into anastomosing capillaries which flow into lymphatic
collecting vessels. The largest lymphatic vessels empty into the left subclavian vein in the base of the
neck as left and right thoracic ducts. Unlike blood vessels, lymphatic vessels do not form a circular
system but carry lymph in only one direction, that is towards the base of the neck. So lymphatic ves-
sels recover fluids that escape into connective tissue spaces from blood capillaries and venules and
return them to the blood.
Lymphatic vessels anastomose with one another and tend to travel with blood vessels (mostly
veins). The flow of lymph is achieved by movement of body muscles particularly by muscle respira-
tory movements, and, surprisingly, by force of gravity, unlike blood which is pumped by heart. The
direction of flow is controlled by a series of one-way valves along the lymphatic vessels, ensuring
that lymph flows only in one direction. The valves are responsible for the beaded appearance of
lymphatic vessels.
Antigens that enter through the skin and gastrointestinal/respiratory tracts is picked up
by lymphatic vessels (which drains the fluid-surrounding tissue of the body) and is transport-
ed to lymphoid tissue such as lymph nodes. These strategically placed lymph nodes “sample”
the lymph for the presence of foreign antigen. Subsequent an immune response is elicited
from lymph nodes where organized lymphoid tissue interact with trapped pathogens and
undergo activation.
This chapter gives a detailed view of various cells, tissues and organs that are important for the
immune system. Different types of cells such as eosinophils, basophils, neutrophils and platelets
are involved in non-specific responses, while B and T lymphocytes provide specific protection
as well memory to the immune system. Blood-borne antigens are filtered off in the spleen while
antigen-reaching tissues are nudged into the lymph and finally into the lymphnodes. The lym-
phoid organs act as meeting point for antigens and effector lymphocytes (B and T lymphocytes).
After interaction with antigens, B and T lymphocytes unleash their specific arsenal (antibodies and
Tcyt cells) that captures and clears the invading pathogen from the system.
S U M M A R Y
• Stem cells are cells that can reproduce themselves and generate are tumour cells. They usually recognize target cells by decreased
differentiated cells. They are of two main types—embryonic stem expression of class I MHC molecules on the target cell surface.
cells and adult (tissue-specific) stem cells.
• Cells such as neutrophils, eosinophils, dendritic cells (for example,
• All blood cells, including erythrocytes, leukocytes and platelets, Langerhans cells) and macrophages are phagocytic in nature, while
are derived from haematopoietic (adult) stem cells (HSC). basophils and mast cells are non-phagocytic cells.
• HSC gives rise to the lymphoid progenitor cell which is a precur- • Phagocytosis is the ability of cells to actively endocytose
sor of B, T and NK cells. Myeloid progenitor cells also arise from particulate antigen (such as bacteria). The coating of antigens by
HSC, and give rise to neutrophils, basophils, eosinophils, monocytes complement components or antibodies (opsonization) leads to
and mast cells. increased phagocytosis.
• The number of cells formed or added is balanced by the • The organs of the immune system can be classified into primary
programmed removal of cells by apoptosis. (bone marrow and thymus), secondary (lymph nodes, spleen) lym-
phoid organs and tertiary (cutaneous-associated lymphoid tissues)
• The signals for programmed cell deaths could be from within the lymphoid tissue.
cell or may come from outside.
• Primary lymphoid organs are sites where B and T lymphocytes
• These signals activate caspases, a family of cysteine proteases mature. Secondary lymphoid organs are sites where naïve lympho-
within the cell, that leads to cell death. cytes interact with antigen and are activated.
• B lymphocytes arise in the bone marrow and mature in the bursa of • Bone marrow is the site where all blood cells are synthesized and
Fabricus (in birds) or in the bone marrow (in mammals). mature (except T cells which mature in the thymus). The thymus is
B lymphocytes are the only cells that produce antibodies. a lymphoid organ where T cells mature and self-reactive T cells are
• T lymphocytes arise in the bone marrow and mature in the thymus. removed.
T lymphocytes and B lymphocytes differ in their function and phe- • Lymph nodes constitute filtration beds where lymph arriving from
notypic markers: for example, T cell express T-cell receptors and tissues bring antigens. The spleen efficiently traps blood-borne
the CD3 complex, while B cells express B-cell receptors. antigens. Antigens reaching the lymph nodes or the spleen activate
• The T cells that express CD4 surface markers are CD4+ TH cells. B and T lymphocytes, initiating antibody synthesis and activating
T cells expressing CD8 surface markers are CD8+ Tcyt cells.TH T cells.
cells help or stimulate B cells to produce antibodies. Tcyt cells are • Tertiary lymphoid tissues include cutaneous-associated lymphoid
involved in the killing of altered or infected cells. tissues. The latter are composed of intraepidermal T cells, Langer-
• NK cells are those lymphocytes that bear neither B-cell receptors hans cells, TH and Tcyt cells that combat antigens that enter through
nor T-cell receptors. They lyse those cells which contain viruses or the skin.
K E Y W O R D S
• antigen-presenting • cutaneous-associated • lymphatic vessel 48 • red pulp 50

cells 35 lymphoid tissue 52 • medulla 48 • secondary lymphoid
• apoptosis 29 • dendritic cells 41 • nasal-associated lymphoid organs 48
• basophils 41 • follicles 48 tissue 51 • spleen 49
• B cells 34 • germinal centre 49 • negative selection 47 • stem cells 25
• bone marrow 46 • gut-associated lymphoid • paracortex 48 • T cells 34
• bronchus-associated tissue 51 • Peyers’ patches 52 • thymic corpuscles 47
lymphoid tissue 52 • high endothelial venule 47 • primary lymphoid • thymocytes 47
• bursa of Fabricus 32 • lymph node 48 organ 46 • white pulp 50
1. How can you differentiate between B and T lymphocytes pheno- 3. What is the missing self hypothesis? Are NK cells part of innate or
typically and functionally? Do they generate memory cells? If yes, adaptive immunity?
why? 4. How is a primary follicle different from a secondary follicle? Where
are these follicles located? Why do you think the body needs two
2. Why do you think the body retains a small yet active population of filtration sites for an antigen (that is, the spleen and lymph node
stem cells? What do you think will happen if it loses this store by performing similar function)?
accident?
5. What are M cells? What role does an M cell play in inducing mu-
H I N T —To replenish cells lost during wear and tear,and normal cell death. cosal immunity?

1. Stem cells that can differentiate into all cell lineages is: 6. Plasma cell formation is initiated in the:
(a) Adult stem cell (a) Primary follicle
(b) Embryonic stem cell (b) Secondary follicle
(c) Progenitor cell (c) Germinal centre
(d) Megakaryocyte (d) Marginal zone
2. The given cells are antigen-reactive lymphocytes, except: 7. Waldeyer’s ring is a:

(a) T cell (a) Primary lymphoid organ
(b) B cell (b) Secondary lymphoid organ
(c) NK cell (c) Tertiary lymphoid organ
(d) Macrophage (d) Gut-associated lymphoid tissue
3. A lymphocyte that is not antigen-specific is: 8. Which of these cells are usually found in the tissues?
(a) B cell (a) Red blood cells
(b) T cell (b) Neutrophils
(c) Macrophage (c) Mast cells
(d) NK cell (d) Platelets
4. Granulocytes that cannot phagocytose are: 9. CD3 complex is normally expressed on:
(a) Neutrophils (a) B cells
(b) Basophils (b) T cells
(c) Eosinophils (c) Granulocytes
(d) None of the above (d) NK cells
5. The thymic education of T cells ensures the elimination of cells 10. The movement of neutrophils towards or away from chemical
that are reactive towards: stimuli is an example of:
(a) Foreign MHC (a) Neutrophilia
(b) Self-MHC + MHC (b) Chemoattraction
(c) Foreign antigen (c) Chemotaxis
(d) Foreign antigen + self-MHC (d) Diapedesis

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part of lymphocytes. T-cell rich region is constituted by __________________.
2. Blood-borne antigens enter ___________________, while lymph 5. Epithelial cells found in Peyers’ patches that are involved in
brings antigen from tissues to __________________. inducing immune response are____________________.
3. ______________________ are the only class of dendritic cells
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In the absence of knowledge of causes or pathogenesis of disease, cen- “You have to be
turies back it was believed that diseases were caused due to poisons first, different or
that were somehow generated inside the human body. These poisons great. If you are
were thought to disturb the four vital humors of the body resulting in one of them you
the manifestation of the disease. It was in 1546 that, for the first time, may make it.”
— L O R E T TA LY N
Girolamo Francastro propagated the idea that a disease could be trans-
mitted from one person to another in the form of small seeds called
seminaria. For the first time, the concept of a discrete entity that could
transmit diseases was put forward. Though this novel concept was by
and large unacceptable at that time, it however, laid the foundation
for the idea of the occurrence of pathogens and, hence, antigens. By
the end of the 19th century, the first antigens that were identified were
After studying this chapter,
pathogens—bacteria, viruses, parasites (and their toxins). Figure 3.1 you should be able to:
shows one of the best studied antigens—sperm whale myoglobin. • Explain the difference between
antigen and immunogen
• Describe antigenic
determinants and explain their
arrangement in antigens
• Distinguish between linear and
conformational determinants
• Explain which properties of
antigen are necessary for its
antigenicity
• Describe, with example, the
difference between B-cell and
T-cell epitopes
• Define superantigen and
explain why it is such a strong
stimulator of the immune
response
• Describe hapten and its
characteristics
• Demonstrate an understanding
of the role of adjuvant and its
mechanism of action
Antigens 3
3.1 INTRODUCTION
The term antigen is derived from two terms: anti from antibody and gen from generates. So, techni-
cally, any substance that can generate antibodies (that is, elicit an immune response) may be classi-
fied as an antigen. Classically, an antigen is defined as a substance that has the (a) ability to induce
Antigens /Immunogens
the formation of specific antibodies, (b) ability to react specifically with the generated antibodies, They are macromolecules that can
and not with other antibodies that are also present in the system. elicit an immune response.
An antigen must have both the properties, as depicted in Figure 3.2. A molecule that has only
one of the properties will not be termed an antigen. For example, non-specific B-cell stimulators
IL
such as IL-4 that stimulate antibody production but cannot bind to generated antibodies are not an- IL or interleukins constitute a
tigens. Small molecular-weight substances termed as haptens (discussed later) can react specifically group of cytokines produced by
with preformed antibodies but cannot induce the generation of antibodies on its own. Hence hap- and regulate cells involved in
immune response. The generic term
tens are not antigens either. Some commonly known antigens and haptens are listed in Table 3.1. interleukin was introduced in 1979.
A more recent term used is immunogen. An immunogen may be defined as any chemical
substance that either elicits the production of specific antibodies or binds to T-cell receptors. Thus,
an immunogen stimulates both B cells that form antibodies and T cells that mount an effective
immune response. This may be represented as:
Immunogen + B cells ⎯→ Antibodies
Immunogen + T cells ⎯→ Activated effector T cells (for example, Tcyt, TH, etc.)
(145) 146-151
Haeme
COOH
56-62
15-21 (22)
Figure 3.1
Sperm whale myoglobin showing
NH2 113-119
configurational and sequential
determinants. (Reproduced from M.
Z. Atassi and A. L. Kazim (1978). “First
Consequences of the Determination
of Entire Antigenic Structure of
Sperm Whale Myoglobin”, Advances in
Experimental Medicine and Biology, 98: 9.)
Antigens Approximate Molecular Mass (Da)

Bovine serum albumin 69,000
Chicken ovalbumin 42,000

Human gamma globulin 150,000
Table 3.1 Keyhole limpet haemocyanin >2,000,000
Some commonly used experimental
antigens used in immunology. Horse apoferritin 465,000
Although technically less precise, the more common term antigen is still widely used. In this
book, antigen and immunogen are used interchangeably.
The name antigenic determinant
The complete antigen molecule is not immunogenic. Instead small regions of the antigen
originated from “ a region on the an- are antigenic or immunogenic. These regions are termed antigenic determinants or epitopes
tigen that determines its antigenic- (Greek: epi—non-self and topos—map or place).
ity”. It refers to a small region of an
antigen that is capable of eliciting an
Antigenic determinants, as depicted in Figure 3.3, are regions of an antigen that can bind an
immune response and is capable of antibody or an antigen-specific membrane receptor on a lymphocyte. The size of an antigenic de-
combining with a specific antibody terminant usually ranges from 6 to 8 amino acids in protein antigens and 6 to 8 monosaccharide
or specific receptors of T lympho-
cytes produced by such a response.
units (molecular weight: 750 Da) in the case of polysaccharide antigens. It is believed that antigenic
The term is synonymous with the determinants have not evolved to sizes larger than this particular size because it will not fit in the
word epitope. antigen-binding site of the antibody.
Almost all major macromolecular substances are capable of provoking an immune response.
» In 1960, it was discovered that Virtually all proteins, polysaccharides and nucleoproteins are capable of inducing an immune
the immune system had two
arms—B cells that produced anti-
response.
bodies and T cells that produced It is interesting to
effector T cells and cytokines. It note that DNA/RNA is
was then that the term “immuno-
gen” was brought into currency to
either non-antigenic or,
include both B- and T-cell-mediated at best, weakly antigenic.
immunity. This could be possible
due to the fact that a set
» Some well-known examples of
of five nucleotides (five
polysaccharide antigens include
bacterial capsule and human blood Antibody nucleotides will fit the
group antigens formed antigen-binding site on
an antibody) generates
» DNA can become antigenic in only 625 different pos-
some diseases such as systemic
Antibody binds sible combination (54),
lupus erythematosus—a severe
antigen all of which may occur in
autoimmune disease.
Antigen cleared from self. Hence, most native
the system DNA molecules are non-
antigenic. Compare this
Antigen
with proteins compris-
enters
ing 20 different naturally
occurring amino acids.
There are one thousand
million possible com-
binations of only seven
residues. With these
many alternatives, many
combinations will not
occur on self-tissues and
so will act as antigenic
determinants when pres-
Figure 3.2
ent on foreign substances.
Antigen stimulates antibody generation. Lipids are generally non-
Note that the antibody is specific for the antigenic as well.
antigen and binds to it to destroy it.
ANTIGENS 61
Macromolecules acting as antigens typically contain

Antigenic
multiple determinants on their surface, that is, they are determinant
multivalent. This means that either the same antigenic
determinant occurs many times on the surface of the
antigen (as in the case of polysaccharides) or that several
different antigenic determinants can occur on the antigen
surface as in most proteins (see Figure 3.4). In nature,
both types of multiplicity exist, that is, some determi-
nants may be repeated on antigen surface interspersed
with some different antigenic determinants.
Antigenic determinants are normally well-separated
Figure 3.3
spatially and two separate antibody molecules can bind Schematic representation showing
to the same antigen molecule without influencing the protein antigen and antigenic
binding of the other. Such determinants are called non- Antigen determinant.
overlapping determinants. In other instances, antigenic

determinants may
be located so close
that the binding of
one antibody to a
determinant may
sterically interfere Figure 3.4
A schematic diagram illustrating the
with the binding varying pattern of arrangement of
of another anti- antigenic determinants.
body molecule. In a) b) c)
such cases, deter-
minants are said
to be overlapping.
An antigenic determinant on a protein antigen may involve elements of the primary, sec-
ondary, tertiary and even quaternary structures. In proteins, epitopes formed by adjacent amino
acid residues in the covalent sequence are called linear or sequential determinants. Sequential
antigenic determinants of the sperm whale myoglobin are depicted in Figure 3.1. Linear determi-
nants may be accessible to antibodies in the native protein if they appear on the surface. More often,
some linear determinants are buried inside the protein core and appear only when the protein is
denatured. Linear determinants are not lost after denaturation, as shown in Figure 3.5.
Conformational
determinant
Conformational lost
determinant
Figure 3.5
Temperature increase The effect of temperature on
Denaturation conformation and linear antigenic
Linear determinants. An increase in
determinant temperature denatures the protein
intact causing it to lose its three-dimensional
structure. Thus the conformational
determinants are lost while the linear
Linear determinant determinants remain intact.
In contrast, conformational or non-sequential determinants are formed by parts of a poly-

peptide chain that are far from each other in the linear sequence but have been brought close to
form an antigenic determinant by folding of the polypeptide. Conformational determinants are
lost after the protein is denatured and unfolded. Conformational determinants of the hen egg
lysozyme are depicted in Figure 3.6.
A single protein may

contain both linear and
conformational epitopes,
30
115 for example sperm whale
myoglobulin. Neoantigenic
determinants (Greek: neo—
127 new) are those determi-
6 nants that are introduced
COOH into the antigen by covalent
modification procedures
Figure 3.6 NH2 such as phosphorylation
Hen egg lysozyme showing one
conformational antigen stablized by and/or proteolysis of pro-
disulphide bonds. Note the disulphide tein antigen. Such modifi-
linkages between the cysteine residues 94 76
that help to keep the three-dimensional
cations alter the covalent
structure intact. (Reproduced from structure of the antigen
the Journal of Biological Chemistry by and can produce new
Canfield and Liu. © 1965 by American 80 64
Soc for Biochem Molecular Biology.
antigenic epitopes. Such
Reproduced with permission of epitopes are called neoan-
American Soc for Biochemistry and tigenic determinants. The
Molecular Biology via Copyright
Clearance Centre.)
formation of neoantigenic
determinants is depicted
in Figure 3.7.
Neoantigenic
determinant formed
Proteolytic
cleavage
Protein showing
Native protein neoantigenic
determinants
Haptenic
determinant formed
Chemical
Figure 3.7
+
modification
The formation of neoantigens by
proteolytic cleavage. The addition of
haptens to native structures leads to the
formation of haptenic determinants. Native protein Hapten
3.2 GENERAL PROPERTIES OF ANTIGENS

3.2.1 MOLECULAR SIZE
All the major types of macromolecules (proteins, polysaccharides and nucleoproteins) are capable,
to different extents, of eliciting an immune response. However, it is believed that the immune system
originally developed with the capacity to recognize any molecule as an immunogen as long as it met
ANTIGENS 63
certain criteria pertaining to size and structure. There is a certain minimum molecular size below
which a molecule is not immunogenic. A relative molecular mass of about 1,000 is the lowest limit
below which molecules are found to be non-immunogenic. Molecules between 5,000–10,000 Da Da or Dalton
are weakly immunogenic though better than 1000 Da molecule. Immunogens of relative molecu- A unit of molecular mass, named
after the chemist John Dalton who
lar mass above 10,000 can generally induce a sufficiently strong immune response, and molecules proposed that the mass of one
above 100,000 are most effective in triggering an immune response. hydrogen atom is one atomic mass
unit (or 1 dalton).
3.2.2 SELF OR FOREIGN

In the early foetal stages, a distinction is made between self and non-self, so that self components can
no longer serve as immunogens. In order to elicit an immune response, a molecule must be recog-
nized as non-self by the biological system. Generally, the body’s own antigens are recognized as self
and hence cannot act as antigen/immunogen. However, homologous antigens from another species
will be recognized as an immunogen and will be dealt with accordingly. For example, bovine serum
albumin is not immunogenic when injected into a cow but is strongly immunogenic when injected
into a rat. Generally, the greater the phylogenetic distance between two species, the greater the struc- Phylogenetic Distance
tural (and hence antigenic) disparity between them; and hence, stronger the immune response. The phylogenetic distance is a
measure of the evolutionary distance
between two species. At the molecular
3.2.3 CHEMICAL COMPLEXITY level, the number of nucleotide or
Apart from molecular size and foreignness, chemical complexity of the immunogen is also a key fac- amino acid substitutions as well
as immunological cross-reactions
tor. Synthetic homopolymers (polymer of one amino acid) fail to elicit an immune response. Heter- is a more popular measure of the
opolymers (polymers of different amino acids) are the best immunogens as they are structurally evolutionary distance.
complex. A complex of lipids (that are themselves non-antigenic) associated with polysaccharides is
called a lipopolysaccharide. This is a chemically complex molecule that is strongly immunogenic.
3.2.4 ROUTE OF ENTRY

The route, or portal of entry (Latin: porta—gate) as it is commonly called, refers to the route taken
by the pathogen (or antigen) to enter the body of the host. The immune response to an antigen
varies according to the portal of entry of that antigen. Protein antigens that enter subcutaneously
or intradermally are usually immunogenic as these antigens are taken up by the antigen-present-
ing cells (Langerhans cells) and transported to lymph nodes, where the immune response occurs.
In contrast, large doses of protein immunogen, administered intravenously, often induce specific
irresponsiveness. Such irresponsiveness could be due to tolerance induction in lymphocytes or Tolerance
suppression of reactive T-cell clones. The inability of the immune system
to elicit an immune response despite
the presence of an antigen is termed
3.3 B-CELL AND T-CELL EPITOPES tolerance.
The mechanism of recognition of antigens by T cells and B cells is basically different. Studies have
revealed that B and T cells recognize different antigenic determinants on the same antigen mol-
ecule as is schematically depicted in Figure 3.8. For example, when mice are challenged by a small
protein such as glucagon, the antibody is elicited against epitopes present on the amino-terminal,
whereas T cells respond only to epitopes located at the carboxyl terminal. This suggests that distinct
B- and T-cell epitopes occur in an antigen. However, such a sharp demarcation between B-cell and
T-cell epitopes may not be there in every antigen. Some important characteristics of B- and T-cell
epitopes are listed in Table 3.2.
Antibody T cell
Figure 3.8
B cells and T cells usually recognize
different epitopes. When a protein such
as glucagon is injected into a mouse,
Binds Responds antibodies are formed against the
epitopes located at the N terminal, while
T cells respond to epitopes present at
H2N COOH the C terminal. Such a sharp difference
between B-cell and T-cell epitopes may
Glucagon not exist in all antigens.
Characteristics B Cell T Cell

Epitopes recognized by B-cell receptor T-cell receptor (Membrane-bound)
(Membrane-bound
antibody)
Epitopes presented by — MHC molecules

Epitopes properties Accessible, hydrophilic, Accessible or hidden, usually internal
sequential and/or non sequential peptides, produced by antigen processing
Table 3.2
B- and T-cell epitopes. Nature of antigen Protein, polysaccharide, lipids Mostly protein
3.3.1 PROPERTIES OF B-CELL EPITOPES

The properties of B-cell epitopes are as under:
• generally composed of hydrophilic amino acids;
• located on the surface of the native protein so that it is topographically accessible to the
antibody or antibody-like B-cell receptor;
• can contain linear or conformational epitopes;
• tend to be located on the flexible region of the immunogen, thus maximizing its easy
binding with the antibody that might not otherwise react with it, if it was rigid; and
• may contain overlapping and non-overlapping determinants. Bovine serum antigen (BSA)
has 25 overlapping antigens on its surface.
Some determinants induce a more pronounced immune response than other epitopes in a given
Immunodominant animal. Such epitopes are called immunodominant. These are usually those epitopes that project
The antigenic determinants (or distally from the central mass of the immunogen.
their subunits) that are most easily
recognized by the immune system.
3.3.2 PROPERTIES OF T-CELL EPITOPES
The properties of T-cell epitopes are as follows:
• Processing of an antigen is required before it can be presented to a T cell. This processing
yields peptides which bind to class I or II MHC molecules and this complex is then
presented to T cells. (The details of antigen processing and presenting are described in
Chapter 10.)
• T-cell epitopes are always presented together with MHC. T-cell receptors do not recognize
any epitopes that are presented alone.
• Antigens recognized by T cells have two regions—epitope, that interacts with T-cell recep-
tor, and agretope, that interacts with the MHC molecule. Interactions between the epitope
and T-cell receptor, and the agretope and MHC are purely non-covalent.
• Epitopes recognized by T cells are often internal and usually sequential.
3.4 SUPERANTIGENS
Recently, a class of protein antigens with “super” antigenic properties has been characterized and
Superantigen
named superantigens. Superantigens bind simultaneously to T-cell receptors and class II MHC
An antigen, usually a protein, which molecules. This non-specific binding activates into the lymphoproliferative phase in T cells and in-
can bind simultaneously to T-cell duces pronounced cytokine production by T cells in vivo, thereby causing a variety of pathological
receptor and a class II MHC molecule
on an antigen-presenting cell is
consequences such as fever, malaise, diarrhoea, etc. Unlike conventional antigens, superantigens
called a superantigen. This binding are not internalized and degraded by antigen-presenting cells. Instead, they bind directly to class II
of superantigen may cause T-cell MHC molecules outside of the antigen-binding cleft.
proliferation and release of cytokines
from the bound cells. Sometimes,
Superantigens bind on the side of the T-cell receptor, far from the normal antigen-binding site
superantigens may also activate on the receptor. Most superantigens activate an impressive 5–25 per cent of T cells although most
mast cells. benign protein antigens can activate less than 0.01 per cent of T cells. To date, the most extensively
studied superantigen is Staphylococcal enterotoxin B (SEB). Among other important bacterial
» Staphylococcal enterotoxin causes superantigens are pyrogenic exotoxins from Streptococcus pyrogens, Mycoplasma arthritidis (MAS) and
food poisoning in humans. Staphylococcal exfoliative toxin. The general mode of action of superantigens is depicted in Figure 3.9.
ANTIGENS 65
A new class of superantigens has recently been

reported that specifically activates B cells, leading
to apoptosis of B cells. Protein A of Staphylococcus
aureus is representative of this class.
3.5 HAPTENS
T cell
The term hapten (Greek: aptein—to grasp or fasten)
was introduced by Ehrlich who considered the
antigen (he was studying the diphtheria toxin then) T-cell receptor
to be a distinct molecular entity, containing two
active groups—haptophore, the group for binding Superantigen
and toxophore, the group for toxicity.
A hapten can be defined as any substance of Class II MHC
molecular wieight less than 1,000 Da, that, though
incapable of stimulating antibody formation by it-
self, is able to react with pre-formed antibody mole- Carrier
cules. Examples of haptens include 2,4 dinitriphenol This term refers to an immuno-
genic protein to which the hapten is
p-aminobenzenearsonate, monosaccharides, amino coupled to enable it to generate an
acids, etc. Many biologically important substances immune response. Carrier proteins
such as sugars, amino acids, drugs, peptide hor- act as the substrate for the antigen-
processing pathway while the
mones, etc. function as haptens. A large number of hapten moiety contributes the
chemicals such as dinitrophenol (DNP), and drugs epitope.
such as penicillin, are classified as hapten.
A hapten becomes immunogenic when it is Figure 3.9
Antigen-presenting
attached to an immunogenic carrier molecule such Superantigen-induced activation of
cell immune response.
as a protein, yielding an immunogenic hapten–
carrier conjugate.
Consider the case of DNP. DNP is a hapten and is unable to induce antibody formation when
Haptenic determinants
injected in test animal, alone. However, when DNP is linked to an immunogenic carrier molecule Hapten molecules that are attached
such as bovine serum albumin (BSA), it results in the formation of anti-DNP antibodies. to carrier molecules and capable of
Although anti-hapten antibodies are usually predominant, animals immunized with such a eliciting an immune response are
called haptenic determinants.
conjugate produce antibodies, not only against hapten, but also against epitopes of the carrier
molecule. Moreover, antibodies are also formed against neoantigenic determinants formed by
parts of both carrier (BSA) and hapten molecules. The formation of antibodies against antigen,
neoantigen and hapten-linked antigen is shown diagrammatically in Figure 3.10.
3.6 A D J U VA N TS
Adjuvants (Latin: adjuvare—to help) are substances which when mixed with antigen and injected with « Adjuvants were first described
it, accelerate, enhance and prolong the immunogenicity of the antigen without altering the chemical by G. Ramon in 1925. He gave the
name substance stimulantes et
composition of the antigen. Adjuvants are agents that enhance the immune response and are usually adjuvantes de l immunite.
used when either the concentration of the antigen or its immunogenicity is low. In both cases, the addi-
tion of adjuvant to the antigen augments the immune response manifold. Chemicals commonly used as « A. T. Glenny in 1926 demonstrated
the adjuvant activity of aluminium
adjuvants are alum (aluminium potassium sulphate), Freund’s complete and incomplete adjuvants. compounds for the first time.
Freund’s incomplete adjuvant is an emulsion of oil in water (aqueous solution of mineral oil and
mannide monooleate which acts as emulsifier). Freund’s complete adjuvant contains heat-killed myco- « In 1937, Jules Freund, a professor
in preventive medicine in Budapest,
bacterium in oil-and-water emulsion. Freund’s complete adjuvant, though a highly effective adjuvant, is developed a very effective adjuvant
never used in human vaccines. Another interesting molecule, ISCOM (an acronym for immune stimu- that was an emulsion of water in
lating complex), was suggested by Morien in 1984 as an adjuvant. ISCOM is a cage-like molecule that mineral oil and heat-killed myco-
bateria. This came to be known as
is made up of cholesterol and Quil A. Quil A is a terpenoid, extracted from the bark of the tree Quillaja complete Freund’s adjuvant and is
saponaria. This molecule spontaneously assembles in the presence of antigens to form cage-like struc- highly effective in animals.
tures. ISCOM is highly immunogenic and is used in a number of animal vaccines. For the vaccination of
human beings, the most commonly used adjuvants are still common ones such as aluminium hydroxide,
aluminium phosphate or alum precipitate. Table 3.3 provides summary of commonly used adjuvants.
Antibodies
Generates
antibody
Antibodies bind to
Native protein antigenic determinant
Generates
antibody
Native protein
modified with hapten
Anti-hapten
antibodies
Neoantigen
Generates
antibody
Figure 3.10
Schematic representation showing
how antibodies are generated against Protein showing neoantigen Anti-neoantigen
antigen, hapten and neoantigen. formed by hapten cross-linking antibody
Adjuvant Type Composition Mode of Action

Alum Aluminium phosphate gel Slow release of antigen;
Aluminium hydroxide gel increased uptake of antigen
by APC
Freund’s complete adjuvant Oil-in-water emulsion with Slow release of antigen;
killed mycobateria stimulates macrophages;
increased uptake of antigen
by APC
Freund’s incomplete adjuvant Oil-in-water emulsion Slow release of antigen
Alum
+BCG Aluminium phosphate + BCG Slow release of antigen;
stimulates APC
+Muramyl dipeptide Aluminium phosphate + Slow release of antigen;
muramyl dipeptide stimulates APC
+Lipopolysaccharide Aluminium phosphate + Slow release of antigen;
bacterial lipopolysaccharide stimulates APC
+Diphtheria and tetanus toxoid Aluminium phosphate/ Slow release of antigen;
hydroxide + toxoid stimulates APC
Table 3.3
Glucans/Dextrans Glucans and dextrans Non-specific stimulator of APC
Some common adjuvants.
ANTIGENS 67
Based on the above discussion, it may be said that the use of adjuvants is beneficial in improving an-
tigen delivery to antigen-presenting cells as well as processing and presentation by antigen-presenting
cells. The mechanism by which adjuvants exert their biological effect appears to be as follows:
• Antigen mixed with oil/water emulsion is slowly released, prolonging the time of expo-
sure to the immunogen from days to a few weeks.
• Adjuvants that have gel (for example, alum) or emulsion (for example, Freund’s) associ-
ate with the antigen and facilitate the transport of the antigen to the draining lymph node
where the immune response occurs.
• Adjuvants (such as alum) bind antigens and increase antigen size, thereby increasing the
chances of phagocytosis.
• Adjuvants may increase the non-specific proliferation of committed lymphocytes thereby
increasing the chances of antigen-specific clonal proliferation.
• Adjuvants increase the efficiency of macrophage-processing of antigen by inducing a
local inflammatory response, which attracts macrophages. This is especially important in
Freund’s adjuvant.
A large number of adjuvants are being developed and clinically tested for use in human vaccines.
These are gel-based (aluminium hydroxide, calcium phosphate), oil emulsion-based (Freund’s incom-
plete, MF-59, SAF), particulate (ISCOM, biodegradable microspheres, saponins,) synthetic (polphos-
phazene, non-ionic block polymer,) and microbe-based (muramyl dipeptide) adjuvants. Freund’s
incomplete adjuvant,which lacks mycobacteria, has been used in influenza vaccine in human subjects.
Gel Filtration Chromatography

Mixture of small
Gel filtration chromatography or molecular sieving is and large proteins
one of the most commonly used techniques in protein
Loaded onto the column
biochemistry. This chromatographic method, which
was invented by G. H. Lathe and C. R. Ruthiven in 1955,
involves the separation of a mixture of proteins on
the basis of molecular weight or, more precisely, their
Small proteins
Stokes radius. This separation of proteins is achieved migrate slowly
on an apparatus called column. A column consists of a
glass cylinder with a nozzle at one end (see Figure 3.11). Large proteins
migrate faster
A glass column is packed with sieving material which
consists of porous beads made up of highly hydrated
polymers such as polymer of glucose (sephadex),
agarose (sepharose) or polyacrylamide (sephacryl).
The beads of these polymers are extremely small
Separation of mixture of small and large
~100 μM in diameter. When a solution containing proteins by Gel Filtration Chromatography
proteins of different molecular weight is applied to
the column, the larger proteins cannot enter the po-
rous beads. These large molecules migrate outside
the gel beads and emerge first. The smaller protein
molecules have to travel through the channels in the
bead as well as outside the bead and hence take the
longer path. Consequently smaller proteins exit later Large molecules cannot
enter beads, migrate Small molecules pass
than large proteins. The result is separation of the small faster, and hence elute early through the beads, take the longer
proteins from the large ones. Gel chromatography is a path, migrate slowly
and elute later
method classically used for protein (including antibody)
purification, desalting (removal of salts) of proteins and
determination of molecular weight of proteins.
Figure 3.11
Gel filtration chromatography.
To summarize, an antigen or, more precisely, immunogen may be defined as that molecule ca-
pable of eliciting an immune response. Antigens are usually large and foreign molecules. Proteins
and polysaccharides can act as antigens while lipids and DNA cannot. Haptens, which are low-
molecular weight chemical compounds such as glucose and amino acids, are non-antigenic. Those
regions on the antigen that can stimulate antibody production and react with it, or are presented
on MHC, are called antigenic determinants or epitopes. B cells and antibodies recognize antigenic
determinants located on the surface while T-cell epitopes are usually internal or buried inside the
protein structure. Those antigens that are not strongly antigenic are mixed with adjuvants. Adju-
vants are chemical substances such as aluminium hydroxide that enhance the immunogenicity of
the antigen without altering its chemical composition.
S U M M A R Y
• An antigen, strictly speaking, is defined as that molecule that can epitopes are presented together with MHC, and are not recognized
stimulate antibody generation by B cells. by T-cell receptors when presented alone.
• A broader and more recent term is immunogen. Immunogens are • The antigens that can stimulate a large number of T cells, irrespec-
substances that stimulate both B and T cells. tive of their antigen specificity, are termed superantigens. Superan-
tigens bind outside of class II MHC, cross-linking MHC and TCR,
• Antigenic determinants are small regions usually on the surface of and activating T cells.
the antigen that bind antibody or antigen receptors of T cells.
• Hapten is a low-molecular weight substance that is incapable
• Antigenic determinant or epitopes on protein antigen can be of stimulating antibody formation on its own. Hapten linked to
formed either by adjacent amino acids (sequential) or those amino macromolecules can elicit the formation of antibodies and can bind
acids that have been brought close by the tertiary conformation of antibodies.
the protein (non-sequential).
• Adjuvants enhance the immunogenicity of antigens without alter-
• Antigen should be large, foreign and of a chemically complex ing their chemical composition, when administered with antigens.
structure, to elicit an immune response. The principal mechanism of adjuvant activity includes aggregating
• B and T cells recognize different epitopes on the surface of the antigens for increased uptake by phagocytes, prolonging the time
antigen. A B-cell epitope is hydrophilic and located on the protein of exposure to the antigens of the immune system and inducing
surface. These epitopes can be sequential or non-sequential in na- a local inflammatory response.
ture. T-cell epitopes are often internal and usually sequential. T-cell
K E Y W O R D S
• adjuvant 65 • conformational antigenic • linear antigenic • sequential antigenic

• alum 65 determinant 61 determinant 61 determinant 61
• antigen 59 • epitopes 60 • neoantigenic • superantigens 64
• antigenic determi- • hapten 65 determinant 62 • T-cell epitopes 63
nants 60 • haptenic determinant 65 • overlapping antigenic
• B-cell epitopes 63 • immunogen 59 determinant 61
1. What do you foresee if the body starts inducing an immune re- H INT —They induce similar immune responses. When antigen is an
sponse against haptens? inactivated pathogen, or its part, and is introduced with the intention of
H I N T —Chaos, very tired immune system and short life. Imagine every meal rendering protection, it is vaccination.
you take pushes millions of haptens (e.g., glucose, amino acids) into the 4. What do you think will happen if some exogenous antigen shares
blood and if the body starts responding to it! antigenic determinants with an eye lens protein?
2. Do you think that the antigen-processing machinery really dif- H INT —Nothing. Eye lens is a privileged site, not exposed to the immune
ferentiates between B- and T-cell epitopes and then processes only system.
T-cell epitopes? Comment.
5. Can you describe how antigen generates an enhanced immune
H I N T— No, but who is complaining! response when mixed with adjuvants. Search to find out whether
3. Bovine serum albumin is used for immunization while tetanus tox- adjuvants have been tested on humans.
oid is used for vaccination. If both are antigens, why do they induce
two different responses?
ANTIGENS 69

1. Immunogen will stimulate: 6. The least antigenic substance among those given below is:
(a)B cell only (a) Protease
(b)T cell only (b) Nuclease
(c)Both B and T cells (c) Glycoprotein
(d)Neither B nor T cell (d) Nucleic acid
2. B cell recognizes: 7. The size of antigenic determinants is approximately:

(a) Internal epitopes (a) 3 amino acid residues
(b) Surface epitopes (b) 7 amino acid residues
(c) Hydrophobic epitopes (c) 20 amino acid residues
(d) Agretope (d) 40 amino acid residues
3. Non-sequential antigenic determinants are usually 8. T-cell epitope will encounter all except:
recognized by: (a) MHC
(a) B cells (b) Antibody
(b) T cells (c) TCR
(c) Macrophages (d) Antigen-presenting cell
(d) None of the above
9. Superantigen activates:
4. For a molecule to be antigenic, it should have all of the (a) T cell
following properties except: (b) B cell
(a) Foreign (c) Antigen-presenting cell
(b) Complex structure (d) NK cell
(c) Large size
(d) Rigid conformation 10. Adjuvant increase all of the following, except:
(a) Number of antigen molecules
5. Neo-antigenic determinants could be introduced into (b) Time of exposure of immunogen
antigen by: (c) Processing of antigen
(a) Proteolysis (d) Inflammatory response
(b) Haemolysis
(c) Cell lysis
(d) Denaturation
State true or false against each statement. If false, give reason(s)

1. Hapten cannot elicit antibody formation but can bind to it. 4. Non-sequential epitopes are retained after denaturation.
2. An equal number of antibody molecules will bind similar over- 5. In glycoproteins, antigenic determinants will only be contributed
lapping and non-overlapping antigens. by amino acids.
3. Nucleic acid can become antigenic if chemically modified.
Amit, A. G., R. A. Mariuzza, S. E. V. Philips and R. J. Poljak Rothbard, J. B. and M. L. Oefter (1991). “Interaction Between
(1986). “Three Dimensional Structure of an Antigen-Antibody Immunogenic Peptides and MHC Proteins”, Annual Review of
Complex at 2.8Å Resolution”, Science, 233: 747–53. Immunology, 9: 527–65.
Arnon, R. (1968). “A Selective Fractionation of Antilypozyme Sela, M., B. Schechter, B. I. Schechter and E. Borek (1967).
Antibodies of Different Determinant Specificities”, European “Antibodies to Sequential and Conformational Determinants”,
Journal of Biochemistry, 5: 583–89. Quantitative Biology, 32: 537.
Demotz, S., H. M. Grey, E. Appella and A. Sette (1989). “Charac- K. G. Garcia, L. Teyton, I. A. Wilson (1999). “Structural Basis of
terization of a Naturally Processed MHC Class II Restricted Cell T-cell Recognition”, Annual Review of Immunology, 17: 369–97.
Determinants of Hen Egg Lysozyme”, Nature, 342: 682–84.
Laver, W. G., G. M. Air, R. G. Webster and S. J. Smith Gill (1990).
“Epitopes on Protein Antigens—Misconception and Realities”,
Cell, 61: 533–36.
The term antibody owes its origin to an ancient German word “Two heads are
Antikorper. The term Antikorper was first coined by Von Behring and better than one.”
Kitasato around the 1890s to describe the agent in the blood that —HOMER,
Iliad, X, 225
was capable of passively transferring immunity. They only suggested
that there exists a discrete entity, or body, capable of carrying
immunologicals pecificity, and thus able to act against (anti) the
offending toxin. Another term, immunoglobulin, came into focus
around the 1930s. At that time it was known that antibodies are
involved in immune response, they are proteins and belong to the
class of proteins termed as globulins.
The term gamma globulin for antibodies was the result of the
pioneering work of A. Tiselius and E. A. Kabat in 1939. Tiselius, who After studying this chapter,
used electrophoresis to separate proteins for the first time, studied
• Explain the origin of the terms
the separation of proteins present in rabbit serum. Electrophoresis antibody, γ-globulin and
immunoglobulin
of the serum fraction revealed four peaks corresponding to albumin, • Give an account of antibody
structure
and alpha (α), beta (β) and gamma (γ) globulins, shown in Figure 4.1.
• Explain immunoglobulin
Tiselius and Kabat demonstrated that most antibodies are found in the domain, immunoglobulin
fold and complementarity-
determining region
third-fastest migrating group of globulins and hence named gamma
• Distinguish between isotype,
globulins (γ) for the third letter of the Greek alphabet. Advances in allotype and idiotype
• Describe the effector functions
electrophoresis technology has made it possible to resolve human of the antibody
serum proteins into four major non-albumin peaks—α (α1 and α2), • Describe the structure and
functions of different classes of
antibodies
β and γ. We now know that although immunoglobulin G (IgG), the
• Give an account of the
major antibody molecule is indeed found in γ-fraction, a significant structure of hinge regions and
location/number of disulphide
amount of IgG as well other classes of antibody molecules are found in in different antibody molecules
• Describe the immunoglobulin
β and α fractions of globulins. superfamily
Antibodies
4.1 INTRODUCTION
Antibodies can be defined as multifunctional (glyco)proteins produced in direct response against anti-
gens by vertebrates, and essential for the deterrence and resolution of infection by various pathogens.
4
Antibodies initiate their biological effect by non-covalently binding to antigens. Antibodies do
not modify the covalent structure of antigens. The binding of antibody to antigen, although non-
covalent, is extremely specific.
Antibody molecules are referred to as multifunctional molecules as they perform more than
one function. One part of the immunoglobulin is involved in the binding of the antigen, while the « There are about 5 x 1016
other part of the molecule may be involved in binding to receptors on phagocytes, the activation of molecules of antibody per millilitre
complement pathways and the activation of the NK cells of the immune system. of blood.
4.2 L A N D M A R K S I N T H E E L U C I D AT I O N O F
ANTIBODY STRUCTURE
A series of experimental observations stretching over almost two decades has led to the present knowl-
edge of basic antibody structure. The chronology of some of the important events is outlined below: « M. Heidelberger established the
protein nature of antibodies in
• By the 1930s, it was shown that antibodies belonged to a class of protein called globulins. 1928 by showing that nitrogen was
• During the 1940s, Ehrlich introduced the concept of antibody specificity and Karl Landsteiner present in every antigen–antibody
precipitate in which the antigen
demonstrated that precipitation of anti-hapten antibodies by hapten–protein conjugates is was a polysaccharide.
inhibited by free hapten. It was inferred that antibodies were specific in their recognition.
- +
Serum
proteins
Albumin
G
β
IgG
A2 A1
IgA IgM
IgD
Electrophoretic mobility
Figure 4.1
Graph showing the electrophoretic
mobility of the four major
immunoglobulin classes.
• In 1945, L. Pauling and D. Pressman suggested that there are pockets in antibodies where
antigens bind. They also suggested that the interaction between an antigen and a specific
antibody site is purely non-covalent.
• In the late 1940s, Eisen and Karush, using the technique of equilibrium dialysis, showed
that antibodies are divalent.
• In 1952, Campbell and Bulman calculated that the specific combining site of the antibody
could not be larger than 700Å.
• With the development of ultracentrifugation by Svedberg, the molecular weight of an
antibody was found to be 160,000 Da.
• In 1950, Porter selectively cleaved antibody molecules by the proteolytic enzyme papain.
The enzymatic digestion gave three products, as shown in Figure 4.2: two fragments (Fab,
acronym for fragment antigen binding) of 45,000 and one of 50,000 (Fc, acronym for
fragment crystallizable).
• Edelman in 1959 demonstrated that if an antibody molecule is first reduced and then
proteolysed, a different set of products is formed.
• In 1961, Edelman and Poulik showed that immunoglobulins are composed of two
subunits with molecular weight of about 20,000 and 50,000, later termed as light (L) and
heavy (H) chains.
• In 1963, using data on a number of chains and enzyme cleavage results, Porter determined
» The first complete amino acid
sequence of an IgG molecule was how Fab and Fc are related to H and L chains. He injected Fab and Fc from rabbits into goats
announced in 1969. to raise antisera. Antisera to Fab could react to an L or H chain but antisera to Fc reacted
only with an H chain. Porter suggested the Y-shaped prototype structure of an antibody.
Bence Jones proteins • During the 1960s, Bence Jones proteins (free immunoglobulin light chain dimers) were
Free immunoglobulin light chains isolated and sequenced. Based on the differences in a sequence of carboxyl-region, classes
which are secreted in the urine of
myeloma patients are popularly and subclasses of immunoglobulins were identified.
known as Bence Jones proteins. They • Edelman, in 1970, suggested that heavy chains of antibodies are involved in the secondary
are named after Dr Henry Bence or effector functions of antibodies.
Jones who first isolated them. Their
exact nature was elucidated by • In 1970, Wu and Kabat identified the hypervariable region on the antibody molecule and
Edelman and Gally in 1962. suggested the location of specific combining site on the antibody molecule.
• In 1974, the structure of the combining site of the antibody molecule was confirmed by
X-ray crystallographic studies. The schematic representation of the elucidation of
antibody structure is shown in Figure 4.3.
2 6
1 4
S-S S- S-S S-
S S-S S
S
S-S
S
S-
S-
Papain
3 5
IgG
Pepsin
S-S
S-S
3
1 2 S-S
S
S- S-S
S-
S
S-S S-S
Figure 4.2 4 5
The enzymatic cleavage of human IgG by
papain and pepsin. Note that these two Two Fab One Fc Fragments
enzymes cleave IgG differently. fragments fragment One F’ab fragment of F’c
ANTIBODIES 73
Hapten Antibodies are proteins of the globulin family

protein (1930s)
Hapten–protein Karl Landsteiner (1940s)

conjugate can
bind antibodies
Antibody has a Linus Pauling (1945)

binding site
Antibody is divalent Eisen and

Karush (1940s)
Measurement of Campbell and

binding site (700 Å) Bulman (1952)
Estimation of molecular Svedberg

weight (160 kDa)
Molecular weight of Edelman and

chain (50 kDa, 20 kDa) Poulik (1961)
Y-shaped structure of Porter (1963)

antibody
Location of Wu and Kabat (1970)

combining site
Figure 4.3
The landmarks in the elucidation of
antibody structure.
4.3 ANTIBODY STRUCTURE

An antibody molecule is a Y-shaped structure that comprises four polypeptide chains (see Figure 4.4),
two identical light chains (L) of 25,000 Da (approximately 214 amino acids) and two identical heavy
chains (H) of 50,000 Da (approximately 450 amino acids). One light chain is attached to each heavy
chain and the two heavy chains are attached to each other to form a Y-shaped structure (see Figure 4.5).
VH NH2
NH2
NH2 Heavy chain
- NH2
-S
-S
-S
CH1 -S
-
-
-S
-S
-S
- -S
-
-S
-S
-S
-S
VL
-
-
-S
Light chain -S
-S
-S -S
-
-S
-
-S
- CL
-S
S-S
HOOC COOH
S-S
-S-S-
-S-S-
Complement-activating CH2
site -S-S-
-S-S-
Cell-binding CH3
site
Figure 4.4
A schematic representation of an
antibody molecule showing the bulging
immunoglobulin domains. HOOC COOH
Variable
region of
Antigen-binding heavy chain Variable
site
region of
light chain
Hinge region
-S
-S
-
S
S- -S-S- Constant
-S-S- regions of
light chain
Constant
regions of
heavy chain Disulphide bridges
Figure 4.5
The immunoglobulin structure.
ANTIBODIES 75
The first 110 or so amino acids of amino terminal of light or heavy chains have a variable se- « It is interesting to note that camels
and llamas naturally lack heavy
quence and composition among antibodies of different specificities. These segments of the highly chains. Their function is performed
variable sequence are called variable (V) region of light (VL) or heavy (VH) chain. The regions by unusually folded single light
beyond the variable region (from 110–214 amino acids in L or 110–450 amino acids in H chain) chains.
have the same composition and a relatively constant sequence in different antibodies. This seg-
ment is referred to as constant (C) region of light (CL) or heavy (CH) chain. A typical antibody
molecule has two intra-chain disulphide bonds in the light chain—one in the variable region and
one in the constant region. There are on an average four covalent disulphide bonds linking the
heavy chains.
Both the light and heavy chains have several repeating homologous units, each of about 110
amino acid residues in length, which form an independent common globular motif called as
immunoglobulin domain [see Figure 4.6(a)]. Domain
Within each domain, there is an intra-chain disulphide linkage which forms a loop of about A structurally and functionally
discrete portion of proteins. A
60–70 amino acids. Light chains contain one variable domain (VL) and one constant domain domain usually contains 50–100
(CL). Heavy chains contain one variable domain (VH) and three or four constant domains (CH1, amino acids. The residues comprise
CH2, CH3 and CH4) depending on the class of antibody. the domain fold independent
of the remainder of the proteins.
All immunoglobulin domains contain two large β-pleated sheets, each with three or four Donald B. Wetlaufer was the fist
strands of anti-parallel polypeptide, connected by loops of variable length. A conserved disulphide to describe domains as “discrete
bond links the two β sheets. This characteristic protein motif found in immunoglobulins is referred structural regions” of globular
proteins in 1973.
to as the immunoglobulin fold.
The β strands within the sheets are stabilized by hydrogen bonding while β sheets within the
immunoglobulin fold are held together by hydrophobic interaction and disulphide linkage. Though Immunoglobulin fold
immunoglobulin domains of the variable and constant regions are very similar, the domains of the Two ß sheets linked by a disulphide
bond and tightly packed
variable region have two extra β strands within the β sheets structure [see figure 4.6(b)]. against each other comprise the
Within the variable region of both heavy and light chains, some short polypeptide sequenc- immunoglobulin fold. This motif
es show exceptional variability. These are termed hypervariable regions (see Figure 4.7) and are is found in the immunoglobulin
domain. It has been suggested that
located near amino acids 25–35, 50–55 and 95–100. the immunoglobulin fold could
Three such hypervariable regions are located in the variable region of both the light have been a pattern-recognition
and heavy chains constituting about 20 per cent of the domain structure in each. The molecule, capable of recognizing
self-components that evolved
to recognize non-self or foreign
antigen.
Hypervariable
region
N-Terminus
Figure 4.6(a)
Ribbon diagram of immunoglobulin
fold and immunoglobulin domain,
C-Terminus together with a schematic diagram for
explanation. (Reprinted, with permission,
from the Annual Review of Immunology,
Volume 6 ©1988 by Annual Reviews
CL VL www.annualreviews.org).
NH2 NH2
S-S
S-S Figure 4.6(b)
Schematic representation of the folding
pattern of β sheets in C and V domain.
COOH COOH (Reprinted, with permission, from the
Annual Review of Immunology,
Volume 6 ©1988 by Annual Reviews
C domain V domain www.annualreviews.org).
Light chain
Antigenic determinant Hypervariable

regions
Figure 4.7
The antigen-binding site is formed by
the hypervariable regions of light and
heavy chains. Heavy chain
150
Variability
100
50
Figure 4.8
Wu and Kabat plot showing variability
of amino acids in immunoglobulin 0
light chain. It represents the amino acid
25 50 75 100 120
variability in the variable region of light
chain. CDR1 CDR2 CDR3
remainder of the intervening peptide segments of VL and VH domains show less variation (and
Framework region
The invariant peptide sequence more conservation). These segments are called framework regions (FRs).The Wu and Kabat plot
within the variable region that shown in Figure 4.8 depicts the variability of aminoacids in the light chain of immunoglobulins.
supports the CDRs is termed the A framework region acts as a scaffold, holding the hypervariable region in place. In intact an-
framework region. Framework
regions act as the scaffolding to tibodies, three hypervariable regions of light chain and three hypervariable regions of heavy chain
which CDRs are fixed. They are highly are brought together to the three-dimensional antigen-binding site. Because these segments form a
conserved. surface complementary to the structure of antigenic determinants, these hypervariable regions are
called as complementarity-determining regions (CDRs). In general, more amino acid residues of
CDR CDRs of heavy chains (VH domain) make contact with antigen and hence confer specificity than
The region within the variable region the light chain CDR.
that generates the antigen-binding
site is called the complementarity-
Numerous studies by Landsteiner and others have shown that a single saccharide, a substituted
determining region or CDR. It is benzene ring or even a dipeptide can occupy an antibody-combining site. These studies appear to
synonymous with the hypervariable have set a lower limit on the size of combining site. Kabat and his colleagues prepared antibodies
region. This definition was proposed
by Kabat.
against glucose and its polymer. They experimentally proved that the upper limit of the determi-
nant size was a heptasaccharide of size 39⫻12⫻7Å. These findings substantiate that antibody-
combining sites can accommodate six to seven amino acids or saccharide residues.
4.3.1 HINGE REGION

The region of the immunoglobulin heavy chain located between the CH1 (first domain on constant
region of heavy chain) and CH2 domains is called the hinge region. It is either an open rod-like helical
» Hinge regions are present in structure or an extended peptide sequence with a flexible conformation (see Figure 4.9).
heavy-chain γ (IgG), δ (IgD) and α
(IgA) chains, but not in μ (IgM) and The hinge contains from 10 (in α1, α2, γ1, γ2 and γ4 chains) to about 60 (in γ3 and δ chains) ami-
ε (IgE) chains. no acid residues. Although the greatest differences between the constant regions of the heavy chains
ANTIBODIES 77
close together
far apart
Hinge region Hinge region
Figure 4.9
a) b) The flexible hinge region.
are concentrated in the hinge region (in IgG subclasses), this region is rich in proline, cysteine, lysine
and aspartic acid which promotes the exposure of this region without tight folding. Among the sub-
classes of IgG, IgG3 is unique in having a long hinge region (about four times as compared to IgG1),
having about 62 amino acids. The region of polypeptide that acts as a hinge in the μ chain (IgM) is
not rich in proline, but instead has an attached oligosaccharide which similarly promotes exposure
to a solvent. The relatively open structure of the hinge region makes it particularly susceptible to
proteolytic cleavage. It is this region that is cleaved by proteases such as papain and pepsin. The flex-
ibility is important in allowing the two antigen-binding sites to bind epitopes placed at varying angles.
The presence of the hinge region allows the Fab arms to twist and align to tightly bind the displayed
epitopes. Most hinge regions are encoded by single exon (except the δ chain which is encoded by two
exons and the γ3 chain which is encoded by four exons).
4.3.2 J CHAIN
These are small proteins that connect two or more basic units (that is, Y-shaped molecules) in poly- « It is believed that the J chain is a
meric immunoglobulin. They are named as J (joining) chains. The J chain is encoded by a separate primitive peptide that arose before
the antibody evolved. It is found in
gene. The J chain is a glycopeptide of a molecular weight of approx 15 kDa and is disulphide-bonded both vertebrates and invertebrates
to the carboxyl terminal portion of α and μ heavy chains. such as molluscs, arthropods and
echinoderms.
4.3.3 DISULPHIDE BONDS « Disulphide bonds between light
Disulphide (-S-S-) bonds that hold together the four polypeptides are of two types—inter-chain bonds chains are reported to occur in
IgA2.
and intra-chain bonds. Inter-chain bonds occur between heavy chains (H–H) and between heavy
and light chains (H–L). The H–H bonds vary from 1 to 15 depending on class and subclass of im-
munoglobulin. H–H bonds occur primarily in the hinge region of the antibody molecules. H–L
chains are connected by only one disulphide bond. IgA2 lacks the H–L bond. Disulphide bonds
between light chains are rare and usually found under pathological condition.
Intra-chain bonds occur within an individual polypeptide, as follows:
• Light chains have two.
• Human γ, α and δ heavy chains have four.
• Human μ and ε heavy chains have five.
It should be clarified that these intra-chain disulphide bonds are the same as those that connect the
β sheets of the immunoglobulin domain. So, as is evident, the number of disulphide bonds on heavy
or light chains is the same as the number of domains.
The antibody form that has been dealt till now is of the secreted immunoglobulin (slg) type.
Secreted immunoglobulins have a charged hydrophilic amino acid sequence at the carboxyl terminal
end. In a membrane-bound antibody, a distinct carboxyl terminal sequence that includes 26 hydro-
phobic amino acids is present, followed by a short cytoplasmic, charged (usually basic) amino acid
sequence. Hydrophobic residues form an α helix that spans the membrane, while the charged tail
of amino acids is present on the cytosolic side.The rest of the Y-shaped antibody molecules point
towards extracellular side.
4.4 CLASSES OF IMMUNOGLOBULIN

Based on the amino acid differences in the carboxyl terminal of the heavy chain in humans
and the higher mammals, five major groups (termed as classes or isotypes) have been defined:
Immunoglobulin G (IgG), IgA, IgM, IgD, IgE (see Figure 4.10). In humans, there are four subclasses
of IgG, and two subclasses of IgA.
The five different classes of antibodies are determined
by the type of heavy chain involved (termed γ, α, μ, δ and ε
for IgG, IgA, IgM, IgD and IgE respectively). The nomen-
clature for antibodies is not readily apparent. The names
γ heavy chains IgM and IgG arose because of the method used to sepa-
rate antibodies into different classes by electrophoresis.
IgG Initially two distinct forms of antibodies were recognized:
high molecular weight macroglobulin and low molecular
» IgA was first described by Joseph
F. Hermans and his collegues in
weight gammaglobulin (gamma refers to electrophoretic
1959 as β2A globulin. It was mobility). When a further new class was discovered, it was
T. B. Tomasi and H. M. Grey who decided to systematize the nomenclature and call this new
gave the complete structure and α heavy chains
function of IgA in 1972.
class of antibody as IgA and it was suggested that IgM and
IgG might be called as IgB and IgC. The discovery of the
» IgD was first identified by John L. IgA next new class of antibodies gave us IgD.
Fahey and David S. Rowe in 1965. IgE was identified by the husband-and-wife team of
Kimishige and Teruko Ishizaka. They were interested in find-
ing out the type of antibody that was responsible for hypersen-
μ heavy chains sitivity to weeds. They obtained the serum from allergic indi-
viduals and immunized rabbits to prepare antisera. The rabbit
antisera was then allowed to react with each class of human an-
IgM
tibody known at that time (IgG, IgA, IgM and IgD). This way
each of the known anti-isotype antibodies were precipitated
and removed from the rabbit anti-serum. What was left was
anti-isotype antibody specific for the unidentified class of anti-
δ heavy chains body. This anti-isotype (actually anti-IgE) was able to neutral-
ize the transferability of allergy with patient serum. (The serum
of the allergic individual was known to transfer allergy to the
IgD recipient which strongly suggested the presence of a new class
» Kappa (k) and lambda (l)
represent, in Greek, the equivalent
antibody that was involved in allergy). Upon purification of
of the first alphabet of the names this new class of antibody, it was named IgE (E for erythema)
of the scientists who discovered since it was known to be involved in weed-allergic reaction,
them—Korngold and Lipari.
ε heavy chains which is characterized by “wheal and erythema” reaction.
Human immunoglobulin light chains are of two types—κ
Figure 4.10
The differences in the chain structures in or λ—based on their distinct structural (antigenic) differences.
IgE
the five major classes of antibodies. The five different classes of immunoglobulins are discussed
briefly.
4.4.1 IMMUNOGLOBULIN G
The chief characteristics of immunoglobulin G are:
• Molecular weight—150,000 Da.
• Sedimentation coefficient—7 S.
• Number of basic four-peptide units—one.
• Heavy chain—γ type.
» It is important to note that the • Four human IgG subclasses are identified: IgG1, IgG2, IgG3 [see Figure 4.11(a), (b), (c)]
IgG subclass nomenclature of and IgG4. The subclasses are distinguished by differences in the amino acid sequence of γ
one species bears no particular
relationship with that of IgG chains and numbered according to their decreasing average serum concentration; that is,
molecules of another species. For IgG1 has the highest serum concentration and IgG4 the lowest.
example, human IgG2 resembles • 80 per cent of the total serum immunoglobulin.
mouse IgG3 and not mouse IgG2.
ANTIBODIES 79
VH
VL
CH1
CL
Hinge
CH2
CH3
a) b)
VH
VL CH1
CL
CH2
CH3
Figure 4.11
Domain structures of IgG1, and IgG2
in (a) and (b); (c) shows the detailed
c) structure of IgG3.
• Functions—most abundant immunoglobulin of internal body fluids where it combats

microorganisms and their toxins. IgG1, IgG3, IgG4 can cross the placenta and impart
« About 60 per cent of the total
neonatal immunity. The complex of IgG and bacteria activates the complement path- serum IgG is IgG1 and 25 per cent
way. IgG3 is the most potent in activating complement, followed by IgG1. IgG enhances is IgG2.
phagocytosis by opsonization. IgG1 and IgG3 are the most important opsonins. IgG4 and « IgG is the only immunoglobulin
IgG2 have low affinity for Fc receptor and hence are not good opsonins. that can cross the placenta.
4.4.2 IMMUNOGLOBULIN A
The chief characteristics of immunoglobulin A
VH are as under.
VL
CH1 • Monomeric (160,000 Da) and sometimes
CL
dimeric, trimeric or tetrameric.
• Sedimentation coefficient—9 S
(monomeric).
CH2
• Number of basic four-peptide
CH3 units—one (in monomeric form).
• Heavy chain—α type.
• Two human IgA subclasses known—IgA1
a)
and IgA2. IgA2 constitutes about 20 per
cent of IgA in serum, but 50 per cent in
VH secretory fluids.
VL
• Valency for antigen binding—
VH1 CL 2 (monomeric form).
• 10–15 per cent of the total immunoglobulin
in serum.
• Functions—the predominant immuno-
CH2 globulin in external secretions such as
CH3 saliva, tears, mucous, breast milk,
secretions of the bronchial, genitourinary
Joining chain
and digestive tracts, where it defends
Secretory the exposed external surface of the body
component
against pathogenic organisms. The bind-
ing of the IgA to bacterial and viral surface
antigens prevents the attachment of the
pathogens to mucosal cells. It provides an
important defence against viruses of polio
and influenza, and certain coliform bacte-
» IgA molecules have about 18 extra ria such as Salmonella. In serum, IgA exists
residues (as compared to IgG) at
the carboxyl terminal. The secretory
as a monomer. In contrast to the serum
form of IgA is a dimer joined by the b) form, in secretory fluids IgA exists as a
J chain and is called secretory IgA. dimer containing the J chain and secretory
component. The structures of IgA1,
secretory IgA and IgA dimer are shown in
Figure 4.12(a), (b) and (c).
s-
-s-s-
-s
s-
-s
-s-s-
4.4. 3 IMMUNOGLOBULIN M
The characteristics of immunoglobulin M are as
J chain follows:
Secretory
component
• Number of basic four-peptide units—five.
The five monomer subunits are arranged
with their Fc region in the centre of the
-s-s- - pentamer and ten antigen-binding sites
-s
-s
-s-s-
-s
-s
on the periphery of the molecule. Each

-
pentamer contains additional Fc-linked

polypeptide called the J chain, which is di-
sulphide linked to two of the ten μ chains.
Figure 4.12 The domain structure and arrargement of
(a) Domain structure of Ig A1;
Heavy chain monomers of the IgM molecule is shown in
(b) secretory component of IgA; and
(c) schematic representation of
Figure 4.13(a) and (b).
secretory IgA. c) • Heavy chain—μ type.
ANTIBODIES 81
VL
CH1
CL
VL CH2
CH3
CH4
J
Chain
a)
Heavy chain
Light chain
J Chain
Figure 4.13
Arrangement of monomeric IgM in
b) pentameric structure.
• No human IgM subclasses known.

• Valency for antigen binding is 10. However, because of steric hindrance only five or fewer
molecules of large antigens can be bound.
• 5–10 per cent of the total immunoglobulin.
• Functions—the first immunoglobulin class that is produced in a primary immune
response; and also the first immunoglobulin to be synthesized by a newborn; very
effective agglutinin; and effective first line of defence against bacterial or viral invasion. Agglutinins
More efficient in activating complement than IgG. Antibodies that cause agglutination
or aggregation of cells or bacteria
• It is believed that IgM is the first to appear in ontogeny and is phylogenetically are called agglutinins.
the oldest antibody.
4.4.4 IMMUNOGLOBULIN D
The characteristics of immunoglobulin D are:
VH VL
CH1 CL
Disulphide linkage
CH2
CH3
Carbohydrate
Figure 4.14 side chain
Domain structure of IgD.
» IgD is acquired on B cells during • Number of basic four-peptide units—one. (The domain structure of IgD is shown in
maturation. Immature B cells Figure 4.14.)
express only IgM.
• Heavy chain—δ-type.
» Secretory IgD also exists in serum • No human IgD subclasses known.
though its concentration is very low • Valency for antigen binding—2.
(around 1–11 μg/ml).
• Approximately 0.2 per cent of total immunoglobulin.
• Functions—function not established; a major immunoglobulin expressed
on mature B cells, apart from IgM; probably involved in lymphocyte activation and
suppression; IgD antibodies cannot activate complement, cross placenta or cause
mast cell degranulation.
4.4.5 IMMUNOGLOBULIN E
The characteristics of immunoglobulin E are:
• Number of basic four polypeptide unit—one. (The structure of IgE is represented in
Figure 4.15.)
• Heavy chain—ε-type.
• Valency for antigen binding—2.
• Represents 0.002 per cent of total immunoglobulins.
• Functions—protection of the external mucosal surface; a major immunoglobulin
that is involved in type 1 hypersensitivity reaction; IgE binds to Fc receptors
Fc receptor
Fc receptor protein found on a present on the surface of mast cells and basophils. The aggregation of Fc receptor
number of cells, such as NK cells, when IgE binds the provoking antigen, results in degranulation of mast cells.
neutrophils, mast cells and macro- Pharmacological active mediators that are present in these granules mediate allergic
phages, that binds to the Fc region of
the antibody. manifestations (see Chapter 12).
A brief summary of the different classes and subclasses of antibodies is given in Table 4.1.
ANTIBODIES 83
VH VL
CH1
CL
Carbohydrate
side chain
CH2
Disulphide linkage
CH3
CH4
Figure 4.15
Domain structure of IgE.
Designation IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 IgM IgE IgD
Heavy chain γ1 γ2 γ3 γ4 α1 α2 μ ε δ
component
Light chain κ⫹λ κ⫹λ κ⫹λ κ⫹λ κ⫹λ κ⫹λ κ⫹λ κ⫹ λ κ⫹λ
Molecular weight (kDa) 150 150 150 150 150–600 150–600 900 190 150
Sedimentation 7 7 7 7 19 8 7
coefficient (Svedberg unit)
Concentration range in 8 3 1 0.5 3.4.0 0.5 1.5 0–.4 17–450
normal serum (mg/ml)
H-chain domain number 4 4 4 4 4 4 5 5 4

Hinge Yes Yes Yes Yes Yes Yes No* No* Yes
% carbohydrate 3 3 3 3 8 8 12 12 13
Complement fixation ⫹⫹ ⫹ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺
Cross-placenta ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺
transfer
Opsonization ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺
Transport across mucosa ⫺ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹ ⫹ ⫺ ⫺
Binding to mast cells ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺
Binding to ⫹⫹ ⫹⫹ ⫺ ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺
staphylococcal
Table 4.1
protein A A summary of the features of the various
*They lack hinge region but have an additional domain with hinge-like feature. classes and subclasses of antibodies.
4.5 A N T I B O D Y - M E D I AT E D E F F E C T O R
FUNCTIONS
In general, the primary function of an antibody is to bind antigen. This binding is sufficient
to inactivate bacterial toxin or the entry of some virus into the cells. However, in most other
instances, simple binding has almost no effect on the invading pathogen. The pathogen has to
be removed from the system. While one part of an antibody molecule (the Fab portion) binds to
antigen, the other part (constant region of heavy chain—Fc) interacts with the other elements
of the immune system, resulting in the effector function of the antibody which eliminates the
pathogen from the system.
4.5.1 A C T I VAT I O N O F C O M P L E M E N T S Y S T E M B Y I g G
AND IgM
The activation of the complement system (discussed in Chapter 9) is one of the most important
Complement System
This comprises a group of effector mechanisms of immunoglobulins (IgM, IgG1 and IgG3, specifically).
approximately 20 proteins/enzymes The complement system consists of a complex series of at least 20 proteins that can be activated
present in plasma, that are involved
by proteolytic cascade to generate effector molecules, which can perforate target cell membrane.
in the body’s defence. This system is
activated when an antibody binds The complement system mediates many of the cytolytic and inflammatory reactions. The response
antigen. is triggered when the complement protein called Clq binds to IgG1, IgG3 or IgM antibodies. IgG2
is less effective in activating the complement while IgG4, IgA, IgD and IgE are ineffective. Antibody-
induced activation of the complement is important for inactivation and removal of antigens, and
killing of pathogens, as is shown in Figure 4.16.
C4a C4b C2b C2a
Bacteria Antibody
C4 C2
Figure 4.16
A general representation showing C1 component
the binding and activation of the of complement
complement system by antibody. pathway
Target cell Target cell NK cell

Lysis
Figure 4.17 Antibody-coated NK cell
of antibody-coated
Antibody-dependent cell-mediated cell Binding
cytotoxicity. target cell
ANTIBODIES 85
4.5.2 C E L L - M E D I AT E D C Y T O T O X I C I T Y TA R G E T E D B Y
IMMUNOGLOBULINS
Cell-mediated cytotoxicity is an essential defence against intracellular pathogen, including viruses, « The number of lymphocytes in the
some bacteria (such as Mycobacterium tuberculosis) and parasites. Several different leukocyte pop- brain is ten times higher than the
number of neurons.
ulations can execute this activity, including Tcyt, neutrophils, eosinophils, mononuclear phagocytes
and NK cells. NK-cell-mediated killing of target cells requires that target cells be precoated with
the specific IgG. They then bind to the antibody already attached to the target cell using their Fc Antibody-dependent
receptors (specifically, Fcγ RIII or CD16). The binding of the Fc region to the Fc receptor on NK cell-mediated cytotoxicity
This refers to the killing of antibody-
cells activates these cells which start secreting cytokines (such as IFN-γ), as well as granular proteins coated target cells by the cells of the
that cause cytolysis of the target cells. This lytic process is called antibody-dependent cell-mediated immune system such as NK cells and
cytotoxicity (see Figure 4.17). marcophages.
4.5.3 O P S O N I Z AT I O N
Opsonization (Greek: opsonein—to render palatable) is a process whereby a particle (often a bacte-
rium) is rendered more attractive (palatable) for ingestion by phagocytes, by coating it with a specific
antibody and/or complement components. Both mononuclear phagocytes (such as macrophages)
and granular leukocytes (such as neutrophils) express receptors for the Fc portion of IgG molecules.
These are called Fc receptors. All three classes of receptors for IgG (called FcγR, implying Fc receptor
for γ chain) are found on leukocytes. The bound IgG is recognized by FcγR molecules present on the
phagocytes. While the binding of the single Fc region with the Fc receptor is weak, the simultane-
ous binding of the Fc regions of several IgG molecules creates an interaction of sufficient strength
between bound antibodies and phagocytes. The cross-linking of Fc receptors (due to the binding of
several IgG molecules on phagocytes) activates a phagocytic pathway that results in the degradation
of ingested pathogen. Figure 4.17 shows the binding of antibody-coated bacteria to phagocyte, which
is the first step in phagocytosis.
4.6 MUCOSAL IMMUNITY « Sir Almroth Wright was the

first to describe the ingestion of
IgA plays a major role in mucosal immunity. The IgA molecule is synthesized inside the body Staphylococci by leukocytes. He
mainly in the mucosal lymphoid tissue. This antibody has to be exported to the mucosal surface of suggested that opsonins present in
the blood were responsible for the
the gastrointestinal, urogenital and respiratory tracts. The transport requires that the IgA cross the phagocytosis of the bacteria.
epithelial layer. The export of the IgA depends on the Fc region of the IgA molecule. Epithelial cells
such as those of the intestine have specific Fc receptors for IgA molecule, called poly-Ig receptor
or secretory component. The first Ig receptor binds the IgA present in the blood. The bound IgA-
receptor complex then passes “through the cell” to the mucosal surface by vesicular transport. Once
in the mucosal surface, the poly-Ig receptor is specifically cleaved. This cleavage is not complete and a
portion of the secretory component peptide (secretory piece) remains attached to the IgA molecule.
The secretory IgA molecule released from the mucosal surface is now free to neutralize the pathogens.
4.7 N E O N ATA L I M M U N I T Y
Neonatals do not have a developed immune system that can mount an effective defence against pathogens. « Once activated, a plasma cell
Some mammals, including humans, produce antibodies that can be transported from may produce more than 10 million
antibodies per hour.
mother to foetus. This transfer of antibodies occurs transplacentally. The repertoire of protec- « The immune system is poorly
tive maternal antibodies tends to neutralize the pathogenic organisms that attempt to colonize developed at the foetal stage and
a neonatal’s body. The transplacental transport depends on properties of the Fc region of the continues to be so in infants up
to twelve months of age. The full
antibody and hence is one of the effector functions. Maternal IgG (as also IgA) are also present functionality of the immune system
in breast milk. is attained after the age of four to
Interestingly, this IgG can be transported from the gut lumen into the blood of the newborn. five years.
This unusual transport is mediated by another receptor for IgG, called FcRN (Fc receptor for neo- « Secretory IgA and IgG are present
in breast milk. These help protect
natal). This receptor binds to the constant region of the IgG molecule in the gut and mediates the the newborn as it lacks an effective
transfer of antibodies. This is another very interesting effector function of antibodies. immune system
4.8 ANTIBODIES CAN BE ANTIGENS TOO

Immunoglobulins are protein molecules and therefore can be immunogens too. Immunolo-
gists have used this property to generate anti-immunoglobulins and this anti-antibody is used to
characterize epitopes present on the antibody. Anti-antibodies were discovered in 1898, when Kossel
(Germany), and Camus and Gley (France) simultaneously demonstrated that an antibody could
Toxins
be formed against a toxin (haemolytic toxin of eel serum). The antibody (they referred to it as
Toxic proteins produced by living antitoxin) was similar to those formed against diphtheria or tetanus toxins. This observation was
organisms are termed as toxins. immediately seized upon by Bordet, Ehrlich and Morgenroth who concluded that if antibody could
Toxins can act on and lyse red
blood cells (haemotoxin), affect the
be prepared against eel toxin it should surely be possible to prepare a similar antibody against
gastrointestinal tract (enterotoxin), hemotoxic antibody.
affect the nervous system Soon, these scientists immunized animals with whole anti-erythrocyte serum and reported the
(neurotoxin), and a number of other
cells and systems.
finding of the “anti-antibody” that would inhibit the destructive lysis action of a haemolytic anti-
body. This finding proved that antibodies can be formed against antibodies too, that is, antibodies
can behave as antigen.
Haemotoxic antibodies The antigenic determinants or epitopes on immunoglobulin have been classified into three
Haemotoxic antibodies are those
antibodies that are found in the
major types (a) isotypic (b) allotypic (c) idiotypic determinants.
serum of animals immunized with
red blood cells. They bind red blood 4.8.1 ISOTYPE
cells and cause haemolysis.
Antibodies are divided into various classes (IgG, IgA, etc.) and subclasses (IgG1, IgG2, IgA1, IgA2),
on the basis of differences in the amino-acid sequences of the constant region of heavy chains.
Light chains can also be of two types (that is, κ or λ) because of differences in their constant region.
These different amino acid sequences gives rise to antigenic structures or epitopes. These epitopes
are called isotypic (Greek: iso—same and topos—place) determinants. Each isotype is encoded
by a separate constant region and all members of species carry the same constant-region genes.
Moreover, antigenic determinants on IgG, for example, that are characteristic for IgG1 (that is,
isotypic determinants) will be there at the same position whether the antibody is against antigen
X or antigen Y. IgA, IgM, etc. will have different antigenic determinants that are characteristic for
their class.
4.8.2 ALLOTYPE
All members of a species inherit the same isotype genes and hence same isotypic determinants.
However, there are determinants on immunoglobulin molecules that differ among individual of the
same species. These epitopes are called allotypic determinants or allotopes (Greek: allos—other
and topos—place). All antibody molecules that show a particular allotope are said to belong to the
same allotype. Most allotopes are located in the constant region of light or heavy chains but some
are also found in framework portions of variable regions. These allotopes are encoded by different
alleles within the species.
Allele
The nomenclature for allotopes (also called as allotypic markers) has been established for
The term allele refers to an alternate γ, α and κ chains. Markers on γ heavy chain are designated as Gm markers (for γ, referring to
copy of the gene present at a single IgG). At least 25 Gm markers or allotypes have been identifi ed. They are designated by class and
locus. Immunoglobulin alleles
for allotopes show Mendelian
subclass, followed by the allele number, for example, G1m (23), G4m(4a) and so on. All IgG
inheritance. molecules do not carry allotopes, they seem to be restricted to some subclasses. Markers on α
heavy chains are designated as Am (for α, referring to IgA). Of the two IgA subclasses only the
IgAZ subclass has allotypes: AZm(1) and AZm(2). κ light chain has three allotype designated
Km(1), Km(2) and Km(3).
Allotypic differences at a given Gm locus usually involves one to four amino acids. For example
G1m(a) locus on IgG1 has the peptide sequence Asp-Glu-Leu-Thr-Lys on each of the IgG1 mole-
cules. Another person whose IgG1 is a-negative will have the sequence Glu-Glu-Met-Thr-Lys ~
two amino acids different.
4.8.3 IDIOTYPE
In continuing the search for new allotopes, three laboratories stumbled on the same result.
Oudin and Michel (France), Gell and Kelus (England) and Kunkel and his colleagues showed
that some of the anti-antibodies generated reacted with the immunogenic combining site of the
ANTIBODIES 87
IgA IgD IgG IgG IgA IgA
Figure 4.18
Antigenic variation in the
Isotype Allotype Idiotype immunoglobulin molecule.
antibodies. Oudin coined the term idiotype (Greek: idios—own or private and topos—place), a
private antigenic determinant, to distinguish it from more common determinants constituted
by allotopes.
An idiotype is another type of antigenic determinant found on the antibody that can be recog-
nized as foreign by other individuals of the same species. It is formed largely by unique amino-acid
sequences of hypervariable regions of VH and VL domains of an antigen-binding site. Such deter-
minants on individual antibodies are called idiotypic determinants or idiotopes, and all antibody
molecules that share an idiotope are called idiotypes. The term idiotype is also used to describe Idiotopes
Idiotopes are epitopes or antigenic
all the idiotopes that are present on the antibody molecule. Idiotopes are usually specific for in- determinants formed by the antigen-
dividual antibody molecules (private idiotopes) but are rarely shared among different antibody binding site.
molecules (public, cross-reacting or recurrent idiotopes).
Idiotopes usually comprise antigen-binding sites but some may include a part of the variable
region outside the binding site (that is, in the framework region). A schematic representation of
isotype, alloptype, and idiotype is shown in Figure 4.18.
Protein superfamily
4.9 I M M U N O G L O B U L I N S U P E R FA M I LY Proteins that share less than 50 per
cent homology in an amino acid
A superfamily is group of proteins that share similar amino acid sequences, where sequences are less sequence are said to be part of
the same superfamily. However, if
than 50 per cent identical. The conserved sequences of the members of a superfamily have a similar they share more than 50 per cent
folding pattern and are related in evolution. If sequences show more than 50 per cent homology, homology, they are said to belong to
proteins are said to belong to one family. the same family.
The immunoglobulin superfamily (IgSF) is a large and diverse group of proteins compris-
ing more than 100 different polypeptides that contain segments of conserved sequence of ~100
amino acids—an Ig homology unit which have significant similarities to those of immuno-
globulin domains. Immunoglobulin domains are classified as V-like or C-like, on the basis of
their homology to either Ig variable or constant region domains. Each immunoglobulin do-
main is composed of two sheets of β strands, each sheet made up of three to four anti-parallel
strands, and each strand is made up of 5–10 amino acid residues. These strands are connected
by flexible peptide loops. The two β sheets are connected by a conserved disulphide bond be-
tween them. V and C region domains are slightly different. V domains of immunoglobulin are
formed from longer polypeptides and contain an extra two strands of β strands as compared
to C domains, even though both of them have a characteristic globular tertiary structure called
the immunoglobulin fold. The two β sheets are held together by hydrophobic interactions
formed by an hydrophobic amino acid side chain facing inwards of two sheets. These resi-
dues alternate with the hydrophilic residue facing outwards. Since inward-facing hydrophobic
residues are necessary for the maintenance of the tertiary structure, they are more conserved
among the residues of the domain. Lower sequence homology is found among loops that con-
nect these strands.
The structures of some of the most identified members of an immunoglobulin are shown
in Figure 4.19. Most identified members of this large superfamily are involved in diverse func-
tions ranging from recognition, adhesion or binding processes of the cells, to proteins involved in
cartilage formation reflecting the versatility of the conserved structure. All members of immuno-
globulin superfamily are either found at the cell surface or in the secreted form. There is no single
discrete function of the members of the immunoglobulin superfamily. A part from their obvious
roles in antigen recognition by its members such as immunoglobulin, and MHC antigens, other
members of this superfamily are known to mediate many cell to cell interaction. These include
C γ δ
ζ
C C C v v
C β 2m η
MHC Interleukin CD3 CDQ/CD2Q

(class I) receptor complex
(Type 1)
C v
C
C v
C C
C
C C C
v v C
v C C C
C C
C C C C
Figure 4.19 T-cell CD4

The members of the immunoglobulin receptor
CD2 IgM
superfamily. NCAM
adhesion molecules such as intercellular adhesion molecules (ICAM) and vascular cell adhesion
molecule (VCAM). Some members of this family act as receptors for viruses (such as CD4 for the
human immunodeficiency virus) or cytokines (interleukin 1 receptor-type family), or are recep-
tors for NK cells (killer inhibitory receptor or KIR), while the functions of the other members of
the superfamily such as contactin and myelin-associated glycoprotein are not known.
Recently, immunoglobulin superfamily members have been identified in invertebrates.
These include 142 IgSF proteins (including fasciclin and amalgam) of Drosophila and 80 IgSF
proteins (twitchin, DIM-1) in the nematode C.elegans. It is believed that present-day immu-
noglobulins evolved from a primitive gene coding for polypeptides with characteristic do-
mains of V and C regions in a single chain. In this scheme, the next evolutionary event was
the duplication of genes as well as their separation into V-and C-region genes. This origin
is reflected by the discovery of the Pap D protein (a molecular chaperone) in the bacterium
E.coli, which has two immunoglobulin-like 3-D structures but shows no sequence similarity
to immunoglobulins. With the acquisition of the ability to undergo DNA rearrangement, this
relatively simple and primitive protein probably evolved into diverse and complex members
of the immunoglobulin superfamily.
To conclude, we can say that an antibody is a multifunctional glycoprotein involved in adap-
tive immunity. It is produced by the immune system of vertebrates in response to antigenic stimuli.
An antibody is a Y-shaped molecule that has antigen-binding sites (Fab segment) located at the tips
of the arms. The stem of an antibody is known as the Fc region and is involved in effector func-
tions such as phagocytosis, complement activation, etc. Antibodies has been classified into
various classes (IgG, IgA, IgM, IgD, IgE) and subclasses based on the amino acid differences
in the constant region of heavy chains. Antigenic determinants have also been located on the
antibody molecules.They can be isotypes (that separate one class of antibody from other),
allotypes (arising because of allelic difference within members of same species) or idiotypes
(formed by the antigen-binding site). Despite the fact that antibodies are restricted to the
vertebrate immune system, proteins belonging to the immunoglobulin superfamily have been
found in a number of invertebrates, suggesting that a primordial protein found in inverte-
brates, capable of antigen recognition, has evolved into large multifunctional defence proteins
of vertebrates.
ANTIBODIES 89
Antigen–Antibody Interactions:
Immunoprecipitation
Antigen–antibody interactions can occur both in vivo as well as in vit-
ro. These interactions when they occur inside the body are called im-
mune reactions. On the other hand, antigen–antibody interactions Antigen
can occur outside the cell, that is, in vitro or in laboratory conditions.
The study of antigen–antibody interactions occurring under con-
trolled laboratory conditions is referred to as serology. A number of
serological reactions/tests are commonly employed in the diagno-
sis of a variety of diseases. In this section of the following chapters,
we familiarize ourselves with some common serological techniques
that are extensively used in diagnostic testing. Precipitate
Multivalent antigens react with bivalent or multivalent anti-
bodies to form insoluble antigen–antibody complexes. The network
of antigen and antibody, called lattice, is so large that it settles out
of the solution as a visible precipitate. The precipitin reaction (short
for precipitation reaction) was first reported by Kraus in 1897. This
precipitation technique has been employed for the qualitative iden-
tification of antigens and antibodies. Immunoprecipitation is the
Antibody/
technique of precipitating out antigen molecules with the help of Antiserum
a specific antibody. A rapid form of immunoprecipitation is the ring
test that is used for the detection of an antigen (or antibody). In this
test, an antigen is layered over the immune serum (containing an-
tibodies) in a test tube. The antigen and antibodies diffuse towards
each other. If the antigen reacts with the antibodies that are present Immunoprecipitation
in the immune serum, a visible precipitating ring is formed at the Figure 4.20
interface (see Figure 4.20). Immunoprecipitation.
S U M M A R Y
• The term antibody signifies the existence of a discrete body that • Immunoglobulins of the same class manifesting allelic differences
can act against a pathogen or its product. Other names include between themselves, even of one or two amino acids, are called
gamma globulin, immunoglobulin. allotypes. Allotypes of IgG in person A will be different from IgG
in person B. Idiotype determinants are formed largely by amino
• Antibodies are defence proteins (glycoproteins) produced by the adap- acid sequences of hypervariable regions of the antigen-binding site
tive immune system of vertebrates to combat invading pathogens. of antibody.
• Antibody is a Y-shaped molecule made up of four polypeptides, • IgG is the most abundant antibody of internal body fluids. It combats
that is, two chains of high molecular weight (heavy chain) and two microbes and their toxins. IgA is the predominant antibody in exter-
chains of low molecular weight (light chain). nal secretions where it defends the surface against viral and bacterial
assault. IgM is a very effective agglutinator, while IgE is the major
• N terminal region of about 110 amino acids in both light and heavy
antibody of allergic reactions. IgD is primarily found on the surface
chains differ (vary) among different antibodies and is called vari-
of mature B cells and is probably involved in lymphocyte activation.
able region. This region forms the arms of Y-shaped antibodies and
binds antigen. The tail of the Y is constituted by the constant region • An immunoglobulin superfamily, of which antibody is a member,
and is involved in receptor binding and complement activation. is a large and diverse group of protein comprising antibodies,
class I and II MHC molecules, T-cell receptors, CD3 molecules
• Antibodies have been divided into different classes or isotypes and adhesion molecules. The functions of the members of this
based on the difference in the constant region. The classes IgG, superfamily range from antigen recognition and mediation is cell
IgA, IgM, IgD and IgE have different functional properties. to cell interaction to acting as receptors for viruses and cytokines.
K E Y W O R D S
• allotype 86 • effector function 84 • immunoglobulin D 81 • J chain 77

• antibody 71 • fragment antigen binding • immunoglobulin • kappa light chain 78
• antibody-dependent cell- (Fab) 72 domain 75 • lambda light chain 78
mediated cytotoxicity 85 • framework region 76 • immunoglobulin E 82 • light chain 75
• complementarity- • gamma globulin 78 • immunoglobulin fold 75 • neonatal immunity 85
determining region 76 • heavy chain 74 • immunoglobulin G 78 • opsonization 85
• constant region 75 • hinge region 76 • immunoglobulin M 80 • variable region 75
• crystillizable • hypervariable region 75 • immunoglobulin
fragment (Fc) 72 • immunoglobulin 78 superfamily 87
• disulphide bonds 77 • immunoglobulin A 80 • isotype 86
1. If the variable region of the antibody is localized towards the c ter- Do you think it will add another step of complication during its
minal region, do you think it is going to affect antibody function in assembly? Comment.
any way?
H I N T —Say bye-bye to antigen-binding activities to almost infinite antigens.
4. A student isolated a mutant Y-shaped antibody that lacked light
chains. What functions of the antibody do you think will be affect-
2. How would you experimentally prove that Fc and not Fab region ed? Will it manifest isotypic, allotypic and idiotypic determinants?
mediates transplacental transport? H INT —Antigen-binding function and idiotypic determinants will be
H I N T —Isolate mouse IgG, generate Fab and Fc. Attach radioactive label to affected.
both. Inject in pregnant rabbit or mice and see whether Fab / Fc can cross
placenta.
5. Why do you think the body needs five different classes of antibod-
ies, when an enormous repertoire could be generated even in a
3. Do you think that having two dissimilar antigen-binding sites on single class?
antibody molecules will be advantageous to human beings or not? HINT —Different classes of antibody have different “micro” functions as well.
Choose the Appropriate Option 5. In the nomenclature of antibody naming, letter G of IgG
suggests:
1. Antigen-binding site of antibody is constituted by: (a) Heavy chain
(a) n-terminal region of heavy chain and light chains (b) Electrophoretic mobility
(b) c-terminal region of heavy and light chains (c) High-molecular weight antibody
(c) n terminal of heavy chain only (d) Light chain
(d) n terminal of heavy chain and c terminal of light chain
2. The number of immunoglobulin domains in the heavy chain of 6. Antibodies that impart neonatal immunity are:
antibody are: (a) IgG
(a) 2 (b) IgA2
(b) 4 (c) IgM
(c) 6 (d) IgD
(d) 8
7. Antigenic determinants that will remain same on antibody mol-
3. Antigen binding is associated with: ecules of different species will be:
(a) Immunoglobulin fold (a) Allotypic determinants
(b) Hypervariable region (b) Isotypic determinants
(c) Framework region (c) Idiotypic determinants
(d) Immunoglobulin domain (d) None of the above
4. The most effective complement activating antibody is: 8. Hinge region is absent in:
(a) IgG4 (a) IgE
(b) IgE (b) IgG
(c) IgM (c) IgA
(d) IgA (d) IgD
ANTIBODIES 91
9. Which of the following is not directly involved in opsonization? 10. All members of an immunoglobulin superfamily share:
(a) Fab (a) Antigen-binding characteristics
(b) Fc (b) Y-shaped structure
(c) Hinge region (c) Domain structure
(d) Fc receptor (d) Similar functions

1. The hinge region is rich in ____________ amino acid and/or 4. The secretory component is associated __________ antibody.
_________.
5. The number of CDRs of light chain and heavy chain involved in
2. Most of the allotypic determinants are located in ___________ of antigen binding are _____________ in antigen-binding site.
light or heavy chains.
3. An effective agglutinator, efficient complement activator and the
first immunoglobulin class to be synthesized in primary response
is _____
Ahmed, R. and D. Gray (1996). “Immunological Memory and Harris, L. J., S. B. Larsen and A. McPherson (1999). “Comparison
Protective Immunity: Understanding Their relations”, Science, of Intact Antibody Structures and Implications for Effector
272: 54–60. Functions”, Advances in Immunology, 72: 191–208.
Burnet, F. M. (1959). “The Clonal Selection Theory of Immunity”. Kerr, M. A. (1990). “The Structure and Function of Human IgA”,
Nashville, Tennessee: Vanderbitt University Press. Biochemical Journal, 271: 285–296.
Burton, D. R. (1987). “Structure and Function of Antibodies”, Miller, G. (1981). “Putting Lady Mary in Her Place: A Discussion
in F. Calabi, and M.S Neuberger (eds), Molecular Genetics of of Historical Causation”, Bulletin of History of Medicine, 55: 2.
Immunoglobulins. Amsterdam: Elsevier Science Publishers.
Ploegh, H. L. (2004). “Immunology: Nothing Against Time’s
Carayannopoulos, L. and J. D. Capra (1993). “Immunoglobulins: Scythe Can Make Defence,” Science, 304: 1262–63.
Structure and Function” in W. E. Paul (ed.), Fundamental
Immunology, New York: Raven. Rajewsky, K. (1996). “Clonal Selection and Learning in the
Antibody System”, Nature: 751–58.
Creighton, C. (1894). A History of Epidemics in Britain, Vol. II.
Cambridge: Cambridge University Press.
One of the distinguishing features of vertebrates is their specific “Variety is the
immunity. The cornerstone of specific immunity is the production of an very spice of
extraordinary repertoire of highly specific antibodies with which ver- life.”
tebrates defend themselves against a myriad of infectious agents. In —COWPER,
(Task, II, 606)
1897, Paul Ehrlich put forward his now famous side chain theory
(see Figure 5.1). He proposed that antibodies (which he called side
chains) were natural constituents of the cell surface, formed within the
cell. Moreover, antibodies possessed from the start the structural
configuration that determined their specificity for a given antigen. The
purpose of antigens is to select, from among all of the side chains (an-
tibodies) available, only those which are able to interact specifically.
The cell will then produce more of these molecules for export into the
blood. In other words, cells inside the body have a large number of you should be able to:
antibody molecules (of different specificity) on their surface. Antigens • Give an account of various
theories of antibody formation
simply bind or select appropriate antibody molecules and the cell • Describe the genetic
organization of λ and κ chains,
starts producing that particular antibody. Although this theory had and heavy chains in humans
and mice
several shortcomings, it held its ground for several years. It was the
• Demonstrate a knowledge of
gene rearrangement of heavy
forerunner of our present understanding of antibody synthesis and chains at the DNA and RNA
levels
the existence of antibody diversity.
• Explain the light chain gene
rearrangement at DNA and
RNA levels
• Describe the concept of 12/23
rule and explain the role of
recombination signal sequence
in joining V, (D), J gene
segments
• Describe the allelic exclusion of
heavy and light chains
• Briefly summarize seven known
mechanisms that generate
antibody diversity
• Describe how membrane-
bound and membrane-secreted
immunoglobulins are formed
• Explain the phenomenon of
class switching
Generation of Antibody
Diversity 5
5.1 INTRODUCTION
Antibodies are secreted by the B cells of the immune system and are unique in their unlimited
potential for diversity. The estimated variability runs into billions. How does our body generate
such a huge diversity? Ideas about the specificity and diversity of antibodies were put forward from
time to time, some far-sighted, others myopic but still thoughtful. Let us take a brief look at some
of the ideas.
• Around 1930, it was understood that antibodies were globular proteins. In view of this
discovery, Linus Pauling redefined the direct template theory (initially put forward by
Oskar Bail and co-workers in 1914). He postulated that antigens would serve as the
template for the final step of protein formation. The nascent polypeptide chains of the
antibody fold around the template offered by the surface determinants of the antigen
molecule (see Figure 5.2). Once this conformation is attained, it is stabilized by the
appropriate weak bonds.
• In 1941, F. Macfarlane Burnet proposed an indirect template theory of antibody forma-
tion. He called this theory adaptive enzyme theory. Burnet proposed that all proteins
(including antibodies) are synthesized and broken by proteinase enzymes. Antigen, once
Figure 5.1
Diagramatic representation of Ehrlich’s
side chain theory.
introduced into the host body,

will find its way into the cells
where it would come in con-
tact with proteinases. The
antigen will then induce an
adaptive modification of the
Antigen Antibody polypeptide enzymes. This newly adapted
Figure 5.2 folds around antigen
Pauling’s direct template theory. enzyme would then be able to
template to form
According to this theory, the antigen
Nascent “unfolded” specific antibody
synthesize an antibody mole-
serves as the template in the final step
of protein formation. antibody polypeptide cule, specific for the interacted
antigen.
» Till about 1950, it was believed Some other theories such as immunocatalysis theory by Sevag (1951), template-inducer theory of
that antibody-producing cells Schweet and Owen (1957) were also put forward which presented only a slightly modified picture
synthesized antibodies in the pres-
ence of antigen, and in the absence of Burnet’s indirect template theory
of antigen these cells synthesized
normal globulins! • In 1955, Neils K. Jerne proposed the natural selection theory of antibody formation that
finally paved the way for the clonal selection hypothesis. Jerne proposed that antibod-
» N. K. Jerne, a Danish immunologist,
suggested that each single special- ies of all possible specificities are normally formed by the vertebrate host and delivered
ized cell (lymphoid cell) synthesized in small amounts into the blood. Any antigen that chances to enter the circulation
antibodies of a single specificity. reacts with the antibody specific for antigenic determinant. Jerne suggested that
He won the Nobel Prize in 1984
for his theoretical contributions to antigen acted as a selective carrier of antibody and carried the antibody to special-
the understanding of the immune ized cells capable of reproducing (synthesizing) this antibody. The specialized cells start
system. producing this particular antibody in large amounts and pouring antibodies in
the blood.
• In 1959, Burnet, Talmage and Lederberg proposed the widely accepted clonal selection
Clonal selection theory
The clonal selection theory predicts theory (see Figure 5.3). They acknowledged Jerne’s suggestion of the presence of pre-existing
that antibody specificities are antibodies as the targets of antigen selection. They suggested that the “natural antibody”
preformed in such a way that each
is located on the surface of lymphoid cell. The interaction of antigen with these anti-
cell produces antibody molecules
of a single specificity. The antigen bodies (receptors) triggers (by some unknown mechanism) the signal for cellular
simply stimulates (selects) the differentiation and antibody production. The cell to which antigen is bound also starts
appropriate clone of an antibody-
proliferating to form clones of daughter cells possessing identical receptors and capable
producing cell and that cell starts
producing antibodies. of identical immunological response. Thus, the antigen would serve to select and
activate specifically the appropriate clonal precursor from a much larger population of
lymphoid cells.
» Nossel and Lederberg in 1958 One problem remained, that of antibody diversity. It was well established that the body could
proposed, for the first time, that generate antibody against almost any antigen that entered the body. One easy solution was to
one B cell produces antibody
molecules of only one specificity. In postulate the existence of a separate gene for each specific antibody. However, for this an organ-
1952, J. Lederberg also introduced ism would need to contain all the genetic information necessary to produce antibodies of about a
the now famous term in molecular million different specificities, even though each plasma cell would eventually produce antibod-
biology, plasmid.
ies of only a single specificity. This would need at least 500 times more total DNA in a single cell.
As the structure of antibodies became known, it was noted that half of a light chain has a variable
amino acid sequence and other half is constant. Similarly with heavy chains, a quarter of the chain
is variable while the rest is constant. It was argued that if there are millions of genes (~108 genes) of
different antibody specificities present in the DNA, how is it possible to maintain this constancy
of the sequence in the constant region?
• In 1965, W. Dreyer and J. Bennet proposed a solution to this problem. They suggested
that the constant and variable regions of an immunoglobulin chain (heavy and light
chains) are coded for by two separate genes, one gene for the variable region and the
One gene–one polypeptide
principle
other for the constant region. They further clarified that diversity could be generated
As per the one gene–one because hundreds or thousands of variable-region genes are present in the DNA of
polypeptide principle, one gene the cell, while one or a few genes code for the constant region of the immunoglobulin
codes for one polypeptide. This
theory was proposed by Veron
chain. At this point, the theory only had to account for multiple variable regions. The
Ingram in 1962. This theory is an two genes for the single polypeptide theory were received with suspicion as it con-
offshoot of the one gene–one tradicted the then-accepted one gene–one polypeptide principle. Dreyer and Bennet
enzyme theory of Beadle and Tatum.
suggested that these two genes must somehow come together at the DNA level to
GENERATION OF ANTIBODY DIVERSITY 95
Selection of
appropriate clone
of B cell
Expansion of
selected clone
Maturation
Figure 5.3
The clonal selection theory. Proposed by
Lederberg and his colleagues, this theory
Antibody synthesis established the mechanism for antibody
diversity.
form a continuous message that is transcribed and translated into a single heavy or « Susumu Tonegawa, a Japanese
light chain. scientist, was awarded the Nobel

Prize in 1987 for discovering the
• In 1978, S. Tonegawa and his co-worker working on genetically identical (syngenic) mechanism of the generation of
strain of mice isolated DNA from a 13-day-old embryo, and two different myeloma antibody diversity.
tumour cells—one which produced homogenous λ light chains (strain H2020) and
one that produced κ light chains (strain MOPC321). They treated the DNA from
these three sources with the same restriction enzyme EcoRI. These three types of
DNA were electrophoresed, and then transferred to a nitrocellulose filter. The trans-
ferred DNA was denatured and then checked for sequences for the λ chain. The fully
differentiated (λ-chain producer) myeloma cell DNA A B C
(H2020) gave four bands of 8.6 kb, 7.4 kb, 4.8 kb and
3.5 kb. The EcoRI digest of the fully differentiated
κ-chain producer myeloma cell DNA (MOPC321) and
embryonic cell DNA gave three bands (7.4 kb band was
missing) (see Figure 5.4). These results suggested that 8.6 kb
during the development from embryonic state to 7.4 kb
a fully differentiated state, DNA undergoes some
rearrangement. Further analysis was done with a probe 4.0 kb Figure 5.4
that recognized specifically the constant region of 3.5 kb Analysis of DNA fragments containing
λ1 gene on agarose gel electrophoresis.
λ gene (Cλ probe) or the variable region of λ gene (Cell by Brack et al. ©1978 by Elsevier
(Vλ probe). These results clearly showed that the Science & Technology Journals.
Cλ probe hybridized with 8.6 kb and 7.4 kb fragments. Reproduced with permission of Elsevier
Science & Technology Journals in the
Vλ probe hybridized with 3.5 kb, 4.8 kb and 7.4 kb format Textbook via Copyright Clearance
fragments. Center.)
This implied that the 8.6 kb fragment contains the constant region only of λ chain. The other
two, 3.5 kb and 4.8 kb fragments, contain the variable region only of λ chain. And the 7.4 kb frag-
ment (which is not present in the embryonic and mature κ-chain producer cells) contains both
constant and variable regions on the same fragment.
Further analysis showed that the 7.4 kb fragment originates from a recombination (DNA
rearrangement) event between 3.5 kb fragment and 8.6 kb fragment. These experiments demon-
strated that Dreyer and Bennet’s two-gene model, that is, the two gene–one polypeptide theory
was true.
Subsequently researchers applied a similar approach together with the newly developed
southern blotting techniques and demonstrated that Dreyer and Bennet’s two-gene model (one
gene coding the variable region and one gene encoding constant region) was true for both light
and heavy chain genes.
5.2 G E N E T I C O R G A N I Z AT I O N O F
IMMUNOGLOBULIN GENES
As sequencing and cloning techniques developed, it was found that Dreyer’s and Bennet’s two gene –
Intron
The intervening non-coding one polypeptide model offered an oversimplified view of the actual picture. It was found that
sequence that occurs within a gene there were two types of light chain genes (λ and κ) and several types of genes for heavy chains
is known as an intron. Introns are (gene coding for α, γ, μ, δ, ε). Moreover there was a multiplicity in each gene segment, that is,
transcribed and removed from
the primary RNA transcript during several λ or κ genes existed. Table 5.1 lists the chromosomal location of immunoglobulin genes in
splicing. Walter Gilberts put forward mice and humans. It was also found that these genes were separated by non-coding regions, that
an intron-only hypothesis in 1978. is intron. The genetic organization of three different immunoglobulin gene pools that is, (λ, κ and
He suggested that expressed
sequences (exons)were complete heavy chains) is depicted in Figure 5.5.
genes which were brought at one
place during evolution. Introns are
actually relics of that gap between V J C
genes.
κ Light chain gene pool
V1-V80 J1 - J5
V JCλ
λ Light chain gene pool
V1-V80 JCλ1 - JCλ6
V D J C
Figure 5.5
The organization of immunoglobulin Heavy chain gene pool
germ-line gene segments in humans
represented by λ-chain DNA, κ-chain
DNA, heavy-chain DNA. V1 - V90 D1-D30 J1 - J6 μ δ
Gene Murine Human

Heavy chain 12 14
Table 5.1 Kappa (κ) light chain 6 2
Chromosomal location of Lambda ( λ) light chain 16 22
immunoglobulin genes.
5.2.1 LIGHT CHAIN LOCI

Dreyer and Bennet proposed that the variable region of light chain (and heavy chain) is coded by a « At times, for unknown reasons,
single coding sequence (gene or gene segment to be precise). It was found by S. Tonegawa, and also κ light chains are overproduced
resulting in a disease called light
by Philip Leder and his colleagues, that the V region of the light chain had more amino acids than chain deposition disease.
could be coded by the then known V gene segment of light chain. This implied that some additional
DNA segment was needed for coding the rest of the V region of the light chain. Tonegawa and his
colleagues soon located the missing DNA segment, which was designated as J (for joining). Thus κ and
λ light-chain families contain V, J and C gene segments. So the variable region is coded by two gene
segments. V and J segments and the constant region of light chain are coded by C gene segment.
- C H A I N FA M I LY
In the mouse germ line, genes for the λ chain family are located on chromosome number 16.
The λ chain is coded by two Vλ gene segments (each about 300 base-pair (bp) long, coding for « The λ chains make up 5 per cent
of the light chains in normal mouse
approximately 95 amino acids), four Jλ gene segments (each about 39–40 bp long, coding serum. The κ light chain accounts
for 13–15 amino acids) and four Cλ gene segments. One each of the Jλ (Jλ4) and Cλ (Cλ4) gene for the rest.
segments is a pseudogene, that is, a defective gene that does not code for anything. The two Vλ
genes, three Cλ genes and three Jλ genes code for variable chains. It should be made clear that one
mature gene will have only one Vλ gene, one Jλ gene and one Cλ gene. There are three Cλ genes that
code for three types of constant regions. There are three λ subtypes (λ1, λ2, λ3). Each Cλ gene has
an associated Jλ gene with it. On the other hand, each Vλ gene has an upstream exon that codes for
20–30 amino acid residues, called leader or signal peptide. A signal sequence directs the proteins
to endoplasmic reticulum. The signal sequence is rapidly cleaved inside the endoplasmic reticulum
and never appears on the secreted antibody molecule.
In humans, there exist about 80 Vλ genes, six Jλ segments and six Cλ segments.
κ - C H A I N FA M I LY
Locus
In mice, the κ-chain locus (pl., loci) is located on chromosome 6. There are about 200 Vκ gene This refers to the position of a gene
segments. 5′ upstream of each variable κ gene segments is located the leader sequence encoding in a chromosome.
leader, peptide. There are four functional Jκ gene segments and one Jκ pseudogene (ψ1 Jκ 3)
that lie upstream of the single Cκ gene. In humans, the κ-chain family contains 80 Vκ genes, « In humans the κ light chain
five functional Jκ gene segments and a single Cκ segment. Since there is only one Cκ gene seg- accounts for 60 per cent of the total
light chains and λ chain for the
ment that codes for only one type of constant region, there are no subtypes of κ light chain in remaining 40 per cent.
either mice or humans.
« Hilschman reported the first
amino acid sequence of light chain
5.2.2 H E AV Y C H A I N LO C I in 1965.
The heavy chain loci contains gene(s) that code for both the variable region and constant region of
the heavy chain. In studying the assembly of genes for the heavy chains of antibody, Tonegawa and
Leroy Hood compared the heavy-chain variable region amino acid sequence with the VH and JH
nucleotide sequences. The VH gene encodes amino acids 1 to 44 and the JH gene segment encodes
amino acids 98 to 113. Neither of these gene segments carries information about amino acids 45 to 97.
These investigators found that in addition to the VH and JH segments, a third group of DNA seg-
ments termed as D (for diversity) segment was involved. The nucleotide sequence corresponded
to amino acids 45 to 97 of the heavy chain. Tonegawa and his colleagues located D gene segments
within mouse germ-line DNA with cDNA probe. This segment was found to be located between
VH and JH gene segments.
In humans, there are 90 VH gene segments and 30 functional DH gene segments. The D seg-
ments encode amino acids within the third complementarily determining regions (CDRs). Each
VH gene segment had a leader sequence located a short distance upstream from it. Six functional
JH gene segments are located downstream of DH genes. Downstream from the JH gene segments are
located a series of CH gene segments. Each CH gene segment encodes the constant region of an im-
munoglobulin heavy chain isotype. The genes in the CH gene segment consist of coding exons and
non-coding introns. In humans (and in mice), CH gene segments (exons) are arranged sequentially
in the order Cμ, Cδ, Cγ, Cε, Cα.
Gene Segment Murine Human

Vλ 2 40–80
JCλ 4 6
Vκ ~200 ~80
Jκ 5 5
VH 200–1500 75–90
Table 5.2 D 12 20–30
Approximate number of variable region
gene segments. JH 4 6
In mice, there are approximately a 1,000 VH region segments, 12 DH region segments and 4 JH
region segments. Downstream of JH gene segments are CH gene segments. Table 5.2 summarizes V,
(D) and J segments of mouse and human immunoglobulin genes.
5.3 REARRANGEMENT OF GENES

As we have seen in the previous section, the variable and constant regions of light and heavy chains
are usually coded by more than one gene. Moreover, the variable region itself is coded by two
types of gene segments in the light chain and three types of gene segments in the heavy chain.
During the maturation of B cells, gene re-arrangement occurs at the DNA level and the primary
transcript level. These rearrangements produce a single functional variable-region DNA sequence
for its heavy chain and a single, functional variable-region DNA sequence for its light chain. Thus,
mature immunocompetent B cells are committed to produce antibodies that carry only one type of
variable-region sequence of light and heavy chains, and hence have a single type of antigen-binding
site. Cells other than B lymphocytes also contain immunoglobulin genes in the germ-line DNA.
However, only B lymphocytes express these genes in properly rearranged forms, capable of giving
rise to functional proteins. DNA rearrangements in immunoglobulin genes occur in a precise or-
der, that is, it is NOT a random rearrangement event.
» IgM and IgD are co-expressed on
B cells when they leave the bone In developing B cells, the gene rearrangement first occurs in the heavy-chain variable region
marrow. and then in the light-chain variable region genes (see Figure 5.6).
5.3.1 H E AV Y - C H A I N G E N E R E A R R A N G E M E N T
The generation of functional immunoglobulin heavy chains requires two separate rearrangement
events: one occurring at the DNA level and other occurring at the RNA level.
R E A R R A N G E M E N T O F G E N E S AT D N A L E V E L
• Within the variable region, the DH gene segment first joins to a JH segment. This forms
the DHJH segment. Additional D segments upstream of DH or additional JH located down-
stream of the
Rearrangements Rearrangements
in Pro-B cell in Pre-B cell Immature B cell
Successful Successful Successful Successful
Figure 5.6
VDJ
The order and regulation of M chain Rearranged heavy
Rearrangement of VJ rearrangement K chain
Ig gene recombination as expressed M chain (VDJ) and light
they appear in various stages heavy chain of light (K ) chain expressed
of a B cell. (1st allele) K chain gene
JH (undergoing rearrangement) are not affected by this. However, those DH or JH occur-

ring in between the rearranged DH – JH are deleted when these two genes join.
• This DHJH gene segment then moves to join one VH segment to generate a VHDHJH unit that
encodes the entire variable region. At this stage, all D segments upstream of the rearranged
DH gene are deleted and the VH is ligated next to the DH. This formation of VHDHJH gene
segments occurs only in cells committed to become B lymphocytes. The V-sequence gene,
that is, VHDHJH, and the constant-region genes are subsequently transcribed. The C-region
genes remain separated by the intron (also the unrearranged J segments). The rearranged
gene now consists of the following sequence starting from the 5′ end: short L exon – intron
joined VDH segment–unrearranged J segments – intron – series of C gene segments.
R E A R R A N G E M E N T O F G E N E S AT R N A L E V E L
• Once the heavy-chain gene rearrangement is accomplished, the RNA polymerase can
bind to the promoter sequence and transcribe the entire heavy chain, including the in-
trons, to generate primary RNA transcript.
• The primary RNA transcript has the same sequence as rearranged exon, that is, starting
from 5′: a short leader sequence – intron –VDJ gene – unrearranged J segments – intron – series
of C-gene segments.
• The primary RNA transcript is subsequently processed. RNA splicing removes the intron
located between 5′ leader sequence and the VDJ gene segment, and the intron located
between the VDJ complex and the first C-region segment(Cμ gene segment in our case).
(The region downstream of the VDJ may contain unrearranged J segments but, for the
purpose of clarity, is not shown here). Initially both Cμ and Cδ are transcribed. The RNA
processing machinery processes the primary transcript to generate mRNA, including
either Cμ or Cδ transcript. Multiple adenine nucleotides called poly-A are added to one of
several consensus polyadenylation sites located 3′ of the Cμ RNA. Gene coding for other
CH classes also have 3′ polyadenylation sites, which are utilized when these C regions
are expressed. It is believed that RNA splicing and addition of poly-A tail to mRNA are Poly (A) tail
tightly coupled.These two mRNAs are then translated and leade r sequence is cleaved to The homopolymer of adenosine
monophosphate added
generate finished μ and δ chains. Since two different heavy chains of mRNA are pro-
to eukaryotic mRNA post-
duced by the heavy-chain variable-region gene rearrangement, mature B cells expresses translationally by polyadenylated
both IgM and IgD with identical specificities. A brief summary of genetic rearrangement polymerase is called the poly (A)
tail. The length of poly (A) tail varies
in making μ and δ heavy chain is given in Figure 5.7.
from 20 to 250 adenine moieties
depending on the mRNA.
Figure 5.7
The heavy-chain rearrangements
showing VDJ recombination in humans.
5.3.2 LIGHT-CHAIN GENE REARRANGEMENT

As in the generation of a heavy chain of immunoglobulin, the formation of a functional light chain
requires the rearrangement of genes both at the DNA and RNA levels.
The generation or expression of light chains (κ or λ) follows an essentially similar sequence involv-
ing V and J gene segments (The D segment is absent in the V region of the light chain). In humans
or mice κ light-chain DNA, any one of the functional Vκ gene segments join to any one of the
functional Jκ gene segments. This forms the Vκ–Jκ gene segment that forms the functional V gene.
However, in the formation of functional V genes, recombination events are not that simple. In
humans, any one of the functional Vλ gene segments can combine with any one of the functional
Jλ gene segments to generate a Vλ–Jλ complex. However, in mice, a Vλ1 gene segment can join only
with a Jλ1 or Jλ3 gene segment where as a Vλ2 gene segment can combine only with a Jλ2 gene seg-
ment. As in heavy-chain gene rearrangement, any of the Vλ or Jλ gene segments located between the
rearranged Vλ and Jλ gene segment are deleted. The rearranged κ and λ genes contain the following
regions in order from 5′ to 3′end: a short leader (L)–exon – a intron – joined VJ gene segment –
second intron–constant region. This rearranged light chain sequence is transcribed by the RNA
polymerase using the promoter sequence located upstream of the leader gene sequence.
R E A R R A N G E M E N T O F G E N E S AT R N A L E V E L
The primary transcript of the light chain formed contains the following sequence starting from
the 5′end: short leader (L)–exon—non-coding sequence intron—joined VJ gene segment—second
intron—the constant region. The introns (some located between the leader sequence and the joined
VJ gene segment, others located between the VJ gene and the constant region) and unrearranged
J gene segments located downstream of the J segment are spliced out by the RNA-processing en-
zymes. The mature mRNA contains the leader sequence VJC gene segment. The poly-A tail is added
at the consensus polyadenylation site located at the end of the C exon.
The light chain mRNA binds to ribosome and is translated into the light-chain protein. The leader
sequence guides the newly synthesized light chains to the lumen of the rough endoplasmic reticulum.
The leader peptide is finally cleaved and does not appear in the final form of protein. The sequence of
events involving rearrangements of κ and λ genes are summarized in Figures 5.8 and 5.9.
VK JK CK
Germ-line DNA
V1 V2 V3 Vn J1-J5
V-J joining
B-cell DNA
V2 V3J4-J5 CK
Transcription
Primary RNA transcript

V3J4-J5 CK
RNA splicing
mRNA
V3J4 CK
Translation
Figure 5.8
A schematic diagram showing κ-chain
rearrange ment in humans at the DNA Kappa chain
and RNA levels. VK CK
VL JCL
Germ-line DNA
V1 V2 Vn JCL 1 - JCL 6
V-J joining
B-cell DNA
V2 J2 CL
Transcription
Primary
RNA transcript
V2J2 CL
RNA splicing
mRNA
V2 J2 CL
Translation
Figure 5.9
A schematic representation of λ-chain
Lambda chain
production in humans at the DNA and
VL CL RNA levels.
The light chain assembles with the previously synthesized γ or μ chain (or any other heavy chain)
in the endoplasmic reticulum to form a complete IgG or IgM molecule (or any other Ig molecule).
5.3.3 R E A R R A N G E M E N T O F V, ( D ) , J G E N E S E G M E N T S
The gene rearrangements that take place during the generation of the functional variable region re-
quire a system to ensure that the DNA rearrangement takes place at the correct locations relative to
the V, D or J gene segments. That is, the V gene segment must join the D segment and not the other V
gene segments. DNA sequencing studies have revealed that DNA rearrangement are in fact guided
by conserved non-coding DNA sequences flanking each germ-line V, D and J gene segments. These
Recombination signal
are called recombination signal sequences (RSS).
sequence
V gene segment uses its 3′ side to join with D or J (in light chain gene) gene segment so the RSS This refers to the sequence in
is located on 3′ side of V gene. For the same reason it is found on 5′ side of the J gene segment and immunoglobulin genes at which
on both sides of the D gene segment. RSS consists of conserved block of seven nucleotides—the somatic recombination takes place.
RSS consists of two conserved
heptamer 5′ CACAGTG 3′ followed by the non-conserved region known as spacer which is either palindromic sequences. Of these,
12 or 23 nucleotides long. This is followed by a second conserved block of nine nucleotides—the one is a heptamer while the other is
nonamer 5′ ACAAAAACC 3′ (see Figure 5.10). The spacer varies in sequence but its conserved a nonamer. These two stretches are
separated by spacer that is 12– 23
length corresponds to one turn (12 bp) or two turns (23 bp) of the DNA double helix. nucleotides long.
v 7 23 9 9 12 7 J
5’ 3’ Light chain κ
v 7 12 9 9 23 7 J
5’ 3’ Light chain λ
v 7 23 9 9 12 7 D 7 12 9 9 23 7 J Figure 5.10
The recombination sequence (heptamer
5’ 5’ and nonamer) and its arrangements in
Heavy chain λ chain, κ chain and the heavy chain.
» Recent studies have shown that 12/23 RULE

sometimes the 12/23 rule can be The recombination of gene segments generally follows the rule that only a gene segment flanked by
violated. In humans, as well as
some other species, D–D fusion is an RSS with a 12-bp spacer can be joined to other gene segment that is flanked by an RSS with a
found in approximately 5 per cent 23-bp spacer. This is referred to as the 12/23 rule or one-turn/two-turn rule.
of antibodies. This results in the for- In the κ light chain DNA,
mation of an unusually long CDR3
loop. This violation of the 12/23 rule 5’ -GGTTTTTGT-3’ 5’ -CACTGTG-3’ the Vκ RSS has a one-turn spac-
however, is an exception. 3’ -CCAAAAACA-5’ 3’ -GTGACAC-5’ er and the Jκ RSS has a two-
Nonamer C G turn spacer. One-turn/two-turn
Heptamer
C G rule is exemplified by pairing
A T
segments of κ chain as shown in
A T
A T Nonamer Figure 5.11. In the Vλ, the order
T T is reversed. The Vλ has a two-turn
A T signal sequence while the Jλ has
C G a one-turn spacer. In the heavy
A T
12-nucleotide 23-nucleotide chain DNA, the signal sequene
spacer spacer of the VH-gene segment has two
G C
T A turns spacer, the DH-gene seg-
G C ment is flanked on both side by
A T Heptamer
one-turn RSS. The JH-gene seg-
C G
A T
ment again has a two-turn signal
C G sequence. Thus for a heavy chain,
V-J recombination the DH gene segment can join to
the JH gene segment and the VH
VK G C JK gene segment can be ligated to the
T A
G C Heptamer
DH segment. But the VH gene seg-
A T ment cannot be ligated to the JH
C G gene segment directly, as both the
A T VH and JH gene segments has have
12-nucleotide C G 23-nucleotide spacer
23-bp spacers.
spacer
T A R E C O M B I N AT I O N
G C SEQUENCE-DIRECTED
T A JOINING OF GENE
A A
SEGMENTS
T A Nonamer
T A There are a number of V, (D) and
T A J segments in the immunoglobu-
G C
Figure 5.11 lin gene. One of the V, (D) and J
G C
Recombination signal sequence and segments is selected and joined
example of 12/23 rule in pairing of the
κ chain.
together to form V, (D), J genes
which code for the variable region
of the light or heavy chain. Usually V, (D), J sequences are situated far apart in the DNA sequence.
These gene segments are recombined with the help of the guiding RSS. This joining of V, (D), J gene
segments is catalysed by enzymes collectively called V(D)J recombinase. Two recombinases have
» Human RAG-1 and RAG-2 genes now been identified — RAG-1 protein and RAG-2 protein.
are located on chromosome RAG-1 and RAG-2 proteins are products of recombination-activating genes 1 and 2, identi-
11. RAG-1 protein mediates the
recognition of the signal sequence fied by David Schatz, Marjorie Oettinger and David Baltimore. RAG-1 and RAG-2 proteins and
and recruitment of RAG-2 in V,(D),J terminal deoxynucleotidyl transferase (TdT) are the three identified enzymes involved in the V,
recombination event. (D), J rearrangement.
The joining of gene segments is a multi-step process as shown in Figure 5.12 and is out-
lined below. V, (D), J gene segments are brought close together by the interaction between signal
sequences. The V, (D), J segments are cleaved by lymphocyte specific recombinases and the cut
ends of the V, (D), J gene segments are sealed by the formation of a hairpin loop. The details of
genetic events are outlined below.
• A hairpin loop is cut to generate a single strand at both ligating ends. Nucleotides added
to this region generally form palindromic sequence and are referred to as P-region
nucleotides.
V J C
Germ-line DNA
DNA looping
Site-specific recombination
V J C
RAG-1/RAG-2
Figure 5.12
Model depicting the general process
Rearranged DNA of recombination illustrated by V-J
V J C recombination.
• Sometimes, an additional 15–20 nucleotides are added to single strands present at both
the ligating ends by the enzyme terminal deoxynucleotidyl transferase. These nucleotides
are called n nucleotides because they are non-template coded.
• Repair enzymes then trim off any non-matching bases and add complementary bases to
fill in the remaining single-stranded DNA.
• Finally ligases such as DNA ligase IV join the V, (D), J gene segments to form the rear-
ranged gene.
The rearranged gene segments have coding joints between the V, (D), J sequence, while the two
signal sequences which are spliced out are also joined by a signal joint.
M E C H A N I S M O F V, ( D ) , J G E N E - S E G M E N T R E A R R A N G E M E N T
The mechanism of the V, (D), J gene segment rearrangement is outlined below.
• The recombination sequence flanks the 3′ region of the V region, 5′ region of the J seg-
ments and both sides of the D-gene segment. As mentioned previously, it may be of the
7-23-9 type (23-bp spacer) or the 7-12-9 type (12-bp spacer). Heptamer is always towards
the coding sequence.
• RAG-1 binds the nonamer sequence on the 12-bp spacer sequence (for example, flank- « RAG-1 and RAG-2 were first
ing V segment). This is followed by the binding of RAG-2, on the RAG-1 DNA complex reported by the American
molecular biologist, David
near the 12-bp spacer. Another molecule for RAG-1 binds to nonamer of 23-bp spacer Baltimore in 1995.
(for example, flanking the J segment). These two signal sequences (12-bp and 23- bp spacers)
are then juxtaposed towards each other forming a synaptic complex between the signal
sequences. The net result is that the two coding regions (V-and J-gene segments), which
were previously distant from each other, are now broughtcloser together.
• One strand of DNA is nicked by the RAG-1/RAG-2 recombinase at the junction of the « Somatic recombination such as
coding-gene segment and the signal sequence. So there will be one single-strand break the VDJ recombination occurs prior
to antigen contact.
at the junction between V and signal sequence and another between the signal sequence
and the J-gene segment. This signal strand creates free 3′ OH on the coding-gene seg-
ment. The free 3′ OH then attacks the phosphodiester bond linking the opposite strands
of the same DNA helix. This breaks the remaining single-strand linkage between the cod-
ing-gene segment and the signal sequence. It produces a hairpin structure at the cut end
of the coding sequence (of both V and J regions).
• The signal sequence now gets separated from the coding gene segment and has a flush
5 phosphorylated double-strand break on the side facing the coding gene segment. This
happens at both the ends, that is, the 3′ end of V and J segments. The two coding gene
segments do not float apart, but are held tightly via a complex comprising RAG proteins
and other associated DNA repair enzymes until the joining is complete. This reaction is
catalysed by RAG-1/RAG-2.
• Now, the two coding regions (for V and J) are facing each other. Each of the segments has
a hairpin loop at the recently cleaved end. This hairpin loop at the ends of both the coding-
gene segments is cleaved by endonuclease activity of the RAG protein complex.
• The hairpin loop is usually cleaved at or near the original point at which the hairpin was first
formed. This occurs in both the juxtoposed coding segments (V-and J-gene segments). In
P-nucleotides most light chain gene rearrangements, the DNA repair enzymes fill in the complementary
P-nucleotides are palindromic nucleotides on the single stranded tails which would leave short palindromic sequences at
sequences of nucleotides added at
the joint. These added nucleotides which make up the palindromic sequences are called
either the V-J or the V-D-J splice site.
p nucleotides (see Figure 5.13).
In heavy-chain and some light-chain gene rearrangements some extra nucleotides are added after
N-nucleotides
the addition of P nucleotides. These are called n nucleotides. These nucleotides are added during
N-nucleotides are non-template the D–J and V to D–J joining processes by the enzyme terminal deoxynucleotidyl transferase (TdT)
nucleotides added at the splice site to the single-stranded ends of the coding segments. This addition of nucleotides occurs after the
after the addition of P nucleotides.
The only known function of TdT is to
hairpin cleavage and p-addition. The TdT enzyme adds up to 15–20 nucleotides. The added nu-
create diversity at the V(D)J junction. cleotides at the ends of the gene
It is believed that another protein, segments form a base pair over
Ku-80, regulates TdT activity.
a short region. Repair enzymes
A-T A-T trim off any non-matching bases,
D J Hairpin loop
T-A T-A at ends synthesize complementary bases
to fill the gaps in base-paired
single-stranded DNA and ligate
Single-strand it to P nucleotides. The addition
cleavage of N-nucleotides is referred to as
A-T-T-A
n-addition (see Figure 5.14).
D J n nucleotides are not en-
T-A-T-A
coded by germ-line V, D or J
Resolution of single strand gene segments. They are added
Repair enzymes add
complementary nucleotides
during gene rearrangement. The
location of p and n nucleotides
on the antibody molecule is
Figure 5.13 A-T-A-T-A-T-A-T
Diagram showing the details of D J shown in Figure 5.15.
T-A-T-A-T-A-T-A
P-addition. In this way, coding gene
segments are ligated. Apart from
RAG proteins that are involved in
gene rearrangements, other pro-
teins include enzymes DNA ligase
A-T A-T
D J IV and DNA-dependent protein
T-A T-A
kinase (DNA-PK). Protein Ku
(heterodimer of 70 and 80 kDa)
Single-strand
that associates with DNA-PK also
cleavage plays an equally important role in
gene recombination.
A-T-A-T
D J
T-A-T-A D E F E C T S I N V, ( D ) , J
Hairpin resolution R E C O M B I N AT I O N
Addition of non-template The recombination process of
(N) nucleotides by TdT V(D)J joining was further clari-
fied by the detection of defects
Figure 5.14 A-T-A-T-C-T-T-A-T-A-T in the various genes involved in
Diagram showing the details of D J
N-addition.
T-A-T-A-G-A-A-T-A-T-A recombination.
• Mice in which either RAG-1 or RAG-2 Variable region of Variable region

is knocked out are unable to cleave the heavy chain of light chain
junction between the signal sequence and
coding-gene sequence in germ-line Ig
DNA. As a result of this defect, germ-line
V, (D), J sequence remains unrearranged
and the development of lymphocytes is P and N
completely inhibited. Since the same set addition

of recombinase is also involved in T-cell
development, such mice suffer from J region of Constant region
heavy chain of light chain Figure 5.15
severe combined immune deficiency—
Diagram showing the location of P- and
SCID. Several humans with SCID have N-addition on antibody molecules.
also been detected with mutated RAG
genes.
• Mice rendered deficient in TdT do not add extra nucleotides to the joints between gene
segments and hence the diversity of B-cell (and T-cell) repertoires is considerably less « Severe combined immunode-
than in normal mice. ficiency is a primary immunode-
• Mice having defects in DNA-dependent protein kinase (DNA-PK) are unable to efficient- ficiency disease. This disease is
ly rejoin DNA gene segments. Such mutation also gives SCID phenotype. characterized by a severe defect in
humoral and cell-mediated immu-
nity. It is also known as the “bubble
boy disease” because it was noticed
in the 1970s when David Letter—a
5.4 ALLELIC EXCLUSION SCID patient lived for 12 years
inside a plastic bubble in a
germ-free enviroment.
During the maturation of B lym-
phocytes only one of several V, Maternal Paternal
(D) or J gene segments is selected
and joined together to make func-
tional light-or heavy-chain genes. Light (L)
chain allele
B cells, like all somatic cells are Light (L)
diploid and contain both maternal chain allele
and paternal chromosomes. So
there will be two sets of rearranged
heavy-chain genes and two sets of
light-chain genes, that is, there are
alleles of light/heavy chains — one
situated on the maternal chromo- Chosen (H)
some and the other on the pater- Pro-B Cell
chain allele
nal. In B cells only one light/heavy
chain gene is expressed, that is, ma-
ternal or paternal. The other allele
is excluded. This process, called
allelic exclusion (see Figure 5.16),
ensures that functional B cells Allelic exclusion
never contain or express more Allelic exclusion is a situation in
Chosen which only one of the pair of alleles
than one VH DH JH and one VLJL Kappa chain Pre-B Cell
of immunoglobulin heavy and light
unit. allele chains is expressed in a diploid cell.
The phenomenon of allelic The other allele either remains in
germ-line configuration or is an out-
exclusion suggests that once a
of-frame rearranged gene.
productive VH DH JH rearrange-
ment and VLJL rearrangement has
taken place, the recombination
machinery that rearranges gene
segments is turned off, so that Paternal Kappa chain
other gene segments remain un- Maternal H chain
rearranged. Figure 5.16
Allelic exclusion.
» Allelic exclusion of immunoglobu- 5.4.1 A L L E L I C E XC LU S I O N O F H E AV Y C H A I N S

lin (Ig) genes ensures the expres-
sion of the antibody molecules of It is believed that once a productive rearrangement is attained, its encoded protein is expressed and
single specificity in B cells. the presence of this protein acts as a signal to prevent further gene rearrangement only. The μ heavy
chain protein that is synthesized in a developing B cell is the key regulator of gene rearrangement.
The μ heavy chain produced by the rearranged gene present on one chromosome inhibits gene
rearrangement on the other homologous chromosome. Heavy-chain gene on the other chromo-
some will rearrange only if the first rearrangement is non-productive. If both the alleles of a heavy
chain undergo non-productive recombination, the cells cannot synthesize immunoglobulin and
apparently die of apoptosis. This occurs quite frequently and a large number of cells that arise
from B-cell progenitors eventually die. Only a small fraction that have one expressed μ heavy chain
develops into mature B lymphocytes. It is believed that the membrane form (not the secretory
form) of μ heavy chain is responsible for allelic exclusion for some unknown reason.
5.4.2 ALLELIC EXCLUSION OF LIGHT CHAINS

The μ heavy chain also stimulates light-chain gene rearrangement. The exact mechanism by which
μ heavy chain stimulates light chain production is still not clear. It was initially thought that a μ
heavy chain turns on the rearrangement of κ-chain genes first. If a productive κ rearrangement
occurs, the κ chain which pairs off with a μ heavy chain is produced. If, however, κ-chain gene rear-
rangement fails, λ chain gene starts rearranging. However, recent studies have suggested either κ or
λ genes may rearrange first. However, the production of either light chain inhibits rearrangements
at the other light chain locus, explaining why an individual B cell clone can produce only one type
of light chain during its life (light chain isotype exclusion). As in heavy chain, if one allele under-
goes non-functional rearrangement, DNA recombination can occur on the other allele. However, if
both alleles of both the λ and κ chains are rendered non-functional, the cell apparently dies.
μ heavy chain is not the only signal that stimulates the rearrangement of a light-chain gene as is
indicated in mice in which μ gene is knocked out. Such mice do have some light chain gene rearrange-
ment indicating that μ chain is not an obligatory signal for the recombination of light chain genes.
5.5 T H E G E N E R AT I O N O F D I V E R S I T Y I N
IMMUNOGLOBULINS
It is well known that virtually any substance can elicit an antibody response. Almost limitless types
of antibodies can be formed by the human body as well as by other vertebrates. The total umber of
antibody specificities available to an individual is known as antibody repertoire. Since antibodies
are proteins and proteins are encoded by genes, it follow that such huge diversity must come from
the genome of the organism. Previously two main hypothesis were put forward to explain the gen-
eration of antibody diversity.
Germ-line theory GERM-LINE THEORY. This theory states that each antibody is coded by separate gene. These
Germ-line theory states that every
repertoires of antibody genes are present in the genome of the individuals. Since the genome is
B cell has all the genes to make all
types of antibodies. B cells, however, contributed by eggs and sperm cells (germ cells), it follows that the entire repertoire of anti-
express only one type of antibody- body genes is coded by the germ line of the organism and is inheritable. This theory was ultimately
forming genes.
rejected when it was found that individuals can generate far more different types of the antibody
than all the genes present in the body.
SOMATIC DIVERSIFICATION THEORY. This theory argues that the repertoire of antibody
genes is limited (that is, the number of variable genes is limited). The limited number of genes is
diversified in the somatic cells by mutational and/or recombination events during an individual’s
lifetime. With cloning and sequencing of immunoglobulin genes, it is now clear that somatic
diversification was essentially correct, although the concept of the presence of multiple germ-line
genes embodied in the germ-line theory also proved true to some extent.
It is believed that there are seven known mechanisms that operate for the generation of
antibody diversity in vertebrates:
• multiple germ-line gene segment;
• V–J and V–D–J recombination, that is, combinatorial diversity;
• junctional diversity;
• p-addition and n-addition;
• gene conversion;
• somatic hypermutation; and
• association of varied light and heavy chains.
5.5.1 M U LT I P L E G E R M - L I N E G E N E S E G M E N T S
For simplicity, we have so far discussed the formation of the complete immunoglobulin V region as
if there is only a single copy of each gene segment. In fact, there are multiple copies of all of the gene
segment in germ-line DNA. There are about 90 genes segments for VH, 30 DH and 6 JH in humans.
There are 80 Vκ and 5 Jκ gene segments, and 80 Vλ and 4 Jλ gene segments found in humans.
In mice there are approximately 1,000 VH, 12 DH and 4 JH region segments. There are about
200 Vκ and 4 Jκ gene segments, as well as two Vλ and three Jλ genes also found in murine genome. « CDR1 of heavy chain is coded by
a V segment. The diversity in CDR1
Thus, as can be seen, each variable region is encoded by several (sometimes hundreds) genes, of heavy chain occurs because of
which by recombination can generate hundreds and sometimes thousands of different types of multiple V segments.
light or heavy chains.
5.5.2 V – J A N D V – D – J R E C O M B I N AT I O N
The gene rearrangement combines two-genes (VL and JL) or three-gene (VH, DH, JH) segments to
form a complete V region exon. There are multiple copies of the V, (D), J gene segments each of
which is capable of contributing to an immunoglobulin V region. Many different V regions can
therefore be made by the random rearrangement of these segments. In humans, any of the 90 VH
segments can combine with any 30 DH and any 6 JH gene segments. This combinatorial diversity « A CDR3 of heavy chain is formed
allows considerable heavy chain diversity (90 × 30 × 6 = 16,200 possible combinations). Similarly by the V–D junction, D segment
80 Vκ gene segments can combine with 5 Jκ segments to potentially generate (80 × 5) 400 possible and D–J segment. Here, a diversity
arises because of multiple V,D,J
combinations at κ locus. 80 Vλ and 4 Jλ gene segments can also generate 320 possible combination. segments. A light chain CDR3 is
So, in all, 720 (400 + 320) different light chains can be made as a result of combining different formed by the V–J junction. CDR1s
light-chain genes. It should be realized that this is the minimal possible number of combinations as of light chains and heavy chains are
encoded entirely within variable
we have not taken into account p- and n-additions, somatic hypermutations and gene conversions gene segments and hence no rear-
(in rabbits and chicken). It may not be possible to predict the exact umber of antibody-binding rangement is needed.
sites that can be generated. It is safe to say that at least as many as 1011 different antibody-binding
sites can be generated.
5.5.3 JUNCTIONAL DIVERSITY

Even the same set of germ-line V, D and J gene segments can generate different amino acid sequences
at the junctions. This results in variable gene segments that bind different antigenic determinants. So
the same V, (D), J gene segment can generate different variable regions in different B cells.
Junctional diversity arises from: (a) imprecise DNA rearrangement and (b) p- and n nucleo-
tide addition
(a) Imprecise DNA rearrangement: As described previously, signal sequence and coding « It is believed that junctional
sequence are cleaved and the two coding gene segments are then joined. However, the diversity can triple the diversity
generated by V–J and V–D–J
joining does not usually occur at the precise point of cleavage. Imprecise DNA rearrange- joining.
ment occurs because the 3′ end of a V gene and the 5′ end of a J segment in a light chain, or
the ends of V, J and D gene segments in the heavy chain can each recombine at any of the
several nucleotides in the coding gene segments in pre-B-cell lines. This imprecision in the
joining process helps generate antibody diversity (see Figure 5.17). When gene segments
are joined in phase that is, the reading frame is preserved, the resulting VJ unit or VDJ unit can
be translated in its entirety, yielding a complete antibody. This is called productive rearrange-
ment. It also has an ugly side. The gene segment may be joined in such a way that this junction
might contains one or more stop codon, that is, triplet reading frame is not preserved. Such
“frameshifts” will lead to the formation of non-functional protein. DNA rearrangements that
lead to such disruptions and the production of non-functional proteins are known as non-
productive rearrangement. Roughly two in every three rearrangement will be non-productive.
Many B cells never succeed in producing functional immunoglobulin molecules.
(b) p- and n-addition: When two coding gene segments join, the hairpin at the joining end
is cleaved. This is followed by filling up the gap by nucleotide addition, giving rise to
a palindromic sequence. A hairpin cleavage usually occurs near the original point at
which the hairpin was formed. The same V, (D), J gene segment can generate a different
» Junctional diversity induces p-nucleotide region if the cleavage occurs at a different point from the initial break. This
diversity in the CDR3 of a heavy
chain and in the center of the
will leave a single-stranded tail on one strand plus a small single-stranded tail on the comple-
antigen-binding site. mentary strand. Gaps are filled, and this creates a new variable region from the same V, D, J
gene segments.
In some heavy-chain (and
Pro Heptamer Trp
some light-chain) gene rear-
-CCTCC -GTGGC rangements, n nucleotides are
-GGAGG -CACCG added at the coding joints.
The n nucleotides are added
V J without a template, that is,
these nucleotides are not cod-
ed by germ-line V, D, J gene
segments. Since these nucle-
otides can be added in any
Possible combinations at the junction (amino acid no. 95-96) sequence, and these encode
amino acids, the junction of
Pro Trp V–J, or V–D–J can have any
1) number of random sequence
-CCT TGG- of nucleotides (15–20 n nucle-
Pro Arg otides) and hence a random
2) sequence of amino acids. In
-CCT CGG- heavy-chain gene segments,
this diversity is located in
Pro Pro
3)
V–D–J and is over and above
-CCT CCG- that generated by p-addition.
This additional n heavy-chain
Figure 5.17 Pro Pro diversity is quite large and is
Junctional flexibility in joining Ig gene 4)
segments. -CCT CCC-
localized in the CDR3 of the
heavy chain genes.
5.5.4 GENE CONVERSION

In contrast to humans and murine members, birds, rabbits, cows and sheep possess a limited
number of genes that code for immunoglobulins. There is only one each of V, J and C segments for
light chain in birds. The heavy chain has one V and one J, and about 16 D segments. Despite this
limitation, birds can generate a huge repertoire of diverse antibodies.
Gene conversion This unique adaption of generating antibodies is by the mechanism called a gene conversion.
Gene conversion is a non-reciprocal Bird (chicken to be precise) genome has only one VL segment. There are about 20 pseudogene
transfer of genetic information. It
implies that the DNA is transferred sequences upstream of the VL segment. Though the base sequence of the pseudogene is similar
from one DNA strand (which remains to the VL gene segment, it has no flanking signal sequence so it cannot be rearranged. A pseu-
unchanged) to another DNA strand dogene lacks the promoter region
which is altered in gene/DNA About 30 pseudogenes
content. It was first defined in and hence cannot be transcribed;
yeast as a type of homologous and as it lacks the exon leader
recombination in which the donor VL J CL Germ-line DNA sequence, it can never be trans-
sequence does not change.
ported to ER to generate func-
V-J rearrangement tional antibodies. Inspite of all
these anomalies, pseudogenes are
VL J CL B-cell DNA not wasted. During recombina-
tion a portion of the pseudogene
Gene conversion
taken and inserted into a viable
VL region (see Figure 5.18). This
Figure 5.18 J CL B-cell DNA generates an additional functional
Gene conversion. VL gene. This process continues
even after B cells have left the bursa, and several of these gene conversions can occur during the
lifetime of the B cells. The same process occurs with a heavy-chain gene which has 100 identified
VH pseudogenes that act to increase the diversity by a similar conversion mechanism.
5.5.5 S O M AT I C H Y P E R M U TAT I O N
It is generally believed that all the diversity that can be generated in the antibody structure occurs « Somatic hypermutations are
during gene rearrangement. This implies that once the functional variable region is formed, it is not always single-residue substitutions
and never insertions or deletions.
altered. This is not true: there is an additional mechanism that generates diversity throughout the
V-region exons of both heavy and light chains, after functional immunoglobulin genes have been
assembled. This process, that operates on B cells in peripheral lymphoid organs, is referred to as Hypermutation
somatic hypermutation. Hypermutation is a process that
occurs after B cells are exposed
Somatic hypermutation can be defined as a high rate of point mutation occurring in the to antigen by dendritic cells in
V regions of the rearranged heavy and light chain genes, giving rise to mutant B cell receptors (and the germinal centres of lymphoid
secreted antibodies) on the surface of the B cells. The purpose of hypermutation is to create new organs.
B-cell receptors that can bind the antigen more strongly and specifically than their precursor. This
allows the host body to respond quickly and effectively to pathogens. Since in somatic hypermuta- Somatic hypermutation
Somatic hypermutation refers
tion, the affinity of B-cell receptors (or antibodies) is increased, it gives rise to phenomenon called
to the mutation that is 104 times
affinity maturation of antibodies (see Figure 5.19). It should be clarified that somatic hypermuta- higher than the rate of spontaneous
tion does not change the specificity of a V region. These small degrees of mutations only influence mutation occurring in V region of
H and L chains. Somatic mutations
the binding affinity of the antibodies that occur in pre-B cells in the central lymphoid organs.
induce diversity in the periphery of
Hypermutation occurs only in B cells that have been stimulated with antigen. During the the antigen-binding site.
development of B cells, genetic recombination forms the basic antibody encoding genes. Variable-
diversity joining or V(D)J recombination creates different heavy- and light-chain variable regions
Affinity maturation
and the joining of essentially any heavy with any light chain results in millions of different specific Affinity maturation refers to
antibodies. The antibodies are of low affinities. Once B cells bind antigen (that is, an antigen binds the increase in the affinity of
antibody molecules due to somatic
the surface receptor of a B cell) these cells starts mutating. B cells with useful mutation survive; that
hypermutation. It is a unique
is, B cells that exhibits higher affinity for antigen survive, and give rise to long-lived memory cells. property of B cells stimulated by
It is believed that the generation of uracil from cytosine in B cells by activation-induced cytosine T-dependent antigens.
deaminase (AID) protein sparks hypermutation.
Antigen stimulation
B cell
Somatic hypermutation
Activation induced
Cytosine deaminase
active
Reduced No antigen High-affinity

antigen binding antigen
binding binding
Cell death Cell death Clonal proliferation of this Figure 5.19

by by B cell and increased Somatic hypermutation and affinity
apoptosis apoptosis antibody synthesis maturation.
In hypermutating cells, all four nucleotides are mutated as cells try to repair the damage. This
occurs in the variable region of the Ig genes. The mismatch repair proteins MSH2 and MSH6, and
DNA polymerase I and Z may also be involved. The hotspots of mutation show a preference for
the short motif of four to five nucleotides and tend to be clustered within the CDRs of VH and VL
sequences. The mutation rate is estimated to be of the order of 10ⴚ3 to 10ⴚ4 per base per cell generation.
This rate is 104 times higher than the rate of spontaneous mutation (which is 10ⴚ8 bp/generation)
in other genes and hence the name hypermutation.
5.5.6 A S S O C I AT I O N O F VA R I E D L I G H T
A N D H E AV Y C H A I N S
The specificity of the binding site on the antibody molecule is determined by the variable region
of both heavy and light chains. The presence of hundreds and/or thousands of potential light and
heavy chains can generate additional diversity of binding sites of the antibody. In humans, there is
a potential to generate 16,200 heavy-chain genes and 720 light-chain genes. Since any light-chain
gene product can possibly pair with any one of the 16,200 heavy-chain gene product, potentially
a heavy chain–light chain combination can occur 11,664,000 (16,200 × 720) times. This number
reflects the maximum permutation/combinations, as not all heavy chains and light chains will give
viable antibody molecules. It is well known that not all VH or VL gene segments are used at the same
frequency; some are used more often, others only occasionally. Apart from combinatorial associa-
tion of very large number of light chain and heavy chains, a large number of additional sequences
is created by junctional diversity, p- and n-nucleotide additions, somatic hypermutation and/or
gene conversion. It is estimated that as many as 1011 different receptors of B cells (that is, antibod-
ies) could be generated.
5.6 MEMBRANE-BOUND AND SECRETED

IMMUNOGLOBULINS
A membrane-bound immunoglobulin and its secreted form is identical in antigen specificity. These
two forms differ from each other by extra amino acids located at the C terminus of each heavy
chain in the membrane-bound immunoglobulin. The secreted immunoglobulin has a hydrophilic
sequence of about 15–20 amino acids in the C-terminal domain. Membrane Igs are slightly larger
than their secreted counterparts. They have a 40 amino acid hydrophilic regions (similar to the
secreted Ig but slightly longer) that lie outside the cell, followed by hydrophobic amino-acid
sequences that span the membrane. The hydrophobic region is followed by a short hydrophilic
cytoplasmic segment at the carboxyl terminus of membrane Ig. The hydrophobic residues are thought
to form a stretch of α helix within the membrane that anchors the antibody into the membrane.
5.6.1 G E N E R AT I O N O F M E M B R A N E - B O U N D
OR SECRETED IMMUNOGLOBULINS
The DNA sequencing of the Cμ gene segment reveals that it consists of four exons (Cμ1, Cμ2, Cμ3,
Cμ4) corresponding to the four domains of the IgM molecule. At the 3′ end of the fourth exon, is a
sequence of nucleotide (S region) that codes for the hydrophilic sequence found at the C-terminal
region of secreted IgM. Two additional gene segments TM (transmembrane) and CT (cytoplas-
mic tail) are located downstream from 3′ end of the Cμ4 and S exons. TM encodes transmembrane
segment and CT encodes the cytoplasmic tail. We now know that all CH gene segments (that is,
those encoding Cδ, Cγ, Cα, Cε) have additional TM and CT exons than encode transmembrane and
cytoplasmic segments.
The primary transcript produced by the transcription of the rearranged μ-heavy-chain genes
contain Cμ1, Cμ2, Cμ3 and Cμ4 exons, and the S gene segment. There are introns between C1, C2,
C3 and C4. There is no intron between the C4 and S gene segments. At the end of S gene seg-
ment is a translation termination triplet followed by a site where polyadenylation can occurs (poly-A
site-1). There is another translation termination triplet located at the end of the CT gene segment,
followed by second poly-A Site.
When activated B cells “want” to make secretory antibodies, the cleavage of primary tran-
script and addition of poly-A tail occurs at site-1. TM and CT exons are lost. From this shorter
primary RNA transcript, introns are excised and the remaining exons are spliced together to form
mRNA that encodes for the secretory form of the heavy chain. When the cells “want” to make
a membrane antibody, cleavage and polyadenylation of the primary transcript occurs at site-2. « In 1980, L. A. Herzenberg and
This primary transcript is then differentially spliced. The introns between Cμ1, Cμ2, Cμ3, Cμ4, TM coworkers showed for the first
time that secreted and membrane-
and CT are removed. Apart from this, the S segment (together with translation termination triplet) bound antibody are generated
located adjacent to Cμ4 is also excised out. The mature mRNA formed contains a VDJ unit from the same DNA sequence by
and Cμ1, Cμ2, Cμ3, Cμ4 (without S), TM and CT, followed by a poly-A tail. The genetic re- alternate mRNA splicing.
arrangements that occur in the formation of secreted IgM and membrane IgM are shown
in Figure 5.20.
Mature naïve B cells produce only membrane-bound antibodies. As differentiation proceeds,
more and more of mRNA of secretory Ig is formed, so differentiated plasma cells produce secreted
antibodies. The secretory form of IgD is rarely made, so IgD is usually present as membrane-
bound proteins.
5.6.2 CO-EXPRESSION OF MEMBRANE-BOUND

IgM AND IgD
Mature B cells express both membrane-bound IgM and IgD on their surface. As mentioned previ-
ously, in mature B cells the primary transcript containing both Cμ and Cδ segments is formed. This
is probably because Cμ and Cδ gene segments are located close together (5 kb apart) and there is no
switch-region between Cμ and Cδ gene segments. From the Cμ–Cδ-containing primary transcript (of
about 15 kb), two types of mRNA (Cμ mRNA and Cδ mRNA) can be generated by alternative splicing.
This long primary RNA transcript contains four translation termination triplets with four
associated poly-A sites. Sites 1 and 2 are associated with Cμ (as described in the previous section,)
and sites 3 and 4 are located in the Cδ gene segments at a similar location (one after the Cδ3-S
exon and other at 3′ end of the CT exon). If introns are excised out in such a way that the VDJ
complex is attached to the Cμ RNA and the heavy-chain transcript is cleaved and polyadenylated
at site 2 after the Cμ exon, it will give rise to a μ mRNA. If however, Cμ RNA is also spliced out so
that the VDJ complex become contiguous with Cδ exon, a δ mRNA is produced and subsequent
translation results in the synthesis of complete δ heavy-chain protein.
Thus, alternative splicing allows B cells to simultaneously produce mature mRNA of two
different heavy-chain isotypes. Thus mature B cells express both IgM and IgD on their mem-
brane. The precise mechanism that regulates the choice of polyadenylation and/or splice accep-
tor sites by which the rearranged the VDJ is joined to Cμ and Cδ are not known, nor is fact that
why B cells expresses both IgM and IgD since both have exactly same specificity (same VDJ
segment, remember!).
It should be remembered that if the cleavage and polyadenylation of a primary heavy-chain
transcript occurs at 1 or 3 poly-A site in mature a B cell, it will produce the secretory form of μ or
δ heavy chain respectively.
CH gene segments CH gene segments
V D J 1 2 3 4 M M DNA V D J 1 2 3 4 M M
Poly-A Transcription Poly-A sites

sites
Primary V D J 1 2 3 4
V D J 1 2 3 4 M M
transcript
RNA splicing
Figure 5.20
V D J 1 2 3 4 MM mRNA V D J 1 2 3 4 The gene segments of the secreted IgM
and membrane-bound IgM.
Translation A membrane-bound IgM has two extra
gene segments that enables it to remain
Membrane-bound form of heavy chain Secreted form of heavy chain attached to the membrane.
5.7 A S S E M B LY A N D S E C R E T I O N O F
IMMUNOGLOBULINS
» Light chain and heavy chain
are translated separately at a The translation of both mature light-chain and heavy-chain mRNAs start on free ribosomes.
very rapid rate. It takes about
25 seconds to translate light The leader sequence is first synthesized, which directs the growing polypeptide chain into the
chain and about a minute to lumen of the ER. The leader sequence is cleaved by signal peptidase and the light chain and heavy
translate heavy chain. chains are assembled and glycosylated in the rough endoplasmic reticulum.
» The light chains are usu- The complete Y-shaped immunoglobulin molecule is then transported to a distribution cen-
ally not glycosylated. Heavy tre, the Golgi apparatus. In the Golgi apparatus, the antibodies are packaged in secretory vesicles
chains are glycosylated at the
asparagine residues, that is,
which finally fuse with the plasma membrane and antibodies are secreted out (see Figure 5.21).
N-linked. Glycosylation at the The exact pattern of assembly of light and heavy chains varies with the type of antibody formed.
hydroxyl group of serine and In the case of IgM, the H and L chains form H and L half-molecules and two half-molecules ag-
threonine occurs rarely, and
that too, only in IgA and IgD.
gregate and form a mature Y-shaped immunoglobulin in rough ER. In the case of IgG, the first two
H chains assemble, followed by the L chain to form an H2L intermediate. The addition of L chain
» The order of assembly can
either be H → HH → LHH →
completes the H2L2 molecule.
LHHL or by H → HL → LHHL.
The assembly depends on Ribosome
random collision of chains
in the rough endoplamic mRNA
reticulum.
Heavy chain Light Chain
Endoplasmic reticulum
Synthesis and assembly

of light and heavy chains
Golgi body addition
Transport vesicles
Figure 5.21 Secreted antibody

The synthesis, assembly and secretion of
an immunoglobulin molecule.
Inter-chain disulphide bonds are formed and polypeptides are glycosylated as they move
through the RER and Golgi apparatus. If the molecule contains the transmembrane sequence of
the membrane form, it becomes anchored in the membrane of RER and finally in the secretory
vesicles. These secretory vesicles then fuse with the plasma membrane in such a way that both the
membrane and membrane bound antibody are incorporated into the plasma membrane. Antibod-
ies always face towards the extracellular side. Almost all the extracellular secretions including Ig,
have to pass through the Golgi apparatus.
5.8 CLASS SWITCHING

Immature B cells express both IgM and IgD on its surface. They, however, have all CH gene seg-
ments. After antigenic stimulation, B cells start to differentiate. During this differentiation, some
B lymphocytes switch from producing antibodies of class IgM or IgD to producing antibodies of
another class (IgG, IgA or IgE) but of the same specificity. This phenomenon is called class switching. Class switching
Since classes (or isotypes) of antibodies are defined by their heavy-chain gene segment such as Cγ, Class switching is the switch
Cα, Cε, class switching involves only the constant region of the heavy chain. The variable region of (change) in the class of antibodies
produced by B cells. After antigen
the heavy chain (hence, specificity) remains the same. exposure, IgM-type antibodies
Class switching occurs in the following way: are produced first by B cells. IgM
antibodies are later replaced by
• Class switching takes place in the DNA instead of the RNA. another class of antibodies such as
• The principal mechanism of class switching is a process called switch recombination in the IgG that has the same specificity
but stronger affinity for the antigen.
which rearranged VHDHJH gene segment recombines downstream with any one C region
and the intervening DNA is deleted; for example, in a switch from IgM to IgG, VHDHJH gene
moves from a site near Cμ gene to the one near Cγ gene. The intervening DNA sequence is
looped out and deleted (see Figure 5.22).
This switch recombina- « Antibody specificity remains
tion involves nucleotide unchanged during class
Switch regions switching.
regions/sequences called
switch regions (S). The « Class switching is one-way event.
switch region is located B-cell DNA It implies that once class switching
μ δ γ3 γ1 α1 γ2 γ4 ε α2 has occurred to, say, IgA, the cell
2–3 kbp upstream of each VDJ loses the capacity to make antibod-
CH segment (see Figure DNA rearrangement and looping ies such as IgE because
δ gene-segment coding for IgE is lost
5.23) (except Cδ which during the
does not have a switch re- μ γ3 recombination event.
gion). A switch region oc- B-cell DNA
cupy a distance of about VDJ γ1 α1 γ2 γ4
« Class switching can never give rise
1–10 kb and contains nu- to IgD antibodies because the Cδ
merous tandem repeats region lacks switch region.
of highly conserved DNA B-cell DNA after
of about 25–80 base pairs VDJ γ1 α1 γ2 γ4 class switch
characteristic to each
Transcription
switch region. They in-
clude many stretches of Primary RNA transcript
nucleotide GAGCT and VDJ γ1
GGGT. Knocking out of
the switch region for any
of class heavy chain leads mRNA
to an inability to switch to VDJ γ1
that class. Translation
Figure 5.22
The Cμ gene is invariably IgG1 chain Class switching IgM to IgG1. The light
the first to be used, followed V Cγ1 chain is not involved in class switching.
by switching to Cγ, Cε or Cα
genes (producing IgG, IgE
or IgA respectively). Class switching involves the recombination and deletion of intervening
DNA. (CH gene segments remaining downstream, 3′ end, of switched CH segment are
retained and can be used later.) Switch recombination proceeds from the switch region of Sμ
region to any of the Sγ, Sε or Sα regions. This results in the formation of a hybrid between Sμ
and anothers isotype (Sμ-Sε) sequence. Switch region joining is not site specific and locates
to various sites within the S region. Since this occurs in the intron, switching does not affect
the reading frame of the heavy-chain gene.
Sμ Sγ3 Sγ1 Sα1 Sγ2 Sγ4 Sε Sα2
Figure 5.23
Schematic diagram highlighting the
switch region found near the heavy
Cμ Cδ Cγ3 Cγ1 Cα1 Cγ2 Cγ4 Cε Cα2 chain.
Heavy-chain class switching is not a random process but is believed to be directed by TH

cells and their secreted cytokines. For example interleukin-4 (IL-4) induces class switch-
ing from Cμ to Cγ and from Cγ’ to Cε. Similarly γ-interferon selectively induces switching
to IgG2a in mice. The Cδ gene lacks a switch region because of which switching to the IgD
isotype does not occur after antigenic stimulation.
5.9 R E G U L AT I O N O F I M M U N O G L O B U L I N
GENE TRANSCRIPTION
The immunoglobulin genes are expressed only in B cells. Moreover, genes are expressed at different
rates during different developmental stages. The transcription of Ig gene is controlled by three types
of DNA sequences.
• Promoters: There are short nucleotide sequences extending about 200 bp. There are usually
located upstream of the transcription start. This is the site at which RNA polymerase
binds.
• Enhancers: They are the cis- acting regulatory sequence of DNA, that activate the transcription
activity from the promoter sequence. They can be located upstream or downstream of the
promoter. Enhancer elements of Ig genes are located in the introns between J and C gene
segments.
• Silencers: These are nucleotide sequences that down regulate transcription. They can act
from both directions, that is, upstream or downstream over a distance.
A promoter lies upstream of every V gene but is inactive. There is no separate promoter for J,
D or C gene segment as these gene segments are joined to form a single V(D)JC complex having
only one promoter upstream of V region.
Unrearranged V genes in germ-line embryonic DNA are not actively transcribed into RNA
(by RNA polymerase II). It was observed that when the C region is productively joined to the V
region, the resulting unit is transcribed actively. Since, the promoter region which is upstream
of the V gene is not altered by the joining reaction, and the the same promoter is found in unre-
arranged and productively rearranged genes, it was suggested that some sequences of DNA
located downstream in the DNA are brought closer by the DNA rearrangement and this somehow
stimulates high level transcription of this immunoglobulin gene. These sequences were found to
be enhancers. Enhancer sequences are located about 250–300 kb away from the promoters. Gene
rearrangement brings the promoter and enhancer sequences within 2 kb of each other. As a result,
the rate of transcription of rearranged VL JL or VHDHJH unit is 104 times faster than the rate of tran-
scription of unrearranged VL or VH segments.
The position of enhancers of some heavy and light chains has recently been mapped. The
enhancer for heavy-chain μ is located near the switch site of the constant region of μ, that is, 5´ of
Cμ. It could be located downstream of the heavy chain as in Cα. The enhancer in the α heavy chain
is located downstream, that is, 3′ of Cα segment. Silencers have been identified in the heavy chain
and κ-chain DNA but still not found in the λ chain.
One of the arms of the immune system is humoral immunity.The key player of humoral
immunity is the antibody.The extreme effectiveness of the antibody as a defence protein is its
extraordinary repertoire. An antibody is a product of recombined genes. This extraordinary num-
ber of highly specific antibodies are generated by a variety of mechanisms. The mechanisms in-
clude the presence of a number of variable (diverse) joining fragments (all which constitute the
antigen-binding site of the antibody), DNA rearrangements such as junctional diversity, p- and
n-additions and gene conversion.The affinity of the antibody is increased by somatic hypermuta-
tion that occurs in gene coding for the variable region. The antibody can be synthesized in a soluble
or membrane-bound form. Moreover the class of antibody can be changed without any change of
specificity. All these mechanisms ensure that antibodies combat millions of pathogens that indi-
viduals encounter during their lifetime.
Single Radial Immunodiffusion

Single radial immunodiffu- Antibody in agar gel
sion is an analytical immu-
nological technique used
Precipitin ring
to quantify antigens. This
technique, though originally
developed by Oudin, was
extended by Mancini (as well
as Fancini and Carbonara) by
incorporating monospecific
antibodies into a thin layer Unknown
concentration of antigen
of agar. Briefly, monospecific
antibodies are mixed with
molten agar and layered
onto slides and allowed to
set. Three or four wells are Known concentration of antigen
made in the set agar and Figure 5.24
Single radial immunodiffusion.
different concentrations of
standard antigens are added
in separate wells. The test antigen (whose concentration is to be wider the ring. Since concentrations of standard antigen are known,
quantified) is added to a separate well. The slide is left untouched a standard plot is constructed with the x-axis depicting the diam-
for about 20–25 hours. During this period, the antigen diffuses out eter of rings and the y-axis depicting the concentration (mg/dl).
of the well into the agar medium. The antigen reacts with antibod- From this standard plot, by measuring the ring diameter of the
ies present in the agar and a ring of precipitation forms around precipitin ring formed by the interaction between the antibody and
the well. The size of the precipitin ring varies with the concentra- the test antigen, the test antigen’s concentration can be quantified
tion of the antigen. The larger the concentration of the antigen, the (see Figure 5.24).
S U M M A R Y
• The distinguishing feature of the specific immunity of chain. During gene rearrangement at the RNA level, the VLJL is
vertebrates is their extraordinary number of highly specific juxtaposed next to the constant region of the light chain (CL).
antibody molecules. Each antibody is specific for one particular
antigenic determinant. • Each V, D and J gene segment is flanked by conserved recombina-
tion signal sequences. The signal contains heptamer sequence and
• Several theories were put forward from time to time to explain the nonamer sequence with either 12-bp or 23-bp spacers. RAG-1,
process through which such a large diversity is generated. RAG-2 and TdT enzymes are involved in V, (D), J rearrangement.
• Antibody molecules are the products of recombined genes, which • In B cells (which are diploid), H-chain and L-chain genes are
are themselves formed by joining of multiple gene segments. located both on paternal and maternal chromosomes. However,
only one parental light-chain gene and heavy chains are rearranged
• The variable region of the light chain is formed by joining VL to form a functional antibody. This phenomenon, allelic exclusion,
(variable) and JL (joining) gene segments. The variable region of ensures that B cells express antibodies of single specificity.
the heavy chain is formed by joining VH, DH (diversity), JH gene
segments. The constant regions of light and heavy gene segments • After antigenic stimulation, (a) somatic hypermutation can occur in
are encoded by CL and CH respectively. The variable genes, J seg- the variable regions of light and heavy chains leading to increased
ments and C gene segments of light and heavy chains are different. affinity for the same antigen; (b) B cells can switch from IgM- or
IgD-producing cells to IgG-, IgA -or IgE-producing B cells (class
• The V, D, J gene segments are selected randomly from multiple switching); (c) B cells can switch from synthesizing
V, D, J gene segments present in the germ-line DNA. membrane-bound antibody to the secreted form.
• During heavy-chain rearrangement in B cells, the DH segments join • Extraordinary diversity in antibody specificity is achieved by the
one JH gene segment. The VH gene segment then joins DHJH. The following: (a) multiple germ-line gene segments; (b) V–J and
VHDHJH codes for the entire variable region of the heavy chain. V–D–J recombinations; (c) P- and N-additions at the splice site;
Gene rearrangement at the RNA level puts the VHDHJH unit next to (d) somatic hypermutation; (e) gene conversion (in species other
the CH gene segments (Cμ and Cδ). than human or mouse).Through these genetic processes, a limited
• During light-chain rearrangement in B cells one VL gene segment number of genes can generate the diversity needed for the
joins one JL gene segment to generate the entire V region of a light recognition of a plethora of varied antigens.
K E Y W O R D S
• recombination signal sequence 101 • junctional diversity 107 • heavy-chain synthesis 112
• RAG 102 • somatic hypermutation 109 • alternative splicing 111
• light-chain rearrangement 115 • class switching 113 • J segment 97
• heavy-chain rearrangement 115 • affinity maturation 109 • V segment 97
• terminal deoxynucleotidyl • membrane immunoglobulin 110 • D segment 97
transferase 102 • activation-induced cytosine • antibody diversity 106
• p additions 107 deaminase 109 • clonal selection theory 94
• n additions 105 • gene conversion 107 • switch recombination 113
• allelic exclusion 105 • 12/23 Rule 102
• combinatorial diversity 106 • light-chain synthesis 112
1. For coding four polypeptides of antibody molecules, nature has 4. Recent reports suggest that terminally differentiated cell can be
assigned innumerable gene segments to code for their different artificially coaxed to become adult stem cell. Can you suggest why
parts. Why? this will not hold true for B cells that have undergone a class switch
HIN T—To generate enormous diversity from IgM to IgA.
2 Does the change from the membrane-bound to the secretory form H INT —Switch from IgM to IgA will result in deletion of all heavy-chain
of antibody occur at the DNA level or the RNA level? genes.
3. Affinity maturation is the product of somatic hypermutation. Com- 5. Predict the scenario if allelic exclusion does not occur and both
ment. Can you guess why mutation does not lead to a change in paternal and maternal Ig genes are co-expressed.
antigen-binding specificity? Do all classes of heavy-chain genes H INT —Two types of light chains and two types of heavy chains will form
have their own transmembrane gene segment? four different combining sites. A single B cell produces an antibody of single
HIN T —Yes, they have their own transmembrane gene segment. specificity.
Choose the Appropriate Option.

1. According to the clonal selection theory: (c) Ligation DJ gene segment to V gene segment
(a) Antigen selects antibody that is to be produced. (d) None of the above
(b) Antigen selects clone of cells that initiate B-and T-cell
response 6. Nucleotides not coded by germ-line DNA of the Ig gene are:
(c) Antigen selects clone of cells that produce antibodies of (a) Vgene segment nucleotides
varying specificity (b) n nucleotide
(d) All of the above (c) p nucleotide
(d) j gene segment nucleotides
2. In human beings, one mechanism NOT involved in generating
antibody diversity is: 7. During somatic hypermutation, antibody undergoes:
(a) Presence of multiple V, D, J segments (a) Mutation in entire heavy and light chain genes
(b) Gene conversion (b) Mutation in V region of light chain
(c) V–D, and V–D–J recombination (c) Increase in affinity towards antigen
(d) Association of varied light and heavy chain (d) Change in antigen specificity
3. Variable regions of light chains will NOT have: 8. The reason that a B-cell cannot switch from IgM to IgD is:
(a) V gene segment (a) There is no switch region upstream of Cμ gene segment
(b) D gene segment (b) There is no switch region upstream Cδ gene segment
(c) J gene segment (c) Cμ and Cδ gene segments are located different chromosome
(d) C gene segment (d) None of the above
4. After class switching one antibody that will NOT be formed by 9. In developing a B cell, the first gene rearrangement occurs in:
B cells is: (a) Variable region of heavy chain
(a) IgM (b) Variable region of λ gene segment
(b) IgE (c) Variable region of κ gene segment
(c) IgG (d) In both variable and constant region of light chain
(d) IgA
10. Two classes of antibodies that can be expressed simultaneously are:
5. Heavy chain rearrangement of genes at the RNA level involve: (a) IgM and IgG
(a) Ligation of D gene segment to J gene segment (b) IgG and IgE
(b) Ligation of VDJ gene segment to C gene segment (c) IgD and IgM
(d) IgG and IgD
Fill in the Blanks with Appropriate Terms:

1. Three gene segments that constitute the light chain gene include 4. __________, __________ and __________ are the three enzy-
_______. mes involved in the rearrangement of Ig gene segments.
2. Recombination signal sequences are located _______ of V seg- 5. One other protein that is assembled similar to Ig gene is _______
ments, and ______ and _________ of D segments. in vertebrates.
3. _________ heavy chain produced by the rearranged gene on
one chromosome inhibits gene rearrangement on homologous
chromosome.
Gearhart, P. J. (1993). “Somatic Mutation And Affinity Maturation” in Lieber, M. (2000). “Antibody Diversity: A Link Between
W. E. Paul (ed.), Fundamental Immunology. New York: Raven Press. Switching and Hypermutation”, Current Biology, 10: R798–800.
Gearhart, P. J. (2002). “The Roots of Antibody Diversity”, Nature, Matsuder, F. and T. Honjo (1996). “Organization of the Heavy
419: 29–31. Chain Locus”, Advances in Immunology, 62: 1–29.
Gellert, M. (1997). “Recent Advances in Understanding V(D)J Schissel, M. (2002): “Allelic Exclusion of Immunoglobulin Gene
Recombination”, Advances in Immunology, 64: 39–64. Rearrangement And Expression: Why and How?” Seminars in
Immunology, 14: 207.
Jung, D., C. Giallourakis, R. Mostoslavsky, and F. W. Alt (2006).
“Mechanism and Control of V(D)J Recombination at the Wood, R. D., P. J. Gearhart and M. S. Neuberger (2001). “Hyper-
Immunoglobulin Heavy Chain Locus”, Annual Review of mutation in Antibody Genes”, Phiosophical Transaction of Royal
Immunology, 24: 541–70. Society, London, 356: 1–125.
Lieber, M. (1996). “Immunoglobulin Diversity: Rearranging by
Cutting and Repairing”, Current Biology, 6: 134–36.
George D. Snell, working in the Jackson Memorial Laboratory in 1935,
“invented” the congenic strain in mice. Two mice are congenic if they
are genetically identical at all genetic loci except one. Hence, any
phenotype or physiological or immunological differences observed
“Things don’t
between these two are related to the genetic region or locus that is dif-
turn up in this
ferent among them. world until
Syngeneic mice strains refer to inbred mice that are completely ho-
somebody turns
mozygous at every genetic loci; that is, any two syngeneic mice are like
them up.”
—J. A. GARFIELD
identical twins. Syngeneic strains are prepared in laboratory by repeti-
tive mating of siblings (brother–sister mating). After about 20 genera-
tions, every individual animal of a given inbred mouse strain will have
identical nucleic acid sequences at all locations on all chromosomes.
Such strains were developed by Clarence C. Little and his colleague,
and later established at the Jackson Memorial Laboratory at Bar Har-
bor, Maine in 1929. After studying this chapter
Snell discovered that tumour transplants (he was studying ways to • Define major histocompatibility
complex (MHC) and MHC
prevent cancers or tumours) on mice from their congenic cousins were molecules
immediately rejected. However, these transplants were successful in • Describe the structure and
function of class I and class II
MHC molecules
syngeneic strains (see Figure 6.1).
• Explain the differences
between peptide-binding
Snell quickly discovered that this single locus (on which congenic mice clefts of class I and class II MHC
molecules
differ) is related to the rejection of tumour grafts and he labelled that • Describe class III MHC
molecules
locus as H (for histocompatibility). As transplantation research con- • Explain the role of anchor
residues of antigenic peptide in
tinued, during the 1930s, it was found that the H locus (named H-2 in peptide–MHC interaction
mice) contained several different genes as well as polymorphism at • Describe and illustrate murine
class I and class II MHC loci
each locus. Peter Gover in England showed that antibodies are formed • Describe and illustrate human
class I and class II MHC loci
in mice when a tumour is rejected. The antigen against which antibodies
• Explain the codominant
expression of maternal and
are formed was named antigen II or antigen 2. The gene for the pro- paternal MHC molecules
duction of this antigen was found to be located in Snell’s H locus and
hence the term H-2 originated. This eventually defined the entire com-
plex of murine histocompatibility genes. Later, it was established that
antibody response was not a general feature of graft rejection; how-
ever, the name of the locus is still in currency.
Major Histocompatibility
Complex 6
6.1 INTRODUCTION
The major histocompatibility complex (MHC) refers to those genetic loci that code for antigens
Major histocompatibility
(MHC antigens) which determine whether transplanted tissue is compatible (Greek: Histo—tissue, complex or MHC
+ compatible) and is accepted or is histo-incompatible and is rejected. This refers to the genetic loci
The histocompatibility complex codes for histocompatibility antigens (synonymous with that impart uniqueness to an
individual. The MHC cluster of genes
transplantation antigens) that are mainly protein molecules (antigens) present on cell or tissue are spread over four megabase
surface that determine the compatibility or incompatibility of transplanted tissue. Those cell regions of the short arm of the
surface antigens that elicit the most rapid tissue graft acceptance or rejection are called major human chromosome 6 and on the
mouse chromosome 17. These
histocompatibility complex antigens and genetic loci that code for these antigens are referred to proteins code for MHC antigens.
as the major histocompatibility complex. MHC molecules are found only in
The MHC is a closely linked complex of genes that governs the production of the major his- vertebrates.
tocompatibility antigens. The MHC is termed as H-2 complex in mice and as HLA complex in « In 1965, Daussat identified HLA as
humans (see Figure 6.2). the H-2 equivalent loci in human
In humans, some histocompatibility genes are located on Y chromosomes, autosomes as beings.
well as on the mitochondrial genome that are also, though less frequently, the cause of graft rejec-
tion. These are referred to as minor histocompatibility antigens (mH). The mH proteins could
Minor histocompatibility
be normal household proteins, transcription factors and GTPase-activating proteins. The mH
antigens
Minor histocompatibility antigens
are proteins that are responsible
for graft rejection in those cases in
which MHC are compatibile. These
Tumour transplant
proteins are encoded by genes
outside the MHC locus. Examples of
mH genes include HA-1 and HB-1.
+
Syngeneic mouse
Genetically identical at
all loci Transplant successful
Transplant between syngeneic strains
Tumour transplant
Congenic mouse
Genetically identical at
all but one locus Transplant rejected
Transplant between congenic strains

Figure 6.1
Diagram of Snell’s experiment that led to
the name histocompatibility.
proteins are encoded by genes

Human located outside the MHC locus.
Chromosome 6 Peptides derived from the mH
antigens are presented by nor-
mal class I and II MHC mol-
ecules like any other peptides.
The MHC genes encode
three classes of molecules:
• Class I MHC molecules—
found on the surface of nearly
all nucleated cells; it is in-
DP DC DR B C A
volved in presenting foreign
epitopes to CD8+Tcyt cells.
• Class II MHC molecules—
» MHC proteins belong to the
immunoglobulin superfamily. Class II MHC locus Class III Class I MHC locus found on the cell surface
MHC locus of the cells of the immune
system, primarily on an-
tigen-presenting cells (for
Cell-membrane Complement Cell-membrane example, macrophages, den-
Figure 6.2
Schematic map of MHC regions proteins found on components, proteins found on dritic cells); they present
of human genome showing the cells of the hydroxylase all nucleated cells antigenic determinants to
classes I, II and III regions. immune system enzymes CD4+TH cells.
• Class III MHC molecules—comprise varied molecules including certain complement com-
ponents (C2, C4 and factor B of alternative pathway), tumour necrosis factors α and β
(TNF-α and TNF-β), some heat shock proteins and two hydroxylase enzymes; class III
MHC molecules do not participate in MHC graft rejection.
6.2 CLASS I MHC MOLECULES

» β2 microglobulin is neither poly- The following are the chief features of class I MHC molecules:
morphic (in humans) nor encoded
within the MHC loci. However, like • Class I MHC molecules are composed of two polypeptide chains α and β, held together by
MHC proteins, it is a member of non-covalent bonds (see Figure 6.3).
the immunoglobulin superfamily.
• The α chain is an MHC-encoded integral membrane glycoprotein of molecular weight 45,000 Da
(in humans) or 47,000 Da (in mice).
Membrane-spanning proteins
• The extracellular non-MHC coded chain of molecular mass~12 kDa (both in humans and
Also known as transmembrane mice) is called β2-microglobulin (β2-m). It is named for β2 which is its electrophoretic mo-
proteins, membrane-spanning bility, micro for small size and globulin because of its globular structure and solubility.
proteins refer to those integral
membrane proteins that traverse the
• The membrane-spanning α chain is approximately 350 amino acids in length. It contains
plasma membrane. These proteins three globular domains α1, α2 and α3, each containing about 90 amino acids. α1 is located at
have hydrophobic amino acids on the N terminal followed by α2 then α3. The α2 domain has an intra-chain disulphide bond
their surface which help them to
“dissolve” in the hydrophobic interior
of the lipid bilayer.
Peptide-binding groove
Peptide of
(63 amino (90 amino length 8-10
A2 A1
acids) acids) amino acids
(86 amino B 2 microglobulin

acids) A3 Anchor
CD8-binding A2
A1 residues
site A3
Hydrophobic stretch Plasma
Plasma membrane
(26 amino acid) membrane
Figure 6.3 Cytoplasmic tail
Schematic diagram of class I MHC and its
(30 amino acids) Cytosol
peptide-binding cleft.
MAJOR HISTOCOMPATIBILITY COMPLEX 121
forming a loop of 63 amino acids and α3 has a disulphide bond enclosing 86 amino acids.
Apart from these three domains, the α chain has a stretch of 26 hydrophobic amino acids
that anchor the α chain into the plasma membrane. This transmembrane segment is in the
form of α helices that pass through the hydrophobic region of the plasma membrane. The
carboxyl terminal of the α chain has about 30–40 amino acids that follow the hydrophobic
transmembrane segment and is located inside the cell (in cytosol) and is phosphorylated
in vivo.
• The α1 and α2 domains interact to form peptide-binding units of class I MHC molecule.
• The platform of peptide-binding unit is formed by four strands of β-pleated sheet and
one α helix of amino acid residues of α1 domain. Four strands of β-pleated sheet and one
α helix are contributed by the α2 domain. A groove of approximate size 25Å × 10Å × 11Å
is formed, whose floor is formed by β-pleated sheet and sides by α helices. As is evident « Mutant mice lacking β2-m do not
from the size of the peptide-binding cleft, it is too small to bind intact globular protein, but express class I MHC molecules,
implying that β2-m is essential for
is large enough to bind 8–10 amino acid residues of foreign epitopes. The small size of the the expression of class I molecules
cleft of MHC requires that native globular proteins be “processed” to smaller fragments on the cell surface.
(that is, form epitopes) which can bind MHC and be recognized by T cells.
• β2-m chain is of a single type (non-polymorphic) in humans and is of two types (dimor- « Class I MHC molecules present
phic) in mice with a single amino acid change at position 85. α3 and β2-m are structurally antigens to Tcyt cells. Class I mol-
ecules commonly present peptides
homologous to the structure of immunoglobulin C domain and contain immunoglobulin- derived from endogenously synthe-
like disulphide loop. From X-ray crystallography, it is predicted that α3 and β2-m domains sized proteins, such as viral proteins
are located at an angle under α1 and α2 domains. α3 and β2-m domains interact with each synthesized during infection.
other. A peptide-binding platform is formed by β-pleated sheets of α1 and α2 domains. These
interactions are essential for the stability of the molecule (and peptide-binding groove in
particular) and its proper folding. « Most amino acid substitutions are
• Tcyt cells show strong specificity for cells displaying peptides associated with class I MHC localized in the α1 and α2 helices
molecules. This is because the CD8 antigen present on the surface of Tcyt cells show a strong while the α3 helix remains relatively
conserved in class I MHC molecules.
affinity for the non-polymorphic α3 domain of class I MHC molecule.
• The class I MHC molecule is a glycoprotein. It contains one (human) or two (mouse) N-
linked oligosaccharide. The degree of glycosylation varies among different class I MHC
antigens depending on the species and haplotype.
• The class I MHC molecule can be cleaved by papain into two units—one comprising
α1, α2, α3 and β2-m units, and the other comprising the transmembrane domain with a short
cytoplasmic tail. The papain cleavage site is located between α3 and transmembrane α helix
regions of the class I MHC molecule.
6.3 CLASS II MHC MOLECULES

The following are the chief features of class II MHC molecules:
• All class II MHC molecules are heterodimers of two non-covalently associated polypep-
« Class II MHC molecules are found
tide chains (see Figure 6.4). The heavy chain (α) has a molecular weight of 30–34 kDa, on the surface of B cells, dendritic
while the β chain ranges from 26–29 kDa. cells and macrophages, as well as
• Like class I MHC molecule, class II MHC molecules have an extracellular amino terminal other specialized cells of the
immune system.
domain, a transmembrane domain and an intracellular carboxyl terminal tail. An extracellular
Peptide-binding
groove
Glycosylation 13-18 amino
SS Peptide-binding acid long
(90 amino acids) B1 A (90 amino peptide
SS groove
acids)
SS B1 A
(90 amino acids) B2 A (90 amino acids)
SS A
Extracellular space
Hydrophobic stretch Plasma CD4-binding Anchor
site Plasma membrane residues
(25 amino acids) membrane
Figure 6.4
Cytoplasmic tail Schematic diagram of class II MHC and
Cytosol
(10-15 amino acids) its peptide-binding cleft.
Feature Class I MHC Class II MHC

Constituting polypeptide chains α chain (45 KDa in humans) α chain (30–34 KDa)
β2 chain (12 KDa in humans) β chain (26–29 KDa)
Antigen-binding domains α1 and α2 domains α1 and β1 domains

Binds protein antigens of 8–10 amino acid residues 13-18 amino acid residues
Peptide binding cleft Floor formed by β sheets and Floor formed by β sheets and
sides by α helices, blocked at both sides by α helices, open at both
ends ends
Antigenic peptide motifs involved Anchor residues located at amino Anchor residues located almost
in binding and carbon terminal ends uniformly along the peptide
Presents antigenic peptide to CD8+ T cells CD4+ T cells
Region that binds T-cell receptor Non-polymorphic α3 domain Non-polymorphic β2 domain
Found on Nearly all nucleated cells Cells of immune system; primarily
Table 6.1 on antigen–presenting cells
Characteristic features of class I and
Involved in Cytotoxicity by CD8+ T cells T-cell mediated helper activity
class II MHC molecules.
» Class II MHC molecules have two region of two domains (α1, α2 or β1, β2) of about 90 amino acids each, are linked by
transmembrane polypeptides short connecting regions to a transmembrane region of 25 hydrophobic amino acid
whose outer extracellular ends fold
to form a peptide-binding groove. residues that span the membrane. In both the chains, hydrophobic transmembrane
Class II MHC molecule displays region ends with a cytoplasmic domain of about 10–15 hydrophilic amino
those antigens that are taken up acid residues.
from the surroundings.
• The peptide-binding region of class II molecule is formed by both chains α and β
involving α1 and β1 segments respectively. This is different from class I MHC molecules
in which only the α chain is involved in peptide binding. The peptide-binding region in
class II MHC is formed by α1 and β1 regions and comprises an eight-stranded, anti-parallel
β-pleated sheet platform supporting two α helices. Four strands of β-pleated sheets and
one of the helices are contributed by α1, and the other four strands of β-pleated sheet and
other helices are formed by the β1 segment.
• Both α2 and β2 domains possess the structural characteristic of immunoglobulin C domain
(such as class I MHC- α3 and β2-m domain) and belong to the immunoglobulin superfamily.
• Immunoglobulin-like regions are probably important for non-covalent interaction
between the two chains.
• Both α2 and β2 domains contain a disulphide bond. Apart from these two domains,
β1 domain contains a disulphide bond generating 64 amino acid loops.
• The CD4 molecules present on T cells bind to class II MHC molecules. Mutagenesis
studies suggest that CD4 molecules bind to the projecting loop of the non-polymorphic
β2 domain of class II molecules.
• Both the chains, α and β2 are n-glycosylated. α1, α2 and β1 domains are glycosylated but
β2 domain is not. The difference in molecular weights of class II α and β chains is
primarily because of the difference in glycosylation.
The characteristic features of class I and class II MHC molecules are listed in Table 6.1.
6.4 CLASS III MHC MOLECULES

The following are the chief features of class III MHC molecules:
• Class III MHC molecules include several serum proteases which are components of the
» Class III MHC molecules encode
complement cascade as well as two enzymes— steroid 21 hydroxylases (21-OHA and
proteins such as enzymes, cytokines
and heat shock proteins that are 21-OHB).
not involved in antigen presenta- • Unlike class I and class II MHC antigens, class III MHC molecules have no role in antigen
tion. In humans, the gene cluster presentation. Since these molecules are structurally and functionally different from class
of class III MHC molecules lie on
chromosome 6, between class I and I and class II MHC molecules, it is suggested they should be considered as MHC-linked
class II MHC molecules. genes which are “almost” involved in immune regulation.
MAJOR HISTOCOMPATIBILITY COMPLX 123
• The complement components include C2 (serine protease), C4A and C4B (pro-proteins
that are finally processed into multichain forms) and factor B (serine protease in the alter-
native complement pathway). They also include certain cytokines such as tumour necrosis
factors α and β (TNF-α and -β) and two heat shock proteins.
6.5 STRUCTURE OF PEPTIDE-BINDING

CLEFT
As discussed previously, the peptide-binding clefts of class I and II MHC molecules share some
similarities and a few differences. Their gross structure is the same. In both classes of MHC, the
peptide-binding cleft is formed by an eight-stranded β-pleated structure forming the floor of the
site and two α helices forming the sides of the cleft. In class I MHC molecules, the binding cleft is
formed by only the α chain (α1 and α2 domains), but in class II MHC molecules, both α and β chains
contribute. The same building protein motifs (β sheets and α helices) are arranged to make two dif-
ferent types of peptide-binding clefts, one in class I MHC molecules other in class II MHC molecules.
The peptide-binding cleft is closed on both ends in class I MHC molecules (see Figure 6.5), whereas
the cleft is open in class II MHC molecules (see Figure 6.6). Because of this difference, class I MHC mol-
ecules bind peptides of smaller length, typically 8–10 residues in length, while class II MHC mol-
ecules accommodate slightly longer peptides of about 13–18 amino acid residues (rarely peptides
up to 30 residues). Peptides that bind class I MHC and class II MHC molecules have characteristic « It was the pioneering work of
conserved features and so do the peptide-binding clefts to which these peptides are bound. Doherty and Zinkernagel that
The peptide-binding sites on MHC molecules have a variety of structural features such as indiacated that T cells recognize
antigens only when they are
pockets, intrusions, depression, clefts and ridges that help in binding and determining the speci- presented together with MHC.
ficity of the peptide. These ridges and pockets are actually spaces between the peptide backbone
of β-pleated strands that make the floor of the cleft. The presence or absence of such pockets is
determined by the amino acid sequence of the β-strands. When a pocket is formed, the amino acid
A 1 domain
A 1 domain
B sheets
A helices
COOH
Figure 6.5
The peptide-binding pocket of class I
MHC molecule. (Reprinted, with
NH2 A 2 domain
A 2 domain permission, from the Annual Review
of Biochemistry, Volume 59 ©1990 by
Schematic representation Ribbon diagram Annual Reviews www.annualreviews.org)
A 1 domain
A 1 domain
B sheets
Peptide-binding
groove A helices Figure 6.6
Class II peptide-binding pocket.
(Structure by Murthy and Stern. Copyright
1997 by Elsevier Science & Technology
Journals. Reproduced with permission of
B domain B domain Elsevier Science & Technology Journals
in the format Textbook via Copyright
Schematic representation Ribbon diagram Clearance Center.)
Figure 6.7
Peptide-binding groove of class I MHC
-HLA-A. (a) This shows the difference
between HLA-A2 and HLA-Aw68;
(b) The polymorphic residues in
HLA-A2 are highlighted, (Reprinted, with
permission, from the Annual Review of
Biochemistry, Volume 59 © 1990
by Annual Reviews. www.annual
reviews.org). (a) (b)
residues that line the pocket determine the nature of the peptide side chain that can be accommo-
dated there (for example, charged or hydrophobic). The conserved peptide residues that fit into the
Anchor residues pockets of the MHC peptide-binding clefts are termed as anchor residues, because these amino
The peptide residues that anchor acid residues “anchor” the peptide in the MHC molecule.
the antigenic peptide into the
MHC groove are termed as anchor The anchor residues are usually (but not always) located at the ends of the peptides. The inter-
residues. Changing a single anchor actions between the residues of peptide-binding cleft that bind anchor residues of the peptides are
residue may prevent it from not the sole basis of attachment to MHC molecules. Some contacts between the non-polymorphic
interacting with the MHC. Anchor
residues that bind a particular MHC amino acid residues (of α helices) of peptide-binding clefts and the peptide backbone of bound
need not be identical but are always peptides contribute to the stability (not specificity) of binding.
similar (such as aromatic or acidic). Amino acid variations within the groove can vary with the positions and number of pockets,
which in turn lead to differences in peptide-binding specificity and affinity, hence influencing the
immune response. This is best exemplified by comparing peptide- binding grooves of two allelic
forms of human MHC-HLA: HLA- A2 and HLA-Aw68 [see Figure 6.7(a)].
HLA-A2 and HLA-Aw68 differ from each other at 13 positions located primarily in the α-
chain. There were six difference in α1, six in α2 and one in α3 domains. These differences in amino
acids gives rise to differences in the binding site of an MHC molecule. The difference in binding
site implies that MHCs will bind different peptides.
For the sake of understanding, let us view the antigen binding cleft of HLA-A2 [see Figure
6.7(b)]. It consists of eight-stranded β-pleated structures forming the floor of the cleft, topped by
two α helices. Five residues on the β-strand (9, 95, 97, 114, 116) point up between two helical re-
gions and make contact with bound antigenic peptides. Six residues on α helices face (at positions
66, 70, 71, 77, 80 and 156) into the peptide-binding site and probably contribute to specificity of
the incoming peptide. Three residues are on the top face of helices make direct contact with the
TCR. The rest of the amino acids do not contribute to the binding of the antigenic peptide.
6.6 PEPTIDE–MHC INTERACTION

Each class I/II MHC molecule binds only one peptide at a time. However, peptide binding by class
I/II MHC molecule does not have the fine specificity exhibited by antibodies or T-cell receptors
for antigen binding. In other words, a given MHC molecule can bind different peptides. Moreover,
» Several thousand protein
molecules must be degraded to some peptides can bind more than one, different MHC (allelic variant) molecule. It should be made
generate a single MHC–antigenic clear that even though MHC molecules can bind different peptides (for example, A bound to MHC
peptide complex! and A′ bound to MHC), the unique T cell will recognize only one peptide–MHC complex (that is,
a T cell will recognize A–MHC but not A′–MHC).
Since the peptide-binding cleft of class I and class II MHC molecules are slightly different,
peptides that bind to these clefts and their way of interaction with them are also different. The peptide-
binding clefts in class I molecule are blocked at both ends by slightly curved α helices at the end of
the clefts. This limits the size of peptide that can bind or be accommodated within the cleft to be
8–10 amino acid residues while the open groove of class II MHC molecules allows peptides from
13–18 amino acids to accommodated easily (see Figure 6.8).
8-10 amino acid residue peptide 13-18 amino acid residue peptide
usually curved to make (not curved) C-terminal
contact with TCR Peptide-binding groove anchor residues
blocked at both ends (hydrophobic)
N-terminal N C
anchor
residue
C-terminal
(at position
anchor
2 or 3)
residues Middle anchor Peptide-
Peptide-binding cleft
(hydrophobic residue binding
residue) cleft (open at
Class I MHC Class II MHC
both ends) Figure 6.8
N-terminal anchor residues Anchor residues of class I and class II
(aromatic or hydrophobic) MHC binding region.
Peptide binding to class I MHC molecule requires the presence of characteristic residues at N and
C terminals of the peptide. These residues are called anchor residues. These anchor residues bind (an-
chor) the peptide into the pocket of the binding cleft, where they interact with amino acids of the bind-
ing cleft pockets. It is these amino acids (amino acids in the binding cleft) that vary in different alleles,
resulting in the binding of different peptides (Peptides eluted from H-2Dd, H-2Kd, H-2Kb are all 9 resi-
dues long). However, the N-terminal anchor residue in H-2Kd is tyrosine, while in H-2Dd it is glycine,
both located at 2nd position. In H-2Kb, anchor tyrosine is located at 3rd position from the N terminal.
In peptides that bind to class I MHC molecules, the anchor residues can be located at the carboxyl-
terminal, or amino-terminal, or at both ends. The conserved and more common anchor residues
are carboxyl anchor residues (located around residue 9) and usually comprise hydrophobic amino
acids (e.g. leucine, isoleucine etc). The anchor residues that are present at the amino terminal are
usually located at position 2 or 3. The anchor residues located at N and C terminals of the peptide
are usually buried inside the binding cleft, interacting electrostatically with counter-charges on the
MHC molecule. The middle of the peptide makes significant contact with the binding cleft of class
I MHC molecules, but arches away from the floor of the cleft. Since the middle of the peptide projects
« The interaction between MHC–
away from the floor of the binding cleft, peptides slightly longer can also be accommodated by bulg-
antigen complex and T-cell
ing away the peptide chain from the cleft. It is believed that amino acids that bulge away from MHC receptor is short-lived, usually for a
molecules are more exposed and therefore can interact directly with T-cell receptor. few seconds.
Unlike class I MHC molecules which bind and present peptides to CD8+ T cells, class II MHC
molecules are involved in binding and presenting peptides to CD4+ T cells. Since the binding
cleft of class II MHC molecules are not blocked at the ends, longer peptides can bind to class II
molecules as they can extend out of the ends of the groove. The bound peptides generally contain
13–18 amino acids.
The characteristic motif of anchor residues that are common in peptides bound to class I
MHC molecules is absent in class-II-MHC-bound peptides. Peptides that bind the class II MHC
molecules have about 7–10 amino acids that provide major contact points. It usually includes
aromatic or hydrophobic residues at amino terminal, three additional hydrophobic residues in the
middle portion and carboxyl-terminal end of the peptide. Peptides bound to class II MHC mol-
ecules maintain a roughly constant elevation on the floor of the binding cleft, that is, does not arch
away from the middle as in class I MHC–peptide binding. Moreover, hydrogen bonds between the
backbone of the peptide and class II MHC molecule are distributed throughout the binding site
rather than being clustered predominantly at the ends of the site as for class-I-bound peptides.
6.7 GENE MAP OF THE MAJOR

HISTOCOMPABILITY COMPLEX
Before understanding a simple picture of a MHC genomic, let us get familiarized with some fre-
quently used terms:
• Locus (plural, loci) refers to the position of a gene on a chromosome. Sometimes, this
term may also be used for chromosomal location of any characterized DNA sequence.
• Gene is the basic unit of heredity. A contiguous stretch of DNA which codes for a given
protein and/or RNA molecule.
• Allele refers to the variant or alternative form of a given gene, for example, yellow and
green peas carry different alleles of the gene determining pea colour.
• Polymorphism is the existence of a character in two or more variant forms in a population
and where the least common form is present in more than 1 per cent of individuals. A
genetic locus is considered to be polymorphic if the variant form (allele) is found in more
than 1 per cent of the population.
• Haplotype denotes the alleles that are inherited together on the same chromosome
(Petersdorf, 2007). Each diploid individual inherits one haplotype from the mother and
the other from the father (which are situated on homologous chromosome). In MHC,
alleles that are present on maternal and paternal haplotypes are codominant that is, both
maternal and paternal haplotypes are expressed in the same cell.
Certain inbred mouse strains are designated as prototypes. These are obtained by brother and sister
mating. The MHC haplotype expressed by these prototype strains is designated by arbitrary italic
superscript (H2a, H-2b, etc.). For example, H-2d is the “short” way of referring to the entire set of H-2
inherited alleles such as H-2Kd, H-2Dd, I-Aαd, I-Aβd and so on within a strain. There may be two (or
more) inbred strains having the same prototype; for example, AKR, C3H and B10 all have the same
» MHC genes span about 2,000 kb
of mouse DNA and about 3,500 kb MHC haplotype-K, that is, they are all H-2k. These strains can differ in genes outside the H-2 com-
of human DNA. plex. The entire MHC is located on chromosome 17 of mice and on chromosome 6 of humans.
6.7.1 MURINE MHC LOCI

The mouse MHC is referred to as H-2 locus (see Figure 6.9). The region encoding the class I and
class II genes are given letter designations (K, L, etc.).
CLASSICAL MURINE CLASS I LOCI

The class I region in mice is not a continuous stretch of bases. It exists as two separate regions. This
region is separated by intervening class II and class III regions.
Class I molecules (that is, the α chains of class I MHC, as the β chains are coded elsewhere)
are encoded in three separate classical (transplantation), serologically defined H-2 loci, that is, H-2K,
H-2D and H-2L. Between different strains of mice, MHC loci are different or polymorphic. They
differ in the number of genes, gene loci and their structure (DNA sequence).
The organization of the H-2K region is similar in all strains that have been studied. It contains
two class I genes termed as K and K2. The H-2K gene encodes the H-2K antigen. It is expressed
on most cell types and is recognized serologically. H-2K2 gene exhibits varied patterns of expression
depending on the strain. The number of class I genes at the other loci such as H-2D/H-2L region
varies among different haplotypes, for example, BALB/c (H-2d) mice have five class I MHC genes
in the D/L region while only one class I MHC gene has been identified in H-2D region of B10 mice.
There are about 30 class I MHC genes in the haploid genome of mice. The number of genes
varies among different haplotypes. Class I genes include classical or transplantation antigens such
as H-2 loci, that is, H-2K, H-2D and H-2L or non-classical antigens such as H-2Q (Qa), H-2T (Tla)
and H-2M region downstream from the H-2 complex (see Figure 6.10).
H-2 Tla
K IA Ie S D L Qa Tla M
Telomere
Centromere
Figure 6.9 Classical Classical Class III Classical Non-classical class I

Gene map of murine class I and class I class II MHC class I MHC
class II loci. MHC MHC molecule MHC
Class II MHC region Murine Chromosome 17
K D L Qa Tla M
1 2 1 2 n 1 2 n 1 7
Centromere Telomere
Figure 6.10
Genomic map showing sub-location of
Classical Non-classical class I MHC murine classical and non-classical
class I MHC class I regions.
NON-CLASSICAL MURINE CLASS I LOCI

« Non-classical murine class I MHC
H-2Q (Qa), H-2T (Tla) and H-2M regions encode molecules that have structures similar to class I molecules present specialized
MHC molecules. These are sometimes referred to as non-classical (non-transplantation) class I peptide fragments. Some of the
molecules and are located on chromosome 17. non-classical molecules can either
be secreted in the serum
The murine Tla region (initially defined as encoding Thymus Leukaemia) antigen is located (Q 10 molecules) or can be
distally to loci of Qa. It has been shown to contain the largest number of non-classical class I membrane-bound(Qa-2).
genes. The M region is located distally to the Tla region, and contains a number of genes termed as
M1–M7. These genes exhibit a low degree of polymorphism.
These non-classical MHC molecules function as specialized peptide receptors. For example,
non-classical MHC H2–M3 is especially equipped to present N-formylated peptides, while Qa-2
is specialized for presenting histidine-containing peptides and Qa-1 preferentially presents signal
sequences. Unlike other classical MHC products, these antigens are expressed only on some cell
types. Qa molecules are expressed on cells of heamatopoietic lineage, while Tla antigens are ex-
pressed on leukaemic cells and thymocytes. These non-classical MHC antigens exhibit transmem- « CD1 molecules are non-MHC
molecules that present lipids and
brane polypeptide of Mr-45,000 joined non-covalently to β2-m. These antigens exhibit the domain other bacterial antigens such as
structure and show structural similarity to classical MHC antigen. formylated peptides to T cells for
There are some molecules that are NOT coded by MHC but are still involved in antigen pre- scrutiny. In the human CD1 family,
there are five genes from CD1A to
sentation for, example, the family of molecules called CD1(found in both humans and mice) which CD1E. CD1 molecules are expressed
present lipid antigen derived from bacteria. on B cells, dendritic cells, activated
monocytes and Langerhans cells.
6.7.2 MURINE CLASS II MHC LOCI
There are two defined classical class II MHC molecules in mice—IA and IE, encoded by two sepa-
rate loci in the I region of the H-2 complex. Almost the whole H-2I region has been mapped and is
linked to the class I H-2K subregion.
The I region has several subregions such as Aa (written as Iaa), Ab (written as Iab). The Ab
and Aa genes encode β and α chains of the A molecule (see Figure 6.11). Similarly, Eb and Ea genes
encode β and α chains of the E molecule. The genes for α and β subunits of IA complex as well as
β subunit of IE complex (not shown in the figure) are located in the IA region of the H-2 complex.
The IE complex encodes Eα and Eβ subunit of the IE complex. Several other class II a and b genes
have been mapped, for which no protein product is known. Gene- Pb(not shown in Figure 6.11),
located in the I locus, is a pseudogene.
There are several α- and β-chain genes located in murine class II locus, that is, the I region.
All genes may or may not be expressed in different haplotypes. Mice with a particular haplotype
IA IE S
TNF TNF
β α β β2 α CYP C4 CYP C4 BF C2 hsp hsp
α α
Centromere Telomere Figure 6.11

Genomic map showing sub-location of
Class II MHC Class III MHC murine class II and class III MHC regions.
(such as b and s type) may fail to transcribe the Eα gene; that is, the Eα chain may not be formed at
all but this mice makes normal level of Eβ chain. Mice of f and q haplotypes fail to make both Eα
and Eβ chains. All the α-chains and β-chain genes can be expressed in the same cell. This increases
the number of different antigen-presenting molecules on the cell surface, as the α chain of one lo-
cus (say A) can combine with the β chain of a variety of other loci to create several types of class II
molecules. However, α chains associate in the cell primarily with β chains of their own loci, that is,
Aα, will tend to associate with Aβ rather than with Eβ which belongs to another locus, E.
» There is indirect evidence that Gene encoding non-classical class II MHC molecules have also been identified in mice. The
suggests the presence of MHC-like class II non-classical MHC molecules are encoded by genes Oa, Ob, Ma and Mb. These MHC mol-
molecules in an invertebrate
Botryllus schlosseri, a tunicate. ecules show limited polymorphism and different patterns of expression as compared to classical
class II MHC molecules.
6.7.3 I-GENE IN H-2 LOCUS

McDevitt and Sela in 1965 noted that when a synthetic antigen such as TGAL (copolymer of tyro-
sine, glutamic acid, alanine, lysine) and similar compounds were injected into random-bred guinea
pigs, some of them showed a good immune response while others were poor responders. In 1969,
McDevitt and Chinitz showed that genes determine the level of immune response, that is, whether
the animal is a poor or good immune responder. They called them Ir genes (immune response
genes). They later mapped the genes to a new region in the H-2 complex called I gene, located near
the K region. We now know that I genes are murine class II MHC genes.
6.8 HUMAN MHC LOCI

» In 1965, Dausset and his co-workers The human MHC loci is referred to as HLA loci (see Figure 6.12).
identified 10 human antigens located
on the surface of a leukocyte. They
termed these antigens human
6.8.1 HUMAN CLASS I MHC LOCI
leukocyte antigens (HLA). The human class I region contains three loci called HLA-A, HLA-B and HLA-C (see Figure 6.13).
Each locus encodes the heavy chain (α chain) of the classical class I MHC antigen. The whole loci
of class I MHC extends to about 2 million bases.
» The most common HLA type in The β2 chain is non-polymorphic and is encoded by a single gene located on a different chro-
Indians is HLA-A1/B17. mosome. Additional class I MHC genes have also been located in the class I region. There are
about 19 non-classical class I human genes including HLA-E, HLA-F, HLA-G, HLA-H, HLA-J
and HLA-X loci, as well as the family of genes called MIC (major immunogene complex).
HLA
Complement
components
Heat shock
proteins
Cytokines
DP DQ DR B C A
Centromere Telomere
Figure 6.12
Simplified genomic map of HLA. Class II MHC Class III MHC Class I MHC
CYP C4 CYP C4 Bf C2 hsp hsp B C A
Figure 6.13 Class III MHC Class I MHC

Genomic map showing human class I
and class III MHC regions. Centromere Telomere
The physiological functions of the classical HLA molecules (HLA-A, HLA-B and HLA-C) are « HLA-A and HLA-B genetic loci
to present peptides to T cells and to inhibit the activity of NK cells. were first identified by van Rood in
1963. After almost a decade,
In contrast, the functions of non-classical HLA molecules such as HLA-G, HLA-E, HLA-F the third locus, HLA-C, was
remain to be established. It is believed that HLA-G, which is predominantly expressed on placental identified.
trophoblast, might mediate protection of the foetus from assault by maternal NK cells. « The highest number of class I MHC
molecules are expressed on the
surface of lymphocytes.
6.8.2 HUMAN CLASS II MHC LOCI
The class II MHC molecules are coded by D subregion of HLA which spans about 1000 kb « Neurons never express class II
of DNA. Within the D region of class II loci, three loci, DR, DQ and DP, encode major expressed MHC molecules.
products of the human class II region (see Figure 6.14).
DR region: This region comprises a single α gene (DRA) and up to nine β genes (DRB1-9)
including pseudogenes. DRB1, DRB3 and DRB4 are usually expressed on the cell while DRB2 is a
pseudogene of class II molecule. The number of DRB loci varies with the haplotype. Even though
DRB loci contain up to nine β genes, all of them are not present on every haplotype. Moreover,
all loci that are present in a given haplotype may or may not be functional; for example, the DR1
group contains B1, B6 and B9 β-chain genes while the DR8 group has B1 and B9 β-chain gene.
DQ region: This region is constituted by one expressed α gene and one expressed β gene. It
also contains additional α-and β-chain genes which may or may not be functional. DQB3 is a pseu-
dogene, while DQB2 and DQA2 genes are usually functional.
DP region: The region comprises one expressed gene, each for α and β chains. As in the DQ
region, additional α- and β-chain genes may or may not be functional. DPA2 and DPB3 are gener-
ally pseudogenes while DPA1 and DPB1 are usually expressed.
In the human class II region, non-classical genes are designated as DM, DW and DO. While
DM helps in the binding of antigenic peptides to class II MHC molecules, DO is suggested to be a
regulator of class II antigen processing. Some important characteristics of classical and non classi-
cal MHC molecules are listed in Table 6.2.
The gene map of class II MHC region reveals the presence of two genes that are not expressed
on the cell surface—LMP2 and LMP7 that encode proteosomal subunits and two genes TAP1 and
TAP2 that encode peptide transporters. These four genes are found in both mice and humans. LMP2
and LMP7 are subunits of the proteosome, a protein-chopping machinery that mediates cytoplasmic
degradation of endogenous proteins into small peptides which can bind peptide transporters (TAP1
and TAP2). These peptides are then transferred to the newly synthesized class I MHC molecules.
DP DQ DR
β2 α2 β1 α1 β2 α2 β3 β1 α1 β β β β α
Figure 6.14
Centromere Telomere Genomic map showing sub-location of
Class II MHC human class II MHC regions.
Human Mouse
Classical HLA-A, HLA-B, HLA-C H-2K, H-2L, H-2D
Class I molecules
Classical HLA-DP, HLA-DQ, HLA-DR I-A, I-E
Class II molecules
Non-classical HLA-E, HLA-F, HLA-G, HLA-H H-2Q, H-2T, H-2M
Class I Molecules (about 19 genes)
Non-classical HLA-DM, HLA-DN, HLA-DO* Oa, Ob, Ma, Mb
Class II molecules
Class III molecules (collection of Complement components, steroid hydroxylases, heat shock proteins,
about 60 genes) cytokines
Table 6.2
*These non-classical molecules play a role in antigen processing and presentation. Classical and non-classical; MHC loci.
6.8.3 HUMAN CLASS III LOCI

The class III region of MHC in murine and human genome comprises a diverse collection of 57–60
genes. The gene products of most of the genes are still unknown. Some of the well characterized
genes of class III loci include genes for complement components 2, 4A, 4B, B, two steroid 21 hydroxy-
lases, two heat shock proteins and two cytokines (TNF-α and TNF-β).
6.8.4 WHY THE NAME HLA?

In 1950s, Jean Dausset of France found that antibodies were formed against some antigens present
on the surface of the human leukocyte when blood is transfused from one individual to another.
In 1965, Dausset and co-workers identified 10 human antigens, located on the leukocyte surface,
whose genes were located in region similar to H-2 region of mice, which they called HLA (hu-
man leukocyte antigen). Unfortunately, they considered that genes of both H-2 and HLA region
played a role only in tissue and blood transplantation. A few years later, Benacerraf and co-workers
demonstrated that many of the genes located within the MHC also control active immune response
to various antigenic stimuli.
6.9 M H C P O LY M O R P H I S M
» MHC polymorphism is different The hallmark of the MHC is its extreme diversity. This polymorphism (presence of multiple allele
from polymorphism observed for at a given genetic loci within a species) of MHC molecules stems from two sources.
antibodies or T-cell receptors.
Antibody/T-cell diversity is
generated by somatic recombina-
• There are a large number of alleles (that is, alternative forms) of each MHC locus. In hu-
tion while MHC diversity resides man, HLA molecule, there are approximately 60 HLA-A alleles, 110 HLA-B alleles and 40
in germ line DNA, for example, HLA-C alleles. In mice, class I locus H-2K has 55 alleles and 60 alleles are known for the
the existence of a large number of
alleles.
D locus. Similar is the case with class II MHC molecules in both humans and mice.
• Some of the genes within a locus may exist as several copies. For example, β-chain genes
in HLA-DR region may vary from one to nine, though two to five are usually expressed.
Thus, diversity of MHC molecule in an individual results not only from having different alleles
of each gene but also from the presence of several copies of one gene at same locus.
The alleles differ in their DNA sequences from one individual to another by 5–10 per cent.
Hence, their gene products (that is, MHC molecules) have differences in amino acid sequence
and composition. The number of amino-acid differences between MHC alleles can be as high as
20. Different amino acid residues impart a unique nature to each allele (product). The amino acid
sequence variability in class I antigens (both human and mice) is clustered in three main regions
of the α1 and α2 domains.
The non-classical class I-like antigens in mice (H-2Q, H-2T and H-2M) are much less poly-
morphic than classical class I antigens.
In human class II molecules, most variability occurs in the β chain of HLA-DR and HLA-DQ
» The α3 domain of class I MHC in regions, while in HLA-DP, the β chains are less polymorphic. Similar is the case of α chain. The α
both human and mice appears to chain encoded by HLA-DQ region is polymorphic whereas HLA-DR encoded α chains are virtu-
be more or less conserved.
ally invariant.
Most of the variations in amino acids in class I and class II MHC molecules are clustered
in and around the peptide-binding cleft (that is, within α1/α2 domains in class I MHC and α1/β1
domain in class II MHC molecules). The variation is almost always centred in the floor of the
antigen-binding cleft or pointing in from the sides of the α helix region. A number of researchers
have suggested that such differences in the class I or II molecule expressed by antigen-presenting
cells may influence the cells’ ability to recognize peptides.
6.10 DISTRIBUTION OF CLASS I AND

CLASS II MHC MOLECULES
In general, class I molecules are present on virtually all nucleated cells and platelets but the number
of molecules vary among different cell types. The highest number of class I MHC molecules are
expressed on the surface of lymphocytes (approximately 1 per cent of the total cell membrane
protein). On the other hand, neural cells, muscle cells and hepatocytes have a very low number of
class I MHC molecules.
The class II MHC molecules are expressed by antigen-presenting cells such as macrophages, B
cells and dendritic cells. Macrophages express only low levels of class II molecules until stimulated
by γ-interferon. The expression of class II MHC molecules by endothelial cells is antagonized by α
and β interferons. Most cell types express class II MHC molecules when exposed to high levels of
« MHC genes are inherited as a
γ-interferons. Neurons are class II MHC negative cells. haplotype, one from each parent.
The transcription and expression of class I genes are coordinately regulated. So is the coordi- A heterozygous individual will carry,
nation of class II genes. However, the transcription of class I and class II MHC molecules may be and can potentially express, one
paternal set and one maternal set of
independently regulated inside the same cell. β2-microglobulin is regulated with class I α chain MHC proteins. However not all cells
even though it is not located on the same chromosome. will express both class I and class II
As noted previously, several different types of class I MHC molecules are expressed on the MHC molecules.
surface of a cell. Let us see how this is made possible. Suppose parent mice have Ka, Da, La and
Kb, Db, Lb alleles, one set from each parent. In F1 generation, the mouse cell will express both the
alleles since MHC alleles are codominant. This F1 generation will have Ka, Kb, Da, Db, La and Lb
alleles. So the cell will express six different types of class I MHC molecules (Figure 6.15a) while
each homozygous parent will have only three types of MHC molecules. Since class II MHC mol-
ecule is composed of two different polypeptide chains, a heterozygous individual expresses not
only parental class II molecule (that is, MHC molecules from both the parent) but also molecules
containing α and β chains from different chromosomes. For example, H-2k mice express I-Ak
(containing genes for α and β chains) and IEk (containing genes for α and β chains). Similarly
H-2d mice, express IAd and IEd molecules (containing both α and β genes). In F1 progeny, the
α chain of Ak sublocus can combine with the β chain of Ak locus or the β chain of Ek locus. It
can even combine with the β chain of Ad or Ed locus. Thus four types of α β chain combination
can occur and four types of class II molecules can be formed, by keeping the αk chain common
[see Figure 6.15(b)]. Similarly another combination can be generated using αk of E subloci. Hence
a large number of class II molecules can be generated due to the presence of multiple β chains in
mice and humans, and multiple α chains in humans. The presence of multiple α- and β-chain genes
increases the polymorphism of antigen-presenting molecules (MHC) on the cell. The diversity
generated by these mechanisms increases the number of different antigenic peptides that can be
presented and thus is advantageous to the organisms.
Ka Da La Kb Db Lb IAA k IAB k IEA k IEB k IAA d IAB d EA d EB d
Ka Da La Ka Da La AA k AB k EA k EB k AA dAB d EAdEB d
Maternal Paternal
class I MHC class I MHC IAA k IEB k IEA k IEB k IAA d IEBd IEA d IAB d
IAA k IAB k IEA k IEBk IAAdIAB d IEA d IEBd

Ka Da La
F1 generation
Kb Db Lb
IAA k IEBk IEA k IABk IAAdIEBdIEA dIAB d
Co-dominantly
expressed Codominant expression of
class I MHC molecules class II MHC molecules
Figure 6.15
Codominant expression of class I and
a) b) class II MHC molecules.
6.11 T R A N S C R I P T I O N A L R E G U L AT I O N
OF MHC MOLECULES
As noted previously, differential expression of MHC genes occur. Class I MHC molecules are ex-
pressed on all cell types but their expression varies among different cells. Class II MHC molecules
are expressed only in a limited number of cell types.
6.11.1 CLASS I MHC: CONSTITUTIVE EXPRESSION

In most cells, class I MHC genes are constitutively transcribed. The DNA segment that is required
Enhancer for constitutive transcription are enhancer A and enhancer B, which are located 5' upstream to the
An enhancer is a short sequence transcriptional start. In a cell at resting stage, that is, in unstimulated cell, enhancer A is occupied
of DNA that bind proteins (called
activators) and enhances the
by a homodimer of p50 (NFκβ-1) subunits. An unknown protein binds at enhancer B. The binding
basal level of gene transcription of of these proteins on enhancers A and B allows the basal transcription of class I MHC genes to occur
occurring at some distance from the and its product to be expressed on the cell surface.
enhancer.
6.11.2 CLASS I MHC: CYTOKINE-INDUCED EXPRESSION
The expression of MHC molecules is also increased or affected by various cytokines. The pres-
ence of interferons (α, β, γ) and tumour necrosis factor increase the rate of expression of class I MHC
molecules.
When cells are exposed to TNF, the p50 homodimer is replaced by p50/p65 (NFκβ-1/ReIA)
heterodimer. The binding of this heterodimer at enhancer A upregulates gene expression of class I
molecules (see Figure 6.16).
However, when cells are treated with interferon (α, β, γ), a slightly different route of gene
activation is followed. Interferons (γ in particular), induce the formation of a protein-
Cis-acting elements transcription factor called interferon response factor (IRF-I). The IRF binds to a consensus
These are genetic elements or DNA sequence upstream to class I MHC genes. The binding of this transcription factor to the consensus
sequences that affect only the DNA sequence upregulates the class I MHC genes.
molecule in which they occur. The
converse of cis-acting element is a
trans-acting element. A trans-acting
6.11.3 CLASS II MHC EXPRESSION
element codes for a product that can Much less is known about the regulation of class II MHC molecules.
diffuse across the cytoplasm and act The promoters of many class II genes have several cis-acting conserved DNA sequences. These
on neighbouring DNA molecules.
include (in the order of 5' to 3'):
• seven nucleotides—S box
(or H box)
A B • pyrimidine-rich,
Class I MHC 15 nucleotides—X1 box
p50/p50 gene • eight nucleotides—X2 box.
(Two nucleotides of X2
Enhancer ? Promoter Transcription
overlap with X1.)
mRNA • ten nucleotides—Y box
Constitutive expression of class I MHC gene The S, X1, X2 and Y boxes

with appropriate alignment are all
that is required for class II molecule
synthesis. These sequences bind
Enhanced stimulation protein complexes which in turn
activate class II genes. The X1 box
is occupied by a protein complex
A B called regulatory factor X (RFX),
Class I MHC the X2 box is occupied by a bind-
p50/p65 gene ing protein (X2BP) while the Y box
Enhancer Promoter Enhanced
binds protein complex called as nu-
transcription clear factor-Y (NF-Y). Proteins that
Figure 6.16 bind the S box are not completely
Diagram showing action by enhancers mRNA
A and B on transcriptional regulation of
characterized. The binding of these
class I molecules. Cytokine-induced enhanced class I MHC gene expression protein complexes is cooperative
and requires all three complexes to be present in the cell for any of these proteins to bind efficiently. Once
these proteins have bound their respective boxes, class II genes are actively expressed. The γ interferon has
been shown to induce expression of class II genes. In fact, the γ interferon induces the production of a pro-
tein called class II transcription activator (CIITA). CIITA binds to the assembled RFX, X2BP, NF-Y complex
and then activates the basal transcription machinery. Class II genes are hence upregulated.
Affinity Chromatography
OH NH2 OH
OH Chemical cross-linking
+
NH2
NH2 Cross-linking mediated by
OH cyanogen bromide OH
Affinity ligand
Affinity matrix formed
Preparation of Affinity Matrix
OH OH
Specific ligand binds,
+
Impurities are washed away
OH OH
Mixture of proteins
Affinity matrix Matrix-ligand complex formed

Binding of Ligand
Competing ligand
HO HO OH
Elution Elution
+
Addition of Change of pH
HO competing ligands HO OH
Matrix-ligand complex
Figure 6.15
Elution of Ligand from the Matrix.
Affinity chromatography is a chromatographic method used for sepa- the macromolecules of interest. The desired macromolecule binds
rating biological macromolecules based on their highly specific bio- to the ligand and is retained on the matrix. Non-specifically-bound
recognition. The method exploits the specific interaction that is present impurities are washed out by the excess buffer. The bound ligands
between the desired macromolecule and its physiological ligand, such can be eluted by a change of pH of buffer (see Figure 6.17) since the
as the interaction between antibody and antigen, enzyme and sub- interaction between the macromolecule and ligand is largely ionic.
strate or substrate analogue, enzyme and coenzymes (such as NAD+), The elution of the macromolecule can also be achieved by the addi-
and receptor and hormone. This chromatographic technique involves tion of biospecific competing ligand (usually ligand analogue). How-
the preparation of an affinity matrix first. An affinity matrix is prepared ever, if the macromolecule fails to elute under the above conditions,
by attaching the ligand or its analogue to an inert matrix such as increase in ionic strength of the buffer or the addition of chaotropic
sephadex or sepharose. This can be achieved by chemically cross-link- agents such as urea or guanidium-hydrochloride is recommended.
ing the ligand to the matrix, by a bifunctional agent such as glutaral- Affinity chromatography can be used for isolating proteins and
dehyde, or attaching the ligand to cyanogen-bromide-activated ma- enzymes from biological samples, purifying antibodies and even
trix (such as agarose). Once the affinity matrix has been prepared, it is antibody isotypes from the serum, purifying hormones or hormone
washed to remove unbound ligands (or ligand analogues). This matrix receptors, and metal-ion-containing proteins etc. Affinity chromatog-
is then incubated with a mixture of biological molecules containing raphy provides at least 10-fold purification of the desired molecule.
HLA Typing HLA typing (serological) can be easily carried out by comple-
ment- dependent microlymphocytotoxicity test (MLCT). In this test,
A large number of alleles code for MHC antigen in humans. The lymphocytes from a potential donor are coated onto the wells of a
testing process by which we can determine which particular MHC microtiter plate. Human polyclonal antisera or monoclonal antibod-
antigen (MHC in humans is called HLA, human leukocyte antigen) ies of predetermined specificity are then added. These antibodies will
variant is present in an individual is called HLA typing. Once the type react with specific HLA antigen variant (if present) on the donor cell.
of polymorphic HLA variant(s) present on the donor cell is deter- Complement proteins (usually of rabbit) is then added to the reaction
mined, HLA antigens between donor and recipient are matched. system. These complement proteins will lyse the cells that had been
Such “matching” is carried out prior to organ/tissue/cell transplan- coated with anti-HLA antibodies. Such lysed cells are detected using
tation from one individual to another. The greater the similarity colour stains such as eosin or trypan blue which permeate the dead
between HLA antigen of donor and recipient, the higher are the cells. The MLCT test confirms the presence (or absence) of various
chances of survival of transplanted tissue. MHC antigen variants (that is, various MHC alleles) on the donor cell.
S U M M A R Y
• The major histocompatibility complex (MHC) refers to genetic loci The binding groove is constituted by α1 and α2 domains of class I
that codes for antigen which determines whether transplanted tis- MHC and α1 and β1 domains of class II MHC molecules. Exog-
sue is compatible and will be accepted, or histo-incompatible and enous antigens are endocytosed, degraded, and presented in as-
will be rejected. sociation with class II MHC molecules while endogenous antigens
(for example, proteins from infecting viruses) are processed and
• MHC molecules are actually involved in the presentation of anti- presented with class I MHC molecules.
genic fragments to T cells for the appropriate immune response.
• Every individual is endowed with a distinct array of MHC proteins.
• MHC is referred to as H-2 loci in mice and HLA in humans. This enormous diversity stems from the fact that every individual
• MHC codes for three subsets of molecules. Class I MHC pro- has a distinct and different set of class I and II MHC alleles.
tein consists of large glycoprotein α chain (having α1, α2 and α3 • Some common classical class I gene in mice include H2K, H2D
domains) and a smaller β2 microglobulin chain. MHC class II pro- and H2L. Non-classical class I genes in mice are H2D, H-2T and
teins are composed of two non-covalently associated α (having α1 H-2M. Unlike classical class I MHC molecules, non-classical class I
and α2 domains) and β (having β1 and β2 domains) chains. The third MHC products are not expressed on all cells. Murine classical class
class of MHC (class III MHC) proteins are not involved in antigen II genes are encoded in two separate loci—IA and IE. Non-classical
presentation and include some complement components, enzymes, class II MHC molecules are encoded by Oa, Ob, Ma and Mb loci.
tumour necrosis factors and heat shock proteins.
• Classical human class I MHC loci contain HLA-A, HLA-B and
• Class I MHC molecules are expressed on all nucleated cells and HLA-C. Non-classical class I MHC genes in mice include HLA-E,
+
present antigen to cytotoxic CD8 T cells. Class II MHC molecules HLA-F, HLA-G and others. Classical class II MHC loci include
are expressed on immune cells and are involved in presenting HLA-DR, HLA-DQ and HLA-DP.
antigens to TH (CD4+) cells.
• The expression of class I and class II MHC molecules is transcrip-
• The outer regions of class I and II MHC molecules contain tionally regulated and can be increased/changed by the action of
peptide-binding grooves that bind and present antigenic peptides. various cytokines.
K E Y W O R D S
• anchor residues 124 • gene conversion 132 • invariant chain 130 • non-classical MHC 127
• β2-microglobulin 120 • haplotype 121 • I gene 126 • peptide-binding cleft 121
• class I MHC 120 • histocompatibility 119 • major histocompatibility • peptide–MHC
• class II MHC 121 • HLA 119 complex 119 interaction 118
• class III MHC 122 • human class I MHC 128 • MHC restriction
• endogenous antigen 121 • human class II MHC 129 • murine class I MHC 126
• exogenous antigen 122 • H-2 complex 119 • murine class II MHC 127
1. Why do you think nature evolved two MHC “plates” for presenting successful strategy in generating alternative enzymes or ligand
antigens to T-cells? Does it offer any advantage to the individual binding proteins? Comment.
other than having one MHC that can present to all types of 4. Why is MHC needed on almost all cells? Can an MHC knockout
T cells? animal survive?
2. Which region in the whole MHC molecule shows the highest ami- 5. What role do anchor residues play in peptide binding to class I
no acid variation? Why is it needed? MHC molecules? How is peptide binding to class II MHC different
3. Amino acid variation is a very useful tool in generating a repertoire from class I MHC? Which one makes more contact with the cor-
of antigen-binding proteins. Do you think it will be an equally responding TCR?

1. MHC plays important role in all, except: 6. Peptide-binding cleft or groove of class II MHC molecules is
a. Tissue transplantation formed by:
b. Blood transfusion a. α1 and α2 domains
c. Antigen presentation b. α1 and β1 domains
d. T-cell response c. β1 and β2 domains
d. β2-microglobulin and α1 domain
2. Class II MHC is involved in antigen presentation to:
a. Cytotoxic T cell 7. Non-MHC molecules involved in antigen presentation is/are:
b. TH cell a. H2Q and H2T
c. B cell b. HLA-H
d. NK cell c. DR product of HLA
d. CD1
3. β2-microglobulin is a part of:
a. Class I MHC 8. Non-MHC genes located within the MHC code for:
b. Class II MHC a. Tumour necrosis factor
c. Class III MHC b. Components of complement
d. All of the above c. Proteosomal subunits
d. β2-microglobulin
4. Human MHC gene products show all of the following
characteristics, except: 9. H-2 loci include all, except:
a. Diverse cellular distribution a. Class I MHC
b. Heat shock proteins b. Class II MHC
c. Involved in tissue acceptance or rejection during c. TAP1
transplantation d. β2-microglobulin
d. Contain β2-microglobulin in classes I, II, III MHC
10. Peptide-binding cleft is larger and open at the ends in:
5. During interaction with T cells, MHC molecules will bind all a. class I MHC molecules
except: b. class II MHC molecules
a. Antigenic peptide c. class III MHC molecules
b. T-cell receptor d. antibody molecule
c. Proteosome
d. CD8/CD4 molecule
State true or false against each statement. If false, give reasons.

1. Class I MHC molecules are present on all types of cells. 4. β2 domain of class II MHC molecule is not glycosylated.
2. All classes, that is, classes I, II and III are involved in antigen 5. Characteristic anchor residues are located at n and c terminals of
presentation. peptides presented on class II MHC.
3. Both class I and class II MHC molecules have two disulphide
bonds in α chain.
Benaceraf, B. (1981). “The Role of MHC Gene Products in Petersdorf, E. W., M. Malkki, T. A. Gooley, P. J. Martin and
Immune Regulation”, Science 212: 1229–38. Z. Gou (2007). “MHC Haplotype Matching for Unrelated Haema-
topoietic Cell Transplantation,” PLoS Medicine, 4(1): e8
Diehl, M., C. Munz, W. Keilholz, S. Stevanovic, N. Holmes,
Y. W. Loke, and H. G. Rammensee (1996). “Non-classical HLA-G Rammensee, H. G., K. Falk and O. Rotzchke (1993). “Peptides
Molecules Are Classical Peptide Presenters”, Current Biology, Naturally Presented by Class I MHC Molecules”, Annual Review
6: 305–14. of Immunology, 11: 213–44.
Karltunen, J. T., J. Trowsdale, and P. J. Lehner (1999). “Antigen Reizis, B., M. Eisenstein, F. Mor, and I. R. Cohen (1998). “The
Presentation: TAP Dances with ATP”, Current Biology, 9: R820–24. Peptide Binding Strategy of the MHC Class I I-A Molecules”,
Immunology Today, 19: 212–16.
Lee, J. and J. Trowsdale (1983). “Molecular Biology of the Major
Histocompatibility Complex”, Nature, 304: 214–15. Steinmetz, M. (1986). “The Major Histocompatibility Complex:
Organization and Evolution”, Clinical Immunology News, 7: 134–37.
Madden, D. R. (1995). “The Three-Dimensional Structure of
Peptide–MHC Complexes”, Annual Review of Immunology, Stern, L. J. and D. C. Wiley (1994). “Antigenic Peptide Binding by Class
3: 587–622. I And Class II Histocompatibility Proteins”, Structure, 2: 245–51.
Palmer, E. G., M. J. Bevan and K. F. Lindahl (1994). “Do Non- Wang, J. H. and E. L. Reinherz (2002). “Structural Basis of T-Cell
Classical Class I MHC Molecules Present Bacterial Antigens to Recognition of Peptides Bound to MHC Molecules”, Molecular
T-Cells”, Trends in Microbiology, 1: 35–38. Immunology, 38: 1039.
“To attain
knowledge,
add things
every day, to
attain wisdom,
In 1974, R. M. Zinkernagel and P. C. Doherty proved that while B cells
remove things
see antigen alone, antigen recognition by T cells is specific not only for everyday.”
antigens but also for MHC molecules. In other words, T cells —LAO TZU
interact with antigen only in the presence of MHC molecules. Later it

was proved that T cells “recognize” antigen only when presented on
the membrane of a cell by self-MHC molecules. The T cell does not
recognize antigen alone, it recognizes antigen only when it is
associated with (self)MHC. This attribute, called self-MHC restriction, After reading this chapter you
should be able to:
distinguishes the recognition of antigen by T cells from that by B cells
• Describe dual receptor and
(see Figure 7.1). In 1975, studies conducted by Kornberg and altered-self model of TCR
co-workers clearly demonstrated that T cells do not contain mRNA for • Explain the structure of TCR
• Describe the organization of
immunoglobulin chains and made it amply clear that a completely mouse α, β, γ, δ gene loci in
germ line DNA
different system, which does not involve immunoglobulins, governs • Give an account of the
organization of human α, β, γ, δ
T-cell specificity. genes loci in human germ line
DNA
• Explain the rearrangement of
TCR α and β genes at DNA and
RNA levels
• Explain the allelic exclusion of
TCR gene
• Briefly summarize how such
enormous T-cell diversity is
generated
• Describe TCR complex, CD3
protein and CD4 and CD8
accessory molecules
• Give a detailed description of
ternary antigen–MHC–T-cell
receptor complex
T-cell Receptors
7
7.1 INTRODUCTION
The search for T-cell receptors started way back in 1976, when J. J. Marchalonis and R. E. Cone
claimed that a T-cell receptor is a monomeric IgM. The inability to a isolate T-cell receptor (TCR)
by classical protein purification techniques gave rise to more speculations. Marrack and Kappler
(1986) pointed out in their review of antigen-specific T-cell receptors, that early attempts to isolate
TCR relied on the fact that TCR might be similar, if not identical to immunoglobulin. However,
immunofluorescence with anti-immunoglobulin antisera failed to demonstrate the presence of
antibody-like molecules on the T-cell surface.
The three main observations pointed that TCR is not an immunoglobulin.
• Parish (in 1971), and Schirrmacher and Wigzell (in 1972) showed that B-cell receptors « B-cell receptor is a membrane-
(that is, antibodies) and TCRs do not recognize the same determinant on a given antigen. bound antibody. Membrane-bound
antibody together with Ig-α(CD79a)
• Hoffman and Kappler (in 1972) demonstrated that even when TCR and B cells are specific and Ig-β(CD79b) is referred to as
for the same antigen, they showed different cross-reaction patterns with other antigens. the B-cell receptor complex.
• Finally it was shown by Benacerraf and co-workers in 1973 that genes associated with the
major histocompatibility complex affected T-cell function and not B cells.
Antigen
No direct
recognition
Antigen of antigen by
Antibody T-cell receptor
Antigen
T cell
B cell
T-cell receptor
MHC not needed for recognition
Antigen
by B cells and antibodies MHC
Antigen-presenting
cell
T cell recognizes antigen when

presented together with MHC
(MHC-restricted) Figure 7.1
T cell is MHC-restricted. It responds only
when the antigen is presented together
with MHC molecules.
All the above observations suggested that TCR is a distinct antigen-recognition system, inde-
pendent of the B-cell receptors or antibodies.
From the studies of Zinkernagel and Doherthy (1974), it became clear that TCRs recognize
antigen associated with MHC and that they do not appreciably interact with either component
alone. This led to the proposal of two models for T-cell recognition. These two models were,
(a) dual receptor model and (b) altered-self model.
Dual receptor model predicts that TCR has two receptors. One receptor recognizes the antigen
present on the cell surface and the other recognizes MHC molecules located at some distance. Thus,
antigen and MHC molecules are recognized separately and that too by two separate receptors.
Altered-self model suggests that antigen binds the MHC molecule and is located at almost
one place. The TCR recognizes this antigen ⴙ MHC complex which is actually altered MHC com-
plex (self-MHC altered by binding of antigen). Altered-self model suggests that the T cell has
only one receptor and that is designed to recognize both the components—antigen and self-MHC
molecules. Figure 7.2 highlights the differences between dual receptor and altered-self model of a
T-cell receptor.
Before going further, let us discuss the specificity of TCR. If a TCR recognizes an antigen asso-
ciated with a particular MHC molecule (say, H-2k), it will recognize only this combination (antigen
A-H-2k). Other combinations (say, antigen B-H2k) that have a different antigen bound to the same
MHC molecule will not be recognized by this T cell. Similarly the same antigen bound to different
MHC molecule (say H-2d) will also not be recognized by that TCR (say, antigen A-H-2d).
The debate between the two models was clarified by experiments of J. Kappler and P. Marrack.
They prepared two sets of T-cell population. One set of T cells recognized antigen ovalbumin when
it was associated with H-2k class II MHC molecules. The other set of TCRs recognized the antigen
keyhole limpet haemocyanin in association with H-2f class II MHC molecules. They fused the two
cells to produce a hybrid cell that produced a receptor for both types of antigen–MHC complex. If
the dual receptor model is correct, the hybrid cell should have separate receptors for ovalbumin-
» Keyhole limpet haemocyanin is an H-2k, keyhole limpet haemocyanin and H-2f. When hybridoma cells were presented with either
extremely large metalloprotein of ovalbumin on H-2f or keyhole limpet haemocyanin on H-2k, the hybrid cell did not recognize any
molecular weight 6–8 million Da.
It is present in the haemolymph of combination and did not respond. This suggested that the dual receptor model was incorrect.
the giant keyhole limpet Mega- However, the hybrid cells responded immediately to ovalbumin–H-2k complex and keyhole
thura crenulata, a mollusk. It is an limpet haemocyanin–H-2f complex. This suggested that the receptors on T cells are not separate
excellent carrier molecule used for
coupling to haptens for generating for antigens and MHC molecules; in fact they recognized the antigen–MHC complex and these
antibodies. receptors for antigen–MHC complex are the actual receptors present on hybrid cells. This finding
supported the altered-self model.
Antigen-presenting
Antigen cell
MHC
Antigen
MHC receptor T cell recognizes
Antigen receptor
T-cell receptor MHC which is altered
by binding of the
T cell antigen
T cell
Figure 7.2
Schematic diagrams showing the
difference between dual receptor and
altered receptor model of T-cell binding. Dual Receptor Model Altered Receptor Model
T-CELL RECEPTORS 139
7.2 STRUCTURE OF T-CELL RECEPTOR

TCRs were first identified using monoclonal antibodies. T-cell clones were screened using mono-
clonal antibodies raised against them.
Monoclonal antibodies raised against T-cell clones (called clonotypic antibodies) bound only Monoclonal antibodies
one T-cell line (clone of cells exhibiting the same TCR) but not others, and that binding could These are antibodies directed
against a single antigenic
specifically inhibit antigen recognition by that clone of T cells or specifically activate them. This determinant produced by a single
approach of identifying TCR was based on the assumption that since the TCR is specific for both clone of cells. The novel studies
an antigen and MHC molecule, there should be significant structural differences in the receptor of Milstein and Kohler in 1975
led to the development of the
from clone to clone. So each T-cell clone must have unique antigenicity that could be character- technique for producing monoclonal
ized using monoclonal antibodies. This approach was adopted by J. P. Allison in 1982, and TCR antibodies with defined specificities.
was identified.
Subsequent studies by other researchers using clonotypic antibodies revealed that TCR is a « Normally T cells develop in the
thymus. In nude mice (mice that
heterodimer of two polypeptide chains, α and β [see Figure 7.3(a)]. Later, a second type of TCR was lack thymus and hair!), T cells
identified, consisting of δ and γ chains. There are at least three different subtypes or isoforms of γδ develop very slowly and reach
T cells. These isoforms differ from each other in the size of γ chain and/or presence of disulphide only up to 10 per cent of normal
numbers.
bond between γ and δ chains [see Figures 7.3 (b), (c) and (d)].
7.2.1 T - C E L L R E C E P T O R S A R E R E L AT E D
TO IMMUNOGLOBULINS
Amino acid sequencing of the αβ and γδ chains as well as studies of cloned cDNA-encoding recep-
tor chains has revealed that both the chains of the TCRs (αβ or γδ) have an amino terminal variable
(V) region with homology to immunoglobulin variable (V) domain, and a constant (C) region with
homology to constant region of immunoglobulin C domain. Hence αβ TCR and γδ TCR are clas- « TCR is a member of the immuno-
sified as members of the immunoglobulin superfamily. globulin superfamily.
The α and β chains of the αβ-TCR, and γ and δ chains of γδTCR have a variable region of
102–119 amino acids and constant region of 83–113 amino acids, a transmembrane region of
18–24 residues and a small cytoplasmic tail of 4–12 amino acids. The membrane-spanning heli-
cal region is unique in that it contains positively charged amino acids (1 or 2) in α, β, γ and δ
chains. These amino acids could be lysine (β chain) or a lysine and arginine (α chain). The structure
of each variable and constant region is similar to the immunoglobulin domain characterized by
multi-stranded anti-parallel β sheets and intra-chain disulphide loop that span 60–75 amino acids.
α β
102-109 γ δ γ δ γ δ
s s
amino V V Variable
acids s s region s s Variable s s s s Variable
V V V V V V
s s region s s s s region
s s Constant
C C
amino s s s s region s s s s Constant
Constant C C C C C C
acids region s s Extra s s Extra s s region
constant constant
Carbohydrate s-s region region
residue
s-s
18-24
amino Transmembrane
acids region
Transmembrane Cytoplasmic Transmembrane
4-12 Cytoplasmic tail
amino region region
tail
acids
αβ 4#2 γ 1 subtype γ 2 subtype γ 3 subtype
A B C D
Figure 7.3
(a) Schematic diagram of TCR of αβ cell; (b), (c) and (d) Schematic diagram of TCRs of the three subtypes of γδ cells—γ1, γ2 and γ3 respectively.
Each chain has cysteine located at the small hinge region (outer side) proximal to transmembrane
segment that forms an inter-chain disulphide bond, that is, between α-β or γ-δ chains. The close
similarities of TCR chains to the heavy and light immunoglobulin chains first enabled researchers
to see structural resemblance of TCR to an Fab fragment of immunoglobulin.
The variable domain having an antigen-binding site is constituted by both α and β (or γ and
δ chains) chains. There are three hypervariable regions in the variable domain of each α chain and
β chain. These hypervariable regions are involved in antigen binding and appear to be equivalent
to the complementary determining regions (CDR) in the immunoglobulin light and heavy chains.
» The fourth hypervariability region The fourth hypervariability region (found in β chain) does not make a contact with antigen and
of a β chain is not a CDR since it hence is not equivalent to a CDR.
does not make contact with
antigen. It is believed to be involved The domain found in Cα is not exactly like an immunoglobulin domain. One half of the domain
in interaction with superantigens. has β sheets similar to that found in immunoglobulin-like domain while the other half is formed of
loosely packed strands and a short segment of α-helices. The intramolecular disulphide bond, which
in immunoglobulin-like domain joins two β strands, in Cα domain joins the β strand to the segment
of α helices. In TCRs, the interaction between Cα and Cβ domains is mediated by carbohydrate
residues. The sugar moieties in the Cα domain make extensive hydrogen bonds to the Cβ domain. A
» γδ T-cell receptors are present on brief summary of comparison between immunoglobulins and TCRs is given in Table 7.1.
less than 5 per cent of T cells. TCR of αβ type is found on 95 per cent of T-receptor bearing lymphocytes. It is synthesized
later in T-cell development than γδ. It becomes apparent on the cell surface at 15–17 days after
» The immune system, which can gestation in mice. By birth, it is the predominant form of receptor present on T cells. T cells with
virtually recognize a limitless αβ receptors are divided into several subtypes depending on their functions, which in turn are
number of antigens, has all the
gene segments (of TCR and determined by the interaction with different types of cells involved in the immune response. TCR
antibodies) localized in around αβ receptor is responsible for the TH function in humoral immunity and for the Tcyt function in
500 gene segments or cell-mediated immunity.
approximately in only 1 per cent of
the genome. Tcyt cells possess the capacity to lyse an infected target cell. TH cells (also expressing the αβ
receptor) assist Tcyt cells in mediating target-cell killing or assist B cell-mediated antigen–antibody
interaction. The γδ receptor is found on less than 5 per cent of T lymphocytes. It is expressed on a
» γδ T cells do not have CD8 or CD4 small subset of αβ-negative peripheral T cells and immature thymocytes. It is synthesized only at
antigens on their surface. These
cells develop earlier than αβ T
an early stage of T-cell development. In mice, it is the only receptor detectable before 15th day of
cells in the thymus and move to gestation but is virtually lost by birth at day 20. Since γδ T cells are predominant in various epithe-
epithelia (skin, lungs, intestines) lial cells (in mice and chicken), it suggested that these γδ T cells recognize frequently encountered
where they reside for most of their
lifetime. They do not circulate in
antigens at epithelial boundaries between the host and external environment and initiate immune
blood, like conventional T cells. response prior to more specific αβ T cells.
T-cell Receptor Immunoglobulin

Constituents α/β or γ/δ chains Heavy and light chains
Number of immunoglobulin α chain—one variable and one Heavy chain-1 variable domain,
domains constant domain 3–4 constant domains
β chain—one variable and one Light chain-1 variable domain,
constant domain 1 constant domain
Number of CDRs Three each in α and β chains Three each in heavy and light
chains
Molecules associated with signal CD3 and ζ chain Ig α and Ig β
transduction
Multiple germ line V, D, J ⫹ ⫹
segments
Junctional diversity ⫹ ⫹
Isotype switching — ⫹
Table 7.1 Somatic hypermutation — ⫹
Comparison of TCR and immunoglobulin
molecules. Production of secreted form — ⫹
The function of γδ T cell is not well characterized. It is believed that γδ TCRs expressed on « Knockout mice that lack γδ T
cells demonstrate slow healing of
αβ-negative T cell, may be specialized to bind certain kinds of ligands such as mycobacterial lipid wounds and are more susceptible
antigens, non-protein ligands such as phosphocarbohydrate as well as specialized protein antigen to cancer.
such as heat shock proteins. It is believed that these cells may bind to peptides or other antigens
presented on non-classical MHC-like molecules or they may even bind to free antigens in the same
way as immunoglobulin.
The γδ molecules are expressed on αβTCR- negative cells. Like α and β chains, γ and δ chains « γδ T lymphocytes have been
are transmembrane glycoproteins. Both γ and δ chains include extracellular V and C regions, short reported to occur in all mammalian
species.
hinge region, hydrophobic transmembrane segment (containing positively charged amino acid)
and short cytoplasmic tails. The hinge region contains interchain disulphide linkages. In humans,
γδ heterodimer may or may not be disulphide-linked.
7.2.2 G LY C O S Y L AT I O N O F T C R C H A I N S
The TCR chains are glycoproteins. The number of attached glycosyl chains attached to the subunit « High-mannose type and complex
of TCR and the extent of glycosylation are given below. type are two important types of
glycosyl side chains attached to the
• Human TCR α chain has 5 N-linked complex oligosaccharide side chains. Human protein. Proteins are glycosylated in
endoplasmic reticulum and the
TCR β chain has 2 carbohydrate chains. One of the carbohydrate side chains in the trimming of these chains starts in
β chain is of high-mannose type and other is N-linked carbohydrate moiety of a the endoplasmic reticulum and may
complex type. continue in the golgi apparatus
where proteins are finally packed
• In mice, TCR α chain contains four N-linked oligosaccharides of the complex type. The and dispatched to the final
TCR β contains 2–3 carbohydrate moieties of the high mannose type. destination.
7.3 O R G A N I Z AT I O N O F T - C E L L R E C E P T O R
GENES IN THE GERM LINE
Functional TCR α and β chain genes as well as γ- and δ- chain genes are expressed as polypeptides
only in cells of T-lymphocyte lineage. The functional TCR genes of α, β or γ and δ are formed by
somatic rearrangement of germ line DNA sequences, a process that is similar to immunoglobulin Somatic rearrangements
Somatic rearrangements are the
gene rearrangement. recombination events occurring in
The α chain can be compared to a light chain of immunoglobulin as it is coded by V, J and C somatic cells. Gene rearrangements
segments. The β chain can be compared to a heavy chain as it is coded, by V, D, J and C gene seg- that occurs during antibody chain
synthesis or TCR synthesis that occur
ments. Similarly the γ chain is encoded by the V and J and C segments while the δ chain is encoded in somatic cells are referred to as
by V, D, J and C segments. somatic rearrangements.
7.3.1 MOUSE TCR -GENE LOCUS

The chief features of the mouse TCR α-gene locus are as follows.
• It is located on chromosome 14.
• There are about 100 Vα gene segments located 5′ upstream of 50–100 Jα segments. There is
single Cα genes of four exon located 3′ downstream of Jα segments.
7.3.2 MOUSE TCR -GENE LOCUS

The chief features of the mouse TCR β-gene locus are as follows.
• Its organization is slightly complex. There are about 20–30 Vβ gene segments. Dβ, Jβ and Cβ
segments are arranged in two groups, B1 and B2.
• The B1 segment comprises a single Dβ1 segment. Downstream of this segment is located 6
Jβ1 segments, followed by a single Cβ1 segment.
• B2 segment comprises a single Dβ2 segment followed by 6 Jβ2 gene segments. Downstream
of 6 Jβ2 gene segments is a single Cβ2 gene segment. The germ line organization of murine
α and β is shown in Figure 7.4.
6A 1 6A 100 D -chain locus *A1 *A100 CA
5’ 3’
Murine TCR A -gene locus
B1 group B2 group
VB1 VBn DB1 JB1 CB1 DB 2 JB 2 CB 2 VB14

Figure 7.4
Germ line gene arrangement of mouse
5 3’
αβ TCR genes. The α-chain locus is
located on chromosome 14 while the (n=30) (n=6) (n=6)
β-chain locus is located on
chromosome 6. Murine TCR B -gene locus
7.3.3 HUMAN TCR α-GENE LOCUS

The chief features of the human TCR α-gene locus are as follows.
» In mice and humans, TCR locus
sequences of the α chain show • There are about 40 Vα gene segments located 5′ of about 75–100 Jα gene segment. There is
about 70 per cent similarity. only one Cα gene segment that has four exons of which the last one is entirely non-coding.
7.3.4 HUMAN TCR -GENE LOCUS

The chief features of the human TCR β-gene locus are as follows.
• There are about 57 Vβ gene segments. D, J and C segments are present in two sets B1 and
B2 as in mouse TCR β-gene locus.
• The B1 segment comprises a single Dβ1 segment. Downstream of this region is located the
6 Jβ1 segment followed by a single Cβ1 segment. The B2 segment has a single Dβ2 segment,
6 Jβ2 segments and a single Cβ2 segment. The whole B2 unit is located downstream of B1
unit. The Vβ segments are shared between B1 and B2 gene segments. The germ-line gene
organization of human α and β chain loci is given in Figure 7.5.
7.3.5 MOUSE TCR γ-GENE LOCUS

The chief features of the mouse TCR γ-gene locus are as follows.
• There are seven Vγ gene segments. There are four sets of JγCγ gene segments. One set of
Vγ and one set of JγCγ gene segments is non-functional. Here Vγ gene segments are not
segregated and located at the 5′ end. The Vγ gene segments are interspersed with four JγCγ
clusters. The functional Cγ gene segments have differences in sequence length and the
number of exons encoding the hinge region among different Cγ gene segments. Multiple
C segments found in the TCR α and β loci are almost identical.
VA1 VA 40 D -chain locus JA1 JA 100 CA
5’ 3’
Human TCR A -gene locus
B1 group B2 group
VB1 VB n DB 1 JB1 CB1 DB 2 JB 2 CB 2
5 ’
3’
Figure 7.5 n=75) n=6) n=6)

Germ line gene arrangement of human
αβ TCR genes. Human TCR B -gene locus
VD1 VD10 DD1 DD 2 JD1 JD 2 CD

5 ’
’
Murine D -chain locus
Pseudogene
vG vG vG vG JG CG vG JG CG CG JG vG vG JG CG
’
3’
Figure 7.6
Murine G -chain locus Gene arrangement of mouse γδ genes.
7.3.6 MOUSE TCR δ-GENE LOCUS

The chief features of the mouse TCR δ-gene locus are as follows.
• The δ-chain gene locus is located on chromosome 14 between Vα and Jα segments.
• It consists of about ten Vδ gene segments, two Dδ and two Jδ segments and a single Cδ seg-
ment. The gene arrangements of murine γ and δ gene are shown in Figure 7.6.
7.3.7 HUMAN TCR γ-GENE LOCUS

The chief features of the human TCR γ-gene locus are as follows.
• It is located on chromosome 7 in humans.
Pseudogenes
• There are 14 Vγ gene segments (including six non-functional pseudogenes). There are five Pseudogenes are gene-like
Jγ gene segments and two Cγ gene segments. They are arranged as Vγ –3Jγ –Cγ– 2Jγ – sequences of DNA that are non-
Cγ –2JC clusters. functional. They are regarded as
defunct evolutionary relatives of
normal functional genes. The name
7.3.8 HUMAN TCR δ-GENE LOCUS pseudogene was coined by C. Jacq
in 1977.
The chief features of the human TCR δ-gene locus are as follows.
• The δ-chain gene locus is located on chromosome 14 between Vα and Jα segments.
• There are about three Vδ gene segments and three, Dδ and three Jδ segments. It contains
only one Cδ gene segment. The gene arrangements of human γ and δ genes are shown in
Figure 7.7.
The location and number of V,D,J gene segments of α, β, γ and δ on human chromosomes are
shown in Table 7.2.
JG JG
VG VG n CG CG
5
’
3’
(n=14; includes n=3) n=2)

6 pseudogenes)
Human G -chain locus
VD 1 VD 2 VD 3 DD 1 DD 2 DD 3 JD 1 JD 2 JD 3 CD
5' 3’
Figure 7.7
Human D -chain locus Gene arrangement of human γδ genes.
Gene Chromosome Gene Segment

V D J C
α 14 40 75–100 1
β 7 57 2 13 2
γ* 7 14 • 5 2
δ 1 4 3 3 3 1
Table 7.2
*D, J, C segments of β chain genes are present in two sets. There are 6 non-functional pseudogenes. Human TCR gene segments.
7.4 REARRANGEMENT OF GENES TO FORM

M AT U R E α A N D β G E N E S
As mentioned previously, the α chain, like the immunoglobulin light chain, is encoded by V, J and
C segments, while the β chain, like the immunoglobulin heavy chain, is encoded by V, D, J and C
gene segments. The rearrangement of TCR α and β chain genes occurs, as the T cell matures, which
results in the VJ joining for the α chain and VDJ joining for the β chain. The TCR α chain has a
single C gene segment while β the chain DNA has two C gene segments. Either of the C gene seg-
ments can join the VDJ complex to generate a mature β chain gene. The two β chains differ only by
a few amino acids and have no functional differences.
7.4.1 G E N E R E A R R A N G E M E N T T O F O R M M AT U R E
TCR GENE
Genes for TCRs are arranged in a non-functional state which is characterized by spatial separation
of V, D, J and C segments in pre-T cells. During the maturation of T cells, V, D, J and C gene seg-
ments are rearranged in an orderly manner to form functional TCR β genes.
The following are the chief features of the rearrangement of genes at the DNA level.
• As in immunoglobulin gene, V, D and J have a flanking recognition signal sequence.
• The recognition sequences for TCR gene rearrangements include a conserved heptamer
and nonamer separated by either a 12-base pair (one turn) or 23-base pair (two turns) non-
conserved spacer sequence.
• However, the location of signal sequences (heptamer, nonamer and spacer sequences)
flanking V, D, J gene segments is such that it generates the usual V, D, J joining and some-
times the unusual V–J joining in the β chain. As a result, all the β chains may not always
contain the D sequence. Somatic rearrangements of TCR V, (D) and J gene segments are
mediated by RAG-1 and RAG-2 recombinases. RAG-1 or RAG-2 recognizes the flanking
heptamer and nonamer signal sequences and catalyse the gene rearrangement. The recog-
nition sequences are essentially the same in the immunoglobulin and TCR and the same
» Both RAG-1 and RAG-2 are recombination mechanism operates in both types of rearrangements. RAG-1/-2 introduces
essential for antibody and T-cell a nick on one DNA strand between the coding and signal sequences. The attack of free 3′-
gene rearrangement. Knockout
mice that lack either RAG-1 or RAG-2 OH on the complementary strand produces a hairpin at the ends of the coding sequence
do not undergo antibody or T-cell and a free 5′ flush phosphorylated double-strand break at the signal sequence. The double-
gene rearrangement. stranded breaks are repaired by the DNA ligase IV and the double-strand repair enzymes,
which join the V–D–J gene segments.
• The β-chain genes are rearranged prior to α-chain genes. The functional, rearranged β chain
gene is needed for signalling the subsequent maturation of T cells.
• First, one Dβ segment joins one Jβ segment then Vβ adds to the Dβ Jβ complex forming the
VβDβJβ rearranged gene. The Vβ and Dβ segments located between the rearranged Vβ and Dβ
gene segments are deleted and so are extra the Jβ located 5′ to the rearranged Jβ gene when
VβDβ joins the Jβ gene. The rearranged gene with the VβDβJβ complex, along with Jβ and Cβ
segments present on the 3′ side, is transcribed to give a primary RNA transcript for the
TCR β chain. The β chain promoters have been identified in the 5′ flanking regions the of
V genes.
S P L I C I N G A N D M AT U R AT I O N O F P R I M A R Y T R A N S C R I P T
The splicing and maturation of the primary transcript is as follows.
• The primary nuclear transcript of the β chain of the TCR contains the rearranged VβDβJβ
gene complex followed by the Jβ segments (if any) on the 3′ side, intron and Cβ genes.
The genomic sequences between the VDJ complex and the Cβ gene are then spliced out
to form mature mRNA having VDJCβ segments. The rearrangement uses the Cβ1 gene
segment; if a non-productive gene rearrangement occurs, a subsequent rearrangement
involving Cβ2 can occur. The use of Cβ1 or C β2 is completely random and there are no reports « T cells cannot switch from one
constant region to another.
that a T cell can ever switch from one C gene to other.
• A functional β chain is formed prior to the formation of an α chain. The newly formed
β chain pairs with a molecule called pre-T α chain (pTα); pTαβ heterodimers are
expressed on the surface of the thymocytes in association with CD3 proteins. The pTαβ
receptor (pre-TCR) is unable to recognize and bind any antigen (antigen–MHC) but
is supposedly involved in initiating intracellular events which lead to β chain allelic Allelic exclusion
exclusion (discussed later).The gene arrangements and RNA processing that occur during The expression of only one allele at
the synthesis of a human β chain is shown in Figure 7.8. a particular locus in a single cell is
called allelic exclusion.
7.4.2 REARRANGEMENT OF α-CHAIN GENES

The rearrangement of α-chain genes occurs as follows.
• One of the consequences of the expression of pre-TCR is that it signals the cells to start
dividing or proliferating. Once this proliferative phase of pTαβ cells is over, α-chain gene
rearrangement starts.
• Once α-chain gene rearrangement starts, it proceeds for four days. It is not clear whether
pre-TCR signal contributes to gene rearrangements at the α-chain locus. The α-chain gene
rearrangements consists of the joining of one Vα gene segment and one Jα gene segment. All
the Vα and Jα segments between the rearranged Vα and Jα segments are deleted. The pres-
ence of a large number of Jα segments allows several attempts to produce a productive VαJα
segment, thereby increasing the chances that a functional α chain is formed.
• Once the VJ joining has occurred, α chain genes are transcribed.
• The primary RNA transcript contains VJ segment ⴙ unrearranged Jα segments ⴙ intron ⴙ
one Cα segment. Since there is only one Cα gene, the RNA processing of a primary tran-
script gives rise to only one possible complete α chain mRNA, which is translated to give a
mature α chain. These α and β chains of the TCR which are translated and transferred to the
Vβ 1 Vβ n Dβ 1 Jβ 1 Jβη C β1
Unrearranged
germ-line DNA
DNA rearrangement
D-J joining
Vβ 1 Vβ n D β 1 Jβ 1 Jβη Cβ 1
Rearranged
T-cell DNA
DNA rearrangement
V-D-J joining
Vβ 1 D β1 Jβ 1 Jβη Cβ 1
Rearranged
T-cell DNA
Transcription
Vβ 1 D β 1 Jβ 1 Jβη Cβ 1
Primary RNA
Transcript
RNA splicing
V-D-J-C joining
Vβ 1 Dβ 1 Jβ 1 Cβ 1 Mature mRNA
Translation
β -chain Figure 7.8

NH2 Vβ 1 Dβ 1 Jβ 1 Cβ 1 COOH Gene rearrangement of human β gene of
polypeptide
DNA and RNA level.
Vα1 Vα 40 Jα1 Jα100 Cα
Unrearranged
germ-line DNA
DNA rearrangement
V-J joining
Vα1 Jα1 Jα2 Jα100 Cα
Rearranged
T-cell DNA
Transcription
Vα1 Jα1 Jα2 Jα100 Cα
Primary RNA
transcript
RNA splicing
V-J-C joining
Vα1 Jα1 Cα
Mature mRNA
Translation
Vα1 Jα1 Cα
Figure 7.9
Gene rearrangement of human α gene of NH2 COOH α -chain polypeptide
DNA and RNA level.
endoplasmic reticulum are packaged and sent to the cell surface plasma membrane where
the α-β heterodimer is expressed in membrane-bound form. Unlike immunoglobulin,
which can be in the membrane-bound or secreted form, there is no differential processing
of the RNA transcript to produce membrane-bound and secreted forms. Only membrane-
bound TCRs are produced. The gene arrangements and RNA processing that occur during
synthesis of human α chain are shown in Figure 7.9.
7.5 REARRANGEMENT OF γ AND δ GENES

γ and δ genes are rearranged by the same mechanism as is used for immunoglobulin and TCR
α- and β-chain genes. They employ same recombinases and same recognition signals as is used in
αβ TCR or immunoglobulin gene rearrangements. Since δ gene locus lie between Vα and Jα gene
segments, rearrangements of α locus that lead to joining of VαJα gene segments, also leads to the
deletion of the δ locus.
7.6 ALLELIC EXCLUSION OF TCR GENES

» Pre- TCR is unable to bind
antigen! When pre-TCR is expressed on thymocyte membrane, it generates several intracellular signals.
One of the signals is that since the productive rearrangement of one β-chain allele has occurred
(that is why β is expressed with pTα), the rearrangement of the other β allele is inhibited. This
results in β-chain allelic exclusion, that is, each mature T cell expresses identical copies of one β
chain on all αβTCR. If both alleles of the β-chain locus are non-productively rearranged, the de-
veloping T cell dies.
There is no evidence that the production of an α-chain protein suppresses further rearrange-
ments of the α-chain locus. It is found that only one type of α-chain genes are rearranged because
recombinases RAG-1/RAG-2 are expressed for a short time only when α-chain gene rearrange-
ment starts. Usually by the time the α chain at one allele has been rearranged, the RAG-1/RAG-2
production is shut off (in mice) causing a halt in further α-locus rearrangement. Under some « About 30 per cent of the mature
peripheral T cells express two types
circumstances, productive rearrangements occur on both the chromosomes (alleles) causing of TCRs, each type with the same β
expression of two α chains and hence two types αβTCRs. However in such cells, only one type of chain but a different α chain.
αβ TCR is likely to be functional. If no productive rearrangement of the α chain occurs on either Researchers believe that only
one of the two types of αβ-TCR is
chromosome (allele) the thymocyte dies. functional.
7.7 INHIBITION OF IMMUNOGLOBULIN

GENE REARRANGEMENT IN T CELLS
We know that the genomes of all the cells, including B and T cells, contain genes for both immu-
noglobulins and TCR. However, the immunoglobulin genes are not normally rearranged in T cells
and the TCR genes are not rearranged in B cells. The rearrangement of both the immunoglobulin
and TCR genes is mediated by recombinases RAG-1/RAG-2 and signal sequences. Both of the
requirements are found in both B and T cells, yet the rearrangment of immunoglobulin genes is
shut off in T cells and the rearrangement of TCR genes is turned off in B cells.
It was detected by Hitachi Sakano and co-workers that the enhancer of κ chains can serve
two opposite purposes in B and T cells. The κ-chain enhancer E2 located 3′ of Cκ, regulates and
enhances Vκ to Jκ joining in B cells.
In T cells, the same enhancer binds a particular protein (not yet identified) present only in T
cells. This protein enhancer complex shuts off the joining of Vκ and Jκ genes in T cells. A similar
mechanism may operate and shut off the rearrangement of λ chain and heavy chain genes in T cells.
7.8 G E N E R AT I O N O F S T R U C T U R A L T - C E L L
RECEPTOR DIVERSITY
The TCR repertoire ranges from 1010 to 1015 different types. This enormously large diversity (which
is greater than that of antibodies) is generated by the following mechanism.
7.8.1 P R E S E N C E O F M U LT I P L E G E R M - L I N E V, D , A N D J
SEGMENTS
The variable-region gene segments generate diversity by random gene combination for all TCR
chains. There are few V genes in TCR α and β loci but this is compensated by the presence of a
large number of J segments. There are four Jλ genes for the λ chain (of immunoglobulin) in mouse
germ line, while the TCR gene segments contains 50–100 Jα segments. During the TCR gene rear-
rangement, any one of the different V,(D) and J gene segments can combine to generate diversity,
for example, ~100 Vα and 100 Jλ can generate 104 different possible combinations for the TCR α
chain. Thirty Vβ can combine with 12 Jβ and two Dβ segments to generate 720 (30 × 12 × 2) possible
combinations or 720 different β chains.
7.8.2 JUNCTIONAL DIVERSITY

Junctional diversity is the diversity of different nucleotide sequences generated at the V–J (in an α
chain) junction or the V–D and D–J junctions (in a β chain). So the TCR having the same V and
J sequences can have different nucleotide sequences (at the junctions) which results in different
amino acid sequences (at the junctions). These diverse nucleotide sequences at the junctions can be
generated by: (a) imprecise recombination of coding (V, (D), J) sequences, (b) P and N nucleotide
addition at the junctions and (c) joining of multiple D segments.
7.8.3 I M P R E C I S E R E C O M B I N AT I O N
Usually when V–J or V–D–J joining occurs, the 3′ last nucleotide of V joins with 5′ first nucleotide
of J or D gene segments. Sometimes the joining is not so precise; for example, more than one 3′
nucleotide of a Vα gene (that is, the last nucleotide or the second last and so on) can join the first
few nucleotides on J segments (that is, the first 5′ nucleotide or the second 5′ nucleotide). In the
β chain, apart from V–D diversity that can be generated in the same way, additional diversity can
be generated by the imprecise recombination of D–J junction; for example, more than one
nucleotide at 5′ nucleotides at the end of the J segment can join the last or second last (and so on)
nucleotide of the D segment.
7.8.4 P-AND N-NUCLEOTIDE ADDITION

p nucleotides (p for palindromic sequence) sequence addition occurs in all TCR genes and fol-
» The final step of recombination is lows the same mechanism as that which is found in immunoglobulin genes. Briefly, the hairpin
the joining of two coding ends. This
is mediated by several enzymes turn that is formed at the ends of the coding sequence is cleaved to generate a single-strand DNA
and factors, the most important of and both the joining ends (that is, of V and J or V and D or D and J). Nucleotides are filled or added
which is (Ku subunits of) the in these single-stranded DNA which results in the generation of palindromic sequence at V–J or
DNA-dependent protein kinase.
V–D or D–J junctions. These p nucleotide sequences can generate additional diversity depending
» N-addition occurs in both α- and on the place at which the nucleotide region is cut.
β-chain genes. The random addition of nucleotides that are not coded by the germ-line DNA sequence oc-
curs at VD, DJ and VJ junctions. The addition of these n nucleotides (non-template nucleotides),
» It is estimated that the combined
effects of P- and N-region nucleotide
catalysed by terminal deoxynucleotidyl transferase enzyme, generates additional diversity.
addition and junctional diversity N-region nucleotide addition occurs in both α and β chain genes (in contrast, in the immuno-
can generate as many as 1013 globulin gene n-addition occurs only in the heavy-chain genes and not in the light-chain genes).
possible amino acid sequences in
the TCR junctional regions alone.
As many as six nucleotides can be added at the junction by this mechanism at each junction
(as in an immunoglobulin). Some of the rearrangements or additions can generate non-productive
» The joining of multiple DD rearrangements because of the generation of an in-frame stop codon. The stop codon prematurely
segments occurs only in the TCR terminates TCR chains.
genes and not in the
immunoglobulin genes. Additional diversity can be
J1 created in the β chain by joining
multiple Dβ segments (in δ chain).
V J
This is an unusual phenomenon
V1 J2
V J that is not found in immunoglob-
V J ulin genes. Moreover, it has been
V2 J3 found that the D segment can be
V J
translated in all three possible
reading frames, an uncommon
V? J4
feature in immunoglobulin heavy-
Imprecise recombination chain genes.
J5 at V,(D),J sequence
7.8.5 PAIRING OF
AND β CHAINS
Multiple V, (D), J segments
Since the binding site of TCR is
V formed by the variable region of
V D both α- and β-chain genes, the pair-
D ing of α and β chains generates an
P/N addition
J J additional diversity. As noted previ-
Joining of multiple
ously, there could be 104 types of Vα
P and/or N addition D segments chains and 750 types of β chains. So
the number of possible VαVβ com-
binations is 750 × 104 = 7.5 × 106. If
» Theoretically, the number of β1 we include the diversity generated
potential antigenic determinants by p- and n-nucleotide–addition as
that an individual may encounter
during his or her lifetime is 1017. well as by the imprecise recombina-
β2
α1 tion and the joining of multiple D
segments (in δ-gene segments), the
β3 potential size of an immature T-cell
α2
repertoire may be around 1015 or
even more different specifications.
α3 β4 This is far more than that gen-
erated by the rearrangement of im-
β5 munoglobulin genes. Figure 7.10
Figure 7.10
The different mechanisms for generating
shows some of the mechanisms of
T-cell diversity. Presence of multiple α and β chains generating T-cell diversity.
7.9 CO M P L E M E N TA R I T Y D E T E R M I N I N G
REGIONS (CDR)OF T-CELL RECEPTOR
There is a big difference in the way that a TCR and an immunoglobulin see an antigen. Immu-
noglobulins recognize almost an infinite variety of different antigens and so the antigen-binding
sites of imunoglobulins must conform to a wide variety of shapes and chemical properties of the
antigen. TCR, however must recognize a large number of processed antigenic peptides, occupying
the centre of a small number of self-MHC molecules. So TCRs must have enough diversity to rec-
ognize varied antigens, while restricting its “MHC”-recognition diversity. A fine balance indeed.
Nature has solved this problem by equipping T cells with two types of CDRs in each of the α
and β chains. Each α and β (or γ and δ) chain has three CDRs or hypervariable regions (actually the
β chain has four hypervariable regions, but the fourth one is not involved in antigen recognition
and is not a CDR). The number of CDR1 and CDR2 which are coded by a small number of germ-
line V genes is relatively small. The third structurally hypervariable loop CDR3 is coded by the
highly variable D and J gene segments. The diversity in CDR3 region of α and β chains is generated
by the junctional diversity in the joining of V–J segments (in the α chain) or V, D, J combination
(in the β chain) or V–D–D–J combination in the δ chain, as well as by the introduction of n and p
nucleotides at the appropriate junction.
It is believed that the less variable CDR1 and CDR2 loops of TCR (of both α and β) will mainly
contact the relatively less variable MHC component of the antigen–MHC complex. The CDR3 (of
α and β chains) will recognize the highly variable portion of the presented antigen–MHC complex,
which is an antigen (or antigenic determinant to be more precise).
Recent evidence suggests that some contacts with CDR1 and antigen also occur and hence
CDR3 is not the only CDR that is in contact with the bound peptide.
7.10 TCR GENES DO NOT UNDERGO

S O M AT I C H Y P E R M U TAT I O N
Somatic hypermutation does not occur in the TCR genes so that the functional TCR genes (includ-
ing three CDRs) generated during germ-line rearrangement in the thymus have the same sequence « Somatic hypermutation in TCR
as those found in mature T cells. On the other hand, somatic hypermutation increases the diversity does not occur because these
mutations might make TCR
of all three complementary determining regions of both immunoglobulin chains. recognize self-MHC as foreign or
Why have T cells evolved in such a way that they lack somatic mutations? We know that non-self !
somatic mutations increase the variability of CDRs, which in turn increases the potential of the
antibody to recognize (with higher affinity) a wide variety of antigen molecules. Since T cells
already interact with a self-component, the MHC molecule and the bound antigen, it is argued
that somatic mutation might make self-MHC molecule be recognized as foreign, which will have
disastrous consequences for antigen-presenting cells and the organism in particular. On the other
hand, somatic mutation in T-cell genes might result in expression of the mutated TCR that does
not recognize self-MHC at all, and hence will not have any ability to respond at all.
7.11 PROMOTERS, ENHANCERS AND

SILENCERS OF T-CELL RECEPTORS
7.11.1 CHAIN
There are 5′ promoters of each Vα gene that have low levels of T-cell non-specific activity. The
α-chain enhancers are located 3′ of the Cα gene and are brought in close proximity to the promoter
sequence after gene rearrangement. There are some consensus-binding sequences in the α chain
enhancer that bind junctionally important nuclear-binding proteins. There are silencer sequences
located 5′ of the α chain enhancer. These silencers are responsible for shutting off α-chain expres-
sions in non-T cells and in cells of the γδ receptor type.
7.11.2 CHAIN
There are promoters 5′ of each Vβ gene like its α counterpart which has minimal non-T cell spe-
cific activity. A powerful β-chain enhancer is located 3′ of the Cβ2 gene segment. The TCR β-chain
transcription is T-cell specific probably due to the T-cell specific rearrangement that brings β the
chain enhancer in close proximity to the β chain promoter. This induces a high level of T-cell spe-
cific activity of Vβ promoters. There are no known silencers of β chain genes, yet.
7.11.3 γ AND δ CHAINS

The promoter for each Vγ or Vδ gene is located 5′ upstream of the V gene. The γ-chain enhanc-
er is located 3′ to the Cγ1 gene segment and the δ-chain enhancer is located between Jδ2 and Cδ
in mice.
The γ silencer is located 3′ of the Cγ1 gene segment that is partly responsible for shutting
γ-gene expression in the αβ-expressing T cells. This is important because one-third of the αβ
T cells have undergone potentially functional γ-gene rearrangement. δ silencer (if any) has not yet
been identified.
7.12 T-CELL RECEPTOR COMPLEX

We have seen till now that the T cell αβ heterodimer is involved in recognition of antigen–MHC
complex. The TCR is not alone in the cell-surface membrane of T cells. In fact, it is associated with
up to five other transmembrane proteins that are non-covalently associated with the αβ heterodimer.
CD The αβ heterodimer together with these associated proteins termed as CD3 proteins form a fully
CD stands for cluster of functional TCR complex.
differentiation or cluster designation. The αβ heterodimer is involved in antigen–MHC complex recognition. The CD3 protein com-
This system of nomenclature is used
to designate leukocyte-surface plex has a role in cell surface expression of the αβ heterodimer as well as the signal transduction
antigens. Most of the leukocyte- inside the cells which results in T-cell activation. The constituent polypeptides of TCR-CD3 pro-
surface molecules are identified teins are given in Table 7.3.
due to their reactivity with a cluster
of monoclonal antibodies that
recognize different parts of one 7.12.1 CD3 PROTEINS
antigen. This cluster is then given Immunological studies on the αβ T-cell receptor led to a “surprise” finding. When anti-αβ fluores-
a number by convention which
becomes the trademark (or name) of cent antibodies were used to aggregate proteins of αβ TCR, they were found to cause aggregation
that particular antigen. of another group of proteins namely CD3 proteins. This observation suggested that the αβ het-
erodimer of TCR is associated with CD3 proteins.
Later, it was demonstrated that TCR and CD3 are located quite close to each other. Controlled
cross-linking of T-cell membrane proteins was induced and a cross-linker that was only 12 Å long
was used. Precipitation of TCR by anti-TCR monoclonal antibodies also precipitated CD3 mol-
ecules. This implied that CD3 molecule lie within a 12 Å distance of the TCR complex. This fact
was later confirmed byAllison and Lanier (1987).
7.12.2 CD3 COMPLEX

It is a complex of five non-polymorphic polypeptide chains, namely CD3 gamma (CD3γ), δ (delta),
ε (epsilon), ζ (zeta) and η (eta) proteins, which are identical in all T cells. These five proteins as-
sociate to form three types of dimers—dimer of gamma and epsilon chains (γε), dimer of delta and
Protein Function Size (kDa)

TCRα Recognizes antigen + MHC 40–60
TCRβ Recognizes antigen + MHC 38–50
TCRγ Recognizes antigen 40–55
TCRδ Recognizes antigen 40–60
CD3γ Signal transduction 25–28
CD3δ Signal transduction 20–28
CD3ε Signal transduction 20–25
CD3ζ Signal transduction 16
CD3η Signal transduction 21
Table 7.3 CD4 Receptor for MHC class II 55
Constituting polypeptides of TCR–CD3
CD8 Receptor for MHC class I 34
receptor complex.
T-cell receptor
NH2 NH2
α chain β chain
CD 3
γ ε ε γ
NH2 NH2 NH2 NH2
ζ ζ
NH2 NH2
-s-s-
HOOC COOH
HOOC COOH HOOC COOH

Figure 7.11
ITAMs Schematic diagram of TCR–CD3 complex
(ITAMs stand for immunoreceptor
T-cell receptor complex COOH COOH tyrosine-based activation motifs).
epsilon chains (δε) and a homodimer of two zeta chains (ζζ) or a heterodimer of zeta and eta chains
(ζη). The schematic diagram of TCR-CD3 complex is shown in Figure 7.11.
The CD3 complex comprises five different proteins and is involved in generating activating
signals in the T cells, after the TCR binds the antigen–MHC complex.
γ CHAIN OF CD3 COMPLEX
• The CD3γ chain is 25 kDa in humans and 21 kDa in mice. It is a glycoprotein having « CD3 antigen is a member of
N-linked oligosaccharide side chains (two carbohydrate chains for CD3γ in humans and immunoglobulin superfamily.
one in mouse).
• It contains an N-terminal extracellular domain followed by a transmembrane region
containing negatively charged aspartic acid group. This negatively charged group enables
the CD3 complex to interact with one or two positively charged amino acids in the trans-
membrane region of the TCR. The transmembrane region is followed by a cytoplasmic
tail of about 44 to 81 amino acid residues.
• The cytoplasmic tail of the γ chain contains one copy of a motif called the immuno-receptor
tyrosine-based activation motif (ITAM) or antigen recognition activation motif
(ARAM). ITAM is composed of approximately 24–30 mostly unconserved amino acid
residues. ITAM contains a sequence tyrosine–x–x–leucine which occurs twice, separated
by six to eight amino acid residues (x would be any amino acid). The tyrosine residues of
this motif of ITAM becomes phosphorylated as a result of TCR–antigen recognition. This
initiates the binding of several proteins and triggers an intracellular signalling cascade.
• The N-terminal extracellular region of the γ chain contains immunoglobulin-like
domains (including a single disulphide bond) and therefore this protein is a member of
the immunoglobulin superfamily. There is no polymorphism or variability exhibited by
extracellular domains and it is not involved in antigen recognition.
δ CHAIN OF CD3 COMPLEX
• The CD3δ chain is a glycoprotein (20 kDa in humans and 25 kDa in mice) having two
n-linked carbohydrate chains in humans and three n-linked carbohydrate chains in mice.
• Like its sister chain γ, the δ chain has N-terminal immunoglobulin-like extracellular
domains, a transmembrane region containing negatively charged amino acids and a
» The γ chain and δ chains that are cytoplasmic tail of more than 40 amino acid residues. The cytoplasmic tail contains a
part of the CD3 complex are distinct
polypeptides and are coded by
single ITAM motif.
distinct genes which are completely • It is non-polymorphic.
different from the γ and δ chains
that form γδ TCR.
ε CHAIN OF CD3 COMPLEX
• CD3ε chain is 20 kDa in humans and 25 kDa in mice. The ε chain is not glycosylated.
• The ε chain has the familiar N-terminal immunoglobulin-like extracellular domain, a
transmembrane segment (containing negatively charged aspartic acids) and a cytoplasmic
tail containing a single ITAM sequence.
• It is not polymorphic and, like other members of CD3, it is not involved in antigen
recognition.
ζ CHAIN OF CD3 COMPLEX
• The zeta (ζ) chain is a 16 kDa non-glycosylated protein which has no sequence or
structural homology to γ, δ, or ε chains of the CD3 complex. The ζ chain exists as a
homodimer interlinked by a disulphide bond.
• The ζ has a very short extracellular domain of six to nine amino acids. The transmem-
brane region has a negatively charged amino acid. It has a very long cytoplasmic tail of
about 113 amino acids. The cytoplasmic tail of the ζ chain contains three ITAMs.
η CHAIN OF CD3 COMPLEX
• The η chain is a 21 kDa non-glycosylated protein, which is similar in structure to the ζ

chain.
• The η chain contains nine amino acids long extracellular domain, transmembrane
domain containing a negatively charged amino acid and a long cytoplasmic tail of 155
amino acids.
• The η chain contains three copies of ITAMs.
7.12.3 S U R FAC E E X P R E S S I O N O F TC R CO M P L E X
The synthesis of the components of the TCR complex, that is, αβ heterodimers as well as CD3γ,
δ, ε, ζ and η surface expressions is tightly and coordinately regulated; for example, if T cells have
mutated one of the genes of CD3 proteins, it will not express other CD3 proteins or αβ heterodim-
ers on their surface.
The genes encoding the CD3η, δ, ε, and ζ/η proteins are expressed before TCR α and β chain
genes in the immature thymocyte. The protein products, CD3γ, δ and ε, are synthesized but remain
inside the cell. The proteins form a core γδε that remain within the ER, held by transient bind-
ing protein, the CD3ω or T-cell receptor associated protein (TRAP). Upon further maturation
of thymocytes the β chain is synthesized, followed by the α chain within the ER. After the syn-
thesis of TCR chains (αβ), the full receptor is assembled within the ER, after association with the
CD3 proteins. The processing of N-linked side chains of TCR and CD3 chains occurs in the Golgi
apparatus. The complete TCR complex, that is, TCR-CD3 complex is then transported to the plasma
membrane. The incomplete TCR complex is directed from the ER to the Golgi and finally to lyso-
some where they are rapidly degraded.
7.12.4 FUNCTION OF CD3 COMPLEX

We usually say that the antigen–MHC complex is recognized by the TCR complex. It should be made
clear that the αβ heterodimer of the TCR is the only part that is involved in antigen recognition, and
signals that initiate the activation of T cells are transduced not by the αβ heterodimer but by the
associated CD3 protein complex. When the antigen binds to the TCR, the associated CD3 proteins
transduce the signals to the cytoplasm of the T cells which leads to the functional activation.
Several lines of evidence support this concept:
• The antibodies against CD3 proteins are non-specific polyclonal activators of T-cells. Un-
like antigen, which stimulate only specific T cells, anti-CD3 antibodies stimulate all T cells
in a mixed population. This suggests that the stimulation of CD3 proteins is necessary for
T-cell activation whether it occurs via antigen binding to αβ heterodimer or direct binding
of antibodies to CD3 protein.
• Chimeric CD3ε or CD3ζ chain can transduce the signal necessary for T-cell activation.
Genetically engineered chimeric molecules containing cytoplasmic portions of CD3ε or ζ
protein fused to extracellular and transmembrane domains of other cell-surface receptors
for soluble ligands such as IL-2 receptor were constructed. Ligand binding, that is, IL-2 bind-
ing to such chimeric receptors expressed in T-cell tumour lines, resulted in T-cell activation
identical to ones induced by stimulation through the normal T-cell receptor complex.
7.13 ACCESSORY MOLECULES ON T CELLS

The proteins of TCR-CD3 complex are the molecules that are involved in the recognition of anti-
gen–MHC complex and antigen-induced T-cell activation. In addition, T lymphocytes express sev-
eral other transmembrane proteins that have accessory (additional) role in antigen recognition and
T-cell activation. These proteins, often collectively called T-cell accessory molecules are often in-
volved in T-cell adhesion to antigen-presenting cells, signal transduction into the T cells and T-cell hom-
ing and retention in tissues. A brief summary of accessory molecules on T cells is given in Table 7.4.
T cells can be subdivided into two populations depending on whether they express accessory mol-
ecule CD4 or CD8. We have T cells expressing the TCR complex as well as CD4 molecules, and CD4+ « CD4 molecules are also expressed
cells. CD8+ T cells have a TCR complex and CD8 accessory molecules on their surface. CD4 and CD8 on cells on monocyte/macrophage
lineage.
accessory molecules are mutually exclusive and both are not normally found on the T-cell surface.
7.13.1 CD4 ACCESSORY MOLECULES

CD4 molecules are cell-surface glycoproteins, that are expressed on those T cells which recognize
antigens complexed with class II MHC molecules. These CD4+ T cells function as cytokine-secreting
TH cells.
Structure: The CD4 molecule is a single-chain transmembrane glycoprotein of a molecular weight
« CD4 antigen was initially named
of 55 KDa. It has four extracellular immunoglobulin-like domains, a hydrophobic transmembrane as T4/lLeu-3 in the 1970s.
segment and a highly basic cytoplasmic tail of 38 amino acids, including three serine residues that
can be phosphorylated.
« The CD4 molecule recognizes β2
Functions: The invariant CD4 protein binds via its two N-terminal immunoglobulin-like domains domain of class II MHC. It is
to the non-polymorphic β2 domain of class II MHC complex of the antigen-presenting cells. This because of CD4 antigen that TH
adhesive function of CD4 molecules allows antigen-presenting cells to be bound long enough to be are class-II-MHC-restricted.
influenced by effector functions (such as lymphokine secretion) of the T cells.
Function
T-cell
Name Size (kDa) Ligand Gene Family Adhesion Signalling
CD4 55 Class II MHC Ig ⫹ ⫹
CD8 34—α chain, 78—β Class I MHC Ig ⫹ ⫹
chain or dimer of α
chains
CD2 50 LFA-3 Ig ⫹ +
CTLA-4 34 (usually occurs as B7-1, B7-2 Ig ⫺ +
dimer of 34 kDa)
LFA-1 180—α chain, 95—β ICAM-1, 2 Integrin ⫹ ?
chain
CD28 Dimer of 40 kDa chain B7-1, B7-2 Ig ⫺ +
CD45R 200 CD22 Ig ⫺ +
CD44 100–200 Matrix protein ? ⫹ ?
Table 7.4
CD5 67 CD72 ? ⫺ ⫹
Some important accessory molecules
Source: The Leucocyte Antigen Facts Book (Academic Press, London) on T cells.
CD4 antigens participate in the activation of T cells upon T-cell recognition of the peptide–
Lck
Lck is lymphocyte-specific protein MHC complexes on antigen-presenting cells. It is believed that the cytoplasmic tail of the CD4
kinase. It is expressed in a variety of antigen binds non-covalently with T-cell-specific tyrosine kinase called Lck. It is likely that this
lymphoid and non-lymphoid tissues non-covalently-bound kinase phosphorylates the nearby tyrosine residues of ITAM of the γ
as well as in some cancers.
chain of CD3 protein and thereby promotes the T-cell activation cascade. There is ample evidence
» HIV gains entry into the host that suggests that individual CD4 (or CD8) molecules make a physical association with the TCR
cells via CD4 antigens present complex which aids in the phosphorylation of the TCR complex needed for T-cell activation.
on the surface of TH cells.
7.13.2 CD8 ACCESSORY MOLECULES
» The CD8 molecules recognize Structure: The CD8 molecules are cell-surface glycoproteins of a molecular weight 34 kDa, found
the α3 domain of class I MHC, on T cells that recognize antigens complexed with class I MHC molecules. These CD8+ T cells are
which makes Tcyt(CD8+) cells
class-I-MHC-restricted. Tcyt cells.
Most CD8 molecules exist as disulphide-linked heterodimers of CD8α and CD8β or homodi-
» CD8 molecules belong to the mers of CD8α molecules. Both CD8α and CD8β have a single extracellular immunoglobulin-like
immunoglobulin superfamily.
They were was previously
domain at its N-terminal, a hydrophobic transmembrane region and a highly basic cytoplasmic
named Leu-2 /T8 antigen. tail of about 25 amino acids at its C terminal.
Functions: The functions of CD8 accessory molecules are similar to those of the CD4 molecules.
They promote the adhesion of the TCR complex to the antigen–MHC complex (class I MHC)
present on antigen presenting cells. The CD8 molecule that is present with the TCR complex binds
to non-polymorphic immunoglobulin-like α3 domain of class I MHC molecules, thereby, stabiliz-
ing the interaction of a class-I-MHC-restricted T cells (Tcyt-lymphocytes). The ability of CD8 (or
CD4) molecules to interact with MHC molecules to mediate cell–cell adhesion has been dem-
onstrated. Anti-CD8 antibodies block the formation of conjugates between class-I-restricted Tcyt-
lymphocyte and class-I-MHC-expressing target cells. In addition, it has been demonstrated that
normal fibroblasts do not “bind” class-I- or class-II-MHC-expressing cell lines; however, if CD4
or CD8 genes are transfected into fibroblasts, they
Binds to β 2domain of
attain the ability to bind cell lines which have class II
class I MHC complex
or class I MHC molecules on their surface.
NH2 The CD8 molecules participate in the sig-
nal transduction that lead to the activation of T
s cells. It is suggested that once T cells have bound
Binds to α 3 domain of
class I MHC complex s the antigen–class I MHC complex, the cytoplas-
NH2 NH2 mic tail of the CD3 complex protein(s) is phos-
s
phorylated by protein tyrosine kinase called Lck.
α β It is believed that this tyrosine kinase initially re-
s
s s mains associated with the cytoplasmic tail of the
s s CD8 (or CD4) molecules. When T cell binds the
s antigen–MHC complex, the cytoplasmic tails of
s
the CD8 (or CD4) molecules are brought in close
proximity to the cytoplasmic tails of CD3 mol-
s ecules which are then phosphorylated, promoting
s T-cell activation cascade. The schematic represen-
tation of CD4 and CD8 molecules is depicted in
Figure 7.12. It should be made clear that CD4 and
CD8 molecules are not the only T-cell surface ac-
Figure 7.12
Schematic diagrams of accessory cessory molecules. There are a variety of T-cell sur-
molecules of T lymphocytes, CD4+
HOOC COOH COOH face accessory molecules that are involved in other
and CD8+. CD8 CD4
T-cell adhesions or signal transduction in T cells.
7.14 ANTIGEN–MHC–T-CELL RECEPTOR

COMPLEX
There is a distinct difference in the mechanism by which antigen is recognized by TCR and/or
B-cell receptors antibodies. Antigen recognition by B cells involves the direct binding the of im-
munoglobulin (located on the B-cell surface) to the intact antigen. Antibodies (or B-cell receptors)
typically bind to the antigenic determinant present on the surface of the intact protein (or any
other antigen). As we have seen previously, these antigenic determinants are formed by the amino
acids (in protein antigen) that are located far apart in the primary sequence but are brought close
together in the folded protein. Strangely, T cells were found to respond to short contiguous amino
acid sequences in proteins. These sequences were often buried within the native structure of the
protein and hence could not be recognized directly. TCR cannot “see” the antigen unless protein is
unfolded and processed to generate small peptide fragments. It soon became clear that “antigen”
that is recognized by T cells is actually small peptides that is bound to the MHC complex. The in-
volvement of the MHC in antigen recognition has been conclusively proved by stimulating T cells
using purified peptide–MHC complex. The TCR makes contact with both the MHC and bound
antigen as we shall see below.
Site-directed mutagenesis and X-ray crystallographic studies have revealed the three-dimensional
structure of TCR receptor–antigen–MHC ternary complex. This structure shows that the TCR is
situated diagonally over the peptide and peptide-binding groove of class I MHC molecule. The
CDR3 loops of both TCR α and β chains meet over the central amino acids of the peptide. The
CDR1 loop of the TCR α chain is at the amino terminal of the peptide and the CDR2 of the TCR
α chain is over α2 domain of MHC molecule. The CDR1 loop of the TCR β chain is at the car-
boxyl terminal of the peptide and the CDR2 of the TCR β chain is over the α1 domain of MHC
molecules. Looking from above the peptide-binding cleft, it can be seen that the TCR is threaded
through a valley between the walls of the peptide-binding cleft (of the MHC) formed by α helices.
The peptide is usually sandwiched between the MHC and TCR and is not easily visible from above.
However, it should be noted that contacts of α and β chains of TCRs are not symmetrically dis-
tributed over the MHC–peptide complex. The CDR1 and CDR2 loops of the α chain (of the TCR)
are in close contact with the helices of the MHC–peptide complex around the amino terminus of
the bound peptide. The β chain CDR1 and CDR2 loops of β chain interact with the complex at the
carboxyl terminus of peptide and have variable contributions to the binding. The CDR3 of both
α and β chains, however, make almost equal contributions in peptide recognition. The peptide is
buried more deeply in the MHC molecule than it is in the TCR. The TCR molecules fit across the
peptide-binding groove between the two high points on the two surrounding α helices that form
the walls of the peptide-binding cleft. « Mutational analysis has shown
It is difficult to predict whether interactions that hold the T cell and target cell/antigen- that changing even a single amino
acid residue in the antigen-
presenting cell are contributed by TCR’s contact with the bound peptide or by TCR’s contact with presenting MHC molecule can
the MHC molecule alter T cell response from strongly
However, the specificity of T-cell recognition involves both the peptide and its presenting cytotoxic to absolutely no response
at all. Studies have shown that a
MHC molecules. This dual specificity of T-cell recognition forms the basis of MHC restriction of change of a single amino acid in the
T-cell responses. bound peptide can have a similar
Since class I and class II MHC molecules have similar peptide-binding regions, the model pre- effect.
sented above of TCR–class I MHC molecule–peptide complex may have features common to both
class I and class II TCR interactions.
7.15 CROSS-REACTIVITY OF T CELL

WITH ALLOGENIC MHC
The T cells are self-MHC-restricted; they recognize and respond to antigen presented by antigen-
presenting cells, only if the antigen-presenting cells express MHC molecules that the T cell recog-
nizes as self. The self-MHC does not refer to MHC molecules expressed by the T cells themselves.
The MHC molecules that the T cells recognize as self are those that the T cells encountered during
their maturation from precursors. Under normal conditions T cells would only be exposed to self-
antigen-presenting cell. The MHC molecules on these cells are called as self-MHC molecule.
When a foreign tissue or an organ is transplanted to a (self) individual, it is immediately
rejected. This was found to be due to histocompatibility antigens (discussed in Chapter 6). The his-
tocompatibility antigens of the donor and the recipient were found to be different. One of the main
Alloantigens
or major antigen that determines histocompatibility is the major histocompatibility complex. Al- Alloantigens are different antigens
most every member of the same species has its unique set of MHC molecules. These unique MHC that are expressed by genetically
molecules are recognized as foreign antigen in the recipient body and result in immune response different individuals of the same
species.
against these alloantigens.
The immune response that follows tissue transplantation is mainly mediated by T cells. Gen-
erally CD4+ T cells (TH cells) respond to class II alloantigen and CD8+ T cell (Tcyt cells) respond to
class I alloantigen.
» Cross-reactivity of T cells with Cross-reactivity of T cells is an unusual problem. T cells can respond to foreign peptides dis-
allogeneic MHC is called played on “foreign” MHC molecules. This appears to contradict the fact that T cells can respond
alloreactivity.
only to foreign antigen plus self-MHC only. The explanation for this is not very simple. T-cell rec-
ognizes foreign peptide presented on self-MHC, however it can sometimes recognize (cross-react
with) foreign peptides presented on allogeneic MHC molecules (though with low affinity), when
the structure of allogeneic MHC–peptide complex resembles that of processed antigen plus self-
MHC (see Figure 7.13). These alloreactive T cell will bind with high affinity its “original” antigen
displayed on self-MHC. Grafted allogeneic cells display class I MHC molecule of about 105 mol-
ecules per cell. Those T-cell bearing low-affinity receptors (that is, cross-reacting) might to able to
bind this high density of membrane alloantigen (foreign peptide on foreign MHC). On the other
hand, foreign antigen would be sparsely displayed on the membrane of an antigen-presenting cell
or altered self-cell associated with self class I or II MHC molecules, inviting wrath of same T cells
having high affinity receptors for this original antigen plus self-MHC.
TCRs are unique antigen receptors that are expressed only on T cells. These receptors recog-
nize antigen only when they are associated with MHC molecules. The majority of receptors are of
the αβ type, comprising α and β polypeptides. TCRs have both variable and constant regions just
like antibodies, and are formed by somatic rearrangements that occur in germ-line DNA. The
α chain of TCR is constituted by V, J and constant gene segments while the β chain is coded by V,D, J
and constant gene segments. T-cell receptors in vivo are associated with five other polypeptides of the
CD3 complex to form a fully functional T-cell receptor complex. Moreover, this TCR complex can be
associated with either of its two co-receptors, CD4 or CD8 proteins, making them either CD4+ T cells
(TH cells) or CD8+(Tcyt cells). TH cells recognize only those antigens that are presented on class II MHC
molecules while Tcyt cells recognize antigenic peptides associated with class I MHC molecules.
Tcyt cell
Reacts Cross-reacts
Tcyt cell
Foreign peptide Foreign peptide

displayed on displayed on
Figure 7.13 foreign MHC
Cross-reactivity of T cell with foreign
self-MHC
MHC. T cell sometimes cross-reacts
with allogeneic MHC displaying foreign
peptide. Allogeneic MHC molecules are
displayed on foreign cells that enter the Self-cell Foreign cell
host body during tissue transplantation.
Transmission Electron Microscopy

The transmission electron microscope (TEM) was built in
1931 by two German engineers—Ernest Ruska and Max
Electron gun
Knoll. Ernst Ruska was awarded the Nobel Prize for his Heated tungsten
contribution. filament
In TEM, an image is formed by those electrons which are

transmitted through the specimen, and hence its name. A
heated tungsten filament acts as source of electron (see Electromagnetic lens
Figure 7.14). Electrons are then accelerated by very high (Anode, condensor lens)
voltage and passed through a very small hole to form a

electron beam. Doughnut-shaped electromagnets (con-
denser lens—anode) are used to focus the electron beam
on the specimen. The specimen is supported on a small, Specimen plate
thin metal grid which is put on a grid holder present in
the electromagnet. Some of the electrons striking the
Specimen
specimen are scattered by it, according to local density
of the specimen and the remaining electrons that pass
Objective lens
through the specimen are focused by magnetic lens to (Anode)
form a magnified image of the specimen on a fluorescent
screen. To avoid collision between speeding electrons and
air molecules, the entire “column” containing electromag-
netic lens, specimen and electron gun are kept in vacuum. Intermediate lens
The sample is put in an electron microscope through an (Anode)
airlock and then exposed to a focused beam of electrons.
Specimen Preparation Projector lens

Since the basic framework of a cell or a cell organelle con- (Anode)
sists of atoms of relatively low atomic mass such as car-
bon, hydrogen, oxygen and nitrogen, these atoms have
very little capacity to scatter electrons and hence become
visible in an electron microscope. To enhance the elec-
tron-scattering property of the specimen, thin sections of Image on cathode-ray
the specimen are coated with salts of heavy metals such tube (video screen)
as uranyl acetate or lead citrate.
The first step in TEM sample preparation is fixation. Fixa-

tion is done to stabilize or cross-link the protein or lipid Figure 7.14
The principle of transmission electron microscope.
molecule in its native state. Common fixative agents used
are osmiun tetraoxide or glutaraldehyde. After fixation, the
sample is completely dehydrated by treating the sample with increas- (20–80 nm thick) sections of samples. These samples are then mounted
ing concentration of organic solvent (ethanol or acetone). Next, the on tiny copper grids and then viewed in the electron microscope. The
sample is transferred to monomeric, unpolymerized resins so that limit of resolution of TEM is around 2 nm and, hence, can reveal more
empty spaces in the sample are filled with resin material. The specimen details than scanning electron microscopy (SEM). TEM, being more sen-
is then placed in an oven to form a hardened block. The solid block is sitive than SEM, can provide information about the internal structure of
sectioned by a very fine knife—an ultramicrotome—to obtain very thin the cell as well as cell organelles which cannot be viewed by SEM.
S U M M A R Y
• Every individual expresses a diverse array of different T cells. J sequence, (c) addition of P and N nucleotides at the junction,
Each T cell bears unique antigen receptor called TCR. (d) joining of multiple D segments, (e) pairing of different α and β
chains.
• Unlike B-cell receptor which can recognize antigen alone, TCR
can recognize antigen only when it is presented bound to an MHC • TCR is not alone on cell surface membrane of T cells. It is associ-
molecule on the surface of a host cell. ated with up to five transmembrane proteins called CD3 to form
fully functional TCR complex.
• TCR is a two-chain (αβ or γδ) transmembrane glycoprotein.
A majority of T-cell express αβ TCR. • CD3 complex include five non-polymorphic polypeptide chains,
namely, γ, δ, ε, ξ, η. These chains form three types of dimers—γε,
• The TCR comprises of variable and constant region similar to anti- δε, ξξ.
body molecule. The variable region of TCR is involved in antigen
binding as in antibody molecule. • α and β chains are involved in antigen–MHC recognition while
CD3 complex act as signal transduction unit.
• Functional TCR genes of α and β or γ and δ are formed by rear-
rangement of germ-line DNA similar to immunoglobulin gene • TCR complex is also associated with co-receptor molecules CD4
rearrangement. or CD8, dividing T cells into CD8+ T cells and CD4+ T cells. These
co-receptor molecules serve several functions–such as (a) binding
• α chain (or γ chain) is encoded by V, J (joining) and C (constant) of MHC molecules (b) adhesion between antigen–presenting cell
segments while β chain (or δ chain) is coded by V, D (diversity), J, and T cell (c) activation of T cell upon T-cell recognition of
and C gene segments. peptide–MHC complexes on antigen-presenting cells.
• TCR germ-line DNA is arranged in non-functional state character- • Site-directed mutagenesis has shown that CDR3 (and CDR1 in
ized by spatial separation of V, (D), J and C segments. By mecha- some cases) of α/β chains of TCR are in close proximity of
nism and enzymes similar to B-cells, these V, (D), J and C gene peptide–MHC complex and play critical role in antigen recognition.
segments are rearranged to form functional TCR (α and β) genes.
• T cells can cross-react. T cells can sometimes recognize and
• The enormously large diversity of TCR (which is greater than anti- respond to foreign antigens presented on foreign MHC (foreign
body diversity) is generated by (a) presence of multiple germ-line tissue) instead of recognizing foreign antigen on self-MHC.
V, D and J segments, (b) imprecise recombination of V, (D),
K E Y W O R D S
• accessory molecules 153 • CD3 145 • gamma-delta (γδ) • variable domain 140
• allelic exclusion 145 • CD4 153 T-cell 140 • p-addition 148
• alpha-beta (α/β) TCR 141 • CD8 153 • gene rearrangement 141 • n-addition 148
• alpha-beta (α/β) T-cell 150 • CDR 140 • hypervariable region 140 • silencer 149
• alpha (α) chain 139 • delta (δ) chain 139 • junctional diversity 147 • ITAM 151
• antigen-MHC-T cell • enhancers 149 • RAG-1 144 • ζ and η chains 151
receptor complex 155 • gamma (γ) chain 139 • RAG-2 144
• beta (β) chain 139 • gamma-delta (γδ) TCR 139 • ε-chain 152
1. How can T cells generate diversity greater than antibody? What are 4. An intelligent student wrote “class switching in TCR would have
its mechanisms? List three events that can occur during antibody been a wasteful exercise if it had evolved in T cells.” Do you agree?
synthesis but not during TCR generation. Think and answer.
HINT —Somatic hypermutation, class switching and switch from making HINT —Is constant domain of TCR free and exposed to the immune system?
membrane-bound antibody molecule to secretory form.
5. RAG-1 and RAG-2 mutant was isolated by a hard-working immu-
2. Allelic exclusion of α-chain gene does not occur in αβ TCR. Does it nologist. What is wrong with this observation? Why do you think it
offer any advantage to the T cell expressing TCR with two different an erroneous result?
α chains? Does a T cell express TCRs with two different α chains at HINT —If no α- or β-chain rearrangement occurs, T cell dies.
the same time on the same cell?
3. Do you think that a T cell would have been more “powerful” had it
been bivalent like an antibody?
Choose the appropriate option.

1. Which of the following is not a part of TCR? 6. One event in TCR generation that is not common with Ig
(a) Antigen binding site formed by α chains. generation:
(b) Transmembrane region (a) Ligation of V, D, J gene segments
(c) Constant region (b) p- and n-nucleotide additions
(d) Cytoplasmic tail (c) Somatic hypermutation
(d) Multiple germ-line gene segments
2. γδ receptor are:
(a) Found on more than 5 per cent of lymphocytes 7. CD3 complex is involved in:
(b) Found on less than 5 per cent of lymphocytes (a) Activation of phosphotyrosine phosphatases
(c) Expressed later than αβ receptor (b) Phosphorylation of CD4 or CD8 molecules
(d) Expressed on αβ positive T cells (c) T-cell activation
(d) Antigen recognition
3. One of the molecules that is always present on a T cell is:
(a) TCR 8. The antigen-binding site of TCR is formed by all, except:
(b) CD4 (a) CDRs of α and β chains
(c) CD8 (b) Variable region of γ and δ chains
(d) CD45 (c) Variable region of α and δ chain
4. The reason why Ig genes are not rearranged in T cell is:
(a) Ig genes are deleted in T cells 9. CDR that recognizes antigen in TCR is:
(b) RAG-1 and RAG-2 are absent in T cells (a) CDR1
(c) Ig gene are rearranged but non-functional (b) CDR2
(d) Ligation of Ig gene is inhibited (c) CDR3
(d) CDR4
5. Cell lacking CD8 molecule on T-cell surface will:
(a) Not bind antigen 10 Diversity chain gene segment is present in:
(b) Not have rearranged TCR gene (a) α chain gene
(c) Not bind class I MHC (b) β chain gene
(d) Not bind class II MHC (c) γ chain of CD3 complex
(d) δ chain of CD3 complex
State true or false against each statement. If false, give reason(s).

1. CD4+ and CD8+ molecules are associated with antigen binding. 4. Majority of T cells express TCR of αβ type.
2. Like immunoglobulins, TCR genes can undergo switch of 5. T-cell lacking CD4 molecule mediates antigen recognition.
constant region.
3. Allelic exclusion of α and β chains occurs during T-cell
development.
Acuto, O. and E. L. Reinherz (1985). “The Human TCR, Structure Hennecke J., and D. Wiley, (2001). “T-cell Receptor–MHC Inter-
and Function”, New England Journal of Medicine, 312: 1100–11. actions Up Close”, Cell, 104: 1–4.
Allison, J. P. and M. F. Krummel (1995). “The Yin and Yang of Lenschow, D., T. L.Walunas and J. A. Bluestone (1996). “CD28/B7
T-cell Costimulation”, Science, 270: 932–33. System of T-cell Costimulation”, Annual Review of Immunology,
14: 233–58.
J. P. Allison and L. L. Lanier (1987). “Structure Function and Se-
rology of the T-cell Antigen Receptor Complex”, Annual Review Linsley, P. S. and J. A. Ledbetter (1993). “The Role of the CD28
of Immunology, 5: 503–40. Receptor During T-cell Responses to Antigen”, Annual Review of
Immunology, 11: 191–212.
Chein, Y. H., R. Jores and M. P. Crowley (1996). “Recognition by
γ/δ T cells”, Annual Review of Immunology, 14: 511–32. Matis, L. A. (1990). “The Molecular Basis of T-cell Specificity”,
Annual Review of Immunology, 8: 65–82.
Garcia, K. C., L. Teyton and I. A. Wilson (1999). “Structural Basis
of T-cell Recognition”, Annual Review of Immunology, 17: 369–97. Wang, J. and E. L. Reinherz (2002). “Structural Basis of T-cell
Recognition of Peptides Bound to MHC Molecules”, Molecular
Grey, H. M., A. Sette and S. Buus (1989). “How T-cells See Immunology, 38: 1039–49.
Antigen”, Scientific American, 261: 56–64.
“Very few can
be trusted with
an education.”
The existence of two distinct subsets of lymohocytes was first —LOUISE IMOGEN GUINEY
suggested in 1966 by a French scientist, Jacques Francis Alberts Pierre

Miller. In the 1960s, Miller was studying the effect of thymectomy on
mice when he noticed that thymectomized mice developed a wasting
syndrome and died prematurely. These mice also lacked circulating
lymphocytes. It led him to propose that the thymus played a central
role in immune response and that it was the “source of lymphocytes”.
Miller, together with his student G. F. Mitchell, went on to discover the
existence of two subsets of lymphocytes—one that produced After studying this chapter,
antibodies (B lymphocytes) and was derived from bone marrow; and
• Describe the process of T-cell
the other set that helped antibody-producing cells (T cells) and was development
derived from the thymus. The existence of two subsets of lymphocytes • Give an account of positive and
negative selection of T cells
was independently arrived at by H. N. Claman, E. A. Chaperon and • Describe the mechanisms of
positive and negative selection
R. F. Triplett in 1966. The existence of TH cells was first suggested by
• Describe the process of T-cell
activation
N. Avrion Mitchinson in 1969 who showed that T- and B-cell
• Briefly summarize the role
cooperation was needed in antibody formation. Figure 8.1 shows a of costimulators in T-cell
activation
scanning electron micrograph of two very important lymphocytes, • Explain how superantigens
activate T-cells
B and T lymphocytes.
• Describe the structure and
function of γδ T cell
• Briefly describe NKT cell
T-cell Development
and Activation 8
8.1 INTRODUCTION
The initiation of the immune response usually requires an interaction between antigen-presenting
cells and T cells. The specificity of this interaction depends on the interaction between the T-cell
receptor and the antigenic peptide ⫹ MHC complex displayed on the antigen-presenting cells.
This complex of foreign antigenic peptide ⫹ self-MHC binds to the T-cell receptor and initiates
T-cell response. Thus, the T-cell receptor recognizes contributions from both self-MHC and for-
eign peptide. So a repertoire of T cells has to be selected that recognizes both self-MHC molecules
as well as foreign peptides. Understanding how this T-cell repertoire develops and matures will
help us understand how T-cell specificity for “two” ligands—self-MHC and antigenic foreign pep-
tide arises. T cells acquire their functional competence during the process called T-cell matura-
tion. This process is a dynamic series of events by which bone-marrow-derived T-cell precursors
are converted into mature T lymphocytes.The thymus is the principal site of maturation of the T
cells. The maturation process involves several important events that include:
• the expression of T-cell receptors and a number of accessory and associated molecules on
the cell surface;
• the ability to respond to antigen, bound only to MHC (MHC restriction); and
• in the final stages, the development of T-cells into either of two pathways—one leading to
CD4⫹ TH cells and other to CD8⫹ Tcyt cells.
These changes are brought by programmed sequential gene expression that leads to the develop-
ment of a particular functional phenotype, the acquisition of functional competence and stringent
selection events that ensure that only those lymphocytes mature that are useful to the host and can
act on foreign antigen and not against self-antigen.
Mature T cells get activated following the interaction of the T-cell receptor with the antigen
displayed on an MHC molecule. This activation event, which also involves a number of accessory
membrane molecules, triggers a series of intracellular biochemical events that culminate in the
division and differentiation of T cells. Mature T cells (Tcyt or TH) differentiate into various types of
effectors cells and/or memory T cells.
Figure 8.1
Scanning electron micrograph of B and
T cells. (Reproduced with permission
from The Journal of Experimental
Medicine, 1973, 138: 607–624. Copyright
1973. The Rockefeller University Press).
8.2 T-CELL DEVELOPMENT

The precursors of T cells originate from haematopoietic stem-cell precursors in the bone mar-
row. These cells have the lineage of CD44⫹CD117⫹CD25⫺. With this marker, precursors of T
cells emigrate from the bone marrow and arrive at the foetal thymus in waves of colonization.
This colonization seems to be essential for the proper development of the immune system, as
newborn mice whose thymus has been removed do not have mature T cells throughout their
adult life. These precursors of T cells which arrive at the subcapsular cortex of the thymus do
not have the distinctive characterstics of T cells. In mice, however, these cells display the surface
marker Lyt protein.
From the subcapsular cortex, these cells migrate into the cortex where the rest of the matu-
ration events takes place. Once in the cortex, these cells undergo high mitotic activity to form a
population of large, proliferating cells. The large pool of cells generated is necessary; as a large rep-
» T-cell precursors migrate towards
the thymus under the influence of ertoire of antigen-specific T lymphocytes needs to form that ultimately recognizes a large variety of
thymotaxin, a chemotactic factor antigens. The proliferative activity of these early lymphocytes seems to be driven by IL-7. This large
secreted by thymic epithelial cells. population of rapidly dividing T-cell precursors present in the thymus (called thymocytes) which
» Lyt markers are believed to be
lack CD4 and CD8 surface molecules are CD4⫺ CD8⫺ and hence referred to as double-negative cells
differentially expressed on various (DN cells). These cells are also called lineage negative cells.
subsets of T cells. Lyt-1 is The immature thymocytes or pro-T cells as they are commonly called give rise to all subsets
preferentially expressed on TH cell
precursor, while lyt -2 is expressed
of T-cell lineage. Pro-T cells do not express CD3 molecules. These cells however display other sur-
on precursors of Tcyt cells. face molecules such as c-kit (receptor for stem-cell growth factor), CD44 (an adhesion molecule),
CD25 (part of interleukin-2 receptor) and CD2. Mouse pro-T cells express high levels of c-kit
» Behcet’s disease is characterized and CD44 but not CD25. These pro-T cells still have unrearranged T-cell receptor genes. These
by an increased percentage of
double-negative thymocytes in cells proliferate extensively. The next stage of maturation is the pre-T-cell stage. In this stage,
the blood. thymocytes stop proliferating and start rearranging their TCR genes which are present in pieces
in the germ line genome. The first set of rearrangement occurs as the T-cell receptor β, γ and δ
» In humans, X-linked severe
combined immunodeficiency
genes rearrange. If a cell makes productive γ- and δ-chain gene rearrangements, it can express the
disease, characterized by blocked heterodimeric γδ receptor. The functional gene rearrangement of the β-chain locus occurs prior
T-cell (and not B-cell) development, to the α-chain locus. The rearrangement of the β-chain locus occurs on the 14th day of gestation
has a mutation in the IL-7 receptor.
in the foetal mouse thymus. If the cell makes a productive β-chain gene rearrangement, it makes a
pre-T-cell receptor (see Figure 8.2) that consists of the β chain and the pre-T-cell receptor α chain
» Pre-TCR α chain and TCR α chain (MW-33 kDa, an invariant protein), and CD3 proteins.
are two different molecules. A small percentage (less than 5 per cent) of thymocytes makes productive rearrangement
» The α chain of the pre-T-cell
of both γ- and δ-chain genes and gives rise to CD3⫹ γδT cells. Cells that produce neither the γδ
receptor is an invariant protein. receptor nor the pre-T-cell receptor usually die at this stage of development. Thus pre-T cells
Pre-αT-cell receptor does not bind have TCR (αβ or γδ) as well CD3 molecules on their surface. These pre-T cells have receptors that
antigen. It plays a crucial role in the
early stages of thymocyte
is believed to recognize intrathymic ligand which causes proliferation and further maturation of
development. Mice, deficient in double-negative T cells. It is believed that the stimulation of pre-T-cell receptor by ligand binding
pre-TCR α chain, show arrested activates the signal transduction pathway that involves Src family tyrosine kinase—lck.
thymocyte development.
» The β chain rearranges first,

followed by the α chain. Once the !NTIGEN BINDING
β chain is arranged on one allele, its ,IGAND BINDING
rearrangement on the other allele A CHAIN B CHAIN
is suppressed. This is called allelic B CHAIN
exclusion. Thus, allelic exclusion
allows each T cell to express a TCR S S S
of a single specificity. S S S
3IGNAL TRANSDUCING 3IGNAL TRANSDUCING
S S S S
» Cells that are mitotically active G E D E G E D E
S S S S
have low concentrations of RAG-1
0RE 4 A ζ ζ ζ ζ
and RAG-2 proteins. S S S S
S S
)4!-S
Figure 8.2
Line diagram of T-cell receptor and pre- )4!-S
T-cell receptor. (ITAMs—immunoreceptor 0RE 4 CELL RECEPTOR 4 CELL RECEPTOR
tyrosine activation motifs).
T-CELL DEVELOPMENT AND ACTIVATION 163
This signal transduction:

• suppresses the further rearrangement of TCR β chain;
• coaxes pre-T-cells to express both CD4 and CD8 antigen on the surface of thymocytes;
• enhances the rearrangement of TCR α chains; and
• induces allelic exclusion of β chains.
The thymocytes that express both CD4 and CD8 antigens on their surface are called double-
positive (DP) thymocytes. TCR α-chain gene rearrangement and the expression of the α chain
does not occur until the cells are double-positive.
Once the thymocytes are double-positive, they stop dividing and the level of recombination-
specific RAG-2 (and RAG-1) recombinases increase, which induces α-chain rearrangement. « 98 per cent of DP
So the net result is that the body has a large collection of cells of a single type of β-chain thymocytes die because they do
not recognize any MHC.
rearrangement, and within this clone different α chains rearrange, generating additional diversity
within the same β-chain cells. Once the α-chain gene has rearranged, the T cell expresses complete
αβ TCR and CD3 complex on its surface. It is believed that the complete T-cell repertoire is formed
by 17th day of gestation in mice. By the virtue of their expression of complete TCR complexes,
double-positive cells become responsive to antigen and are subjected to positive and negative se-
lection (discussed in the following section).
As T cells mature, they move deeper into the cortex and 98 per cent of the thymocytes die for
reasons not understood (possibly by neglect because they fail to recognize any available MHC and
consequently do not receive survival signal).
Cells die of apoptosis within the thymus possibly because (a) they fail to make produc-
tive TCR gene rearrangement; or (b) they fail to survive thymic selection. Th e double-positive
thymocytes that express αβ TCR and CD3 complex and survive thymic selection, develop into
either single-positive CD4⫹ thymocytes or single-positive CD8⫹ thymocytes. Figure 8.3 shows
the two checkpoints that
determine the destiny #$
#$
of T cells. This pheno- #$
typic maturation is ac- D G B GENE REARRANGING
companied by functional
ST #HECKPOINT
specialization. CD4⫹ T
cells acquire the abil-
ity to produce cytokines 0RODUCTIVE B 0RODUCTIVE
and express effector REARRANGEMENT GD REARRANGEMENT
molecules such as CD40
that activate or “help” B
lymphocytes. CD8⫹ cells
become equipped to pro- %XPRESSION OF NEITHER G D
0RE 4#2 G D 4 CELL
duce perforin, the pro- RECEPTOR NOR 0RE 4 CELL B 0RE 4 A
tein that lyses other cells. RECEPTOR !POPTOSIS
The exact mechanism by
4 CELL RECEPTOR COMMITMENT
which CD4⫹ or CD8⫹
mature into TH or Tcyt #$
respectively is still not #$
Figure 8.3
known. Mature single- Line diagram showing the
positive thymocytes leave checkpoints in T-cell development.
the thymus from the deep ND #HECKPOINT
The commitment to either α, β,
γ, or δ lineage occurs at the first
cortex or medulla by way A GENE
checkpoint and commitment
REARRANGEMENT
of blood vessels or lym- SUCCESSFUL to either CD4⫹, CD8⫹, CD4⫺
phatics. Figure 8.4 illus- or CD8⫹ occurs at the second
#$ checkpoint. It is believed that the
trates a simplified view of #$
“Notch” protein might be involved
#$
T-cell development. #$
A B 4 CELL at the checkpoints. (Current
A B 4 CELL
The different stages Biology by Harold von Boehmer.
Copyright 1999 by Elsevier Science &
of T-cell development &AILED A GENE REARRANGEMENT Technology Journals. Reproduced
and maturation are de- &AILED THYMIC SELECTION 4 CYT CELL with permission of Elsevier Science &
4 ( CELL
picted in Table 8.1. Technology Journals in the format
Textbook via Copyright Clearance
,INEAGE COMMITMENT Center).
Bone marrow
lyt+
CD44+ T-cell precursor
CD117+
CD25-
lyt+
Pro-T-cell
c-kit+
CD44+
CD25+
DN cell (CD3-, CD4-, CD8- TCR)
Pre-T cell
CD4-
CD8-
CD3+
γδ TCR+ CD3+ (low) pre TCR+
CD8+ CD4+
DP cell
Positive and
negative selection
Apoptosis of cells that
have failed positive
and negative selection
CD4+
CD8+
CD3+ (high)
TCR+
Figure 8.4 CD4- CD8-

Thymus CD8+ CD4+
Simplified view of T-cell development. TCR+
(DN—double-negative cells; DP—double- TCR+
positive cells). Tcyt cells TH cell
Double-
positive Immature Naïve Mature
Pro-T Cell Pre-T Cell Thymocyte T Cell T Cell
Location Thymus Thymus Thymus Thymus Peripheral circulation
Surface CD3, CD3, C25 CD3, CD4, CD8 CD4 or CD8CD4 or
markers CD25, CD8 TCR CD8 CD4, CD8CD4, TCR,
CD44 TCR, CD3 CD3
DNA Unrear- β- or γ/δ- α-gene rear- Rearranged α/β Rearranged α/β

ranged gene rearrangement gene gene
germ- line rangement
DNA
Expression None Membrane Membrane Membrane αβ Membrane αβ TCR
of antigen pre-TCR (pre- αβ TCR TCR
receptor Tα/β chain)
or γδTCR
Table 8.1 Stimulated No No Yes (self- Yes (self-antigen) Yes (foreign antigen)
Stages of T- cell maturation. by antigen antigen)
8.3. P O S I T I V E A N D N E G AT I V E
SELECTION
The large repertoire of immature T lymphocytes, that is, double-positive cells undergo positive
and negative selection before they can mature into CD4⫹ or CD8⫹ T cells. The double-positive imma-
ture thymocytes may potentially recognize via its receptors any peptide antigen (foreign or self)
and those displayed on any MHC (foreign or self).
Moreover, some cells may have receptors that do not recognize any peptide–MHC complex.
The positive and negative selection of T cells ensures that only those T cells survive who can re-
spond to foreign antigens displayed on self-MHC and who do not respond to self-antigens (that is,
are self-tolerant). Cells that fail the selection process within the thymus are eliminated by apoptosis.
The thymic environment provides the stimuli and environment for positive and negative
selection of thymocytes. These include thymic epithelial cells, bone-marrow-derived dendritic
cells and macrophages. Thymic epithelial cells are mainly present in the cortex though some are
also found in the medulla. Dendritic cells are present at the cortico-medullary junction and within
the medulla. Macrophages are present primarily within the medulla.The migration of thymocytes
through the meshwork of these cells allows physical interaction between the thymocytes and other
cells. Cortical macrophage, epithelial cells and dendritic cells express high levels of class II MHC
molecules. Medullary macrophages express only class I MHC, while medullary epithelial and den-
dritic cells express both class I and II MHC molecules that display hundreds of antigenic peptides.
A brief overview of positive and negative selection is given in Table 8.2.
8.3.1 POSITIVE SELECTION

Recent researches have suggested that double-positive thymocytes are programmed to die (within « Positive selection rescues about
3 days) unless rescued through positive selection. 3–5 per cent of thymocytes. Thymo-
Those double-positive thymocytes that express TCR which bind self-MHC+self-peptide cytes that are saved are those that
recognize only self-MHC, that is,
complex with low affinity are rescued through positive selection and differentiate into MHC- they are self-MHC-restricted.
restricted TH and Tcyt cells. Positive selection takes place in the cortical region of the thymus.
It is believed to involve the interaction between thymocytes and epithelial cells. When double-
positive thymocytes enter the thymus, their αβ T-cell receptor encounter self-peptides (only
peptides normally present in the thymus) displayed on self-MHC molecules (only MHC molecules
available in the thymus). Positive selection ensures that only those T cells survive that recognize with
low affinity, self-peptides displayed only on self-MHC molecules (that is, they become self-MHC-
restricted). Thymocytes that bind weakly (low affinity) to self-peptide+self-MHC molecules, are
permitted to survive by epithelial cells by sending a signal that switches off apoptotic machinery
in the thymocytes.The thymocytes whose TCR are not MHC-restricted (that is, do not recognize
self-MHC), die. Because positive selection “instructs” or “teaches” a population of thymocytes to
recognize only self-MHC-associated antigens/peptides, the process has also been called thymic « Positive selection occurs in the
education (see Figure 8.5). cortical region of the thymus.
Positive Selection Negative Selection

Location Medulla of thymus Cortical region of thymus
Cells involved Thymocytes, dendritic cells and Thymocytes and epithelial cells
macrophages
Selection mechanism Thymocytes that recognize self- Thymocytes that recognize
MHC survive. Thymocytes that self-MHC ⫹ peptide with high
recognize self-MHC ⫹ peptide affinity die
with low affinity survive. Thymo-
cytes that do not recognize any
MHC die.
Net result T-cell repertoire become self- Development of tolerance.
Table 8.2
MHC-restricted Positive and negative selection.
E X P E R I M E N TA L E V I D E N C E O F P O S I T I V E
Thymic epithelial SELECTION
cell Positive selection works by promoting the selec-
Self-MHC tive survival and proliferation of thymocytes that
+peptide recognize with low affinity, self-antigen–self-MHC
(that is, have self-MHC-restricted TCR). Thymo-
CD4+, CD8+, cytes lacking the ability to recognize self-MHC are
Thymocyte allowed to die by apoptosis. Immunologists have
used a wide variety of experiments to understand
and deconstruct the process that involve selection
via MHC molecules.
If T cells or thymocytes are allowed to ma-
TCR recognizes ture in an environment that contains other
self-MHC MHC (foreign) molecules, these thymocytes
(+antigen)
recognize the foreign MHC as self and hence
become tolerant. This was verified by creating a
TCR does not chimeric animal (say, mouse A) which was first
recognize lethally irradiated to remove all T cells and then
self-MHC (+antigen) transplanted with bone-marrow-derived stem
cells from a mouse of another strain (say, mouse
B). The T cells that developed in mouse A from
the stem cells of mouse B were tolerant to MHC
molecules of mouse A, that is, they recognized
Thymocyte MHC of mouse A as self. This implies that T cell
Thymocyte dies
survives
self-MHC-restriction occurs because T cells rec-
ognize the MHC present at the thymocytes’ ma-
Figure 8.5 turing stage as self. If foreign MHC molecules
Thymic education or the positive are present at that time, cells may recognize
selection of T cells. Those CD4⫹CD8⫹
thymocytes are selected that recognize them as self and cells will then be foreign-MHC-
self-MHC–antigen complex. Those Thymocyte restricted. Normally, however, the thymus and
thymocytes that fail to recognize “positively” developing T cells will express only those MHC
self-MHC are deleted.
selected molecules which are “self ” in the individual.
Positive selection was also clearly demon-
strated by using mice transgenic for already rear-
ranged α- and β-T-cell receptor genes. In these experiments, they took already rearranged αβ TCR
genes from CD8⫹ T cells and injected them into fertilized eggs from two different mouse strains
(one of H-2k class-I-MHC haplotype and other of H-2d class-I-MHC haplotype).The αβ TCR trans-
gene that Kiselow et al. took was specific for the H-2k class I MHC (⫹ peptide). Because the trans-
genes were already rearranged, they suppressed the rearrangement of endogenous TCR genes in
the fertilized eggs and hence the majority of thymocytes expressed this single (transgenic) TCR
on their surface. A large number of mature T cells can be found only in the transgenic mice with
H-2k class I MHC haplotype and not in H-2d class-I-MHC haplotype.This finding suggested that
interaction between TCR and self-MHC molecules (H-2k class I MHC was self-MHC molecule for
transgenic TCR) is needed for positive selection.
Though the process is more of selection or deletion rather than training, positive selection
also fixes the class I or class II MHC restriction of T-cell subsets. This ensures that CD8⫹ T cells
are specific for peptides displayed by class I MHC molecules and CD4⫹ T cells for class-II-MHC-
associated peptide antigens.
Class I and class II molecules are required for positive selection, which produces self-MHC-
restricted CD8⫹ or CD4⫹ T cells. Experiments conducted in knockout mice lacking either class I or
II molecules reveal the involvement of these specific MHC in positive selection. Class-I-deficient
mice have normal distribution of CD4⫹ lymphocytes but failed to produce CD8⫹ thymocytes. Ap-
parently, double-positive (CD4⫹ CD8⫹ )T cells are able to bind only class II molecules present in
class-I-MHC-deficient mice and these cells are selected to become CD4⫹ T cells. Apparently the class
II MHC molecules present, picks up and coaxes the CD4⫹ CD8⫹ T cells to the CD4⫹ pathway. Similarly
class-II-deficient cells mice have normal distribution of CD8⫹ T cells but lack CD4⫹ T cells.
Peptides bound to MHC molecules are also required for positive selection of T cells. The
MHC-associated peptides on thymic antigen-presenting cells (thymic epithelial cells to be precise)
serve two main functions:
• Peptides cause the normal expression of MHC molecules. A normal MHC expression is
necessary for positive selection.
• Peptides influence the specificity of the T cells that are selected. The role of peptides in
positive selection is depicted in Figure 8.6.
Since the full range of foreign antigenic peptides to which an individual can respond is never
present in the thymus, foreign peptides cannot be involved in positive selection of T cells that ulti-
mately may recognize these peptides. Self-peptides are involved in positive selection. This is proved
using the following approach. In TAP1-deficient or β2-microglobulin-deficient mice, class I mol-
ecules are not fully loaded. Empty class I molecules that are erroneously displayed can be “loaded”
exogenously. If the thymus from such deficient mice is cultured in the absence of added peptides,
only a few mature CD8⫹ T cells develop inspite of the presence of near-normal expression of class
I MHC (empty) molecules. The addition of peptides drastically increases the development of CD8⫹
T-cells. This implies that peptides plus MHC complex is needed for positive selection.
Peptides
available Peptides
Antigen-presenting not
cells available
Class Class
I MHC II MHC
Proper MHC expression Peptide not Improper MHC

loaded expression
Effect on MHC expression
CD8
Thymocyte expressing
TCRs of different
specificity
CD4
Thymic
epithelial cells
Thymocytes Figure 8.6

(Positively selected) Role of peptide in positive selection.
Though thymocytes are positively
selected for recognizing self-MHC,
peptides play an important role not
only in the proper expression of MHC
molecules but also in selecting the
Thymocytes self-MHC restricted but exhibiting TCR of varying specificity repertoire of thymocytes.
Apart from MHC and peptides present on antigen-presenting cells, CD4 and CD8 molecules
are also required for the efficient selection of class-II-MHC-restricted/class-I-MHC-restricted
T cells as well as for transition from double-positive (CD4⫹ CD8⫹ ) T cells to CD4⫹ CD8⫺ or
CD4⫺ CD8⫹ T cells. These molecules provide both adhesive and signalling functions, as the blocking
of CD8 or CD4 molecules by non-cytolytic antibodies results in the lack of development of CD8⫹or
CD4⫹cells respectively.
8.3.2 N E G AT I V E S E L E C T I O N
The positive selection allows only those thymocytes to survive that recognize self-antigens bound
to self-MHC molecules with low affinity. Though positive selection is a rigorous “exercise”, the
population of thymocytes that remains after positive selection will almost certainly contain some
» Negative selection ensures thymocytes with autoreactive TCRs. This population of MHC-restricted thymocytes comprises
survival for those T cells that have a thymocytes that bind strongly (with high affinity) to self-peptides and self-MHC molecules and
weak affinity for self-antigen–
self-MHC complex. T cells that
hence are potentially autoreactive.
exhibit a strong reactivity/affinity During negative selection, which occurs in the medulla, the interaction of medullary dendritic
towards self-MHC or self-antigen– cells and macrophages expressing class I and class II MHC molecules with thymocytes that have a
self-MHC complex are deleted.
These cells can potentially cause
high affinity for self-antigen–self-MHC or self-MHC alone results in apoptosis. Thus, negative
autoimmune diseases in an selection potentially eliminates self-reactive T cells, hence allowing the maturation of T cells that
individual. have weak affinity for self-antigen–self-MHC. The deletion of T-cell clones that are self-reactive
(also called clonal deletion) makes the individual self-tolerant. It is now believed that tolerance to
self-antigens is largely because of the fact that T cells are tolerant towards self-antigens. One of the
mechanisms of T-cell tolerance is the deletion of self-reactive T-cell clones which occurs during
negative selection. A brief overview of negative selection is given in Figure 8.7.
» Negative selection occurs in the E X P E R I M E N TA L E V I D E N C E O F N E G AT I V E S E L E C T I O N

medulla of the thymus. Several experimental approaches allow researchers to observe the deletion of thymocytes that
bind with high affinity to self-antigen ⫹ self-MHC molecules. In mice, H–Y antigen is a mol-
ecule that is expressed in male mice only. In one such study, transgenic mice were created that ex-
pressed class-I-MHC-restricted T-cell receptors specific for H–Y antigens. Female mice with these
transgenic T cells have a normal number of thymocytes in the medulla and a normal number of
T cells in the periphery. In contrast, male mice which express H–Y antigens have few single-positive
T cells in the medulla and few very mature peripheral T cells Apparently developing T cells in male
mice expressed transgenic TCR that reacted with H–Y antigen (presented by dendritic cells) and
» There is evidence to suggest that caused clonal deletion of developing T cells.
thymic epithelial cells are involved The best evidence of negative selection comes from the studies of interaction between superan-
in positive selection; and dendritic
cells and macrophages are mainly tigens and double-positive thymocytes. Superantigens are those proteins that bind to the TCR which
involved in negative selection.
Self-peptide
Low-affinity
CD4+
CD8+
binding Thymocyte
Class I/II
MHC
CD4+
CD8+ Thymic epithelial Thymocyte survives
Thymocyte cell
Figure 8.7
Negative selection of T cells. The High-affinity
CD4⫹CD8⫹ thymocytes that recognize binding
self-MHC–antigen complex with high
Class I/II
affinity are deleted ensuring that the
MHC Apoptosis
body does not mount an immune
Thymocyte dies
response against its own antigens.
expresses particular Vβ chain on T cell and class II MHC molecule present on antigen-presenting
cells. Some of these superantigens are produced by retroviruses. These retroviruses can infect germ
cells (such as egg or sperm cells) and get integrated into the animal genome. Most strains of mice
carry such superantigen genes without any ill effect on the animal. Superantigen-encoded protein
are therefore self-antigen in such mice (see Section 8.5 for details).
The superantigen cross-links Vβ chain (of the TCR) and class II MHC molecule. If the supe-
rantigen is present during T-cell development, this superantigen induces cross-linking (in other
words, binding with high affinity) of thymocytes that express a particular Vβ chain (TCR) and
antigen-presenting cells. Hence, all thymocytes that express a particular Vβ chain (TCRs)
specific for that superantigen are deleted from the mature T-cell repertoire. The final out-
come of this selective procedure is a population of mature T cells that are tolerant towards
that superantigen.
The importance of deletion of autoreactive clones of T cells by negative selection can be
seen in lpr mice. lpr mice are genetically predisposed to develop autoimmunity. These mice have
a defect in the apoptotic pathway because they have mutated Fas proteins. Defective Fas pro-
tein cannot transmit the apoptotic “death” signal to autoreactive T cells destined to negative se-
lection. As a result these T cells survive and mice develops autoreactivity against self-antigens
(autoimmunity).
CD4 and CD8 antigens also play a crucial role in the negative selection of thymocytes. Ex-
periments conducted in mice have shown that when anti-CD4 antibodies are administered to double-
positive thymocytes, elimination (that is, negative selection) of these thymocytes is blocked.
Not much is known about the exact mechanism of negative selection. Unlike death by default
which occurs in the absence of positive selection, active death-promoting signals are generated
when immature thymocytes bind with high affinity to antigen. Most of the evidence suggests that
epithelial cells are uniquely effective in inducing positive selection while macrophages and den-
dritic cells as well as thymic epithelial cells can induce clonal deletion.
8.3.3 M E C H A N I S M S O F P O S I T I V E A N D N E G AT I V E
SELECTION
Both positive and negative selection processes involve recognition of self-peptides associated with
self-MHC molecules. In one case (positive selection), TCR showing low affinity of self-peptide–
self-MHC are spared and these T cells survive. In the other case (negative selection), TCR show-
ing high affinity of self-peptide–self-MHC are eliminated. What worried scientists was how two
different signals could be initiated through the same TCR. Two hypotheses have been proposed to
resolve this paradox of MHC-dependent positive and negative selection.
One proposal, called receptor occupancy model or avidity theory states that a low number
of peptide–MHC complexes will induce positive selection (survival) whereas a high number of the
same peptide–MHC complex will cause negative selection (death). This is because high-affinity
ligands would tend to occupy more TCRs than would low-affinity ligands. A developing thymo-
cyte could distinguish a low from a high-affinity ligand by “counting” the number of receptors
occupied over a given period of time. The avidity theory is supported by studies on transgenic
mice. Transgenic mice were created that had T-cell receptors for self-antigens found in the myelin
sheath (myelin basic protein). Under natural circumstances, the antigens bind weakly to self-MHC
(class II) and hence escape negative selection. High-affinity analogues of the same antigens when
injected into the same transgenic mice bind strongly to MHC molecules and generate a large num-
ber of antigen analogue–MHC complex on antigen-presenting cells.The binding of a large number
of these analogue–MHC complexes, localized on antigen-presenting cells induces negative selec-
tion of such thymocytes.
The second hypothesis, called kinetic proofreading distinguishes ligand affinity by “reading”
its occupancy time. It is suggested that low-affinity ligands will have a short occupancy time on
TCRs and thus will lead to positive selection. High-affinity ligands, on the other hand, contact the
TCRs for a longer timescale and induce different signals that initiate negative selection (Werten
et al. 2003).
A diagrammatic representation of receptor occupancy and kinetic proofreading model is
given in Figure 8.8.
CD4+/CD8+
Large number
Thymocyte
of peptide-MHC
ClassI/II MHC+ complex occupied Apoptosis
peptide on thymic epithelial cells Negative selection
Thymic
epithelial cells
CD4+/CD8+ Small number
Thymocyte of peptide-MHC
complex occupied
on thymic epithelial cells
Survival
Positive selection
Thymic
epithelial cells
Receptor occupancy model
Short Longer
Positive contact contact Negative
selection time time selection
of of
thymocyte thymocyte
Figure 8.8 Thymic

Models to explain positive and negative Thymocyte Thymic
epithelial cells
selection. Line diagram explaining epithelial cells
avidity model and kinetic proofreading
model of positive and negative selection.
Kinetic proofreading model
These two theories are now believed not to be mutually exclusive. Thus the overall avidity of
TCR is determined by the:
• affinity of specific TCR for its ligand;
• number of peptide–MHC complexes;
• time of occupancy of ligands on TCR; and
• number of TCR as well as other relevant co-receptor molecules expressed on
the T cells.
What causes double-positive CD4⫹CD8⫹ cells to mature into either CD4⫹TH cells or the CD8⫹Tcyt cell?
The exact pathway and signals that make double-positive T-cells transform to single positive
CD4⫹ or CD8⫹ lineages are not well understood. It could be the strength of the signal, which in
part is determined by the co-receptors. TCRs that have co-engaged CD4 are thought to deliver
a stronger signal because the protein tyrosine kinase, lck, is bound to CD4 more tightly than it
is to CD8 molecule. Such double-positive cells mature into CD4⫹ TH cells [see Figure 8.9(a)]. By
altering the levels of lck in the thymocyte the lineage decision can be modified. It is believed that
maturation to CD4⫹ CD8 lineage might require a stronger receptor signal than CD4⫺ CD8⫹
lineage [see Figure 8.9(b)].
Thymic epithelial cell
Class I MHC CD4+ engaged

CD8
engaged
CD4+ CD8+
lck protein kinase
T cell
lck protein Activation
kinase (Strong signal)
Activation CD8
(Weak signals)
CD4
Faint signal from Strong signal from

weakly bound Ick lck protein kinase interacting Figure 8.9
protein kinase, CD8- with CD4 molecule, (a) CD8⫹ lineage commitment and CD4⫹
lineage commitment lineage commitment. (b) Line diagram
CD4+ lineage commitment
showing how double-positive
T cells evolve into CD4⫹ and CD8⫹
T cells. It is believed that TCRs of DP cells
that have co-engaged CD8 are thought
to deliver a weak signal making the DP
T cell, a CD8⫹ T cell. TCRs of DP T cells
that have co-engaged CD4 are thought
to deliver a stronger signal because
CD4- CD8+ T cell CD4+ CD8+ T cell protein kinase lck is more tightly
associated with CD4. A stronger signal
a) b) makes DP T cell, a CD4⫹ T cell.
8.4 A C T I V AT I O N O F T LY M P H O C Y T E S
The activation of T-lymphocytes refers to changes that occur in T-lymphocytes following antigen
recognition. Antigen recognition by naïve T lymphocytes initiates cell proliferation as well as dif-
ferentiation while antigen recognition by T cells pre-exposed to antigen (effector T cells) triggers
the effector functions of respective T cells that tend to eliminate the pathogen. Effector function
of Tcyt is to specifically lyse the antigen-bearing cells and that of TH cell is to secrete cytokines that
stimulate other T or B cells. Tha activation of T lymphocyte also generates memory T cells which
remain in circulation for a long time. The activation of T cells can be broadly divided into the fol-
lowing steps:
• Recognition of antigen–MHC complex by the T-cell receptor and signal transduction by
the TCR complex;
• Secretion of cytokines by the activated T cell;
• T-cell proliferation and division;
• Differentiation of newly divided cells into effector cells and memory cells; and
• Fall of T-cell response.
These steps are now discussed in detail.
8.4.1 RECOGNITION OF ANTIGEN–MHC COMPLEX

AND SIGNAL TRANSDUCTION BY TCR
The recognition of the peptide–MHC complex by the TCR and the co-receptors CD4 or CD8
provides specificity to the subsequent T-cell response. The TCR recognizes specific peptide ⫹ MHC
complex while CD4 and CD8 co-receptors recognize class II and class I MHC molecules respectively.
The specificity for co-receptor for different MHC molecules accounts for differing specificity of CD4⫹
and CD8⫹ T cells. CD4⫹ cells recognize only class-II-MHC-associated antigens while CD8⫹ T cells
recognize class-I-MHC-associated antigens. The events that occur after antigen recognition consists
of two discrete stages: (a) Immediate events occurring within seconds, which include TCR clustering
and activation of protein tyrosine kinases (PTKs); (b) Early events occurring within minutes, which
involve cytoplasmic transduction pathways.
The TCR complex consists of ligand-binding α and β chains, MHC-binding CD4 or CD8
» The lck protein tyrosine kinase is molecules and a signalling unit that includes a CD3 complex and a ζζ (zeta) chain homodimer.
a member of the Src-family of tyro- The initiation of T-cell activation starts by ligand binding and TCR clustering. The clustering of
sine kinases that is involved in the
T-cell signal transduction pathway receptors and co-receptors brings lck, (an Src-family tyrosine kinase that is associated with the
Its gene is located on chromosome cytoplasmic tail of CD4 and CD8) close to ITAMs (immuno-receptor tyrosine-activated motifs) in
1 in humans and on chromosome the CD3 complex and zeta (ζ) chains.
4 in mice.
ITAMs are nine conserved peptide sequences present on the cytoplasmic portions of CD3
» Knockout mice lacking lck show proteins that include δε, εγ and ζζ chains. Lck phosphorylates tyrosines in the ITAMs of the CD3
defects in T-cell development, while complex and ζ chains. Thus within seconds of receptor clustering, many tyrosine residues within
mice lacking both lck and fyn show
more severe defects than lck-
the ITAMs are phosphorylated. Another protein, tyrosine kinase (fyn), that is associated with CD3
deficient mice. plays a similar role as that of lck.
The phosphorylated ITAMs in the ζ chain become specific “docking sites” for ZAP-70 (for zeta-
associated protein of molecular weight 70 kDa) kinase. ZAP-70 belongs to the Syk family of tyrosine
» Two signalling proteins that are kinases. ZAP-70 contains SH2 (Src-Homology-2) domains that binds to the phosphotyrosines of
involved in T-cell activation are LAT
(linker for activation of T cells) and
ITAMs of the ζ protein. Once bound to ITAM, ZAP-70 is tyrosine-phosphorylated by the lck which
SLP-76 (SH2-domain-containing acquires its own tyrosine kinase activity. Activated ZAP-70 can autophosphorylate itself as well as
leukocyte protein of 76 kDa) which phosphorylate several other cytoplasmic signalling molecules such as adapter proteins LAT and
serves as the docking site and
activator of phospholipase C.
SLP-76 which themselves do not have any enzymatic activity. Once these adapter proteins are phos-
phorylated, they serve as docking sites for other proteins that are involved in a variety of signalling
pathways. A summary of the immediate activation events of T cells is shown in Figure 8.10.
The activation of signalling proteins by ZAP-70 proteins leads to the activation of several signalling
pathways.These pathways include phospholipase-C-initiated pathways, and Ras and Rac pathways.
Antigen-presenting cell
MHC
CD4/CD8 TCR ζ ζ
ε γ ε δ
Adaptor protein
P P P
P P lck P (LAP, SLP-70
P P P P
phosphorylated)
Phosphorylates Phosphorylates ZAP phosphorylates
Figure 8.10
Line diagram showing intracellular
signalling in T-cell activation—an
overview. Activated lck phosphorylates
ITAMs of ζ chains of CD3 complex. Ras pathway Activation of
Phosphorylated ITAMs bind ZAP-70
Rac pathway phospholipase
which itself gets phosphorylated and
Cγ
activated. ZAP-70 phosphorylates
adaptor proteins. These adaptor proteins
Pathways activated
activate three main signal transductions
pathways leading to T-cell activation.
LAT-linker of activation of Tcells; SLP 76 –
SH2 domain containing lecocyte protein
of 78 kDa. ITAMs, immunoreceptor
tyrosine-based activation motifs.
One of the signalling pathways that is initiated by phosphorylation is the phospholipase Cγ

(PLCγ) pathway. Once bound to the phosphorylated adapter proteins, PLCγ is phosphorylated by
ZAP-70 kinase, thus activating it. Once activated, PLCγ hydrolyses phosphatidylinositol (PIP2), a
membrane phospholipid, into two molecules—diacylglycerol (DAG) and inositol 1, 4, 5 triphos-
phate (IP3). These two signalling molecules initiate two important signalling pathways of T-cell
activation. In one pathway, IP3 triggers the release of Ca2⫹ from the endoplasmic reticulum and
there is an increased entry of Ca2⫹ in T cells by an unidentified mechanism, which results in the
rise of intracellular Ca2⫹ (from a resting level of 100 nM to 1,000 nM). The free calcium acts as sig-
nalling molecules by binding and subsequently activating calmodulin-dependent serine/threonine
phosphatase called calcineurin. Calcineurin has an “important” role in the activation of transcrip-
Calcineurin
tion factor NF-AT(nuclear factor of activated T cells). The NF-AT transcription factor is required Calcineurin is a calmodulin-
for the expression of IL-2 and other cytokines needed for T-cell activation and differentiation. An dependent phosphatase. Calcineurin
is responsible for activating the
overview of the signalling pathway initiated by phospholipase C is shown in Figure 8.11.
transcription of the IL-2 gene
In the other pathway, the DAG which is formed activates the enzyme protein kinase C which that stimulates the proliferation
phosphorylates various cellular substrate including the cytoplasmic inhibitor of NF-κB, called IκB. and differentiation of T cells. This
calcium–calmodulin controlled
The phosphorylated inhibitor can no longer bind the transcription factor NF-κB, hence the tran-
protein was originally identified in
scription factor translocates into the nucleus. the extracts of the mammalian brain.
In addition, the phosphorylated and activated adapter proteins also activate the Ras pro-
tein pathway. The Ras protein is a GTP-/GDP-binding protein that connects T-cell receptors
with downstream signalling pathways. When the ZAP-70 Kinase is activated, it phosphorylates
the adapter protein LAT. LAT binds another SH2-domain-containing the protein Grb-2. Once
bound to LAT, Grb-2 is phosphorylated by ZAP-70. Phosphorylated Grb-2 recruits a GTP/GDP
exchange factor Sos.
Sos
Sos catalyses the exchange of bound GDP to GTP, generating active Ras-GTP. Ras-GTP is an Sos is the mammalian
allosteric activator of a cascade of enzymes called mitogen-activated protein (MAP) kinases. This homologue of the Drosophila
protein, son of sevenless.
cascade involves the sequential phosphorylation and activation of three different kinases each of
which phosphorylates and activates the next. MAP kinase pathway which finally leads to the acti-
vation of the extracellular signal-regulated kinase (ERK) enzyme. ERK induction ultimately leads
PIP2 DAG
Activated
adaptor P
protein P
Protein kinase C
Phospholipase CG P P
IP3
ZAP-70 activation
P
P P
IP3 receptor
Endoplasmic
Intracellular Ca2+ store
reticulum
Ca2
CaM Phosphorylation of
Figure 8.11
Ca2+ multiple cellular Detailed diagram of intracellular
substrates events following T-cell activation
showing involvement of ZAP-70, Ras
Calcineurin and Rac pathway. Phospholipase C is
phosphorylated by adaptor proteins.
Active phospholipase C γ hydrolyses
PIP2 to DAG and IP3. IP3 stimulates the
release of calcium from the ER. DAG
Activation of transcription
activates PKC-Ca2⫹. CaM and active
factor, eg. NF, NF-KB, AT PKC stimulate various transcription
factors ( through two different
pathways) that activate T cells. (PIP2—
phosphatidylinositol 4,5-bisphosphate.
DAG—diacyl glycerol, IP3—inositol
Transcription and expression of cytokine and Cytokine-receptor genes trisphospate, PKC—protein kinase C,
CaM—calmodulin, NF-AT—nuclear factor
of activated T cells.
to the activation of transcription factor AP-1. AP-1

is a transcription factor that physically associates
Activated adaptor
ZAP-70 protein-LAT with other factors such as NF-AT and stimulates
P
kinase Grb-2 cytokine synthesis. The details of the activation of
activation Recriuts
P Ras pathway are shown in Figure 8.12.
Phosphorylates Sos-GTP/GDP Another pathway, called Rac pathway is
exchange factor
also activated by the TCR-associated phosphory-
Ras-GDP Ras-GTP lated adapter molecules. In this pathway another
MAP-kinase
GTP/GDP exchange protein, Vav, gets activated
cascade after binding to these activated adapter molecules.
activated Vav acts on the GTP/GDP binding protein, Rac.
ERK activation Rac–GTP once formed, initiates another MAP
Figure 8.12
Activation of Ras pathway. Active ZAP-70 kinase cascade resulting in the activation of c–
kinase phosphorylates adaptor proteins. Jun–NH2–terminal kinase (JNK). Rac–GTP phos-
Phosphorylated adaptor protein binds
Activates transcription factors phorylates and activates c-Jun, a component of the
SH2 domain protein Grb2. Active Grb2,
AP-1,NF-AT
through a series of reactions, activates AP-1 transcription factor. The activation of AP-1
Ras-GDP to Ras-GTP. Ras-GTP activates activates the transcription of the cytokine and
MAP-kinase leading to the activation
of the transcription factors NF-AT and cytokine-receptor genes.
AP-1 that activates T cell. (LAT—linker of Transcription and activation of cytokine The activities of JNK and ERK are eventually
activation of Tcells; ERK—extracellular
and cytokine-receptor genes shut off by tyrosine/theonine phosphatases. The
signal-regulated kinase, AP1—activation
protein-1). activation of Rac pathway and related events is
shown in Figure 8.13.
» c-Jun actually stabilizes the bind-
ing of NF-AT to DNA. Each of these four signal transduction path-
» IL-2 gene transcription, which is
ways initiated by ligand-binding to TCR, contributes to the expression of proteins needed for
a key event in the activation and T-cell proliferation, differentiation and, of course, their effector functions.
proliferation of T cells, is regulated Within hours of T-cell activation, the gene coding for cytokines in T cells undergoes stimu-
by the action of several factors,
including NF-AT, AP-1, NF κB.
lated transcription. This results in the expression and secretion of cytokines from activated T cells.
The principal cytokine produced by naïve T cells is IL-2 which stimulates the growth and differen-
tiation of the T cells. Concomitant with the release
of this cytokine from T cells, activated T cells also
ZAP-70 Adaptor protein
increase their expression of cytokine receptors.
kinase P
Vav
One such antigen is CD25 (α chain of IL-2). The
activation expression of both cytokine and its receptors after
the activation of the T cell makes the T cell more
Rac GTP
receptive to the autocrine growth pathway.
Rac GDP
The T-cell proliferation that occurs after an-
tigen recognition, is mediated primarily by the
JNK
autocrine growth pathway. The principal cyto-
kine involved in T-cell proliferation is IL-2. The
responding T cell secretes its own cytokine which
Activates transcription factor binds to its own cell surface receptor resulting in
Figure 8.13
Activation of Rac pathway. Active GDP/
the proliferation of T cells (clonal expansion). This
GTP activation protein Vav stimulates Rac clonal expansion generates a large number of anti-
protein. Rac–GDP is converted to Rac– Transcription and activation of gen-specific T cells required to capture and elimi-
GTP, which initiates another MAP-kinase cytokine and cytokine-receptor genes nate the pathogen.
cascade that activates JNK. Activated
JNK stimulates transcription factor AP1 The progeny of antigen-stimulated CD4⫹ or
which activates T cells. MAP—mitogen- CD8⫹ T cells differentiate into effector (and/or
activated protein kinase.
memory) cells. CD4⫹ T cells differentiate into
» It is estimated that each naïve
cytokine-secreting TH cells which may secrete a
or virgin T cell circulates at least variety of cytokines. CD8⫹ T cells differentiate into functional Tcyt lymphocytes with the ability to
once in 24 hours from blood to the lyse the target cell. CD4⫹ and CD8⫹ T cells leave the thymus and enter the circulation as resting
lymph nodes and back. This recircu-
lation increases the chances of their cells in the Go stage of the cell cycle.
encounters with specific antigens. When naïve T cells encounter antigen (with MHC) on an appropriate antigen-presenting cell,
Naïve T cells usually survive for a primary response is initiated. T cells enlarge in this response and undergo multiple cell divi-
about 4–6 weeks in the absence of
an encounter with antigen. sions. This multiplication and activation of T cells is brought about by the TCR and co-stimulating
signals. These signals push T cells in the G1 phase and induce the expression and secretion of IL-2.
T cells also respond the to IL-2 by binding IL-2 to the receptors expressed on T cells themselves.
This results in the proliferation of clones of T cells (clonal expansion) that generates a large num-
ber of antigen specific cells required to capture and eliminate the pathogen.The progeny of anti-
gen-stimulated CD4⫹ or CD8⫹ T cells differentiate into effector (or memory) cells.
CD4⫹ T cells differentiate into cytokine-secreting TH cells. There could be two subpopulations
of TH cells, depending on the panels of cytokines they secrete. TH1 subset secretes IL-2, γ-IFN and
TNF-β, and is responsible for a delayed type of hypersensitivity and other cell-mediated functions,
as well as the activation of Tcyt lymphocyte. TH2 subset secretes IL-4, IL-5, IL-6 and IL-10. This sub-
set function as a “helper” for B-cell activation. Cytokines secreted by TH2 cells also stimulate other
TH2-cell development and stimulate a defence against parasitic infections.The two populations of
TH cells and their secreted cytokines are shown in Figure 8.14. About 5–8 per cent of cells TH do not
belong to either TH1 or TH2 cells.These cells, which are CD4⫹ cells are called T regulatory (Treg) cells.
These regulatory cells express the cell surface marker CD25 and Fox3, a transcription factor. These « TH1 cells are involved in cell-
cells suppress or inhibit the action of TH1 and TH2 cells. Tregs recognize antigen associated with class mediated immunity while TH2 cells
II MHC and receive costimulation from B7 molecules present on antigen-presenting cells. “help” in humoral immunity. Tregs
secrete IL-9, IL-10 and the trans-
CD8⫹ T cells differentiate into Tcyt cells.These cytotoxic T cells kill the host cells that are in- forming growth factor β (TGF β )
fected with intracellular pathogens such as virus or intracellular bacteria. The Tcyt cells make direct that inhibit TH1 and TH2 cells.
contact with the target cell and lyse it in an antigen-specific manner. Tcyt -mediated killing may
involve the secreted protein, perforin that “perforates” the target cell membrane or may involve the
stimulation of the target cell’s Fas receptor by T cells’ Fas ligand leading to the apoptosis of the tar-
get cell. Effector T cells, whether TH or Tcyt have a short lifespan, ranging from few days to weeks.
Some of the progeny of antigen-stimulated T cells develop into antigen-specific memory
T cells. (memory TH or memory Tcyt cells). Antigen-stimulated effector T cells last only for a few
days or a few weeks and their response quickly wanes as the antigen is eliminated. Memory T cells
survive for long periods, apparently without a need for continuous antigen exposure. The mecha-
nism of memory cell survival is not yet known. Memory cells accumulate with time, reflecting
encounters with varied pathogen and antigen. The memory T-cell population is responsible for
rapid and enhanced secondary immune response. Memory T cells express high levels of surface
molecules such as CD44 that help in homing these cells to peripheral sites were they encounter
antigen. These cells do not express receptors characteristic of activation, such as the IL-2 receptor.
Unlike naïve T cells, which are activated by dendritic cells, memory T cells can be activated by
dendritic cells, macrophages and B cells. The underlying mechanism by which antigen-stimulated
CD4⫹ or CD8⫹ differentiates into effector cells or memory cells is not currently known.
8.4.2 C O S T I M U L AT O R S A N D T - C E L L A C T I VAT I O N
T cells require two sets of extracellular signals for complete activation and differentiation. The first sig-
« In the absence of costimulation,
nal is the binding of the TCR complex (TCR, CD4⫹ or CD8⫹, CD3 molecules) to the peptide–MHC T cells that encounter antigens
complex displayed on antigen-presenting cells. The second antigen-non-specific costimulatory signal fail to respond and die by
is provided by the interaction of the costimulator expressed on antigen-presenting cell. apoptosis or enter the state of
unresponsiveness.
The best characterized costimulatory molecules expressed on antigen-presenting cell include
B7-1 (CD80), B7-2 (CD86), CD58, CD40. B7-1 and B7-2 bind CD28 and CTLA-4 (cytotoxic
T lymphocyte-associated molecule-4) present on the T-cell surface. CD58 binds CD2 of the T cell,
and CD40 binds CD40L on the surface of the T cell.
G -IFN IL-4
B-cell
TNF-B IL-2 proliferation IL-5
and activation
Activation Involved in
of Tcyt delayed-type IL-6
hypersensitivity
B-cell Activation of Needed for IL-10

proliferation macrophages synthesis of IgE,
IgM, IgA, IgG
Figure 8.14
TH1 cell and its functions TH2 cell and its functions Two populations of TH cells.
» T-cell activation is initiated by B7 molecules, which are expressed on professional antigen-presenting cells such as dendritic
the interaction of the TCR–CD3
complex with antigen–MHC
cells, macrophages and B lymphocytes interact with CD28 and CTLA-4 present on T cells and
molecules on the surface of the cell. induce the production of IL-2, which induces the differentiation of naïve T cells into effector cells,
This interaction initiates a cascade and augments T-cell response to the binding of B7 molecules.
of biochemical events in the T cell,
occurring primarily through an in-
The binding of CD58 (LFA-3) to CD2 which is a T-cell surface protein enhances T-cell
crease in IL-2 secretion by the T cell response to antigens. The binding of CD40 to CD40L (present on the T-cell surface) activates the
and an increase in IL-2 receptors on antigen-presenting cell (that bears CD40) to secrete IL-12 that promotes T-cell differentiation.
the T-cell surface.
However, the binding of costimulators to its ligand on the T-cell surface may not always induce
T-cell activation. Mature dendritic cells express the highest levels of costimulators and hence are
» IL-2 is a potent T-cell growth
cytokine which, in T-cell activation,
the most potent stimulators of naïve T cells.
acts in an autocrine fashion to pro- Apart from dendritic cells, all other professional antigen-presenting cells require activation for
mote the growth, proliferation and expression of costimulatory B7 on their surface. Resting macrophages (that is, not activated) which
differentiation of the T cell recently
stimulated by antigen.
do not express these B7 molecules are unable to activate naïve T cells. Activated macrophages (ac-
tivated by γ-IFN, phagocytosis of bacteria) upregulates B7 (as well class II MHC) molecules and
» Dendritic cells express the highest hence become the activator of naïve T cells (as well as of effector and memory T cells). Similarly
level of costimulators.
resting B cells which do not express B7 molecules fail to activate the naïve T-cell population. Upon
activation, B cells upregulates the expression of B7 molecules, and class II MHC molecules, and
hence acquire the capability to activate the T-cell population.
Costimulation may function in T-cell activation by increasing the level of the same signal trans-
duction pathway that is triggered by the TCR. Alternatively costimulators may activate distinct sig-
nalling pathways that ultimatly converge with those activated by the TCR, or they activate a unique
signal transduction pathway that is totally unrelated to TCR signals. For example, CD28 molecule of
T cells are connected to at least two pathways: Ras-activated MAP kinase pathway and Vav-activated
Rac pathway. However, which of the two is actually used by T cells in vivo is not currently known.
8.4.3 T H E FA L L O F T - C E L L R E S P O N S E
The activation of mature T cells causes them to proliferate and differentiate into effector cell popu-
lation. These large and expanded populations of T cells are no longer needed once the antigen is
eliminated by effector cells. The fall of T-cell population occurs because the majority of antigen-ac-
tivated T-cells die by apoptosis. The reason for this is that as the antigen is eliminated, lymphocytes
» It is estimated that the expanded
population of virus-specific CD8⫹ T are deprived of survival stimuli that is provided by the presence of antigen as well as by cytokines
cells decreases (by apoptosis) by 95 and costimulators.
per cent as the antigen is cleared. Mature T cells, that are present in the peripheral blood are deleted by activation-induced
cell death (AICD). AICD,which is actually apoptosis, is induced through the Fas pathway which
require the presence of Fas protein and ligand of Fas, FasL. The mechanism of Fas–FasL has been
» Fas knockout mice, as well as
discussed in Chapter 12. AICD happens after repeated stimulation, when T cells secrete a soluble
children with Fas mutation develop form of FasL which binds to the Fas present on the same cell or adjacent T cells. This binding
a lymphoproliferative disorder that activates a cascade of intracellular cysteine proteases–caspases resulting in apoptotic cell death of
results in swollen lymph nodes and
early death, suggesting the impor-
T cells. Apoptotic cells are rapidly removed by phagocytes and do not elicit inflammation. It should
tance of Fas in activated, induced be remembered that, through AICD, T cells specific for the antigen is decreased, and there is no
apoptosis. general decrease in T-cell population.
8.5 SUPERANTIGEN-INDUCED
T - C E L L A C T I VAT I O N
» Superantigens show some T-cell Superantigens are usually bacterial or viral proteins that bind simultaneously to T-cell receptors
specificity even though it binds and class II MHC molecules. The cross-linking of T-cell receptors with class II MHC molecules
outside the antigen-binding site.
triggers T-cell activation and proliferation. These superantigens bind to the specific Vβ region of
T-cell receptor (outside the antigen-binding site) and to the α chain of a class II MHC molecule.
Their importance lies in their ability to activate many T cells, leading to the production of a
large amount of cytokines and the induction of pathophysiological abnormalities that act similar
Mls to septic shock. A variety of endogenous and exogenous superantigens are known. These include
Mls or MLS minor lymphocyte-
stimulating antigen are actually exogenous superantigens such as Staphylococcal enterotoxin (SEA, SEB, SEC), Streptococcus
cell-membrane proteins that are enterotoxin and Staphylococcal exfoliative toxin as well as endogenous superantigens such as
encoded by certain viruses.
proteins encoded by certain mammalian viruses inside the mammalian cells; for example, mouse
mammary tumour virus (MMTV) encodes protein Mls.
Antigen-presenting Antigen-presenting Antigen-presenting

cell cell cell
Class II MHC Class II

Superantigen
MHC
Antigen
Non-specific
A chain B chain A chain B chain
antigen
B chain A chain Figure 8.15
Superantigens and their mode of
T cell T cell T cell action. Diagram showing binding of
normal antigens, and exogenous and
Binding of conventional Binding of exogenous Binding of endogenous endogenous superantigen to the TCR
antigen superantigen superantigen and class II MHC molecules.
Endogenous superantigens are not soluble proteins but they are usually membrane-bound
proteins such as Mls1 protein.
Different superantigens show different Vβ regions of T cell specificity and do not indiscrimi-
nately bind to all TCRs. For this reason, these molecules are called antigens and should not be « Superantigens can induce the
called polyclonal T-cell activators. Since they induce a higher than normal antigen response negative selection of thymocytes.
from responsive T cells, these proteins are called superantigens. Table 8.3 compares the immune
response of normal antigens and superantigens.
Superantigens bind to class II MHC molecules outside the antigen-binding site (see Figure 8.15)
on antigen-presenting cells without a need for intracellular processing.This complex is then recog-
nized by the T cell expressing superantigen-specific Vβ segment on its TCR. In other words, the su-
perantigens bind to class II MHC molecules, and this complex has an affinity for T-cell receptor’s
β chains. Superantigens are not only capable of inducing T-cell activation but can also induce
negative selection of thymocytes, if these are present at the time of T-cell maturation.
Superantigens will bind strongly (that is, with high affinity) to the specific Vβ domain of the
« Staphylococcal enterotoxin B (SEB)
TCR and class II MHC on antigen-presenting cells and hence induce a negative selection of spe-
is classified as an exotoxin, since it
cific TCR-bearing thymocytes. If there is a massive deletion of a particular Vβ-domain-expressing is secreted by a pathogen (Staphy-
T cells, these cells will not appear in the mature T cell population. This was easily demonstrated lococcus aureus). Staphylococcus
by the negative selection that occur in mice having endogenous superantigen minor lymphocyte- species thrive and produce toxins
in unrefrigerated meat products,
stimulating antigen (Mls). The mouse strain (AMR strain) has a gene (in fact it is a retrovirus— dairy and bakery products. SEB
MMTV-7) that expresses MIs-1 superantigen and another strain (BIO.BR) has no MMTV integra- normally exerts its effect on the
tion and does not express MIs superantigens. Since MIS-1 superantigen binds to Vβ6, Vβ7, Vβ8.1 intestines and, hence, is termed an
enterotoxin.
and Vβ9 domains in different T cells, these cells are negatively selected and hence do not appear
as mature T cells. BIO.BR mice contain mature T cells bearing, among others, T cells having Vβ6,
Vβ7, V 8.1 and Vβ9 domains.
Antigens Superantigens
Stimulates Usually B and T cells Only T cells
Antigen processing Yes (for cell-mediated response) No
Binds Processed antigen, binds class I Binds only class II MHC molecules
and class II MHC molecules
Location of binding Antigenic determinant binds in Binds outside the antigen-binding
antigen-binding site of MHC site of TCR and with α chain of
class II MHC, cross-linking them
Immune response Specific B- and T cell stimulated Only T cells, that too of particular
specificity (expressing particular Vβ
region) hyperstimulated
Can be involved in Positive and negative selection Negative selection
Examples Proteins, polysaccharides Staphylococcal enterotoxin,
Table 8.3
Minor lymphocyte stimulating Antigens and superantigens—a
antigen-MIs comparison.
8.6 γ δ T LY M P H O C Y T E S
The αβ-expressing thymocytes and γδ-expressing thymocytes are two separate lineages of T cells
» In mice, γδ T cells are in the range
of 1–3 per cent of the total T cells.
derived from a common precursor. In humans around 95 per cent of T cells are αβ T cells while the
remaining 5 per cent of cells are γδ cells.
» Most of the γδ T cells develop in
In foetal thymocytes, TCR gene rearrangement of γ and δ loci occurs first and the surface
extra-thymic sites such as crypto- expression of γδTCR occurs in the third to fourth day after their arrival in the thymus. αβ TCR
patches in the intestine. are expressed about two to three days later. In humans, the expression of αβ receptor begins at 10
weeks, while the γδ receptors are expressed a week earlier.
» The γδ T cells are predominantly The T cells that express functional γδ heterodimer do not express αβ TCRs and vice-versa. The
found in the intestinal epithelium,
pulmonary epithelium and skin. γδT cells represent the major T-cell population in the epithelial tissue of the intestine and lungs,
and are also expressed in the skin. Up to 1 per cent of the epidermal cells are intra-epidermal lym-
» The γδ T cells do not express
phocytes, which are γδ T cells. The γδ T cells exists in three isoforms (see Figure 8.16).
CD4 or CD8 antigens and are not These cells express the γδ TCR–CD3 complex but do not express either CD4 or CD8 molecules.
MHC-restricted. On the other hand, γδT cells of the intestinal epithelium called intra-epithelial lymphocytes
express the γδ TCR–CD3 complex as well as CD8 molecule. Unlike αβ T cells which recirculate
regularly, the γδT cells of the epithelial/epidermal tissues seem to be localized in that region and
do not recirculate. The γδ T cells in different tissues express different Vγ and Vδ genes. However
within the same tissue, γδ T cells are identical; for example, the intra-epithelial lymphocytes found
in intestinal epithelium are monospecific and lack junctional or n-diversity but these cells are dif-
ferent in terms of sequence from γδ T cells found in the skin. Theoretically, the diversity of γδ TCR
repertoire is far greater than the αβ TCR repertoire because in γδ TCR even D–D can join. Actu-
ally, however, the diversity of expressed γδ TCR is very limited, as very few V, D and J segments
are used to make γδ T cells. It is generally believed that the different tissue-specific expressions of
γδ T-cell is actually a specialization of these T cells which have evolved to reside and combat ef-
fectively the antigens encountered in the external milieu of the body surface.
8.6.1 SPECIFICITY OF γδ T CELLS

The γδT cells show an unusual antigen-recognition pattern.They behave like antibodies, that is,
bind to protein antigens without requiring antigen processing and proper presentation together
» It is suggested that γδ T cells can with MHC. The γδ T cells have been shown to directly bind a Herpes-virus protein without the
also recognize antigen presented need of an MHC. It was also reported that γδ T cells can bind to a non-peptide antigen such as
on a non-MHC antigen-presenting
molecule(CD1).CD1 molecules are ligand isopentenyl pyrophosphate (found on some mycobacteria).
expressed on dendritic cells.
8.6.2 FUNCTIONS OF γδ T CELLS
The biologic function of γδ T cells is not known. In absence of a well-defined role, a number of
» PPD antigen is actually a con- functions have been assigned to γδ T cells. It is believed that γδ T cells act like NK cells by me-
served protein found in all organ- diating tumour cell lysis in a non-MHC-restricted manner. A γδ T cell can respond to an antigen
isms including mammals, in which
it occurs as a heat shock protein. called purified protein derivative (PPD) present on mycobacteria. It is now believed that a variety
Heat shock proteins are usually of proteins, including heat shock proteins, class II MHC, bacterial superantigens and CD48 can be
expressed due to a sudden increase recognized by γδ T cells. The schematic representation of γδ T cells with the target antigen/cell is
in temperature, an inflamma-
tory response or a viral infection in depicted in Figure 8.17.
mammalian cells. A comparison between αβ and γδ T cells is shown in Table 8.4.
G chain D chain G chain D chain G chain D chain
Figure 8.16
Schematic diagram showing three
isoforms of γδ T cells. Schematic diagram
showing γδ T cells. Isoform 2 and isoform
3 are characterized by duplication and
triplication of some constant regions of
the γ chain. Isoform 1 GD T Cell Isoform 2 GD T cell Isoform 3 GD T cell
Cellular
Tumour cell, cell
or
harbouring
bacterial
mycobacterium
or
viral antigen CD1 (?)
?
G chain D chain G chain D chain
Induces cell lysis.
Mechanism
unknown
Figure 8.17
Line diagram showing suggested
GD T Cell GD T Cell binding of γδ T cells with the antigen.
Not MHC-restricted Mediates target-cell lysis
Property αβ T cell γδ T cell

T-cell receptor type α/β γ/δ
Percentage of total T cells 95 05
TCR gene rearrangement Late (as compared to γδ T cell Early

in same individual)
TCR expression of membrane Late Early
Express CD4 or CD8 surface Yes No
marker
Recirculation Yes No
MHC-restricted Yes No
Function(s) Involved in cell-mediated and Behaves like antibody. Combats
humoral immunity antigens in external milieu,
involved in tumour cell lysis in Table 8.4
non-MHC restricted manner Comparison of αβ and γδ Τ cells.
8.7 NKT CELLS

A specialized group of T cells has recently been recognized that express both conserved αβ T cell
receptors (TCR) and natural killer (NK) receptors. These cells are appropriately named NKT cells.
These T cells are different from conventional CD4⫹ and « CD1 molecules present glycolipid
CD8⫹ T cells which recognize specific antigens antigens to NKT cells. These anti-
gens are processed in lysosomes.
bound to class I MHC or class II MHC molecules.
NKT cells recognize glycolipid antigens presented by
« NKT cells are believed to be impor-
class I MHC, like molecule CD1 (see Figure 8.18).
tant for suppressing autoimmunity
NK T cells develop in the thymus, where they and promoting tumour immunity.
branch off from the mainstream T-cell precursor,
and randomly generate a TCR that interacts with CD1(antigen-
Glycolipid presenting
CD1. CD1-expressing cells present endogenous
antigen molecule)
glycolipid antigens that have been processed in ly-
sosomes to NKT cells. It is believed that these cells AB TCR
are important for suppressing autoimmunity and
graft rejection. They offer resistance to infection
and promote tumour immunity.
Figure 8.18
T-cell development is a dynamic series of events Line diagram showing NKT cells interact-
through which T cells acquire their phenotypic and NKT cell ing with target cell.
functional competence. It involves undergoing MHC–restriction, expression of receptors and finally

acquiring functional competence as mature Tcyt cell and TH cells. T-cell precursors migrate from the
bone marrow to cortex where they first express T-cell receptors. First the γδ genes are rearranged. If
the γδ genes are rearranged, the T cells express γδ TCR. If β genes rearrange first, T cells ultimately
express αβ TCR. The majority of T cells express αβ TCR. Once αβ TCR is expressed, T cells undergo
both positive and negative selection. In positive selection, those T cells that recognize self-MHC ⫹
self-antigen with weak affinity are saved and the rest die by neglect or apoptosis. Negative selection,
which occurs after positive selection, fine-tunes the selected self-MHC-recognizing cells. This selec-
tion only saves those T cells that have weak affinity for self-MHC plus self-antigen. These cell finally
evolve into either cytokine secreting CD4⫹-TH cells or cytolytic CD8⫹ Tcyt cells that exit in the thymus
and circulate in the blood. Once these cells encounter antigen, they get activated and undergo dif-
ferentiation into effector T cells and memory T cells.
Scanning Electron Microscopy

Probably the first microscopic ob-
servation appears to have been
made by Francesco Stelluti of Electron gun
Heated tungsten
Italy around 1630. However, it was
filament
Antony Von Leeuwenhoek of Hol-
land who constructed a simple
microscope and described a micro-
organism accurately. These simple Electromagnetic lens
microscopes, which could magnify
around 100–300 times, gave a new
direction to studying microscopic
organisms and cells.
Beam deflector
The limit of resolution of any
microscope is governed by its
wavelength. The shorter the wave- Primary electron
length, the lower will be the limit of beam
resolution and the better the mag-
nification. Since electrons also have
radiation-like characteristics, and Electromagnetic lens
much shorter wavelengths (elec-
tron wave has λ ~ 0.004 nm, approx- Back-scattered
imately 100,000 times shorter than and secondary
electrons
visible light), electron wave was
explored to study microscopic speci- Specimen
mens. Electron microscopes provid- Photo-
ed greater clarity and around 100,000 multiplier Cathode ray tube
Specimen
times magnification of microscopic detector for viewing
holder
specimens. It can discern two points
located 4Å (0.4 nm) apart (light has a
resolution limit of only 0.2 μm). Vacuum
The first electron microscope Figure 8.19
The principle of the scanning electron microscope.
prototype was built in 1931 by two
German engineers, Ernst Ruska and
Max Knoll. Electron microscopes are microscopy can be of two main types—transmission electron
highly sensitive scientific instruments that use a beam of accelerat- microscopy (TEM) and scanning electron microscopy (SEM). Around
ing electrons to examine very fine details of an object. Ernst Ruska 1940, Manfred Von Ardenne developed SEM. In light microscopy,
was awarded the Nobel Prize for his contribution in 1986. Electron an image is formed from the radiation that has passed through a
specimen while in SEM an image is formed by the electrons that are beam knock off the electrons from the specimen (secondary elec-
scattered by the specimen. For viewing under SEM, a microscopic trons). These back-scattered and secondary electrons are detected
specimen is first fixed, dehydrated and then coated with a thin layer by a photomultiplier detector which sends these impulses to a cath-
of heavy metal (such as gold), to give a better image. The specimen ode ray tube to form a video image that can be viewed or photo-
is then introduced into the microscope through an airlock, so that graphed (see Figure 8.19).
the vacuum in the microscope is not disturbed. The specimen is The greatest advantage of SEM is that it can provide three-
then scanned with a focused beam of electrons (5 nm in diameter). diamensional images of a range of specimens, from a miniscule cell
As the beam hits the specimen, some of the electrons are reflected to the head of an insect, or even a complete insect. The limit of reso-
back (back-scattered electrons) or sometimes the electrons from the lution of SEM is in the range of 7–10 nm.
S U M M A R Y
• The series of events through which T cells acquire their structural events, triggered by the binding of TCR to the antigen–MHC
and functional competence is called T-cell development. T-cell complex. The various signal transduction pathways activated in the
maturation involves the expression of T-cell receptor and associat- T-cell activation involve phospholipase C-stimulated pathway, Ras
ed accessory molecules, and among other events, MHC-restriction. protein and Rac protein.
• T cells migrate from the bone marrow to the thymus where they • These signal transduction pathways which are initiated by ligand-
become CD4 CD8 (double-negative) pro-T cell. The next stage binding to TCR trigger T-cell activation and their effector function.
is the pre-T cell stage in which the thymocyte undergoes rearrange-
ment of T-cell receptor (TCR) genes. • Antigen-stimulated CD4⫹ T cells differentiate into cytokine-secreting
• If a cell makes a productive γ- and δ-gene rearrangement, it TH cells. CD8⫹ T cells differentiate into Tcyt cells with the ability to
⫹
expresses CD3 γ δ receptor. If it makes a productive β-gene rear- lyse target cells.
rangement it expresses pre-T-cell receptor. • There could be two subpopulations of TH cells—TH1 cells which
• The thymocytes expressing pre-TCR develop into CD4⫹ CD8⫹ function in cell-mediated immunity and delayed hypersensitivity,
double-positive (DP) thymocytes. and TH2 cells which “help” B-cell function and humoral immunity.
• These DP thymocytes undergo positive and negative selection to • Apart from the binding of TCR and CD8/CD4 molecules to the
develop into CD4⫹ αβ or CD8⫹ αβ T cells. peptide–MHC complex, additional stimulatory signal is provided
by costimulatory molecules such as B7 present on antigen-
• Positive selection allows those cells to survive that recognize presenting cell.
self-peptide ⫹ self-MHC with weak affiniy. DP T cells that do not
recognize self-MHC are eliminated. The DP T cells hence become • Superantigen can trigger T-cell activation and proliferation by
self-MHC-restricted. simultaneous binding to Vβ region of TCR outside the antigen-
binding site and the α chain of class II MHC molecules.
• In negative selection, T cells that recognize with high affinity
self-MHC or self-MHC ⫹ self-antigen are eliminated. Negative • About 5 per cent of T cells are γδ CD3⫹ T cells which are different
selection allows only those T cells cells to survive that recognize from the rest of the 95 per cent of αβ T cells. γ δ T cells are present
self-MHC with weak affinity or recognize self-MHC ⫹ self- in the epithelial tissues of lungs, intestine and skin where they
antigen with weak affinity. Finally, DP T cells mature into CD4⫹ or combat effective antigens from the external milieu of the body.
CD8⫹ T cells. They do not show MHC-restriction and can bind non-peptide
antigen.
• Activation of T cells refers to changes that occur in T lymphocytes
following antigen recognition. It is accompanied by cell prolifera- • NKT cells represent another subset of T cells that expresses both
tion and cytokine secretion, and differentiation into effector cells T-cell receptor and NK cell receptor. These cells recognize glyco-
and memory cells. lipid antigens presented on class I MHC, like molecule CD1.
• Activation involves a series of intracellular signal transduction
K E Y W O R D S
• αβ TCR 163 • positive selection 165 • TH2 cells 175

• B7 175 • NKT cell 160 • T-cell differentiation 176
• costimulators 176 • negative selection 168 • T-cell maturation 161
• calcineurin 173 • superantigens 176 • Ras 172
• double-positive cell 163 • T cell 160 • Rac 174
• double-negative cell 162 • T-cell activation 172 • ZAP-70 172
• γδ T cell 160 • TH1 cells 175
1. Though a T cell never encounters foreign antigen during its de- 2. What are γδ T cells? How are they structurally and functionally dif-
velopment, yet the T-cell repertoire that matures can recognize ferent from αβ T cells? What type of antigens do they recognize?
the self–MHC–foreign antigen complex. How is this amazing feat 3. Superantigen should not be called a polyclonal T-cell activator.
achieved? Why? Do you think it also evokes some humoral response?
HINT —Only those T cells are allowed to survive that recognize self-MHC I 4. Which signalling pathways are evoked during T-cell activation?
self-antigen with weak affinity. Self-MHC I self-antigen that shows weak Describe and illustrate your answer.
affinity for TCR mimics self-MHC I foreign peptide that shows high affinity 5. How does a T cell discriminate between peptide–MHC complexes
for TCR. of differing affinities? What are the two most popular hypotheses
for this observation?

1. T cells are positively selected in thymus, if it binds with: 6. Superantigen binds:
(a) Low affinity with self-MHC ⫹ self-peptide (a) Class II MHC molecules only
(b) Low affinity with self-MHC ⫹ foreign peptide (b) Class I MHC ⫹ TCR
(c) High affinity with self-MHC ⫹ foreign peptide (c) Class II MHC ⫹ TCR
(d) High affinity with self-MHC ⫹ self-peptide (d) Class I MHC molecules only
2. Pre-T cell expresses all except: 7. Which one of the following is NOT needed for γδ T-cell
(a) α chain recognition:
(b) β chain (a) γδ TCR
(c) pre-Tα chain (b) MHC
(d) CD3 (c) Glycolipid antigen
3. Negative selection eliminates T cells that:
8. NKT cell can be associated with all of the following, except:
(a) Have high affinity for self-antigen ⫹ self-MHC
(a) αβ T-cell receptor
(b) Have low affinity for self-MHC alone
(b) NK-cell receptor
(c) Have low affinity for self-MHC ⫹ foreign antigen (c) Peptide antigen
(d) Have high affinity for self-MHC ⫹ foreign antigen (d) Lysosome
4. A subset of T cells that plays important role in humoral 9. Positive selection takes place in this region of the thymus:
immunity is: (a) Cortex
(a) TH1 cells (b) Medulla
(b) TH2 cells (c) Subcapsular region
(c) Tcyt cells (d) High endothelial venules
(d) All of the above
10. Which of the following express B7 molecules under resting
5. Which of the following is NOT a costimulator expressed on state:
antigen presenting cell? (a) Dendritic cells
(a) CD80 (b) Macrophage
(b) CD86 (c) B cells
(c) CD40 (d) None of the above
(d) CD2

1. α chain of αβ T-cell receptor is not expressed until thymocytes 4. Memory cells can differentiate into either CD4⫹ or CD8⫹ T effec-
are double-positive. tor cell.
2. Those T cell are positively selected that bind with strong affinity 5. γδ T cell can bind unprocessed antigen.
foreign antigen on self MHC.
3. Both class I and class II MHC molecules are required for positive
selection.
Barton, M. J. and A.Y. Rudensky (1999). “Requirement for Nossal, G. J. (1994). “Negative Selection of Lymphocytes”, Cell 76:
Diverse, Low Abundance Peptides in Positive Selection of 229–239.
T-Cells”, Science, 283: 67–70.
Saint-Ruf, C., K. Ungewiss, M. Groettrup, L. Bruno, H. J. Fehling
Cayabyab, M., J. H. Philips and L. L. Lanier (1994). “CD40 and H. Von Boehmer, (1994). “Analysis and Expression of a
Preferentially Costimulates Activation of CD4⫹ T-Lymphocytes”, Cloned Pre-T Cell Receptor, Current Opinion in Immunology”,
Journal of Immunology, 152: 1523–31. 266: 1208–12.
Godfrey, D. I., D. G. Pellicci and M. J. Smyth (2000). “The Von Bochmer, H. (1994). “Positive Selection of Lymphocytes.”
Elusive NKT Cell Antigen—Is the Search Over”, Science, 306: Cell 76: 219–28.
1687–89. Von Boehmer, H. (1999). “T-Cell Development: What Does
Jameson, S. C., K. A. Hogquist and M. J. Bevan (1999). “Specific- Notch Do for T-Cells”, Current Biology, 9: R186–88.
ity and Flexibility in Thymic Selection”, Nature, 369: 750–52. Werlen, G., B. Hausmann, D. Naeher and E. Palmer (2003).
Janeway, C. A. Jr and K. Bottomly (1994). “Signals and Signs for “Signaling Life and Death in the Thymus: Timing Is Everthing”,
Lymphocyte Responses”, Cell, 76: 275–285. Science, 299: 1859–63.
Merkenschlager, M , D. Graf, M. Lovatt, U. Bommhardt, R. Zamoyska
and A. G. Fisher (1997). “How Many Thymocytes Audition for
Selection?”, Journal of Experimental Medicine, 186: 1149–58.
In early 1960s, J. F. A. P. Miller proposed that the thymus was the “The best
source of immunocompetent lymphocytes. It was initially thought education in the
that the thymus gave rise to a single homogenous population of world is that got
lymphocytes that were involved both in cell-mediated and humoral by struggling to
responses. The first hint that there could more than one sub- get a living.”
—WENDELL PHILLIPS
population of lymphocytes was suggested by F. M. Burnett and his
associates in 1962 when they found out that the removal of the
thymus crippled cell-mediated immunity, while the removal of the
bursa of Fabricus in chickens caused a decrease in antibody
production. It was Miller and his student G. F. Mitchell who, in 1968,
demonstrated that antibody-producing (B) cells arose from the bone
marrow. The deconstruction of the developmental stages of B
After reading this chapter, you
lymphocytes started with the discovery of pre-B cells by M. D. Cooper should be able to:
in 1976. The λ5 and V pre-B chains that constitute surrogate light • Describe the different stages of
B-lymphocyte development
chains of pre-B-cell receptors were discovered by Shiv Pillai and David • Differentiate between B1B and
B2B lymphocytes
Baltimore and associates, and its gene was cloned by F. Melchers and
• Explain the signalling cascades
his collegues in the late 1980s. Over the years, studies in mice with that activate B cell
homozygous deletions in different genes such as RAG, Igα, Igβ, V pre-B thymus-dependent and
thymus-independent antigens
and other proteins that are important in B-cell developments led to the • Briefly summarize
contact-mediated and
elucidation of the development pathway of B cells. Figure 9.1 shows a cytokine-mediated activation
of B cells
scanning micrograph of a B cell—the source of antibodies.
• Describe germinal centre
reactions
• Define the mechanism of
somatic hypermutation
• Explain the varying modes
of regulation of the immune
response
B-cell Development
and Activation
9.1 INTRODUCTION
9
The principal events that occur during the production of plasma cells and memory B cells can be
categorized into three broad stages: development of B lymphocytes, activation of mature B cells,
and differentiation of activated B cells into plasma cells and memory cells.
9.2 B-CELL DEVELOPMENT

The pluripotent haematopoietic stem cells undergo the commitment process that gives rise to the multi- « Pax-5 stands for paired box gene
ple cell lineages which constitute blood cells. It is generally believed that commitment to the B-lymphoid activator 5. Pax-5 is a transcription
factor that commits haematopoietic
lineage depends on the transcription factor, Pax-5. Pax-5 is expressed exclusively in the B-lymphocyte stem cell to the B-cell lineage. In-
lineage that is believed to instill B-lymphoid commitment in the haematopoietic stem cells. activation of Pax-5 precludes B-cell
The maturation of B lymphocytes from committed precursor occurs in the bone marrow. development.
Before birth, B cells are generated and mature in the yolk sac, foetal liver and, to some extent, the
foetal bone marrow.
Figure 9.1
Scanning electron micrograph of
B cell. (Reproduced with permission from
The Journal of Experimental Medicine,
1973, 138: 607–624. Copyright 1973. The
Rockefeller University Press.)
9.2.1 S TA G E S O F B - LY M P H O C Y T E D E V E L O P M E N T
B-lymphocytes, during their development, undergo sequential stages, each of which is character-
» Pro-B cells have tyrosine ized by the expression of a specific pattern of immunoglobulin genes as well as the expression
phosphatase CD45R marker ex- of other phenotypic markers. The earliest bone marrow cell committed to B-cell lineage is called
pressed on the membrane (B220 progenitor B cell or pro-B cell. In the pro-B cell stage, the μ (mu) heavy chain rearrangement oc-
in mice). They also have B-specific
markers such as CD19 and CD10. curs. The first rearrangement of DH to JH gene occurs, followed by ligation VH to DHJH. The enzyme
terminal deoxyribonucleotidyl transferase (TdT), that catalyses the insertion of N nucleotides at
DH–JH and VH–DHJH coding joints, is active in the pro-B-cell stage.
Pro-B cells require direct contact with stromal cells for their development. This direct contact
» IL-7 plays an important role in B-
is mediated by the cell adhesive molecule VLA-4 on the pro-B cell and its ligand VCAM-1 on stro-
cell development. Knockout mice mal cells. Once the contact is made, the stem cell factor (SCF) present on stromal cells binds to its
lacking IL-7 show blocked B-cell receptor on Pro-B cell called c-kit, which is a tyrosine kinase. The binding activates c-kit tyrosine ki-
differentiation at the pro-B-cell
stage.
nase activity, which in turn induces proliferation and differentiation of pro-B cells into pre-B cells.
Pro-B cells divide within the bone marrow filling the extra-vascular spaces in sinusoids in the
shaft of the bone. The pre-B cell represents the next stage of development. The pre-B cell expresses
the receptor for cytokine IL-7 which is secreted by stromal cells. IL-7 downregulates cell adhesion
molecules on the pre-B cells, and the pre-B cells no longer require direct contact with stromal cells
» Max Cooper and his associates for their growth but continue to require IL-7 for growth and proliferation.
discovered pre-B cells in 1976. Hence, the pre-B cells have a detectable cytoplasmic μ heavy chain. Some of the μ heavy chains
associate with surrogate light chains, namely, λ5 and pre-B chain. They are called surrogate light
chains because they associate with heavy chains just as light chains (λ or κ) but are themselves
invariant, that is, they do not have a variable region. The complex of μ heavy chain and λ5/pre-B
chain is called pre-B cell receptors (see Figure 9.2). These receptors are expressed on the cell sur-
face at low levels in association with Igα and Igβ.
» Surrogate light chains, λ5 and The pre-B-cell receptors are required for stimulating the proliferation and continued matura-
pre-B chains, are immunoglobulin-
like peptides. These were tion of the developing B cells. The critical role of the pre-B-cell receptor is illustrated by studies of
discovered by Shiv Pillai, David knockout mice and, in rare cases, of human deficiency of these receptors. In mice, if gene encoding
Baltimore, N. Sakaguchi and other μ or surrogate light chain λ5 is knocked out, B-cell development was shown to be blocked. This
members of their team in the
mid-1980s. suggests that μ-λ5/pre-B chain complex delivers signals required for B-cell maturation and if either
gene is disrupted, pre-B cells do not proceed to the mature B-cell stage. It is not known what the
» V pre-B resembles the VL domain
μ-λ5/pre-B chain complex recognizes or what actual stimulus it provides to pre-B cells, but it is
and λ5 resembles the CL-like
immunoglobulin folding domain speculated that it might be involved in shutting off VH to DHJH rearrangement on the other allele
thus leading to allelic exclusion.
» It has recently been suggested Once a pre-B-cell receptor is expressed, each pre-B cell divides to produce 32 to 64 progeny
that heparin sulphate or Galectin-1 cells. These 64 pre-B cells step into the next stage of maturation which is the early immature
may be the ligand for pre-B-cell
receptor. These ligands bind
B-cell stage. In this stage, each developing B cell produces either a κ or λ light chain and, because
pre-B-cell and signal cell survival of allelic exclusion, only one light chain isotype is expressed on the membrane of B cells. The light
and proliferation. chain complexes with the μ heavy chain and the assembled IgM molecules are expressed on the
cell surface (together with Igα and Igβ). The membrane-bound IgM-expressing B-cells are called
Ligand-binding Antigen-binding
site ( ) site
L or Light
Vpre-B chain
Surrogate
light chains L5
Disulphide bond
M Heavy Disulphide bond M Heavy chain
chain
ITAMs ITAMs
Ig B IgA Ig B Ig A
Figure 9.2
Signal transducing Signal transducing
Line diagram explaining the difference
between pre-B-cell receptor and B-cell
receptor. Pre-B-cell receptor B-cell receptor
B-CELL DEVELOPMENT AND ACTIVATION 187
or
Pre-B-cell IgM IgG
receptor Effector IgA
VLA-4 B cell IgE
Cytokine receptor
Stem Pro-B cell Pre-B cell Immature Mature
cell B cell B cell
Figure 9.3
Memory Line diagram of maturation pathway of
B cell B cells in the bone marrow.
immature B lymphocytes. The schematic representation of the different stages of B-lymphocyte

development is shown in Figure 9.3.
As expected, since the heavy chain rearrangement occurs in the pro-B-cell stage and the light-
chain rearrangement occurs in the pre-B-cell stage, both stages express recombinase enzymes
RAG-1 and RAG-2. The different stages of B-cell maturation as visualized by different phenotypic
and functional changes is listed in Table 9.1.
9.2.2 N E G AT I V E S E L E C T I O N O F B C E L L S
The immature B lymphocytes expressing membrane IgM do not proliferate and differentiate in
response to antigens. In fact, their encounter with antigens (which are usually self as only self-
« It has been established that about
antigens are normally present at that time) in the bone marrow may lead to death or functional 90 per cent of the total B cells pro-
irresponsiveness. This property, called negative selection of immature B cells, leads to the elimina- duced each day die by apoptosis
tion (or clonal deletion) of immature B cells specific for self-antigens present in the bone marrow. because of negative selection.
However, this is not a simple single-step mechanism. Recent works of Nemazee and Burt using
transgenic mice show that the following steps occur in the negative selection of B lymphocytes:
• Once the immature B cells encounter the self-antigen, their development is arrested. Receptor editing
Receptor editing refers to the gene
Self-antigens that mediate negative selection are polyvalent, and deliver strong signals to rearrangement of the secondary
IgM-expressing immature B-lymphocytes. antigen receptor that allows B cells
• The B cells try to save themselves by changing or “editing” their receptors by changing to replace an inappropriate receptor
by a new receptor. Receptor editing
their light chains (but not heavy chains), a phenomenon called receptor editing. In this usually involves κ light chains.
process, RAG(s) are reactivated, generating an additional light chain V–J recombination
Immature Mature Naïve

Pro-T Pre-T B cells B cells
Location Bone marrow Bone marrow Bone marrow Peripheral circula-
tion
Surface markers CD19⫹ CD43⫹ B220, CD43 IgM, CD43⫺ IgM, IgD*
CD10⫹, CD45R⫹
DNA, RNA Rearranged Rearranged Light chain (κ or Rearranged heavy
H-chain gene, H-chain gene, λ) rearrangement and light chains,
μmRNA μmRNA occurs, κ or λ Cμ/Cδ mRNA
mRNA
TdT expression ⫹⫹ ⫺⫺ ⫺⫺ ⫺⫺
Antigen receptors None Pre-B-cell receptor Membrane IgM Membrane IgM,

IgD*
Stimulation by No Not known Yes (self-antigen) Yes (foreign
antigen antigen)
*IgD are not expressed on B1B cells; (TdT—Terminal deoxyribonucleotidyl transferase). Table 9.1
Stages of B-cell maturation.
Self-antigen
can bind
Receptor editing
failed
Light chain unchanged
Apoptosis
IgM
of B cell
+ Self-antigen No binding of
self-antigen
Immature
B cell
Receptor editing
successful
Figure 9.4 Light chain changed

Line diagram explaining the process of
receptor editing. B cell survives
» David Nemazee, an immunologist and consequently new immunoglobulin light chains. Figure 9.4 shows the process of
working in Denver, discovered receptor editing that occurs in B cells.
receptor editing in the 1990s.
• The edited light chain together with the original heavy chain is then expressed. These
newly edited IgM-expressing immature B cells usually convert self-reactive immature
B cells into cells that are not self-reactive, thereby rescuing the cells from an otherwise
confirmed cell death by negative selection.
• Cells that do not succeed in replacing the light chains to change their specificity, die.
After this stage of negative selection, cells co-express μ and δ heavy chains in association with κ or
λ light chain and therefore produce both membranes IgM and IgD. Both classes of antibodies have
the same specificity. The expression of IgM and IgD by B cells is accompanied by the acquisition of
functional competence and such cells therefore referred to as mature naïve B cells. These mature B
cells leave the bone marrow and enter the circulation and peripheral lymphoid organs. These IgM+
IgD+ cells which have not yet encountered antigen are called naïve B cells. Once the transformation
is made into the mature naïve B cell stage, antigen recognition leads to proliferation and differentia-
tion and not apoptosis or receptor editing.
9.2.3 B1 SUBSET OF B CELLS

» The B2B subset of B cells, which There are two common subsets of B cells. The B1B cells and the more common B2B cells.
constitute about 90 per cent of the
total B cells in humans, are com- The B1B subset of B cells, have some unique features of immunoglobulin gene expression and
monly referred to as B lymphocytes. maturation. B1B cells develop earlier during ontogeny than B2B cells. In humans as well as in mice,
B1B cells constitute 5–10 per cent of B1B cells appear to originate from the foetal liver and to a lesser extent from bone marrow. B1B
the total population of B cells.
cells express IgM but no IgD, and also express CD5 molecules, but CD5 negative B1B cells are also
» Depending on the expression found in normal individuals.
of the CD5 marker, B1B cells have In animals having B2B cells as the major B cells, the B1B population is found as the self-
been subdivided into two types: renewing B-cell population of the peritoneum. They are found in low concentrations in secondary
B1Ba cells which express CD5 and
B1Bb cells which do not express the tissues such as the lymph nodes and spleen. B1B cells express a limited repertoire, and have a low
CD5 antigen on their surface. affinity for variable chains with less junctional diversity than conventional B cells. This is because
in B1B cells there is less somatic hypermutation and class switching than in the B2B subset of
B cells, which generates limited antigen-receptor repertoires. These B1B cells spontaneously secrete
IgM antibodies that interact with invading microorganisms and rarely with self-antigens. These IgM
antibodies secreted by B1B cells are present in the individual without any antigenic stimulation « B1B cells can spontaneously
secrete IgM antibodies. These
and hence are sometimes called natural antibodies. antibodies are “normally” secreted
The B1B population responds to carbohydrate antigens in a much better way than to protein by an individual without any overt
antigens. Since there is not a great deal of information about whether B1B cells serve a special func- antigenic stimulation and hence are
called natural antibodies. These IgM
tion in the immune response, it is safe to assume that the IgM produced serves as a major line of antibodies contribute most to the
defence against microbes in a specific site such as the peritoneum. IgM antibodies found in the serum.
9.3 A C T I VAT I O N O F B C E L L S « B1B cells are considered to be a

part of the innate immune system
The proliferation and differentiation of mature B cells in response to foreign antigens occurs in the while B2B cells are considered to
peripheral lymphoid organs. These antigen-driven processes which result in the generation of antibody- be a part of the adaptive immune
system. These cells are found mainly
producing cells (and memory cells) are referred to as activation of B lymphocytes. The activated ma- at mucosal sites and provide the
ture B cells proliferate and differentiate into antibody-secreting plasma cells and non-antibody-secreting major line of defence against bacte-
memory cell (see Figure 9.5) that can last for weeks, months or even years. In the absence of any activa- rial invasions at these sites.
tion, naïve B cells die within a few weeks. The process of activation of B cells with the resultant genera-
tion of antibody-producing plasma cells follows a distinct sequence:
• the recognition phase initiated by the interaction of antigens with receptors IgM or IgD
expressed on mature B cells;
• the proliferation phase which results in the clonal expansion of the antigen stimulated
B cells; and
• the differentiation of the B cells with the resultant generation of effector cell-Plasma cell,
(that actively secrete antibodies) and memory B-cells.
9.3.1 ANTIGEN
RECOGNITION Antigen
The activation of antigen-specific B
lymphocytes is initiated by the bind-
ing of antigen to membrane immu- IgM or IgD
noglobulin molecules which act as Antigen recognition
antigen receptors of mature B cells.
The binding of antigen to the
receptor results in two events: Resting B cell
• It induces the clustering

of membrane receptors
that initiates the process
of activation.
• The bound antigen is
endocytosed by B cells,
hydrolysed, processed
and presented on the
surface of B cells.
B-cell
The clustering of membrane recep- proliferation
tors brought about by the binding
to multivalent antigen is respon- B lymphoblast
sible for one of the ways of B cell
activation. The other way, which
does not require antigen mem-
brane receptor clustering, needs
B-cell
TH cells, and is discussed separate- + differentiation
ly in this chapter. It is believed that
these membrane receptors, that is, Plasma
mIgM and mIgD, have a short (3 cell
amino acids: lys, val and lys) cyto- Memory Figure 9.5
B cell Line diagram showing the activation of
plasmic tail which is too short to B cell into effector and memory cell.
CR2(CD21)
be able to transduce
mlg
activating signals. The
Ig domains
membrane immuno-
CD 19 globulin is associated
with the disulphide-
IgA Ig B IgA Ig B CD81 (TAPA-1) linked heterodimers
Igα and Igβ. Two
molecules of the Igα/
Igβ heterodimer (also
Figure 9.6 known as CD79α and
Line diagram showing B-cell β) non-covalently as-
antigen-receptor complex. (TAPA—
ITAMs
sociate with mIg to
transmembrane protein; mIg—
form the B-cell recep-
membrane Ig). B-cell receptor B-cell co-receptor
tor (BCR).
B-cell receptor Thus the BCR has functionally two parts—antigen-binding mIg and signal-transducing
One surface antibody plus dimer of the Igα/Igβ heterodimer (see Figure 9.6). The cytoplasmic domains of Igα and Igβ
two molecules of the Igα–Igβ have a tyrosine-rich motif, ITAMs (immunoreceptor tyrosine based activation motifs), that are
heterodimer combine to form a B-
cell receptor. The mIg are primarily of found on CD3 and ζ proteins of T cell receptors. The following events take place after receptor
either the IgM or the IgD type. cross-linking.
Tyrosines in the ITAMs of Igα and Igβ are phosphorylated by the action of tyrosine kinases
» mIgG and mIgE have 28 amino such as lyn, blk and fyn which are associated with the B-cell antigen receptor complex.
acids. mIgA has a 14-amino-acid tail
which is slightly longer yet unable The phosphorylated Igα and Igβ then bind the tyrosine kinase, syk (via SH2 domain, src-
to generate the required signals. homology-2). Syk is then phosphorylated by the B cell-receptor-associated kinases such as blk, fyn
» Lyn, blk and fyn kinases belong to or lyn. It then auto-phosphorylates.
the Src family of tyrosine kinases. Syk as well other B-cell-receptor-associated tyrosine kinases (such as src kinases) activate nu-
Src tyrosine kinases are a family merous other signalling molecules. These include phosphophatidylinositol-specific phospholipase
of non-receptor protein tyrosine
kinases that play an important role Cγ1 (PLCγ1) which degrades membrane phosphatidylinositol bisphosphate (PIP2) to form inosi-
in cell signalling. Tyrosine kinases of tol triphosphate (IP3) and diacylglycerol (DAG). IP3 increases intracellular Ca2+ by augmenting
the Src family can be expressed in a the influx of Ca2+ from outside the cell and mobilizing intracellular stores of Ca2+ (stored in the
variety of cell types and can have a
variety of locations within the cell. endoplasmic reticulum). The increased Ca2+ causes protein kinase C (PKC) molecules to move
They can be associated with either towards the membrane where it is activated by DAG. The activated PKC in turn phosphorylates a
the plasma membrane, the endo- number of substrate proteins such as transcription factor-CREB (cAMP response element/B-cell).
plasmic reticulum, or the nuclear
membrane. The elevated Ca2+ also stimulate other Ca2+-dependent proteins (such as calmodulin) and enzymes
that activate several transcription factors, including NF-AT. Syk kinase from activated BCR also
» Syk is the B-cell equivalent of
causes phosphorylation of the inhibitor of NFκB (IκB) which releases the active transcription fac-
ZAP-70 that is present in T-cells.
Those mice in which the syk gene tor NFκB which can then enter the nucleus.
is knocked out are unable to Syk kinase also activates the Ras protein, which in turn activates the mitogen-activated pro-
transduce signals from the B-cell tein kinase (MAPK) cascade. The phosphorylated MAPK is activated, and translocates itself in the
receptor.
nucleus where it activates a transcription factor, Tfa. The sequence of events of signal transduction
» Src, the first identified protein in the B cell is shown in Figure 9.7.
tyrosine kinase, is the product of All these signalling cascades ultimately activate the transcription factors which stimulate the
the first characterized proto-
oncogene, src. transcription of cytokine and immunoglobulin genes all of which ultimately leads to the activation
of B cell. The same signalling pathways are used by naïve B cells (which have mIgM and mIgD) or
other B cells which have undergone isotype switching and express mIgG, mIgA and mIgE. This is
because all mIg associate with signal transducing Igα and Igβ molecules.
9.3.2 SIGNALLING THROUGH B-CELL CO-RECEPTOR

COMPLEX
The B-cell co-receptor is a complex of three proteins CR2 (CD21), CD19 and CD81 (TAPA-1).
The CR2 component is a receptor of C3d, a breakdown product of the complement system. CR2
binds to the C3d-opsonized microbial surface via its affinity for C3d molecules. CD19 is a mem-
ber of the immunoglobulin superfamily and has a long cytoplasmic tail and three extracellular im-
munoglobulin-fold domains. The cytoplasmic tail of CD19 is rapidly phosphorylated (tyrosine
phosphorylated) upon appropriate stimulation and this augments the signalling pathway set in
motion by BCR cross-linking. CD81 or TAPA-1 (transmembrane-protein-1) is a transmembrane
protein that spans the membrane four times. In addition to this stimulating co-receptor, there is
PIP2
P P P
P P
PLC 1 P Ras protein
SyK
SyK Activates
DAG IP3 MAP-kinase

NF- B cascade
I B
P
PKC Elevated Ca2+
Figure 9.7
CREB Calmodulin Line diagram showing signal
transduction by the B-cell antigen
receptor complex. Syk kinases activate
Activates
Ras protein which in turn activates the
NFAT Transcription NF B MAP kinase cascade. The docking of
CREB Tfa
factors Transcription Syk on the BCR also activates PLC γ1
Transcription
factor that hydrolyses PIP2 to produce DAG
factor and IP3, two second messengers. Syk
Binds
also phosphorylates 1κB resulting in
DNA its dissociation from NFκB. Several
signalling pathways are initiated that
lead to the activation of transcription
factors. (CREB—cAMP response element/
Activation of cytokine and immunoglobulin B cell, NFκB—nuclear factor κ of B cells;
gene leading to B-cell activation. 1κB—inhibitor of NFκB).
an inhibitory molecule CD22, which is associated with BCR. The stimulation of CD22 makes B
« The TAPA or CD81 molecule acts as
cells difficult to activate. a receptor for the Hepatitis C virus.
The signalling through the B-cell co-receptor complex involves the proteolytic fragment of It is estimated that about
the complement C3d which is generated in response (via an alternative pathway) to microbes or the 180 million people are currently
infected with this virus that causes
antigen–antibody complex (classical pathway). When antigen coated with C3d is bound by the B cell, hepatitis and cirrhosis, often lead-
the antigen is bound by mIg and the C3d complement binds to the CR2-receptor present on B-cell ing to hepatocellular carcinoma.
surface (see Figure 9.8). This binding, cross-links the co-receptor complex with the BCR. This re-
« In vitro studies have shown that if
sults in the interaction of the CD19 component of the co-receptor with the Igα/Igβ of BCR, and only BCR (and not co-receptor com-
consequently its phosphorylation. The phosphorylation of CD19 allows a number of signalling plex) is involved in B-cell activation,
molecules such as lyn, to bind CD19. Since CD19 is associated with BCR, the delivery of these 104 molecules of BCR are needed.
Those cells that have a co-receptor
signalling molecules contributes to the hastening of the activation process. It has been shown that complex associated with the BCR
the binding of the C3d to protein antigen makes it at least 1,000-fold more immunogenic than the complex need only 100 molecules
antigen alone. of BCR for B-cell activation.
9.3.3 P R O L I F E R AT I O N P H A S E
The cross-linking of the B-cell receptor (and co-receptor) induces a series of events that leads to
B-cell proliferation and finally the next-phase, differentiation.
Naïve or resting cells are non-
dividing cells that are at the G0 stage of Antigen
the cell cycle. Antigen-induced cross- C3d
linking of BCR pushes the B cells into CR2
G1, S, G2 and finally into mitosis phases Figure 9.8
(M phase). The B cells start expressing Schematic diagram showing cellular
or increasing the expression of costimu- IgB IgB CD19
events induced by antigen-mediated
CD81 cross-linking of BCR complex. Antigens
lators such as B7-2 (CD86) and then later with covalently bound complement
B7-1 (CD80). Because of the expression fragment C3d (or C3b) can cross-link
of co-receptors, the B cells acquire the P P mIg (which binds normal antigenic
determinants) and CR2 (which binds
capacity to activate TH lymphocytes. The P coated C3d). This leads to initiation
Lyn
B cells also increase the expression of of signal transduction pathway from
receptors for several cytokines making both B-cell receptor and co-receptor
complexes leading to enhanced
them more receptive to T-cell help. B-cell activation response.
9.4 THYMUS-DEPENDENT AND

THYMUS-INDEPENDENT ANTIGEN
Depending on the nature of the antigen, B-cell activation may require direct contact (as well as
» TI antigens usually generate a cytokine simulation) with TH cells (thymus-dependent antigen) or they may be independent of any
weak immune response and the participation from TH cell (thymus-independent antigen). The thymus-dependent (TD) antigens
IgM type of antibody production.
No memory cells are formed by TI and thymus-independent (TI) antigens activate B cells by two different mechanisms as will be dis-
antigens and there is no affinity cussed shortly.
maturation of B cells. The TI antigens are two types:
• Antigens such as lipopolysaccharide (LPS) which act as polyclonal B-cell activators are
called TI-1 antigens. These antigens which are usually bacterial cell-wall components are
able to activate B cells regardless of their specificity. Activated B cells multiply and secrete
antibodies. The mechanism by which they activate B cells is not yet understood but they
can stimulate antibody production in athymic mice (mice that lack the thymus and are
T-cell deficient). This suggests that such an immune response is not dependent on T-cell
stimulation. TI-1 antigens will activate both mature B cells and immature B cells. TI-1
antigens stimulate antibody production without any requirement of any other cells. At
» The TI-1 antigen can stimulate B
cells regardless of specificities. In low concentration, TI-1 antigens stimulate specific antibody production. At high concen-
other words, it is a polyclonal B-cell tration, they act as polyclonal B-cell activators stimulating growth and differentiation of
activator. virtually all B cells without binding to the membrane Ig. It should be noted that LPS is a
polyclonal B-cell activator in mice and not in humans.
• TI-2 antigens include antigens such as polysaccharides and glycolipid that display
multiple-repeating identical epitopes. These also include protein antigens such as flagella
of microbes that contains the highly repetitious polymeric protein flagellin. TI-2 antigen
bind and effectively cross-link mIg and hence initiate a B-cell response. TI-2 antigens
are not processed and presented in association with MHC molecules and hence cannot
elicit T-cell-dependent response. TI-2 antigens are not polyclonal B-cell activators and
» TI-2 antigens are not truly T-cell-
independent antigen as B cells activate only specific B cells. Table 9.2 provides a brief account of thymus-dependant and
exposed to TI-2 antigen require thymus-independent antigens.
cytokines derived from TH cells.
Rigorous depletion of T cells abol- However, there are a number of proteins that express only one copy of each antigenic deter-
ishes the response to this antigen. minant per molecule in their native state. Such proteins are unable to cross-link mIg on B-cells in
TI-2 antigens, unlike TI-1 antigen,
activate mature B cells and inacti- vitro and hence unable to deliver the activating signals to B-cell. However, in vivo this is not the
vate immature B cells. case. Small peptide antigens and Fab fragments of anti-Ig antibodies which cannot cross-link mIg,
still induce B-cell response though it requires presence of and help from specific TH cells. There-
fore, in the presence of TH cells, these T-cell dependent antigen (such as protein antigen) need
minimal or even no signals by the B-cell receptor complex to induce a humoral immune response.
In the T-cell dependent response, the main function of BCR is to bind and endocytose antigens
Thymus-dependent Thymus-independent Antigen

Properties Antigen Type 1 Type 2
Antigen Protein Bacterial cell wall com- Antigen-expressing,
ponents (e.g. LPS) multiple-repeating
epitopes (e.g., flagella;
capsular polysaccha-
ride)
Antibody response in No Yes Limited, if at all
athymic mice
Isotype switching Yes No Limited
Affinity maturation Yes No No
Memory B cells formed Yes No No
Table 9.2
Thymus-dependent and thymus- Polyclonal B-cell No Yes (high doses) No
independent antigens. activation
Antigen with
repetitious Antigens with
antigenic multiple,
determinants dissimilar epitopes
Polyclonal
activator Clustering
Cytokine-mediated Contact-mediated
of membrane
T-cell help needed T-cell help needed
B cell B cell lg
Antigen acts Clustering of membrane No clustering of

as polyclonal Ig gives strong stimulus, membrane Ig
activator T-cell help not required T-cell help required
(TI-1) antigen (TI-2) antigen Figure 9.9
Line diagram explaining the
difference between T-dependent and
T-independent antigen induced
T-independent antigen T-dependent antigen activation of B cells.
so that it is subsequently presented to TH cells. The TH cells, through a number of costimulatory

molecules and cytokine, induce B-cell proliferation and differentiation leading to humoral response.
Figure 9.9 shows the interaction of T-cell dependent and T-cell independent antigens with B cells.
9.5 ROLE OF TH CELLS IN

B - C E L L A C T I VAT I O N
The process of B-cell activation by thymus-dependent (TD) antigen is more complex than the acti-
vation induced by TI antigens. It involves:
• contact-mediated activation of B-cells; and
• cytokine-mediated activation of B-cells.
« Without recirculation, the chance
The chances that a particular antigen-specific B-cell will meet its antigen and specific T-lymphocyte that a B cell will meet a specific TH
(for help) in peripheral lymphoid organs is very low. It is as low as one chance in 106 encounters; cell is as low as 1 per million
hence it seems highly unlikely that the right cells will be at the right place at the correct time. This encounters.
problem is solved by nature by continuous recirculation of lymphocytes.
The transport of antigens occurs from peripheral tissues to the lymph nodes and spleen, and
the recirculation of lymphocytes brings B and T lymphocytes to these lymphoid organs raising the
« TD antigen induces a strong
possibility of their chance encounter.
immune response, memory cells are
The fundamental feature of T-dependent B-cell activation is the interactions that occur be- formed and IgG is the main type of
tween antigen-stimulated specific B and T cells. This ensures that the ensuing response is specific antibody formed.
for that particular antigen. This T–B cell interaction is sometimes termed as cognate interaction
(because it involves specific recognition events), and involves specific recognition of antigen to-
gether with several other specific surface molecules on the interacting T and B cells.
9.5.1 A N T I G E N P R E S E N TAT I O N B Y B C E L L S
TO TH CELLS
Membrane immunoglobulin (mIg) is a high-affinity receptor for the antigen. Antigen-specific B
cells bind to the native antigen via mIg, internalize and process the molecules into small peptides.
These small peptides are then complexed to class II MHC molecules and presented on the surface
of B cells. Thus the B cells themselves function as antigen-presenting cells. In fact, they are better
antigen presenting cells as antigen is specifically recognized by B cells. It has been shown that B
cells are able to endocytose and present antigen to TH cells at 102 to 106 times lower than that is
needed by professional antigen-presenting cells such as dendritic cells or macrophages which do
not express specific receptors. This is because B cells recognize, endocytose and present specific an-
tigens to TH cells. Once a TH cell recognizes peptide antigen displayed by a class II MHC molecule
on the membrane of a B cell, these cells interact to form a T-cell–B-cell conjugate.
During the time that antigen is being processed and presented by B cells, B cells also enhances
the expression of costimulators that increase the ability of B lymphocytes to activate TH cells. These
include B7-1 and B7-2, both of which bind to CD28 and CTLA-4 on the TH cells. TH cells recognize
the peptide–MHC complex as well as costimulators which stimulate TH cells to perform their ef-
fector function.
CD40–CD40 LIGAND INTERACTION

» CD40, CD40L, Fas and FasL are CD40 is a member of family of cell surface proteins that includes Fas and receptors for tumour
all members of TNF/TNF-receptor necrosis factor (TNF). CD40 is constitutively expressed on the surface of B cells. CD40 ligand
superfamily. The TNF superfamily
consists of about 20 transmem- (CD40L or CD154) is expressed on TH cells after its activation by antigen and costimulators. CD40L
brane proteins with a conserved N is structurally related to the receptors of Fas and tumour TNF.
terminal cysteine-rich domain in When CD40, a B-cell protein interacts with CD40L on TH cells, oligomerization of CD40 mol-
their extracellular ligand-binding
region. ecules occurs. This oligomerization of CD40 molecules causes the association of the cytoplasmic
TNF receptor associated factor (TRAFs) to the cytosolic domain of CD40. The TRAF bound to
CD40 initiates an enzyme-cascade that ultimately leads to the activation of a number of intracel-
lular signalling pathways such as, (a) the activation of tyrosine kinases such as lyn and syk, (b) the
activation of phospholipase C which results in the formation of IP3 and DAG.
These events lead to the activation and binding of several transcription factors such as NF-
κB and AP-1. The expression and action of these transcription factors provide the first signal for
TH-cell mediated B-cell proliferation. The binding of CD40 to CD40L also leads to an enhanced
expression of B7-1 and B7-2 molecules on B cells, with greater T-cell activation. Since, the expres-
sion of B7 molecules on B cells, and that of CD40L on T cells is induced only when lymphocytes
are specifically stimulated, specific B-cell proliferation is induced.
Several experimental evidences provide concrete proof of the involvement of CD40–CD40L
interaction in contact-dependent help of TH cells. For example, when antigen-stimulated B cells
are treated with anti-CD40 monoclonal antibodies (which mimic CD40L of the TH cells), in
the absence of TH cells, B cells are activated and start proliferating. Moreover, the incubation
of B cells with plasma membrane prepared from activated TH cells (and not resting TH cells)
induces B-cell proliferation. Activated TH cells express C40L on their surface. Similarly, the
treatment of TH cells with antibodies that block CD40L, blocks B-cell activation despite the
presence of TH cells.
T H C E L L’ S C Y T O K I N E I N B - C E L L P R O L I F E R AT I O N
A N D D I F F E R E N T I AT I O N
» Upon binding CD40L, CD40 mol- CD40–CD40L-stimulated B cells start proliferation but fail to differentiate into antibody-secreting
ecules expressed on B cells gener- plasma unless cytokines are also present. Cytokines are soluble proteins secreted by T lymphocytes
ate a stimulus that results in clonal
proliferation of B cells and class as well as other cell types in response to activating stimuli. Activated TH cells secrete cytokines.
switching of antibodies. As mentioned before, the engagement of CD40–CD40L activates B cells at one end and TH
cells on the another, which starts the secretion of cytokines. Cytokines released by TH cells serve
two functions:
• They augment B-cell proliferation and differentiation. Three TH cell-derived cytokines
IL-2, IL-4 and IL-5 contribute to B-cell proliferation.
• They promote class switching and thus determine the type of antibodies produced, for
example, IL-4.
Moreover, activated B cells enhance the expression of receptors for cytokines such as
IL-2, IL-4 and IL-5 making B cells more receptive to cytokines secreted by TH cells. As a result,
antigen-specific B cells respond to cytokines more than bystander B cells that have a different
antigen specificity. The role of TH cells in contact-mediated and cytokine-mediated T-cell help is
shown in Figure 9.10. Moreover, TH cells release cytokines in a directional manner towards the
interacting B cells. This was shown by the work of C. A. Janeway. He isolated the TH-cell clone
that secreted cytokine IL-4 in response to binding monoclonal antibodies to the T-cell receptor.
The TH cells were adsorbed on a membrane having 3-micron pores. This membrane was sus-
pended between two chambers in a tank. On addition of monoclonal antibodies in one chamber,
TH cells bound to the membrane released IL-4 cytokines towards the chamber containing the
stimulatory monoclonal antibodies.
Surface antibody
(CD80,CD86)B7 CD28
LFA-3 CD2
(CD58)
Class II MHC T-cell receptor
CD4
ICAM-1 LFA-1(CD11a/CD 18)
CD40 CD40L(CD154)
Cytokine receptor
B cell T cell
Cell surface molecules involved in T–B cooperation
Cytokines
TH cell TH cell released
CD28 TCR CD40L CD28 TCR CD40L IL-2,IL-4,

IL-5
Cytokine
B7 CD40 B7 CD40 receptor
B cell MHC+peptide B cell MHC+peptide
TRAF
PLC
Activation Activation of
Lyn,Syk kinase
Promotes class Augments B-cell Figure 9.10

IP3 DAG
switching proliferation and Line diagram explaining the role CD40–
Expression of NF-KB, AP-I
differentiation CD40L interaction and TH cytokines in
B-cell activation (DAG—diacyl glycerol;
PLC—phospholipase C; TRAF—TNF
B-cell proliferation Cytokine-mediated help
receptor-associated factor; IP3—inositol
CD40-CD40L-mediated help triphosphate).
9.5.2 B - C E L L D I F F E R E N T I AT I O N I N T O E F F E C T O R
PLASMA CELLS
B cells, activated and proliferated in response to antigens differentiate, either into effector cells that
actively secrete antibodies or memory cells that survive for long periods of time and are not anti-
body secretors.
Antibody synthesis and secretion in response to protein antigens are stimulated by CD40–
CD40L interaction and by the action of cytokines on B cells. Both stimuli enhance the transcription
of immunoglobulin genes (via activation of transcription factors) and increase antibody synthesis
and secretion. The differentiation of mature B cells into antibody-secreting plasma cells requires
changes in the processing of RNA transcriptions in such a way that the shorter secretory form
of membrane-bound rather than the longer membrane-bound form of antibody is synthesized.
Membrane-bound immunoglobulin and secretory immunoglobulin molecules differ in their C
terminals of heavy chains; for example, in the membrane-bound form of IgM, the Cμ4 domain
is followed by 26 hydrophobic transmembrane segments (meant for anchoring immunoglobulin « Astrid Elsa Fagraeus-Wallbom, a
into membrane) and a short (3 amino acids) tail inside the cytosol. In the secretory form, the Cμ4 professor at Karolinska Institute,
domain is not followed by any hydrophobic acids but instead by a short charged amino acid tail. Stockholm, was the first to dem-
onstrate in her doctoral thesis (in
The transition of antibody from its membrane-bound form to the secretory form is brought about 1948) that antibodies were
by the processing of heavy-chain mRNA. produced in plasma cells.
Alternative splicing of the primary heavy chain transcript determines whether the exon con-
tains transmembrane and cytosolic coding segments or not. If the exon contains transmembrane
segments, mIg is formed as it contains hydrophobic transmembrane segment that anchors the
immunoglobulin in the lipid bilayer of the plasma membrane. If, however, the hydrophobic trans-
membrane segment and cytosolic charged segment are excluded, it cannot be anchored in the cell
membrane and is secreted. All CH genes contain separate membrane exons and all heavy chains
can usually be expressed as membrane-bound (except δ heavy chain, which is always membrane-
bound) or secretory forms. Several cytokines, including IL-4, IL-2 and IL-6, stimulate immuno-
globulin synthesis and secretion. Some of the cytokines are also believed to facilitate the formation
of the secretory form of antibodies. As differentiation proceeds, more and more of the secretory
form of antibodies is produced. Some of the progeny of antigen-activated IgM or IgD expressing
B cells undergo heavy-chain class switching, leading to the production of antibodies of different
classes such as IgE, IgG and IgA. Cytokines also play an important role in regulating heavy-chain
class switching. IL-4 induces class switching from IgM/IgD producing cell to IgE and IgG (IgG4)
producing B cells in humans. Figure 9.11 shows the role of cytokines in class switching of antibod-
ies in B cells. γ-IFN promotes class switching of B cells to IgG2a isotype (in mice) while TGF-β is
believed to be the IgA switch factor in both mice and humans. γ-IFN knockout mice have greatly
reduced IgG2a serum concentration. It should be mentioned that for some antigens (thymus-
dependent) the interaction between CD40 and CD40L also plays an important role in class switch-
ing. The mechanism of the induction of isotype switching by CD40 signal is still not clear.
Mature B cell
IL-2,IL-4,IL-6
Proliferating
mature
B cell
IL-6 Differentiation
IL-2
γ -IFN TGF-β into plasma
IL-4 IL-4
IL-2 IL-5 cell, and
IL-5
IL-4 class switching
Plasma cell
Figure 9.11
Line diagram showing role of cytokines
in B-cell differentiation into IgM IgG IgA-
and IgE-secreting plasma cells.
IgM IgG2 IgE IgA
9.5.3 B - C E L L D I F F E R E N T I AT I O N « In B cells lacking CD40, or in

specific TH cells that lack CD40L (in
INTO MEMORY B CELLS mice or humans), no class switching
Some of the antigen-activated B cells do not differentiate into antibody secreting plasma cells. In- occurs. In these cases, the antibody
stead they acquire the ability to survive for long periods of time without any overt antigenic stimu- response to an antigen is dominat-
ed by IgM antibodies and little or
lation (see Figure 9.12). no switching to other isotypes.
These cells are called as memory cells and they “remember” the antigen encountered. These
cells mount a rapid response when they subsequently encounter the same antigen. It is still not « Memory cells can express IgM, and
understood why some progeny of antigen-stimulated B cells differentiate into antibody-secreting IgD and even class-switched IgG,
plasma cells with a short lifespan while others differentiate into long-lived memory cells. Some IgA and IgE on their surface. There
are separate memory cells for TH
memory B cells may reside in the lymph node, while others may take up residence in other lym- cells and Tcyt cells.
phoid tissue such as the spleen. Memory cells bear high affinity immunoglobulin molecules on
their surface. The mIgD are usually expressed at lower concentrations on memory cells. It is still
not clear how memory cells survive for such a long time. It is possible that memory cells are ac-
tually continually generated and maintained by low-level stimulation provided by antigen over
months or years, or memory cells express bcl-2 gene (a proto-oncogene) whose gene product al-
lows the cells to avoid apoptosis. Overexpression of bcl-2 causes B-cell lymphoma. Differences
between memory B cells and naïve B cells are outlined in Table 9.3.
Naive B cell
Primary immunogenic exposure
Plasma cells Memory cell
Secondary immunogenic exposure

Figure 9.12
Line diagram giving an overview of
differentiation of B cells into memory
and plasma cells. Repeated antigenic
exposure increases the population of
memory cells. It is believed that the bcl-2
gene that is not expressed in the plasma
cell but expressed in the memory cell is
Plasma cells Memory cells responsible for its long life.
Properties Memory B cell Naïve B cell

Derived from Naïve B cell after antigen Progenitor B cell
stimulation
Resides in Lymph node, spleen, bone Spleen
marrow
Surface antibody IgM, IgD, can also express IgG, IgM, IgD
IgA, IgE
Lifespan Long Short
bcl-2 gene (survival gene) Expressed Short-lived or absent
Membrane proteins
Adhesion molecules ICAM (high) ICAM (low)
Complement receptor High Low Table 9.3
Memory B cells and naive B cells—a
Surface antibody affinity High Low
comparison.
9.6 PRIMARY AND SECONDARY HUMORAL

IMMUNE RESPONSE
The primary immune response occurs following the first exposure to antigen. It involves the
activation of naïve B lymphocytes which generates antibody-secreting plasma cells and memory
B cells. IgM is a primary antibody that is secreted initially, often followed by a shift (or switch)
to IgG. The primary response depends on various factors underlined below and can last for few
days to few weeks:
• nature of antigen;
• route of administration of antigen;
• type of host being immunized; and
• presence (or absence) of adjuvant;
The kinetic study of primary response reveals that it has an initial lag phase during which naïve B
cells undergo activation and proliferation, and subsequently differentiation into plasma (or memory)
cells. The lag phase duration varies with antigen and is usually longer for soluble antigen such as
proteins. The lag phase is followed by a peak phase, in which antibody serum concentration reaches
the highest point. In this phase, specific plasma cells are at the highest level and when these cells
» Primary response occurs after secrete antibodies, serum antibody concentration rises and also reaches a peak. This is followed by
four days and peaks at seven to ten a gradual decline of serum antibody and, within a few days (or weeks), the return to normal. The
days. Subsequent exposure to the
same antigen results in a secondary
memory B cells that are formed during the primary response persist in an individual. These cells
response which has a shorter lag have a longer lifespan and sometimes persist for the life of the individual.
period and high antibody titre. The Secondary immune response results from the activation of these memory cells. The activation
main antibody of primary response
is IgM and that of secondary
of memory B cells, formed from a previous antigenic encounter with the same antigen, results in the
response is IgG. rapid proliferation of memory cells into plasma cells and memory cells with the concomitant produc-
tion of a large amount of specific antibodies. The secondary immune response (also called booster
response, memory response) is different from primary immune response in the following ways:
• It has a short lag period.
• The peak phase is of greater magnitude and longer time.
• The antibodies of IgG or other isotypes (other than IgM or IgD) predominate.
• The antibodies produced are of higher affinity than those formed in primary response.
The greater magnitude of secondary response occurs because:
• The population of memory B cells is larger than that of the original naïve B cells. The activa-
tion of a large number of memory cells leads to much higher levels antibody production.
• Memory B cells can be activated more easily (within less time) than naïve B cells and
hence exhibit a shorter lag period.
• The process of class switching which shifts the response from IgM to IgG (or any other
isotype) and affinity maturation (discussed later) are responsible for the higher affinity of
antibodies produced during the secondary response.
The high level of antibody coupled with a higher affinity for the antigen produced within a short
period of time provides an effective host defence against re-infection by the same pathogen. Differ-
ences between primary and secondary immune responses are briefly summarized in Table 9.4.
Primary Response Secondary Response

Responding B cells Naïve B lymphocytes Memory B lymphocytes
Lag-phase duration 4–8 days 1–3 days
Antibody isotype Predominantly IgM Predominantly IgG
Stimulating antigen All antigens Thymus-dependent antigens
Somatic hypermutation No Yes
Table 9.4 Class switching No Yes
Primary and secondary immune
Antibody affinity Lower Higher
response.
9.7 ROLE OF TH CELLS IN HUMORAL

RESPONSE
Various studies on cellular interactions of humoral responses have revealed that TH cells play an
important role in a humoral response. B cells recognizes native, unprocessed antigen while TH cells
recognize antigenic peptides displayed on MHC molecules. Thus, TH cells and B cells recognize dif-
ferent antigenic determinants on the same antigen, resulting in a more effective immune response.
This property of T- and B-cell interaction in the humoral response is called associative or linked
recognition. This property is best highlighted in the humoral response to hapten–carrier conju-
gates. Hapten, by virtue of its small size, is unable to elicit an immune response if injected alone in
an experimental animal. Hapten is to be chemically linked to a large carrier molecule to induce a
humoral response. If the animal is immunized with both hapten and carrier separately, little or no
specific immune response is elicited. Once a primary immune response to hapten has been gener-
ated, the secondary immune response can be raised in the animal by immunizing again with the
same hapten–carrier conjugate used for the primary immunization. If the secondary immuniza-
tion is done by using the same hapten but a different carrier, no secondary anti-hapten response
occurs. This phenomenon is called carrier effect. It has been experimentally shown that CD4+
T cells are responsible for the carrier effect. It is now known that B cells specific for hapten produce
antibodies against hapten only when stimulated by TH cells specific for carrier epitopes. Some B
cells are, however, also generated against the carrier epitopes.
9.8 SITES FOR INDUCTION OF HUMORAL

RESPONSE
Protein antigens are recognized by specific B and T lymphocytes in peripheral lymphoid organs
such as spleen and lymph nodes. Blood-borne antigens are taken care of by T and B cells present in
the spleen, while the antigens entering tissues are drained into lymphatic system and are finally “fil-
tered” by lymph nodes. For the sake of simplicity and clarity, the generation of a humoral response
in the lymph node is discussed below.
The structure of the lymph node has already been discussed (in Chapter 2). Briefly, it is a bean-
shaped structure present in areas such as the neck and groin, among other places. It has an outer
dense collagenous capsule. The lymph node has an outer region rich in B cells (cortex), a middle
T-cell rich region (paracortex) and the innermost region (medulla) which extends towards the « Immunoglobulin variable, diver-
sity and joining gene fragments
hilus, and has both T and B cells. Afferent lymphatics empty into the lymph node while efferent have been selected by evolution for
lymphatics leave the node from the hilus. 200 million years.
Antigens (or antigen–antibody complex) enter the lymph node through afferent lymphatics.
The initial activation of both B and T cells is believed to occur in the paracortex. Naïve B-cells from
the B cell rich cortex migrate to the T-cell rich paracortex. B cells have membrane immunoglobu-
lins that bind the antigen, endocytose, degrade and display on class II MHC molecule present on
its surface. On entering the paracortex, B cells present antigen + MHC complex to specific TH cells,
forming a T-cell–B-cell conjugate. This causes B-cell (as well as TH-cell) activation and prolifera-
tion. Once B-cell activation has taken place, a small region of rapidly dividing B cells is formed at « In hyper-IgM disease, patients
the edge of the paracortex (secondary follicle) within two to four days of antigen exposure. express only IgM and no other class
of antibody.
These B cells differentiate into antibody-secreting (IgM, IgG isotype) plasma cells and
memory cells.
Antibodies produced during the primary response come from the plasma cells present in the
secondary follicles. A few days after the formation of secondary follicles, a few activated B cells and
TH cells migrate to the cortex towards the primary follicle (which is a spherical structure contain-
ing tightly packed naïve lymphocytes and follicular dendritic cells). These primary follicles then
develop into secondary follicles having primed activated B-cells, activated TH cells and special-
ized antigen-presenting follicular dendritic cells. Secondary follicles contain a central pale staining « Follicular dendritic cells do not
area, germinal centre (discussed in the next section). Follicular dendritic cells which present anti- express class II MHC and do not
gen to only B cells (and not TH cells) do so in a very specialized manner. Follicular dendritic cells present antigen to TH cells. Instead
they bind antigen–antibody com-
attract B cells towards themselves by secreting chemokine CXCL-13 which binds to the chemokine plexes on their surface via FC recep-
receptor on B cells. tors and present them to B cells.
Follicular dendritic cells express Fc receptors, and do not express class II MHC molecules
(thus no TH activation). These Fc receptors can bind and retain the antigen–antibody complex for
a long period of time, sometimes for weeks or even months. These antigen–antibody complexes
displayed on long plasma membrane extensions of follicular dendritic cells stimulate the B cells
» Immune complexes are associ-
in very interesting way. Follicular dendritic cells release small particles from their cell membrane.
ated with follicular dendritic cells These particles are derived from its cell membrane and are heavily coated by the immune complex
via receptors such as complement and sometimes called iccosomes (immune complex coated endosome/bodies).
receptors CR1 and CR2 and Fc
receptor FcγRII.
Iccosomes are then bound by membrane receptors on specific B cells, which then endocy-
tose, process and present the antigenic determinants bound to class II MHC to activated TH cells.
This response which usually occurs during the secondary response (when antigens bind previously
present antibodies and generate iccosomes) results in more B-cell activation and proliferation,
with the net result of production of antibodies in large quantities and for a longer period of time,
a characteristic of the secondary immune response.
9.9 GERMINAL-CENTRE REACTIONS

As mentioned previously, a few days after the formation of secondary follicles, B cells are activated.
TH cells migrate towards the centre of the secondary follicle forming a pale staining region called as
germinal centre. It is in this germinal centre that T-cell dependent response, affinity maturation (dis-
cussed in the next section) and generation of plasma and memory B cells occurs. Germinal centres
usually arise within six to eight days of antigen exposure. Each fully formed germinal centre contains
one or more antigen-specific B-cell clones. The proliferating B cells present in the germinal centre are
large cells having no-membrane immunoglobulin called centroblast. These centroblasts rapidly di-
vide to give rise to small B cells called centrocytes which express surface immunoglobulin molecules.
Proliferating B-cell centroblasts accumulate at a histologically identifiable central dark coloured zone
called dark zone which has a few follicular dendritic cells. The small, non-dividing B-cell-centrocytes
move to an adjacent light zone where they come in close contact with the processes of abundant fol-
licular dendritic cells. Centrocytes which have surface immunoglobulin bind to antigen presented
by the follicular dendritic cells in the light zone and undergo differentiation into memory B cells
and pre-plasma cells or plasmoblasts. Plasmoblasts and memory cells undergo isotype switching
in the germinal centre. Plasmoblasts leave the germinal centre and migrate to the medulla where
they mature as plasma cells and start secreting antibodies released into the circulation via efferent
lymph. Germinal-centre reactions which takes places in the lymph node is shown in Figure 9.13.
The memory B cells so formed may leave the germinal centre and lymph node and recirculate, or
may stay in the lymph node waiting for the next encounter with antigen. The majority of centrocytes
that cannot bind the antigen presented, or cannot bind with high affinity the antigen presented by
follicular dendritic cells, die of apoptosis within the basal light zone. The debris of these dying cells is
cleared by specialized phagocytes called tingible body macrophages.
CD40–CD40L interactions are required for the interaction between B cells and TH cells that
leads to the formation of the germinal centre. If the CD40–CD40L interaction is blocked or either
CD40 or CD40L is inactivated, the germinal centre formation fails. CD40–CD40L interaction is
also needed by B cells in the process of differentiation within the germinal centre.
9.9.1 A F F I N I T Y M AT U R AT I O N O F B C E L L S
Affinity maturation involves the selection of those B cells that have a high affinity for the antigens. It is
a T-dependent process and is a result of somatic mutation of immunoglobulin genes followed by the
positive selection (survival) of the B cells producing antibodies with high affinity (see Figure 9.14). The
average affinity of antibody towards the same antigen increases 80–110-fold during the course of hu-
moral response. TH cells and CD40–CD40L interactions are required for affinity maturation and hence
this occurs only in antibody response to TH-cell-dependent antigens. As discussed previously, TH-cell-
independent antigens produces low affinity antibody (usually of IgM type) that shows no affinity maturation.
Affinity maturation occurs in germinal centre B cells as a result of somatic hypermutation.
9.9.2 S O M AT I C H Y P E R M U TAT I O N
After infection (or vaccination), the body first produces antibodies of relatively low affinity for the
antigen. As the immune response progresses, these antibodies become “hypermutated”. The purpose
of hypermutation is to create new protein sequences that can bind the antigen more strongly and
G
Centroblasts E
B cells R
Dark M
zone I
N
A
Somatic hypermutation L
High
Low affinity affinity Low affinity Centrocytes
(Small B
Light
cells)
zone
Apoptosis
Apoptosis of low-affinity
B cell C
E
Follicular N
Selection of dendritic cell T
high-affinity centrocytes E
R
Antigen-antibody
complex
High-affinity
plasmoblasts survive Iccosome (derived from
follicular dendritic cells)
T-cell help
M Figure 9.13
Lymph node E Line diagram explaining the
D development of B cells in the
U germinal centre. The centroblasts
L divide and mutate in the dark zone
L of the germinal centre giving rise to
A
high- and low-affinity centrocytes.
High-affinity centrocytes interact with
iccosomes/follicular dendritic cells and
are transformed into plasma cells and
High-affinity antibodies Memory B cell memory B cells. Low-affinity centrocytes
die of apoptosis.
specifically than their precursors. This allows the body to respond quickly and effectively to pathogen
that the body has encountered previously.
Proliferating B cells present in the germinal centre show extensive mutation (point mutation)
rate in their rearranged variable regions of heavy and light chain genes. The rate of mutation in
the V region of immunoglobulin gene is estimated to be around 1 in 103 gene base pairs per cell
division which is about a million times higher than the spontaneous rate of mutation in other
mammalian genes. It is for this reason that the high rate of somatic mutation in the variable region
of the immunoglobulin gene is called somatic hypermutation.
Somatic hypermutation that occurs in the immunoglobulin gene probably takes place when
centroblasts divide in the dark zone of the germinal centre. These mutations which are usually
point mutations, deletions and insertions that occur in rearranged immunoglobulin genes, are fo-
cused in the gene segment coding for three complementarily-determining regions or CDRs. They
occur at the κ- and λ-light chain loci as well as at the heavy-chain locus. The mechanism of somatic
hypermutation is not yet understood. Apparently, the rearranged variable immunoglobulin region
VDJ gene segment binds a number of DNA binding proteins that make this segment susceptible
to mutations. Though TH cells are found in the germinal centre, it is not clear whether they pro-
vide contact signals or cytokine-mediated stimulus to somatic hypermutation, or, if they are at all
involved in this event.
Centroblasts
B cells
Somatic hypermutation
polymerase η,AID
Antigen (on FDC or iccosome)
Enhanced antigen
binding
Low-affinity No binding of High-affinity

binding antigen binding
B cell survives
and proliferates
Apoptosis Apoptosis
Figure 9.14
Line diagram showing the process of
somatic hypermutation and affinity
maturation in a lucid way. (AICD— High-affinity antibody-producing
activation-induced cytosine deaminase; B cells selected for survival
FDC—follicular dendritic cell). (Affinity maturation)
During hypermutation, a large number of nucleotide substitutions (some are small deletions or
insertions) are introduced into the rearranged V gene to produce variant antibodies with increased
affinity for cognate antigen. It is speculated that DNA polymerases particularly polymerase η and
activation-induced cytosine deaminase (AID) participate in somatic hypermutation in B cells.
Since VDJ segments total about ~1000 nucleotides (base pairs), it has been estimated that mu-
» DNA polymerase η and activation- tation will accumulate in expressed variable regions at an approximate rate of almost one mutation
induced cytosine deaminase par- per cell division. This implies that the nucleotide sequence of the variable region of immunoglobu-
ticipate in somatic hypermutation lin derived from one clone of B cells can diverge to about 5 per cent from the original germ-line
that results in increased affinity of
the antibodies during a secondary sequence, which in turn implies a difference of 10 amino acids substitution.
response. Somatic hypermutation has been confirmed by several experimental evidences:
• Kelsoe et al. working on clones of B cells isolated from the germinal centre of the spleen of
mice that were immunized with a hapten-carrier conjugate (4-hydroxy-
3-nitrophenylacetyl-chicken gammaglobulin) showed that the progeny of single B-cell
clones progressively accumulate mutations with the tissue after immunization.
Another study conducted by analysing immunoglobulin genes of B cells clones isolated at different
stages of humoral response showed:
• an accumulation of point mutation in the VDJ regions of immunoglobulin gene
segments;
• clustering of point mutations in three complementarily-determining regions of the
variable regions;
• the presence of mutation correlated with an increasing affinity of the antibodies for the
antigen that induced the response; and
• isotype specificity; IgG showed more mutation than IgM type.
These studies point out that some somatic hypermutation that occurs in immunoglobulin genes are
likely to be useful because they will generate high affinity antibodies. Since mutation is a random event,
it is also likely that many mutations may result in the decline or even loss of antigen binding. Therefore,
the next important step in affinity maturation is the selection of useful, high affinity B cells.
9.9.3 SELECTION OF HIGH-AFFINITY B CELLS

Somatic hypermutations take place in the dark zone of the germinal centre, and the selection takes
place in the light zone among the non-dividing centrocytes. These B cells are programmed to die by
apoptosis unless they are rescued. This rescue is mediated by follicular dendritic cells which have
on their surface, antigen-antibody complex, antigen–C3d and C3b complex, as well as Fc receptors
and complement receptors. A centrocyte whose membrane immunoglobulin binds and cross-links
antigen (bound on the surface of follicular dendritic cells) receives a signal that rescues the B cells
from default cell death. Those B cells which do not bind and receive such a signal, die by apoptosis.
However, since only a small amount of antigen is available for a large number of centrocytes, only
those centrocytes that express high affinity membrane immunoglobulin are selected to survive.
The net result of this selection process is a population of centrocytes (B cells) capable of producing
antibodies with significantly higher affinity for antigen than antibodies produced by the same clone
of B cells earlier in the immune response. It should be remembered that even though the antigen
displayed on follicular dendritic cells provide essential signals that rescue the B cell from almost
certain death, another very important signal should also be received from the complete rescue. It is
the interaction between specific TH cells and B cells (centrocytes).
This interaction between B and TH cells involves the engagement of:
• CD40 on B cells and CD40L on TH cells;
• B-7 molecules on B cells with CD28 on TH cells; and
• processed antigen displayed on class II MHC molecules on B cells and TCR of TH cells.
B cells that fail to receive either antigen-mediated immunoglobulin signal or TH cell interaction
undergo apoptosis in the germinal centre. Cells that survive are high affinity B cells that have
undergone affinity maturation. These surviving B cells migrate from the basal light zone of the « In X-linked hyper-IgM, an immu-
germinal centre to an apical light zone where they undergo additional isotype switching. The class nodeficiency disorder, TH cells do
not express CD40L. In this disease,
switching of immunoglobulin, as mentioned previously, also requires the interaction of CD40 and patients express only IgM and no
CD40L (apart from cytokines). other class of antibodies.
Memory B cells are also generated from high affinity centrocytes in the light zone of the ger-
minal centre. Some of the memory cells remain in the lymph node while others exit the germinal
centre and circulate in the blood, homing up on other lymphoid tissues. The high-affinity antibody
secreting, B cells and some memory B cells, then exit the germinal centre and circulate in blood,
secreting antibodies for the future.
9.10 R E G U L AT I O N O F I M M U N E
RESPONSE
The immune mechanism is regulated by a variety of control mechanisms. These regulating mecha-
nisms restore the immune system to its original resting state once the pathogen/antigen has been
cleared up. The regulation of the immune response takes place in both the humoral and the cell-
mediated branches of the immune system.
On a purely theoretical basis, Victor Najjar in 1955 speculated that the interaction between
antibody and antigen leads to a change in the conformation of immune complexes. The formation « The first speculation about the
possible existence of an immuno-
of the immune complex triggers the formation of an anti-antibody which reacts with the immune regulatory mechanism was
complex and somehow keeps it in a steady state. This theory about the internal control of the im- provided by Victor Najjar in 1955.
mune complex formation was a result of the fertile imagination of Najjar, but still it provided the
first hint of the existence of an immuno-regulatory network within the body. Today, we know
that several aspects of the immune response can be regulated by a variety of ways which includes
(a) antigen (b) antibody (c) lymphocyte (see Figure 9.15).
9.10.1 R E G U L AT I O N
BY ANTIGEN
The presence of antigen trig-
Intracellular Polyvalent Soluble antigen gers an immune response. Dif-
pathogen antigen ferent antigens activate differ-
ent branches of the immune
system. Intracellular pathogens
Cell-mediated Humoral Cell-mediated Humoral such as mycobacteria induce a
response response response response cell-mediated response. Soluble
antigens such as proteins trigger
Regulation by antigen
both cell-mediated and humoral
responses. Moreover, some an-
Cross-linking of B-cell tigens are more specific in the
mlg and Fc feceptor
immune response. Polyvalent
mlg Fcγ RIIB polysaccharides of various bacte-
B cell ria generally induce humoral re-
Antibody synthesis sponses having IgM antibodies.
inhibited An effective immune response
kills and inactivates the patho-
gen, and removes it from the
Antibody-mediated regulation system, and the immune system
returns to the resting state. Not
Idiotype only the type of antigen but also
Anti-idiotype antibody (of an antibody) the dose and route of adminis-
Antigen enters tration of antigen determines the
immune response. Large doses of
Specific antibodies
antigen usually induces tolerance
formed, equilibrium while small doses of antigen elicit
disturbed both humoral and cell-mediated
immune responses. Similarly,
Anti-idiotypic
antibodies formed
antigens administered intrader-
maly evoke an effective immune
response while oral or nasal ad-
System returns
to equilibrium ministration of the same antigen
Figure 9.15 may cause tolerance or a very
Line diagram explaining the various ways weak immune response. This is
by which immune response is regulated. Idiotypic-anti-idiotypic network
clearly seen in a classic case of
immune response against lym-
phocyte choriomeningitis virus (LCMV). Intra-peritoneal injection of peptides of LCMV together
with incomplete adjuvant causes tolerance in mice, while intradermal injection of the same antigen
evokes a strong immunity against LCMV.
9.10.2 A N T I B O D Y - M E D I AT E D R E G U L AT I O N
It has been proved that antibodies exert feedback-control on their own synthesis and hence regu-
late the immune response. The antibody-mediated suppression which is best exemplified by IgG
can be induced by various ways.
Blocking of antigen: Passive administration of specific antibodies just before or just after antigen prim-
ing of B cells results in a subdued immune response. Passively administered antibody binds to antigen
and thus prevents the binding of antigen to the B cells. The specific B cells do not get exposed to anti-
gen and hence cannot clonally expand. Only high affinity B cells compete successfully for the antigen.
This type of IgG-mediated suppression does not involve the Fc portion of the antibody.
Cross-linking of B-cell Ig and Fc receptors: It has been experimentally proved that IgG can downregu-
late IgG production by B cells if there is simultaneous engagement of both B-cell immunoglobulin
(suggesting the presence of antigen) and Fc receptor (suggesting that antibody is already present).
If both the receptors are engaged, B-cell activation is inhibited and antibody production is down-
regulated. Fc receptor (FcγRIIB or CD32) mediated antibody (synthesis) feedback inhibition is a
physiologic control mechanism of the humoral response because it is triggered by secreted anti-
bodies and blocks further antibody production.
9.10.3 R E G U L AT I O N B Y LY M P H O C Y T E S
Since T-cells, particularly TH cells, help various effector mechanisms of B- and T-cells, they help
mediate humoral and cell-mediated immune response. Though the role of CD4+ or CD8+ T cells in
the regulation of an immune response is still under scrutiny, it is safe to say that the secretion of cy-
tokines by different TH cells (TH1or TH2 ) regulates the immune response, favouring either humoral
or cell-mediated immunity.
9.10.4 R E G U L AT I O N B Y I D I O T Y P I C – A N T I - I D I O T Y P I C
NETWORK
The antigen-combining site of each immunoglobulin molecule itself represents an immunogenic
epitope known as an idiotope. An idiotype is the sum total of all the idiotopes present on the
variable (VL and VH) domains of an antibody. The idiotype can be recognized by other antibodies
termed as anti-idiotypic antibodies. The idiotypic network theory which was put forward by Neil K.
Jerne in 1985, stated that the whole immune system is a network of interacting idiotypes and anti-
idiotypes. However, when the external antigen enters the system, a particular clone of B-secreting
antibodies gets clonally expanded. The network system which was previously in “equilibrium” gets
disturbed. As a result, anti-idiotypic antibodies are also synthesized, which plays a regulatory role
in modulating the immune response. This network theory, though complex, is very appealing but
the role of such an idiotype network in regulating a normal immune response is still debated.
The sequential development of B cells occurs in the bone marrow. Each development stage is char-
acterized by the expression of specific immunoglobulin genes and phenotypic markers. The earliest
cell committed to a B-cell lineage is a pro-B cell. Pro-B cell differentiates into pre-B cell which in turn
develops into an immature B lymphocyte. Negative selection eliminates the immature self-reactive
B-cells and the cells that survive express surface antibody molecule of IgM or IgD type. Once a B cell
binds an antigen it gets activated to antibody-secreting plasma cell and memory cell. The differentiation
of B cell into plasma cell and memory cell occurs in the germinal centre of the lymph node and spleen.
Antibodies then enter the circulation and bind the challenging pathogen, which is neutralized or killed
or phagocytosed by cells of the immune system. The immune system is believed to be kept at a steady
state by antigen, antibody, lymphocytes and idiotypic network, among several others.
Double Diffusion Agar Assay (Ouchterlony Technique)

Antigens
1 1 1 2 1 1,2
Anti 1 Anti 1 and 2 Anti 1 and 2
Antibody
Reaction of identity Reaction of non-identity Reaction of partial identity
Figure 7.16
The Ouchterlony technique.
Double diffusion agar assay technique was developed by Orjan interface. The visible line of precipitation provides the details of anti-
Ouchterlony, a Swede, in 1948. It is called double diffusion because in gens present in different wells. A smooth arch of precipitation sug-
this method both antigen and antibody diffuse towards each other, gests that antibodies bind to the same antigen determinants on both
and a precipitin line is formed where the antigen and antibody meet. samples and hence both the antigens are identical (see Figure 9.16).
In this technique, molten agar is poured over a slide or petri-plate and If two completely unrelated antigens are added to the wells a reac-
allowed to solidify. Wells are punched onto the agar plate. Test solution of non-identity occurs, creating an X-shaped arch. A reaction of
tions of antigen and antibody are added to the separate wells. This partial identity occurs if the antigen in the well shares some but not all
plate is then incubated at least for 24 hours. The antigen and antibody antigenic determinants. A small extension of the precipitation band, a
solutions diffuse outwards and a line of precipitation is formed at their spur, is formed towards the well having “extra determinants”.
S U M M A R Y
• B cells develop in the bone marrow from a committed precursor • B-cell activation involves the stimulation of signalling cascades
that undergo sequential stages, each characterized by the expres- that ultimately activate transcription factors leading to the changes
sion of specific immunoglobulin genes and phenotypic markers. in the expression of specific genes.
• The earliest committed cell to B-cell lineage is the pro-B cell. • B-cell activation by thymus-dependent antigen involves contact-
Heavy-chain (μ) rearrangement occurs in the pro-B cell. The bind- mediated (for example, CD40–CD40L interaction) or cytokine (for
ing of pro-B cell to stromal cells differentiates it into pre-B cell. example, IL-2)-mediated activation of B cells.
• Pre-B cell expresses pre-B-cell receptor that are μ (heavy) chains • Antigen-activated B cells differentiate into antibody-secreting
associated to surrogate light chain λ5 and V pre-B chain. plasma cells (B cells without surface antibody) and long-lived
memory B cells (which expresses surface antibody).
• These pre-B cells differentiate into immature B lymphocytes that ex-
press B-cell receptors comprising μ heavy chain and κ or λ light chain. • The primary immune response occurs following the first exposure
to antigen, which results in the generation of IgM-secreting plasma
• Negative selection eliminates immature, self-reactive B cells. Cells cells and memory B cells. The activation of these memory cells
that survive, then co-express IgM and IgD to become mature during the secondary response leads to the production of a large
B cells which exit the bone marrow and enter the circulation. amount of specific antibodies (IgG type).
• There are two subsets of B-cells—B1 cells and the more common • The differentiation of B cells into plasma cells and memory cells
B2B cells. occurs in the germinal centre of the lymph node and spleen. B cells
• The activation of antigen-specific B cells involves the binding of are transformed from proliferating centroblasts to centrocytes,
antigen to surface antibody receptor, endocytosis of bound antigen plasmablast and finally into plasma cells.
and presentation of processed antigen on class II MHC molecules • Somatic hypermutation is an unusually high rate of mutation that oc-
on the B-cell surface. curs in the variable region of immunoglobulin genes. Hypermutation
• B-cell activation may require help from TH cells (thymus- increases the affinity of the antibodies without changing its specificity.
dependent) or it may be independent of any participation from TH • The immune response can be regulated by a variety of ways, in-
cells (thymus-independent antigen). cluding antigen, antibody and lymphocytes as well as idiotypic and
anti-idiotypic networks.
K E Y W O R D S
• affinity maturation 192 • germinal centre 200 • memory B cell 185 • surrogate light chain 186
• B cell 185 • immature B cell 186 • primary immune • somatic
• B-cell activation 191 • Ig-α 184 response 198 hypermutation 199
• B-cell differentiation 186 • Ig-β 184 • pro-B cell 186 • thymus-dependent
• B-cell receptor 190 • ITAM 190 • plasma cell 185 antigens 198
• centroblast 200 • iccosomes 200 • pre-B-cell 184 • thymus-independent
• centrocyte 200 • idiotypic network 205 • receptor editing 187 antigens 192
• CD40 194 • λ 5 186 • secondary immune
• CD40 ligand 194 • negative selection 187 response 198
1. During development, T cell undergoes both positive and negative 2. What will happen if a B cell does not undergo negative selection?
selection while B cell undergoes only negative selection. Why? Which spectrum of diseases are likely to develop and why?
HINT— B cell recognizes antigen only, i.e., without MHC help. During positive H INT —Autoimmunity diseases
selection, T cells that react with self-MHC are selected. Now can you guess?
3. What does receptor-editing mean? Which type of receptors are 5. Somatic hypermutation is a random event that may result in the
involved in it? Why does a cell attempts it? loss of the antigen-binding capacity of an antibody-producing
4. How are contact-mediated and cytokine-mediated activation of B cell. Yet such cells are never observed in circulation. Why?
B cells different from each other? Are they equally important?

1. Which one of the following is not found in germinal centre: 6. Memory B cells are generated in the lymph node in the:
(a) Plasmablast (a) Primary follicle
(b) Centroblast (b) Secondary follicle
(c) TH cells (c) Germinal center
(d) Immature B cells (d) Dark zone
2. Mature B cells, upon antigen encounter, can undergo: 7. Affinity maturation occurs in immune response:
(a) Apoptosis (a) Against any antigen
(b) Receptor editing (b) Lipopolysaccharide antigen
(c) Class switching (c) Protein antigen
(d) Allelic exclusion (d) All of the above
3. One molecule that is a part of the B-cell co-receptor is: 8. Follicular dendritic cells are involved in all, except:
(a) Membrane Ig (a) Generating iccosome
(b) CR2 (b) Activating TH cell
(c) Igα (c) Presenting antigen to B-cell
(d) Igβ (d) Binding antigen–antibody complex on its surface
4. Memory cells are formed in all, except: 9. B-cell co-receptor binds:
(a) Primary immune response (a) Antigen
(b) Secondary immune response (b) Antigen–antibody complex
(c) Thymus-independent response (c) C3d
(d) Thymus-dependent response (d) C3a
5. Blood-borne antigens encounter specific B and T cells in: 10 “Natural” antibodies present in an individual without any
(a) Lymph node antigen stimulation are the product of:
(b) Thymus (a) B1 cell
(c) Spleen (b) B2 cell
(d) None of the above (c) B cell
(d) All of the above

1. Heavy-chain rearrangement occurs at the pro-B-cell stage. 4. Affinity maturation occurs during secondary immune response.
2. B-cell co-receptor is a complex of three different proteins— 5. Processed antigen displayed on follicular dendritic cell activates
CD21, CD19 and CD18. centrocyte in the germinal centre.
3. Memory B cells are formed in both primary and secondary responses.
Dustin, M. L. and L. B. Dustin, (2001). “The Immunological Matsuuchi, L. and M. R. Gold, (2001). “New Views of BCR Struc-
Relay Race: B-Cells Take Antigen by Synapse”, Nature Immunology: ture and Organization”, Current Opinion in Immunology, 13: 270.
480–82. Meffre, E., R. Casellas, and M. C. Nussenzweig (2000). “Antibody
Enver, T. (1999). “B-Cell Commitment: Pax5 Is the Deciding Regulation of B-cell Development”, Nature Immunology, 1: 309–16.
Factor”, Current Biology, 9: R933–35. Pillai, S. (1999). “The Chosen Few? Positive Selection and Gen-
Fearon, D. T. and R. H. Carter (1995). “The CD19/CR2/TAPA-1 eration of Naïve B-Lymphocytes”, Immunity, 10: 493–502.
Complex of B-Lymphocytes. Linking Natural to Acquired Rajewsky, K. (1996). “Clonal Selection and Learning in the Anti-
Immunity”, Annual Review of Immunology, 13: 127–49. body System”, Nature, 381: 751–58.
Gearhart, P. J. (2002). “The Roots of Antibody Diversity”, Nature, Storb, U. (2001). “DNA Polymerases in Immunity: Profiting from
419: 29–31. Errors”, Nature Immunology, 2: 484–85.
Gellert, M. (1997). “Recent Advances in Understanding Wardemann et al. (2003). “Predominant Autoantibody Produc-
V(D)J Recombination”, Advances in Immunology, tion by Early Human B-cell Precursor”, Science, 301: 1374–77.
64: 39–64.
In 1888, Grohmann Nutall observed that the serum of normal animals “Let us wet our
possesses a natural toxicity for certain microbes. This observation whistles.”
was confirmed by Buchner in 1889, who named the bactericidal fac- —PETRONIUS,
S AT Y R I C O N , 3 4
tor as alexin. (Greek: alexein—to ward off ). Shortly afterwards, Paul
Ehrlich replaced alexin with the German term komplement (now called
complement). This bactericidal activity was to aid or complement the
antibacterial activity of antibody. The complement components are
prefixed with a C. They are numbered by numerals (C1–C9) in the order
of discovery, which, unfortunately is not their order of action. Ferrata,
in 1907, discovered two complement components called midpiece and
endpiece, which were later renamed complement components one and
two respectively. The third complement component (C3) was discov-
ered by Ritz in 1912 as that component which could be inactivated by you should be able to:
cobra venom. Gordon and a co-worker in 1926 reported C4, the fourth • Explain the nomenclature of
complement components and
component of the complement, which was found during studies exam- their peptide fragments
• Describe the classical
ining the ability of ammonia to inactivate the haemolytic activity of complement pathway
fresh serum. • Describe the structure and
function of C1 components
• Comprehend and explain
The classical C3 pathway was discovered by Rapp, Linscott and alternative pathways
• Describe the the mannan-
Mayer in the beginning of the 1960s. They showed that the classical binding lectin pathway
pathway is the result of a cascade of enzymatic alteration of the • Give an account of the
formation of the membrane-
different components. Soon, biologically important effector functions attack complex
• Explain the biological functions
of the complement were investigated and the role of the complement of the complement pathway
• Describe how complement
was deciphered. Some of the important effector functions of the regulatory proteins control
the complement cascade at
complement are given in Figure 10.1. different points
• Briefly summarize various
acquired and genetic
complement deficiencies
The Complement
System
10.1 INTRODUCTION
10
The complement system comprises a group of more than 30 plasma and cell-surface proteins « Bordet correctly reasoned that
(mainly enzymes, proenzymes and other proteins) that interact with other immune system com- bacteriolytic activity requires two
ponents to induce a series of inflammatory responses that help fight infection. different components of the serum.
The first component includes
A large number of complement proteins are proteinases that occur in the inactive zymogen specific antibodies which survive
form. At the site of activation, however, they are activated locally and trigger a cascade of potent the heating process, and the
inflammatory events. The complement system activates through an enzyme-triggered cascade. In second component is heat-sensitive
and termed alexin.
this series of events, an active complement enzyme is generated by cleaving the zymogen precursor
which then cleaves its substrate, another complement zymogen, to its active enzymatic form. This
in turn cleaves and activates the next, and in this way the activation signal is amplified. The pep-
tide fragments formed by cleavage of complement components are designated as a and b. In most
cases, the smaller fragment is designated as a and the larger fragment as b, for example, C5a and
C5b. C2, however, is an exception. C2a is the larger fragment and C2b, the smaller. The complexes
that have enzymatic activity are designated by a bar over the number or symbols; for example,
C4b2a has enzymatic activity and is designated as C4b2a.
Figure 10.1
Schematic representation of various
effector functions of complement.
» The complement system, which is There are three converging pathways of complement activation, each one initiated by a specific
a part of the host defence system
is not antigen-specific and, hence,
set of stimuli. All the three pathways converge to form one common thing, a membrane-attack
is considered to be a part of the complex that forms pores in the membrane of a pathogen causing its lysis and eventually death.
innate immune system.
10.2 C L A S S I C A L P AT H W AY
The activation of complement by the classical pathway begins with the formation of the antigen–
» IgA, IgE and IgD cannot activate
the classical pathway because they
antibody complex or the binding of antibody on a suitable large antigen, such as a cell, in the pres-
cannot bind the C1q component of ence of complement proteins. IgM and certain subclasses of IgG (human IgG1, IgG2 and IgG3) are
the complement. IgG4 cannot acti- known to activate the classical complement pathway. The complete reaction sequence of classical
vate the complement system while
IgG3 is most active in activating the
pathway is depicted in Figure 10.2.
complement system. The binding of antibody to antigen induces conformational change in the Fc portion of the
» The initial name of a complement antibody. This exposes a single C1 complement-binding site on the IgG and five such sites on the
was alexin. pentameric IgM. A single C1 molecule must bind simultaneously to at least two Fc portions of an
» Free IgM cannot activate the immunoglobulin (Ig) to activate the complement system. Since secreted IgM is a pentamer con-
complement even though it is a taining five Fc regions, even a single IgM attached to an antigen can bind the C1 component and
pentamer. This is because C1q has
six binding sites for the Fc region activate the complement system.
of the antibody. Two of these sites In contrast, IgG is a monomer. So several IgG molecules must be aggregated, bringing close
must be filled to activate C1q, multiple Fc regions so as to activate the C1 component. Such an aggregation is common when
which implies that at least two Fc
regions must be located near each IgG binds multi-determinant antigens such as bacterial cell surface. For this reason, only an
other. This happens when two or antigen–antibody complex and not free or soluble antibodies can activate a complement. Free IgM
more IgG molecules or a single IgM cannot activate the C1 component even though it is pentameric because the Fc regions are far apart
binds the antigen. The IgM
molecule gets distorted, bringing and do not have exposed C1-binding sites. As mentioned previously, they are exposed only after
the two Fc regions close and expos- the antibody binds the antigen.
ing the complement-binding site. The C1 in plasma is a large multimeric protein complex of approximately 750 kDa composed
of C1q, C1r and C1s subunits. There is one molecule of C1q and two each of C1r and Cls held to-
» C1 can also be activated without
the participation of the antigen– gether to form the C1 component (C1qr2s2) which is stabilized by Ca2+.
antibody complex. Non-immuno- The C1q subunit is composed of six collagen-like chains arranged like an inverted umbrella.
logical mediators of the classical The tips of the chains have a globular head and the whole Clq molecule looks like a bunch of six
pathway include proteolytic
enzymes such as plasmin, lipid A of tulips. The tips of the globular head performs the recognition function of the molecule by binding
bacterial endotoxin and staphy- to Fc regions (specifically the CH2 domain of the Fc region).The Clq is a lectin (sugar-binding pro-
lococcal protein A (a constituent tein) which contains a collagen-like domain and hence is also termed as collectin.The schematic
of the cell wall of Staphylococcus
aureus). representation of the C1 component is shown in Figure 10.3.
The catalytic function of C1 is initiated by C1s–C1r–C1r–C1s (C1r2–C1s2) tetramer that is
Collectin non-covalently associated with the C1q subunit. C1s and C1r are proteolytic enzymes (serine es-
Collectin is a lectin containing a terases) that remain inactive till the C1 molecule binds the immune complex.
collagen-like domain. Collectins are
mainly soluble pattern-recognition
The binding of two or more globular heads of C1q to IgM or IgG molecules induces a confor-
molecules that are members of mational change that leads to an enzymatic activation of the associated C1r to active serine prote-
the collagen superfamily. Another ase, C1r. The activated protease then cleaves C1s to a similar active enzyme, C1s. The C1s has two
important example of collectin is
the mannan-binding lectin that
substrates of classical complement pathways—C4 and C2.
initiates the lectin pathway of the The C4 component is the next complement component that is activated. C4 consists of three
complement. polypeptide chains α, β and γ. The α chain contains an internal thiolester bond similar to that
found in C3.
» Complement proteins are
produced in the liver and by extra-
hepatic sources such as macro-
phages and fibroblasts.
Antigen-antibody
complex
Thiolester bond
A thiolester bond is the bond C1 Activated C1
C2b C3a
between the sulph hydryl group (C1qr2s2)
(of cysteine) and carbonyl group
C4 C4b C4b2a C4b2a3b
(of glutamic acid). It has the structure (C3 convertase) (C5-convertase)
of a thiol group, esterified with a C2 C3
carbonyl group. that is,—S–C=O.
C4a
Figure 10.2
C5678(9)n C(9)n+C8+C7+C6+C5b C5
Overview of various cascades of a
classical complement pathway. Lytic complex C5a
THE COMPLEMENT SYSTEM 211
The (α chain of) the C4 compo-

nent is cleaved by C1s to yield a
« Fragments “a” and “b” are formed
smaller fragment of C4a and a during complement activation (for
large C4b molecule. The smaller Immunoglobulin receptor example, C3a, C3b). Of these two,
C4a diffuses away from cell sur- site fragment “a” is smaller in size.
Collagen-like triple-stranded helices
face and functions as a mediator 200
of inflammation. amino 80 amino acids
acids in each
C4a does not participate di- Anaphylatoxins
rectly in complement cascade but Anaphylatoxins are fragments
Intact C1q of complement components
functions as an anaphylatoxin (C3a,C4a,C5a) that can trigger mast-
and will be discussed later. cell or basophil degranulation. These
As a result of the cleavage of fragments can also act on several
Immunoglobulin other target cells such as smooth
the C4 component, the thiolester C1s receptor site muscles, neutrophils and endothelial
which is present in the C4b part cells. Anaphylatoxins play an
(C4a is only a small part of the important role in inflammatory
response and defence against
amino terminal and does not in- parasites.
clude the thiolester) gets activated C1r
and converted into a less stable, –
more reactive state. Some of the
activated C4b molecules decay C1r
after reacting with water mole- Triple-stranded
cules which cleave their thiolester helices
bond. Some C4b molecules form C1s Figure 10.3
covalent bonds via the thiolester Schematic diagrams of a various
subunits of C1 component. The C1q
with hydroxyl or amino groups component is made up of six identical
of the nearby target surface in the C1r2,C1s2 subunits and each subunit is composed
vicinity of the C1. subuint Intact C1 of three different polypeptide chains.
The C-terminal halves form the
The next soluble plasma com- immunoglobulin-receptor site that binds
ponent is the C2. It is a single- IgG or IgM antibody. Two C1r and C1s
chain polypeptide that attaches to molecules lie across the C1q.
the exposed binding site on the C4b. The C2 is then cleaved by C1s which is located nearby. The cleavage
of C2 generates a smaller fragment, C2b, which is of unknown importance and diffuses away. The
larger C2a fragment remains physically associated with C4b on the cell surface. The resulting C4b2a
complex is the classical pathway, C3 convertase, having the ability to bind and proteolytically cleave
the C3 proenzyme.
The C3 is sequentially the fourth soluble serum component of the classical complement « At a concentration of about
pathway. The native C3 component consists of two polypeptide chains, α and β. The classical C3 1.2 mg/ml, C3 is the most abundant
complement protein in the plasma.
convertase (C4b2a) removes a small C3a fragment from its amino terminal. The other larger C3b
generated contains an unstable thiolester bond. As with C4b, some C3b molecules react with
water molecules to cleave their thiolester bond and are rendered inactive (see Figure 10.4) and
hence do not participate in the complement pathway. Other C3b molecules react with the cell
surface molecules near C4b2a to form a new complex, C4b2a3b, which functions as the classical
C5 convertase.
A single C3 convertase can generate over 200 molecules of C3b resulting in tremendous am-
plification of this step. Around 1,000 molecules of C3b can bind in the vicinity of a single active C3
convertase. The main effect of deposition of C3b is of two types. The first is that the binding of C3b « C3b is an important opsonin that
to C3 convertase results in the formation of C4b2a3b, a C5 convertase, which catalyzes the enzy- promotes the phagocytosis of
pathogens.
matic cleavage of C5 which, in turn, signals the formation of a membrane attack complex. Another
effect of C3b deposition (when it binds at a place distant from C4b2a) is the C3b-mediated destruc-
tion of the pathogen by phagocytes (that is, the C3b molecules function as opsonins).
The C5 convertase generated by classical (or alternative) pathway initiates the binding and
cleavage of the C5 component. The C5 is a disulphide-linked heterodimeric plasma protein which
shares structural homology with C3 and C4. The C5, however, lacks an internal thiolester bond.
It binds to the C3b component which alters its conformation so that the C4b2a component (of
C5 convertase) can cleave C5 into C5a and C5b. The smaller fragment C5a diffuses away and the
larger fragment C5b attaches to C6 and initiates the formation of a membrane-attack complex. The
diagrammatic representation of classical complement pathway showing the role of C1, C3 and C5
in the formation of a membrane-attack complex is shown in Figure 10.5.
A summary of the characteristics of the components of the classical pathway are given in
Table 10.1.
C3/C4
Activation by proteolysis Decay to inactive form
conformational Change +H2O
CH2
CH CH2 CH2 CH2
2 S CH2 CH2
C
CH2- SH C= C=
CH
=
O O OH O
2
Thiol ester Reactive
SH
carbonyl group
Figure 10.4
Diagram showing activation of C3 or C4.
Proteolytic activation of either C3 or C4 CH2
elicits a conformational change in the Covalent binding to membrane CH2
protein or polysaccharide C=
CH 2
proteins, breaking the thiolester bond.
The carbonyl group generated may react O O
SH
with water or nearby membrane proteins
or polysachharides.
C3
C4a C2a Convertase
C4b C4b
C2b
Antigen +
C4 C2 C5
Convertase
Antigen-antibody C1 Complement binding C3a
C2a
interaction
C3b C4b
C3
C2a C5a
Formation of C3b C4b C5b
membrane-attack
Figure 10.5 complex
Diagrammatic representation showing
formation of C3 and C5 convertase
complex in the classical pathway.
C6,C7,C8,C9 C5
No. of Polypeptide Human Serum Conc.

Name MW (kDa) Chains Chromosome (μg/ml)
C1q 400 3 1 80
C1r 83 1 12 50
C1s 83 1 12 50
C2 102 1 6 20
Table 10.1 C3 185 2 19 1300
The components of the classical
complement pathway
C4 200 3 6 600
10.3 A LT E R N AT I V E P AT H W AY
The existence of an alternative pathway was suggested by Pillemer and Ecker in 1941.This pathway
was a variant (alternative) of complement activation and could be activated (historically) by yeast.
It has since been shown that various bacterial polysaccharides can also initiate the alternative path-
way of the complement cascade.
This pathway of complement activation can occur in the absence of antibody and hence can
be considered a component of the innate immune system. The alternative pathway also generates
C5b, the same product that initiates the assembly of a membrane-attack complex in the classical
pathway. The alternative pathway of complement activation involves four serum proteins—C3,
factors B and D, and properdin.
The alternative pathway components are known as factors and designated by capital letter
symbols (for example, factor B) or by trivial names (for example, homologous restriction factor)
or by acronyms (DAF, for decay accelerating factor). As with the classical pathway, their cleavage
products are designated by the addition of small (lower case) a and b. Thus the large fragment of B
will be Bb and the smaller one, Ba.
In contrast to the classical pathway, it does not depend on antibody for its initiation. The alter-
native pathway is initiated through the spontaneous hydrolysis of C3 (see Figure 10.6). C3 which
is present in high amounts in plasma undergoes a low level of spontaneous, continuous cleavage
(also known as tickover) to generate C3b (or C3(H2O)) and C3a.
This occurs because of hydrolysis of C3 by fluid phase H2O. This C3b which has an altered
conformation binds on a suitable surface such as the outer membrane lipopolysaccharide of bac-
teria. Once bound to such a surface, C3b binds another alternative factor-plasma protein B. The
binding of B by C3b allows another alternative complement protein D, a serine protease, to cleave
B into Ba and Bb in which the smaller fragment Ba diffuses away. The latter remains associated
with C3b to form the C3bBb complex. This complex is highly unstable and decays unless it is sta- « The alternative complement
bilized by another alternative pathway member, properdin or factor P which prevents spontane- pathway is activated by teichoic
ous decay of Bb from the enzyme. This relatively stable C3bBb complex is a potent C3 convertase acid, a lipopolysaccharide present
on the surface of pathogens that
capable of cleaving C3 into C3a and C3b. bind C3b generated in the plasma.
Once this stable complex (C3bBb) is formed on microbial surface, this complex again starts
recruiting factor B and factor D. The cascade then continues its cycle generating many more C3b « In 1958, H. J. Rapp suggested a
mathematical model of
molecules which are deposited on the same surface, forming more C3 convertase which generates complement lysis and demonstrated
more C3b molecules. In fact, because of this amplification mechanism, several million C3b molecules that multiple components must
can be generated on a cell surface within a few minutes of initiation of the alternative pathway. be involved in this process. This
spurred rapid advances and the
Some of the C3b molecules generated by the alternative pathway C3 convertase, bind to the remaining components and their
C3 convertase itself to form a new complex C3bBb3b. This is referred to as alternative pathway conversion products were soon
C5 convertase which functions to proteolyticaly cleave C5 into C5a and C5b. The C5b component discovered by Linscott and Nishioka
(1963), Inove and Nelson (1966) and
binds the microbial surface and initiates the formation of a membrane-attack complex. A sche- Mayer (1972).
matic representation of the alternative pathway is shown in Figure 10.7.
Some characteristics of important components of the alternative pathway are given in Table 10.2.
C3a Ba C3a C5a

Microbes Factor D
C3 C3b C3bBb C3bBbC3b
Cell-envelope B [C3 convertase] [C5 convertase] C5
polysaccharide
C3bBbC3bC5b
Formation of C6,C7,C8,C9 Figure 10.6

membrane-attack Overview of reactions of alternative
complex pathway of complement activation.
C3 convertase
Deposition Ba
Microbial C3b C3b Bb
polysaccharide
+
C3 C3b C3a C3 convertase
Pathogen
Factor B Factor D
C5 convertase
C5b C3b Bb C3b C3a
C5a C3b Bb C3b
Formation of
C3
membrane-attack
complex
C6,C7,C8,(C9)n
Figure 10.7
Diagrammatic representation of an
alternative pathway. C5
No. of Polypeptide Human Serum Conc.

Name MW (kDa) Chain (s) Chromosome (μg/ml)
Factor D 24 1 ? autosome 1
Factor B 90 1 6 210
C5 204 2 9 70
C6 120 1 5 65
C7 120 1 5 50
C8 150 3 1/9 55
C9 70 1 5 56
Regulatory components
C1-inhibitor 110 1 11 185
Factor H 150 1 1 450
Table 10.2 Factor I 88 1 4 35
The components of the alternative
Factor P – 1 – 15
pathway and regulatory components.
10.4 THE MANNAN-BINDING LECTIN

P AT H W AY
The mannan-binding pathway which has recently been discovered, is an additional pathway by
which complement system can be activated.
» MBL is an acute-phase protein Mannan (mannose)-binding lectin (MBL) is an acute-phase protein. It is present at low con-
whose concentration increases centration in normal plasma of most individuals and its production is increased during inflam-
during inflammation. A deficiency
in MBL is associated with increased matory response. MBL (also called defence collagen) has a collectin (C1q)-like structure and is
vulnerability to infections. six-headed.
It specifically binds to mannose residues present on the surface of microorganisms. After MBL
binds to the surface of a cell or pathogen, two MBL-associated serine protease zymogen or MASP
bind to it. MASP1 and MASP2 share homology with Clr and Cls respectively, and mimic their
activities. When MBL complexes with MASP and this MBL–MASP complex binds to the pathogen
surface, MASP1 and MASP2 are activated to cleave C4 and C2 to form C4bC2a. This C4bC2a
complex which is classical C3 convertase, gets deposited on the surface of microorganisms. Thus
lectin pathway initiates the complement activation in almost the same way as the classical pathway.
It is considered as another important arm of the innate defence mechanism, as C3 convertase is
C3 Convertase
C4a C2a
C2b
C4b C4b
C4 C2
MASP1 MASP2
C5 Convertase
MBL C3a C3
C3b C2a C2a
C5b C4b C5a C3b C4b
Formation of
membrane-attack
complex
C6,C7,C8,(C9)n
Figure 10.8
Schematic diagram of a lectin pathway.
formed without the need for a specific antigen–antibody complex. It is suggested that mannan-
binding lectin pathway provides “innate” protection to a newly born child during that “gap period”
of about 12 months in which there is loss of the protective cover of maternal antibodies and the
development of child’s effective immune system. A diagrammatic representation of mannan-bind-
ing lectin pathway is shown in Figure 10.8.
10.5 T H E F O R M AT I O N O F M E M B R A N E -
AT TA C K C O M P L E X
The C5 convertases generated by any of the pathways described above can initiate the activation of
the terminal components of the complement system which results in the formation of a cytocidal
membrane-attack complex. This last sequence of events, often referred to as terminal sequence,
involves C5b, C6, C7, C8 and C9 which interacts to form the membrane-attack complex. The end
result is the formation of pores in the lipid bilayer membrane of the target cell that destroys the
membrane integrity and kills the pathogen, possibly because of the loss of osmotic stability due to
the influx of water and loss of electrolyte.
The C5 serum protein binds to the C3b molecule of C5 convertase. The C5 convertase cleaves
C5 into small C5a that is released, and C5b fragment that remains bound to the cell surface. The
C5b component is unstable and transiently maintains a conformation capable of binding the next
protein in the cascade, C6, which stabilizes the C5b6 complex on the cell surface.
As soon as C5b6 binds C7, the resulting complex undergoes conformational change to the
highly lipophilic C5b67 complex as this structural transition exposes the hydrophobic regions on
this complex. It is a structural transition. The complex which was till now residing on the cell surface
of the pathogen, inserts into the lipid bilayer of the cell membrane where it becomes a high-affinity
integral membrane receptor for C8. If this binding of C7 to C5b6 occurs on an immune complex,
and not on any cell surface, the hydrophobic C5b67 complex is liberated from there and binds to
the cell surface of an innocent cell standing nearby. This could kill the cell by innocent-bystander Innocent bystander effect
The killing of innocent non-target
lysis and produce tissue damage. This occurs in a number of autoimmune diseases, as will be
cells by accidental (unintentional)
discussed in later chapters. formation of membrane-attack
The binding of C8 to membrane-bound C5b67 induces the conformation change in C8 so that complexes on their cell membranes,
resulting in tissue damage, is termed
it becomes hydrophobic and inserts into the bilayer. The C8 molecule is a trimer composed of α, β
as the innocent bystander effect
and γ chains. The β chain binds to the C5b67 complex and the γ chain inserts into the lipid bilayer.
This stably inserted C5b678 complex (C5b-8) creates a pore of 10Å in diameter. The formation
of such a pore can lead to cell lysis, though such pores do not have potent bactericidal activity.
The formation of a highly lytic and microbicidal membrane-attack complex is accomplished « Membrane-attack complex
by the binding and polymerization of C9, the final component of the complement cascade to the punctures the target cell membrane
with 100 Å pores.
C5b678 complex. The formation of a membrane-attack complex on the surface of a membrane is
Transmembrane
aqueous channel
C7 C8 C9
C5b C6
Figure 10.9
Schematic diagram showing the
formation of a membrane-attack
complex.
» C9 is a 70 kDa, 537-residue-long As many as 10–17 molecules of C9 can be bound and polymerized by a single C5b678 even
protein. Though It is soluble in an though just four C9 molecules (C5b-894) have full lytic capabilities for many microorganisms and
aqueous environment, it also
contains hydrophobic domains eukaryotic cells.
that aggregate to form an ion- During polymerization, hydrophobic sites on C9 molecules are exposed so that they can in-
conducting doughnut-shaped sert into membranes, forming a pore-like structure. The pores have an internal diameter of about
channel in the plasma membrane
of the target cell. ~100 Å and appear similar (but smaller) than the membrane pores formed by the perforin protein
secreted by Tcyt lymphocytes.
» As few as four C9 molecules can Since small molecules and ions can diffuse freely through the central channel of the mem-
bind the C5b-8 complex and cause brane-attack complex, it is assumed that a passive exchange of small soluble molecules, ions and
lysis of microorganisms.
water dissipate the energy-dependent gradient across the outer membrane of the microorganism
that is required for cell survival. The death of the microorganisms usually results from osmotic lysis
because of the influx of water or by toxic effects of high concentration of influxed ions, particularly
Ca++. Since membrane-attack complexes with as few as four C9 are fully bactericidal even though
such structures do not form pores, it is suggested that the killing of cells could also be due to some
disturbances of the membrane lipid bilayer caused by the insertion of the hydrophobic portion of
the C5b-9 complex.
10.6 BIOLOGICAL FUNCTIONS

OF COMPLEMENT PROTEINS
The complement system comprises a set of proteins that are designed to eliminate microorganisms
and other antigens from the tissue and blood. The biological functions of the complement system fall
into five main categories: (a) cytolysis, (b) inflammation, (c) opsonization, (d) immune complex
clearance and (e) induction of immune response. Some important biological functions of comple-
ment proteins are shown in Figure 10.10.
10.6.1 C Y T O LY S I S
The complement-mediated cell lysis is capable of lysing a broad spectrum of microorganisms. Spe-
cific humoral responses to microbes generate antibodies that bind to the invading microorganisms;
these antibodies activate the complement on the surface of microbes and lead to their lysis by the
formation of membrane-attack complexes. Since lectin and the alternate pathway of complement
activation does not require initial antigen–antibody interactions, these pathways serve as an im-
portant arm of the innate system for non-specific defence against infectious microorganisms.
Apart from its well-known bactericidal activity, membrane-attack-complex-meditated lysis
may also be an important defence against viral infections. Most of the enveloped viruses are sus-
ceptible to complement-mediated lysis. These include Retroviruses, Herpesviruses, Orthomyxo-
viruses, Paramyxoviruses. Certain viruses express regulatory inhibitory molecules that are used
to evade the complement response; for example, Herpes simplex viruses express the Fc receptor
that binds antibody at the “wrong” (Fc region) end and hence hide its complement-activation site.
Similarly, certain fungi like Candida albicans express complement receptor (CR-2 and CR-3)-like
molecules, which bind and block the complement components, preventing their further activa-
tion. More importantly, certain viruses have evolved mechanisms to use complements to enhance
their pathogenicity. These pathogens use complement receptors to gain entry into the host cells.
Echovirus uses decay-accelerating factor, measles virus uses CD46 and Epstein–Barr virus uses
CR2 to gain entry into the host cells.
Anaphylatoxin
C3a,C4a,C5a
Increased
chemokinesis Prostaglandins
Adhesion Prostaglandins
C5b-C9 Adhesion
molecules ROS molecules
Bacteria RNS ROS,RNS
Vasoactive Enzymes IL-1,IL-6 Enzymes

Membrane-attack substances
complex Mast cells/basophils Neutrophils Monocytes/macrophages
Cell lysis Inflammation
To liver
and spleen
Neutrophils,macrophages C3b
CRI Red blood cells
C3b C3b
CRI,CR3,CR4
C3b,iC3b,C4b C3b Fc
Antigen-antibody complex Antigen-antibody complex Figure 10.10

disrupted and cleared Schematic diagrams showing various
biologic functions of complement
Promotion of phagocytosis Immune complex clearance proteins.
Similarly, certain bacteria have evolved a variety of mechanisms to evade complement attack.
Some Gram-negative bacteria possess surface cell-wall lipopolysaccharides component, which
directs the covalent binding of C3 and the attachment of the membrane-attack complex away from
« Gram-negative bacteria with an
the bacterial cell membrane so that their opsonization and lysis is impossible. In some other Gram- exposed outer membrane are
negative bacteria such as E. coli and Salmonella, lipopolysaccharides resist complement activation by generally more susceptible to
preventing the sticking (insertion) of membrane-attack complex into the bacterial membrane. The complement-mediated cell lysis
than Gram-positive bacteria
membrane-attack complex is released from the bacterial cell rather than forming a pore. However, it covered with a thick peptidoglycan
should be noted that most Gram-negative bacteria are susceptible to complement-mediated lysis. layer.
Gram-positive bacteria are generally more resistant to complement-mediated cell lysis be- « Some bacteria have evolved
cause of the tough nature of thick peptidoglycan layer in their cell wall, which prevents insertion novel mechanisms for preventing
of the membrane-attack complex into the inner membrane. the onslaught of complements.
These microbes possess elastase,
A membrane-attack complex can also lyse host red blood cells, and nucleated cells leading a proteolytic enzyme that cleaves
to tissue injury and disease. When the complement is activated, the membrane-attack complex pharmacologically active C3a and
is finally formed on the surface of the pathogen as well as on the innocent bystander host cells. C5a, preventing them from
inducing inflammation.
The formation of membrane-attack complex on host-nucleated cell is usually not very lethal. This
is partially due to the presence of the regulatory membrane protein such as CD59 (or protectin-
inhibitor of membrane-attack complex), surface sialic acid (inactivates bound C3b) but mainly
because of the majority of host cells can endocytose a membrane-attack complex. If a membrane
attack complex is removed as soon as it is formed, cells can repair any damage done and survive.
However, red blood cells are highly sensitive to pore-forming membrane-attack complexes, and
even a single membrane-attack complex can lyse a red blood cell. « In 1968, Shin and co-workers
unveiled a new dimension of the
complement story. They reported
10.6.2 A N A P H Y L AT O X I N S A N D I N F L A M M AT I O N that the byproducts of the
Anaphylatoxins are small cationic peptides generated by the complement cascade, that induces the activation of C3 and C5 are signifi-
cant pharmacological contributors
release of pharmacologically active mediators from mast cells which cause inflammatory responses to inflammation. The larger
characteristic of anaphylaxis. fragment binds to the target near
The activation of complement cascade results in the generation of anaphylatoxins—C3a, C4a the site of activation and the
smaller fragments diffuse away
and C5a. C3a (9 kDa) and C5a (11 kDa) peptide can be generated from classical or alternative from the target site to initiate a
pathway while C4a (8.7 kDa) is generated from classical pathway only. The formation of anaphyla- localized inflammatory reaction.
toxins from C3, C4 and C5 is shown in Figure 10.11.
All three anaphylatoxins can bind to receptors

s s
s s on mast cells and basophils causing their degranu-
C3 lation and release of vasoactive mediators such as
sc
Thiolester histamines. Histamines induce smooth muscle
o
contraction and increased vascular permeability.
s s These combined effects result in the influx of fluid
s s
that carries antibodies and phagocytic cells to the
C3b Active
» Out of C3a, C4a and C5a, C5a is the C3a SH c=o site of antigen entry. C5a is a potent activator of
most potent anaphylatoxin. C3a is neutrophils, monocytes, macrophages, basophils
20-fold less potent and C4a is 2500 Activation of C3
times less potent than C5a. and mast cells.
s s Neutrophils respond to C5a by stimulation of
s s
C4 chemokinesis and chemotaxis, increased surface
C4a s c s s expression of adhesion molecules, stimulation of
Thiolester o s s
Eicosanoids the production of eicosanoids, respiratory burst
Eicosanoids (Greek: eikosi—twenty) and production of reactive oxygen species.
refer to polyunsaturated fatty acids Upon stimulation by C5a, monocytes and mac-
containing 20 carbon atoms. These
include products of arachidonic acid s s rophages show similar responses and secrete IL-1
metabolism such as prostaglandins,
s s and IL-6, while basophils and mast cells release his-
C4b Active
leukotrienes and thromboxanes. C4a s s form tamine and other vasoactive substances. The combina-
These molecules play an important SH c=o
s s tion of C5a action on mast cells, endothelial cells and
role in inflammation and allergy
and also serve as key intercellular neutrophils contributes to the process of inflam-
mediators. Activation of C4
mation. The biologic effect of the C5a is mediated
C5a s
by the presence of C5a receptor on various cell types.
s
» The rhodopsin family has seven s s The C5a receptor belongs to rhodopsin family of
α-helical transmembrane segments C5 receptor.
and uses heterotrimeric G protein Thiolester
for coupling to the signal absent
C5a receptor is expressed on many cell types
transduction pathway. including neutrophils, eosinophils, basophils,
C5a s s monocytes, macrophages, mast cell, endothelial
Figure 10.11 s s
Structure of C3, C4 and C5 and formation C5b Active cells, smooth muscle cells and astrocytes. C3a is
Activation of C5 form much less potent than C5a and is recognized by a
of anaphlatoxins C3a,C4a and C5a.
different receptor (though of the same rhodopsin
superfamily). C3a induces weak neutrophil aggre-
» C3a and C4a receptors are found gation and stimulation of the respiratory burst. It is a weak stimulator of movements (chemokinesis
on basophils, mast cells, lympho- and chematoxis).
cytes and smooth muscle cells.
The activities of these anaphylatoxins are regulated by plasma protease called as
carboxypeptidase-N which cleaves carboxyl-terminal arginine residues common to C3a, C4a and
C5a, resulting in slightly truncated forms of anaphylatoxin termed as des-Arg forms. The des-Arg
form of C5a retains about 10 per cent of its chemotactic activity while C3a des-Arg and des-Arg
C4a are completely inactive.
10.6.3 C 3 b G E N E R AT I O N A N D P R O M O T I O N
OF PHAGOCYTOSIS
All classical, alternative and lectin pathways lead to the generation of C3b. C3b is the major
» C3b, iC3b and C4b can act as
opsonin of the complement system although iC3b (cleaved product of C3b) and C4b also coat the
opsonins.
microorganism and immune complex, and have opsonizing activity.
Neutrophils and macrophages, as well as a few other cells express complement receptors (of
CR1, CR-3 and CR-4 type) that bind the complement component C3b, C4b and iC3b.The details
of complement receptors, their ligands and their activities are given in Table 10.3.
Thus complement activation on the surface of microbial cells promotes the adherence of the
microbes to host leukocytes that are competent at phagocytosing and killing the microbes. Not
only this, the coating of the immune complexes or phagocytes by C3b/iC3b increases the phago-
cytosis mediated by Fcγ receptors. For example, if a microbe is coated by IgG, it is phagocytosed
albeit slowly via the Fcγ receptor. If the same particle bears iC3b apart from coated IgG, it is found
that iC3b binds CR3 receptor on leukocytes and IgG binds Fcγ receptor on leukocytes. The simul-
taneous binding on both the receptors greatly enhances the process of phagocytosis.
Molecular
Weight (kDa)/
Receptors Location Ligands Cellular Distribution Activity
Complement receptor—CR1/ 190–280/ C3b, C4b, iC3b, Erythrocytes, B cells, neutro- Acts as cofactor for factor-1-
CD35 Located on cell C3b > iC3b phils, monocytes macro- mediated cleavage of C3b/C4b;
surface, plasma phages, glomerular epithelial Immune complex clearance;
cells, follicular dendritic cells, involved in Fc-receptor-dependent
eosinophils and independent phagocytosis
Complement—receptor 145/Cell surface iC3b, C3dg, B cells, follicular dendrilic Promotes humoral response, binds
(CCR2/CD21) Epstein–Barr Virus, cells, epithelial cells of cervix Epstein–Barr virus
interferon-α and nasopharynx
Complement receptor— α-165, β-95/ iC3b, certain Follicular dendrilic cells, Surface adhesion molecule re-
(CCR2/Mac-1) CD11bCD18 cell surface bacteria, fibrinogen NK-cells, monocytes/ quired in enhancing phagocytosis,
factor-X. ICAM macrophages, some T cells chemotoxis
Complement receptor— α-150, β-95/ iC3b Platelets, monocytes, Involved in Fc -receptor-dependent
(CCR4-/CD11c/CD18/P-150, 95) cell surface neutrophils and independent phagocytosis
Clq receptor ? cell surface Clq Leukocytes, platelets endo- Enhances the phagocytic activities
thelial cells, smooth muscle of leukocytes and thereby en-
cells hances clearance of microbes
C3a/4a receptor ? cell surface C3a, C4a Mast cells and basophils Induces degranulation of
basophils and mast cells causing
release of histamines
C5a receptor 40 cell surface 5a Endothelial cells, mast cells, Degranulation of mast cells/
neutrophils, monocytes, basophils, promotes chemotaxis
macrophages basophils,
smooth muscle cells
Table 10.3
Receptors for complement fragments.
10.6.4 S O L U B I L I Z AT I O N O F I M M U N E C O M P L E X E S
OR IMMUNE COMPLEX CLEARANCE
Aggressive immune response towards an antigen leads to the production of a large amount of im-
mune complexes in the circulation. These immune complexes can potentially deposit in any vital
organs such as kidney, liver, etc., or on the vessel wall. These deposited complexes can activate the
complement and lead to inflammatory responses that damage the neighbouring and affected tis-
sues. Interactions between Fc regions of different molecules of antibodies lead to the aggregation
of the immune complex and formation of a large lattice. The binding of complement components
on antibody molecules sterically blocks the Fc–Fc interactions, preventing the formation of a large « Approximately 5 ⫻ 102 molecules
of CR1 are present on each red
lattice structure, and dissociating the already formed immune lattices. blood cell. In comparison, each neu-
Another function of the complement system is the rapid clearance of the immune complex. trophil has 5 ⫻ 104 molecules per
This function is best exemplified by the receptor of complement (CR1) present on the surface of cell. However since the number of
red blood cells is about 1,000 times
red blood cell. more that the white blood cells, the
The immune complex thus formed activates the complement, and the C3b that is generated, number of CR1 of red blood cells
forms covalent bond with the antibody. The C3b absorbed on the complex has high affinity for forms the majority (more than
92 per cent) of CR1 in the blood.
CR1 present on the surface of red blood cell. It is believed that red blood cells bind these immune
complexes and take them to the liver and spleen, where phagocytic cells remove this load (of
immune complex) from the red blood cell as they pass through the sinusoids of these organs. It is
suggested that this uptake of immune complex by phagocytes of the liver and spleen is mediated
by Fc receptor present on phagocytes. In addition, some of the C3b is converted to iC3b on the
immune complex (since CR1 is a cofactor for factor-I-mediated conversion of C3b to iC3b) and
this iC3b is recognized by mononuclear phagocytes in the liver. The immune complex bearing free
Fc regions of antibodies and iC3b is taken up by these phagocytes.
10.6.5 N E U T R AT I Z AT I O N O F V I R A L I N F E C T I O N
Certain viruses such as Retroviruses can activate the alternative, lectin or even classical pathways in
the absence of antibody. For most viruses, the complement system is activated because antibodies
bind to repeating subunits of the viral protein coat, a pattern ideally suited for complement activa-
tion by the classical pathway.
The neutralization of viral infection by a complement can be achieved by several ways. C3b
complement components facilitate antibody induced clustering of virus particles. Aggregation of
viral particles reduces the number of effective infectious viral particle.
The binding of antibody/and or complement to the surface of a virus may block its attach-
ment to the susceptible host cell or aid in binding to phagocytic cells bearing a CR1 complement
receptor or an Fc receptor. These phagocytes ingest such bound complement/antibody-coated viral
particles and destruct the ingested viral particles.
10.6.6 INDUCTION OF IMMUNE RESPONSE

The complement system helps in phagocytosing antigen. The phagocytosed antigen is presented
together with MHC to naïve B cells. Naïve B cells present in the periarteriolar lymphoid sheath of
Centroblasts
lymph nodes starts dividing rapidly after being assisted by TH cells. A few centroblasts are formed
Centroblasts are rapidly after this exposure. However, when antigen is presented together with C3dg (cleaved product of
proliferating B cells. C3b), a large number of centroblasts are formed.
This evidence suggests that a complement has an accessory, if not crucial, role in the efficient
induction of antibody responses.
10.7 R E G U L AT I O N O F C O M P L E M E N T
CASCADE
Given the non-specific, potent and damaging effects of complement activation and the way
activation is rapidly amplified, several elaborate mechanisms have evolved to restrict its activity
to designated targets. The general mechanism of regulation includes the presence of complement
components such as zymogens (inactive enzyme forms), and getting activated only after binding
» The complement system is to pathogen surface. Moreover, there are several labile components which undergo spontaneous
regulated by controlling the serum inactivation as they diffuse away from the target cell.
concentrations of components, Even so, several complement components are activated spontaneously at a low rate in the
spontaneous inactivation of
complement fragments, the plasma and these activated complement components can sometimes bind to the cell surface of a
presence of inhibitors of serum host cell. The potentially damaging consequences are prevented by a series of complement-control
proteinases and specific proteins which regulate the complement cascade at different points. Some characteristics of a few
complement inhibitors and
regulatory proteins. important components that regulate the complement pathways are given in Table 10.2.
10.7.1 R E G U L AT I O N O F C 1
The activation of C1 is controlled by a plasma serine proteinase inhibitor, C1 inhibitor (C1Inb). It acts
in two ways. It binds free C1 in the serum (the concentration of free C1Inh is greater than that of C1)
and inhibits spontaneous activation of C1 ( it is released on activation of C1 by immune complexes). It
also limits the activation of C4 and C2 [see Figure 10.12(a)] by binding and inhibiting the Clr and Cls
proteases. Clr–Cls proteases dissociate from Clq which remains bound on the pathogen. In this way,
C1Inh limits the time during which active C1s is able to cleave C2 and C4 components.
10.7.2 R E G U L AT I O N O F C 3 C O N V E R TA S E S
The C3 convertase is formed in the classical, alternative and lectin pathways. It serves as a major
amplification step in complement activation, generating hundreds of molecules of C3b. The gener-
ated C3b is extremely reactive (and has no mechanism to distinguish between host cell surface or
» During infection, the concentra-
tion of plasma C3 falls. This is due to pathogen) and has the potential to bind to nearby host cells and damaging them.
the fact that it is used up at a faster The damage to normal host cells is prevented because of the short lifetime of C3b as it under-
rate than it is formed. goes spontaneous hydrolysis. Hydrolysed C3b can no longer bind to the target site. Moreover, the
lifetime of activity of C3 convertase is reduced by several ways.
• Classical C3 convertase is formed by C4bC2a. Regulatory proteins such as C4b binding
protein (C4BP), complement receptor-1 (CR1), and membrane cofactor protein (MCP)
bind C4b and prevent Spontaneous-activation

its association with C2a, inhibited
and hence no C3 conver-
tase is formed (see Figure
10.12b). Not only this,
C1 C1Inb
the C4b bound to these
proteins (CR1, MCP, Activation of C4 and
C2 inhibited
C4bBP) is rapidly cleaved
into C4d (bound form) a) Regulation of C1
and soluble C4c forms,
inactivating it. All these
C4BP No
regulatory proteins are Binding inhibited
C4bC2a
found in the host body. + CRI [C3
All these proteins are en- convertase
coded on chromosome C4b MCP C2a formed]
1 in humans, known as
regulators of comple- b) Regulation of C3 convertase—classical pathway
ment activation (RCA)
gene cluster. Some of these
RCA proteins present Promotes dissociation
on the cell surface such C2a
C2a of C2a
as decay accelerating Inhibits binding
factor (DAF), and CR1,
act on both classical and C4b C4BP Figure 10.12
C4b DAF Schematic diagrams of (a) regulation of
alternative C3 conver- Cell surface Cell surface C1; (b) classical pathway of regulation of
tase. CR1 and DAF pro- C3 convertase; and (c) classical pathway
mote the dissociation of c) Disruption of C3 convertase—classical pathway of disruption of C3 convertase.
the C3 convertase (both

classical and alternative). Other serum proteins such as C4BP and factor H act on C4b and
C3b respectively and block the formation of C3 convertase (see Figure 10.12c). Interest-
ingly some parasites use similar molecules to prevent complement activation; for example,
Trypanosomes express a DAF-like molecule while Schistosomes achieve same results by
adsorbing host DAF.
• Alternative C3 convertase (C3bBb) is prevented from forming on the host cell surface by
a similar reaction mechanism. In this case CR1 or factor H binds C3b.The binding of
CR1 to C3b prevents its association with factor B and hence prevents the formation of
alternative C3 convertase. Moreover, once C3b is bound to these regulatory proteins
(CR1, factor H), it is cleaved by factor I (plasma protease) into bound iC3b and solu-
ble C3f fragment. Further cleavage of iC3b generates free C3c and C3dg (bound) form
(see Figure 10.13), which is covalently attached to the membrane surface.
Factor H
+
Factor I C3c
C3b + iC3b C3f
CR1 CR1 C3dg
HS 0=C SH 0=C SH C=O

Figure 10.13
N-H Regulation of alternative C3 convertase.
N-H N-H The formation of an alternative C3
convertase C3bBb is prevented by
binding and cleavage of factor C3b by
factor I, factor H and CR1. The cleaved
Membrane surface Membrane surface Membrane surface product of C3 cannot bind protein B and
(Non-activating) hence no C3 convertase is formed.
S-protein C9 monomers
Protectin
C567
C5678
Binding to
target membrane Binding of C9 to C5b
inhibited HRF
678 is inhibited
Figure 10.14
Regulation of the membrane-attack
complex. No formation of membrane-attack complex
It should be clarified that factor I binds to C3b bound on the host cell as it has an affinity for
sialic acid present on the host (mammalian) cell. By contrast pathogen surfaces (for example, bacteria)
lack these sialic acid residues and hence lack the binding of these regulatory proteins. Hence C3bBb
convertase persists on pathogen and is active.
10.7.3 R E G U L AT I O N O F M E M B R A N E - AT TA C K C O M P L E X
In addition to the mechanisms we have studied till now, there are also inhibitory mechanisms
that prevent the insertion of the membrane-attack complex into innocent bystander cells. Several
plasma proteins bind to the C5b67 complex and thereby inhibit its random insertion in the cell
membrane. A serum protein called S protein can bind to C5b67 and prevents its insertion into
the membrane of nearby cells. Another protein, intrinsic membrane protein-CD59 or protectin,
» Protectin and S protein prevent
the formation of a membrane- and HRF (homologous restriction factor) also a membrane protein, inhibits the binding of C9 to
attack complex on nearby C5b678 complex and hence prevents the formation of a fully functional membrane-attack complex.
host cells. The role of S protein and protectin in shown in Figure 10.14.
10.8 COMPLEMENT DEFICIENCIES

» In 1923, R. R. Hyde described The first case of complement deficiency in a human adult was reported by A. Silverstein in 1960,
complement-deficient guinea pigs following which numerous cases of complement deficiencies have been described.
for the first time. Unfortunately, the
precise nature of the defect could There are deficiencies of the components of the classical and alternative pathways, or in soluble
not be worked out as the whole or membrane-bound regulatory proteins. Some important complement deficiencies are given in
colony of guinea pigs died before Table 10.4. Most of the complement deficiencies cases reported till now are attributed to acquired
further studies could be carried out.
or genetic deficiency of complement.
• Acquired complement deficiency diseases are acquired during the lifetime of an individual.
They may have normal gene and its product i.e., complement components are normal but
are non-functional or depleted because the patient might have produced an autoantibody
to the component; for example in partial lypodistrophy, an autoantibody is formed against
the C3 complement factor which becomes non-functional and is depleted, manifesting C3
deficiency.
• Genetic complement deficiencies involve change(s) in genetic loci coding the comple-
ment components. As a result a specific complement component is not synthesized and
hence is deficient. Since these gene-based complement deficiencies result from congenital
defects in the gene, they are heritable. Some deficiencies require only one defective gene
while others require both alleles to be defective. The deficiencies involve all the compo-
nents of the alternative and classical pathways, membrane-attack complex, and soluble
and membrane-bound proteins and receptor.
1. Genetic deficiency in classical component, including Clq, Clr, Cls, C2 and C4, pre-
vents activation of the classical pathway. The single most important clinical conse-
quence of these deficiencies is the manifestation of systemic lupus erythematosus
(SLE). This could be because of the fact that these classical pathway components
may be involved in clearance and solubilization of circulating immune complexes.
If the immune complexes that are generated normally are not routinely cleared from
circulation, they may deposit in blood vessel walls and tissues where they produce
Deficient Complement Clinical

Component Inheritance Function Affected Manifestation
C1 Autosomal recessive No activation of SLE, renal disease, hypo-
classical pathway gamma globulinemia,
bacterial infections
C2, C4 Autosomal recessive No activation of Rheumatoid arthritis,
classical pathway SLE, glomerulonephritis
C3 Autosomal recessive Opsonic fragments Glomerulonephritis,
(C3b not produced), recurrent bacterial infec-
terminal components tions
not activated
Mannose Binding Autosomal recessive No activation of lectin Recurrent bacterial
Lectin, Terminal pathway. No membrane (Neisseria) infection, SLE
components C5, C6, attack complex formed,
C7, C8 no bacterial cell lysis
C9 Autosomal recessive No membrane pores Asymptomatic or
formed Neisseria infections
Factor H, Factor I ? Activation of C3 is not Hemolytic-uremic
regulated syndrome, recurrent
bacterial infection
Properdin, Factor D X-linked (properdin) No activation of alter- Recurrent pyogenic
nate pathway infection Table 10.4
Complement deficiencies.
inflammation. Such inflammatory reaction may promote the breakdown of peripher-

al tolerance and produce autoantibodies against vital organs/tissues. Surprisingly, the
deficiency of the above-mentioned complement components is not associated with
increased infections, suggesting that an alternative pathway is sufficient to combat the
onslaught of most of the bacteria. All these deficiencies of Clq, Clr, Cls, C2 and C4 are
« Factor I is a serine protease that
autosomal recessive.
cleaves both C3b and C4b into
2. Deficiencies of C3 as well as of factor H and factor I cause recurrent pyogenic bacterial smaller fragments in the presence
infection that may be fatal. In addition, the patient may also be susceptible to glomer- of factor H. A deficiency of factors H
ulonephritis where immune complexes are deposited in the kidney. The deficiencies and I leads to excessive consump-
tion of the complement compo-
of C3, factor H and factor I show autosomal recessive inheritances. nent C3 and recurrent pyogenic
3. The deficiencies of components C5, C6, C7 and C8, which prevent the formation of infections.
membrane-attack complex, have also been reported. As expected, the patient with
this deficiency of the terminal components would not generate any membrane-
attack complex and therefore would not be able to lyse invading microbes. Individuals
would be therefore expected to have recurrent microbial infections. Surprisingly, the
only constant problem in these patients is recurrent Neisseria infections, including
N.meningitidis and N.gonorrhoeae, suggesting that complement-mediated bacterioly-
sis is an important combat tool against these microbes. Individuals with deficient C9
complement components are usually asymptomatic even though the absence of C9
blocks the complement-mediated pore formation. Deficiencies of C5, C6, C7, C8 and
C9 are autosomal recessive.
4. The deficiency of the alternate pathway component properdin and factor D inhibits
the activation of the alternate pathway. It is usually associated with recurrent Neisseria
infections. The deficiency of properdin is X-linked.
5. Some complement deficiency diseases are also manifested due to defective regula-
tory proteins. Hereditary angioneurotic edema (HANE) is an autosomal dominant
deficiency of C1Inh. C1Inh, as mentioned previously, limits the spontaneous activa-
tion of C1 in plasma. Chronic spontaneous complement activation of C1 leads to
the production of cleaved fragments of C4 and C2 from which C2 kinin is formed.
C2 kinin causes extensive swelling in the larygeal, facial and intestinal mucosa.
» In the absence of regulatory Serious symptoms caused by the swelling of tissue results in abdominal pain, trachea
complement components, comple-
ment components are decayed
choking, nausea, vomiting and diarrhea. HANE also produces another C2-kinin-like
at a very fast rate. The deficiency mediator, bradykinin, which is generated by kallikrein. Kallikrein is also regulated by
of factor H and DAF can result in C1Inh. The deficiency of C1Inh is an autosomal dominant trait.
complement-mediated hemolysis
of red blood cells and the appear-
6. Another deficiency of complement-related proteins is paraoxysmal nocturnal hae-
ance of haemoglobin in the urine of moglobulinuria (PNH). In this disease both CD59 and DAF fail to function. This is
the patient. because CD59 and DAF need to be anchored to the lipid bilayer of the host cell surface
for its proper functioning. In PNH, the (lipid) anchor is not formed and these proteins
are secreted and not anchored. Normally, CD59 functions to inhibit the binding of
C9 to C5b678 complex, hence protecting host cells, while DAF functions to inhibit
C3 convertase formation on the host cell surface. The inactivation of these proteins
in PNH is characterized by episodes of red blood cell lysis by the complement. Red
blood cells that lack CD59 are also susceptible to lysis as a result of spontaneous acti-
vation of the complement pathway. This leads to haemolytic anaemia, pancytopenia
and thrombosis.
7. Deficiencies of complement receptors include the absence of CR3 and CR4 result-
ing from mutation in their gene. This disease, called leukocyte adhesion deficien-
cy (LAD), is characterized by frequent pyogenic infections. This is probably due to
inadequate adherence of phagocytes to the site of infection and probably impaired
iC3b-dependent phagocytosis of bacteria.
The complement system is group of about 30 proteins comprising both soluble and mem-
brane-associated components that functions as the innate arm of immunity. These proteins cause
bacteriolysis, phagocytosis of invading pathogens and induction of inflammatory responses that
control microbial invasion. Of the three complement pathways, (that is, classical, alternate and lec-
tin) only the classical pathway is initiated by antigen and antibody complex. The alternate pathway
is activated by bacterial lipopolysaccharides and lectin pathway is triggered by mannose residues
found on certain bacterial cells. All three pathways converge to form the cytocidal membrane-
attack complex that form pores in the cell membrane that kills the pathogen. The lethal enzymatic
cascades of complement are kept in check by a variety of regulatory proteins such as C1 inhibitor,
factor H, I and S proteins and several others. Complement deficiencies have also been reported to
occur following deficiency of either classical components, alternate pathway components, regu-
latory components or due to the lack of receptor(s) for complement components. Complement
deficiencies results in several clinical manifestations such as recurrent microbial infections, hae-
molytic anemia, increased inflammatory response and thrombosis.
Complement Fixation Test

When an antibody binds to an antigen, its complement-binding site his serum will carry specific antibodies against the pathogen. When
is exposed. Complement proteins then bind to antibody molecules antigen is mixed with antiserum, antibodies will react with the path-
and lyse the antigen. In this process, the complement is said to be ogen. This is followed by addition of complement proteins from guinea
fixed or used up. The complement fixation test exploits this property pigs (these proteins have high cytolytic activity). Since antibodies
of the complement system to detect the presence of specific anti- are bound on pathogen, the complement system is activated and
body in the serum and even measure serum antibody level. This test the complement proteins are used up. This is followed by addition
was designed by Bordet and Gengou in 1901. of “sensitized indicator cells”. These indicator cells are red blood cells
The complement fixation test is performed by mixing antigen of sheep, coated with antibody. Since all complement proteins are
(say a pathogenic bacterium) with antiserum (that has been de- used up, the antibody-coated sheep RBCs remain intact and the test
complemented) of the individual suspected to be infected by that solution appears as a cloudy red-coloured suspension (Figure 10.15).
particular pathogen. If the individual is infected with the pathogen, If the individual lacks the specific antibody in his serum, pathogen
Addition of
Mixed together antibody-coated
+ +
sheep RBC
(indicator cells)
Pathogen Test antiserum Complement
contains proteins Specific antibody
specific antibodies (from guinea pigs) binds pathogen, No haemolysis, cloudy red
(Complement proteins complement fixed or used up cell suspension
removed) (positive result)
Positive result
Mixed together Addition of

antibody-coated
+ +
sheep RBC
Pathogen Test serum does Complement Antibody does not

not contain proteins react with pathogen, Unused complement causes
antibody against (from guinea pigs) so complement is not lysis of antibody-coated sheep
pathogen fixed or used up RBC. Red solution formed
(negative result)
Negative result
Figure 10.15
Complement fixation test.
will not bind any antibody and hence no complement protein will them. This will make the test solution a transparent red. The comple-
be fixed. So when indicator cells are added in the test solution, free ment test is currently used to diagnose a number of bacterial, viral,
complement proteins will bind antibody-coated sheep RBCs lysing fungal and protozoan diseases.
S U M M A R Y
• The complement system is a group of functionally related proteins • There are several biological functions of complement proteins.
found in plasma (and on cell surface) that functions as an innate These include (a) cytolysis, (b) inflammation (due to production of
arm of immunity. anaphylatoxins), (c) opsonization (due to C3b, C4b), (d) immune
complex clearance (due to C3b, C4b) and (e) induction of immune
• A complement causes (a) lyses of the invading antigen, (b) phago- response by enhancing B-cell response (due to C3dg).
cytosis of the pathogen and (c) triggers inflammatory reactions.
• Due to the potent damaging effect of complement activation, the
• There are three converging pathways of complement activation–– activity of complement components is tightly regulated. Regula-
classical, alternative and lectin, each initiated by a specific set of tory proteins include C1 inhibitor, C4 binding protein, factor H,
stimuli. I, and S. proteins found in plasma, and CR1 and DAF found on a
• The classical pathway is initiated by the binding of C1 component cell surface. A pathogen’s surface does not express or bind these
to an antigen–antibody complex. The alternative pathway is trig- regulatory proteins.
gered by a variety of agents, including cell wall of bacteria, and • Complement deficiencies can be genetic or acquired during the
occurs in the absence of antibody. The lectin pathway is stimulated lifetime of an individual. The deficiency could be of classical com-
by carbohydrate (mannose) residues present on the surface of a ponents (Clq, Clr, Cls, C2, C4), alternative pathway components
pathogen. (D, properdin), regulatory proteins (C1 Inh, DAF) or receptor for
• All the three pathways result in the formation of cytocidal complement (CR3, CR4). Complement deficiencies may result in
membrane-attack complex comprising complement components the promotion of inflammatory reaction, recurrent microbial infec-
C5b to C9.The membrane-attack complex forms a pore in the lipid tions, haemolytic anaemia and thrombosis.
bilayer of the target cell, killing the pathogen.
K E Y W O R D S
• alexin 209 • complement regulatory • immune complex • opsonization 218

• alternative complement proteins 208 clearance 216 • phagocytosis 211
pathway 213 • C1 inhibitor 220 • lectin complement • regulatory complement
• anaphylatoxins 211 • Cytolysis 216 pathway 214 proteins 221
• classical complement • C4BP 220 • mannan-binding pro- • S protein 222
pathway 210 • decay-accelerating teins 214 • paraoxysmal nocturnal
• complement 209 factor 216 • MASP 214 haemoglobinuria 224
• complement • Factor B 213 • membrane-attack complex • leukocyte adhesion
components 209 • Factor D 213 (MAC) 210 deficiency 224
• complement deficiencies 222 • hereditary angioneurotic • membrane cofactor
• complement receptor 216 edema 223 protein 220
1. Can a single type of bacterial cell invoke a response from all the 3. The deficiency of complement components involved in classical and
three complement pathways? Think and answer. alternative pathways results in repeated bacterial infections. Why?
HINT —Yes, if it has bound IgM, lipopolysaccharide and surface mannose Why is the body not more susceptible to viral infections?
residues. 4. Imagine you are one smart bacteria! What strategies would you like
2. Why does the human body have three separate complement to evolve to evade cell lysis by the complement pathway. Figure out
pathways when all of them converge to a single result—cell lysis? whether any bacteria has evolved these strategies.
Comment. 5. What are anaphylatoxins? How are they formed? What role do they
HINT —They get activated after “seeing” different danger signals. play in inducing inflammation?

1. Which of the following is not associated with the classical 6. Factor C3dg is not derived from:
component pathway? (a) C3b
(a) C3 (b) C3c
(b) IgM (c) i
C3b
(c) C4 (d) C3
(d) Properdin
7. Leukocyte adhesion deficiency is associated with the
2. Which of the following is not a part of the membrane-attack absence of:
complex as an intact molecule? (a) C1, C2, C4
(a) C9 (b) C3, factor I
(b) C7 (c) CR3, CR4
(c) C5 (d) C5, C2, CR3
(d) C6
8. All of the following are proteases except:
3. Which one of them is not a anaphylatoxin? (a) Cls
(a) C3a (b) C3b
(b) C2a (c) Bb
(c) C4a (d) Clr
(d) C5a
9. Complement receptor expressed on macrophage that binds
4. Which of the following free antibody can activate comple- C3b is:
ment? (a) CR-1
(a) IgA (b) CR-2
(b) IgG4 (c) CR-3
(c) IgM (d) CR-4
10. Which of the following cannot function as a regulatory protein
5. Thiolester bond is not present in: of complement system?
(a) C4 (a) CR-1
(b) C3 (b) C1 Inb
(c) C5 (c) C1
(d) None of the above (d) MCPex

1. All components of complement are proteases. 4. Red blood cells, unlike nucleated cells, are more sensitive to
complement-mediated cell lysis.
2. C3a des-Arg can act as a chemotactic factor.
5. Hereditary angioneurotic oedema is a result of deficiency of C1
3. Lipolysaccharides can activate both classical and alternative component.
pathways.
Di Scipio, R. (1991). “The Relationship Between Polymerization Association with a Novel Cls-like Serine Protease”, Journal of
of Complement Component C9 and Membrane Channel Forma- Experimental Medicine, 176: 1497–502.
tion”, Journal of Immunology, 147: 4239–47.
Muller-Eberhard, H. J. (1986). “The Membrane Attack Complex
Frank, M. M. and L. F. Fries (1991). “The Role of Complement in of Complement”, Annual Review of Immunology, 4: 503–28.
Inflammation and Phagocytosis”, Immunology Today, 12: 322–26.
Panburn, M. K., D. C. Morrison, R. D. Schreiber and H. J.
Joiner, K. A. (1968). “Complement Evasion by Bacteria and Para- Muller-Eberhard (1980). “Activation of the Alternative Comple-
sites”, Annual Review of Microbiology, 42: 201–30. ment Pathway: Recognition of Surface Structures on Activators
by Bound C3b”, Journal of Immunology, 124: 977–82.
Lachman, P. J. (1980). “Complement Deficiency and Pathogenesis
of Autoimmune Complex Disease”, Chemical Immunology, 49: Porter, R. R. and K. B. M. Reid (1978). “The Biochemistry of
245–63. Complement”, Nature, 275: 699–704.
Matsushita, M. and T. Fujita (1992). “Activation of the Classical Walport, M. J. (2001). “Complement”, New England Journal of
Complement Pathway by Mannose-binding Protein in Medicine, 344: 1058–140.
In the early 1980s, K. Ziegler and E. R. Unanue suggested that T cells “What the
recognized only processed antigens. They observed that when a caterpillar
bacterial protein antigen is exposed to antigen-presenting cells that calls the end
are later exposed to TH cells, the TH cells are activated. If antigen- of the world,
presenting cells were first chemically modified (fixed) by paraformal- the master a
dehyde (so that neither endocytosis nor internalization occurs) and butterfly.”
—RICHARD BACH
then exposed to antigen, no TH cell activation occurred. However, if the
antigen-presenting cells were allowed to ingest the antigen and fixed
After studying this chapter, you
with paraformaldehyde after four hours of antigen exposure, TH cell should be able to:
activation occurred. This suggested that during the four-hour interval, • Give evidences for the presence of
two antigen-presentation pathways
the antigen-presenting cells had processed the antigen and displayed
• Distinguish between professional
it on the cell membrane to activate TH cells. It was found that T-cell and non-professional antigen-
presenting cells
activation did not occur if antigen presenting-cells were subjected to • Describe the processing and
presentation of peptides from
temperatures below the physiological temperature or to a metabolic membrane and secreted proteins
• Describe the structure and function
inhibitor such as azide that made antigen- presenting cells of proteasome
metabolically inert. All these conditions inhibit the processing of • Explain the process of
ubiquitination of proteins
antigen in antigen-presenting cells. Further studies showed that there • Explain the processing and
presentation of endogenous
were two processing pathways—one that dealt with exogenous (intracellular) antigen via class I
MHC pathway
antigens (or antigens captured from outside the cell) and the other
• Give an account of the different
ways by which endogenous
with endogenous antigens. A brief overview of the two processing pathways can be blocked by
viruses
pathways is shown in Figure 11.1.
• Describe in detail the processing of
exogenous antigen via class II MHC
processing pathway
• Explain the role of invariant chain,
CLIP, HLA-DM and HLA-DO in class
II MHC processing pathway
• Give a brief account of the
presentation of non-peptide
bacterial antigens on CD1
molecules
Antigen Presentation
and Processing
11.1 INTRODUCTION
11
The antigen recognizing part of the immune system has two main arms: the B lymphocytes which
produce antibodies against the invading antigen and T lymphocytes which are mainly involved
killing virus-infected and tumour cells. However, there is a distinct difference between how B and
T cells recognize the antigens.
B cells and secreted antibodies bind soluble antigens in the body fluid, whereas T cells recog-
nize and respond to foreign peptide antigens only when they are displayed on MHC molecules at
the surface of the cell. Each cell in our body is covered with around 10,000 tiny protein fragments.
These peptide fragments which comprise 8–18 amino acids are glued to peptide receptors, that is,
MHC molecules, and convey information to T cells of the immune system. This display of anti-
genic peptides together with MHC complex on the cell surface that can be recognized by T cells is
called antigen presentation.
What are the evidences for antigen processing and presentation?
• The processed forms of most protein antigens that T cells can recognize, can be artificially
generated by proteolysis of antigen in vitro. Antigen-presenting cells (macrophages) that
are fixed or that are treated with chloroquine before exposure to antigen can effectively
present pre-digested peptide fragments of that antigen to specific T cells. R. P. Shimon-
Kevitz showed that gluteraldehyde-treated antigen-presenting cells can bind ovalbumin
fragments (generated in a test tube) and present it successfully to specific TH cells. Intact
ovalbumin bound to glutaraldehyde-treated antigen-presenting cells failed to activate spe-
cific TH cells. These results clearly suggest that TH cells recognize only processed antigens.
Exogenous antigen
Endogenous antigen
Degradation
Endocytosis
Class I MHC
Antigen Class II MHC

degradation
Antigen presentation Figure 11.1

Schematic diagram showing a broad
overview of antigen presentation.
• A similar observation was made by A. Townsend and his colleagues. They were identifying
the proteins of influenza virus that were recognized by Tcyt cells. They were surprised to find
that internal proteins such as matrix and nucleocapsid proteins were often recognized by
Tcyt cells better than the more exposed capsid proteins. Moreover, Townsend observed that
Tcyt cells recognized the short linear peptide sequence of the influenza protein on the target
cell and lysed it. He observed that when non-infected target cells were incubated in vitro
with digested peptides of internal influenza proteins, the peptide-displaying target cells
were recognized by Tcyt cells and were subsequently lysed just as target cells that had been
infected by live influenza virus.
• C. R. Parish (1971) and V. Schirrmacher and H. Wigzell (1972) demonstrated that B and T
cells recognized different antigenic determinants on the given antigen; for example, mouse
B cells recognize an antigenic determinant located at the n terminal of glucagon, whereas T
cells responds to determinants near the C terminal.
The net result of processing of a protein antigen is the generation of peptides of about 8–18 amino
acids that bind MHC molecules. The requirement for antigen processing prior to T-cell stimula-
tion explains why T cells recognize linear (and often internal) sequences but not conformational
determinants of protein.
11.2 ANTIGEN-PRESENTING CELLS

All cells which bear class I or class II MHC molecules can present peptides to T cells, yet all cells
Antigen-presenting cells are not referred to as antigen-presenting cells. Historically, only those cells that display peptides
Those cells that display peptides associated with class II MHC molecules to TH cells (CD4+ T cells) are referred to as antigen-
associated with class II MHC
presenting cells.
molecules to TH cells are referred to
as antigen-presenting cells. These The antigen-presenting cells should also have the ability to endocytose, and process the endo-
include cells such as dendritic cells, B cytosed antigens. Most mammalian cells do have the property to endocytose antigens but not all
cells and macrophages.
the cells have class II MHC molecules, and hence all the cells are not antigen-presenting cells.
Those cells that display peptides associated with class I MHC molecules to CD8+ Tcyt cells are
» In the 1950s, it was demonstrated
referred to as target cells. They are so called because these cells are the targets of cytolytic action
that fluorescent or radioactive
antigens injected into animals were of Tcyt cells.
found in mononuclear phagocytes A variety of cells can function as antigen-presenting cells. There are three pre-requisite properties
or follicular dendritic cells and not that allow the antigen-presenting cells to function efficiently: (a) expression of class II MHC molecules
in lymphocytes. This suggested
that only a few cells can take up on the cell surface; (b) ability to endocytose/phagocytose and process antigens; (c) ability to deliver
antigens present outside the cell. costimulatory signal to T cells. The antigen-presenting cells not only present antigenic peptides on class
» The stimulation of T cells can be
II MHC molecules to TH cells, but also provide costimulators (could be membrane-bound or secreted
measured by assaying the products) for full physiologic activation of the T cells. The characteristics and representatives of antigen-
production of cytokines by T cells or presenting cells are de-
by proliferation of T cells.
picted in Figure 11.2.
There are three
Endocytosis/phagocytosis types of antigen-present-
ing cells: (a) dendritic
cells, (b) mononuclear
phagocytes such as mac-
rophages, and (c) B lym-
Class II MHC molecules phocytes.
These classes of
Costimulators antigen-presenting
cells are referred to as
» Among dendritic cells, macro- Characterstics of professional antigen-presenting cells professional antigen-
phages and B cells, B cells are least
efficient as antigen-presenting cells.
presenting cells. Anti-
gen-presenting cells are
found in lymph nodes,
spleen, skin, within or
Figure 11.2
Schematic diagram of professional
underneath most mu-
antigen-presenting cells—dendritic cells, cosal epithelia, and in
macrophage and B-cells. Dendritic cell Macrophage B cell the thymus.
ANTIGEN PRESENTATION AND PROCESSING 231
11.2.1 DENDRITIC CELLS

Dendritic cells or dendrocytes are irregularly shaped cells usually found in spleen and lymph
nodes.They are derived from the bone marrow and are related to mononuclear phagocytic line-
age. Dendritic cells are best designed for presenting proteins to TH cells. This is due to the fact that
dendritic cells constitutively express class II MHC molecules as well as costimulatory molecules
that are needed for TH-cell activation (for details see Chapter 8). It is believed that dendritic cells
are also important for inducing T-cell immune response to foreign (allogeneic) MHC molecules
in an allograft.
11.2.2 MONONUCLEAR PHAGOCYTIC CELLS

Macrophages and other mononuclear phagocytic cells play an important role in presenting anti-
gens derived from extracellular sources such as bacteria and parasites. Macrophages, however, do « Apart from their role as antigen-
not constitutively express class II MHC molecules (if they do, it is at a very low level) or costimu- presenting cells, macrophages
latory molecules. Macrophages have to be activated by phagocytosis (or IFN-γ) before they can can secrete a variety of cytokines,
enzymes and free radicals which
express these two molecules required for antigen-presenting functions. Since macrophages are the help them play a diverse role in
only antigen-presenting cells that have ability to phagocytose (not be confused with endocytosis), inflammation, lymphocyte activa-
and hence ingest challenging bacteria and parasites, they are definitely the best antigen-presenting tion and tumour immunity.
cells available.
11.2.3 B LY M P H O C Y T E S
B cells are rich in class II MHC molecules, especially after activation. B cells bind antigen on surface
immunoglobulin molecules, internalize, process and then present the proteins to activated TH cells. B
cells do not constitutively express costimulators but can be induced to express it by surface antibody « Endothelial cells form a one-
cross-linking or by cytokines from T cells. When a B cell functions as an antigen-presenting cell, TH cell-thick lining in blood vessels and
lymphatic vessels. In humans,
cells express new surface molecules (CD40 ligands) and cytokines. These two new molecules of T cells vascular endothelial cells c an
bind to the receptor on B cells and activate antibody production and isotype switching in B cells. express class II MHC molecules
Even though macrophages, B cells and dendritic cells are antigen-presenting cells and have the (induced by IFN-γ) and hence can
act as antigen-presenting cells to
ability to endocytose and process antigens, there may be subtle differences among them: for example, TH cells. It has been suggested that
macrophages contain a higher concentration of proteinases than B cells, and are more aggressive in they play a role in cell-mediated
internalizing and processing large particulate antigens such as bacterial cells, etc. It is also possible immune reactions and during graft
rejection.
that different antigen-presenting cells display different sets of peptides from the same native pro-
tein because of the differences in their endosomal proteinases and their MHC molecules. Different « Dendritic cells and B cells always
express class II MHC molecules.
antigen-presenting cells may present different sets of peptides because the set of class II MHC molecules Macrophages also express these
expressed by one antigen-presenting cell may not be identical to that expressed by another. molecules, albeit at low levels.
Costimulatory molecules (such
as B7 molecules) are expressed at
high levels only on dendritic cells,
while B cells and macrophages
constitutively express low levels
of costimulators. However, upon
Interferon cytokine activation, the levels of
costimulators can be increased.
Class II MHC
Characteristics of non-professional antigen-presenting cells
Figure 11.3
Schematic diagram of non-professional
antigen-presenting cells such as
Fibroblast Glial cell Epithelial cell fibroblasts, epithelial cells, glial cells.
It is generally believed that an antigen-presenting cell presents the antigen to a T cell and the
T cell gets activated. The relationship between a T cell and an antigen-presenting cell is, however,
bi-directional. Antigen presentation by antigen-presenting cells to specific TH cells induces TH cells
to secrete cytokines. Cytokines secreted by stimulated T cells (which includes IFN-γ) upregulates
the expression of class II molecules as well as costimulator molecules on antigen-presenting cells.
Both MHC and costimulators are required for activation of TH cells.
11.2.4 NON-PROFESSIONAL ANTIGEN-PRESENTING

CELLS
Cells such as fibroblasts, glial cells, epithelial cells, pancreatic β cells and mesenchymal cells also
express class II MHC molecules in response to γ interferon. Since, these cells do not constitutively
express costimulators, it is unlikely that these non-professional antigen-presenting cells play a cen-
tral role in most T-cell response. It is suggested that non-professional antigen-presenting cells may
play a secondary role in cell-mediated immune response. The characteristics and representative
examples of non-professional antigen-presenting cells are shown in Figure 11.3.
11.3 T W O P R O C E S S I N G A N D P R E S E N TAT I O N
P AT H W AY S
Keeping in mind the way in which antigens are dealt with in antigen-presenting cells, antigens
can be classified as intracellular (endogenous) and extracellular (exogenous) antigens. Endogenous
antigens are those that are synthesized (or introduced) in the cytosol of the target cell; for example,
viral proteins are synthesized in cytosol when virus replicates inside the cell. If antigens are artifi -
cially introduced directly into the cytosol (using electroporation) they behave as endogenous an-
tigens. These endogenous antigens are then processed through what is called as cytosolic pathway
and presented on the membrane with the class I MHC molecules. These endogenous antigens on
» In 1979, Doherty and Zinkernagel class I MHC molecules are recognized by Tcyt cells. Exogenous antigens are extracellular antigens
demonstrated that while B cells (such as bacteria coated with specific antibodies) that are endocytosed and presented on the mem-
see antigen alone, T cells recognize
the antigen only in the presence of brane together with class II MHC molecules. These exogenous antigens presented on class II MHC
MHC molecules. molecules are recognized by TH cells.
Several experimental evidences support the existence of two processing and presentation
pathways that direct antigen fragments to either class I MHC or class II MHC molecules.
• If an antigen is artificially introduced into the cytoplasm of a cell by a technique such as
electroporation (which makes a plasma membrane transiently permeable), or by fusing
lipid vesicles containing the proteins, the antigen is processed and peptides are presented
in association only with class I MHC molecules and not with class II MHC molecules, even
though both class I and II MHC molecules are present on the surface of the cell.
• If a protein is introduced in the extracellular milieu of cells that expresses both class I and
II MHC molecule, the antigen is endocytosed, processed and presented only in associa-
tion class II MHC molecule and not with class I MHC molecules. The exogenously added
antigens displayed on class II MHC molecules sensitize only TH cells and not Tcyt cells.
However, if the gene encoding the same protein is transfected into the cells so that protein
is synthesized in the cytoplasm on polyribosome, the peptides derived from these protein
associate only with class I MHC molecules and hence these cells are subjected to lysis by Tcyt
cells (see Figure 11.4).
It is thus clear that what determines whether an antigen will be presented by class I or class
II MHC molecules, is if the cell is challenged by intracellular (endogenous) or extracellular (exog-
enous) antigens.
11.3.1 P R E S E N TAT I O N O F E N D O G E N O U S PAT H O G E N S

TO CLASS I MHC MOLECULES
The prerequisite for entry of protein into cytosolic pathway is its location. Antigen should be lo-
cated in the cytosol. This antigen is degraded into small peptides within the cytoplasm and these
small peptides associate with class I MHC molecules inside the cell. Finally, peptides attached to
class I molecules are displayed on the cell surface.
The pathway by which endog- Antigen entry

enous antigens are degraded in the Electroporation,fusion of lipid vesicles
cytosol is the same pathway that is
involved in the normal turnover of
intracellular proteins, and is dis-
Protein degrading machinery
cussed below.
PROTEASOME COMPLEX
Proteins in the cell are continually
being degraded and replaced with
newly synthesized proteins. Each Binds Does not
protein has its own unique half-life class I MHC bind class II MHC
after which it is “marked” for de-
X-Antigen added in
struction and finally degraded. The
extracellular medium
degradation of cytosolic protein is
carved out by a large, multicatalytic Endocytosis
proteinase complex called protea-
some. «The existence of proteasomes was
A proteasome is a large (26 S, lysosome pointed out by A. L. Goldberg in
~1500 kDa), cylindrical multipro- the early 1970s, when he showed
that cells lacking lysosomes, such
tein complex that contains many as bacteria and immature red blood
proteases that degrade specifically No display of Class II MHC-peptide cells, can destroy abnormal proteins
targeted proteins in small pieces. It peptide on class complex rapidly, via an energy-dependent
I MHC molecule process. In 1988, two groups— one
has 28 subunits arranged as a cyl- led by Goldberg and the other by
inder in the form of a stacked array Gene of X-Antigen Martin C. Rechsteiner—discovered that
of four inner and four outer rings cytosolic proteins are degraded by
injected into cytoplasm
a large multi-enzyme complex. This
with each ring composed of seven complex was named proteasome
Transfected gene-X
distinct subunits (see Figure 11.5). by the Goldberg group.
The four stacked rings, each Intracellular synthesis of
composed of seven subunits, sur- X-Antigen
round the central channel or tun- Protein degrading
nel of 10–50 Å. The outer stack machinery Figure 11.4
of rings appears to act as gates to Experimental evidence showing that
same protein may be processed in two
keep stray proteins from acciden- Class I No display of peptide on different ways by two different pathways,
tally tumbling into the degradation MHC-peptide class II MHC molecules if introduced at two different sites in
chamber. Each proteasome has one complex the cell.
or two 19 S regulatory subunits po-

sitioned at both its end like caps. These regulatory proteins recognize proteins that are targeted for
destruction and then actively unfold and inject the proteins into protein shredders, proteasomes,
where they are cut into small pieces.
U B I Q U I T I N AT I O N O F P R O T E I N S
Most proteins are continually replaced by newly synthesized proteins. However, the half-life varies
from protein to protein. Some proteins such as transcription factors and cyclins as also some ab-
normal proteins may have a half-life of 20 minutes whereas others in the same cell may last for days
or even weeks. Those proteins that are targeted for proteolysis often have several copies of the 76-
amino-acid protein, ubiquitin, attached to the lysine residues of target proteins. These ubiquitin « Ubiquitin derives its name from
its ubiquitous presence across the
tails act like postal codes that speed doomed proteins towards proteasomes. Proteasomes specifi- prokaryotic and eukaryotic king-
cally recognize the ubiquitin tail on the protein and degrade only ubiquitin-tagged proteins and doms. It has been found in a wide
not any random protein. variety of organisms, from bacteria
to human beings, having an almost
The ubiquitination process has several steps and involves three enzymes named E1, E2 and E3. identical sequence and the same
Briefly, E1 activates ubiquitin which binds to the second enzyme E2. The third enzyme E3 transfers function—tagging proteins to
ubiquitin from E2 to the target proteins. It is suggested that this transfer of ubiquitin to target pro- doom.
teins is not a random process. Proteins undergoing turnover have phosphate groups attached to
them, which are recognized by E3. Denatured proteins and damaged proteins are also recognized
» LMP-2 and LMP-7 subunits of the by E3 (by some unknown mechanisms)

proteasome are encoded within the
MHC loci, and their expression is Ubiquitin and a ubiquitin tail is added, and proteins
upregulated by interferons. are finally targeted for degradation.
The immune system uses this general
Ubiquitination pathway of protein degradation to pro-
duce small peptides for presentation with
class I MHC molecule. The immune sys-
Antigen Antigen tagged tem appears to modify the proteasome by
with ubiquitin the addition of two subunits to the protea-
some: LMP-2 and LMP-7 (low molecular
mass polypeptide 2 and 7).
An additional subunit, LMP 10 (pre-
viously called MECL-1), is also added
to proteasomes. LMP-10 is not encoded
within MHC. The addition of these inter-
α α feron-inducible subunits to proteasomes,
changes the specificity of the proteasomes.
Such proteasomes generate peptides of
β β about 8–15 amino acids. Moreover, after
the addition of these subunits there is an
Figure 11.5
Diagram of proteasome complex increased cleavage of polypeptide after
showing its various subunits. The
β β hydrophobic and basic residues, and re-
proteasome complex is a hollow duced cleavage after acidic residues. This
cylinder made up of four stacked rings
of seven subunits each. The active sites produces peptides of about 8–15 amino
for proteolysis located in β subunits α α acids having a hydrophobic or basic resi-
are shown in blue. CURRENT BIOLOGY due at the carboxyl terminal. The peptides
by Robin Pals-Rylaarsdam. Copyright
1998 by Elsevier Science & Technology that are released into the surrounding
Journals. Reproduced with permission of cytosol are cleaved by aminopeptidase en-
Elsevier Science & Technology Journals zymes which remove amino acids from the
in the format textbook via Copyright
Clearance Center Current Biology, 1999, amino terminus. These truncated peptides
Vol. 8: N25, R902. Peptide fragements which are generated are now 8–10 amino
acids long, and fulfil all the requirements
that are needed to bind class I MHC molecules. The only problem is, these peptides are generated
in cytosol, while class I MHC molecules are located within the ER. How are these peptides trans-
located into the ER?
There are evidences to suggest that proteasomes and ubiquitination are involved in the pro-
cessing of antigens for class I MHC pathway. These are:
• The inhibition of enzymes involved in ubiquitination (E1, E2 and E3), inhibits the display
of antigenic peptides on class I MHC proteins.
• Proteins are modified in such a way that they are recognized by ubiquitin-conjugating
enzymes, leading to their ubiquitination and more rapid class-I-MHC-associated display
of peptides.
• The inhibitors of proteasomal activity, inhibit the presentation of cytoplasmic proteins on
class I MHC molecules. Experimentally tagging proteins with ubiquitin also results in more
efficient presentation of their peptides by class I MHC molecule.
PEPTIDE TRANSPORT FROM CYTOPLASM TO ER

Since peptides are generated in the cytosol and class I MHC molecules are assembled in the ER,
a mechanism must exist for delivery of cytosolic peptide into the ER. The first clue came from
the mutant cell line RMA-S, with a defect in antigen presentation by class I MHC molecules. In
these mutant cells, both α- and β2-microglobulin chains of class I MHC molecules are synthesized
normally, but these molecules remain intracellular (that is, not displayed on cell surface) or are
displayed, if at all, at abnormally low levels on the cell surface. This defect was corrected by “feed-
ing” the cells, that is, adding peptides to the medium bathing the cell, and class I MHC molecules
appeared on cell surface together with added peptides. The investigators suggested that peptides
might be required to correctly express class I MHC molecules on the cell surface as peptides might « ABC transport proteins are found
in both eukaryotes and prokary-
stabilize the interactions between α- and β2-microglobulin chains of class I MHC molecules. The otes. They mediate ATP-dependent
mutation responsible for this defect in RMA-S cell line turned out to involve two genes which were transport of low molecular weight
homologous to a family of genes that encode ATP-binding cassette proteins or ABC proteins. compounds such as ions, amino
acids, peptides and sugars
When mutant RMA-S cells were transfected with these two functional genes encoding trans- across membranes in a variety of
porter proteins, the cell started expressing class I MHC molecules on the membrane and started cell types.
presenting peptides normally. These proteins are now called TAP1 and TAP2 (transporter associ-
ated with antigen processing). TAP1 and TAP2 combine to form peptide transporter localized in « TAP only transports peptides that
ER and cis Golgi. Each TAP1 and TAP2 has a hydrophobic domain which projects through the are longer than 7 amino acids!
outer membrane of rough endoplasmic reticulum (RER) into the lumen of RER and two ATP
binding domains which projects into the cytosol. Although TAP1 and TAP2 dimers (often called « Mice with targeted disruption of
genes encoding TAP1 and TAP2
TAP-heterodimers) have a broad range of specificity, TAP has the highest affinity for peptides of show defects in class I MHC antigen
8–10 amino acids, having hydrophobic or basic carboxyl terminal amino acids. presentation.
These peptides are ideal for display on class I MHC molecules. Thus, the TAP transporter is
uniquely designed for delivering peptides of the right size generated by proteasome for binding to « Both TAP1 and TAP2, as well as
class I MHC protein. LMP-2 and LMP-7 genes map within
the MHC. Both the transporter
genes and LMP genes are
polymorphic.
P R E S E N TAT I O N O F P E P T I D E S D E R I V E D F R O M M E M B R A N E - B O U N D
AND SECRETED PROTEINS
« Peptides that fail to bind class I
It was generally believed that secreted, membrane-bound proteins, proteins resident inside the MHC molecules exit ER within 2–4
ER, were processed by less well-defined mechanisms that did not require ubiquitination or pro- minutes, utilizing ATP through a
teasomes. Newly discovered mechanisms of processing and presenting peptides of secretory and pathway that is suggested to be
independent of TAP. It involves
membrane-bound proteins have proved the above assumption to be incorrect. It is now believed another protein, Sec61, an ER
that secretory, membrane-bound or misfolded proteins are transported from ER to cytosol. This channel protein.
transport, known as retrograde translocation may be the normal mechanism by which these pro-
teins in the ER are “thrown” out and degraded.
Once in the cytosol, these proteins are degraded by the proteasome, resulting in the peptides
being transported back into the lumen of the ER via TAP, loaded on class I MHC molecules and
displayed on the cell surface.
E V I D E N C E F O R C Y T O S O L I C D E G R A D AT I O N O F M E M B R A N E - B O U N D
AND SECRETED PROTEINS
Asparagine-linked carbohydrate moieties are commonly present in the secreted and membrane-
bound proteins. These glycosyl residues can be removed only in the cytosol by enzymatic reaction
that changes the asparagine residue to aspartic acid. This diagnostic sequence change (Asn→Asp)
is visible in some peptides presented by class I MHC molecules. This suggests that secreted and
membrane-bound proteins are degraded in the cytosol.
BINDING OF CLASS I MHC MOLECULES AND PEPTIDES

The α-chain and β2-microglobulin components of class I MHC molecule are synthesized on ribos-
omes attached on RER.
Inside the RER, the following processes occur:
Calnexin
• The α chain binds a chaperone protein, calnexin, which prevent degradation and promotes Calnexin, an ER resident protein,
proper folding of the proteins. Another chaperone called BiP, a member of the heat shock is involved in the proper folding,
assembly and retention of a variety
protein family, also binds the α chain of class I MHC molecule inside the ER lumen. of proteins.
• When a β2-microglobulin binds to the partially folded or completely folded α chain, the
chaperones dissociate. The newly formed α-chain–β2-microglobulin dimers are unstable
and now bind a complex of chaperone proteins. One of the proteins, calreticulum, is simi-
lar to calnexin and carries out a similar function.
• The second component of the complex is TAP-associated protein—Tapasin. Tapasin is en-
coded by a gene within the MHC. Tapasin forms a bridge between class I MHC molecules
and TAP1 and TAP2 peptide transporters, to transport the suitable antigenic peptide from
cytosol. The physical association of class I α–β2 proteins with TAP proteins promotes pep-
tide-capture by class I MHC molecules.
• The third component of the complex is a protein disulphide isomerase, Erp 57. The role of
Erp57 protein is not clear and may involve breaking and reforming disulphide bonds in the
α2 domain of class I MHC molecule that may occur during peptide loading.
• The binding of a peptide to class I MHC molecule greatly enhances its stability and it is
released from the complex of TAP–tapasin–calreticulum–Erp57.
• The fully folded stable peptide–class I MHC complex is now able to leave the ER, move
» In mutant cells, where TAP trans-
through the Golgi and is then transported to the cell surface. Thus, a class I MHC molecule
porter is absent or defective and is must bind antigenic peptide to complete its folding and be transported onward from the
unable to transport peptides, class ER. Figure 11.6 shows the loading of an antigenic peptide onto a class I MHC molecule.
I molecules in the ER are unstable
and are eventually translocated
• Peptides that fail to bind class I MHC molecules are rapidly cleared out of the ER, possibly
back into the cytosol where they transported back to the cytosol by an active transport mechanism which is different from
are degraded. that used by TAP.
» The half-life of peptide–class I In normal uninfected cells, self-proteins fill the waiting class I MHC molecules and are car-
MHC molecules on the cell surface ried to the cell surface. It is suggested that in normal cells, some class I MHC molecules are always
depends on the cell type and the present in the ER. This might be important for the functioning of class I MHC molecules because
MHC allele, and ranges from 2 to
20 hours. they must be immediately available to transport viral peptides to a cell, in case virus infects the cell.
When the cell is infected, pathogen-derived peptides are immediately placed on “plates” of class
I MHC molecules and shown to T-cell surveillance. A generalized schematic representation of
endogenous antigens on class I MHC pathway is shown in Figure 11.7.
B L O C K A G E O F E N D O G E N O U S PAT H W AY B Y V I R U S E S
Since the display of viral peptides on class I MHC molecules of target cell implies that they will
be recognized and ultimately killed by Tcyt cells, some viruses have evolved mechanisms to evade
» Nef protein of HIV-1 downregu- recognition by preventing the appearance of peptide class I MHC complexes at the cell surface. The
lates class I MHC molecules. herpes simplex virus produces a protein ICP-47, that binds and inhibits TAP, thereby inhibiting
transport of viral peptides to class I MHC molecules which occurs via TAP transporter. Cytomega-
lovirus produces some chemical substances that translocate class I MHC molecules back into the
cytosol of the cell, that is, accelerate the retrograde translocation of class I MHC molecules from
ER to cytosol.
Other viruses, such as human papilloma virus (HPV), have evolved ways to hijack some
process of endogenous pathway for their own nefarious purposes. When HPV infects the cells,
cell responds by producing defence protein p53, one of the body’s tumour suppressor proteins.
8-10-residue-long
antigenic fragments
TAP
BiP Calnexin Calreticulum

ER
Tapasin Cytosol
Lumen
Erp 57
A chain B 2chain
class I MHC class I MHC Calreticulum
Tapasin
Erp 57
Class I MHC-peptide complex
Figure 11.6
Loading of peptides on class I MHC
complex—a schematic diagram showing
the role of TAP, Erp57 and tapasin. To Golgi complex
Outside cell
LMP-2
E1,E2 LMP-7
E3
Proteasome
Endogenous
antigen Protein tagged with ubiquitin
LMP-10
destined for degradation
8-10-
residue-long
Cytosol
TAP-1,TAP-2 peptide
Rough ATP
endoplasmic
reticulum Class I MHC-peptide complex
Golgi apparatus
Figure 11.7
Class I MHC–peptide complex Simplified view of class I processing
pathway.
p53 blocks the transformation of an infected cell to cancer cell. HPV produces a protein that binds
p53 to E3, the ubiquitinating enzyme. p53 is ubiquitinated and put on a fast track to doom by the
action of proteasomes. The poor defenceless cells become cancerous.
11.3.2 P R E S E N TAT I O N O F E X O G E N O U S A N T I G E N
TO CLASS II MHC MOLECULES
The main steps in this pathways are as follows :
« Endocytosis is the process of
• endocytosis/phagocytosis of native protein antigens from the extracellular environment intake of extracellular material
into the cytosol of antigen-presenting cell; without passing through the cell
• degradation of protein antigens into peptides within the acidic endosomes or lysosomes; membrane. The cell membrane is
infolded to enclose the surrounding
• transport of newly formed class II MHC molecules to endocytic vesicles and binding of substances which are then brought
peptides to class II MHC molecules; and inside the cell. Ingested material
• surface display of antigenic peptide–class II MHC molecule complex. can either be liquid, substances
dissolved in a liquid medium
These complexes are finally recognized by TH cells that are specific for the foreign peptide and self- (pinocytosis), or particulate matter
MHC molecule. that binds specific receptors on
ENDOCYTOSIS OF PROTEINS phagocytes so that only specific
ligands are ingested (receptor-
The antigen-presenting cells can internalize the extracellular antigen by phagocytosis or endocyto- mediated endocytosis).
sis (receptor-mediated endocytosis or pinocytosis).
Macrophages and dendritic cells internalize antigens by both the processes. Macrophages « Phagocytosis is derived from
mediate the uptake of extracellular antigen in a selective as well as non-selective manner. Macro- Greek word phagein which implies
to eat, cytos which is cell or con-
phages and dendritic cells bind many proteins with little or no specificity. These proteins bind on tainer, and osis, a process.
currently undefined receptors on the cell surface of macrophages and dendritic cells. Proteins are
internalized by phagocytosis or endocytosis.
Macrophages also have receptors that mediate very specific binding and internalization of
antigen: for example, specific receptors for Fc portion of immunoglobulin or receptor for comple-
ment component C3b. Antigens are coated by immunoglobulin or C3b, and hence are very effi-
« A phagocyte is the most primitive
ciently and specifically internalized inside the macrophage.
type of immune cell present in
B cells which can also act as antigen-presenting cells have very specific receptors on their sur- invertebrates and vertebrates. It is
face, that is, immunoglobulins. Surface immunoglobulins have high affinity for antigens and can present in all invertebrates from
very efficiently bind and internalize the antigen. The other non-professional antigen-presenting sponges upwards. Phagocytosis
in a unicellular animal represents
cells are not phagocytic, or are poorly phagocytic, and therefore endocytose antigenic proteins by the most ancient defence against
receptor-mediated endocytosis or pinocytosis. foreign material.
PEPTIDE PROCESSING IN ENDOCYTIC/LYSOSOMAL VESICLES. Once a protein antigen is

internalized, it is degraded into peptides comprising 13–18 residues within the compartments of
» The internalized antigen takes
about three hours to traverse the the endocytic processing pathway. Proteins that enter cells through endocytosis become enclosed
endocytic pathway and appears at in endocytic vesicles which become increasingly acidic as they progress into the interior of the cell,
the cell surface as antigenic eventually fusing with lysosomes.
peptide–class II MHC complex.
The endocytic pathway appears to involve three increasingly acidic compartments: early en-
» Studies suggest that chemi- dosomes (pH 6.0–6.5), late endosomes (pH 5.0–6.0) and, finally, lysosomes (pH 4.5–5.0).
cal agents, such as chloroquine The internalized antigen moves from early to late endosomes and finally to lysosomes. The
and ammonium chloride, which mechanism by which internalized antigen moves from one endocytic compartment to the next
increase the pH of endosomes and
lysosomes are also potent inhibitors is still not very clear. Scientists have suggested that small vesicles carry antigens from early
of the processing of antigens that endosome to late endosome. Similar vesicles transport antigen from late endosome to lysosome.
are endocytosed/phagocytosed by (A similar mechanism transports proteins from trans-Golgi to medial-Golgi and from medial
the cell. The endosome/lysosome is
an acidic compartment where to cis-Golgi.)
antigen processing takes place in Peptide processing starts in the lysosome where an antigen encounters low pH and hydrolytic
acidic pH, which is a enzymes optimally active at low pH. Lysosomes contains a unique collection of more than 50 acid-
necessary condition for proper
antigen processing. dependent hydrolases including nucleases, proteases, glycosidases, lipases, phospholipases, glyco-
sidases and phosphatases. Proteinase inhibitors (such as leupeptin) as well as drugs that raise the
» Vacoular H+ ATPases present in pH of endosome such as chloroquine, which make lysosome less acidic, inhibit the presentation of
the membrane of endosomes are class II antigens. This suggests that both acidity (acidic environment) and proteinases are respon-
responsible for the acidification of
the endosomes. sible for the “cutting” of internalized antigen. Among acid proteinases are cysteine proteinases,
cathepsins B, D, S and L, that are actively involved in antigen processing.
» Mice that lack cathepsin B or D Cathepsin L is also supposed to be very active in antigen processing. Peptides generated by
show normal antigen processing, proteolysis of antigens in endosomes and lysosomes bind to class II MHC molecules within intra-
whereas mice with no cathepsin
S cannot process antigens. cellular vesicles and this peptide–class II MHC complex is displayed.
BINDING OF PROCESSED PEPTIDES TO CLASS II MHC MOLECULES

The function of class II MHC molecules is to bind peptides generated in the endosomes/lysosomes
of dendritic cells, macrophages, B cells and other antigen-presenting cells and present these pep-
tides to CD4+ TH cells. The “loading” and presentation of lysosome-degraded antigen is performed
by class II MHC molecules through the following steps.
The α and β chains of class II MHC molecules are coordinately synthesized in the RER. As
the ER is richly endowed with unfolded and partially folded polypeptide chains as well as peptides
generated (for class I MHC molecules), a general mechanism is needed to prevent their binding in
a class II MHC peptide binding groove. Though the size of peptides that bind class I and class II
MHC molecules are broadly different, the lumen of ER is filled with peptides of all sizes. The bind-
ing of these peptides is prevented by the assembly of newly synthesized class II MHC molecules
together with a protein known as class II MHC associated invariant chain (Ii). This protein is a
non-polymorphic 30 kDa member of the Ig superfamily . The native invariant chain is a homotrimer.
Three pairs of the class II αβ chains associate with a pre-assembled Ii trimer. Ii binds to a class II
MHC molecule with a part of its polypeptide chain lying within the peptide binding groove of the
class II MHC, blocking the groove and preventing the binding of either free peptides or partially
folded protein.
Calnexin functions as molecular chaperone, ensuring that α and β chains are properly folded.
Only when an invariant chain binds class II MHC molecule to produce a nine-chain complex (that
is, three αβ heterodimer bound to one invariant chain trimer), calnexin is released and the class II
MHC molecule-invariant chain complex is transported out of the ER to Golgi and finally to early
endosomes. A simplified schematic representation of class II antigen processing pathway is given
in Figure 11.8.
Since antigenic peptides are generated in lysosomes and class II MHC molecules are synthe-
» The invariant chain performs two
important functions. First, it blocks sized in the ER, there must be some site within the cell where these two associate.
the peptide-binding site of class II It is believed that invariant chain directs the newly formed class II MHC molecules to spe-
MHC molecules so the non- cialized low pH endosomal vesicles where peptide loading can occur. The invariant chain per-
antigenic peptides present in the ER
do not bind MHC; second, it targets forms this targeting function by virtue of certain amino acid sequences in its aminoterminal
the assembled MHC molecules to cytoplasmic tail.
the endosome that carries antigenic The complex of class II MHC and α, β heterodimers with invariant chain is retained
peptides so that peptide loading
can occur. up to four hours in the endosomal vesicle. During this time, the invariant chain is gradually
Antigen
Outside the cell
Lysosome
Endocytosis
Antigen degradation in lysosome

13-18-residue-long fragments formed
Class II MHC molecule
Invariant chain
Class II
Digested Golgi Rough endoplasmic reticulum
MHC-
invariant complex
peptide
chain
complex
Displayed Class II MHCnpeptide complex Figure 11.8
Simplified view of class II processing
pathway.
cleaved by acid proteases such as cathepsin S in a multi-step process. A 24-amino-acid frag-

ment of invariant chain termed as CLIP (for class II-associated-invariant chain peptide)
remains bound to class II MHC mole-
Class II MHC Invariant Rough
cules. Class II MHC molecules associ-
molecule chain endoplasmic
ated with CLIP still cannot bind other reticulum
peptides. CLIP must either be dissociated
or be displaced to allow a peptide to bind
to the MHC molecule.
A non-classical class II MHC mol-
Transport
ecule, HLA-DM in humans (H-2M in
vesicle
mouse) plays a key role in the removal of
CLIP and subsequent loading of class II
Digested
molecules with antigenic peptide. HLA- antigenic
DM catalyses the exchange (degrada- peptides in
tion) of CLIP with an antigenic peptide endosome
as is shown Figure 11.9. The proteolytic « A non-classical class II MHC
molecule, HLA-DM removes the
cleavage of Ii and its subsequent binding CLIP peptide from the class II MHC
of antigenic peptide occurs in a particu- groove and replaces it with anti-
lar endosomal compartment called MIIC genic peptide in an endosome.
(class II MHC compartment). This endo-
some has properties of a vesicle in transi- Cathepsin-S Antigenic peptide
tion between late endosome and lysosome,
CLIP
including high density and characteristic HLA-DM Class II MHC
multi-vesicular appearance. In some B compartment
cells, such an exchange of Ii with antigenic
peptide on class II MHC molecules occurs
in vesicle CIIV (class II vesicles) which is
Class II MHC-
similar to MIIC. peptide complex
As with class I MHC molecules, class
II MHC molecules in uninfected cell bind
peptides derived from self-proteins. It is,
therefore, not surprising that self-peptides Figure 11.9
Plasma membrane Diagram showing loading of antigenic
peptides onto class II MHC molecules.
form a large proportion of the peptides presented by class II MHC molecules in normal un-
infected cells. Class II MHC molecules that do not bind peptide after dissociation from the
invariant chain are unstable and are rapidly degraded within the lysosome.
ROLE OF SPECIALIZED CLASS II MHC-LIKE MOLECULES—HLA-DM

The removal of CLIP from class II MHC molecule and its subsequent loading with suitable anti-
genic peptide is catalysed by HLA-DM.
Like other class II MHC molecules, HLA-DM is a heterodimer of α and β chain. Unlike class II
» The HLA-DM genes are found near
MHC molecules, they are not polymorphic, they do not associate with invariant chain, their sub-
TAP and LMP genes in the class II
region of the MHC. cellular distribution is distinct from class II MHC molecules, and they are not expressed on the
cell surface. HLA-DM is predominantly found in MIIC compartment where it binds and stabilizes
empty class II MHC molecules that would otherwise aggregate. In addition, it acts as a peptide
exchange molecule, catalysing both the release of the CLIP fragment from class II MHC–CLIP
complex and binding suitable peptides to empty class II MHC molecules. HLA-DM also catalyses
the release of unstably bound antigenic peptide from class II MHC molecules. HLA-DM will con-
tinuously bind and rebind antigenic peptides till the best fit is found. This ability of HLA-DM to
remove unstably bound peptide, sometimes called peptide editing, ensures that the peptide–class II
MHC complex is tightly bound and will survive a tempestuous wait of several days on a cell surface
before encountering T cells that are able to recognize them.
Another non-classical and non-polymorphic class II molecule HLA-DO (H-20 in mice) is also
» HLA-DO is produced in thymic involved in the binding of antigenic peptide to class-II MHC molecules.
epithelial and B cells only. HLA-DO HLA-DO is found only in intracellular vesicle and does not appear to bind peptide. Instead, it
is not present on the cell surface
and is found in the endosomal
acts as a negative regulator of HLA-DM. The reaction between HLA-DM and class II MHC–CLIP
vesicle. HLA-DO inhibits the action complex that facilitates the exchange of CLIP for another peptide is inhibited in the presence of
of HLA-DM. HLA-DO. HLA-DO binds HLA-DM and blocks the exchange reaction.
How is this inhibition overcome? This inhibition of HLA-DM by HLA-DO is overcome by
the overexpression of HLA-DM during inflammatory response. The expression of HLA-DO is
not increased by γ interferon, whereas the expression of HLA-DM is. Thus, during inflammatory
responses, in which IFN-γ is produced by T cells and NK cells. IFN-γ binds antigen-presenting
cells causing an increased expression of HLA-DM. This enhanced expression of HLA-DM is able
to overcome the inhibitory effect of HLA-DO. Why the antigen presenting ability of only thymic
epithelial cells and B cells is regulated in this way is not currently known!
As with class I MHC molecules, peptide-binding to class II MHC molecules stabilizes the αβ
heterodimer and is required to maintain the structure and stability of class II molecules. Once a
peptide is bound, peptide–class II MHC complex is transported to the plasma membrane, where it
is displayed for T-cell screening. In rare cases, when a peptide dissociates from class I or class II mol-
ecule once it is displayed on the cell surface, an MHC molecule changes its conformation and hence
cannot bind suitable peptide from the surrounding media. The class I or class II MHC molecules are
internalized and degraded. Thus, both class I and II MHC molecules under usual circumstances are
effectively prevented from acquiring peptides from the surrounding extracellular fluid.
11.4 P R E S E N TAT I O N O F N O N - P E P T I D E
BACTERIAL ANTIGENS
The actual mechanism of presentation of non-peptide bacterial antigen is not currently clear. It has
been shown that antigenic lipid and glycolipid molecules derived from pathogenic microbes such
as Mycobacterium tuberculosis, M. leprae and M. avium are presented by family of non-classical
class I molecules, CD1.
The CD1 family of molecules has the same general structure as class I MHC molecule, includ-
ing its association with β2-microglobulin. However, α c hain of CD1 molecule bears little similarity
to α chain of class I molecule (for example, the α3 domain of CD1 molecule is closer in sequence
similarity to β2-microglobulin than to α3 of classical class I MHC molecule).
Antigen such as M.avium, M.leprae
Third (?) processing pathway
CD1
Phospholipid and
mycolate antigens Transport Golgi
Rough
vesicle complex
endoplasmic
reticulum
CD1-lipid antigen complex
Figure 11.10
Schematic diagram showing
presentation of non-peptide antigens by
the CD1 pathway. The third processing
pathway implies that this pathway is
different from the presentation and
processing pathways of class I
γδ T cells and II MHC.
There are five gene-encoding human CD1 molecules (CD1a–CD1e) which are not located
within the MHC, but on a different chromosome. Only four (CD1a, CD1b, CD1c and CD1d)
are antigen-presenting elements. CD1 gene products are found on antigen-presenting cells, in all
mammalian species. Antigens associated with CD1 molecules are recognized by T cells (γδ T cells,
but not αβ T cells). Porcell and co-workers (2000) directly purified and biochemically character-
ized antigens bound to CD1 molecules. They revealed that glycosylated mycolates and phosphati- « Mycobacterium tuberculosis down-
regulates the expression of CD1
dylinosotides are prominent CD1b-presented antigens and polyisoprenoid phoshoglycolipids are molecule for its survival.
CD1c-presented antigen. Lipid antigens are presented by CD1a and CD1d, but their exact details
are still being worked out. The peptide-binding groove of CD1 resembled class I MHC molecules
more closely than class II MHC molecules. CD1 peptide binding groove of mouse CD1 was found
to be narrower but deeper with two major binding pockets that are hydrophobic, suggesting that
this molecule may be well adapted in presenting hydrophobic lipid antigens to T-cell scrutiny. The
pathways by which these non-peptide antigens are processed and bind CD1 molecules for presen-
tation are different from class I MHC molecule (endogenous pathway) as TAP-deficient cell which
cannot process antigen for endogenous pathway can still process antigens for CD1 pathway.
The data concerning CD1 antigen presentation points to the existence of a third pathway for
processing non-peptide antigens. Exactly how different or how similar this third pathway is to
class I and class II pathways remains to be understood. A brief overview of the presentation of non
peptide antigens on CD1 molecules is shown in Figure 11.10.
Another pathway for processing and presenting non-peptide antigens have been suggested
by Morita and co-workers in 2002. This newly discovered novel presentation pathway is distinct
from those used by MHC and CD1 proteins. This pathway seems to be involved in presenting
at least two non-peptide antigens—prenylpyrophosphates and alkylamines. Prenylpyrophos-
phates are small molecules required to make compounds such as cholesterol while alkylamines
are small molecules produced by a variety of cells, including bacteria and non-Hodgkin B-cell
lymphoma cells. Alkylamines are also found in certain foods. It is believed only a particular subset
of γδ T cells (Vγ2 Vδ2 TCRs) are able to recognize the non-peptide antigens on these novel antigen-
presenting molecules.
Gel Electrophoresis
The movement of charged Loading syringe
Protein
molecules under the influence Buffer
of electric field is called electro- Tracking
phoresis. Electrophoresis was dye
_ Cathode _ Cathode
first demonstrated by Reuss
in 1809 when he showed that
fine sand particles would mi-
grate under the influence of Protein migrates
electric current. This analytical as per charge
technique was first applied for and mass
separation of proteins by Arne Cross-linked gel
Tiselius and Elvin A. Kabat in such as Tracking dye
1939. They separated serum polyacrylamide gel migrates faster
proteins into α, β and γ frac-
tions on the basis of their mo-
+ Anode Anode
bility under the influence of an
electric field. Electrophoresis Native PAGE
can be performed to separate
any charged molecule, be it
protein, DNA, ion, etc. Clas- _ Cathode
Negative charge
sically, electrophoresis was
performed in a liquid medium
Large protein,
but later better separating more steric
media such as gelatin, paper, hinderance,
kieselguhr, starch, agarose and less mobility
polyacrylamide were used. The
Small protein, less steric
commonly seperating media hinderance, more mobility
used include polyacrylamide
(in the case of proteins) and
agarose (in the case of DNA)
as it provides a molecular siev- + Anode
ing effect on the separated SDS-PAGE
molecules. For the sake of rel- Figure 11.11
The experimental set-up for gel electrophoresis.
evance, we shall focus only on
the electrophoresis of proteins.
Since proteins are built from
positively charged, negatively charged and neutral amino acids, for the conduction of electricity and maintains a pH so that pro-
proteins may have positive, negative or neutral charge at any giv- teins remain negatively charged. Once proteins are applied onto
en pH. In commonly used electrophoretic set-ups, proteins carry the gel, the power is switched on and proteins start migrating.
a negative charge and have anodic mobility. This is achieved by Once the tracking dye (which is visible) reaches the bottom of
first mixing the protein sample with a sample dye that contains a the separating gel input voltage is switched off. The gel (which
buffer and tracking dye. The sample buffer has a pH which makes contains the separated proteins) is stained with protein stain-
almost all types of proteins negatively charged. The blue tracking solution such as coomassie brilliant blue. The gel is then
ing dye helps keep track of protein movement and it migrates destained revealing blue protein bands against the transparent
as a visible blue band ahead of the protein sample. The protein background.
sample is then applied on the separating media, polacrylamide To separate proteins on the basis of molecular weight, a slightly
gel. The gel is cast in a mould (see Figure 11.11) and presoaked different procedure is followed. The protein is first boiled in a solution
in “reservoir buffer” that serves two main functions: provides ions of detergent—sodium dodecyl sulphate (SDS). SDS binds to the protein,
denatures it and renders it negatively charged. Once the SDS-treat- meshwork of the gel. Small proteins, inspite of carrying less charge,
ed protein is applied onto the gel and electrophoresed, low molec- easily pass through the gel and hence, exhibit higher mobility. There
ular weight proteins migrate faster, followed by large proteins. The is widespread use of gel electrophoresis in the analysis of consti-
reason for this is that large proteins, though they carry a higher (neg- tuting proteins in a sample for determining the homogeneity of
ative) charge, exhibit slower mobility because of steric hindrance, protein preparation, and for determining the molecular weight and
as large protein molecules find it difficult to pass through the number of constituting subunits in protein.
S U M M A R Y
• B cells and secreted antibodies recognize and bind soluble class I MHC–peptide complex moves through the Golgi
antigen whereas T cells recognize and respond to antigenic to the surface of the cell and are shown to Tcyt cell
peptide displayed on MHC. surveillance.
• Antigens, either exogenous or endogenous, are first processed and • The presentation and processing of exogenous antigens starts with
then presented on MHC to stimulate T cells, that is, TH cells and endocytosis/phagocytosis of antigen from the extracellular milieu.
Tcyt cells. The endosome formed fuses with lysosome, resulting in the deg-
radation of protein antigens. Lysosomes, which contain a diverse
• Cells that display peptides associated with class I MHC molecules array of hydrolases, process the protein antigens to generate short
to CD8+ Tcyt cell are referred to as target cells. Cells that displays peptides.
peptides associated class II MHC molecule to TH cell are called
antigen-presenting cells. • In RER, class II MHC molecules are assembled and then the
peptide-binding groove is blocked by an invariant chain (li).
• There are three types of antigen-presenting cells—dendritic
cells, macrophages, B lymphocytes—called professional • The invariant chain directs vesicles containing class II MHC
antigen-presenting cells. Non-professional antigen-presenting molecules towards endosomes containing antigenic
cells express class II MHC molecules upon stimulation by peptides.
interferons.
• Once the class II MHC – Ii complex encounters antigenic
• There are two main processing pathways leading to either peptide, Ii chain is first cleaved to form CLIP which then
class I or class II MHC molecules. One is a cytosolic pathway dissociates.
used by endogenous antigen leading to class I MHC
molecules. In the second pathway, exogenous antigens are • The removal of CLIP and loading of class II MHC molecules
endocytosed, processed and presented on class II with antigenic peptide is catalysed by non-classical class II MHC
MHC molecules. molecule, HLA-DM.
• Endogenous antigens are first tagged with ubiquitin, and • Peptide–class II MHC molecule is then transferred to the cell
then chopped in the cellular degradation chamber, proteasome, surface for T-cell scrutiny.
into 8–10 amino acid residue peptides. The processed peptides • Non-peptide antigens such as lipid and glycolipid antigens are
then bind peptide transporters TAP1 and TAP2 localized in the presented on non-classical class I molecule, CD1, while prenyl-
ER, and enter the ER. Peptides bind and stabilize α and β chains of pyrophosphates and alkylamines are presented on novel, newly
class I MHC molecules. Loaded and fully folded stable discovered antigen-presenting molecules.
K E Y W O R D S
• ABC protein 235 • CIIV 239 • non-professional antigen-

• antigen processing 229 • dendritic cells 231 presenting cell 232
• antigen presentation 229 • endogenous antigens 232 • processing pathways 232
• antigen-presenting cell 229 • exogenous antigen 232 • professional antigen-presenting
• BiP 235 • Erp57 236 cell 230
• B lymphocyte 229 • HLA-DM 239 • proteasome 233
• CD1 240 • HLA-DO 240 • retrograde translocation 236
• calnexin 235 • invariant chain 238 • TAP 235
• calreticulum 235 • LMP 240 • tapasin 235
• CLIP 239 • macrophage 229 • ubiquitination 233
1. What are the advantages of having two different antigen processing 4. What role does non-classical MHC molecules play in the process-
pathways in a single cell? Do you think both pathways are used for ing of exogenous antigen? Will knockout mouse lacking these
antigen processing in all cells? Think and answer. molecules be able to process antigen properly? What likely conse-
H I N T —Are class I and II MHC molecules present on all cells! quences will occur?
2. Many pathogens (for example, viruses) do not infect antigen- 5. Pathways that orchestrate the destruction of misfolded proteins are
presenting cells, yet they still stimulate TH cells. How? Can you termed ER-associated degradation. What will happen if this deg-
suggest some mechanisms? radation pathway becomes aberrant? What will be its major conse-
quences?
H I N T — Ever heard of cross priming?
H INT —Intracellular accumulation of misfolded proteins resulting in gain of
3. Predict the effect of deficiency of following proteins on antigen function toxicity disorders.
processing: (a) ubiquitin ligases, (b) tapasin, (c) lysosomal hydro-
lases, (d) invariant chain.
Choose the appropriate option. 6. TAP proteins found in ER are associated with the processing of
peptides for:
1. T-cells recognize all except: (a) Class I MHC
(a) Soluble antigen (b) Class II MHC
(b) Antigen displayed on MHC (c) Class III MHC
(c) MHC (d) CD1
(d) Processed antigen
7. Which appears as the odd one out in the context of antigen
2. Cells that display the processed antigen on their surface are all, processing?
except: (a) Invariant chain
(a) Antigen-presenting cell (b) CLIP
(b) Target cell (c) HLA-DM
(c) Immune cells (d) HLA-A
(d) Bacterial cell
8. Antigen-presenting molecules involved in presenting lipid:
3. Dendritic cells display antigenic peptides on: antigens are:
(a) Class I MHC molecule (a) Class I MHC molecules
(b) Class I and III MHC molecules (b) Class I MHCII molecules
(c) Class II and I MHC molecules (c) CD1 molecules
(d) Class II MHC molecules (d) CD2 molecules
4. Proteasome is a: 9. Exogenous antigens stimulate:

(a) Enzyme (a) Tcyt cells
(b) Organelle (b) TH cells
(c) Multienzyme complex (c) Class I MHC
(d) All of the above (d) Class II MHC
5. The subunit of proteasome that is coded within the MHC is: 10. Cell that constitutively express costimulators are:
(a) LMP-1 (a) Glial cells
(b) LMP-2 (b) Dendritic cells
(c) LMP-10 (c) Epithelial cells
(d) LMP-6 (d) Mesenchymal cells
Fill in the blanks with appropriate terms.

1. An invariant chain is associated with __________________ 3. Antigen injected into cytoplasm of a cell will be displayed on
MHC processing pathways. _____________ molecules.
2. In general, antigen-presenting cells express _________________ 4. Two proteins that mediate proper folding of the α chain of class I
and ___________ on their surface. MHC are _________ and __________.
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299: 1330–31.
Hedges, J. F., K. J. Lubick and M.A. Jutila (2005). “γδT cells
Respond Directly to Pathogen-Associated Molecular Patterns”, York I. A. and R. Kenneth (1996). “Antigen Processing and
The Journal of Immunology, 174: 6045–6053. Presentation by the Class I Major Histocompatibility Complex”,
Annual Review of Immunology, 14: 369–96.
Nakagawa, T. Y. and A. Y. Rudensky (1999). “The Role of
Lysosomal Proteinases in Class II MHC-mediated Antigen
Processing and Presentation”, Immunological Reviews,
172: 121–29.
It is interesting that cell-mediated immunity and humoral immunity
have been studied separately. It is as if humoral immunity, which
involves antibodies, does not involve cells. We all know antibodies
arise from B cells after their suitable activation. So “cells” are involved
in humoral immunity too, isn’t it?
During ancient times, following the teachings of Hippocrates and

Galen, disease was considered to be a maladjustment of normal bal-
ance between four vital humors–blood (sanguis), phlegm (pituita), “Those who
yellow bile (chole) and the black bile (melaine chole). To regain health, can command
humors were reset to their original ratio or balance, by the application
themselves,
of leeches, phlebotomy and purgatives which were believed to cure all
types of diseases.
command
others.”
It was in 1858 that Rudolf Virchow advanced the idea that the pathol- —HAZLIT T
ogy of a disease is due to the malfunction of cells rather than the

maladjustment of humors. Although Virchow’s cellular pathology was
first widely acclaimed and then rejected, the association of humoral-
ism with disease continued until the 1870s, when the germ theory of
diseases was advanced. Some years later, in 1884, Ellie Metchnikoff
proposed that the invading organism that causes disease in the body
is ingested and digested by a cell—phagocytic cell—that is present After studying this chapter,
in the host body, and this constitutes the first line of defence against you should be able to:
pathogens. This cellular theory of immunity proposed by Metchnikoff
• Describe the mechanisms of
is the forerunner of the present-day cell-mediated immunity theory. cytolysis used by Tcyt cell
Cellular theory continued for several years as the only mechanism that • Distinguish between perforin/
granzyme pathways and Fas
would explain how the body fought the battle against pathogens. In pathway
1888, Nutall, followed by Buchner, introduced, for the first time, the • Describe the structure and
concept of humoral immunity when he found that normal animals pos- function of NK cell
sess a factor in the serum that can kill bacterial pathogens. This factor • Distinguish between killer-
activatory receptor and killer-
was named alexine (and, later by Ehrlich, complement). This was fol- inhibitory receptor
lowed by several years of debate and a series of experiments to prove • Describe the mechanism of NK-
cell cytotoxicity
which of the two theories of immunity were correct. It was in 1903 that
• Describe delayed-type
Wright and Douglas tried to rationalize the differences between the hypersensitivity as a part of cell-
mediated immune response
supporters of cellular and humoral immunity. They claimed that both
cellular and humoral functions were equally important and interde- sensitization and effector
phases of delayed
pendent. They pointed out that humoral antibody (humoral immunity) hypersensitivity response
interacts with its target microorganisms and renders these susceptible • Explain the protective role and
to phagocytosis by macrophages (cell-mediated immunity). Bound pathological consequences
of delayed hypersensitivity
antibody was later found to activate another cell-mediated reaction reaction
known as antibody-dependent cell-mediated cytotoxicity. This cyto-
toxic reaction can be mediated by a variety of cells including natural
killer cells, macrophages, neutrophils and eosinophils. A schematic
representation of antibody-dependent cell-mediated cytoxicity is
Cell-mediated Immunity
12.1 INTRODUCTION
Cell-mediated immunity refers to those immune reactions that occur on or near the cells, and in
which immune cells are directly involved. Cell-mediated immune response can be antigen-spe-
cific as well as non-specific. The non-specific arm of cell-mediated immunity (shown in Figure
12
12.2) includes cells such as macrophages, neutrophils, eosinophils and natural killer (NK) cells.
These cells bear pattern-recognition receptors (such as Toll-like receptor (TLR) and scavenger re-
ceptor) that recognize conserved structures present on the surface of pathogen, called pathogen-
associated molecular patterns (PAMP). PAMPs include bacterial lipopolysaccharide, peptidogly- « It took about 50 years for scientists
can, double stranded RNA, etc. NK cells also contribute to non-specific defence by binding non- to believe that humoral and cell-
specifically to antibody-coated cells and inducing target cell lysis. mediated immunity can coexist.
It has recently been suggested that someother molecules—Nod proteins (Nod-1 and Nod-2)—
« In 2005, Hedges et al. showed
are also involved in non-specific immunity. Nod-1 and Nod-2 are located intracellularly (not in a that γδ T cells can recognize and
membrane like TLR) and represent an important defence against invasive Gram-negative pathogens respond to PAMPs directly without
such as Shigella flexineri, E. coli, Streptococcus pneumoniae and Psuedomonas aeruginosa. Nod-1 is any involvement of antigen-
presenting cells.
reported to occur in epithelial cells.
The specific cells include Tcyt cells and cytokine-secreting TH cells. Both Tcyt and TH cells need to « A Nod molecule recognizes the
interact directly with the cells they are going to kill (or help, depending on the cell type) and they do tripeptide motif present in Gram-
negative bacterial peptidoglycans.
this through a specific recognition mechanism. Antigen-specific Tcyt cells perform the effector func-
tion by directly lysing the target cells. Tcyt and TH cells are activated specifically by a particular antigen « Mammalian TLR are homologues
(plus MHC) and may cause cell death by activating the target cell or may activate and recruit non- of the Toll protein found in
specific effector cells (see Figure 12.3) such as NK cells, macrophages, neutrophils or eosinophils. Drosophila. The Toll family of
proteins comprises membrane-
The target cells that are killed by non-specific effector cells use bound antibody (to target cell) as a spanning proteins that signal
infection during an innate immune
response. The first Toll protein was
identified in Drosophila.
Perforin
NK cell
TNF
Antibody-coated cell Binding of NK cell Lysis of antibody-coated

cell
Figure 12.1
Antibody-dependent cell-mediated
cytotoxicity by NK cells.
Figure 12.2
Schematic diagram showing the non-
specific arm of cell-mediated immunity.
(PAMP—pathogen-associated molecular
patterns, such as lipopolysachharide,
lipoteichoic acid and mannan; PRR—
pattern-recognition receptors, such as
Toll-like receptor, scavenger receptor;
MBP—major basic protein.)
signal for killing. The activity of both specific and non-specific cells is regulated by a concentration of
a variety of different cytokines.
12.2 LY M P H O C Y T E S A N D C E L L - M E D I AT E D
RESPONSE
Cell-mediated immunity refers to immunity rendered by cells of the immune system. This form of im-
munity can be transferred to a non-immune individual by the transfer of cells and not by serum or
plasma. There are two major subsets of effector cells that are involved in cell-mediated response. One
subset comprises specific effector cell such as Tcyt cells and non-specific effector cells such as NK cells and
macrophages that are involved in the lysis of target cells. The other subset comprises CD4+ TH cells that
mediate delayed-type hypersensitivity reactions. Table 12.1 lists some important cells and their effector
molecules involved in cell-mediated immune response. Representative examples of specific effector cells
(Tcyt cell) and non-specific effector cells (NK cell) are discussed below while the role of TH cells and mac-
rophages is described in this chapter in Section 12.4 (see Chapter 2 for additional information on cells).
12.2.1 C Y T O T O X I C T LY M P H O C Y T E S
Cytotoxic T lymphocytes (Tcyt) are a subset of T cells that express CD8 antigens (together with
TCR) on their surface. This CD8 molecule interacts with class I MHC molecules that present pep-
tides derived from intracellular microbes. The Tcyt cells appear important for the elimination of cells
Cell Type Effector Molecules

Tcyt cell Perforin, granzyme, Fas ligand, TNF-β, IFN-γ
NK cell Perforin, granzyme, IFN-γ, TNF-α, TNF-β, FcγRIII that mediates ADCC
TH1 cell IL-2, TNF-β, IFN-γ
TH2 cell IL-3, IL-4, IL-5, IL-10, IL-13, CD40 ligand.
Macrophage IL-1, IL-6, IL-12, IFN-γ, TNF-α, GM-CSF, enzymes, prostaglandins, complement
Table 12.1
Cells and effector molecules involved in
proteins
cell-mediated immunity Note: ADCC—Antibody-dependent cell-mediated cytotoxicity; IFN interferon; TNF tumour necrosis factor; IL—interluekin.
CELL-MEDIATED IMMUNITY 249
that harbour intracellular bacteria such T-cell receptor

as Listeria monocytogenes, or viruses or
cells that express foreign MHC mol- CD4
ecules (allograft) or tumour antigens.
The majority of Tcyt cells express CD8+ Effector
CD8
molecules on their surface. CD8 binds to cells
TH cell Tcyt cell
the non-polymorphic part of class I MHC
molecule of the antigen–class I MHC
complex present on the target cells.
Tcyt cells undergo maturation and Target cell
selection in the thymus. T cells exiting
the thymus are mature but innocent or
virginal. These cells express CD3 associ- Class I MHC Perforin and
ated αβ T-cell receptor and CD8, but lack granzyme
Antigen peptide
cytolytic functions. These cells are often « A small percentage of CD4+ T cells
referred to as pre-cytotoxic T lympho- also show cytolytic activity. This
cytes (or naïve or pre-Tcyt cells). The dif- cytolytic activity is mediated
Tcyt cell through the Fas pathway that kills
ferentiation of pre-Tcyt to functional Tcyt the target cell without the use
requires at least three separate kinds of Mode of action of Tcyt cell of perforin (used by Tcyt). In vitro
signals: studies have shown that these
cells form a very effective defence
• Recognition of foreign antigen against certain viruses such as the
Antigen-presenting
Epstein–Barr virus.
associated with class I MHC cell
on the target cell surface by the
Class II MHC
TCR complex on Tcyt cell;
Antigen peptide
• Second signal provided by co- CD4
T-cell receptor
stimulators such as members of
the B7 family present on anti-
gen-presenting cells that inter-
TH cell
act with CD28 molecule of the
pre-Tcyt cells; and
• The last signal, cytokines, pro- Mode of action Cytokines
duced by TH cells that act on
high-affinity receptors present Stimulates humoral Stimulates cell-mediated
on activated pre Tcyt. immunity immunity Figure 12.3
Schematic diagram showing specific arm
First, the unactivated pre-Tcyt cells Mode of action of TH cell of cell-mediated immunity.
do not express cytokine receptors. Only
when pre-Tcyt cells are activated by anti-
gen binding, do they start expressing cytokine receptors. Pre-Tcyt cells also start secreting IL-2. IL-2
secreted by pre-Tcyt cells are sufficient for proliferation and activation of Tcyt, particularly of mem-
ory Tcyt. However most of the pre-Tcyt cells require an additional dose of IL-2 which is produced by
TH cells. The precise cytokine spectrum required for Tcyt activation is not known. This type of Tcyt
activation is helper-dependent Tcyt activation. Thus the differentiation and proliferation of pre-Tcyt
to Tcyt depend on cytokines secreted by antigen-activated TH cells (TH1 to be precise) as well as by
pre-Tcyt cells themselves. TH1 cells (which may or may not directly interact with Tcyt) provide para-
crine cytokine stimulus to pre-Tcyt, cells, which in turn leads to differentiation and proliferation to
Tcyt. Additionally, TH1 can increase or upregulate the cell surface concentration of costimulatory
molecules on antigen-presenting cells, stimulating TH cells which indirectly helps in differentiation
and activation of Tcyt cells. A schematic representation of the generation of functional Tcyt cells is
The CD8+ Tcyt could also undergo proliferation and differentiation independent of TH cells
either through direct interaction with costimulators or through other Tcyt-cell-derived cytokines.
These cells are referred to as helper-independent Tcyt cells.
The proliferation and differentiation of Tcyt involve the acquisition of machinery to perform
cell lysis. This includes the development of several membrane-bound vesicles (that appear as gran-
ules) that contains the membrane-pore forming ~65 kDa protein perforin (or cytolysin), a variety
IL-2 secreted by TH1 cells

CD8
IL-2R
Figure 12.4 CD3

Pre-Tcyt cell
Schematic diagram showing generation
of mature Tcyt cells after activation by CD8 CD28
T-helper cytokines and peptide–MHC Fas ligand
Class I MHC
costimulatory molecules. T cells exiting
+ antigen CD8
the thymus are mature but innocent. The
Costimulation
differentiation of these pre- Tcyt cells into
functional Tcyt cells requires the presence Proliferation and CD3
of specific antigens on class I CD8 T-cell receptor differentation of
MHC, stimulation by IL-2 secreted by
Target cell Pre-Tcyt cell to Tcyt cell CD28
TH1 cells, costimulation by members of
the B7 family present on target cells Fas ligand
(IL-2—interleukin 2, IL-2R—interleukin-2 Activation of
receptors). Pre-Tcyt cell pre-Tcyt cell Tcyt cell
of enzymes containing serine at their active sites—granzymes (or fragmentins) and the surface
» In humans, mutation in the
perforin gene leads to the manifes- expression of Fas ligand (other cells of T-lineage also express this ligand).
tation of familial hemophagocytic Fas ligands present on Tcyt cells can interact with Fas protein expressed on target cells and can
lymphohistiocytosis. It is a rare, deliver apoptosis-inducing signals. Under appropriate conditions, differentiated Tcyt cells having
fatal, autosomal recessive immune
disorder, characterized by uncon- the above characteristics can deliver a lethal hit.
trolled activation of T cells. Tcyt and
NK cell activity is reduced or absent
in these patients. MECHANISM OF CYTOTOXICITY
Recent studies have suggested that between the perforin/granzyme pathway and the Fas pathway,
the perforin/granzyme pathway is the key mediator of cytolytic function of Tcyt cells. However, both
the pathways share some common steps, outlined below, that involve the adhesion of target cells
with Tcyt cells.
• The first step is the binding of Tcyt cell to the specific target cell. The target cell should carry
the same antigen that causes differentiation and proliferation of this Tcyt. Tcyt binds the target
cell in such a way that the TCR binds to a specific peptide on the polymorphic part of class
I MHC; The CD8 component of T cells binds to the non-polymorphic part of class I MHC
molecule; LFA-1, a cell adhesion molecule of T cell binds ICAM-1 (or 2); CD2, another cell
adhesion molecule of T cells, binds LFA-3 of the target cell. For proper T-cell activation both
the signals should be received, that is, TCR binding as well as cell adhesion. A detailed view
of T-cell synapse with the antigen-presenting cell is shown in Figure 12.5.
If T cells receive only one signal, that is, TCR
Cytolitic Function
Cytolytic function is the ability to binding and no cell adhesion, the T cell is turned
IFN-G ,IL-4,TNF-B
lyse cells. Examples of cells having off. Those target cells that do not express ICAM-1
cytolytic capabilities include Tcyt cells
CD45 or LFA-3 and hence do not provide effective cell
and NK cells.
CD44 adhesion, are ineffectively lysed.
CD43
CD40L CD40 • The second step involves the activation
CD8/CD4
of Tcyt. The activation of Tcyt is initiated by
CD3 TCR the clustering of TCR on the T-cell surface
because of antigen binding. The signal gen-
CD28 MHC erated by the TCR as well as by various
CD80/CD86
CTLA4 accessory molecules that are costimulated,
LFA1
ICAM-1 initiate a reaction cascade analogous to TH-
CD2 cell activation. The Tcyt cell is now ready to
CD48/CD
59/LFA-3 lyse the target cell.
Figure 12.5
Diagram showing interaction of Tcyt IL-1,IL-6,IL-12,TNF-A
cells and helper T cells with ligands. The
PERFORIN/GRANZYME PATHWAY. After the
molecules on T cells are known to be formation of a Tcyt–target cell conjugate, the
involved in interactions between T cells T cell Antigen- microtubule organization of cytoskeleton
and APC (CTL4—cytotoxic T lymphocyte
presenting changes in such a way that perforin and granzyme
4; LFA1—lymphocyte-associated
function antigen1, IFN—interferon).
cell (granule-associated enzymes) granules move to
the contact area of the T cell with the target cell. The fusion of granule membranes occurs with the Rab protein
T-cell membrane, resulting in exocytosis of granules contents into the extracellular region. The Rab proteins consists of a large
fusion of granules membrane is supposed to be Ca2+-dependent and is thought to involve a Ras- family of GTPases, belonging to the
superfamily of Ras proteins. They
associated binding (Rab) protein. are lipid-anchored proteins. About
The perforin monomer comes in contact with high extracellular Ca2+ where it undergoes po- 60 members of Rab have been
lymerization and conformational change to form cylindrical pores which then insert in the target identified. Rab proteins are involved
in intracellular vesicular transport,
cell membrane. A large number of such pores are formed at the contact point of Tcyt–target cell including vesicle movement and
resulting in osmotic swelling and cell lysis of the target. This method of cell lysis is analogous to that exocytosis.
produced by a C9 component of the complement pathway. In fact, perforin monomer shares sequence
homology and, as we have just seen, functional similarity with the C9 complement component.
Although the puncture of the target cell membrane with a large number of perforin holes
is sufficient to cause osmotic lysis, it has one other very important function. These pores allow
the entry of a large number of proteases, granzymes (liberated from Tcyt cells), into the target
cell. These enzymes cleave the cellular proteins such as interleukin-1 converting enzyme (ICE)
and related cysteine proteases. The products of these enzymes initiate an ICE protease cascade
that results in the activation of caspases and finally apoptotic cell death (see Figure 12.6). During
apoptosis, DNA-cleaving enzymes are activated. These enzymes are non-specific endonucle-
ases that cleave the cellular DNA as well as any viral DNA that may be present inside the cell
into oligomers of 200 base pairs.
It is believed that damage to viral
DNA (as well as cellular DNA)
ensures that viral replication and
assembly is terminated before +
destruction of the target cell; in Tcyt cell Target cell
other words, both the crook and
its safe haven are destroyed at the
same time by Tcyt-cell action. Tcyt and target cell binding
Fas PATHWAY. There is an Perforin

additional mechanism by which monomers
an apoptotic pathway can be Granzyme
triggered in the target cell. It was
found that some CD4+ cytotoxic Tcyt cell Target cell
T cells do not contain perforin–
granzyme but were able to lyse Clustering of TCRs onTcyt cell
the target cell in the absence of
Ca2+. Moreover, “knockout” mice Polymerized perforin
Fas
lacking perforin gene were found to pores Fas is the 48 kDa member of
have reduced, but not nil, cytolytic TNF family of proteins. This
activity. Clearly, these cells must transmembrane protein has three
domains—extracellular, membrane-
have some other mechanism of spanning and intracellular. The
inducing cytotoxicity. This pathway Tcyt cell Target cell intracellular domain has the
which does not involves perforin “death” domain that, upon suitable
Perforin pores activation, delivers intracellular
was found to involve a group of death signal. Fas is used during
molecules which could direct the Release of perforin and granzyme
lymphocyte development to
signal of apoptosis to the target eliminate or control cell population.
cell. This pathway involved the

perforin pores
expression of Fas (CD95) on the
target cell and Fas-Ligand (Fas-L) ICE
on the activated Tcyt (or on CD4+ Caspase Ca2+,H2O
TH) cells.
Apoptosis
The binding of FasL to Fas
molecule on the target cell results in Figure 12.6
its clustering. Fas activates another Tcyt cell detachment Target cell lysis Line diagram showing steps in killing
protein, FADD (Fas-associated of target cell by Tcyt via perforin
Delivery of lethal hit pathway.
protein with death domain) which
» FLICE has now been characterized associates with a second protein,

as a caspase.
T-cell Class I MHC FLICE (FADD-like interleukin
receptor converting enzyme protease).
These Fas-associated pro-
Peptide teases activate caspases. These
caspases cleave and inactivate
FasL Fas the inhibitor of CAD (caspase
Tcyt cell Target cell activatable DNase). Once CAD
is free it enters the nucleus, de-
grades the DNA and initiates an
» Tcyt cells dissociate from target apoptotic cascade in the target
cells before target-cell lysis,
escaping self-destruction. cell (see Figure 12.7).
FADD As can be seen in both the
activated
» NK cells are found mainly in “lethal” pathways, they activate
peripheral blood, where they make
up 10 per cent of the lymphocyte
the caspase family of proteases
population. Tcyt cell Target cell which ultimately causes death of
the target cell by apoptosis. Nor-
» After emerging from the bone T-cell receptor interacts Fas interacts with Fas mally caspases are present in the
marrow NK cells accumulate mainly with class I MHC + peptide ligand
in the spleen, lymph nodes and cells as inactive proenzyme that
tonsils where they mature. require proteolytic activation/
FADD-FLICE cleavage to get them transformed
» A word of caution! Not all LGLs are activated
NK cells in blood. NK cells constitute

into lethal caspases that induces
caspases activated
95 per cent of LGLs and the orderly and systematic cell death.
Target cell
remaining 5 pet cent of LGLs are Tcyt cell apoptosis Tcyt cells are themselves not
Tcyt cells.
killed by lytic process as they
Fas pathway activated
» NK cells kill cancerous/virus-
in target cell dissociate from the target cell
infected cells because they are before the cell lysis. It is believed
deficient in cell surface class I MHC
molecules. that autodestruction of Tcyt is
prevented by some regulatory
membrane proteins as well as by
the proteoglycan—chondroitin
sulphate A that inactivates per-
Figure 12.7 forin. This protective proteogly-
Line diagram showing steps killing of can is secreted from the Tcyt cells
Tcyt cell Target cell lysis
target cell by Tcyt via Fas pathway. themselves.
» NK cells do not develop in the 12.2.2 N AT U R A L K I L L E R C E L L S

thymus and nude mice which do NK cells are an important defence against cells infected with viruses, bacteria and protozoa or
not have T cells have normal and tumour cells. These cells constitute a small subset of lymphocytes found in blood and lymphoid
functional NK-cell population. Also
knockout mice lacking RAG-1 or tissues, about 5–10 per cent of the total lymphocyte population.
RAG-2 genes have normal NK-cell NK cells are derived from the bone marrow but they develop extra-thymically (outside the thymus).
population. These cells appear as large lymphocytes with numerous cytoplasmic granules and hence are some-
times called large granular lymphocytes (LGLs).
They are also called natural killers because they carry an inherent (natural) tendency to kill
infected and cancerous cells. Unlike B or T lymphocytes, they do not need prior antigenic stimula-
tion for their full potential. So when NK cells interact for the first time with tumour cells/infected
cells, they mediate cytotoxicity. NK cells are also referred to as null cells as they do not express
either B-cell or T-cell receptors on their surface.
NK cells form a distinct group of lymphocytes with no immunological memory and are inde-
pendent of the adaptive immune system.
S U R FAC E M A R K E R S O N N K C E L L S
Our understanding of NK cells, particularly of their surface markers and receptors, is still in its in-
fancy. NK cells are neither B nor T cells since they lack membrane-bound antibodies or T-cell
receptors. Inspite of being lymphocytes, NK cells do not undergo productive rearrangement of
T-cell receptor or B-cell receptor genes. It is believed that NK cells share common early pro-
genitors with T cells and, hence, are best assumed to be phylogenetically primitive T cells that
possess perforing ranzyme-medi- KIRs

ated cytotoxicity but lack the abil- p70
ity of specific antigen recognition. p50 p58
They also lack CD3 molecules.
However, NK cells express a number
of membrane molecules, including
CD2, CD16 (receptor for Fc region CD94
of IgG, also called FcγIIIA), LFA-1, NKG2
ICAM-1, CD56 (binds neural
cell adhesion molecule). In
addition, NK cells express recep-
tors for IFN-γ and IL-12 which in- Inhibitory receptors on NK cell
creases their capability of cell lysis.
NK CELL RECEPTORS. NK CD69*

cells express several non-antigen
CD2*
specific receptors which can be
KIKR-P1
activated by a variety of stimuli CD16*
and kill susceptible target cells CD28*
by perforin-mediated lysis. These
are referred to as killer activatory
receptors (KARs). NK cells also
Activatory receptors on NK cell
express inhibitory receptors.
When these receptors are engaged
or stimulated by specific molecule
Classical class Figure 12.8
on non-susceptible target cell, NK KIR
Line diagram of NK cell showing KAR
effector function, that is, cytolysis I MHC
and KIR. In humans, KIR recognize and
HLA-C
induction, is inhibited. These KAR bind directly to the conserved section
Non-classical
are termed as killer inhibitory Ligand of the class I MHC molecules. CD94/
class I MHC CD94 NKG2 recognize peptides from classical
receptors (KIR). HLA-E NKG2 class I MHC molecules displayed on
non-classical class I MHC molecules.
Signal peptide KAR recognize KAR ligand displayed
A C T IV A T O R Y R ECEPTOR S . The
Healthy from classical NK cell Healthy cell on healthy cells (KIR—killer inhibitory
exact nature of activatory cell class I MHC receptor; KAR—killer activator receptor).
receptors localized on NK cell
surface is not completely known.
They probably represent several different receptors that bind to a variety of ligands present on the
cell surface of target cells. The stimulation of these receptors would signal NK cells to kill the target
cells. These “killing” signals can be overridden or vetoed any time by a signal from KIR.
Some of the KARs, including NKR-P1, CD28, and possibly CD2, CD16 and CD69, are depict-
ed in Figure 12.8. NKR-P1 is a lectin molecule (carbohydrate-binding protein) that requires Ca2+
for its activity, that is, it is a C-type lectin. CD28 is a receptor for the B-7 family of costimulatory
molecules. CD2 is a receptor for LFA-3. CD16 is a low-affinity receptor for the Fc region of IgG,
and CD69 is a molecule possibly involved in the activation of NK cells (as well as B and T cells).
Inhibitory receptors: There are three NK inhibitory receptor families known, namely, CD94/
NKG2 family (found both in humans and mice); killer inhibitory receptors (only in humans); Ly49
family (only in mice).
• One of the important inhibitory receptors of NK cells is CD94/NKG2. CD94/NKG2 is a « CD94/NKG2a receptor recognizes
member of the C-lectin family and its genes lie on human chromosome 12 (mouse chro- peptides derived from classical class I
MHC that are displayed on
mosome 6). CD94/NKG2 is a heterodimer of two glycoprotein, one of which is CD94 non-classical MHC–HLA-E.
(encoded by single gene). The other is NKG2 (encoded by one of the four NKG2 genes).
CD94/NKG2 receptors in humans interact mainly with the non-classical class I MHC mol- « HLA-E is a non-classical
class I MHC molecule that presents
ecule, HLA-E. HLA-E carries in its groove a peptide from the leader sequence of classical peptides of degraded classical
class I MHC proteins, HLA-A, HLA-B or HLA-C. class I molecules.
• The killer inhibitory receptor (KIR) family (which includes 12 genes in humans) is a family
of cell surface proteins expressed on NK cells that contain two or three immunoglobulin-
like domains in their extracellular regions and sequence that resemble immunoreceptor
tyrosine activation motifs (ITAMs) in the intracellular region. Any NK cell expresses one
or more of these KIR proteins. Most of the KIR proteins interact with the α1 domain of
the classical HLA–class I molecules complexed with self-peptide. Once KIR binds class I
MHC–self-protein complex, it activates tyrosine phosphatases in the NK cell that shuts off
the NK cell response.
• The Ly49 family of inhibitory receptors is found only in mice, contains at least nine genes
and is expressed on murine NK cells surface as homodimers. Ly49 proteins interact with
α1 or α2 domains of classical class I MHC molecules in mice. The details of this interaction
and how it switches off NK cell response remain somewhat unclear.
We know that an individual B cell expresses, on its surface, receptors of one type only, both in
structure and specificity. Similarly T-cell receptors expressed on one T cell are all of same specific-
ity and structure. NK cells, on the other hand, express an average of five to six receptors of different
» Another immunophenotypically specificity (for example, a CD94/NKG2 receptor and four different KIR receptors). Moreover, dif-
distinct subset of NK cells is NKT ferent NK cells in an individual can express different receptor subsets. A very interesting hypoth-
cells that express markers of both
NK and T cells. Receptors present on
esis has been proposed for the regulation of receptor expression on NK cells. It states that NK cells
NKT cell, however recognize only will randomly express one receptor, then a second and so on until at least one receptor is expressed
limited antigens such as glycolipid that can interact with self-MHC molecules, thus ensuring that the NK cell will not attack the host.
antigen presented on MHC-like
molecule—CD1.These cells are less
This hypothesis called “at least one” hypothesis suggests that the interaction of NK receptors with
than 1 per cent in peripheral blood. self-MHC will prevent the subsequent expression of still more NK receptor types.
MECHANISM OF NK-CELL CYTOTOXICITY

The main function of NK cells is to kill self-cells which contain viruses as well as some cancer-
ous/tumour cells. Though there are several surface molecules on NK cells that can interact with the
target cells (see Figure 12.9), the mechanism used for killing is identical to that used by T cyt cells
and is mediated by perforin and granzymes released by NK cells. Perforin monomers are released
from the vesicles in the NK cell at the junction of the target cell–NK cell. The perforin monomers
polymerize to form transmembrane channels which “punch a hole” in the cell membrane of the tar-
get cell allowing the passage of the granzymes into it. The target cell later dies of apoptosis. NK cells,
like the Tcyt cells, are also able to induce target-cell apoptosis through surface Fas ligand (present
on NK cells) ligating Fas molecules on the surface of the target cell. It should be remembered that
KIR IFN-γ
Perforin
FasL
KAR Granzyme
CD 16
Receptors involved in target Arsenal for cytotoxicity

recognition
NK cell
CD 16
NK cell KAR KIR
Antibody FasL NK cell
Fas KAR ligand
Class I MHC
Target cell Target cell Target cell
Figure 12.9
NK cells and their toxicity and
recognition. Different ways of target cell recognition by NK cell
even though NK cells and T cells share the same mechanism of cell lysis, there are some basic dif-
ferences. In Tcyt cells, granules appear in the cell after its activation, while in NK cells granules are
« Mature NK cells are found in the
constitutively present. Tcyt cells recognize target cells by specifically recognizing antigen plus MHC
bone marrow, spleen, lymph node
complex on the target cells, while NK cells effectively lyse target cells lacking class I MHC mol- and liver. They are present in small
ecules. Additionally, incubation of target cells with IFN-γ increased their sensitivity to Tcyt cells but numbers, if at all, in brain, muscle
diminished their sensitivity to NK cells. and skin.
In order to explain the fact that NK cells lyse non-specifically virus-infected and/or tumour
cell, in 1990 Ljunggren and Karre proposed the “missing-self” hypothesis. They proposed that
NK cells did not search for antigens on virus infected or tumour cells; they searched self-MHC
molecules (class I MHC to be precise) on target cells. Class I MHC molecules are vital to the con-
trol of most viral infections (and also for eradication of many tumours) and several viruses such
as Herpes virus, Epstein–Barr virus, as well as tumour cells can downregulate MHC expression
to “hide” from the onslaught of Tcyt. However, such downregulation of MHC, although it could
protect the infected cells from Tcyt, would make the cells more sensitive to NK-mediated cell lysis.
Thus, Tcyt and NK cells may seem as complementary arms of the immune system that together scan
the MHC expression spectrums for viral infection. With the discovery of activatory receptors on
the NK cells, this hypothesis has also been modified. It is believed that activating receptors engage
ligands on the target cells. These ligands may be abnormal forms or patterns of glycosylation on
the surface of tumour or virus-infected cells or they may be yet uncharacterized molecules on the
target cells. The activation of activatory receptors by these ligands will signal the NK cells to kill the
target cells. So in order to be lysed by NK cells, target cells must lack self class I MHC molecules
and should have ligands for binding activatory receptors, as depicted in Figure 12.10. However,
any of these killing signals can be vetoed by one signal from inhibitory receptors. As we know,
inhibitory receptors, whether CD94/NKG2 or KIR, are triggered only by binding to class I MHC
molecules. This suggests that the target cell is expressing normal class I MHC molecules and hence
will be dealt with by Tcyt cells later. NK cells even if they have been activated by activating ligands
on target cells will be switched off by one signal from inhibitory receptors. The overall result, the
well-being of the cells, is determined by the critical indicator of normal self class I MHC molecules.
Cells expressing class I MHC molecules are spared and those who lack them (or have reduced
levels) are terminated.
NK cells can be activated in vitro by IL-2 to become lymphokine-activated killer (LAK) cells. Lymphokine-activated killer
These LAK cells show a broadened target-cell range and increased cytotoxicity towards tumours. cells (LAK)
LAK cells are white blood cells
A recent approach for the treatment of some forms of cancer is to isolate the patient’s NK cells, treat produced by the cultivation of
them with high concentration of IL-2 in vitro and then reinfuse them. LAK cells are currently peripheral leukocytes with IL-2.
These cultivated cells manifest
undergoing trials for the treatment of cancer in humans.
enhanced cytotoxicity towards
NK cells can also secrete cytokine. When NK cells are “activated” by recognizing a virus- tumours. LAK cells are mainly NK
infected cell they secrete IFN-γ. This helps to protect the surrounding cells from virus infection cells but may also include T cells and,
rarely, macrophages.
and also enhance specific T-cell response directed to virus infected cells.
In general, it is believed that NK cells serve to lyse virally infected cells prior to development
of antigen-specific Tcyt response. When a primary virus infection occurs, that is, a host is exposed
to virus for the first time, it readily induces antiviral antibody and T-cell response, but the timing
is different. T-cell response peaks usually around 7–10 days and the number of T cells decreases
Class I MHC
Perforin
KIR vetoes Class I MHC
KIR KAR downregulated KIR KIR not
activated
KAR
Activation Target cell
KAR KAR activated
blocked NK cell
Target cell NK cell
Virus-infected or tumour cell
KAR ligand
No class I MHC expressed
Target cell lysis
Figure 12.10
MHC present on target cell. MHC absent on target cell Mechanism of NK-cell toxicity. The roles
No target cell lysis Target cell lysis of only KIR and KAR are shown.
» There is only one documented from the second week onwards. Antibody response usually peaks later than T-cell response. An-
human case of absolute NK-cell
deficiency.
tibody concentration or titre is usually small during the acute stage of viral infection and it peaks
over a period of two to four weeks and often lingers for weeks or months or even years depending
on host and virus. Since the body has to be protected from virus and virus-infected cell before the
first specific response (which occurs around day seven) the role of NK cells comes into the picture.
Early in the course of a viral infection, the population of NK cells is expanded and activated by
cytokines of innate immunity such as IFN-1, IL-12 and IL-15. NK cells which form the major line
of defence, lyse the target cells that are virus infected and hence display reduced levels of class I
MHC molecules. In addition to lysing target cells, the NK cells secrete IFN-γ which enhances the
development of specific T-cell response and activates macrophages, a non-specific phagocytic cell.
Examples of individuals with complete NK cell defects are rare, perhaps attesting to the fact that
their complete absence is lethal or almost lethal to the patient.
However, it should be pointed out that NK-cell response is not equally effective or lethal in
eradicating all viruses. Studies in mouse models suggest it might good in controlling some viruses
such as murine Cytomegalovirus but others such as Lymphocytic choriomeningitis virus are not
affected by NK onslaught.
12.3 ANTIBODY-DEPENDENT
C E L L - M E D I AT E D C Y T O T O X I C I T Y
Antibody-dependent cell-mediated cytotoxicity is the killing of antibody-coated target cells by a
non-phagocytic mechanism in which effector cells bind the Fc region of the antibody and lyse the
target cell. Effector cells involved in this form of cell-mediated cytotoxicity are NK cells, macro-
phages, neutrophils, monocytes and eosinophils. The antibody, usually IgG (or IgE, IgA), specific
for a structure on a target cell membrane, “coats” the target cell. The Fc receptor-bearing effector
ADCC cells then bind to antibodies already attached to antigen on a target cell and subsequently cause
The coating of target cells with target cell death. These “cytotoxic” cells are non-specific for antigen since they bind to the Fc region
the antibodies and making them of IgG (specifically IgG1 or IgG3), specificity of the antibody directs to specific target cell. This is
vulnerable towards attack by cells
of the immune system, such as known as antibody-dependent cell-mediated cytotoxicity (ADCC).
NK cells, eosinophils, neutrophils Target-cell killing by ADCC is mediated by a variety of slightly different cytotoxic mecha-
and macrophages is called nisms. The cell damage done by eosinophils and NK cells involves the release of perforin from
antibody-dependent cell-mediated
cytotoxicity (ADCC). these cells, while the binding of macrophages, neutrophils or eosinophils to target cells usually
results in the release of lytic enzymes at the site of Fc receptor binding. Moreover, NK cells and
macrophages also secrete tumour necrosis factor (TNF) after antibody-mediated binding to the
target cell. The binding to TNF to TNF receptor on the target cell may trigger target cell lysis.
TNF
Tumour necrosis factor (TNF) is a
Eosinophils mediate a special type of ADCC against helminths such as schistosomes or blood
pro inflammatory cytokine that is flukes. Schistosome larvae (schistosomules) are too large to be directly ingested by macrophages/
synthesized by a variety of cell types neutrophils and relatively resistant to lysis by neutrophils or the lytic enzymes of other phagocytes.
such as T cells (TH1), NK cells and
macrophages upon appropriate
These larvae are killed by basic proteins secreted by eosinophils. These basic proteins are secreted
stimulation. There are two forms of only when eosinophils bind effector IgE (not IgG !) coating the larvae. The receptor involved in this
TNF—TNF-α and TNF-β. IgE-induced ADCC is FcεRI or Fc α receptor which are different from CD16Fc receptor (FcγRIII)
that bind IgG. The different mechanisms of ADCC used by different effector cells are depicted in
Figure 12.11.
Target cell TNF-receptor Target cell

Perforin TNF
Target cell
Major
basic Lytic
Lytic
protein enzymes
enzymes
Eosinophil Macrophage Neutrophils

Figure 12.11
Different cytotoxic mechanisms of
antibody-dependent cell-mediated
cytotoxicity. Eosinophil-mediated Macrophage-mediated Neutrophil-mediated
12.4 D E L AY E D - T Y P E H Y P E R S E N S I T I V I T Y
Tuberculosis was one of the leading scourges of 19th century industrialized western world. Robert
Koch identified its causative organism as tubercle bacillus in 1882. He and his colleague Louis Pasteur
looked forward to conquer this disease by vaccinating as well as treating infected individuals by
tuberculin, is a product of the tubercle bacilli culture. Tuberculin
The intravenous injection of tuberculin in tuberculosis patients led to severe systemic reaction Tuberculin is a glycerine extract
of tubercle bacilli. This extract
and occasionally death. Accompanying all these disappointing results was one ray of hope. He was first used by Robert Koch in
discovered in 1891 that if tuberculin was applied intradermally, local inflammatory reaction would 1882 for vaccinating tuberculosis
be elicited in those individuals that had this disease. This was termed as delayed-type skin test patients. His effort, however led to
the development of DTH reactions.
(as it took 24–48 hours to develop) or, specifically, tuberculin reaction. This tuberculin reaction Instead of gaining protection, many
helped to isolate and detect patients having tuberculosis during those days. With the discovery that patients succumbed to pathological
similar reaction could be elicited by luetin (extract of treponemes for syphilis), lepromin (extract DTH reactions.
of Hansen’s bacillus for leprosy) as well as other bacterial extracts, these reactions were termed as
delayed skin reactions (and, later, delayed-type hypersensitivity).
Finally in 1942, Landsteiner and Chase demonstrated for the first time that contact hypersen-
sitivity to picryl chloride could be transferred from one guinea pig to another naïve recipient via
live peritoneal cells. The recipient guinea pig was found to elicit positive skin test to picryl chloride
24 hours later. No transfer of delayed hypersensitivity could be obtained using the fluid phase from
the exudates or killed cells. These experiments proved that delayed-type-hypersensitivity reaction
is mediated by cells and not by circulatory antibodies dissolved in the fluid phase.
Delayed-type hypersensitivity (DTH) is a form of cell-mediated immune response that
involves TH cells and non-specific effector cells such as activated macrophages. Since the ultimate
effector cells are non-specific, and since they cannot differentiate between self- and invading patho-
gen, some degree of host damage is done. Symptoms appear after some delay following antigen
« Contact antigens such as poison
exposure (usually after 24–72 hours) and, hence, it is referred to as delayed-type hypersensitivity. ivy and picryl-chloride are small
The term hypersensitivity is attached here because DTH reactions sometimes produce tissue injury molecules of natural or synthetic
without providing any “protective” function. origin that elicit DTH reactions
when they come in contact with
However, it should not be assumed that DTH reactions are always harmful. In fact, this type skin.
of cell-mediated immunity is a part primary defence mechanism against intracellular bacteria such
« Despite being called “hypersensi-
as Mycobacterium leprae, Mycobacterium tuberculosis, Listeria monocytogenes. tive,” DTH reactions are not always
When pathogens enter the host body, they are phagocytosed by cells of innate immunity harmful. It is a primary defence that
such as macrophages. However, some pathogens such as Mycobacterium tuberculosis can actually contains and destroys a number
of intracellular bacteria such as
survive inside the phagocyte’s cytoplasm or lysosome. The killing of such intracellular bacteria M.tuberculosis.
requires switching ON the previously subverted microbicidal mechanism in the phagocyte by TH-
cell-derived cytokines.
12.4.1 S E N S I T I Z AT I O N A N D E F F E C T O R P H A S E S I N D T H
RESPONSE
DTH reactions are produced in two separate phases: (a) the sensitization or activation phase, and
(b) the effector phase.
S E N S I T I Z AT I O N O R A C T I VAT I O N P H A S E
The sensitization phase in DTH response takes about 8–14 days in humans. During this phase the
host comes in contact with antigen and the antigen is endocytosed by antigen-presenting cells and
presented together with class II MHC molecules to naïve TH cells. This results in the activation
and clonal expansion of appropriate TH cells. These TH cells then elicit effector actions directly or
indirectly. A variety of antigen-presenting cells are involved in DTH response, including Langerhan’s
cells, macrophages and vascular endothelial cells. Langerhan’s cells are specialized dendritic cells
found in the epidermis. These cells carry antigens from the skin surface to the lymph nodes or
spleen where they activate specific naïve TH cells. After naïve TH cells encounter antigens, they
start proliferating (clonal expansion) and differentiations into effector and memory TH cells. Mac-
rophages as well as vascular endothelial cells also express class II MHC molecules and hence can
function as antigen-presenting cells in DTH response. A simplified view of the sensitization phase
is depicted in Figure 12.12.
Class I MHC Antigen
Epidermis
Dendritic cells Skin

Dermis
Class II MHC
Lymph vessel
Cortex
(B-cell rich)
T-cell
activation
Naive and
Naive
CD8+ cell differentiation
CD4+ cell
Paracortex
(T-cell rich)
TDTH cells
TH1 Memory Tcyt Memory
CD4+ cell CD8+ cell
Medulla
Lymph node or spleen
Efferent lymphatic
Figure 12.12
Line diagram showing sensitization Differentiated T cells
phase of DTH reaction. TDTH cells enter circulation
comprise mainly CD4+ T cells, but may and migrate to
also include CD8+ T cells. the site of antigen
» In a normal animal, there is only The generation of memory CD4+ T cells (of TH1 subtype) is the primary event of the sensitiza-
one memory cell per one million
naïve T cells. tion phase of DTH response. Sensitization generates TDTH cells that are directed against a particular
irritating antigen.
» The T cells that are involved in The involvement of both CD4+ TH cells and CD8+ Tcyt cells in DTH response provides a double
delayed type of hypersensitiv- ring of security. Cytoplasmic viral proteins are largely presented to Tcyt cells in association with
ity, though named TDTH, are not a
specialized subset of T cells; they
class I MHC molecules; whereas protein antigen entering via the endocytic pathway are presented
are constituted primarily by TH cells to TH cells together with class II MHC molecules. So TDTH cells comprise both the cells (Tcyt and
and, in a few cases, Tcyt cells. TH)—one that recognizes antigen with class I MHC (Tcyt) and the other that identifies it with class
II MHC molecule(TH) cells. One very important event that happens during the sensitization phase
is the formation of a large number of memory T cells that circulate in blood. There is one in 10,000
memory T-cells in the circulation for a particular sensitizing antigen. The same experimental ani-
mal will have one memory cell in 1 million naïve T cells in an unsensitized animal.
EFFECTOR PHASE OF DTH

The effector response of DTH is initiated after secondary exposure of antigen to TDTH cells. After
the second encounter with the antigen, the TDTH cells secrete cytokines that recruit macrophages
and other non-specific cells at the inflamed site where antigen has entered or is localized. The
inflammatory reaction contains and destroys the pathogen-causing infection and gets rid of in-
jured tissue leading to elimination of antigen and resolution of the infection (see Figure 12.13).
The symptoms of DTH response become apparent a day (24 hours) after secondary contact with
IFN-G ,TNF-B ,
IL-2,TNF-A
Interaction of TDTH cells

and antigen presenting cells
Blood vessel
Secretion of cytokines by TDTH cells
induces synthesis of adhesion
molecules, chemotaxis of
monocytes/macrophages
E-selectin
Monocyte
VCAM Monocyte
Macrophages
Enzymes Reactive oxygen and

nitrogen species
Figure 12.13
Line diagram showing effector phase of
Antigen damaged DTH reaction.
antigen, even though earliest histological change can be seen after ~six hours (but it is not apparent
to the naked eye). The DTH response generally peaks 48–72 hours after secondary exposure, when
a large number of lymphocytes infiltrate the tissue and induce localized inflammatory reaction (for
example, skin in contact dermatitis).
It is not clear which cells present antigen to TDTH cells (that is, present antigen for the sec-
ond time). They could be macrophages or vascular endothelial cells (cells that line capillaries)
as both express class II MHC molecules and various costimulatory molecules needed for their antigen- Diapedesis
presenting functions. Once activated, TDTH cells secrete cytokines. These cytokines induce the Trans-endothelial migration of
leukocytes (for example, monocytes,
cells of capillaries (endothelial cells) to secrete a number of adhesion molecules on their surface neutrophils) from the blood
(for example, E-selectin, VCAM). These adhesion molecules bind blood monocytes, causing their capillaries into the tissue is called
rolling along the endothelial surface in the direction of blood flow. This rolling activates the mono- diapedesis.
cytes which then transmigrate through inter-endothelial cell junctions of the capillaries into the
neighbouring tissues where they differentiate into activated macrophages. Macrophages are major
effector cells of DTH response.
Activated macrophages show increased phagocytosis (efficient killing of microorganisms
by generating reactive oxygen and reactive nitrogen species) and become more efficient antigen-
presenting cells.
Generally, inflammation and activation of macrophages in DTH response are sufficient to
eradicate intracellular microbes or any other irritant pathogen which is rapidly cleared with little
tissue damage. However, if DTH reaction fails to eradicate the microbe, activated macrophages
and their product such as cytokines/growth factors generate local inflammatory reactions that
become injurious to normal host tissues. Prolonged activation of macrophages and slow actions of
various growth factors lead to the replacement of differentiated tissue by fibrous tissue, a process
Epithelioid cells referred to as fibrosis. At other times, persistent activation of macrophages causes them to pro-
Epithelioid cells are flattened
macrophages that are found in duce nodules of inflammatory tissue called granulomas. Histologic sections of granuloma show
granulomas. They derive their name chronically activated macrophage-derived cell type such as skin epithelial cells (epithelioid cells)
due to their pink cytoplasm which which can sometimes fuse to form a multinucleated giant cell, which a forms part of the granu-
give them an appearance similar to
squamous epithelium. loma. Granulomas secrete large number of lytic enzymes which damage the surrounding tissue.
12.5 CYTOKINES AND DTH REACTION

A large variety of cytokines play a important role in shaping DTH reaction. In DTH reaction,
cytokines are secreted by TDTH cell, macrophages and sometimes even by keratinocytes (in contact
hypersensitivity).
The cytokines secreted by TDTH cells in DTH reactions and their functions are listed below.
• Interleukin 2(IL-2): This cytokine functions in an autocrine and paracrine manner
to expand the population of antigen-specific TDTH cells, thus amplifying the
response.
• Tumour necrosis factors α and β: TNF-α and TNF-β (lymphotoxin) act on vascular
» γ interferon is secreted only by endothelial cells to induce an activated state. Activated endothelial cells express several
T cells and NK cells. γ interferon can adhesion molecules such as E-selectin, VCAM, ICAM, etc. These adhesion molecules cause
upregulate MHC molecules and leukocyte binding, rolling and recruitment to the site of the reaction.
activate macrophages. Interferons
were discovered by the Scottish T-cell derived IFN-γ serves to activate macrophages. As mentioned previously, the activation
virologist Alick Isaac in 1957.
boosts the machinery of macrophages making them more efficient killing machines. Moreover, the
activated macrophages express an increased amount of class II MHC molecules and costimulators of
the B7 family, making them more efficient antigen-presenting cells. A brief overview of role of cyto-
kines secreted by TDTH cells in DTH reaction is shown in Figure 12.14. In addition, IFN-γ stimulates
macrophages to secrete a number of cytokines, as discussed in the next section. Table 12.2 shows
some important cytokines and their functions that are important in cell-mediated immunity.
Expansion of
TDTH cells population
IFN-G
IL-2
IL-8,IL-12
PDGF
TDTH cell
TNF-A ,TNF-B
Activated macrophages
increases expression of
(a) Class II MHC
(b) B7 co-stimulator molecules
and secretes several cytokines
VCAM ICAM E-selectin
Figure 12.14
Cytokines and delayed-type Expression of adhesion molecules
hypersensitivity reaction. on endothelial cells
Cytokine Sources Major Functions

IL-2 TH1 cells, NK cells Enhanced activity of B, T cells,
NK cells
IFN-γ TH1 cells, NK cells Enhanced antimicrobial activity of
macrophage, NK cells, depresses
viral growth
TNF-α, TNF-β TH1 cells, macrophages Induce apoptosis in tumour cells,

enhances activity of B-cell,
neutrophils.
IL-12 Macrophages, dendritic cells Induce differentiation and prolif-
eration of Tcyt cells, TH1 cells
Table 12.2
Chemokines Macrophages, endothelial cells, Lymphocyte recruitment, active in Cytokines involved in DTH reactions
T- cells inducing inflammation
12.5.1 G R O W T H FAC TO R S / C Y TO K I N E S S E C R E T E D
BY MACROPHAGES
Activated macrophages produce cytokines and growth factors that assist and augment the DTH
reaction. These include, (a) IL-12, which stimulates lymphocyte proliferation and differentiation,
IL-8, which induces adherences of neutrophils to vascular endothelium.
Platelet-derived growth factor (PDGF) produced by activated macrophages stimulates fibro-
blast proliferation while transforming growth factor β promotes tissue fibrosis and angiogenesis.
In contact hypersensitivity reaction, keratinocytes, which provide integrity to the epidermis
and on which antigen gets bound in contact sensitization, release several cytokines. These include
IL-1, GM-CSF, TNF-α, TNF-β, IL-6 and IL-8 which activate Langerhan’s cells involved in the
hypersensitivity reactions.
12.6 DETECTION OF DTH REACTION

The presence of a DTH reaction or TDTH cells in an individual can be experimentally observed. If « Mantoux test is named after the
the individual develops skin lesion, TDTH cells can be experimentally observed. It implies that the French physician Charles Mantoux,
who developed this test in 1907.
individual has TDTH cells for that antigen and is prone to DTH reaction for the test antigen. For
example, to determine whether individuals have T-cell-mediated immunity against tuberculosis,
Mantoux test is performed. In this test a small amount of the purified protein derivative (PPD), PPD
PPD is purified tuberculin that is
obtained from cell wall of Mycobacterium tuberculosis is injected into the skin of the individual and isolated from the cell wall of dead
site is examined between 48–72 hours. tuberculosis bacteria. It differs from
A positive skin test shows up as firm red swelling, maximal at 48–72 hours after injection. classical tuberculin in that it is grown
in a synthetic medium. Classical
This is a clinical evidence of tuberculosis infection in the individual and presence of TDTH cells. tuberculin (used by Koch) was
This test does not allow differentiation between those individuals that carry sensitized TDTH (for extracted from bacteria grown in
M. tuberculosis) cells because of exposure to the pathogen and those that have sensitized TDTH broth medium.
because of the vaccination.
12.7 SIGNIFICANCE OF DTH REACTION

Protective role: The DTH response is one of the types of cell-mediated effector mechanism employed
by our body against a variety of intracellular pathogens and contact antigens. In contrast to Tcyt re-
sponses which are very specific, the DTH response non-specifically destroys cells that harbour in-
tracellular pathogens. The cells that are involved in DTH reactions are mainly TH cells that recruit
macrophages which release the damaging enzymes. Since these enzymes are unable to differentiate
between cells that harbour pathogens and normal cells, a small amount of healthy tissue is also dam-
aged. However, this is a small price to pay for elimination of intracellular pathogen. Similarly, contact
antigens would be difficult to get rid off by a mechanism other than DTH response.
Loss of DTH responses to common intracellular antigen such as Candida albicans is consid-
ered to be an indication of deficient T-cell function. These individuals develop life-threatening
infections from intracellular microorganisms such as mycobacteria, fungi, protozoan that would
otherwise never “harm” individuals whose DTH response is intact. This is best illustrated by AIDS,
a disease in which TH cells are severely depleted.
Pathologial consequence of DTH: Sometimes in DTH reaction, the source of the antigen is
not completely eradicated or degraded. This, in turn, leads to the aggregation and proliferation
of macrophages stimulated by TH cells. Continual stimulation of macrophages and other non-specific
effector cells release lytic lysosomal enzymes meant to destroy persistent chronic infection.
These enzymes cause extensive tissue damage and hence at that particular moment its damaging
pathologic response of DTH far outweighs any beneficial effects. The pathologic aspects of DTH
response are discussed in detail in the next chapter.
Cell-mediated immunity can be best described as those immune reactions that occur on or
near the body cells. It has two main branches—a non-specific part that is constituted by NK cells,
macrophages, eosinophils and neutrophils, and a specific part constituted by Tcells—both Tcyt and
TH cells. Cells involved in non-specific cell-mediated defence recognize pathogens by the pres-
ence of PAMPs present on the pathogens while T cells recognize and execute defence arsenal after
recognizing peptides derived from pathogen that is presented on MHC molecules. Cell-mediated
immunity is by and large beneficial. However, if pathogen persists, defence response continues for
longer time. This excessive immune response leads to tissue damage and development of delayed
type of hypersensitivity (DTH). DTH response is mediated primarily by TH cells. Though DTH
response tends to contain and destroy the pathogen, excessive DTH reactions are harmful for the
body and tend to aggravate into clinical disease.
Agglutination
Cells Antibodies Agglutination

(microbes or cells) (from patient suffering (clumping of cells by antibodies)
from microbial infection)
Direct agglutination test
Antigen- antibody
complex
Dilution of antibody
(antibody/total volume) 1/10 1/20 1/40 1/80 1/160 1/320
Titer of antibody = 80
Determination of antibody titer
Visible antibody–antigen complex is detected at maximum dilution of 1/80. The reciprocal of this value
will give antibody titer of 80
Figure 12.15
The principle of agglutination.
Agglutination reaction refers to the clumping together of particulate clumping or agglutination reaction and hence a positive widal test.
matter such as microbial or eukaryotic cells. This clumping generates In indirect or passive agglutination, antigens are adsorbed onto the
aggregates of cells that are visible to the naked eye. This agglutina- surface of particulate objects such as latex spheres and polystyrene
tion reaction is mediated by antibodies, though other polyvalent mol- beads. Serum antibodies are then detected by observing the agglu-
ecules such as lectin can also mediate such reaction. Agglutination tination of these particulate objects.
reaction. can be of two main types—direct agglutination reaction
and indirect agglutination reaction. In direct agglutination reaction, Agglutination reaction may be performed to determine the pres-
the target cells are directly reacted with antibodies from an individ- ence of antibodies in a patient’s serum (as in widal test) or can be
ual. If antibodies are specific against the antigen, they will react to used to determine the concentration or titre of antibodies. For de-
form a visible aggregate or clump that can be detected. This princi- termining the titre of antibodies, first a constant amount of antigen
ple forms the basis of widal test used for detecting typhoid. Typhoid is added in a series of tubes. Antibodies (that is, serum sample) is
bacilli (inactivated) are mixed in vitro with serum from an individual then serially diluted (1/10, 1/20,1/40,1/80, etc.) and then added to
suspected to have typhoid fever. If the person is suffering from ty- each tube. The maximum dilution of serum that shows agglutina-
phoid, his serum will have anti-typhoid bacilli antibodies. These anti- tion is then noted and a reciprocal of that gives the serum antibody
bodies will react with inactivated typhoid bacilli and give a positive titer (see Figure 12.15).
S U M M A R Y
• Cell-mediated immunity (CMI) refers to immune reaction activate apoptosis of target cell. The Fas pathway is activated by
occurring on or near the body cells. the binding of FasL (present on T cells) to Fas (CD95) on the target
cell, resulting in the activation of the death domain and consequent
• CMI has two branches: (a) non-specific CMI which includes apoptosis of the target cell.
immunity rendered by macrophages, neutrophils and NK cells;
(b) specific CMI rendered by Tcyt and TH cells. • NK cells form a very important defence against cells infected with
viruses, bacteria and protozoa.
• Cells involved in non-specific CMI bear pattern-recognition
receptors that recognize pathogen by binding to its conserved • NK cells express killer activatory receptor (KAR) and killer
PAMPs. inhibitory receptor (KIR) on their cell surface which are
involved in target cell killing. The stimulation of KAR induces
• Cells involved in specific CMI have specific recognition target cell death. The activation of KIR by class I MHC molecules
mechanisms. Tcyt cells recognize peptides from intracellular leads to the shutting off of NK cell response and the veto
microbes that are displayed on class I MHC and kill the of KAR action. Thus the target cell is spared by KIR
target cell. activation.
• CD8 molecules present on Tcyt cells recognize non-polymorphic • Like Tcyt cells, NK cells can employ perforin or the Fas–FasL
part of class I MHC while TCR recognizes the antigenic part pathway for target cell lysis.
displayed on class I MHC molecules.
• Delayed-type hypersensitivity (DTH) is a form of cell-mediated
• Once naïve Tcyt cells recognize antigen plus class I MHC molecules immune response that involves TDTH cells. This type of CMI is a
on target cells, they get stimulated by costimulators and cytokines primary defence response against intracellular bacteria. TDTH cells,
and get transformed into functional Tcyt cells. upon activation, secrete a variety of cytokines that summon
• Activated Tcyt cells can induce target cell lysis by using either the non-specific macrophages. These cytokines and macrophages
perforin pathway or the Fas pathway. initiate inflammatory reactions that destroy pathogens and/or
infected cells. Excessive DTH response, however, can have
• The release of perforin by Tcyt cells punches pores into the target pathologic implications.
cell causing osmotic cell lysis, and makes way for granzymes that
K E Y W O R D S
• antibody-dependent cell-mediated • DTH response 257 • large granular lymphocytes 252

cytotoxicity 256 • Epithelioid cells 260 • NK cell 247
• CD8+ T cell 258 • Fas pathway 250 • NK-cell receptor 253
• cell-mediated immunity 247 • granzyme pathway 250 • perforin 249
• cytotoxic T cell 251 • killer activatory receptor 253 • purified protein derivative 261
• delayed-type hypersensitivity 257 • killer inhibitory receptor 253 • Toll-like receptor 247
1. There is only one documented human case of absolute NK cell

deficiency. This individual was associated with recurrent life- 4. Delayed-type hypersensitivity is often portrayed as a tissue-
threatening bacterial and viral infections. Explain this observation. damaging reaction that causes harm to the individual.
2. Tcyt cells and NK cells are equipped with two cell lysis arsenals— Giving examples, prove that this is not always
perforin and Fas pathway. How are these pathways different from the case.
each other? Why have two pathways evolved, when one will suffice? 5. NK cells and Tcyt cells both induce apoptosis of target cells by a
3. What is the “missing-self ” hypothesis? According to this similar mechanism. How do these two types of cells differ in
hypothesis, which part of self is “missing”? Which class of cells recognizing target cells? Which one is more specific?
search for this missing part? Why? Are these two cells equally important in cell-mediated immunity?

1. Which one of the following is not involved in innate cell- 6. Cell-mediated onslaught by eosinophils against schistosomes
mediated immunity? is primarily mediated by:
(a) Toll-like receptor (a) Perforin
(b) Nod molecules (b) γ interferon
(c) T-cell receptor (c) Major basic protein
(d) Pattern-recognition receptor (d) Granzyme
2. Which one of the following is not involved in Tcyt-mediated 7. Delayed-type hypersensitivity involves:
cytotoxicity? (a) Tcyt cells
(a) Perforin (b) TH cells
(b) Granzyme (c) Macrophages
(c) FasL (d) All of the above
(d) Class II MHC
8. Cells that present antigens to naïve T cells in DTH reaction
3. Which one of the following is not an NK cell? are not:
(a) Null cell (a) Langerhans cells
(b) Large granular lymphocyte (b) Macrophages
(c) Granulocyte (c) Follicular dendritic cells
(d) LAK cell (d) Vascular endothelial cells
4. KIR present on NK cell binds: 9. CD3 negative, CD56 and/or CD16 positive lymphocyte are
(a) Classical class I MHC molecule difficult names of:
(b) Non-classical class I MHC molecule (a) NK cells
(c) Classical class II MHC molecules (b) Tcyt lymphocytes
(d) None of the above (c) TH lymphocytes
(d) B lymphocytes
5. One molecule that is not expressed on NK cell is:
(a) CD2 10. TDTH cells are involved in all, except:
(b) CD3 (a) Production of interleukin-2
(c) KIR (b) Acquired resistance to tuberculosis
(d) CD56 (c) Expression of adhesion molecules on endothelial cells
(d) Generating antibodies against polysaccharide antigen.

1. CD94/NKG2 and NKR-P1 are killer inhibitory receptors. 4. Antibody-dependent cell-mediated cytotoxicity can be mediated
by eosinophils, NK cells and macrophages.
2. Fas pathway is activated by binding Fas present on Tcyt cell to
FasL, present on target cell. 5. Fas pathway is used by both NK cells and Tcyt cells.
3. NK cells are called null cells because they do not express any cell
surface receptor.
Aderem, A. and D. M. Underhill (1999). “Mechanisms of Mak, T. W. and W. C. Yeh. “A Block at the Toll Gate”, Nature,
Phagocytosis in Macrophages”, Annual Review of Immunology, 418: 835–36.
17: 593–623.
Metchnikoff, E. (1893). Lectures on the Comparative Pathology of
Boes, M. and H. Ploegh (2004). “Translating Cell Biology In Vitro Inflammation. London: Kegan, Paul, Trench, Trubner (Reprinted
to Immunity In Vivo”, Nature, 430: 264–71. by Dover, New York, 1968).
Enk, A. H. and S. I. Katz (1995). “Contact Hypersensitivity as a Rouvier, E., M. F. Luciani and P. Golstein (1993). “Fas Involve-
Model for T-cell Activation in Skin”, Journal of Investigative ment in Ca2+-independent T-cell-mediated Cytotoxicity”, Journal
Dermatology, 105: 805–35. of Experimental Medicine, 177: 195–200.
Kranebuhl, O. and J. T. Schopp (1991). “Perforin-induced Pore Rosenberg, H. and J. I. Gallin (2003). “Inflammation”, in W. E.
Formation”, Immunology Today, 12: 399–402. Paul (ed) Fundamental Immunology, 5th ed. New York:
Lippincott-Raven.”
Laneir, L. L. (1998). “NK Cell Receptors,” Annual Review of
Immunology”, 16: 359–93. Sigerist, H. E. (1961). A History of Medicine, Vol. II. New York:
Oxford University Press.
Liu, C. C., L. H. Young and J. D. Young (1996). Lymphocyte-
mediated Cytolysis and Disease”, New England Journal of
Medicine, 335: 1651–59.
In 1890, Koch tried to “vaccinate” those individuals that were already
infected by tuberculosis (vaccination had already been introduced in
1798). The material he employed was a bacterial product, tuberculin.
This attempted vaccination proved to be extremely harmful to patients
and some of them even died. At that time, Koch was unaware that what
he witnessed was a hypersensitive reaction. In fact, he attributed these
damaging reactions to the toxic effect of the injected extract of tuber- “The fire you
culosis bacteria, tuberculin. Again, in 1893, Emil Von Behring reported kindle for your
“hypersensitivity” to the diphtheria toxin in guinea pigs, previously enemy often
immunized with same antigen. It was Von Behring who coined the burns yourself
term hypersensitivity to imply (wrongly) that these animals had be- more than him.”
—CHINESE PROVERB
come more sensitive to direct effect of the injected toxins . Little
attention was paid to these reports or their implication until the work
of P. Portier and C. Richet in 1902. These physiologists were engaged
by the Prince of Monaco to study the mode of action of poison of the
Portuguese man of war (jellyfish) in mammals. This study was under-
taken because bathers in the Mediterranean were plagued by stings
of this jellyfish. These two French scientists carefully investigated and you should be able to:
concluded that the reaction of the bathers to the sting was due to • Define the terms
hypersensitivity and allergen
some toxin present in the sting. Since vaccination had been recently
• Give an account of Gell and
discovered, these scientists isolated toxins from jellyfish and planned Coombs classification of
hypersensitivity reactions
to use them as immunogens to vaccinate bathers. To test the efficacy • Explain the role of IgE, mast
cells and basophils in type I
of this “vaccine”, they injected the purified toxins in dogs. To boost the hypersensitivity
immune response in dogs, they re-injected the toxin after a few days. • Describe the various biological
mediators of type I reactions
What they encountered was shocking to all of them! Dogs, instead of • Describe and illustrate type II
showing effective immune response against the toxin, exhibited
• Explain drug allergies,
clinical shock syndrome together with asphyxia, vomiting and, in some haemolytic anaemia,
erythroblastosis foetalis and
transfusion reactions
cases, even death. They named this “overreaction” as a new phenom-
enon, anaphylaxis (opposite of prophylaxis which implies protection). mechanism of type III reactions
• Explain Arthus reaction and
The credit of discovering anaphylaxis should also go to Theobald serum sickness reaction
Smith who, in 1902, independently studied the analogous anaphylac- • Describe type IV delayed-type
tic shock reaction in guinea pigs. With the discovery of anaphylaxis,
several other types of hypersensitivity reactions came to be
discovered. Later, as we shall see, these reactions came to be classified
into type I, II, III and IV hypersensitive reactions (see Figure 13.1).
Hypersensitivity
13.1 INTRODUCTION
C. Richet, after his observation on jellyfish toxins, postulated that anaphylaxis is the direct result of
the injected toxins. He believed (though incorrectly) that the injected toxin contained two compo-
nents: thalassin, which induced immunity and congestin, a poisonous component which induced
13
hypersensitivity by directly damaging the host. At that time, the role of immunity was considered
sacred, offering protection to the host body. The fact that this immunity could also damage the host
was unimaginable. The fact that an immunological mechanism was indeed involved in hypersensitiv-
ity reaction was pointed out by the Viennese paediatrician, Clemen Von Pirquet. « Clemen Von Pirquet coined the
Hypersensitivity is now defined in a more clear way as the induction of a state of excessive term allergy (Greek: allos ergos—
immune response with resulting damage to normal tissues of host body when exposed to an appar- altered reactivity) to identify these
ently innocuous antigen. In this state the individual is more sensitive (instead of being protective) responses as distinct from the mini-
mal immune reaction that occurs
to antigen exposure. The antigenic substance that induces an allergenic reaction is termed as an after exposure to normal innocuous
allergen. Just as the individuals that are exposed to normal antigen are said to be immunized, in- antigens.
dividuals exposed to the allergens are said to be sensitized. Hypersensitivity reactions are antigen
Allergen Neoantigen
Complement-
Antibody mediated cell lysis
Fc receptor
Mast cell
Opsonization NK Cell-
Degranulation and mediated
phagocytosis ADCC
Tissue damage Phagocyte
Type I Type II
Antigen
Processed and
displayed by
antigen-present cell
Complement
Immune complex
activation
deposition
summons TDTH Cell (TH cell)
phagocytes
Cytokines
Basement membrane
Phagocytes induce Activates

tissue damage macrophage
Type III
Tissue damage
Figure 13.1
Type IV
A line diagram showing all four types of
hypersensitivity.
» Ancient Greeks called hypersen- specific and occur after the immune system has already been primed/exposed to an antigen (that
sitivity reaction as idiosyncrasis.
(Greek: idios—self; syncrasis—a
is, the individual has been sensitized).
mixture of humors). As the prefix Hypersensitivity is not manifested on first contact with the antigen, but usually appears on
idio implies, these conditions were subsequent contacts. Hypersensitivity reactions usually occur at a different time after coming into
unique conditions to an individual
and supposedly not present in all
contact with the offending antigens.
the individuals of a population. Hans Zinsser in 1921 classified hypersensitivity reactions into “immediate” and “delayed” re-
Apparently, Greeks were aware that actions. Sensitized individuals can manifest symptoms within minutes (immediate) to hours (de-
these conditions were unique to an
individual.
layed) after they encounter the antigen. Immediate hypersensitivity generally involves the humoral
arm (that is, antibody-mediated) of the immune system while delayed hypersensitivity reactions
» Gell and Coombs classified allergic are cell-mediated reactions involving TDTH cells. As we have seen in Chapter 12, delayed hypersen-
reactions based on the differences sitivity is a part of the normal immune defence mechanism against intracellular pathogens. However,
in the effector molecules gener- sometimes due to varying reasons severe tissue-damaging reactions are elicited.
ated. This classification, which was
presented in their book Clinical
Aspects of Immunology, proved to
be extremely popular and is still in
13.2 G E L L A N D C O O M B S C L A S S I F I C AT I O N
currency.
In 1968, P. G. H. Gell and R. R. A. Coombs proposed a classification of immunopathological hyper-
» It was Hans Zinsser, an Ameri- sensitivity reactions into four distinct categories—types I, II, III and IV. They classified hypersensi-
can bacteriologist, who, in 1921, tivity reactions based on the differences in the effector molecules generated in the reaction.
applied (the still used) the terms
“immediate” to hypersensitivity skin The type I hypersensitivity involves IgE-mediated reactions and includes anaphylaxis and
reactions that commence within a atopic allergies. Type II reactions include antibody and complement-mediated membrane-de-
few minutes and fade after a few structive immune reactions, Type III reactions involve the effects of immune complexes and in
hours and “delayed” to tuberculin-
type skin reactions that start around type IV or delayed-type hypersensitivity effector cells—TDTH cells and their secreted cytokines are
4–5 hours and peak after around 48 involved. Type I, II and III reactions are antibody-mediated (and immediate, as they manifest
hours. Thus, the terms immediate within a few minutes) hypersensitivity while type IV reaction is a cell-mediated (and delayed type)
and delayed were introduced.
immune response. Table 13.1 shows the four representative types of hypersensitivity reactions as
suggested by Gell and Coombs.
13.2.1 TYPE I HYPERSENSITIVITY REACTION

When a “normal” antigen first enters the host body, it encounters specific-antibody-bearing B cells.
On their first encounter with the specific antigen, these specific B cells get activated into (IgM- or
IgG-secreting) plasma cells and memory cells. Memory cells persist in the body of the individual
and can be converted anytime into effector cells by subsequent exposure to antigen.
However, when an allergen enters the body, it somehow tends to activate B cells to produce
IgE instead of normal antibody IgG. The IgE formed binds to high-affinity Fc receptors of tissue
mast cells and blood basophils. These IgE-coated mast cells/ basophils are said to be sensitized.
» The primary exposure of allergic Now when a sensitized individual is exposed to the allergen again, mast cells and basophils bind
individuals to allergen generates
IgE antibodies that get localized
the allergen on their surface, setting off a series of reactions leading to degranulation and release
on mast cells and basophils. The pharmacologically active mediators stored in mast cells/ basophils. These vasoactive substances
secondary exposure usually results act on the surrounding tissues causing vasodilation, and bronchial and visceral smooth muscle
in allergic reactions. Why some
antigens generate IgE instead of
contraction depending on the extent and place of mediator release, that is, whether systemic or
normal IgG is not currently known.
IL-4 and IL-13 secreted by TH2 cells
are known to mediate class switch-
ing in B cells to secrete IgE. Participating
Type Molecules/Cells Reactions Examples
I IgE, mast cells, basophils IgE-mediated Anaphylaxis, atopy
hypersensitivity
II Antibody, complement, Antibody-dependent Blood transfusion reaction,
NK cells cytotoxic hypersensitivity Rhesus antigen
incompatibility
III Antigen–antibody complex, Immune-complex- Arthus reactions, serum
neutrophils mediated sickness, SLE
hypersensitivity
IV TDTH cells, cytokines, Cell-mediated Contact hypersensitivity,
macrophages, Tcyt cells hypersensitivity granulomatous
Table 13.1
hypersensitivity
The four types hypersensitivity reactions.
local. This reaction is called immediate hypersensitivity as it begins rapidly within a few minutes of Wheal and flare
antigen challenge. The classic manifestation of immediate hypersensitivity reaction is the development A raised red area that develops on
of wheal and flare reaction. the skin after an allergic reaction.
Also called wheal and erythema.
This characteristic reaction usually
W H AT I S A N A L L E R G E N ? develops within 10–15 minutes after
Those antigens that elicit strong hypersensitivity reactions are referred to as allergens. Since in recent the injection of an allergen.
years, the term allergy has become synonymous with type I reactions, allergens are defined as
those chemical substances, proteins or chemicals, free or bound on proteins that elicit strong IgE
Allergy
response or immediate hypersensitivity reactions. Allergy is a maladaptive immune
What makes an antigen an allergen? Unfortunately, we do not know. It is believed that allergens response to an innocuous antigen.
Common symptoms of the allergic
of which almost all are proteins, have molecular weight ranging from 5,000–40,000 kDa. These small
reaction include watery eyes,
proteins are water soluble, carried on small particles such as pollen grain, pet danders and dust par- running nose, itching and sneezing.
ticles, and are very diverse biologically. For example, allergens from cockroach (Bla g1, Bla g2) are
aspartic proteases while Fel d 1 from cats and Amb a 5 from ragweed pollen do not have associated
enzymic activity; allergens identified from filarial parasites are enzyme inhibitors and some of the Allergen
Any antigen that triggers an
food allergens are small highly glycosylated proteins. Important representatives of allergens are high- allergic reaction (typically type
lighted in Table 13.2. Some representative types of allergens are shown in Figure 13.2. I reaction) is called an allergen.
The allergens can enter the host body through inhalation (allergen of pollens, cockroach al- Common allergens include pollen,
pet danders, certain foods such as
lergen, cat dander), ingestion (food and drug allergen) or through invasive method (bee stings). nuts and chocolates, as well as dust
Inhaled allergens enter the host body usually in miniscule quantities (some as little as 1μg/year) particles, drugs such as penicillin,
while allergens that are ingested are often eaten in very large quantities (15–100 g/day). Studies of chemicals, smoke and several
others. Immunogen, on other hand,
various allergens have revealed that each allergen is made up of several components , for example, are those antigens that stimulate
ragweed pollen has five allergic components (Amb a 1 to 5: previously called ε, κ, Ra3, Ra4, Ra5), protective immunity in the host.
ryegrain allergen has three components—Lol p 1 to 5.
The development of allergy depends not only on allergens but also their dose, sensitizing
route, genetic constitution of the recipient and, in the case of experimentally sensitized animals,
the adjuvant used.
IgE
The existence of a “special” type of antibodies involved in allergic reactions was first pointed out
by J. R. Curne in 1907. He cautiously suggested that allergic reaction is because of the formation of
a special type of antibody against an instigating antigen. This antibody, he pointed out is different
from normal protective antibody. The antibodies responsible for allergies were given the special
name, reagins, presumably to separate them from more useful antibodies associated with protective
immune response.
In 1921, C. Prausnitz and H. Kustner demonstrated that human allergic condition could be
passively transfered by serum from one individual to other. Kustner was extremely sensitive to the
cooked flesh of fish while Prausnitz was not. Kustner injected a little of his serum into Prausnitz
Types Examples
Proteins Animal hair, Fel d l (cat), Amb a 5 (pollen)
Enzyme Bla g 1 (cockroach)
Enzyme inhibitor Allergens from filarial parasites
Table 13.2
Drugs Penicillin, sulphonamides Components of allergens.
Glycosyl residues
Enzyme
Active site inhibitor
Enzyme Enzyme
Figure 13.2
Enzyme Enzyme inhibitor Protein Glycosylated protein Some types of allergens.
» The E of the IgE comes from skin and demonstrated that it was possible to passively transfer allergic reaction (this forms the
erythema, since these antibodies
were involved in wheal and
basis of Prausnitz–Kustner or P–K test). The nature of antibody involved in allergy was not known
erythema reactions of ragweed. until 1966, when the husband-and-wife team of Kimishige and Ishizaka prepared a reagins-rich
fraction from the serum of a person showing allergy to ragweed and isolated a new type of antibody
γ-E (E for erythema), later renamed IgE.
Apart from being produced in small quantities, IgE has much a smaller half-life (two days) than
IgG (23 days) or IgM (five days), which accounts for its low serum level. IgE molecules secreted in the
» IgE has the smallest half-life (two
to three days) among all classes of plasma are quickly bound to high-affinity receptor present on tissue mast cells and bloods basophils.
antibodies present in the plasma. The low levels of IgE antibody found in the serum made its isolation and characterization
In the bound state, its half-life is difficult. Johansson and Bennich, who in 1967 discovered IgE myeloma in a patient, made such
about three weeks. IgE does not fix
complement and is heat-labile. studies possible. IgE is a bivalent, isotype of immunoglobulin that contains two heavy chains (of
ε isotype) and two light chains with a combined molecular weight of 190 kDa. IgE is different from
other isotypes because it has an extra constant-region domain that contains binding sites for both
» The concentration of IgE in the
high- and low-affinity IgE receptors. These binding sites enable IgE molecules to bind mast cells
serum of normal individuals is very
low (less than 1 μg/ml) compared and basophils. Once bound to the receptors on these cells, IgE can survive a number of weeks.
to all the other immunoglobulins.
Under pathogenic conditions, such 13.2.2 BASOPHILS, MAST CELLS AND EOSINOPHILS
as allergy or helminth infection, IgE
levels can increase upto 1 mg/ml. Basophils are phagocytic, motile polymorphonuclear granulocytes found in circulation where they
differentiate and mature. Basophils circulate in the blood in a differentiated form.
» Under normal conditions,
These are not normally present in the tissue but can be recruited into the tissue during in-
basophils rarely migrate into the flammation by some cytokines. Basophils contain granules that stain with basic dyes and, hence,
tissue. However, in allergic the name. They constitute less than 1 per cent of the circulating white blood cells. Basophil gran-
disorders, basophils show trans- ules contain heparin and histamine. Heparin, an anticoagulant, is a muco-polysaccharide that is
endothelial migration from the
blood stream to the site of responsible for metachromasia of the granules.
inflammation. Histamine and slow-reacting substances (SRS), are the other pharmacologically active
mediators found in basophils. Histamine, a vasoactive mediator, can cause constriction of bron-
» Some dyes (such as toluidine blue) chial and intestinal smooth muscles and contraction of endothelial cells, apart from several other
give a different colour when bound prompt and transient functional effects. SRS acts in a more sustained manner after a latent period.
by intracellular granules than they
do when staining nuclear DNA, so SRS has diverse actions involving leukocyte activation, aggregation, chemotaxis and vascular per-
that these granules are sometimes meability, in addition to smooth muscle contraction.
referred to as metachromatic
(Spanish: meta—change; Greek:
chroma —colour). MAST CELLS
Mast cells (German: mast—well-fed or fattening) precursors are formed in the bone marrow.
» Leukotriene C4 (LTC4) and its deg- Immature mast cells exit the bone marrow and migrate to peripheral tissues where they undergo
radative products, LTD4 and LTE4,
constitute what was once called
differentiation. Normally mature mast cells are not found in circulation. Mature mast cells are
slow-reacting substance of found throughout the body, particularly near lymphatic and blood vessels, nerves and beneath
anaphylaxis (SRS-A), a major epithelia. Electron microscopy reveals that they are round or oval with a typically round nucleus.
mediator of asthmatic
broncho-constriction.
The cytoplasm of mast cell contains granules like basophils which are known to enclose pharmaco-
logically active mediators. The mediators include histamine, heparin, leukotrienes as well as several
different enzymes such as chymase and tryptase. The exact type of mediators produced by mast
cells varies with their anatomic location and are probably regulated by cytokines.
» Mast cells constitute that part of
the immune system that counters
Mast cells in rodents (as well as in humans) are of two main types—mucosal mast cells (MMC)
parasites, as opposed to bacterial or and connective tissue mast cells (CTMCs). Both in humans and rodents, MMCs show T-cell de-
viral invaders. pendence in growth and development, while CTMCs exhibit a development pattern independent
of T cells. The major differences between these two types of mast cells include:
• MMCs produce mainly tryptase, while CTMCs produce both tryptase and chymase (both
serine proteases).
• The major granule content of MMC is a proteoglycan—chondritin sulphate, while CTMC
has heparin.
• The anatomic location of MMC is mainly the lungs and nasal cavity, while CTMC are
largely located in skin, blood vessels and intestinal submucosa.
Mast cells also secrete a variety of cytokines, including GM-CSF, TGF-β, TNF-α, IL-1, IL-5,
and IL-6 which exert diverse biological effects.
Neutral serine proteases, primarily tryptase and chymase, are the most abundant protein con-
stituents of all mast cells.
The presence of chymase in biological fluids suggests mast-cell activation. Chymase is found « Tryptase is present in human mast
cells and is not known to occur in
in some, but not all human mast cells. The function of these enzymes in vivo is not known, how- any other cell type.
ever, in vitro tryptase has been shown to cleave and activate collagenase. Thus tryptase contributes
to tissue damage by activating collagenase, whereas chymase is known to cleave basement mem-
brane and convert angiotensin I to angiotensin II, thereby adding to pathogenesis.
Proteoglycans, including heparin and chondritin sulphate, are also major constituents of both
types of mast cells. These proteoglycans are strongly negatively charged and hence serve as storage
matrix for positively charged histamines, proteases and other mediators. Once the granules are
exocytosed, these molecules dissociate at different rates from proteoglycans, with histamines dis-
sociating more rapidly than tryptase or chymase.
EOSINOPHILS
Eosinophils are bone-marrow-derived granulocytes. They are motile phagocytes, though not as
good as macrophages or neutrophils. Eosinophils are so named because their cytoplasm contains
granules that stain red with acidic dyes such as eosin.
These cells develop and mature in the bone marrow and after maturation circulate in blood. « Eosinophils are involved in the
Eosinophils are normally located in the mucosa of the lungs, GI tract and genitourinary tract. How- pathogenesis of asthma. Two forms
ever, they can be summoned to any tissue by cytokines secreted by basophils and mast cells. of asthma that cause high eosino-
phil count are bronchopulmonary
Basophils and mast cells release factors that are chemotactic for eosinophils such as eosino- aspergillosis (caused by allergic
philic chemotactic factor of anaphylaxis (ECF-A), tetrapeptides and histamines. Eosinophils are reaction to fungus) and
also recruited into tissues by monocytes, chemotactic protein-5, eotaxin complement component Churg–Strauss syndrome (a rare
asthma that has unknown etiology).
C5a, and leukotrienes such as LTB4. In the body, eosinophils have defence functions. They kill
the larvae of parasites that invade tissues such as in ascariasis, trichinosis and schistosomiasis. In
non-parasitic inflammations such as late-phase reaction of immediate hypersensitivity, eosino- « The number of eosinophils
phils are summoned by basophils and mast cells to release granule proteins that have inflamma- in tissue is usually 100 times higher
than in blood!
tory properties and can injure the normal host tissue. The granule content of eosinophils include
lysosomal hydrolases, platelet-activation factor (PAF), arylsulphatase B, major basic protein, hista-
minase, eosinophil peroxidase and eosinophilic cationic protein. The eosinophil-derived proteins
are toxic to parasites and bacteria, and in their absence, can also inflict harm on normal host cells.
A schematic representation of mast cells, basophils and eosinophils is shown in Figure 13.3.
Nucleus
Granules
Mast cell Basophil
Nucleus
Figure 13.3
False colour representation of mast cell,
basophils and eosinophils. [Adapted
from Cell Fine Structure. An Atlas of
Drawings of Whole-Cell Structure. Edited
Eosinophil by T. A. Lentz (1971) © Elsevier Saunders]
13.2.3 RECEPTORS FOR IgE

There are two types of IgE receptors. High-affinity receptor FcεRI is found on mast cells and
basophils. It has also been found on dermal macrophages, epidermal Langerhans cells, blood
monocytes and activated eosinophils where it is involved in antibody-dependent cell-mediated
cytotoxicity (ADCC). Low-affinity receptor FcεRII (human CD23 antigen) is present on B cells,
platelets, monocytes, eosinophils.
H I G H - A F F I N I T Y R E C E P T O R ( Fc ε R I )
Each FcεRI is composed of three separate transmembrane subunits, comprising four polypeptide
unit, αβ γ2. This receptor has molecular weight ~76 kDa. The α chain is involved in ligand (IgE)
binding and the rest, that is, one β chain and two identical disulphide-linked γ chains, contribute
to signalling. The external amino terminal region of α chain (25 kDa) contains two 90 amino acids
» There are approximately immunoglobulin (Ig)-like domain that forms the binding site for the IgE. The α chain also has a
40,000–90,000 FcεRI molecules on 20 amino acid residue hydrophobic sequence and 20 amino acid C-terminal cytoplasmic tail. The
the surface of human basophils.
FcεRI interacts with CH3/CH3 and CH4/CH4 domain of the IgE via its immunoglobulin (Ig)-like do-
main with very high affinity (KD∼ 12 109M).
The high affinity of this receptor for IgE allows it to bind IgE despite low serum concentration
of IgE (less than 1 μg/ml in serum). The β chain is 26 kDa polypeptide that crosses the membrane
four times and has both n and c terminals inside the cell. It contains a single immunoreceptor
» High-affinity receptors for IgE are tyrosine kinase activation motif (ITAM) in its carboxyl terminal. The two γ chains (7 kDa each)
expressed not only on mast cells
and basophils but also on are disulphide-linked and each chain has five amino acid n terminal extracellular region, a trans-
Langerhans cells and eosinophils. membrane segment and a c terminal that extends a considerable distance into the cytoplasm.
The cytoplasmic portion of each γ chain
NH2 contains one ITAM. The γ chain of FcεRI serves
» FcεRI α-chain knockout mice have as a common subunit for FcγRI, FcαR and
s
normal levels of mast cell but do
s F γRIIIA, and is sometimes referred to as FcεRγ
not develop anaphylaxis. 90 amino Binds CH3 and CH4 c
acids domains of IgE
chain. The cross-linking of FcεRI by invading
s allergen results in the aggregation of FcεRI
s receptors and tyrosine phosphorylation of the
N N
s-s ITAMs of the β and γ chains. FcεRI surface
20 amino expression on mast cells and basophils is up-
» Low-affinity receptors for IgE are acids regulated by binding IgE, thus amplifying IgE-
expressed on B cells, eosinophils, mediated effector functions in allergic reactions.
macrophages, monocytes and 20 amino COOH N B Cytosol
C C C
platelets. acids A chain L O W - A F F I N I T Y R E C E P T O R ( FC ε R I I )
chain ITAM G chains FcεRII is a single, glycosylated polypeptide
Fc E RI chain of 321 amino acid residues having a
High-affinity receptor
total molecular weight of 45 kDa. It comprises
a 274-residue c-terminal extracellular do-
main, a 23-residue n-terminal cytoplasmic
domain and a short 24-residue transmem-
brane segment. It has much lower affinity for
s IgE (KD = 1 106 M) than FcεRI. Recently,
s two isoforms of FcεRII have been detected—
ss FcεRIIA and FcεRIIB. FcεRIIA form is
s
COOH B-cell specific. The expression of FcεRIIB or
Proteolytic CD23 is induced on monocytes, eosinophils
274 residues
cleavage site and B cells by IL-4. The cross-linking of this
Extracellular
domain IgE receptor has been shown to activate
B cells, macrophages and eosinophils.
24 amino acids
FcεRII receptors are present on B cells,
eosinophils, macrophages and platelets. It is
23 amino acids Cytosol
suggested that the cross-linking of low-affinity
NH2 Fc receptors triggers the production of free
Fc E RII
radicals which provides an effective defence
Figure 13.4 against pathogens. A schematic representation
Schematic diagrams of FCεRI and FCεRII
Low-affinity receptor of FCεRI and FCεRII is shown in Figure 13.4.
receptors for IgE.
13.2.4 A C T I VAT I O N O F M A S T C E L L S
AND BASOPHILS
« In individuals manifesting im-
The first step in the initiation of immediate hypersensitivity is the binding of allergen to IgE bound
mediate hypersensitivity against
on the surface of basophils and mast cells. The binding of multivalent allergen to IgE causes an a particular antigen, a significant
aggregation of IgE and the resulting cross-linking of FCεRI molecules. This induces degranulation number of IgE molecules bound
of mast cells and basophils. to mast cells are specific for that
antigen. Exposure to allergen will
If the observation that aggregation induces degranulation is true, then any biomolecule or cross-link sufficient IgE molecules
chemical that can cross-link the adjacent IgE bound on mast cell surface should trigger mast-cell and trigger mast-cell activation. In
degranulation. This is indeed the case as mast cells can be triggered by: contrast, in non-allergic individu-
als, IgE molecules associated with
• anti-IgE antibodies, which can cross-link IgE molecules regardless of antigen specificity, mast cells and basophils are specific
for many different antigens and so
and trigger mast cells in allergic and non-allergic individuals; IgE of the same specificity will be
• cross-linking of IgE receptor by anti-receptor antibodies; and located far from each other. Thus,
• chemical cross-linkage of IgE. no single antigen will cross-link
enough IgE molecules to cause
Moreover, the importance of receptor cross-linking is also clear from the inability of monova- mast-cell or basophil activation.
lent allergens to induce cross-linking of IgE (hence its receptor) to trigger degranulation.
The process of activation of mast cells (and basophils) involves three main types of biological
responses in them:
• secretion of the contents stored in granules inside the mast cells and basophils;
• synthesis and release of lipid mediators such as prostaglandins and leukotrienes from
mast cells; and
• synthesis and secretion of cytokines.
« Certain food additives such as
E V E N T S L E A D I N G T O M A S T - C E L L A C T I VAT I O N aspartame, colouring agents (azo

dye, tartrazine) and preservatives
The cross-linking of FcεRI results in the activation of protein tyrosine kinases (PTKs) associated also elicit symptoms of allergic reac-
with cytosolic domain of β and γ chains of FcεRI. These PTKs (Syk and Fyn PTKs) phosphorylate tions possibly by inducing
β and γ chains and phospholipase C (phosphatidylinositol-specific phospholipase C). These phos- histamine release. These reactions
are not mediated by IgE and are
phorylation events, through a series of reactions, lead to the production of a number of second said to be pseudo-allergic reactions.
messengers. Within a few seconds of cross-linking of IgE, phosphatidylethanolamine of lipid cell
membrane of mast cell is methylated to phosphatidylcholine (by phospholipid methyl transferase
« Drugs such as cromolyn sodium
I and II, PMT I and II) located in the membrane. The increase in phosphatidylcholine content of
(also known as cromoglycate diso-
the membrane causes increase in fluidity that favours the opening of Ca2+ channels (such as membrane- dium) that block the inflow of Ca2+,
bound calcium-release-activated calcium channels). There is a sudden increase in cytosolic Ca2+ also block mast cell degranulation.
due to both uptake of extracellular Ca2+ and release of Ca2+ from stores in the endoplasmic reticulum.
The increase in Ca2+ activates phosholipase A2 which promotes the breakdown of phospha-
tidylcholine into arachidonic acid and lysophosphatidylcholine. The arachidonic acid formed is
converted into two potent lipid mediators—prostaglandins and leukotrienes.
The increase in cytosolic Ca2+ also activates protein kinase C, which causes the disassembly of
the actin–myosin network beneath the plasma membrane, allowing granules and plasma to come
together and fuse. The fusion of vesicles with the plasma membrane is guided by proteins pres-
ent on both vesicles and plasma membrane (such as Rab 3b). The fusion of granules/vesicles with
plasma membrane results in the exocytosis of mediators stored in the granules.
The cross-linking of the IgE receptor also activates adenylate cyclase activity. This results in
a transient increase in cAMP. The increase in cAMP activates cAMP-dependent protein kinase » When cAMP levels are increased
which (also) phosphorylates the granule’s membrane proteins. The increase in cAMP is transient by certain drugs such as theophy-
line, the degranulation process is
and is followed by a sharp decrease in cAMP. It is believed that this decline in cAMP induces de- blocked.
granulation. The details of mast-cell activation are shown in Figure 13.5.
13.2.5 B I O L O G I C A L M E D I AT O R S
OF TYPE I REACTIONS
The clinical manifestations of type I hypersensitive reactions are actually the biological effects of
mediators released by mast-cell or basophil degranulation. It is not necessary that all mast cells and
basophils release the same range of mediators. These mediators are pharmacologically active agents
that act on local tissue or population of cells. These mediators act as a double-edged sword. When
generated in response to parasitic invasion, these mediators initiate a beneficial defence process.
Antigen cross-links IgE
PMT Fc E RI
Ca2+
LPC PC s-s s-s
PIP2
D
Adenylate A
Cyclase G
PLA2 PE
ATP cAMP lyn syk
Ca2+ inflow ITAMs P P
(Transient increase)
PLCG P IP3
Figure 13.5
Line diagram showing the biochemical
Protein
events of mast-cell activation. The
Activated kinase C
cross-linking of bound IgE by allergen Arachidonic Protein
protein P
activates protein tyrosine kinase syk acid kinase C P P

kinase C
and lyn and adenylate cyclase. Protein ER Ca2+
kinases, through a series of reactions,
activate phospholipase C γ (PLCγ) Lipooxygenase Cyclo-oxygenase Histamine
and phospholipase A2. PLCγ causes pathway pathway
the realease of IP3 from phosphatidyl
diphosphate. PLA2 releases arachidonic
acid from membrane PC. This arachidonic
Granule
acid gives rise to lipid mediators.
Stimulated adenylate cyclase causes a Leukotrienes
Prostaglandin
transient increase of cAMP. cAMP and LTC4,LTD4,
D2 Actin and myosin
IP3 activate cytokine gene expression LTE2
and IP3 causes degranulation via a depolymerization
cascade of reactions (PE—phosphatidyl
ethanolamine; PC—phosphatidyl
choline; PMT—phospholipid methyl
Rab 3b
transferase; LT—leukotrienes; LPC—
lysophosphatidylcholine, DAG—diacyl
glycerol; ER—endoplasmic reticulum; Secretion Granule exocytosis
ITAM—immunoreceptor tyrosine-based
activation motifs). Histamine
Allergen Vasodilation and increased vascular permeability

results from the release of mediators and brings
IgE an influx of plasma and cellular warriors to attack
the pathogen. If, on the other hand, mediators are
Degranulating
mast cell released at an “inappropriate” time by allergen,
they start an unnecessary increase in vascular per-
meability and inflammation, resulting in damage
to naïve tissue and organs.
The mediators are of two types—primary (or
pre-formed) or secondary (or newly synthesized)
mediators. The primary mediators are produced
before degranulation and are stored in the gran-
ules. These include histamines, arylsulphatase,
serine proteases, heparin and eosinophil che-
Secretion Synthesis motactic factor. The secondary mediators are ei-
of granules and release ther synthesized during degranulation or during
» Primary mediators are stored
(within seconds) of cytokines
pre-formed, secondary mediators mast-cell/basophil activation but are never stored
are newly synthesized. * Histamine * TNF-α pre-formed. The secondary mediators include
* Serotonin * IL-1 leukotrienes, prostaglandins, bradykinins and
* Cathepsin Synthesis * IL-3
G and release various cytokines. The time course of release of
* IL-4
of lipid * CSF biological mediators from mast cells is depicted in
mediators Figure 13.6. Table 13.3 provides a brief overview of
Figure 13.6
Time course and biological response of (within minutes) important primary and secondary mediators and
mast cell mediators. Schematic diagram
* Prostaglandins their main biological effects.
showing biosynthesis of PGD4, PAF, LTE4
* Leukotrienes
and LTC4.
Mediators Effects
Primary
Histamine Smooth muscle contraction, increased vascular permeability, increased peristalsis
Serotonin Smooth muscle contraction, increased vascular permeability, vasospasm
Neutrophil Chemotactic Neutrophil chemotaxis
Factor-A (NCF-A)
Eosinophil Chemotactic Eosinophil chemotaxis
Factor-A (ECF-A)
Heparin Anticoagulant
Serine proteases Degradation of blood vessel basement membrane; generation of fragments of
complement components
Secondary
Leukotrienes Broncho-constriction, increased vascular permeability
Prostaglandins Vasodilation, broncho-constriction, platelet aggregation
Platelet-activating factor Platelet aggregation, broncho-constriction, retraction of endothelial cells
Bradykinin Smooth muscle contraction, increased vascular permeability
Cytokines (IL-1, IL-3, TNF-α) Variety of effects on immune system, increased expression of adhesion molecules Table 13.3
Primary and secondary mediators
involved in type I hypersensitivity.
P R I M A R Y M E D I AT O R S
« Histamine was discovered by a
Histamine is one of the most important primary mediators of type I hypersensitivity. It is a basic
British chemist, Henry H. Dale
amine produced from the amino acid histidine (and hence the name). Histamine acts by binding
to target cell receptor (for example H1, H2, H3) expressed on different cell types. Upon binding Histamine
to cellular receptors (for example H1), histamine induces constriction of intestinal and bronchial Histamine is the primary mediator
smooth muscles, increased peristalsis, increased permeability of venules and increased nasal secre- produced from histidine. Most of
the histamine formed in the body
tion. It can also inhibit migration and chemotaxis of eosinophils. The binding of histamine to H2 is stored in the granules of mast
receptor present on mast cells has a different effect. It suppresses mast cell degranulation and aug- cells or basophils. Histamine exerts
ments eosinophil migration and chemokinesis. its action by binding to histamine
receptors. There are four types of
Other primary mediators that are exocytosed by mast cells and basophils include heparin, histamine receptors H1–H4, of which
serotonin, carboxypeptidase A, cathepsin G, ECF-A, NCF-A and neutral serine proteases. H1 is primarily involved in allergic
reactions.
S E C O N D A R Y M E D I AT O R S
Leukotrienes, prostaglandins (PGD2) and platelet-activation factor (PAF) are important secondary « Lipid mediators are usually
mediators that are synthesized and released by activated mast cells and basophils. secondary mediators.
In general, the synthesis of prostaglandins is initiated by the action of phospholipase A2 which
releases arachidonic acid from the precursor membrane or lipid body. An ensuing enzymatic cas-
cade converts it into PGD2 by the cyclooxygenase pathway. Basophils do not produce PGD2 in
large quantities. Prostaglandins released from mast cells bind to the receptors on smooth muscle
cells and act as a vasodilator and broncho-constrictor. Leukotriene is another lipid mediator de-
rived from arachidonic acid. It is produced by lipooxygenase pathway in both mast cells and ba-
sophils. Leukotrienes bind to receptors on smooth muscle cells that are different from receptors
for PGD2 and cause prolonged broncho-constriction. Platelet-activation factor (PAF) is the third « Being active at nanomolar levels,
type of lipid mediator. leukotrienes are a thousand times
PAF is found in mast cells and basophils. It is synthesized by the acylation of lyso-glyceryl more potent than histamine.
ether phosphorylcholine which in turn is derived from phospholipase-mediated hydrolysis of
membrane phospholipids. PAF has a strong broncho-constricting action. It causes the retraction « Platelet-activation factor derives
of endothelial cells and relaxes vascular smooth muscles. The effects of secondary mediators PGD2, its name from its original bioassay
LTC4 and PAF are longer lasting than those of histamines. procedure where it was used
as an inducer of rabbit platelet
Histamine is removed from the extracellular milieu by the action of amine-specific transport aggregation.
system or by the action of the enzyme histaminase while PAF is rapidly destroyed by the plasma
enzyme PAF-hydrolase thereby limiting their strong action.
CYTOKINES
Activated human mast cells secrete a variety of cytokines that alter the microenvironment and help
in recruiting inflammatory cells such as eosinophils and neutrophils. The cytokines secreted by
these cells include TNF-α, IL-1, IL-3, IL-4, IL-5, IL-6 and colony-stimulating factors (CSF). IL-4
increases IgE production while IL-5 is important in the recruitment and activation of eosinophils.
Cytokines, which are peptides, are transcribed and synthesized after mast-cell activation. TNF-α
may be stored in granules and released after mast-cell activation. TNF-α activates endothelial
expression of the adhesion molecules that accounts for the binding of polymorphonuclear and
mononuclear cells, followed by infiltration of adhered cells at the inflamed site. The important
events of type I hypersensitivity are summarized in Figure 13.7.
13.2.6 CLINICAL CONSEQUENCES OF TYPE I

HYPERSENSITIVITY
Clinical consequences of type I hypersensitivity response vary greatly in severity and character,
from life-threatening conditions such as systemic anaphylaxis, to asthma and common forms of
allergic reactions such as hay fever and eczema.
F E AT U R E S O F A N A P H Y L A X I S
Anaphylaxis (Greek: ana—away from; phylaxis—Protection) is an immediate hypersensitivity re-
action that is inducible in a normal individual of any species upon appropriate exposure to allergen.
» Hypersensitivity can also be a non-
The response could be systemic (anaphylactic shock) or local but in all cases characterized prima-
immunological phenomenon when
it does not involve the immune rily by smooth muscle contraction and increased capillary permeability. Systemic anaphylactic re-
system. Indigestion/diarrhoea action is a shock-like and often fatal state, induced within minutes of type I hypersensitive reaction.
triggered by lactose (sugar present Systemic anaphylaxis can be induced in a variety of experimental animals and is occasionally mani-
in milk)-intolerance in humans is
an example of non-immunological fested in humans. Each species exhibits characteristic symptoms and has different target organs: for
hypersensitivity. example lungs (in humans and guinea pigs), heart (in rabbits) and liver (in dogs).
Allergen
Outside
Inside the host body
Allergen
Histamine
Serotonin
Serine Hay
Resting B cell protease fever
Heparin
Anaphylaxis
Prostaglandins
IL-4,IL-10, Leukotrienes
Activation,
IL-13 Platelet- Asthma
Synthesis
(from TH2 Binds activating
of IgE
cells) factor Eczema
TNF,IL-2,
IgE IL-3,IL-5, Food
IL-6,CSF allergies
Mast cell
degranulating Mediators
Clinical
Plasma cell effects
Figure 13.7
An overview of type I reaction.
Anaphylaxis can be induced in guinea pigs with ease and the symptoms closely parallel those
in humans. The immediate hypersensitivity response in the guinea pig involves convulsion, itch-
ing, sneezing, urination, defecation and death within minutes due to severe broncho-constriction,
smooth muscle contraction and trapping of air in the lungs. Systemic anaphylaxis in humans is
characterized by a similar sequence of events. Allergens that are known to trigger anaphylaxis in
humans are venom from bee, hornet, wasp and ant, drugs such as penicillin and insulin, foods such
as eggs, nuts, chocolate and seafood. If not treated at the appropriate time, these reactions can be
fatal. The mainstay treatment is systemic epinephrine administration. Epinephrine can be life-saving
as it counteracts the effects of mediators such as histamine and leukotrienes by relaxing the smooth
muscles and reducing vascular permeability. Epinephrine also improves cardiac output and
increases cAMP level in the mast cells, thereby blocking further degranulation. « Atopy occurs only in some
genetically pre-determined
F E AT U R E S O F AT O P Y individuals. Since this allergic
reaction has a genetic overtone, it
Atopy is an immediate hypersensitivity response that occurs only in genetically predisposed indi- was out of place with other known
viduals upon sensitization to a specific allergen. allergic reactions such as serum
Unlike anaphylaxis, atopic reactions are limited to a specific target tissue. Moreover, this con- sickness and systemic anaphylaxis,
and hence was called atopy (Greek:
dition differs from anaphylaxis in that it cannot be induced in normal hosts. atopos—out of place) to suggest it
Atopic reactions which are IgE-mediated include bronchial asthma, allergic rhinitis (hay might be genetically controlled.
fever), urticaria (hives), atopic dermatitis (eczema) and food allergies. Recent studies have shown
« Genes located on the short arm of
clear autosomal transmission of atopy within the family. Various population-based studies using a chromosome are given the suffix
genetic mapping and positional cloning techniques have identified several genes or loci involved p. The letter p is derived from the
in allergy (atopy). These include loci on chromosomes 5q, 11q, 12q and 14q. French word petite meaning short.
If genes are found on the long arm,
the suffix q is attached. The letter
A L L E R G I C R H I N I T I S ( H AY F E V E R ) q was chosen to signify the long
Allergic rhinitis or hay fever is the most common atopic disorder, affecting 10–20 per cent of the US arm simply because it was the next
letter after p.
population. It results from the reaction of inhaled airborne allergens such as plant pollens or dust
mites, fungal spores and animal danders, with sensitized mast cells present in the upper respiratory « About 35 million Americans suffer
tract (conjunctivae and nasal mucosa). The binding of allergen to these cells induces the release from hayfever every spring!
of pharmacologically active mediators from mast cells. These mediators cause mucus secretions,
localized vasodilation and increased capillary permeability. The symptoms of hay fever include
mucosal oedema, watery exudation of conjunctivae, itching and tears in the eyes, as well as sneez-
ing, coughing and difficulty in breathing.
ASTHMA
The patho-physiological sequence of allergic asthma is initiated by mast-cell activation in re-
sponse to the binding of allergen to IgE localized on mast cell surface. The allergens may be
blood-borne (viral antigens) or airborne such as dust, fumes and pollen. Degranulation of mast
cells results in the release of mediators and cytokines. Mast-cell-derived mediators induce smooth
muscle hyperactivity resulting in broncho-constriction. Airway oedema, constriction of airway
and smooth muscles, mucus secretion, and all contribute to bronchial constriction which leads to
shortness of breath. The most important of the broncho-constricting mediators is leukotriene C4
(LTC4 ), and its breakdown-products LTD4, LTE4. Recent evidence also suggests that PAF can be
a mediator of asthma. The release of mediators occurs within minutes of allergen exposure and
hence is termed as early response. Late response occurs hours later and involves cytokines such
as IL-4, IL-5, IL-16, TNF-α and PAF. The major effect of these mediators is the recruitment of
eosinophils, basophils, neutrophils and TH2 cells into bronchial tissue. Neutrophils, eosinophils
and basophils are capable of causing significant tissue injury by releasing toxic enzymes, reactive
oxygen species and cytokines. These events lead to the blocking of bronchial lumen with mucus,
proteins and cellular debris, hypertrophy of bronchial smooth muscles and fluid accumulation
or oedema. All these result in bronchial constriction and increased production of thick mucous,
which in turn leads to bronchial obstruction and exacerbates respiratory difficulties. Histamines
have little role in airway constriction and antihistamines (H1 receptor antagonists) have no role
in the treatment of asthma. Bronchodilators which relax the bronchial muscles, expectorants
which promote the dissolution and discharge of mucous that gets accumulated in alveoli and
« Asthma affects about 10 million
anti-inflammation agents such as steroids which reduce inflammation of the airways are helpful people in the USA and the numbers
in providing symptomatic relief for asthma. are increasing.
FOOD ALLERGIES
In an allergic individual, a variety of food items can induce localized immediate hypersensitivity
reactions. Allergen cross-linking of IgE present on intestinal mucosal and submucosal mast cells
» It is estimated that more than leads to release of mediators that induce enhanced peristalsis, increased fluid secretion from intes-
10 million people have food allergy tinal lining cells, vasodilation and often vomiting and diarrhoea. Allergens might enter the blood
in USA alone.
stream from the gut and, depending on the fate of allergens, various symptoms can ensue.
Some allergic individuals develop urticaria (hives), which may take hours to subside, others
might develop wheal and flare reaction on the skin, a reflection of the inflammatory events of the
body. Wheal and flare response usually subsides after about 15 to 20 minutes. Allergic reactions
can develop to varied types of food. Some of the most common food items that contain allergens
include eggs, peanuts, milk and shellfish. Antihistamines relieve the symptoms of hayfever and
food allergies by blocking the vasoactive action of histamine released from mast cells.
AT O P I C D E R M AT I T I S ( E C Z E M A )
Atopic dermatitis or eczema is an inflammatory disease of the skin that is characterized by skin
» Atopic dermatitis is less common,
affecting 1–2 per cent of the total eruptions that are erythematous and filled with pus. The term eczema is a generic term for all kinds
American population and is associ- of blistering, scaly brownish itching conditions of the skin.
ated with an individual having a Atopic dermatitis is usually manifested during childhood (as infantile eczema) though it can
family history of atopy.
also occur in adult population. The patients usually have high serum IgE level, increased number of
eosinophils and TH2 cells in skin lesion. Atopic dermatitis is often treated with topical corticosteroids.
13.2.7 L AT E - P H A S E R E A C T I O N
In type I hypersensitive reaction, as its symptoms begin to subside, there is onset of late-phase re-
actions. This is “second” round of localized inflammatory reaction induced by mediators released
during the initial phase of inflammatory reaction. This late-phase reaction begins to develop four to
six hours after the initial type I reaction and continues for a couple of days. The localized late-phase
reaction is mediated partly by cytokines and partly by PAF released from mast cells and basophils.
These cytokines summon neutrophils, eosinophils, monocytes, macrophages, lymphocytes and ba-
sophils, that characterize the late-phase response.
Eosinophil chemotactic factor released by mast cells during the early phase of hypersensitive
reaction attracts a large number of eosinophils to the target site. These eosinophils undergo growth
and differentiation at the site due to the presence of IL-3, IL-5 and GM-CSF. Eosinophils express Fc
receptors for IgG and IgE and hence can bind antibody-coated allergen. The binding of antibody-
coated allergens to eosinophils leads to their degranulation (like mast cells), and the release of
inflammatory mediators such as lysosomal hydrolase, major basic protein, PAF and leukotrienes.
The eosinophil-derived proteins are toxic to parasites, bacteria, etc. However, when produced in
response to allergen, these mediators elicit extensive tissue damage in the late-phase reaction.
Neutrophil is another major cell involved in the late-phase reaction, accounting for 30 per
cent of the accumulated inflammatory cells. Neutrophils are attracted by neutrophil chemotactic
factor released from mast cells and basophils during the early phase of type I reaction. These cells
are then activated by a variety of cytokines present at the target site, resulting in degranulation-
releasing lysosomal enzymes, leukotrienes, PAF and consequent tissue damage.
PAF is also important in late-phase reactions where it can activate inflammatory cells. In the
late-phase reaction, a major source of PAF can be basophils or vascular endothelial cells rather
than mast cells. A summary of late-phase events is given in Figure 13.8.
13.2.8 TESTS FOR DIAGNOSIS OF TYPE I

HYPERSENSITIVITY
SKIN TEST
The most common method for identifying and assessing type I hypersensitivity is skin testing.
A small amount of the potential allergen is introduced at the specific skin site either by intradermal in-
jection (0.02–0.03 ml by 25 gauge needle) or by a lancet or by superficial scratching. A test or tests are
usually applied to sites on the forearm or at the back. If the person is sensitive to the antigen, there is
rapid appearance of a raised red area, or wheal and flare reaction. The wheal is caused by extravasation
of serum from capillaries into the skin which results from the direct effect of histamine released by
local activated mast cells. The large red flare is mediated by axon reflex. This skin response takes about
Allergen cross-links IgE
Mast cell
Degranulation
(within secs,mins)
Release of histamine, serotonin, Early or

thromboxanes, prostaglandins, immediate
leukotrienes reaction
Release of cytokines and Late-

platelet-activation factor phase
response
IL-1,IL-4,TNF, Neutrophil Eosinophil

IL-6,PAF chemotactic chemotactic factor
factor,IL-8 IL-3,IL-4,IL-5
B cell T cell Neutrophil Eosinophil
Degranulation Degranulation
Macrophage Basophil
Antibody production, Release of lysosomal Release of major basic

phagocytosis, TH2 enzymes, leukotrienes, proteins,lysosomal enzymes,
cytokine production PAF and phagocytosis leukotrienes,PAF
Figure 13.8
Late-phase reaction of type I
hypersensitivity. The platelet-activating
factor (PAF) causes platelet aggregation
and the release of arachidonic acid,
Tissue damage Tissue damage Tissue damage augmenting mast cell effects.
in 5–20 minutes and may persist for 30 minutes or more. Skin tests are evaluated by the size of the
wheal compared to histamine (positive control) and saline (negative control). In general, a 3 3 mm
wheal in children and a 4 4 mm wheal in adults is considered positive for the allergen. The positive
aspect of this testing is that it is inexpensive and allows a large number of allergens to be screened at
the same time. The disadvantage includes inadvertent sensitization of the individual with a new anti-
gen and, rarely, an anaphylactic shock.
» Not all individuals who show posi- PAT C H T E S T

tive skin tests will always develop This test is carried out to check whether the patient has atopic dermatitis or eczema and hence is
allergy. In fact studies indicate that
one-third of skin-test-positive pa- sometimes referred to as eczematous patch test. In the patch test ~10–12 mg of allergen is applied
tients do not experience symptoms on a gauge pad of 2.5 cm3. The patch of allergen stays on the skin for two days and a biopsy of skin
even when they are exposed to cells is carried out at 24 or 48 hours. A positive patch response induces macroscopic eczema, spon-
allergens in their lifetime.
giosis of the epidermis and infiltration of eosinophils, basophils and lymphocytes into the dermis.
A biopsy of patch tests also sometimes detects T cells specific for the allergen.
RADIOIMMUNOSORBENT TEST (RIST)

This test assesses the total IgE antibody of the patient. The principle of RIST is similar to radioim-
munoassay and is equally sensitive in detecting up to nanogram levels of total IgE. In this method,
a patient’s serum (which contains IgE) is mixed with agarose beads precoated with anti-IgE. The
beads are then washed to remove non-specific binding of IgE. After washing, I125-labelled rabbit
anti-IgE is added and radioactivity on the beads is measured by a gamma counter. The radioactivity
on the beads is directly proportional to the level of IgE in the patient’s plasma.
RADIOALLERGOSORBENT TEST (RAST)

RAST is similar to, but more specific, than RIST. In RAST, the serum level of IgE specific to an
allergen is quantitated as compared to the total serum IgE content in the RIST method. In this
method, the allergen is coupled with agarose beads. These allergen-coated beads are then incubated
with a patient’s serum which contains IgE. Allergen binds specific IgE and unbound antibody is
washed away. Bound specific IgE is then measured by adding I125-labelled anti-IgE and incubat-
ing it with the beads. The IgE-specific bound antibodies are then quantified by counting bound
radioactivity.
13.2.9 THERAPEUTIC MEASURES FOR TYPE I

HYPERSENSITIVITY
The avoidance of responsible allergen is the easiest way to manage allergic disease. This can be
» The term desensitization, though accomplished quite easily with some allergies such as food allergies, pet dander allergies (removal
still prevalent, is actually a misno-
mer. It indicates the removal of of-
of house pets) but is physical impossibility with inhalant allergens. So another approach, called desen-
fending antibodies (sensitization is sitization (or hypo-sensitization) is followed. During desensitization, a gradually increasing quan-
akin to immunization with allergen) tity of allergen is administered subcutaneously over a period of time, extending over weeks or
while it actually is neutralization
of allergen before it can induce an
months.
allergic reaction. This form of immunotherapy is aimed at stimulating IgG synthesis together with decreasing
specific IgE levels. The individual is repeatedly exposed to a gradually increasing dosage of aller-
gens. The repeated introduction of allergen may induce T-cell-mediated suppression that turns
off the IgE response. The offending IgE concentration in serum decreases. The IgG formed against
allergen competes for allergen, binds the allergen and forms a complex that can easily be removed
by phagocytosis. The allergen is hence not available for binding to IgE on the mast cell membrane
and hence the allergic response subsides.
» Daniel Bovet , a Swiss physiologist The unravelling of the entire mechanism of type I hypersensitive reaction has opened up ways
and pharmacologist, was awarded to use drug therapy against allergy. Drug treatment involves the administration of chemical agents
the Nobel Prize in 1957 for the
development of antihistamines in designed to stop or reverse various allergic mechanisms (see Figure 13.9). Antihistamines such
the treatment of allergy. as Allegra act by binding to histamine receptors present on various cells, blocking the binding of
histamine. H1 receptor of histamine is usually targeted, though H2 receptors are also blocked by
newer class of antihistamines. Antihistamines are useful drugs for a variety of allergic reactions,
including hay fever.
Since all pathological reactions associated with type I hypersensitivity are brought about by
pharmacologically active mediators and cytokines, blocking the release of allergic mediators by
interfering with mast-cell activation and degranulation was another therapeutic approach. Some
of the therapeutic approaches are given below:
• Inhibiting influx of Ca2+ into mast cells by sodium cromolyn (to prevent degranulation of
mast cells);
• Administration of corticosteroids which blocks the production of inflammatory cytokines;
Allergen
FcεRI
H1 receptor
Histamines Mast cell
Stablization of membrane by
Binding to
sodium cromolyn
histamine
receptor Stimulating cAMP
blocked Antihistamines
Corticosteroid synthesis by epinephrine
(inhibition of degranulation)
Figure 13.9
Production of inflammatory Therapeutic measures of type I
cytokines (IL-4,IL-5) inhibited hypersensitivity.
Therapeutic Agent Activity

Antihistamines (e.g., Allegra) Blocks H1 and H2 receptors on target cells, inhibits smooth muscle
contraction
Cromolyn sodium Prevents Ca2+ influx into mast cells, prevents mast-cell
degranulation
Corticosteroids (e.g., prednisone) Potent immunosuppressive activity, blocks production of inflam-
matory cytokines
β-agonist (e.g., epinephrine) Inhibits degranulation, relaxes smooth muscles, stimulates
adenylate cyclase, decreases vascular permeability
Theophylline Relaxes smooth muscles of bronchi, inhibits cAMP breakdown by Table 13.4
inhibiting phosphodiesterase, inhibits degranulation of mast cells Therapeutic agents used in the
treatment of type I hypersensitivity
Ziluton Inhibits broncho-constriction, inhibits formation of leukotrienes reaction.
• Stimulating adenylate cyclase, an enzyme that converts adeneosine triphosphate (ATP) to

cAMP (High cAMP inhibits degranulation. This is achieved by administering epinephrine
and related β2-adrenergic agents that stimulate cAMP synthesis.); and
• Inhibiting cAMP breakdown by inhibiting phosphodiesterase enzyme by drugs such as
theophylline.
Though several drugs such as PAF receptor antagonists and inhibitors of leukocyte adhesion such
as antibodies to V-CAM-1 are under clinical trial, it remains to be seen whether these agents are ef-
fective in treating allergic reactions. Table 13.4 lists some of the important therapeutic agents used
in the treatment of type I hypersensitivity reaction.
13.3 T Y P E I I H Y P E R S E N S I T I V I T Y:
ANTIBODY-DEPENDENT CYTOTOXIC
HYPERSENSITIVITY
Type II hypersensitivity reactions are those allergic reactions that are brought about by antibody
reacting with antigen present on the surface of the cell. Antibody binds the antigen via its Fab arms
leaving its Fc tail free to bind complement components, NK cells or phagocytes. The binding of
complement components to antibodies attached to target cells results in the formation of pores
and finally cell lysis. NK cells with their Fc receptors can also bind antibody-coated cells and kill
the cells by antibody-dependent cell-mediated cytotoxicity. Antibody bound to cells can also act as
opsonins, enabling phagocytic cells to bind and phagocytose antibody-bearing cells.
Examples of type II hypersensitivity include drug allergies, transfusion reaction haemolytic

anaemia, erythroblastosis foetalis, Goodpasture’s syndrome and several autoimmune diseases. The
three common examples are discussed here.
13.3.1 DRUG-INDUCED HYPERSENSITIVITY

REACTION
Drugs (or their metabolites) can induce hypersensitivity reaction against blood cells including red
blood cells and platelets.
» Drug-induced blood cell lysis Drug allergies (now synonymous with drug-induced haemolytic anemia) can follow (in cer-
was first reported by J. F. Akroyd tain individuals) after administration of a wide variety of drugs such as penicillin, quinine, and
in 1964, who noted that the drug
Sedormid induced destruction streptomycin. The drugs adsorb non-specifically to red blood cell membrane proteins causing
of platelets and development of them to appear “foreign” to the host immune system. In some cases, these drug adsorbed on red
purple rash, a condition called blood cell membrane proteins (which form neoantigen) induce the formation of antibodies which
thrombocytopenic purpura.
bind to these neoantigens on red blood cells. This binding of antibodies on red blood cells induces
haemolysis by activating the complement system, antibody-dependent cell-mediated cytoxicity
» Penicillin is one drug that can
mediate all four types of hypersen- and phagocytosis of red blood cells (see Figure 13.10). This red blood cell destruction or haemoly-
sitivities. sis causes the onset of anaemia. When the drug is withdrawn, the haemolytic anaemia disappears.
13.3.2 TRANSFUSION REACTIONS

ABO blood groups form the dominant blood group system in humans. The major blood group an-
tigens A and B are expressed on the surface of human red blood cells. The antigenic groups A and B
are derived from the H substance by action of glycosyl transferases encoded by A or B genes respectively.
Individuals with both genes have both antigens on the surface (group AB) and those lacking both
genes synthesize only H substance (group O). Alternatively, red blood cells may carry A or B antigen
depending on which single gene is present. Individuals who exhibit blood group A have antibod-
ies (in serum) to B antigens, those who exhibit blood group B will have anti-A antibodies and who
exhibit AB group type will have neither. Those individuals who have blood group O will have both
types of antibodies. The anti-blood-group antigen antibodies are called isohaemagglutinins.
They are usually of IgM type. Isoheamagglutinins antibodies are thought to arise through
Isohaemagglutinins
Those substances that cause
immunization against antigens of the gut flora which are similar to blood group substances; for
agglutination of red blood cells example, an individual usually has gut microorganism that carries antigens similar to A or B anti-
are termed as isoheamagglutinins. gens. An individual with blood group A will recognize B group antigenic determinants present on
The first report of the existence of
isoheamaglglutinin was made by
microorganisms and form antibodies against it. The same host will recognize microbe antigen A
Karl Landsteiner in 1901. Examples as self and will be tolerant towards it. Hence, a person with blood group A will have anti B isohae-
include anti-blood-group-antigen magglutinins in his plasma. If a type A individual is accidentally transfused with blood containing
antibodies normally found in
B cells a transfusion reaction occurs in which anti-B isohaemoglutinin present in type A individual
human plasma. Now the term
isohaemagglutinins also includes will react with B+ blood cells and induce a massive destruction of red blood cells by complement-
lectins. mediated lysis, a life-threatening haemolysis (see Figure 13.11).
ABO blood group antigens are strong immunogens and induce antibody synthesis of IgM
type. Transfusion reactions with ABO blood group incompatibilities begin immediately and
involve complement-mediated cell lysis triggered by IgM isohaemagglutinins. Red blood cells are
Opsonization and
phagocytosis
Neoantigen
Surface Hypersensitivity
+
Response Antigen-antibody
Figure 13.10 Adsorption
(Antibodies formed) reaction
Line diagram showing various steps Red blood Drug
in type II reactions. Drug-induced cells Neoantigens
reaction to blood cells. Drugs adsorb
formed
non-specifically on the surface of the Complement-mediated
cells causing them to appear foreign cell lysis ADCC mediated
to the immune system. The immune
system gets activated and reacts, by NK cells, phagocytes
causing cell lysis. This results in type II
hypersensitivity. Drug-induced type-II hypersensitivity reaction
Antibody- and complement-

mediated donor RBC lysis
by anti-B antibodies
present in the recepient
Donor
blood cells
expressing
antigen-B Figure 13.11
Understanding blood transfusion
Blood reaction. Anti-blood group antigen
transfusion Anti-B antibodies present antibodies arise in an individual
in the recepient because some gut microbes express
Haemolysis similar antigens. Individuals expressing
Donor
Type II hypersensitivity blood group A antigen will have
cells
Recepient reaction anti-B antibodies in their blood. If a
blood group A individual is transfused
(mismatched) with blood expressing B antigen, a
Recipient’s blood cells hypersensitive reaction occurs, resulting
expressing antigen-A in blood cell lysis.
lysed and free haemoglobins are filtered through the kidney resulting in haemoglobinuria. High
concentration of haemoglobin results in high concentration of bilirubin which is again toxic. This
usually results in chills, nausea, clotting within blood vessels, fever and pain in the lower back due « Antibodies that are formed
to massive intravascular haemolysis. The treatment usually recommended for such reactions is the against incompatible red blood
cells are usually of IgG, IgM or IgA
immediate termination of blood transfusion and flushing out accumulated haemoglobin by urine type.
flow with diuretic.
Transfusion reaction with minor blood antigens usually occurs when individuals receive re-
peated transfusion of ABO compatible blood that is incompatible for other blood group antigens.
Such repeated transfusions generate antibodies against blood cells antigens.This is common in
patients that are given repeated blood transfusions, as in pregnancy or sickle cell anaemia. Such
reactions usually develop two to six days (rarely up to 3 months) after transfusion and involve
antibody-mediated haemolysis of red blood cells. Reactions that occur after a delay of several days
are called delayed haemolytic transfusion. These reactions which are less severe than immedi-
ate transfusion reaction may involve agglutination, opsonization, phagocytosis and complement-
mediated lysis. Symptoms of delayed haemolytic transfusion reaction include haemoglobinuria,
low haemoglobin, high bilirubin, jaundice and anaemia.
13.3.3 R H E S U S A N T I G E N I N C O M PAT I B I L I T Y
The Rhesus (Rh) blood group forms the other major antigenic system. The most commonly in-
volved antigen is RhD. It is carried by red blood cells. Children born to RhD- mother and RhD+
fathers may express RhD on their red blood cells. During the first pregnancy with RhD+ foetus, « Karl Landsteiner and Alexender
RhD- mother is not exposed to enough foetal blood cells to activate B-cell response against RhD Wiener discovered the Rh system
in 1940.
antigen. At the birth of the first child, separation of the placenta from the uterine wall occurs, which
releases a large number of red blood cells carrying RhD antigen into the mother. These foetal cells
activate Rh-specific plasma cells and memory cells in the mother.
These antigen-bearing cells are cleared from the mother’s bloodstream by maternal IgM an-
tibodies but memory cells remain in circulation in the mother’s body. Any subsequent pregnancy
with Rh0D+ foetus will induce anti-RhD–IgG which are able to cross the placenta and destroy Rhogam
Rhogam is anti-Rh factor
foetal red blood cells. Jaundice, hepatosplenomegaly and, in untreated infants, bilirubin encepha- gammaglobulin. It is an injectable
lopathy may occur. The consequences of such transfer is the development of mild or severe anae- anti-Rh antibody that is administered
mia characterized by haemolysis in the foetus. This disease is called erythroblastosis foetalis or (to the mother) to protect Rh-
positive foetus from the antibodies
haemolytic disease of the newborn. This disease is not a problem during the first pregnancy but can generated in a Rh-negative mother.
cause a problem in subsequent pregnancies. However, this disease can now be entirely prevented These antibodies neutralize and
by administering prophylactically anti-RhD antibodies to the mother within 72 hours after the first remove any Rh-positive blood cells
that might enter maternal circulation
delivery. These antibodies called rhogam (gamaglobulin against RhD) bind any stray foetal red and immunize the mother. Rhogam
blood cells that enter the mother’s circulation at the time of delivery. was developed by Vincent J. Freda,
Thus the RhD+ red blood cells are opsonized and cleared away from the system before they can a professor at Columbia University in
the 1960s.
encounter any memory cells and sensitize them. In a subsequent pregnancy with an Rh+ foetus,
a mother who has been treated with rhogam is unlikely to produce anti-Rh antibodies and hence
the foetus is protected from damage. However, anti-Rh–IgG is a blocking agent and therefore must
be given after each pregnancy.
Reaction to other blood group antigens such as K antigen (of Kell system) may also cause
erythroblastosis fetalis. However, reactions to K antigen are less common due to weaker antigenic-
ity and relatively low frequency of antigen.
If haemolytic disease caused by Rh incompatibility is detected during pregnancy, the treat-
ment depends on the severity of the reaction. In severe cases, the foetus requires exchange transfu-
sion. The foetus is given intrauterine blood-exchange transfusion to replace Rh+ cells of the foetus
with Rh- cells. These transfusions are given every 10–20 days until delivery. In less severe cases,
blood-exchange transfusion is not given until after birth.
13.4 T Y P E I I I H Y P E R S E N S I T I V I T Y:
I M M U N E C O M P L E X - M E D I AT E D
HYPERSENSITIVITY
» Type III hypersensitivity reaction Type III reaction involves the interaction between soluble antigen and high levels of circulat-
results from immune complex ing antibodies. The immune complexes formed, precipitate and become lodged in fine capillary
deposition in various organs such
as kidneys, joints, and lungs. networks such as those of kidney, liver, joints and lungs (where there is naturally high pressure
in blood).
As the complement system is activated, C3a and C5a are formed. These complement frag-
ments, termed as anaphylatoxins stimulate the release of vasoactive amines from basophils and
mast cells. These chemotactic factors attract neutrophils, eosinophils and basophils to the target
site. Moreover, immune complexes can bind via Fc receptors to basophils mast cells, and platelets
causing the release of vasoactive amines. These vasoactive amines cause the retraction of endo-
thelial cells of blood vessels, exposing the basement membrane. Hence the immune complexes
are deposited on the basement membrane. Since the immune complexes are deposited on the
basement membrane of the blood vessels, the phagocytes are unable to engulf the complexes (be-
cause of steric hinderance) resulting in the release of lysosomal enzymes onto the site of deposi-
tion. The released lysosomal enzymes from the phagocytes as well as the granules released from
mast cells cause tissue damage (see Figure 13.12). Platelets aggregate on the “exposed” basement
membrane of the blood vessels to form microthrombi which results in ischaemia. Examples of
type III hypersensitivity reactions, include Arthus reaction, serum sickness, staphylococcal infec-
tive endocarditis, farmer’s lung, pigeon fancier lung, as well as a number of autoimmune diseases
such as rheumatoid arthritis, systemic lupus erythematosus (SLE), and other persistent infections
(hepatitis, meningitis and malaria).
13.4.1 HOW ARE IMMUNE COMPLEXES REMOVED

IN NORMAL INDIVIDUALS?
The outcome of the formation of immune complexes in vivo depends on the amount of antigens
and antibodies. In a normal host, large aggregates of immune complexes are formed. These large
complexes are rapidly removed by phagocytes and are therefore harmless.
» There are about 700 molecules Once these large immune complexes are formed, the complement system is readily activated.
of CR1 on a single human red These immune complexes are then opsonized by C3b molecules generated by the complement
blood cell. Though CR1 molecules system. These C3b molecules mediate the removal of immune complexes by binding to the C3b
are expressed in thousands on
neutrophils and lymphocytes, their receptor (CR1) present on human red blood cells. (Red blood cells of primates have CR1; non-
contribution to immune complex primates rely on platelet CR1 for the removal of immune complexes.)
clearance is less because of the low- These immune complexes are then towed by red blood cells to the liver and spleen, thus
er number of these cells in blood as
compared to red blood cells. removing them from the plasma. In the sinusoids of the liver, immune complexes are removed
from red blood cells and safely inactivated by fixed tissue macrophage (see Figure 13.13).
Immune complexes are released from red blood cells in the circulation by enzymatic action of
factor I. Factor I does not hydrolyse the bound immune complexes; in fact, it cleaves the recep-
tor C3b which is then dissociated from red blood cells together with bound immune complexes.
Antigen entry
Immune
Immune system activated
system
Antibodies formed
Large antigen-antibody
complex formed
Complement system Complement system

activated summons effector cells
C3a,C5a formed
Basophils Platelets Neutrophils
Vasoactive amine-
mediated endothelial Immune-complex deposition
cell retraction on basement membrane
Basement membrane
Figure 13.12
Neutrophil-mediated damage Line diagram showing type III
Complement-mediated hypersensitivity. This reaction involves an
platelet aggregation interaction between soluble antigen and
Exocytosed enzymes
forming microthrombi antibody. These immune complexes are
damages tissue
(Ischaemia) deposited in the fine capillary network
Enzymes released ensuring hypersensitive reaction. The
damage is mediated by the complement
system and the summoned basophils,
Blood vessel damage neutrophils and platelets.
Immune complexes
are bound by RBC and
towed away from
circulation
Immune Complement
C3b RBC
response system activated
(C3b binds antibody) CR1
(C3b receptor)
Antigen Antibodies Antigen-antibody
entry formed complex formed
Factor I releases
immune complexes
from RBC
Fc receptor
Kupffer
cell RBC
Sinusoids of liver
(Immune complexes bind Fc
receptor and are Figure 13.13
endocytosed by kupffer cells in the liver) Schematic diagram showing transport
of the immune complex by RBCs and
CR1-mediated removal of immune complexes involvement of hepatic macrophages.
These immune complexes are then removed by phagocytic cells (Kupffer cells of the liver)
bearing Fc receptors.
W H AT G O E S W R O N G I N T Y P E I I I H Y P E R S E N S I T I V I T Y R E A C T I O N S ?
First, large quantities of small immune complexes are formed (soluble small immune complexes
could be formed in either antigen-excess or antibody-excess condition.) Due to the sheer magni-
tude of immune complexes formed, the system is overloaded or overwhelmed; and all the immune
complexes cannot be safely towed away from the site of antigen entry. This results in the release
of some free soluble immune complexes in the plasma which get deposited in the tissue very near
the site of antigen entry (in which case a localized reaction develops) or may circulate in the blood
and eventually get deposited in a range of tissues such as kidneys, joints and skin, eliciting tissue-
damaging type-III hypersensitive reactions. When small or soluble immune complexes are formed
in the blood, they frequently get deposited on blood vessel walls (basement membrane), synovial
membrane of joints, glomerular basement membrane as well as choroids plexus of the brain, result-
ing in varying manifestation of type-III reactions.
13.4.2 MECHANISM OF TYPE III HYPERSENSITIVITY

REACTIONS
Much of the tissue damage is the result of immune-complex-induced complement activation.
When immune complexes activate the complement cascade, C3a and C5a are formed. These
anaphylatoxins stimulate the release of histamine, 5-hydroxytryptamine (which causes vascular
C3b
Antibody
Antigen permeability changes) and chemotactic factors
Antigen-antibody
complex (which induces the influx of polymorphonuclear
leukocytes) from basophils and mast cells. Moreo-
ver, immune complexes may directly trigger (via Fc
Phagocyte receptors) basophils and platelets to induce the re-
(neutrophil, lease of vasoactive amines.
macrophage)
The vasoactive amines released by mast cells, ba-
Fc receptor sophils and platelets cause endothelial cell retraction,
(or C3b receptor) resulting in the exposure of basement membrane.
This results in deposition of immune complexes
on the basement membrane of blood vessel walls
Phagocyte-opsonin
coated pathogen (or kidney glomeruli). Smaller immune complexes,
interaction however, can pass through the basement membrane
and get deposited in the underlying tissue.
Chematactic factors released by complement
activation (such as C5a) as well as by mast cells and
basophils summon neutrophils to the site where im-
mune complexes are deposited. Neutrophils bind to
C3b-coated immune complexes through C3b com-
plement receptors and initiate the process of phago-
Phagocytosis cytosis. Since immune complexes are deposited on
the basement membrane, neutrophils attempt to
» Frustrated phagocytosis results
in the release of lysosomal enzyme
engulf the deposited immune complexes but are
outside the phagocyte. This unable to do so because of steric hinderance as the
phenomenon is also called sloppy complexes are bound to the vessel wall .This process
eating.
of attempted and unsuccessful effort of phagocy-
tosing is sometimes called frustrated phagocytosis.
Antigen endocytosed
and degraded The difference between normal phagocytosis of an
Figure 13.14
Line diagram showing normal
antigen and frustrated phagocytosis is shown in
phagocytosis of antigen. Normal Phagocytosis Figures 13.14 and 13.15.
During their failed attempt Antigen-antibody

to phagocytose the immune com- complex deposited on
plex, phgocytes exocytose their basement membrane
lysosomal enzymes onto the site of
immune complex deposition. The
lysosomal enzymes include col- Endothelial cell
lagenase, neutral proteinase and Basement membrane
kinin forming enzymes. These pro-
Phagocyte
teolytic enzymes damage local tis-
(neutrophil,
sue and intensify the inflammatory macrophage)
responses. The activation of the
complement system also induces
Fc or C3b receptor
platelet aggregation. This, in turn,
results in the release of vasoactive
amines and clotting factor which
can form microthrombi, leading
to local blockage of the capillary
(ischaemia).
The formation and eventual Interaction of
deposition of immune complexes phagocyte
of type III hypersensitivity reac- with opsonin-coated
tions contributes to pathogenesis pathogen
of several diseases such as SLE,
leprosy, malaria, meningitis, strep-
tococcal glomerulonephritis, and
dengue haemorrhagic fever. Antigen-antibody
complex too large to
be phagocytosed or Figure 13.15
13.4.3 LOCALIZED steric hinderance
Diagram showing frustrated
phagocytosis and damage to basement
TYPE III Degraded
membrane. The phagocytes are unable
immune
REACTIONS to endocytose the antigen–antibody
complex complex because of steric hindrance
Nicolas Maurice Arthus, in 1903, Release of lysosomal as the complexes are bound to the
observed that intradermal injec- enzymes endothelial basement membrane. In frustration, the
Lysosomal basement membrane
tion of soluble antigen into sensi- phagocyte exocytoses the lysosomal
enzymes damaged enzymes that degrade the basement
tized rabbit produced haemorrhage
membranes and tissues.
and oedematous reaction reaching
its peak at three to eight hours
after which it slowly resolved. This “local anaphylaxis” reported by Arthus is popularly termed as » Complement activation is essen-
tial for Arthus reaction. Arthus
Arthus reaction. reaction can be blocked by deple-
Arthus reaction takes place at a local site. Injection of antigen intradermally or subcutane- tion of the complement. Without
ously into the animal that has a high level of circulating antibodies leads to the formation of high the complement, neutrophils are
not attracted to the site and only
level of immune complexes. These large complexes precipitate, often within the venule. Subse- mild oedema develops.
quently the complement system binds these immune complexes and anaphylatoxins are gener-
ated which cause mast-cell degranulation. Chemotactic factors generated at the site of antigen
entry leads to the influx of neutrophils and platelet aggregation. The accumulation of complexes
in the capillaries also causes release of histamine from mast cells. This causes oedema and haem-
orrhage. The reaction reaches a peak after three to eight hours and then slowly subsides within
48 hours. After 48 hours, the neutrophils are replaced by mononuclear cells and eventually by
some plasma cells.
INTRAPULMONARY ARTHUS-TYPE REACTION. Arthus type reaction appears to be responsible

for number of hypersensitivity disorders in humans induced by inhaled antigens. Inhalation
of bacterial spores, antigens from dried faeces of pigeon (pigeon fancier disease), thermophilic
actinomycetes from mouldy hay (farmer’s lung) introduces antigen into the lungs. This usually
results in formation of local immune complexes in the alveoli leading to inflammation and alveolitis
associated with severe breathing difficulties.
13.4.4 GENERALIZED TYPE III REACTIONS

Sometimes a large amount of antigen enters the host body and in such case antigen is still in cir-
culation when antibodies are formed. Since antigen is in excess, small (soluble) antigen–antibody
complexes are formed. These soluble immune complexes are not easily removed from the circula-
tory system as they are not easily phagocytosed. Deposition of these complexes at various sites
in the body elicits various tissue-damaging type III reactions at the site of their deposition. One
example of such generalized type III reaction is serum sickness.
Serum sickness is a complication of serum therapy in which a relatively large dose of foreign
serum containing a particular antibody is given against diseases such as diphtheria or tetanus (for
example, horse anti-diphtheria or horse antitetanus serum). Usually within a week of injection of
serum, antibodies are formed against injected horse proteins particularly horse globulin and the
individual begins manifesting a variety of symptoms that are called serum sickness.
The symptoms of serum sickness include fever, swollen lymph nodes, painful swollen joints
and albuminuria, apart from general weakness. The antibodies formed against injected horse
» Von Pirquet and Schik first proteins enter the circulation and complex with antigen to form antigen–antibody complex.
described serum sickness in 1905. Because the reaction occurs in antigen excess, immune complexes are small and very slowly
Antigen
Induction of immune response
(Antibodies produced in excess)
Soluble immune
complexes formed
Bowman’s
capsule
Joints
Deposition in Deposition in Deposition in

blood vessel kidney joints
Figure 13.16
Clinical manifestation of generalised
type III reactions (serum sickness). Vasculities Glomerulonephritis Arthritis
removed by phagocytes and hence cause tissue-damaging reactions at various sites (Figure 13.16).
The precise manifestation of serum sickness depends on the amount of immune complexes
formed and site of their deposition. Clinical recovery usually occurs within 20–30 days. How-
ever, if these complexes are deposited at the site of filtration they may lead to the development of
number of diseases. Deposition on glomerular basement membrane causes glomerulo-nephritis;
on basement membrane of blood vessels, vasculitis, on synovial joints, arthritis and at choroids
plexus of brain, SLE.
13.5 TYPE IV HYPERSENSITIVITY

R E A C T I O N S : D E L AY E D - T Y P E
HYPERSENSITIVITY REACTIONS
Type IV hypersensitivity reactions develop when antigen activates sensitized TDTH cells ( CD4+T
of TH1 subtype or sometimes Tcyt cells). Activation of TDTH cells by antigen leads to the secretion of
cytokines such as IL-2, IFN-γ, migration inhibition factor (MIF) and TNF-β.
These cytokines activate macrophages and draw them to the affected (lesion) area promoting
increased non-specific phagocytosis. The macrophages cause non-specific tissue destruction and
induce inflammation. In delayed type of hypersensitivity (DTH) reactions, tissue injury results
from the products of activated macrophages such as hydrolytic enzymes and cytokines, as well as
reactive oxygen and reactive nitrogen species. The DTH reaction is characterized by erythema and
induration which appear after several hours and reach a maximum at 24–48 hours.
There are three variants of type IV hypersensitivity: contact hypersensitivity, tuberculin-type « Delayed-type hypersensitivity is
hypersensitivity, and granulomatous hypersensitivity. not always harmful. It is the main
immune defence against some
Contact hypersensitivity usually occurs within 72 hours of (secondary) contact with antigen. intracellular pathogens such as
Antigen could be a chemicals such as nickel (found in jewellery), chromate (cement, tattoos), M. tuberculosis.
formaldehyde (various cosmetics), paraphenylenediamine (hair dyes), balsam of Peru (perfumes)
and pentadecacatechal (chemical found in poison ivy).
Tuberculin-type hypersensitivity which also peaks around 72 hours is characterized by
fever, swelling and hardening at the site of antigen injection. It usually manifests after encounter «The term delayed hypersensitivity
with soluble antigen of number of microbes such as Mycobacterium tuberculosis and Leishmania was first time used by Hans Zinnser
in 1921. It was meant to convey
tropica. The tuberculin lesion normally resolves within five to seven days. that the hypersensitivity reaction
Granulomatous hypersensitivity develops over a period of 21–28 days and the granuloma (nod- (Zinnser meant tuberculin-type
ule-like structure) formed may persist for several weeks. The histological appearance of granuloma hypersensitivity) does not com-
mence until four or more hours and
reaction is quite different from that of tuberculin-type like reaction, even though they also result from usually peaks within 48–72 hours.
sensitization by microbial antigen such as antigens of M. tuberculosis, M. leprae.
13.5.1 CO N TAC T H Y P E R S E N S T I V I T Y
A number of small molecules penetrating the skin can give rise to contact hypersensitivity. It is
basically an epidermal reaction that is mediated by macrophages. The allergic chemical (usual-
ly hapten) combines with skin proteins and is internalized by powerful-antigen presenting cell,
Langerhans cells. Epidermal Langerhans cells leave the epidermis and migrate to the lymph nodes.
There they present the antigen (bound to class II MHC molecule) to TH lymphocytes, producing
a population of memory (as well as effector) TH cells. This is called sensitization phase and usually «When an allergen makes skin
takes 10–14 days in humans. contact and the skin immediately
shows a wheal and flare allergic
The elicitation of allergic reaction requires secondary encounter with the antigen. During sec- reaction, it is called contact
ondary contact, antigen presenting cells take the antigen and present it to TH (TDTH) cells in skin urticaria and is IgE- mediated
and lymph nodes. These activated TDTH cells start secreting cytokines. After about 48–72 hours of allergic reaction. It is not a type of
delayed hypersensitivity.
secondany exposure to antigen, cytokines produce a gradient for the movement of macrophages
to the lesion area, infiltrating the dermis and epidermis. Macrophages migrate along the cyto-
kine gradient and release their lytic enzymes into the surrounding tissue, causing localized antigen
(and its associated tissue) destruction. Diagrammatic representation of contact hypersensitivity is
Allergen
Langerhans
cell
Skin
Localized Class I MHC

antigen
(and tissue)
destruction
and
inflammation Class II MHC
Migration of
Langerhans
cell to lymph
node
Figure 13.17
Line diagram showing induction of Allergen
Cytokine secretion Migration of
contact hypersensitivity DTH and the activates
by TDTH cells activated and
way the body responds to it. Activated sensitized
summons macrophages sensitized
TDTH cells secrete cytokines that activate TDTH cells
and summon macrophages to the TDTH cell
lesion site. The non-specific action of to lesion site Lymph node
macrophages causes the destruction
of antigen and tissue, leading to a TDTH cell
hypersensitivity reaction. (sensitized)
»The tuberculin reaction was origi- 13.5.2 TUBERCULIN REACTION

nally described by Koch. He showed The tuberculin reaction (also called as tuberculin test) is still performed to test whether the person
that the subcutaneous injection of
mycobacterial antigens (derived is infected with tuberculosis bacilli. In this test, a small amount of purified protein derivative (PPD)
from tuberculin culture filtrate) in of tuberculosis derived from M.tuberculosis is injected into the skin and the site examined 72 hours
patients with tuberculosis, resulted later. The positive test show swelling and redness at the injected site within 48–72 hours. Histological
in hardening and swelling at the
site of infection. Patients developed analysis suggests that monocytes constitute 85 per cent of the total cellular infiltrate at the lesion site.
fever and generalized sickness.
13.5.3 G R A N U L O M AT O U S H Y P E R S E N S T I V I T Y
Granulomatous hypersensitivity usually results from the persistence within macrophages of in-
tracellular microorganisms or other particles that the cell is unable to destroy. This persistence of
antigen leads to the chronic stimulation of TH cells (TDTH cells) which stimulates the continuous
production of cytokines. These signals summon macrophages at the lesion site. Some macrophages
turn into epithelioid cells under the influence of cytokines and fuse to form giant cells. These mac-
rophages, epithelioid cells and giant cells become surrounded by collagen fibres (caused by fibrob-
last division). This raised or nodule-like structures that is formed is referred to as granuloma and
is believed to occur to “wall off ” or contain the microbes in granuloma.
To conclude, it can be said that hypersensitivity is a hyper-immune response to an apparently
innocuous antigen. Depending on the type of effector molecules generated during hypersensi-
tivity, Gell and Coombs have classified hypersensitivity into type I, II, III, IV. Type I, II, III are
antibody-mediated, while type IV is cell-mediated hypersensitivity. Type I reactions are IgE-medi-
ated; type II is an allergic reaction brought about by antibody reacting with antigens on cell sur-
face; Type III is an immune complex (antigen–antibody complex)-mediated reaction that results
from deposition of such complexes in tissues such as kidneys, liver, joints; Type IV reactions are
cell-mediated hypersensitivity response to persistent antigen. These hypersensitivity responses
which start as normal cell-mediated responses slowly aggravate into inflammatory responses if
antigens are persistent.
Immunoelectrophoresis
Immunodiffusion techniques which involve simple Antigen
diffusion work best when there are one or two an- + _

tigens and their corresponding antibodies present
in the system. When there are a large number of
Agar gel
antigens present in the system that can react with
variety of antibodies, the precipitin line formed gets Electrophoresis of antigen is performed
difficult to resolve and interpret. The efficacy of im-
munodiffusion was further improved by Williams
_
and Graber who, in 1953, invented immunoelec-
trophoresis. Immunoelectrophoresis combines two +
analytical techniques—immunodiffusion and elec-
trophoresis—to make it easier to interpret results
Antibody
with increased resolution. For this, antigens are first
After electrophoresis, a trough is cut and
separated in one dimension by electrophoresis on antibody is poured into the trough
agarose gel. The antigens show movement towards
both positively charged anode and negatively
charged cathode. A long trough is then cut on the
gel next to the well. This trough (rectangular well)
Precipitin
is then filled with antiserum/antibodies. This agar- band
ose plate is then incubated for three to four hours
at 370C. Separated antigens and antibodies diffuse
towards each other and form precipitin bands (see Antigen and antibody react to give a precipitin band
Figure 13.18). This technique is used for studying Figure 13.18
complex proteins in serum. Immunoelectrophoresis.
S U M M A R Y
• Hypersensitivity is the induction of a state of excessive immune • Common examples of type II reactions include drug allergies,
response with resulting damage to the host body. erythroblastosis foetalis and transfusion reaction.
• Hypersensitivity has been classified as immediate or delayed, • Type III hypersensitivity occurs as result of deposition of immune
depending on the time of appearance of symptoms. complex in tissues such as kidney, liver, joints and lungs.
• Gell and Coombs have classified hypersensitivity reactions into • These immune complexes activate the complement system result-
four distinct categories, type I, II, III, IV, based on the differences ing in the synthesis of C3a and C5a. These chemotactic factors
in the effector molecules generated. attract neutrophils, eosinophils and basophils to the target site
causing tissue damage. Examples of type III hypersensitivity reac-
• Type I reactions are mediated by IgE antibodies bound to mast tions include Arthus reaction and serum sickness. The formation
cells and basophils. Bound IgE is cross-linked by allergen, trigger- and deposition of immune complexes contribute to pathogenesis
ing the release of pharmacologically active molecules from mast of several diseases such as SLE, leprosy and meningitis, among
cells/basophils. others.
• These mast cell mediators act on the surrounding tissue to induce • Type IV reactions result from cell-mediated immune response to
pathological oedematous inflammatory reactions. persistent antigen. It starts as a normal cell-mediated response to
• The classic manifestations of type I hypersensitivity include ana- intracellular pathogen such as bacteria, virus and fungi. The per-
phylaxis, atopy and allergic rhinitis. sistence of antigen sensitizes TDTH cells (which are usually CD4+ T
cells of TH1 subtype) which leads to the secretion of inflammatory
• Type II reactions are those allergic reactions that are brought about cytokines. These cytokines activate phagocytic cells that cause
by antibody reacting with antigens present on the cell surface. non-specific tissue destruction and induce inflammation. Symp-
The free Fc region of bound antibody can activate complement toms usually take 24–72 hours to develop and are therefore called
system or bind NK cell via its Fc receptor, and induce target cell delayed hypersensitivity reaction.
lysis. Antibody bound to a cell can also act as opsonin facilitating
phagocytosis.
K E Y W O R D S
• anaphylaxis 267 • drug allergy 269 hypersensitivity 289 • type I

• allergen 267 • eczema 276 • hay fever 276 hypersensitivity 268
• allergic rhinitis 277 • eczematous patch • histamine 270 • type II
• asthma 270 test 280 • late-phase reactions 278 hypersensitivity 281
• atopy 277 • FcεRI 272 • mast cell 268 • type III
• atopic dermatitis 277 • FcεRI 272 • mucosal mast cell 270 hypersensitivity 284
• Arthus reaction 287 • food allergy 278 • rhesus antigen • transfusion
• delayed-type • frustrated incompatibility 283 reaction 282
hypersensitivity 268 phagocytosis 286 • RAST 280 • wheal and erythema 269
• desensitization 280 • granulomatous • RIST 280 • serum sickness 288
1. Two eosinophil-deficient mice (of two different strains) were gener- two reactions different? Why is one called immediate transfusion
ated successfully using transgenic strategies. Only one of the mice reaction and the other, delayed haemolytic transfusion?
showed acute pathophysiological manifestation of asthma when 4. How are immune complexes removed in normal individuals? What
exposed to allergen. Why is asthma manifested in only one strain goes wrong in type III reaction that leads to immune complex
and not in the other? Can you suggest at least one explanation for hypersensitivity?
this difference?
5. What could be the reason that one allergen triggers a type I reac-
H I N T — Background (genetic) strain variability tion while another one stimulates type II or III or IV? What quali-
2. Outline the contribution to the hypersensitivity reactions made by ties should an allergen have to trigger all four types of hypersen-
the complement system. Which particular allergic reaction(s) are sitivity reactions? Can you name one antigen that can induce all
most likely to be affected by complement deficiency? types of hypersensitivity?
3. Transfusion reaction mediated by ABO blood group and MN blood
group systems are type II hypersensitivity reactions. How are these
Choose the appropriate option. 6. The process of desensitization involves:

(a) Administration of antitoxic antibodies
1. A typical allergen could be all of them except: (b) Removal of offending antibodies
(a) Protein (c) Neutralization of antigen before it starts allergy
(b) Enzyme inhibitor (d) None of the above
(c) DNA
(d) Asbestos 7. NK cells are involved in:
(a) Type I reaction
2. IgE receptors can be cross-linked by all except: (b) Type II reaction
(a) Anti-receptor antibodies (c) Type III reaction
(b) Chemical cross-linking agents (d) Type IV reaction
(c) Anti-IgE antibodies
(d) All of the above 8. Nicole Maurice Arthus described a hypersensitivity reaction
that was:
3. One of the following is not released by mast cells: (a) IgE-mediated
(a) Tryptase (b) Mediated by Tcyt cells
(b) Chymase (c) Dependent on immune complex formation
(c) Heparin (d) Antibody-independent
(d) IL-2
9. One of them the following is not a type IV hypersensitivity.
4. TH2 cells are increased in lesion of: Which one is it?
(a) Atopic dermatitis (a) Granulomatous hypersensitivity
(b) Contact dermatitis (b) Tuberculin hypersensitivity
(c) Arthus reaction (c) Transfusion reaction hypersensitivity
(d) Glomerulonephritis (d) Contact hypersensitivity
5. Which one of the following is a primary mediator of anaphy- 10. Type IV hypersensitivity can be differentiated from type I reac-
laxis? tions by all, except:
(a) Arylsulfatases (a) Passive transfer with lymphocyte
(b) PAF (b) Passive transfer with immune complexes
(c) Bradykinin (c) Involvement of mediators
(d) Leukotrienes (d) Time of appearance of symptoms

1. Mast cells and basophils are the key cells involved in type IV 4. Atopic dermatitis and contact dermatitis are examples of type IV
hypersensitivity. hypersensitivity reactions.
2. Hayfever, asthma and serum sickness are all different forms of 5. PPD of Mycobacterium tuberculosis injected into the skin shows
type I hypersensitivity. swelling at the injection site after a few days. This is a wheal and
flare reaction.
3. Arthus reaction can be blocked by depletion of complement
components.
Coca, A. F. and R. A. Cooke (1923). “On the Classification Lindstrom, J. (1985). “Immunobiology of Myasthenia
of the Phenomenon of Hypersensitiveness”, Journal of Gravis, Experimental Autoimmune Myasthenia Gravis
Immunology, 8: 163. and Lambert Eaton Syndrome”, Annual Review of
Immunology, 3, 109–31.
Cooper, A. M. and J. L. Flynn (1995). “The Protective Immune
Response to Mycobacterium tuberculosis”, Current Opinion Metcalfe, D. D., D. Baram and Y. A. Mekor (1997). “Mast Cells”,
in Immunology, 7: 512–16. Physiological Reviews, 77: 1033–79.
Enk, A. H. and S. I. Katz (1995). “Contact Hypersensitivity As a Theofilopoulos, A. N. and F. J. Dixon (1979). “The Biology
Model for T-cell Activation in Skin”, Journal of Investigative and Detection of Immune Complexes”, Advances in Immunology,
Dermatology, 105: 805–35. 28: 89–220.
Fearon, D. T. (1988). “Complement, C Receptors and Immune Turk, J. L. (1975). Delayed Hypersensitivity. 2nd ed., New York:
Complex Disease”, Hospital Practice, 23: 63–72. American Elsevier.
Karp, W. M. and C. L. Karp (2004). “Eosinophils in Asthma:
Remodeling a Tangled Tale”, Science, 305: 1726–28.
Inflammation was described for the first time in the Ist century, based “I begin to smell
on visual observations. The Roman physician Celsus described, for the a rat.”
first time, the host response to an injury in terms of four cardinal signs — C E R VA N T E S
(D ON QUIXOTE I)
of inflammation. These signs were redness (rubor), heat (calor), pain
(dolor) and swelling (tumor). The fi fth cardinal sign of
inflammation—loss of function (functio leasa)—was added later by the
Greek physician Galen in the 2nd century. Initially,
inflammation was considered to be a part of the healing process. This
view was changed later, when it was suggested (incorrectly) that
inflammation was an undesirable immune response that was
harmful to the host. With the discovery of phagocytosis by Metchnikoff
should be able to:
in the 19th century, the contribution of inflammation to the immune
response and the healing process was rediscovered. Now, • Give an account of
different cell-surface adhesion
inflammation is considered to be the keystone of pathology because molecules
• Describe, in detail, the process
changes observed during inflammation are suggestive of disease or of leukocyte migration to the
site of inflammation
injury. A generalized representation of inflammation is shown • Describe chemokines and its
two subgroups
in Figure 14.1.
• Explain the role of mediators—
plasma enzyme mediators,
cytokines, lipid mediators in
inflammatory response
localized and systemic acute
inflammatory response
• Describe chronic inflammation
• Briefly summarize the mode of
action of various
anti-inflammatory agents
Cell Migration and
Inflammatory Response
14.1 INTRODUCTION
14
Under normal conditions, blood cells, including leukocytes, circulate through all the tissues of
the body. The purpose of migration of leukocytes is to give a small number of lymphocytes, which
are specific for any particular antigen, a chance to encounter the antigen. Migration of cells ensure
antigen (entering from lymphatic system) and antigen-presenting cells converge and meet in the
lymph nodes, while blood borne antigens are taken care of in the host’s spleen. Transmigration of
leukocytes from the blood stream into the tissue inflammatory sites involves attachment to several
adhesion molecules present on the endothelial cell which are recognized by receptors on activated
lymphocytes or phagocytes. These lymphocytes or phagocytes then unleash a cascade of damaging
reactions that is primarily aimed at destruction of the invading pathogen or irritant, though some
Tissue injury Mast cells/

Skin basophils
Bacteria
Physical injury brings

Vasoactive
pathogens into the
amines
tissue. Mast cells
and basophils
stimulated.
Capillary
Bacteria Blood vessels dilate

Plasma
Increased vascular
inflow
permeability causes
plasma inflow to
Neutrophil the site.
Macrophage
Neutrophils and
Neutrophil macrophages
migrate to the site of
infection. Assault on
pathogen starts.
Macrophage
Fibrin
Site of injury covered by

fibrin wall that encloses
bacteria,leukocytes,
RBC and RBC that form pus.
Abscess formed.
Macrophage Neutrophil
Pus
Figure 14.1
Abscess relieved. An overview of the process of
inflammation.
unavoidable non-specific tissue damage also occurs.

These reactions that develop in tissues in response
Tissue to pathogen or accidental tissue damage are termed
injury inflammatory reactions.
Inflammation (Latin: flaume—little flame) is a
Inflammation non-specific defence response by the body to an in-
Inflammation has been best jury to the tissue. An inflammatory response could
described as a protective response
that occurs in a living tissue when it
be initiated after mechanical injury (for example,
is injured, provided that the injury Release of pin prick), chemical insult (damage by acid/alkali/
Injury to
is not of such a degree as to at once vasoactive bee venom), physical agent (heat or uv radiation) or
destroy its structure and vitality. nerves
factors living organism (pathogen).
At the cellular level, as a result of tissue in-
jury, basophils or mast cells found in most tissues
are stimulated to secrete mediator molecules—
histamine and leukotrienes. They act on local
vasculature to induce following events: (a) There is
Vasodilation Increased vascular dilation of blood vessels surrounding the injured
permeability tissue, (b) There is increased vascular/capillary per-
meability permitting the escape of fluid (plasma)
into the tissue and fluid accumulation at the site
of injury resulting in swelling, (c) Phagocytes and
lymphocytes tend to stick to the endothelial cells of
Figure 14.2
The development process of four the Dolor Rubor Calor Tumor capillary and assume more flattened morphology
cardinal signs of inflammation. (Pain) (Redness) (Heat) (Swelling) (margination) (d) These phagocytes/lymphocytes
then migrate through the capillary wall from the
blood into the tissue space. These cells start phago-
cytosing the irritant/pathogen. The phagocytes are initially neutrophils that enter the inflamed
area, though later macrophages replace neutrophils. Living, dying, and dead leukocyte (mostly
neutrophils and macrophages) together with the fluid, tissue debris, and living or dead microbes
produce a purulent exudate called pus. The process of pus formation is called pyogenesis.
The inflamed area shows five characteristic signs of inflammation—(a) red colour from blood
accumulation (rubor), (b) warmth from the heat of blood (calor), (c) Swelling from accumulation
of fluid (tumor), (d) Pain from the injury to the local nerves (dolor) and (e) loss of function in the
affected part ( functio leasa). Figure 14.2 shows the development of four common cardinal signs of
inflammation. When inflammation persists only for few days or weeks (that is, subsides quickly),
it is called as acute, however, when it last for months or even years it is referred to as chronic
inflammation. The inflammatory response is a non-specific mechanism by which a host contains
and destroys pathogen and injurious material. However, prolonged inflammation can be harmful
to the host, causing persistent pain, tissue and organ damage.
14.2 C E L L - S U R FAC E A D H E S I O N
MOLECULES
Cell-surface adhesion molecules (CAMs) are membrane-bound proteins that are involved in the
recognition and adhesion of one cell to another. These molecules serve as molecular recognition
molecules that allow them to interact in a specific way with other cells. These molecules also serve
as adhesive proteins that the cells can use to gain traction on other cells or on extracellular matrix.
Cells can modulate their interaction with other target cells by increasing the number of adhesion
molecules or altering their affinity or avidity to the target cell receptor.
CAMs play a very important role in guiding circulating leukocytes to enter the inflamed area.
Vascular endothelial cells express a large number of leukocyte-specific CAMs (constitutive or
inducible)on their surface. These molecules attach and hold on to flowing leukocytes despite ex-
posure to the blood flow pressure. The bound leukocytes slowly stick to the wall of the vessel and
» Blood flow provides the neces-
undergo diapedesis (that is, extravasate or pass through the wall of the blood vessels) towards the
sary force for the transmigration of
leukocytes from blood stream into lesion site. A large number of CAMs have been identified that have a role in leukocyte migration.
the tissues. They have been classified into five major families—immunoglobulin superfamily CAMs, selectins,
CELL MIGRATION AND INFLAMMATORY RESPONSE 297
ICAM,
Ligand Integrins Fucosylated glycans Selectin
Fibronectin
Extracellular
Ig domain
Lectin-like
domain Carbohydrate
Fibronectin- Epidermal-growth-
type factor-like domain side chains
domain Repeat consensus
sequence
Cell membrane Cytosol
COOH
Figure 14.3
Immunoglobulin Mucin family The four families of CAMs and their
Selectin Integrins
superfamily CAM CAM ligands.
integrins, mucin family and cadherins. Four families that play an important role in inflammation
are shown in Figure14.3.
14.2.1 I M M U N O G L O B U L I N S U P E R FA M I LY C A M s
A large number of CAMs contain one or more immunoglobulin domains in their extracellular
region and are thus classified in the immunoglobulin superfamily. They also have a transmem-
brane domain and an intracellular domain that interacts with cytoskeleton. These include ICAM-1
(intercellular adhesion molecule-1), ICAM-2, VCAM (vascular CAM) and MadCAM-1 (mucosal
« ICAMs are widely expressed on
addressin CAM-1). These members are either constitutively expressed or are inducible on vascular endothelial and epithelial cells and
endothelium. Members of this CAM family have variable number of extracellular domain depend- VCAM on endothelial cells.
ing on type of functions they perform.
14.2.2 SELECTINS Selectins

Selectins are a family of mammalian carbohydrate-binding adhesive molecules. They are membrane Selectins are carbohydrate-binding
glycoproteins with an n-terminal extracellular domain of 120 amino acids homologous to C-type proteins that bind fucosylated
carbohydrates, especially sialylated
animal lectin that binds sialylated carbohydrate (and, hence, the name selectin) and a domain re- Lewis and mucins. L-selectin
lated to the epidermal growth factor. They are expressed on the endothelium, leukocytes and plate- mediates the homing of leukocytes
lets. The selectins include E-selectin (found on endothelium), P-selectin (found on platelets), and to the endothelium in the lymph
nodes.
L-selectin (found on leukocytes). Their ligands are expressed on platelets, leukocytes, endothelium.
14.2.3 INTEGRINS
Integrins constitute a large family of adhesion molecules present on many cells, including leuko-
cytes. Each member of the integrin family is a heterodimer with one α and one β subunit bound
non-covalently. Both the subunits span the membrane.
There are more than 20 subfamilies of integrins depending on the type of β subunit they con- « Defects in CAMs are usually associ-
tain. Each class of β chain can associate with any subset of α chain and the specific αβ combination ated with the leukocyte adhesion
disease (LAD). It results from a
determines the specificity of ligand recognition. mutation in the β subunit of inte-
However, broadly speaking, β integrins are involved in the binding of cells to extracellular grins. The patients (suffering from
matrix. A type of β integrin, β2 integrin, is involved in leukocyte adhesion. The α chain of integrins LAD-1) usually succumb to death
unless they receive bone marrow
has a conserved divalent cation binding motif domain. It is for this reason that integrins show transplantation within a few years
dependence on divalent cation for their binding to their ligands, for example, α1β2 integrin present of birth.
on leukocyte gets activated by Mg2+ ions.
« Integrins show both “outside in”
14.2.4 M U C I N FA M I LY and “inside out” signalling proper-
Mucin is a vague term for a group of serine- and threonine-rich glycoproteins. Mucin family is an ties. Outside in signalling is when
ligand binds on the integrin mol-
emerging family of adhesion molecules, made up of glycoproteins (sialomucins ). These molecules ecule and signal is transmitted from
are expressed on cells of haematopoietic system. The members of the mucin family share a common integrin to the interior of the cell.
characteristic—highly glycosylated polypeptide containing predominantly O-linked carbohydrate Inside out signalling is when the
activation of TCR activates integrin,
side chain linked to serine and threonine residues. They have branched and extended thread- which, in turn, binds its ligand from
like structures. The ligands for mucin family are selectins. GlyCAM-1 and CD34 are members the outside the cell.
mucin family that are expressed on certain endothelial cells. These adhesion molecules bind leukocytes
bearing L-selectin.
14.2.5 CADHERINS
Cadherins are cell-surface transmembrane proteins that mediate cell–cell adhesion in Ca2+ depend-
ent way. They have an extracellular domain, a transmembrane domain and an intracellular domain.
The extracellular domain is a rigid rod-like structure that has several Ca2+ bound to it. Cadherins show
homophilic adhesion, that is, cadherins bind cadherins. Cadherins are involved in tissue organiza-
tion and embryonic development. There are three main types of cadherins—Neural(N)-cadherin,
» All vertebrate cells exhibit one or placental(P)-cadherin and epithelial (E)-cadherin, although the less important protocadherin and
more cadherins. desmocadherin are also members of the cadherin family.
14.3 L E U K O C Y T E M I G R AT I O N
There are two major stages in leukocyte migration. The first is the attachment of leukocytes to the
walls of the blood vessel or capillaries, that is, vascular endothelium. The second stage involves the
transmigration of leukocytes from the blood vessels to the site of infection or inflammation, tra-
versing the endothelium under the guidance of chemotactic stimuli. This process is partly control-
led by a large number of adhesion molecules (and their ligands) present on the surface of vascular
endothelium, extracellular matrix tissue as well as leukocytes. Cell migration is partly influenced
by soluble signalling molecules such as chemokines and chemotactic factors.
The process of leukocyte migration to the site of inflammation across the endothelium require sever-
al consecutive steps. This multi-step model of leukocyte migration is based on experimental observation.
INITIAL ATTACHMENT OR TETHERING. Leukocytes become loosely attached or adherent

(tethered) to cytokine-activated endothelium. This attachment is mediated by low-affinity interaction
between selectins present on endothelial cell (E-selectin) and their ligands on leukocytes or via
L-selectin (present on leukocyte). On leukocytes, these adhesion molecules are concentrated on the
tip of leukocytes and because of their low affinity they hold leukocytes only transiently. Figure 14.4
shows how a leukocyte is tethered to the endothelium via different types of adhesion molecules.
ROLLING. In the microvasculature, continuous exposure to the blood flow pushes the tethered
leukocyte and causes the disruption of the weak selectin–ligand interaction. These interactions are
rapidly reformed downstream as the leukocyte contacts the endothelium again. The result of these
events is the rolling of the spherical leukocyte on the endothelial surface. As the leukocyte starts
rolling, it may induce other leukocytes to roll, probably mediated by binding of L-selectin on one
leukocyte (for example, neutrophil) and its ligand on another leukocyte (neighbouring neutrophil).
ACTIVATION OF LEUKOCYTE. As the leukocytes start rolling on the surface of the endothelium,
they become activated. This activation is mediated by chemokines which are bound by heparin
sulphate groups present on the surface of the endothelial cells. In response to chemokines, leukocytes
assume a more flattened shape and become less motile. The spreading allows a large number of
» Chemokine activates leukocytes adhesion molecules present on the cell surface to engage their ligands on the endothelial cells.
by binding and activating serpen- Activation also results in an increase in the affinity of leukocyte CAMs for endothelial ligands.
tine chemokine receptors present
on the leukocytes.
ARREST OR STABLE ADHERENCE OF LEUKOCYTES
TO ENDOTHELIUM. Using their high-affinity
Integrin
CAMs, especially LFA-1, CR-3, Mac-1 (present
LFA
Sialomucin
on neutrophils, monocyte, T cells) and VLA-4
L-selectin Chemokine (on leukocytes other than neutrophils), activated
receptor leukocytes bind firmly to the endothelial surface.
GlyCAM These adhesion molecules recognize their ligand
ICAM VCAM present on the endothelial surface (LFA-1 binds
Figure 14.4 P-selectin ICAM-1, ICAM-2, ICAM-3; VLA-4 binds
Adhesion molecules that tether a
leukocyte to endothelial cells. E-selectin Chemokine
VCAM). The expression of their ligands on the
Lumen of blood vessel

Sialomucin
E-selectin Receptor
LFA ICAM
Rolling
Chemokine
Attachment Activation of Arrest of Diapedesis

leukocyte leukocyte
Chemokine
gradient
Chemokine
Figure 14.5
Directing a leukocyte to the site of
Very late antigen injury. Leukocutes do not bind resting
expressed endothelium. Distressed tissue-resident
cells release cytokines/chemokines
Inflammed that trigger rolling, activation, arrest
tissue releasing and transendothelial migration of
chemokine leukocytes.
endothelium cell is increased by inflammatory cytokines (TNF-α, IL-1 and IFN-γ). Leukocytes
firmly adhere at one place and do not roll. This adherence allows migration across the endothelium
to be fine-tuned.
DIAPEDESIS (TRANSENDOTHELIAL CELL MIGRATION). Once the leukocyte is firmly bound « Diapedesis of leukocytes occurs
to the endothelial cell surface, its leading end begins to move, frantically searching for clues that primarily in the venules, except in
the lungs where it occurs in
direct its extracellular destination. This clue is provided by the chemotactic gradient which is capillaries.
received by a leukocyte chemokine receptor. Once received, activated leukocytes start traversing
between endothelial cells inducing changes in the tight junctions between endothelial cells. When
the leukocyte encounters a basement membrane, it secretes enzymes that digest collagen and
other components of the basement membrane allowing the cell to migrate into tissues. Luscinskas
et al.(2001) proposed that apart from all the factors mentioned above, continuous fluid shear
stress (in vivo contributed by blood flow) is essential for transmigration of leukocytes. Figure 14.5 VLA
summarizes how a leukocyte is directed towards the inflammatory site. Very late antigen (VLA) is a family
Once they cross the endothelium and enter the tissue, leukocytes must interact with the proof adhesion receptor molecules
(of the integrin family), initially
tein of extracellular matrix (collagen, fibronectin, laminin, etc.) as well as tissue cells. As leukocytes identified on the T-cell surface. These
leave the blood vessel, their functional phenotype changes, to allow them to move through the heterodimeric molecules were so
tissues. They lose L-selectin and start expressing very late antigens (VLAs). This group of antigens named because they appeared
“very late” on the T-cell surface
include adhesion receptors for laminin (VLA-3, VLA-6), fibronectin (VLA-3, VLA-4, VLA-5) and after its activation. These adhesion
collagen (VLA-2, VLA-3). The VLA group of proteins expressed on leukocytes allows interactions molecules have now been reported
with extracellular matrix protein making them easy to reach the target site under the influence of to occur on almost all types of cells.
This family of receptors includes six
chemokines or chemotactic factors. Extravascular migration occurs preferentially towards gradi- members (VLA-1 to VLA-6).
ents of chemotactic molecules formed within the tissue.
14.3.1 C H E M OTAC T I C M O L E C U L E S
Right from the very beginning of leukocyte migration (that is, from the tethering of cells to the
transmigration of leukocytes from blood vessel to the lesion site), chemotactic molecules guide
the leukocytes towards the target site. Chemotactic molecules could be chemokines or chemotactic
factors. There is a basic difference between the two phenomenon—that is, chemokinesis and chemotax-
is—they induce. Chemotaxis is the directional migration of cells along a concentration gradient of
chemotactic factors, while chemokinesis is non-directional migration. Chemokinesis is the overall « Taxis is the locomotive response
increase in the motility of the cells in response to chemical stimuli (but not in a particular direc- exhibited by certain cells (or organ-
isms such as bacteria) to an external
tion). For chemotaxis or directional migration to occur, cells detect and respond to the concen- stimulus. Chemotaxis is the re-
tration gradient of chemotactic factors. A difference of as little as 0.1per cent can be detected by sponse to an external concentration
migrating neutrophils and macrophages. In chemokinesis, mediators such as histamine and IL-8 gradient of a chemical molecule.
Chemotaxis could be towards the
enhance the overall motility of the cells. However, if a cell responds to the concentration gradient stimuli (chemoattractant) or away
of chemokines, it is said to undergo a directional movement or chemotaxis. from stimuli (chemorepellant).
14.3.2 CHEMOKINES
» Chemokines are small secreted Chemokines are a group of a large number (more than 50) of small polypeptides containing 90–
proteins of 8–14 kDa, originally 120 amino acids that function primarily as chemoattractants for leukocytes. Chemokines play an
identified to stimulate chemotaxis
in lymphocytes. They are produced important role in recruiting leukocytes to regions of infection and inflammation. Almost all the
by a variety of cell types (consti- chemokines bind heparin. These molecules are released at inflammatory sites and become attached
tutive or inducible) that include to surface groups on a variety of cell: for example, heparin sulphate groups present on the surface of
monocytes, T cells, epithelial cells
and endothelial cells. endothelial cells; Duffy blood group antigen, DARC, expressed on venular endothelium. A variety
of lymphoid and non-lymphoid tissues (such as the endothelium) can produce chemokines often
during initiation or the progress of inflammation. Chemokines are signalling molecules that cause
leukocytes to move into various tissues towards high localized concentrations of chemokines.
» Two additional chemokines that fit All chemokines possess four conserved cysteine residues. Based on these cysteine residues,
into neither of the two chemokine
subgroups have been identified. chemokines have been divided into two subgroups.
The first chemokine has missing
first and third cysteine residues • —C–C subgroup chemokines which contain uninterrupted conserved cysteine residues;
(for example, lymphotactin) and • —C–X–C subgroup chemokines in which conserved cysteines are separated by some
the second one possesses a CXXXC other amino acid.
motif (for example, neurotactin).
Since chemokines are bound to the surface of endothelial cells, they can trigger the leukocytes
nearby, which have been tethered by selectins. The chemokines’ action is mediated by a serpentine
receptor. The binding of chemokine to its receptor (on leukocyte) initiates a signal transduction
that involves the G protein and generates a number of second messengers such as cAMP, inositol
triphosphate and Ca2+.
The second messengers generated through a complex series of events induce changes in the
shape of leukocytes, greater adhesiveness to endothelial cells and activation of microbicidal ma-
chinery that generates reactive oxygen and nitrogen species. A detailed view of the activation of a
chemokine receptor is shown in Figure 14.6. This chemokine–receptor interaction also promotes
other cellular events from leukocytes such as degranulation from basophils and release of cytotoxic
proteins from eosinophils.
D D PS
PIP2 A A
G G
β γ P P
G protein α P Phospholipase C Protein
Kinase C
Chemokine receptor
Protein+ATP protein+ADP+P
P P
P IP3
Protein kinase
Ca2+ P
-CaM
cAMP P P
IP3receptor
Protein+ATP Protein-P+ADP ER
Figure 14.6
Activation of chemokine receptor on
leukocyte. The binding of chemokine
to its receptor on a leukocyte leads
to the activation of the GTP-binding
proteins. The activated G protein
stimulates phospholipase C that cleaves
the inositol triphosphate moiety from
phosphatidylinositol 4-, 5- bisphosphate.
DAG and membrane PS activate protein ROS
kinases, that leading to a variety of Increased adhesion Changes in leukocyte RNS
changes in the target cell. (RNS—reactive shape
nitrogen species, DAG—diacylglycerol, Activation of ROS,
PS—Phosphatidylserine). RNS machinery
Analogous to the two subgroups of chemokines, there are two subgroups of chemokine recep- « Some viruses use chemokine
receptors to gain entry into the
tors, each recognizing a separate subgroup of chemokine. cells. Some strains of HIV use CCR5
and CXCR4 receptors to gain entry
• —CC receptors (CCR) which recognize the CC group of chemokines. into the cells.
• —CXC receptors (CXCR) which recognize the CXC group of chemokines.
The interaction between chemokines and their receptors is of very high affinity (Ka > 109) and
is quite specific. However, there is no absolute specificity. Most receptors can bind more than one
chemokine: for example, CXC5 binds only chemokine BCA-1, while CXCR3 binds chemokines
IP-10, Mig and I-TAC. Similarly many chemokines can bind to more than one receptor: for ex-
ample, RANTES can be bound by CCR1, CCR3, CCR4, CCR5 and CCR10. Different receptors
are selectively distributed on particular populations of leukocytes which partially accounts for the
differential inflammatory response of different tissues. Clearly, a cell can respond to a chemokine
only if it possesses a receptor that recognizes it. Table 14.1 depicts some of the important chemo-
kines, chemokine receptors and their important functions. It includes most but not all chemokine
receptors.
Chemokine Chemokines- Receptors Major

Receptors Bound Expressed on Functions
CCR1 RANTES, MIP-1, MIP-5 Granulocytes, NK cell, Active in inducing
T cells, macrophages inflammation, degranu-
lates basophils
CCR2 MCP-1, MCP-2, MCP-3 Monocytes, macro- Activates basophils and
phages, fibroblasts, macrophages, inflam-
basophils mation
CCR3 RANTES, eotaxin, MCP-2, Eosinophils, basophils, Promotes histamine
MCP-3, MCP-4 NK cells, T cells release from basophils,
degranulates basophils,
inflammation
CCR4 TARC, RANTES T cells Lymphocyte migration
CCR5 MIP-1α, MIP-1, RANTES Monocytes, macro- Promotes T-cell immu-
phages, T-cells nity, co-receptor for HIV
binding
CCR6 LARC B cells, T cells Lymphocyte migration
CCR7 ELC T cells, monocytes, Lymphocyte migration
macrophages, dendritic
cells
CCR8 1-309 Monocytes, macro- Lymphocyte migration
phages, T cells
CCR-10 RANTES, MCP-1, Eosinophils, basophils, Inflammation?
MCP-2, MCP-3 T cell
CXCR1 IL-8 Neutrophils, naïve Activates neutrophils,
T cells inflammation,
angiogenesis
CXCR2 IL-8, Gro, α, β, γ Neutrophils Inflammation
CXCR3 IP-10, Mig TH cells T-lymphocyte migration
CXCR4 SDF, PBSF Neutrophils, T lympho- T-lymphocyte
cytes, B lymphocytes migration, co-receptor
for HIV-1.
CXCR5 BCA-1, BLC B and T lymphocytes Migration of lympho-
cytes (particularly B)
into lymphoid follicle
Note: MIP—macrophage inflammatory protein; MCP—monocyte chemoattractant protein; RANTES—regulated upon activation
normal T-cell expressed and secreted; TARC—thymus and activation regulated chemokine; LARC—liver and activation regulated Table 14.1
chemokine; ELC—EBV-ligand chemokine; Gro—growth related oncogene; IP10—inducible protein 10; Mig- monokine induced by Chemokine receptors, chemokines and
IFN-γ; SDF—stromal cell derived factor. their functions.
14.3.3 C H E M OTAC T I C M O L E C U L E S
A number of chemotactic molecules apart from chemokines, are produced/present at the inflam-
matory site which helps in summoning leukocytes. These include anaphylatoxin C5a, tripeptide
fMet–Leu–Phe (fMLP), LTB4 (leukotriene B4), fibrin peptide B and thrombin. C5a is generated
» fMLP is a methionine–leucine–
from complement activation, while fMLP is expressed on bacterial cell and LTB4 is synthesized
phenylalanine tripeptide expressed
on some bacterial cells. Kashkin following the activation of macrophages and mast cells.
et al. reported, for the first time Macrophages and neutrophils have receptors for fMLP and this provides a simple specific
in 1987, the immunomodulating signal for the presence of bacteria towards which phagocytes should move. These leukocytes also
activity of fMLP Currently, fMLP
peptides are also being explored as have receptors for C5a and LTB4, both of which are generated at the sites of inflammation.
adjuvants. Once the leukocytes have arrived at a site of inflammation, they release mediators which control
and regulate accumulation and activation of other blood cells. The ultimate control of inflamma-
tory response is exerted by the antigen itself. If the antigen or irritant is rapidly cleared from the
host body, the inflammatory response subsides quickly. However, when the antigen cannot be
eradicated (as in chronic infection or autoimmune disease), cellular accumulation at the site of
infection occurs and tissue damage by mediators of inflammation ensues.
14.4 M E D I AT O R S O F I N F L A M M AT I O N
Inflammatory response is controlled by plasma enzyme mediators, inflammatory cytokines and
lipid mediators, apart from chemokines which have already been discussed.
Plasma contains four major plasma enzyme systems that play an important role in inflam-
mation. These are kinin system, clotting system, fibrinolytic system and complement system (see
Figure 14.7). When inflammatory response occurs, these four interconnected systems get activated
and generate a number of mediators of inflammation.
Tissue
injury
Hageman factor Ag-Ab Microbial Blood vessel Fibrinolytic cascade

XII activated reaction cell products damaged activated
Complement Clotting
Kallikrein Plasmin
system system
Activated Activated Activated Activated
Figure 14.7
Plasma enzyme systems that mediate
inflammation. Flow chart of the roles
of kinin, complement, clotting and
fibrinolytic systems in mediating Bradykinin C3a,C4a,C5a Fibrin Fibrinolysis C3a,C4a,C5a
inflammatory response.
14.4.1 KININ SYSTEM Kallikrien

The kinin system is an enzymatic cascade that is brought to life when the Hageman factor (HF factor, Kallikrien is a group of serine
XII) is activated. HF is activated to produce Hfa, a serine protease. Hfa activates pre-kallikrein, forming proteinases whose normal
physiological function is to generate
kallikrein. Kallikrein in turn generates bradykinin from high molecular weight kininogens. small vasoactive peptides called
Bradykinin is an inflammatory mediator that induces smooth muscle contraction, increases kinins from some circulating plasma
vasodilation, increases vascular permeability and also induces pain. Kallikrein can also activate the proteins called kininogens.
complement system by stimulating plasminogen activator to activate plasmin. The activation of

complement components generates anaphylatoxins C3a and C5a, both of which induce release of « Kallikrien is derived from the
inflammatory mediators from basophils and mast cells. Another vasoactive peptide, lysl-bradykinin, Greek word kallikreas, meaning
pancreas. It was initially thought
commonly known as kallidin, is also generated by the activated plasmin system. that kallikrien that was found in
the urine of some patients actually
originated in the pancreas.
14.4.2 CLOTTING SYSTEM
The clotting system is activated as an innate defence response to seal damaged blood vessels. This
enzymatic cascade which is activated by the generation of a large amount of thrombin, produces an
insoluble fibrin clot from the soluble fibrinogen in the plasma or tissue fluid. This fibrin clot which
is formed, acts as a barrier to prevent blood loss, and to limit the spread of infection/microbes into
the bloodstream.
14.4.3 F I B R I N O LY T I C S Y S T E M
The removal of the fibrin clot from the inflamed or injured tissue is achieved by the fibrinolytic system.
The last enzyme of this cascade is plasmin which is a potent proteolytic enzyme. Plasmin not only
breaks down the clot but also releases chemotactic and vasoactive fibrinopeptide from fibrin. Moreover,
plasmin can also activate the complement cascade and, hence, generate damaging anaphylatoxins.
14.4.4 COMPLEMENT SYSTEM

The complement cascade when activated either by antigen–antibody reaction (classical pathway)
or microbial cell products (alternative pathway) results in the formation of a number of chemotac-
tic factors such as C3a, C4a, C5a which are released into the surrounding fluid medium. The bind-
ing of these anaphylatoxins on the membrane receptors of tissue mast cells/basophils causes their
degranulation, releasing histamine, serotonin and other vasoactive compounds. These vasoactive
compounds induce smooth muscle contraction and increase vascular permeability. These ana-
phylatoxins together with the complement protein complex C5b67 act on monocytes/neutrophils
inducing them to adhere/tether to the endothelial cells, extravasate through endothelial lining and « The complement system
migrate towards the inflamed area. originated about 600–700 million
years ago.
14.4.5 I N F L A M M AT O R Y C Y T O K I N E S
A number of cytokines play an important role in the development of an acute or chronic inflamma-
tion. In the initial stage of inflammation, IL-1 and IL-6 are released from the inflamed cell. These
cytokines increase vascular permeability, increases leukocyte adhesion, activate T and B cells and
summon leukocytes to the site of inflammation. These leukocytes can themselves release cytokines
of their own (TNF-α, IL-4, IFN-γ) which further enhances cellular migration, contributing in a ma-
jor way to inflammation. Table 14.2 lists some important inflammatory cytokines, their sources and
effects.
14.4.6 I N F L A M M AT O R Y L I P I D M E D I AT O R S
The stimulation of cells such as macrophages, monocytes, neutrophils, eosinophils and mast cells by
a variety of stimuli (such as cytokines and the immune complex) causes membrane phospholipids of
these cells to undergo degradation and release arachidonic acid and lyso-platelet activating factor.
Arachidonic acid formed and released in the cell can be utilized by two different pathways—
cyclo-oxygenase pathway and lipoxygenase pathway. Metabolism of arachidonic acid by lipoxygen-
SRS- A
ase pathway yields leukotrienes. There are four leukotrienes—LTB4, LTC4, LTD4 and LTE4—and LTC4, LTD4 and LTE4 are together
all of them induce smooth muscle contraction. LTC4, LTD4 are LTE4 are together called SRS-A, called SRS-A. They play a major role
slow reacting substance of anaphylaxis. Leukotrienes are produced by a variety of cells, including in broncho-constriction, a condition
associated with asthma.
macrophages, mast cells and monocytes. They are potent attractants of neutrophils.
Cytokines Size (in kDa) Produced by Biological Effect

IL-1 15–17 Macrophages, endothe- Produces fever,
lial cells, lymphocytes acute– phase proteins,
and many other types chemoattractant,
increases expression of
ICAMs
IL-4 20 (glycosylated) Activated TH2, Mast cells, Growth factor for
T cells B, T and mast cells,
promotes IgE and IgG
synthesis
IL-6 22–29 Activated T cells, fibro- Acute-phase protein
blast, macrophages synthesis, T- and B-cell
activation, stimulates
immunoglobulin
synthesis
IFN- 50 (glycosylated) TH1 cells, Tcyt, NK cells Activation of macro-
phages, endothelial
cells, NK cells; induces
expression of class II
MHC molecules
TNF- 17 Macrophage, eosino- Mediates inflamma-
phils, NK cells tory response, activates
neutrophils, and macro-
phages, induces weight
loss (cachexia), fever,
Table 14.2
Biological effects of selected cytokines.
toxic to tumours
The utilization of arachidonic acid by the cyclooxygenase pathway produces thromboxanes and
prostaglandins (PGs). Thromboxanes induces platelet aggregation and vasoconstriction. Prostaglan-
dins are a diverse group of mediators and hence have diverse function. Their biological effects include
increased vascular permeability, increased vascular dilation and induction of leukocyte chemotaxis.
Different prostaglandins are produced from different cells in different amounts. Mast cells
produce a moderate amount of PGD2, while neutrophils produce PGE2. Macrophages and mono-
cytes produce large quantities of PGF2 and PGE2.
14.5 T H E P R O C E S S O F I N F L A M M AT I O N
Inflammation is a physiological non-specific response to a variety of stimuli such as infection or
tissue injury. It is a protective response that enables the body to overcome infection/injury and
return to normal function. Inflammation can be of two types—acute and chronic. Acute inflamma-
tory response has a rapid onset, reaches a peak and is followed by a rapid decline. It usually lasts for
a short duration. Acute inflammation is generally accompanied by systemic acute response which
involves induction of fever, increased production of white blood cells and production of a large
number of acute-phase plasma proteins. Chronic inflammation has a slow onset, its peak is rarely
reached and it subsides slowly if the irritant is removed. The time course and progress of the two
types of inflammation, acute and chronic, is depicted in Figure 14.8.
14.5.1 A C U T E I N F L A M M AT O R Y R E S P O N S E
Acute inflammatory response involves both localized and systemic responses.
L O C A L I Z E D I N F L A M M AT I O N
Within minutes of injury to a tissue, the blood vessels and capillaries show vasodilation and
increased vascular permeability in the affected area. This results in an increase in the volume of
blood to the lesion site. Increased blood volume reddens the tissue due to presence of red blood
cells (rubor). There is leakage of plasma from the blood vessels due to increased vascular permeability.
Neutrophils,Macrophages
(phagocytosis)
Intensity of Macrophages
inflmmation
Oedema
Erythema
Injury Time (Days)
Figure 14.8
Graph showing acute and chronic
Acute inflammation Chronic inflammation inflammation.
This results in the accumulation of fluid in the inflamed site which leads to the swelling of the
inflamed area (oedema). Tissue injury activates kinin, clotting and fibrinolysis. Many vascular
changes such as vasodilation and increased vascular permeability are due to the direct effects of
mediators such as bradykinin, fibrinopeptide, C3a, C4a and C5a, as well as mast cells histamines.
Within next few hours, vascular endothelium increases expression of E-selectin (or sometimes P)
under the influence of IL-1 or TNF-α. The circulatory neutrophils express mucin CAMs such as
« Neutrophils are the primary
PSGL-1, Sialyl Lewisa and Sialyl Lewisx which bind to E- (or P-) selectin on the endothelium. This phagocytic cells that arrive at the
binding tethers neutrophils to the endothelium, allowing cells to roll in the direction of the blood flow. inflamed site usually within 70–80
Chemokines bound on the endothelium activate neutrophils as discussed previously, induc- minutes.
ing conformational change and finally their transmigration into tissue from the blood. Once in the
tissue, activated neutrophils are guided to the lesion site by a gradient of chemoattractants (such as
C3a, C5a, fibropeptides and leukotrienes). Activated neutrophils (which have stimulated microbi-
cidal machinery, increased Fc receptors and increased granular content) bind and neutralize anti-
bodies or complement-coated microbes via phagocytosis. Since neutrophils and other phagocytes
are non-specific in killing invading pathogens, neutrophils and their products also induce tissue
damage to the host. Neutrophils also release mediators such as macrophage inflammatory protein « Macrophages arrive later than
(MIP-1α, MIP-1β) which attract macrophages to the lesion site. neutrophils, usually 5–10 hours
These macrophages contribute towards phagocytosis, as well as release mediators that aid in after the initiation of inflammation.
the recruitment and summoning of other leukocytes to the inflamed area. The macrophages se-
crete IL-1, IL-6 and TNF-α. TNF-α stimulates the expression of E-selectins which adheres neutro- « IL-1, IL-6, TNF-α are pro-
inflammatory and IL-4, IL-10
phils to the endothelium. IL-1 induces expression of ICAM and VCAM on endothelial cells that and TGF-β are anti-inflammatory
tether monocytes and lymphocytes. IL-6 induces the synthesis of acute-phase proteins. The net cytokines.
result of the action of all these mediators is the adhesion and transmigration of circulating mono-
cytes, lymphocytes and neutrophils from the blood vessels into the tissue spaces. In addition, these
cytokines also activate macrophages and neutrophils, promoting increased phagocytic activity and
increased release of damaging enzymes. These microbicidal activities of phagocytes clear the in-
vading pathogen and hence remove the irritant. However, they also induce significant damage to
the host tissue due to their non-specific mode of action. A schematic representation of localized
acute inflammation is shown in Figure 14.9.
SYSTEMIC ACUTE-PHASE RESPONSE

Localized inflammatory response is usually accompanied by general whole body systemic response
known as acute-phase response. This is marked by induction of fever, increased production of acute-phase « Acute-phase proteins activate
proteins, increased production of hormones (such as ACTH), and increased leukocyte production. the innate immune system.
Chemotaxis
MIP ROS,RNS
MIP
Lytic
enzymes
Fc receptor
Gradient of chemoattractant Bacteria
MIP
(chemokine, anaphylatoxins,
leukotrienes
Transmigration Killing and
Sialyl removal of
lewis IL-1,IL-6 irritant
TNF-α
ICAM/VCAM
Neutrophil expression on Activated
endotheltial macrophage
cells
Figure 14.9
An overview of the mechanism of acute
inflammation.
Within 24 hours of the onset of acute-phase inflammation there is an increase in the levels of
IL-1, TNF-α, and IL-6 which induces the production of acute-phase proteins such as C-reactive
protein, serum amyloid A protein and fibrinogen by heptocytes. C-reactive protein is the best
known example of an acute-phase protein. It has five identical polypeptides held by non-covalent in-
teractions. This protein binds to a wide variety of pathogens and activates complements, leading to
the deposition of C3b on microbes. C3b-coated microbes are readily phagocytosed by phagocytes
as they bear C3b receptors. The induction and role of acute phase proteins in systemic inflamma-
tory response is shown in Figure 14.10.
The increase in body temperature is also a non-specific defence response that helps in
inhibiting the growth of microbes, most of which are temperature-sensitive. Fever response is
induced by the action of IL-6, IL-1 and TNF-α on the hypothalmus.
Once the offending pathogen has been removed, damage-control and tissue-repair mecha-
nisms become activated. Pro-inflammatory cytokines IL-1, IL-6 and TNF-α are neutralized by
binding to their soluble receptors. Anti-inflammatory cytokines such as IL-4, IL-10 and TGF-β
are produced by TH cells. Other anti-inflammatory agents produced include C-protein, hor-
mones such as glucocorticoids and corticotrophin and α-melanocyte stimulating hormone.
These anti-inflammatory agents inhibit the production of nearly all pro-inflammatory media-
tors. As the inflammatory phase is neutralized, the repair of damage begins with accumulation
and proliferation of fibroblasts, which make the collagen required for extracellular matrix and
proper tissue repair.
14.5.2 C H R O N I C I N F L A M M AT O R Y R E S P O N S E
Some microbes such as mycobacteria, protozoa and fungi have survival mechanisms that allow them
to persist either inside the cell or outside the cell. The persistence of pathogens (antigens) leads to
chronic inflammation as the release of macrophage products including proteolytic enzymes, reac-
tive oxygen and nitrogen species results in significant tissue damage. On some occasions chronic re-
sponse is also induced by persistent immune complex, as in allergic alveolitis. Persistent stimulation
of the immune system contributes to significant tissue damage and wasting of tissue which is associ-
ated with many autoimmune diseases, infections (tuberculosis) and hypersensitivity (type IV).
Acute inflammation
IL-1,TNF- A ,IL-6,LIF
Fever
Liver
Bacterial growth
inhibited
Synthesis
Acute-phase proteins
(C-reactive protein,fibrinogen,serum amyloid A)
Bacterial surface coated

with acute-phase protein
Figure 14.10
An overview of the mechanism of
systemic inflammation
(IL-Interleukin, TNF-Tumour necrosis
factor, LIF-Leukaemia
Phagocytosis Complement-mediated lysis inhibitory facor).
Macrophages are probably the main cells involved in chronic inflammation. Under the influ-
ence of cytokines, macrophages assume a large flattened structure that continually secrete TNF,
potentiating inflammation (see Figure 14.11). These macrophages are called epithelioid cells
which may fuse to form a multinucleate giant cell. These cells mediate cellular destruction and
often lead to the formation of granuloma. Granuloma is a tumour-like mass consisting of a central
area of necrosis, surrounded by epithelioid cell/macrophage core, which in turn is surrounded by Granuloma
lymphocytes. Figure 14.12 shows a electron micrograph depicting a granuloma and a giant cell. Granuloma is one of a number
of forms of localized nodular
A granuloma is usually surrounded by collagen fibres (fibrosis) secreted by proliferating fibro- inflammation found in tissues.
blasts and macrophages. IFN-γ and TNF-α play a central role in chronic inflammation. IFN-γ is Granuloma can be induced either by
produced by activated T cells and NK cells. IFN-γ activates macrophages, the major cells involved a persistent antigen or its toxin at a
given site in the body. It is formed
in chronic inflammation. Activated macrophages, are more effective in killing intracellular patho- in a number of diseases such as
gen. They show increased cytokine production, increased class II MHC expression and increase Crohn’s disease, tuberculosis, and
in various proteolytic enzymes content. The accumulation of a large number of activated macro- sarcoidosis.
phages at the site of persistent infection results in aggravating tissue damage. TNF-α is released
by activated macrophages. The combined action of IFN-γ and TNF-α results in greater increase
in CAMs, facilitating the recruitment of a large number of cells in chronic inflammatory response
and aggravating tissue damage.
IL-12
Macrophage
TH cell
IL-3,IFN-G ,TNF-B
Immature
RNS
macrophages
ROS
Lytic enzymes Epithelioid cell

TNF-A
Fusion
Macrophage (activated) TNF
of cells
Multinucleate giant cell

Granuloma
T cell formed
Multinucleate giant cell
Figure 14.11
The process of chronic inflammation.
Macrophage
Persistent antigen
Figure 14.12
Chronic inflammation. Electron
micrograph showing granuloma around
schistoma ova. (From Encyclopedia
of Life Sciences.www.els.net, Kumar
Rakesh K and Dennis Wakefield (2005).
Copyright John Wiley & Sons Limited.
Reproduced with permission).
14.6 A N T I - I N F L A M M AT O R Y A G E N T S
The normal inflammatory response is a non-specific defence response that is beneficial to the host.
However, aggravated inflammatory response or long-term inflammation which is usually asso-
ciated with persistent microbial infection, autoimmune diseases and allergies can sometimes be
detrimental to the body. Hence, various therapeutic measures and agents are available to deal with
complications associated with inflammation. Some of the agents give symptomatic relief while oth-
ers actually interfere with the inflammatory process (see Figure 14.13).
Phospholipids
(macrophages,neutrophils,
mast cells)
Arachidonic acid
Aspirin Cyclo-oxygenase
pathway
inhibited
Binding of leukocytes Prostaglandins Thromboxane

to endothelial cells inhibited
Anti-CAM antibodies Non-steroidal drug
Decreased expression 1L-1
Bacteria Lysosomal
enzymes
Lympholysis Reduced chemotaxis, Decreased expression of

reduced phagocytic ability class II MHC,Release of IL-1
and lysosomal enzymes inhibited Figure 14.13
Mode of action of anti-inflammatory
Action of corticosteroids agents.
14.6.1 ANTI-CAM ANTIBODIES

The binding of leukocytes to vascular endothelial cells of the blood and their transmigration into
the tissue is a crucial step in inflammation. This adhesion is mediated by cellular adhesion mol-
ecules or CAM molecules and their ligands present on leukocytes and vascular endothelium. The
blocking of these adhesion molecules with anti-CAM antibodies will prevent the binding of CAM
with its ligand. Anti-ICAM-1 and anti-LFA-1 antibodies are used to inhibit inflammatory reaction
and increase the chances of kidney-graft survival. Anti-LFA-1 antibodies have been successfully
used to inhibit neutrophil build-up.
14.6.2 CORTICOSTEROIDS
Corticosteroids (prednisone, prednisolone and methylprednisolone) are potent anti-inflammatory
agents that have an immunosuppressive effect. Corticosteroid treatment causes a decrease in the
number of circulating lymphocytes as a result of either apoptosis/lysis of leukocytes (hence decreasing
their count) or inducing striking changes in the lymphocyte circulating pattern (so that less are visible « In some other species, such as rat
in the blood). Corticosteroid treatment causes lymphocytopenia (decrease in lymphocyte concentra- and rabbit, corticosteroid adminis-
tion) and monocytopenia. In humans, the decrease in lymphocyte/leukocyte population is not due to tration induces lympholysis.
apoptosis of leukocytes but due to a decrease in the number of circulating lymphocytes. Experimental
studies suggest that these cells are redistributed in the bone marrow and spleen.
Corticosteroids reduce chemotaxis, which in turn reduces the number of inflammatory cells
migrating to the lesion site. They also reduce the phagocytic and microbicidal ability of macrophages « Corticosteroids which are lipid-
and neutrophils, and this contributes to their anti-inflammatory action. Corticosteroids stabilize the soluble molecules are believed to
shut off genes involved in T-cell ac-
lysosomal membrane, decreasing the level of damaging enzymes released at the lesion site. They also tivation as well as those of cytokine
decrease the expression of class II MHC molecules, reducing the number of antigen displayed. They production.
also reduce the production and release of pro-inflammatory IL-1 by macrophages.
14.6.3 NON-STEROIDAL DRUGS

The most important and commonly used anti-inflammatory drug that is a non-steroid is aspirin
whose active ingredient is acetylsalicylate. Non-steroidal anti-inflammatory drugs (NSAIDS) exert
their anti-inflammatory effect by inhibiting the synthesis of inflammatory mediators–prostaglandins

and thromboxanes (inhibiting the cyclooxygenase pathway). The reduction in the concentration of
these mediators limits that chemotaxis of inflammatory cells, as well vascular permeability. NSAIDs
are effective and generally recommended in the treatment of acute and chronic inflammation.
Inflammation is a non-specific defence response by the body to an injury to the tissue. It is
characterized by increased blood flow to the lesion site, swelling, redness and pain at the affected site.
This non-specific defence is initiated by the process of leukocyte migration to the site of inflamma-
tion. Leukocyte migration requires the tethering of leukocytes to the walls of blood vessels, rolling
of leukocytes on the endothelium, arrest and activation, followed by trans-endothelial cell migration
(diapedesis) from blood vessels into the host tissues. Once these cells reach the lesion site they as-
sault and clear the pathogen, and initiate the repair process. Acute inflammation is of short duration,
has a rapid onset, sharp peak and rapid decline. Chronic reaction has a slow onset, the peak is rarely
reached, and it is followed by a slow decline. Acute inflammation can be localized or restricted to a
particular region of the body or it maybe systemic affecting the whole body. A large number of non-
specific and specific anti-inflammatory agents such as corticosteroids and anti-CAM antibodies are
now available to counter any complications resulting from inflammatory responses.
ELISA—Indirect Assay
Enzyme-linked immunosorbent assay (ELISA) is a widely used im- Antigen
muno-clinical technique for quantifying molecules such as antigen,
hormone, drug, etc., by the use of an antibody that is specific for
that molecule. This specific antibody is linked or conjugated with
an enzyme. The key reagent of ELISA is this enzyme-conjugated an-
tibody. The enzyme conjugated to the antibody can be visualized Antigen adsorbed
on to the well
and quantified by using chromogenic substrate. This technique
was developed independently by two research groups in 1971.
A. Schuurs and B. Weeman (Netherlands) and E. Engvall and
P. Perlmann (Swveden) used an amalgam of techniques to produce Primary antibody
a suitable alternative to radioimmunoassay which used radioactivity.
This new technique was called enzyme immunoassay or enzyme-
linked immunosorbent assay.
There are two main basic types of ELISA—indirect immunosorbent

assay and double antibody sandwich assay.
Antigen bound to the antigen
Indirect Immunosorbent Assay

In indirect immunosorbent assay, an antigen is first coated onto Coloured product
microtitre plates in an appropriate buffer. The antigen concentra- Substrate
tion may vary but is usually 20 μg/ml, and 50 μl of the antigen is

taken in each well. This antigen gets adsorbed onto the wells af- Enzyme-linked
secondary antibody
ter incubation of two hours at 37°C. The excess antigen is washed
away. The test antiserum (diluted,100 μl in each well) is then added
onto the microtitre plates. If this antiserum contains antibodies
(say, IgG) specific for that antigen, it will bind that antigen. This
antibody that directly binds to the antigen is called as primary
antibody. Unbound primary antibody is then washed off. After Enzyme-linked secondary antibody detects
washing, an anti-antibody (anti-IgG) that has a conjugated enzyme primary antibody bound to the antigen
(peroxidase or alkaline phosphatase) is then added (see Figure 14.14). Figure 14.14
The principle of indirect immunosorbent assay.
This secondary antibody which is an anti-IgG enzyme conjugate
will bind to the primary antibody bound to the antigen. The excess Indirect immunosorbent assay is used for testing the presence
enzyme-conjugated secondary antibody is then washed off. The of antibodies in patients with German measles, hepatitis B and
enzyme can then be detected by adding chromogenic substrate HIV. The appropriate antigen is coated in the wells of a microtitre
(p-nitrophenyl phosphate for alkaline phosphatase and hydrogen plate. The serum suspected to contain antibodies is then incu-
peroxide/ABTS for peroxidase). The formation of a coloured prod- bated with the antigen. Enzyme-linked secondary antibodies are
uct in the wells of the microtitre plates suggests the presence of then used to detect any antibodies of the serum that are bound
specific antibodies in the antiserum. Moreover the amount of coloured to the antigen.
product formed is directly proportional to the amount of antibody
present in the test antiserum.
S U M M A R Y
• Under normal conditions, the migration of blood cells through all • Inflammation can be acute or chronic. Acute inflammation is of
the tissues of the body ensures that a small number of a short duration, has a rapid onset, sharp peak and rapid decline.
antigen-specific immune cells that are normally present encounter Chronic reaction has a slow onset, the peak is rarely reached,
antigen entering the host body. followed a slow decline.
• Inflammation is a non-specific defence response by the body to an • Acute inflammatory response can be both localized and systemic.
injury to the tissue. Localized response is initiated by tissue injury that leads to
vasodilation and increased vascular permeability in traumatized
• Transmigration of leukocytes from the blood stream into an tissue. Tissue injury also activates plasma enzyme mediators, and
inflammatory site involves the attachment to several adhesion induces transmigration of neutrophils into the tissue from
molecules present on endothelial cells. the blood.
• Cell surface adhesion molecules (CAM) are membrane-bound • Stimulated neutrophils, released anaphylatoxins, bradykinin,
proteins involved in cellular recognition and adhesion. CAMs are histamine and other inflammatory mediators help clear the
classified into five major families––immunoglobulin superfamily offending pathogen at the inflamed site.
CAM, selectins, integrins, mucin and cadherin.
• Systemic acute phase response is marked by fever, increased
• The process of leukocyte migration to a site of inflammation production of acute phase proteins, hormones and leukocytes.
requires tethering of leukocytes to endothelium, rolling of
leukocytes on endothelium, arrest and activation, followed by • Persistence of an antigen leads to chronic inflammation as the
trans-endothelial cell migration (diapedesis) into the host release of products such as enzymes, reactive oxygen and
tissues. nitrogen species from immune cells results in significant
tissue damage.
• Leukocyte migration to the lesion site is guided by chemokines and
other chemotactic factors. • Persistent stimulation of the immune system accompanies many
autoimmune diseases, infection and hypersensitivity.
• Chemokines are low molecular weight polypeptides secreted by a
variety of cells that mediate chemotaxis of the immune cells. • Corticosteroids, anti-CAM antibodies and non-steroidal drugs
are some agents that deal with complication associated with
• Inflammatory response is mediated and controlled by inflammations.
(a) chemokines (b) plasma enzyme mediators (c) cytokines
(d) lipid mediators.
K E Y W O R D S
• acute inflammation 304 • corticosteroids 309 • lipid mediators 302

• adherence 298 • delayed hypersensitivity 306 • leukotrienes 296
• anaphylatoxins 303 • diapedesis 296 • localized inflammation 305
• anti-CAM antibodies 309 • granuloma 307 • margination 296
• CAM 296 • immunoglobulin superfamily 296 • mucin 297
• cell-surface adhesion molecules 296 • inflammation 296 • non-steroidal drugs 309
• chemokine 298 • inflammatory cytokines 303 • selectin 296
• chemotactic factors 298 • integrin 297 • systemic inflammation 305
• chronic inflammation 296 • kinin system 303 • very late antigens 299
1. Inflammation is an immune response that is considered 4. What are chemokines? How are they different from other
to be beneficial for the host, yet we administer a chemotactic molecules?
number of anti-inflammatory drugs when it
happens. Why?
5. Clinical trial investigating anti-inflammatory agents showed
prednisone (corticosteroid) has a potent immunosuppressive
2. What is the major difference between acute and chronic effect. What is the probable mechanism for this effect?
immune response? Can an acute phase response develop into Can you name a drug that is non-steroidal yet
chronic inflammation? anti-inflammatory?
3. What role do adhesion molecules play in the directional movement
of leukocytes during an inflammatory response?

1. Which one of them is not cardinal sign of inflammation? 6. A factor that play central role in chronic inflammation is:
(a) Rubor (a) IL-2
(b) Colour (b) TNF-β
(c) Tumor (c) IFN-γ
(d) Dolor (d) IL-6
2. One of the factors not important in diapedesis is: 7. Corticosteroids act by:
(a) Leukocyte adhesion (a) Decreasing the level of platelets
(b) Fluid shear stress of blood (b) Inhibiting binding to CAM molecules
(c) Change in tight junction of endothelial cells (c) Inhibiting synthesis of prostaglandins
(d) Expression of VLA on leukocyte (d) Reducing the number of circulating lymphocytes
3. One of the CAMs is not a member of immunoglobulin 8. Two classes of cells important in mediating localized
superfamily: inflammation:
(a) Gly CAM-1 (a) Macrophage and endothelial cells
(b) V-CAM (b) Endothelial cells and neutrophils
(c) MadCAM (c) Macrophages and neutrophils
(d) ICAM (d) TH cells and neutrophils
4. Chemokine triggers its action by binding on: 9. Cells mainly involved in chronic inflammation are:
(a) Serpentine receptor (a) NK cells
(b) Adrenergic receptor (b) Neutrophils
(c) CD23 (c) Macrophages
(d) None of the above (d) T cells
5. One of the factor that is not involved in generation of 10. One cell that does not undergo diapedesis are:
bradykinin is: (a) Macrophage
(a) Hfa (b) Neutrophils
(b) C3a (c) Lymphocytes
(c) Kallikrein (d) Endothelial cells
(d) Kininogen

1. Basement membrane underlying endothelial cells sterically inter- 4. Cyclo-oxygenase pathway that utilizes arachidonic acid
feres in diapedesis and hence is digested by endothelial cells. produces leukotrienes.
2. Chemotaxis is directional movement while chemokinesis is non- 5. TGF-β produced by TH cells is a pro-inflammatory cytokine.
directional migration.
3. Both clotting system and fibrinolytic system are involved in
inflammatory response.
Brubaker, R. R. (1985). “Mechanisms of Bacterial Virulence”, B. Champion (eds), Advanced Immunology, 3rd edn
Annual Review of Microbiology, 39: 21–50. London: Mosby.
Gabay, C. and I. Kushner (1999). “Acute Phase Proteins and Miller, M. D. and M. S. Krangel (1992). “Biology and
Other Systemic Response to Inflammation”, New England Journal Biochemistry of the Chemokines: A Family of Chemotactic and
of Medicine, 340: 448–454. Inflammatory Cytokines, Critical Reviews of Immunology,
Kashkin, K. P. et al. (1987). “Immunomodulatory Activity of a 12: 17–46.
Chemotactic Peptide Conjigated with a Liposomal Antigen”,
` Osborn, L. (1990). “Leukocyte Adhesion to Endothelium in
Immunologiya, 6: 37–40.
Inflammation”, Cell 62: 3–6.
Lawrence, M. B. and T. A. Springer (1993). “Neutrophils Roll on
E-selectin,” Journal of Immunology, 151: 6338–6346. Sameulsson, B. (1983). “Leukotrienes: Mediator of
Immediate Hypersensitivity and Inflammation”, Science,
Luscinskas, F. W., Y. Lim and A. H. Lichtman (2001). “Wall Shear 219: 568–75.
Stress: The Missing Step for T-cell Transmigiration?”, Nature Im-
munology 2: 478–480. Von Andrian, U. H. (2001). “PK-β (1): The Whole Ignition
System or Just a Sparkplug for T-cell Migration?”, Nature
Male, D. K. (1996). “Cell Traffic and Inflammation”, in
Immunology, 2: 477–78.
D. K. Male, A. Cooke, M. Owen, J. Trowsdale and
During ancient times, it was believed that disease was caused by quali- “There is no
tative changes in the four humors that consistute the human body— little enemy.”
blood (sanguis), phlegm (pituita), yellow bile (chole) and black bile —FRANKLIN
(melaine chole) of the body. It was Abu-Bekr Mohammed Ibn Zakariya

al-Razi, more commonly known as Rhazes (CE 880–932 AD), who gave
the first modern clinical description of a infectious disease (smallpox) in
his Treatise on the Smallpox and Measles. He suggested that the person
who had suffered from smallpox once in his/her lifetime will not be af-
flicted again. He, however, wrongly believed that smallpox was caused
due to the fermenting action of blood because of “excess moisture”.
Girolamo Fracastoro, in 1546, gave the idea for the first time that the
disease was caused by small seeds (which he called seminaria) and After studying this chapter,
these seeds could spread from person to another. He thought that
• Describe innate and adaptive
these seeds could arise spontaneously within an individual or from air immune responses to viruses
• Explain the various strategies
or earth or water. The 17th and 18th centuries saw the development of used by viruses to evade the
host immune response.
many interesting hypotheses regarding the occurrence and spread of
• Define and illustrate antigenic
infectious diseases. It was only after the studies of Pastuer (who had drift and antigenic shift
• Give an account of the immune
already experimentally negated the hypothesis of spontaneous gener- response against extracellular
and intracellular bacteria
ation using the famous swan-neck flask experiment in 1859) and Koch • Describe how bacteria can
evade host defences
(work on fowl cholera bacilli in 1880) that it was established that a dis-
• Explain the etiology and
crete etiological agent (bacteria in this case) could cause a disease, and pathogenesis of diphtheria,
tuberculosis and Lyme disease
attenuation of these bacteria could be achieved in vitro. Pastuer fur- • Briefly summarize the host
immune response against
ther showed that the introduction of these attenuated bacteria could Plasmodium, trypanosome and
Leishmania spp.
induce immunity in experimental fowls against cholera. Further work • Give an account of the host
immune response against
on infectious agents and their associated immune response led to the parasitic worms, particularly
against helminths
elucidation, understanding and control of a large number of infectious
diseases. The prevalence of infectious diseases in today’s world is
summarized in Figure 15.1.
Immune Response
to Infectious Agents 15
15.1 INTRODUCTION
The primary function of the immune system is to protect the host against pathogenic microorgan-
isms. Acute infections represent a ceaseless battle between replication of pathogen and host im-
munity; the usual outcome is either termination of infection followed by recovery or death of the
host. The battle of supremacy places pathogens in a difficult situation. If a pathogen is too readily
eliminated by the host, it becomes extinct. On the contrary, if a pathogen is too virulent and cannot
be overpowered by the host, the host dies and, theoretically, the microbes again face extinction.
Thus, infectious agents have developed mechanisms that allow transfer of microbes from infected
to healthy host body, and host defences are tempered in such a way that the host survives long
enough to permit transfer of infectious agents. The development of infection involves a complex
series of events, including entry of the microorganism, colonization of the host tissue, evasion
from host defences, and tissue damage or functional impairment of the host body. The number of
deaths due to infectious diseases is summarized in Figure 15.2. The mechanism and pathogenesis
of microbes is beyond the scope of this book and will not be discussed here. Rather, we will focus
on various host defence reactions that provide immunity to microbes.
The defence against infectious agents is mediated both by innate immunity and adaptive im-
munity. Innate immunity provides a non-specific early defence till more specific adaptive immunity
AIDS
Diphtheria
Lassa fever
Lyme disease
Malaria
Tuberculosis
Typhoid fever
Figure 15.1
World map showing infectious diseases.
Infectious diseases appears at later stages. This chapter focuses on

15 million immune response to four main types of patho-
genic microorganisms—namely, virus, bacteria,
protozoa and helminthes—that sum up the main
Cardiovascular
features of immunity to infectious agents.
diseases 17
million
15.2 IMMUNITY TO
Death due to VIRUSES
other reasons
Viruses (derived from the Latin word virus which
means poisonous liquid) are obligate intracellu-
Neoplastic lar microbes that replicate only within the host
diseases cell, often using the cell’s biochemical machinery
7 million
(nucleic acid, protein and sugar-metabolizing
Injuries machinery). Viruses are extremely diverse in
5 million
terms of structure, nucleic acid machinery, ways
Figure 15.2 Asthma and of replication and proteins they encode. A virus
Pie diagram showing leading causes of pulmonary diseases infects a large population of cells using normal
death. 3.0 million cell-surface molecules as gateways or receptors.
Following entry, the virus uncoats, with release
of nucleic acid. The transcription of viral genome produces proteins that are necessary for the es-
tablishment of infection as well as formation of new viral particles. When a virus replicates in the
host cell, it disrupts the normal cellular machinery and ultimately kills the host cell. This is called
the cytopathic effect of the virus and is usually manifested by those viruses that lyse (lytic virus)
the cell. Moreover, the antiviral immune response that ensues also damages the host cell and tis-
» There are 15 million deaths annu-
ally due to causes that are directly sues together with the virus. Such an immune response that is itself harmful to the host is termed
related to infectious diseases. an immunopathological response.
15.2.1 I N N AT E I M M U N E R E S P O N S E T O V I R U S E S
» Interferons act very rapidly, usu- The early or innate immune defence against virus includes interferons (IFN), natural killer (NK)
ally within a few hours or a few cells and macrophages. The viral infection of a cell directly stimulates the production of type I IFN
days, against a number of viruses.
Interferons were first identified by (that includes IFN-α and a single IFN-β, as well as the less-known IFN-ω and IFN-τ) by infected
their ability to interfere with virus cells.
replication. The type-I IFN functions to inhibit viral replication in both infected and uninfected cells by inducing
an antiviral state. IFN-α/β binds to IFN-α/β receptor present on host cells that trigger IFN signal
transduction pathways. Stimulated signal transduction pathways lead to transcriptional activation
of 30 or more cellular genes whose products are responsible for inducing the antiviral state. Some
of the important gene products that have direct antiviral action are discussed below.
• IFN-induced protein kinase R (PKR; 67 kDa) is an inactive kinase and requires dsRNA
for its activation by autophosphorylation. dsRNA is uncommon except in replicating vi-
» Interferons do not have any
ruses, and it is believed that activation by dsRNA is related to the establishment of an an-
antiviral activity of their own. They
induce an antiviral state in other tiviral state. Activated PKR whose concentration rises by 5- to 10-fold upon interferon
cells by activating or inducing po- stimulation, can phosphorylate eIF-2α to inhibit protein synthesis and block viral replication
tent antiviral genes. in the infected cell.
• IFN also induces the family of dsRNA-dependent 2'-5' oligoadenylate synthase. These enzy
mes include three separate components—2, 5 synthase, endoribonuclease RNaseL and 2,
5 phoshodiesterase. These enzymes synthesize novel 2'-5' phosphodiester-linked oligoad-
enylates (2'–5'A), that function to activate a latent ribonuclease RNaseL. RNaseL degrades
single-stranded RNAs of cellular and viral origin, and induces an antiviral state in the cell.
Since oligoadenylate (2'–5'A) is unstable and does not last long, response of 2'–5'A is tran-
sient.
• IFN also induces the synthesis of the isoform of dsRNA-specific adenosine deaminase
which converts adenosine to inosine in viral and cellular RNA. The consequence of this
» All nucleated cells express recep- IFN-induced RNA editing is amino acid substitution and, hence, the formation of an inac-
tors for α and β interferons. tive viral protein.
IMMUNE RESPONSE TO INFECTIOUS AGENTS 317
Virus
Target cell Viral infection
Interferon
Induces 2’-5’-oligo
Activates adenylate synthase Induces
Protein kinase R adenosine
deaminase
Activates
RNase L
Inhibits protein
Inactive viral
synthesis
Degrades ssRNA protein formed
Antiviral state
induced in
neighbouring cell
Interferon-mediated protection
Viral antigen Fc receptor
Immune
Response
Virus-infected NK cell Perforin

cell
Antibodies Antibodies bind Target cell
formed target cell lysis
Figure 15.3
Various innate defence mechanisms of a
NK cell-mediated cell lysis host body against a virus.
« IFN-γ, a type II interferon, is

The binding of IFN-α, IFN-β and IFN-γ to NK cells activates them for killing virally infected primarily produced by NK cells.
cells. NK cells kill virally infected cells by binding to target-cell-bound-IgG. NK cells attach to
antibody-coated cells via their Fc receptors triggering the release of perforin (pore-forming protein) « Mx proteins are interferon-
and target cell death. The role of interferon and NK cell in limiting viral infection is illustrated in induced GTPases that have a
wide range of antiviral activities
Figure 15.3. The definitive role of NK cells in viral infection has been established using a mouse against a variety of RNA viruses
model. The depletion of NK cells in vivo by administering anti-NK cell-antibodies leads to en- such as bunyaviruses and
hanced susceptibility to viruses such as murine cytomegalovirus (MCMV) and recombinant vac- orthomyxoviruses. Mx GTPases
detect viral infection by detecting
cinia virus, but not to other viruses such as a lymphocytic choriomeningitis virus (LCMV). NK-cell nucleocapsid-like structures inside
activity is initiated two to three days before the specific T-cell response. the cell.
Other anti-viral mechanisms that are more specific in action include the presence of specific en-
zymes with binding activity for dsRNA, called Mx proteins, which are induced by IFN. Mx proteins,
though initially discovered for the influenza virus, interfere with the viral replication of a number of
RNA virus families, including members of Orthomyxoviridae, Paramyxoviridae, Togaviridae.
15.2.2 V I R A L N E U T R A L I Z AT I O N B Y A N T I B O D Y
AND COMPLEMENT
Humoral immunity plays an important role in limiting the spread and re-infection of viral particles.
Antibodies can exert antiviral activity via several different, yet related, mechanisms.
• Antibodies may bind to the surface of a virus, preventing its binding to the cellular receptor.
Hence its entry in the target cell is inhibited: for example, influenza virus binds to the sialic
acid residues in cell membrane glycoprotein and glycolipid. Antibodies, formed against the
viral antigen that binds to sialic acid residues, prevent the entry of the virus into host cells.
• Antibodies may agglutinate viral particles, reducing their number and hence limiting their
» IgM is best suited for inducing
agglutination in vitro because of its spread. Agglutination of viral particles makes them more susceptible to phagocytosis.
pentameric structure. • The secreted antibodies of IgA type may be important for neutralizing viruses that en-
ter via respiratory and intestinal mucosa. The complement cascade which forms a part
of both innate and adaptive immune systems also plays an important role in combating
viral infection. Complement factors neutralize virus by several different mechanisms:
• Complement factors and fragments can blanket the antibody-coated virus rendering it
completely enveloped and incapable of binding to its receptor on the host cell.
» Complement-coated virus is • Like antibodies, complement factors can clump viruses reducing the number of
targeted efficiently at cells that infectious units.
express complement receptors.
This effect has been documented • The binding of C3b on a virus–antibody complex makes it more susceptible to
for CD21 (CR2) and CD35 (CR1) ex- phagocytosis.
pressed on B cells and on follicular • Complement factors can bind antibody-coated viruses that possess lipid envelope, and
dendritic cells.
induce lysis.
• Complement factors alone can inactivate certain viruses in the absence of any specific
antibody: for example, several retroviruses bind Clq which in turn activates the comple-
ment cascade and induces lysis of pathogen. This occurs because viral protein alone
(rather than antigen–antibody complex) can act as a receptor for Clq. Adaptive immune
responses triggered against viruses are shown in Figure 15.4.
15.2.3 T - C E L L - M E D I AT E D A N T I V I R A L M E C H A N I S M
Humoral antibodies act against viral infection by blocking virus-binding and entry into the host
cells. However, once a virus has gained entry into the host cell, antibodies become ineffective and
cell-mediated immune mechanisms come into play to eradicate the infection. In general, Tcyt cells
Virus Viral peptide

Virus Class I MHC
Antibody
Antibody
Fas
Perforin
FC receptor
FasL
Phagocyte
Entry of virus Virus agglutination and Cytolysis by Tcyt

blocked phagocytosis
C3b C3b
Complement
Enveloped
activation
virus
C3b I
CR Antibody
(alternate or
classical
iC3b pathway) Virolysis
Figure 15.4 Coating of complement Opsonization of virus Complement action

Line diagram showing how adaptive proteins prevents binding by complement
immune responses neutralize viral of virus to receptor
infections. on host cell
and TH1 cells are the main components of cell-mediated antiviral defence. Antigen-specific recogni-
tion mediated by the TCR of Tcyt cells and TH1 cells results in the activation of T-cell effector func-
tions. Tcyt cells identify and destroy virus-infected cells. Tcyt cells recognize viral peptides displayed
on the surface of infected cells together with class I MHC molecules. These Tcyt cells release granules
of the protein-perforin on the target cell membrane. These perforin molecules form transmem-
brane pores in the target cell membrane, lysing and killing the target cells. Virtually all the cells
of the body express class I MHC molecules, making it easier for Tcyt cells to identify and eliminate
virus-infected cells. T cells undergo massive proliferation during viral infection, producing T cells
specific for the viral peptide–Tcyt complex. Tcyt cells can also kill virus-infected cell by inducing ap-
optosis in target cells. Fas expressed on virus-infected cells interacts with FasL on T cells, causing
apoptosis of virus-infected cells. The Tcyt cells destroy the virus-infected host cells, ensuring that
new progeny viruses are eliminated before they are released from the cell. Apart from the above
mechanism, Tcyt (as well as TH1) cells also counter viral infection by releasing cytokines. Tcyt cells
release IFN-γ and TNF-α, while activated TH1 cells release IL-2, IFN-γ and TNF-α. Incubation
or contact of target cells with IFN-γ increases their sensitivity to Tcyt cells. IFN-γ also activates
macrophages. IL-2 acts indirectly by recruiting the Tcyt cells towards the virus-infected cell. TNF-α
has several antiviral activities that help in cell mediated response. However, there is a tight
regulation of cytokine secretion by T cells. Virus-specific Tcyt cells produce cytokines only on « CD4+ Tcyt cells have been detected
antigen contact with the infected cell and its production is terminated the instant the contact is in HIV-infected individuals.
broken, ensuring that cytokines, which in excess is toxic to the host, is produced only at a site
where it is needed.
Apart from Tcyt cells and TH1 cells, in measles virus infection CD4+ Tcyt cells (present in minute
numbers) play an important role. CD4+ Tcyt cells recognize the antigenic determinant together
with class II MHC molecules displayed on infected cells and kill the cells. However, these CD4+ Tcyt
cells, which function against a specific virus, do not seem to have a broader role in cell-mediated
immunity against viral antigens.
15.3 V I R U S S T R AT E G I E S F O R T H E E VA S I O N
OF HOST IMMUNE RESPONSE
The ability of a virus to evade host immune or defence mechanism is critical to the survival of the
virus in the host. A virus should save itself from the host defence as well as be able to move effi-
ciently among different hosts. Thus a virus can either avoid the host immunity or inhibit or disrupt
it. Table 15.1 lists some of the strategies/products used by viruses in evading immune response.
Host Response Virus Viral Factor Mechanism

Affected
Class I MHC pathway EBV Gly-Ala repeat Resistance to proteasomal
degradation
HSV ICP-47 Blocks peptide binding to TAP
Humoral/Cell-mediated EBV - Viral presence causes destruction
immunity HIV - of B (EBV) and/or T (HIV) cells
Detection by antibodies Influenza New/mutant surface Mutant surface HA and NA es-

HA and NA antigen capes detection by antibodies
Apoptosis Pox virus MC-159 Inhibits Fas-mediated apoptosis
Adenovirus E1B Delays apoptosis
Production of cytokine Myxoma IFN-γ receptor homo- Competes for IFN-γ and blocks
receptor logue its function
Cytomegalovirus Chemokines receptor Competes for chemokines,
homologues blocks their function
Table 15.1
Note: EBV—Epstein–Barr Virus; HSV—herpes simplex virus; HIV—human immunodeficiency virus; IFN—interferon; HA—haemagglutinin; Selected viral products that interfere
NA—neuraminidase; TAP—transporter associated with antigen processing. with host immune response.
15.3.1 V I R A L AV O I DA N C E O F I M M U N E R E S P O N S E
D O W N R E G U L AT I N G C E L L U L A R P R O T E I N S
»T cells require 10–100 MHC– Since viruses are intracellular parasites, they are detected and killed by Tcyt cells. Tcyt cells detect
peptide complexes to respond to viral peptides together with class I MHC molecules and initiate cytolytic activity.
infected target cells.
Virus can deploy various strategies to escape host immunity. These are:
• Some viruses shut down protein synthesis during the latent period so that no viral pep-
tides are formed and, hence, displayed on infected cell surface, for example herpes virus.
• Some viruses can downregulate manifold expressions of cell surface glycoprotein such
as class I MHC and class II MHC molecules after infection. Targeting of class I MHC
» Studies on mouse models have molecules is done by herpes simplex virus, cytomegalovirus, adenovirus. Class II MHC
shown that the vaccinia virus (used molecules are downregulated by HIV and Cytomegalovirus which renders viral infected
for making small pox vaccine)
causes disruption of the class II cell “immune” not only to Tcyt cells but also to TH cells, thus avoiding both cell-mediated
pathway and humoral immunity.
• Some viruses have evolved peptides that are less digestible by cellular processing of class I
MHC pathway. These include EBV antigen (that contains resistant Gly-Ala repeat) of
Epstein–Barr virus that is resistant to proteasomal degradation.
E VA S I O N O F A N T I B O DY D E T E C T I O N
Viruses, and in particular RNA viruses, respond to the host immune defence by evolving mechanisms
that escape detection by pre-existing antibodies. This strategy which is best explained by an influ-
enza virus, involves generating new mutant antigenic sequences of surface antigens—hemagglutinin
(HA) and neuraminidase (N)—help in escaping antibody detection and provides temporary edge
to the infection. This is more prominent in RNA viruses because of the high mutation rate of RNA
polymerase which provides diverse variants or mutants from which the best form, which is least
noticeable, is selected.
E VA S I O N O F T - C E L L R E S P O N S E
Viruses can also mutate those viral peptides that are presented to the immune system as T-cell epitopes.
Studies with HIV have shown that viruses can mutate in vivo into Tcyt cell-escape variant. The cells are
then not recognized by Tcyt cells and hence escape elimination by T-cell mediated onslaught.
HIDING IN IMMUNE-PRIVILEGED SITES

Certain regions of the body appear to be immune privileged, that is, they have little or no immune
surveillance as immune reactions at this site will be catastrophic to the body. These include the an-
terior chamber of eye and the central nervous system (particularly meninges of the brain). Several
virus families have evolved to survive at these privileged sites to avoid immune-mediated destruc-
tion. These include herpes simplex virus which causes infection of the sensory nerve ganglia, polio
virus, mengovirus and alphavirus which infects neuronal cells.
E VA S I O N BY I N H I B I T I N G H O S T I M M U N I T Y
» In vitro studies have shown that DESTRUCTION OF SPECIALIZED ANTIGEN-PRESENTING CELLS. Antigen-presenting cells of
activated macrophages are 10- to both professional and non-professional types endocytose antigens and present it to effector cells
100-fold less efficient in antigen
presentation than professional
of the immune system. The destruction of antigen-presenting cells usually leads to generalized
antigen-presenting dendritic cells. immunosupression. LCMV variants are known to cause suppression of antibodies and T cells
However, activated macrophages by infecting specialized antigen-presenting cells, interdigitating dendritic cells, which are then
are 10 to 100 times more efficient
than antigen-presenting B cells.
destroyed by Tcyt cells, causing transient immunosuppression.
INHIBITION OF ANTIGEN PRESENTATION. Since class I MHC molecules are expressed on all
somatic cells which make them prone to Tcyt-mediated lysis, a vast majority of viruses have evolved
varied mechanisms to inhibit antigen processing and display of peptides. Herpes simplex virus
and human cytomegalovirus (HCMV) are capable of blocking antigen processing and presentation
pathways at several stages. The herpes simplex virus protein, ICP47, binds to the cytosolic surface
of the human transporter molecule (TAP) present in the ER and inhibits the transport of peptides
in ER. The human cytomegalovirus protein, US3, retains the peptide–MHC complex in the ER, and
protein US6 binds TAP molecules (on the luminal side) which inhibits peptide transport in ER.
A 19 kDa protein encoded by adenovirus binds to class I MHC and prevents its transport to the
cell surface. It should remembered that certain viruses are also capable of inhibiting class II MHC
pathway: for example, Epstein–Barr Virus protein, BZLF2, inhibits antigen presentation by class
II pathway.
DELAYING APOPTOSIS OF VIRUS-INFECTED CELLS. Some Tcyt cells and TH cells eliminate
virus-infected cells by induction of apoptosis. It would be beneficial for the virus if it can devise a
mechanism that delays, if not inhibits, apoptosis till it is ready to leave the target cell. This happens « Molluscum contagiosum virus
with the adenovirus which encodes protein E1B that delays apoptotic process. Epstein–Barr virus codes for two proteins, MC159 and
MC160, that have death effector
protein BHRF1, a bcl-2 homologue, acts in a similar way and allows the survival and proliferation domains resembling those of cel-
of an infected cell. Protein MC159, a product of Molluscum contagiosum virus (pox virus) inhibits lular regulators of apoptosis.
the Fas-mediated apoptosis of the infected cells.
INFECTION OF LYMPHOCYTES. The easiest way to sabotage the immune system is by infecting
immune effector B and T cells. These cells could either be rendered dysfunctional because of the
Virus x x
Resistant viral
peptide
Viral m RNA
Class Class
I MHC II MHC
Dowregulation Proteosome
Shutting down of viral
protein synthesis. No chopping of
No display of viral viral protein
peptides on MHC
Decreased display of viral proteins on MHC
Antigen-presenting CD4+ /CD8+ B cells

cells T cells
Viral destruction of immune cells
No entry for TAP blocked

Peptide x viral peptides
fragments
Viral TAP interacting with viral protein
proteins Retention of
MHC–peptide
complex in x x
ER Prevents transport
to cell surface
Figure 15.5
Line diagram showing how viruses evade
Inhibition of antigen presentation immune responses.
disruption of cellular machinery or may be promptly killed by specific Tcyt cells. Both the ways are
beneficial to viruses. The most common examples include infection of TH cell by HIV-1 causing
their destruction/elimination and B-cell disruption caused by Epstein–Barr virus and LCMV.
The measles virus infects all the cells of the immune system such as B cells, T cells and antigen-
presenting cells, which results in transient immunosuppression. Some of the important strategies
that are used by viruses for evading immune responses are depicted in Figure 15.5.
15.4 VIRAL INFECTIONS: INFLUENZA

» Influenza A viruses are endemic The International Committee for the Taxonomy of Viruses has included the influenza virus in the
gastrointestinal viruses of wild family Orthomyxoviridae (Greek: orthos—correct; myxa—mucus) because of its ability to bind to
water fowls.
the mucus. The word influenza is derived from Latin word influentia which implies epidemic which
was believed to occur due to the influence of stars. Influenza affects the central respiratory system
in humans, birds, horses and even seals.
Influenza is a widespread human disease, affecting about 10–20 per cent of the US population
» Infections in animals that are
transmitted to humans are referred each year, killing up to 50,000 individuals. The 1918 pandemic was the worse influenza pandemic
to as zoonoses. in which 20 million people died worldwide, many of them in the prime of their lives.
15.4.1 THE INFLUENZA VIRUS

The influenza viruses are of three types—A, B and C. The A, B and C types of viruses are distinguished
on the basis of antigenic difference between their nucleocapsid (NP) and matrix (M) proteins.
In general, influenza viruses have a fairly regular spherical appearance of 80–120 nm in dia-
meter. The structure of type A (see Figure 15.6) is discussed here. The virus is composed of 20 per
cent lipid, 70 per cent protein, 5–8 per cent carbohydrate and 1 per cent RNA. It is an enveloped
virus having an outer lipid membrane which is derived from the plasma membrane of the host
cell. Inserted into the lipid envelope are about 500 spikes radiating 10 nm outwards. The spikes
» Influenza A and B viruses encode
three integral membrane proteins, are of two types—rod-shaped spikes of haemagglutanin (HA) and mushroom-shaped spikes of
haemagglutinin (HA), neuramini- neuraminidase (NA) glycoproteins. The viral matrix protein (M) underlies the lipid bilayer and is
dase (NA), and M2 (influenza A virus) associated with the ribonucleoprotein core of the virus. The nucleocapsid core of influenza virus
or NB (influenza B virus). Influenza
C viruses encode only two integral consists of eight segments of single-stranded RNA (ssRNA). The influenza virus C contains seven
membrane proteins, HEF and CM2. segments of ssRNA.
The HA spikes extend in the form of trimers from the lipid envelope. The spikes are involved
in binding to sialic-acid-containing receptor on the host cell surface, which in turn is involved in
fusion of the endocytosed virus with endosomal membrane in the cell. HA is so named because of
the ability of the virus to agglutinate red blood cells by attachment to specific sialic-acid-containing
cell surface molecules. NA is an hydrolase enzyme (acylneuraminyl hydrolase) which catalyses the
cleavage of linkage between terminal the sialic acid and the adjacent carbohydrate moiety. NA is an
integral membrane protein containing a mushroom-shaped head that is enzymatically active and a
stalk region that is attached to the membrane. One function of NA is the removal of sialic acid from
HA and other cell surface molecules, permitting the transport of the virus through the mucus layer
of the respiratory tract, enabling the virus to find target epithelial cells. The matrix (M) proteins
underlie the viral lipid envelope and provide rigidity to the membrane. It is believed that M proteins
interact with HA and NA proteins, as well as viral RNA.
The influenza virus enters the cell by receptor-
» Influenza A virus can cross the mediated endocytosis through the clathrin-coated
species barrier into farm animals pits. After internalization, the virus containing the
and humans. Influenza B virus is
usually found in humans and are coated vesicle, uncoats and fuses with the endosome
Nucleocapsid
less harmful than influenza A virus. having acidic pH. The HA of the virus mediates fu-
Influenza C virus causes only a mild sion of the endosome membrane and the viral mem-
illness in humans and cannot cause RNA
any epidemics. brane and all of the viral RNA enters the cytosol
Matrix protein where it replicates and expresses viral proteins.
The naming of influenza as well as other vi-
Envelope ruses is done according to the nomenclature of
WHO—defined by animal host (specified, if not
Neuraminidase human), geographical origin, strain number,
Figure 15.6
Detailed structure of influenza virus. Hemagglutinin year of isolation and antigenic description of HA
and NA. Strain A/Japan/305/57 implies strain A (isolate 305) that arose in humans in Japan in 1957.
Antigenic description of HA and NA is given within bracket: for example, A/Sw/Iowa/15/30 (H1N1)
designates strain A isolate 15 that arose in Iowa in 1930 and has antigenic subtype 1 of HA and NA.
15.4.2 ANTIGENIC DRIFT AND ANTIGENIC SHIFT

A novel feature of the influenza virus is its ability to make either a little or a drastic change in its sur-
face antigens—HA and NA. The change in virus antigens is sometimes so drastic that the immune
response to infection with the virus that caused a previous viral attack gives little or no protection
against the virus causing subsequent epidemic. Minor changes in antigenic character of influenza vi-
ruses occurs as a result of the accumulation of mutation (and hence amino acids changes) in surface
antigens HA and NA. This process of minor antigenic changes of influenza virus is known as anti-
genic drift and seems to be accumulating with time. The extensive changes in HA and NA that leads
to the appearance of subtypes of human influenza virus is known as antigenic shift. A schematic
representation of antigenic shift and antigenic drift is shown in Figure 15.7. The influenza virus has
eight RNA segments and during co-infection of a single cell by two viruses (for example, A and B),
the segments of both infecting viruses can mix and re-assort. HA-coding segments, for example,
may come from virus A, while the rest of viral segments may come from virus B. Such molecular re-
assortment appears to take place in non-human hosts, most probably in birds or farm animals, where
there is a co-infection of human and avian viruses. Such periodic gene re-assortments are likely to « Periodic gene segment re-
cause antigenic shifts which are responsible for severe influenza epidemics and pandemics. assortments produce antigenic
changes in the influenza virus.
15.4.3 IMMUNE RESPONSE TO INFLUENZA

INFECTION
The immune response of the host to an influenza virus is the production of antibodies specific
for the HA molecule. This antibody protects the human body against influenza infection but its
memory B cells are of almost no use as the influenza virus undergoes antigenic drift. So the next
time a “different phenotype” of influenza virus will infect the host, and new antibodies will be
formed. “Old” antibody molecules will be useless against this new phenotype of virus. Antibodies
Antigenic
drift
Influenza virus New strain of Influenza virus
Antigenic
shift
Figure 15.7
New and very different strain Diagram showing the difference between
Influenza virus
of Influenza virus antigenic shift and antigenic drift.
are usually directed against the conserved sialic-acid-binding cleft of HA, which is necessary for the
binding of virus to the target cells. The antibody titre peaks within few days of infection, and then
declines and plateaus. Antibodies against influenza virus remain in circulation for several months,
even years, and provide resistance against re-infection by the same strain of virus.
15.5 IMMUNITY TO BACTERIAL

INFECTIONS
The immune response directed against bacteria are related to the structure of the invading bacteria
(Gram-negative or Gram-positive), place of its residence in the tissue (for example, respiratory
tract, blood), place of its growth (intracellular or extracellular) and of course their virulence (which
involves toxicity and/or invasiveness).
15.5.1 FIRST LINE OF DEFENCE

» The secretions of sebaceous The physical and chemical barriers of the body from the first and most primitive barrier to the
glands present in the skin are entry of the infection. The skin and the exposed epithelial surfaces have non-specific innate
bacteriocidal.
defence mechanisms, which restrict the entry of pathogen. Intact skin is rarely penetrated by bac-
teria. Moreover, some fatty acids produced by the skin are toxic to some bacteria.
The presence of lysozyme in tears and saliva, the acidic pH of stomach and gastric enzymes
of the gastrointestinal tract provide chemical barrier to pathogens entry. Epithelial surfaces are
constantly cleansed by the flushing of fluids (for example, tears in eyes) and by ciliary action (in
trachea) which removes microbes bound to the mucus. This first line of defence provides a non-
specific defence mechanism to the entry of a variety of pathogens, including bacteria. In practice,
however, these barriers are not inpenetrable. Several species of bacteria can still overcome these
barriers and enter the host body.
Bacteria that enter the host body could be of two types:
• Extracellular bacteria—These bacteria are capable of surviving and replicating outside the
host cell, that is, in tissue spaces such as airways and intestinal lumen. (for example, Vibrio
cholerae, Clostridium tetani).
• Intracellular bacteria—These bacteria are facultative cellular parasites, capable of surviv-
ing and even replicating within the cells such as phagocytes. (for example, Listeria mono-
cytogenes and Mycobacteria).
15.5.2 IMMUNE RESPONSE TO EXTRACELLULAR

BACTERIA
The pathogenicity of extracellular bacteria occurs via two main mechanisms. Bacteria produce a
variety of cytotoxic substances called toxins which harm the host body. These include endotoxin, a
component of cell wall of Gram-negative bacteria (a type of lipopolysaccharide), that is released by
bacterial disintegration; and exotoxin which is proteinic in nature and is actively secreted by bac-
teria. These toxins kill or damage the host cell by interfering with their normal cellular functions
and causing the disease.
I N N AT E D E F E N C E
» TLRs belong to the group of

The principal innate immune response against extracellular bacteria comprises complement activa-
pattern-recognition receptors tion, phagocytosis, inflammation-mediated control and sensing of pathogen-associated molecular
present on cells of the innate patterns (PAMP) by pattern recognition molecules such as Toll-like receptors (TLRs).
immune system.
Both Gram-positive bacteria (via its peptidoglycan in cell wall) and Gram-negative bacteria (via
its lipopolysaccharide) can activate alternate pathways in the absence of antibodies. This results in
bacterial cell lysis, opsonization and enhanced phagocytosis. Mannose-expressing bacteria may bind
mannose-binding lectin that also activates the complement pathway via the lectin pathway. In addi-
tion to enhancing phagocytosis by opsonization (by C3b), complement fragments C3a, C4a and C5a
generate an inflammatory response by inducing degranulation of mast cells which releases histamine
and other pharmacologically active mediators. These mediators induce vasodilation and diapedesis
of inflammatory cells, lymphocytes and neutrophils, from the blood into the tissue spaces. An in-
flammatory response can also be stimulated by bacterial cell wall products such as lipopolysaccharide
and peptidoglycan which acti- PAMP

vate macrophages to secrete Mannose
inflammatory cytokines such Extracellular
as TNF-α and IL-1 which at- bacteria
tract inflammatory cells to the
Skin
site. These inflammatory cells
Entry
tend to eliminate the pathogenic
bacteria and clear the system
of pathogen. TLRs, which are
mainly expressed at the plasma
membrane of most phagocytes,
sense PAMPs such as lipo-
polysaccharide, peptidoglycan,
lipoproteins. This results in Activated phagocytes
phagocytosis or destruction of PRR
bacteria displaying such “dan- Activation of lectin
Activation of pathway of complement
ger signals” (PAMPs). Innate alternate
defence mechanisms against ex- pathway
tracellular bacteria are depicted
Phagocyte
in Figure 15.8. activation C3a,C4a,C5a Bacterial
cell lysis
ADAPTIVE IMMUNE
RESPONSE Bacterial cell lysis
The principal adaptive immune Mast cells
response to extracellular bacte- degranulation
ria is the generation of specific
antibodies. The antibodies se- Diapedesis
creted by plasma cells in the Figure 15.8
lymph node are directed against Neutrophils Diagram showing various innate defence
mechanisms of a host body against
cell wall antigens (protein or extracellular bacteria.
polysaccharide) and bacterial
toxins. The bacterial polysac-
charides, because of their polyvalent nature, induce thymus-independent antibody production.
Once the antibody is bound to the bacterial cell wall, it can induce the following:
• opsonization by IgG and enhanced phagocytosis;
• complement activation by IgM, IgG; and
• neutralization by binding to receptors on bacteria that aid in its entry (by IgA, IgG).
Apart from antibodies, cell wall components of extracellular bacteria also induce secretion of
inflammatory cytokines such as IFN-γ and TNF from TH cells which stimulate antibody produc-
tion and trigger local inflammation. The adaptive immune response against extracellular bacteria
is shown in Figure 15.9.
15.5.3 IMMUNE RESPONSE TO INTRACELLULAR

BACTERIA
Since intracellular bacteria find a place inside the cell, they are inaccessible to circulating antibodies
that might be synthesized after their encounter with the immune system at the time of their entry.
These intracellular bacteria are dealt through cell-mediated immunity.
I N N AT E I M M U N E R E S P O N S E
Innate defence against intracellular bacteria comprises mainly NK cells. Intracellular bacteria
activate NK cells either directly or by stimulating macrophages to secrete IL-12, which activates
NK cells. Activated NK cells produce IFN-γ which activates a previously inactivated bacterio-
cidal mechanism inside the cell (usually macrophages) in which bacteria reside, hence killing the
intracellular or phagocytosed bacteria. It has recently been shown that epithelial cells (which rep-
resent the major line of defence against invasive pathogen) are equipped with a new group of
Bacterial cell
Entry
Adaptive immune response
Receptor for adhesion
and entry blocked
IgG
IgA,
IgG
binding
IgG,IgM binding
Complement activation Binding of opsonized pathogen

to phagocytes
X
Figure 15.9
Diagram showing various adaptive
immune response against extracellular Entry into host Bacterial cell lysis Phagocytosis of opsonized
bacteria. cell blocked pathogen
pattern -recognition molecules, Nod molecules, located in the cytosol that could detect Gram-negative
bacteria inside the cell and kill it. It is believed that these Nod molecules recognize muropeptide from
Gram-negative bacterial peptidoglycans. The muropeptide–Nod molecule complex activates the
» Nod molecules can detect
intracellular Gram-negative bacteria bacteriocidal mechanism inside the host cell, resulting in lysis of pathogen. The innate immune
and lyse it. response against intracellular bacteria is shown in Figure 15.10.
ADAPTIVE IMMUNE RESPONSE

The major adaptive immune response against intracellular bacteria comprises Tcyt lymphocytes and
TH cells. Both Tcyt and TH cells respond to protein antigen of intracellular pathogen which are dis-
played as peptides and MHC conjugate on the cell surface. Tcyt lymphocytes induce lysis/apoptosis
of infected cells displaying protein antigens on class I MHC molecules and hence eradicating the
intracellular infection. TH cells, upon stimulation, differentiate into TH1 cells under the influence of
IL-12 (produced by phagocytes). These TH1 cells secrete IFN-γ and interact via the CD40 ligand to
activate cells (particularly macrophages) to produce reactive species such as reactive oxygen species
(ROS) and reactive nitrogen species (RNS), as well as lysosomal enzymes, that kill the intracellular
pathogen. IFN-γ also activates the complement and antibody production enabling the immune
system to counter the pathogen in a more comprehensive way.
15.5.4 E VA S I O N O F H O S T D E F E N C E S BY B AC T E R I A
Some pathogenic bacteria have an inbuilt structure that allows them to resist bactericidal compo-
nents of the host tissue: for example, the outer membrane of Gram-negative bacteria is not easily
Nod molecules
(cytosolic proteins
of host)
Epithelial cell
Bacterial cell lysis
Innate immune response
Perforin
Bacteria Bacterial
TH1-cell peptide
ROS,RNS Class
Bacteria killed I MHC
γ−IFN Tcyt
Lysis of target cell harbouring intracellular bacteria Figure 15.10

Innate and adaptive immune responses
Adaptive immune response against intracellular bacteria.
permeated by hydrophobic bactericidal agents such as bile salts which are harmful to the bacteria.
The waxy cell wall of mycobacteria resists enzymatic attack by host enzymes. However, most of the
bacteria have evolved additional structural features that allow them to resist the host’s internal im-
mune response against them. These include antiphagocytic mechanisms, inhibition or inactivation
of complement components, donning antigenic disguise and neutralization of antibodies targeted
against them. Some of the important mechanisms used by bacteria to evade the host’s immune
response are shown in Figure 15.11. Table 15.2 depicts some bacterial products that interfere with
host immune response.
ANTIPHAGOCYTIC MECHANISM
Antiphogocytic mechanisms include a number of strategies that avoid phagocytic killing of bacte-
ria. These are:
• Avoidance of contact with phagocytes: This is either by localizing in regions not patrolled
by phagocytes (for example, surface of the skin) or inhibiting chemotaxis of phagocytes
(for example, streptolysin secreted by strains of Streptococci suppresses chemotaxis of
phagocytes at low concentration).
• Avoidance of engulfment: Some bacteria are equipped with surface molecules that inhibit
phagocytic engulfment. These include K antigen of E.coli, Vi antigen of Salmonella typhi,
M protein of Streptococci (Group A).
• Avoidance of digestion inside the cell. These mechanisms include inhibition of fusion of
bacteria containing phagosomes and lysosomes (used by M. tuberculosis and Chlamydiae)
as well as escape from membrane-bound phagosomes into the cell (employed by
Rickettsiae, which has a phospholipase). Even within phagolysosome vesicles, some
bacteria such as Bacillus anthracis and M. tuberculosis have some unknown ways to avoid
bacterial cell killing.
• Killing the phagocytes: The easiest strategy is to nullify or kill the cellular warriors, phago- « Anthrax bacteria can survive
cytes, that inhibit the entry of the bacteria into the host cell. Pathogenic Staphylococci inside the phagolysosome.
Bacteria
Endocytosis
Bacterial Lysosome
M-protein
Phagosome No
ER
fusion
Survival inside
Phagocyte phagolysosome
Escape from phagolysosome
Avoidance of phagocytosis Strategies for avoiding

digestion inside the cell
Leukocidins
Exotoxin A
Bacteria
Lysis of macrophage Lysis of neutrophil
Killing of phagocytes
Host fibrin
Figure 15.11 Antigenic determinant hidden

Diagram showing various mechanisms
used by bacteria for the evasion of host
immunity. Antigenic disguise
produce leukocidins which damage neutrophils, Exotoxin A of Pseudomonas aeringinosa

kills macrophages, and haemolysin secreted by Gram-positive pathogens kills phagocytes.
(Haemolysin also lyse red blood cells, for which they are so named). Some of bacteria can
exert their toxic action even after ingestion. These include species of Brucella and Listeria
that destroy macrophages if they ingest them.
FEIGNING OF ANTIGEN (ANTIGENIC DISGUISE)

Some bacteria are able to hide their own antigenic surface determinants from immunological
surveillance by coating themselves with host proteins (such as fibrin and fibrinonectin) or host
polysaccharides (such as sialic acid and hyaluronic acid). S.aureus produces the enzyme coagulase
that causes fibrin to clot and get deposited on the bacterial surface so that “hidden” bacteria are not
recognized by the host immune system. These disguised bacteria therefore escape the onslaught of
the immune system.
N E U T R A L I Z AT I O N O F A N T I B O D Y
Some bacteria can liberate soluble antigenic surface determinants that combine with neutralizing
antibodies before they can bind a bacterial cell. Streptococcus pneumoniae and Neisseria meningitidis
are known to release capsular polysaccharides during their growth in the host. These released surface
antigens bind the circulating antibodies before these antibodies can act and destroy the pathogen.
Host Response
Affected Bacteria Bacterial Factor Mechanism
Apoptosis Shigella Factor IpaB IpaB, Yop induces apop-
Yersinia Yop (Yersinia outer tosis of phagocytic cells
membrane protein)
Complement Streptococcus M protein Protein M binds C4BP,
Porphyromonas Protease Protease inactivates C3
and C5, complement
system blocked
Evasion of host anti- Staphylococcus Protein A Protein A binding of IgG
bodies Peptostreptococcus Protein L blocks phagocytosis,
protein L binding of
κ light chain blocks
antibody binding
Inhibition of phago- Pseudomonas ExoT, ExoS ExoT, ExoS, YopO targets
cytosis Yersinia YopO proteins involved in
phagocytosis
Survival in phaogcytes Legionella DoT/ICM gene DoT/ICM genes Inhibits
Coxiella phagolysosome fusion Table 15.2
Selected bacterial products that interfere
(Source: Merrell, D. S. and Falkow (2004) Nature, Vol. 430, 250–256. Reproduced with permission.) with the host’s immune response
C H A N G I N G A N T I G E N S O N B AC T E R I A L S U R FAC E
Pathogenic bacteria, like viruses, can change surface proteins that antibodies target. This is the
result of site-specific inversion or gene conversion/rearrangement in bacterial DNA that codes for
surface antigens. Some bacteria also change one type of fimbrae to another to avoid detection by
antibodies. It is for this reason that multiple serotypes of pathogenic bacteria exist. There are 80
different antigen types of Streptococcus pyogenes based on cell surface antigen as well as over a hun-
dred strains of pneumococcus depending on capsular antigen.
15.6 BACTERIAL INFECTIONS

15.6.1 CORNYEBACTERIUM DIPHTHERIAE
The causative agent of diphtheria is a Gram-positive rod-like bacteria Cornynebacterium diphtheriae.
Diphtheria is caused by exotoxin secreted by the bacteria. It was found that bacteria was localized
at its portal of entry (site of entry) in humans. In human beings, C. diphtheriae colonizes the naso-
pharangyl region as this pathogen is acquired by airborne respiratory droplets. The growth of bacteria
in the superficial layer of the respiratory mucosa leads to only a mild inflammation. The damage
associated with diphtheria is caused by its secreted exotoxin. The toxin causes the destruction of
the underlying respiratory tissue resulting in fibrosis and formation of a tough fibrillar membrane.
The presence of this membrane in lung tissues induces the feeling of suffocation associated with « Diphtheria is caused by the
diphtheria. This exotoxin is also responsible for neurological damage as well as myocardial damage exotoxin secreted by the bacteria
C. diphtheriae.
causing congestive heart failure.
The exotoxin which wreaks havoc on an individual is encoded by tox gene coded by bacterio-
phage β residing in C. diphtheriae under the state of lysogeny.
The diphtheria exotoxin consists of two chains—A and B—linked by a disulphide bond.
Through the B chain, the toxin binds to the ganglioside receptors on susceptible cells. The A chain
is then cleaved from the toxin which enters the cell. This A chain has an inhibitory effect on protein
synthesis because this fragment has an enzymatic activity (ADP ribosyl transferase activity) that
catalyses ADP ribosylation and the inactivation of EF-2 (elongation factor-2). EF-2 is necessary
for elongation of the growing polypeptide chain during protein synthesis. As a result, tissue cells
can no longer make proteins and eventually die. The exotoxin is extremely potent as only a single
molecule is needed for killing a single cell. The mechanism of action of the toxin of C. diphtheriae
is shown in Figure 15.12.
COO-
Disulphide bond
NH3+
Diphtheria exotoxin
Endocytosis or
Phagocytosis
Entry
Host cell
Proteolysis NH3 + COO-

Fragment A (toxicity)
in reducing
environment NH3 + COO-
Fragment B (entry into cell)
Endocytic
vesicle
Fragment A
NAD+ + ADP-ribosyl
EF2 EF2
Figure 15.12
The mechanism of action of the exotoxin Protein synthesis inhibited.
of C. diphtheriae. Cell death
Since diphtheria exotoxin is a protein, it can be modified chemically (or by heat) to give an
immunogenic yet non-toxic derivative of the toxin, called toxoid. The diphtheria toxin is cross-
linked with formaldehyde and used as an immunogen together with tetanus toxoid and inactivated
Bordetella pertussis toxin in a combined vaccine termed as DPT (diphtheria–pertussis–tetanus)
vaccine. Immunization with DPT induces the formation of antibodies against the toxin called as
antitoxin. The antitoxins formed react with the B chain of the toxins preventing its entry into the
host cells. If the toxin is already attached to the host cell, antitoxin cannot neutralize the toxin. It
is for this reason the booster doses of DPT are recommended at 10-year intervals to maintain a
constant protective level of antibodies. With increased awareness, and with the immunization with
the toxoid, the number of diphtheria cases has decreased rapidly with only five cases reported since
2000 in the USA.
15.6.2 M YCO B AC T E R I U M T U B E RC U LO S I S
Mycobacterium tuberculosis is the main pathogen that causes tuberculosis (M. bovis being the
second major pathogen). Tuberculosis kills about 2–3 million people every year, most of them in
developing countries. Although tuberculosis had been controlled in the developed countries, it
» Tuberculosis is called the disease slowly began to reappear in early 1990, partially because of complacency about tuberculosis, but
of poverty. It is believed that one mainly because HIV-positive individuals that were infected with mycobacterium slowly progressed
new tuberculosis infection occurs
per second! to active disease. These patients also transmitted the disease to others.
Mycobacterium spreads through the air from one person to another. The bacteria are ejected
out in air when the infected person sneezes or coughs. Inhalation of these small droplets (aero-
sols) containing bacteria results in pulmonary infection and the disease is established. The inhaled
bacteria are phagocytosed by alveolar macrophages. Tuberculosis bacilli are able to survive inside
the macrophages as they inhibit the formation of phagolysosome (that is, fusion of lysosome and
phagosome). These bacilli not only survive but multiply inside the cell causing eventual cell lysis
and the release of a large number of pathogenic bacteria.
The branch of immunity that protects the host body against mycobacterium involves
cell-mediated immunity by TH cells. The specific THcells bind to bacterial antigen displayed on
the surface of macrophages (together with class II MHC molecules) and TH cells get activated.
The activated TH cells produce a number of cytokines (IFN-γ and IL-12) that kill or inhibit the
intracellular pathogen on the one hand and induce infiltration of macrophages to the lesion site
on the other hand. IL-12 which induces TH-cell proliferation (specifically TH1) and activation,
also summons macrophages to the lesion site. IFN-γ binds macrophages, activating microbici-
dal pathways that kill the intracellular pathogen. The induction of infiltration of a large number
of activated macrophages to the lesion site causes the development of granulomatous lesions
called tubercles. The tubercle consists of a central area of necrosis (that has a cheesy constitu-
ency) surrounded by a macrophage/epithelioid core. This structure is surrounded by lympho-
cytes which in turn are enclosed by collagenous fibres. The central necrotic zone is created by
the lysis of cellular architecture by lysosomal enzymes released by activated macrophages. As « Mycobacterium tuberculosis
these lesions heal, they get calcified. However, if there is progression of infection, the tubercles divides every 15–18 hours, an
expand and ultimately rupture, disseminating bacteria into the bronchus, blood, pleural cav- extremely slow rate as compared
with other bacteria. E. coli, for
ity, peritoneum, skin or even bones. Babies and young children are particularly susceptible to example, usually divides every
Mycobacteria because they have weak immune systems. Tuberculosis occurs in these patients 20 minutes.
when their immune resistance is lowered.
There is another type of tuberculosis that is prevalent in USA, known as latent tuberculosis. « In HIV-positive people, because of
People who are infected with latent tuberculosis do not have any symptoms, do not feel sick and a weak immune system, there is an
increased risk of latent tuberculosis
cannot spread the disease. However, bacteria can become active and develop tuberculosis later. infection progressing into an active
Tuberculosis can almost always be cured by chemotherapy. The most common drugs used to disease.
fight tuberculosis are isoniazid, rifampin, pyrazinamide, ethambutol and streptomycin. The in-
tracellular location of Mycobacteria makes them a relatively difficult target for drugs. It is for this
reason, drug therapy must be continued for at least nine months to kill all the pathogens. Usually
within a few weeks of treatment, clinical symptoms subside which tempts patients to stop taking
medication, resulting in the recurrence of symptoms and the emergence of multi-drug resistant
strain of Mycobacteria. It is the non-compliance of the nine-month-long drug therapy of tubercu-
losis, which is the principal factor that compromises complete eradication of this disease.
Tuberculosis is detected by skin test which gives positive reaction to the tuberculin test (which
involves injection of a small amount of tuberculin). However, once an individual is vaccinated
by an attenuated strain of M.bovis called BCG (Bacillus Calmette–Guérin), tuberculosis skin test
cannot be used to test the individual for its exposure to pathogen, as vaccinated individual gives
positive test for tuberculin skin test.
15.6.3 LY M E D I S E A S E : B O R R E L I A B U R G D O R F E R I
Borrelia burgdorferi is a Gram-negative bacteria of spirochete family that causes Lyme disease. It is
so named because in 1975 it suddenly appeared in Lyme, Connecticut.
Lyme disease is characterized by severe neurological complications including loss of memory « Borrelia burgdorferi is a spirochete.
and meningitidis. Patients also experience severe headaches, arthritis, and development of charac- Spirochetes are a group of phyloge-
netically distinct bacteria that move
teristics rashes in the form of a bullseye (used in target practising) (see Figure 15.13). This bacteria by axial filaments or endoflagella
is introduced into the human body by a tick (Ixodes sp., commonly called deer tick) bite. Once in-
troduced into the host body, these pathogens enter various organs via the blood stream, including
spleen, liver and kidney, and even reach the meninges of the brain.
The pathogenesis of Lyme disease is apparently because of antibodies formed against the fla- « Lyme disease was first recognized
gella of B. burgdorferi. These antibodies which ironically do not provide any protection against in the USA in 1975 by Dr Allen
Steere, in Lyme, Connecticut.
this pathogen react with the bacteria, and this antigen–antibody complex formed results in type
III hypersensitive reaction. The deposition of the complex near the original tick bite results in
the formation of a red raised area 20–60 cm in diameter, that resembles a bullseye. Similarly, the
deposition of immune complexes in joints results in arthritis and the deposition on meninges of
the brain leads to neurological disorders. Apart from the deposition of complexes at various tissue
sites, additional host tissue damage by the immune complex is elicited by activation of comple-
ment and generation of inflammatory C3a and C5a which induces chemotaxis of phagocytes, causing
more tissue damage.
Figure 15.13
Photographs showing bull’s-eye rashes
of Lyme disease. Reproduced with
permission from Wormser, G. P., E.
Masters, J. Nowakowski, D. McKenna,
D. Holmgren, K. Ma, L. Ihde, L. F. Cavaliere
and R. B. Nadelman (2005). “Prospective
Clinical Evaluation of Patients from
Missouri and New York with Erythema
Migrans- Like Skin Lesions”, Clinical
Infectious Diseases, 41:958–65.
Apart from pathogenesis elicited by the deposition of immune complexes at various strategic
sites, IL-1 is also believed to contribute to the same. Being a Gram-negative bacteria B. burgdorferi
has lipopolysaccharide in its cell wall. Lipopolysaccharide is a known inducer of IL-1 in macro-
phages. In vitro studies have shown that IL-1 is potent in releasing proteolytic enzymes such as
collagenase from synovial cells. The release of collagenase result in the degradation of tissues in the
joints resulting in a damaging inflammatory reaction.
Studies on B. burgdorferi in mice have led to the development of a vaccine for humans. It was
» Vaccines, based on the outer
surface protein (ospA), were found
observed that mice though a reservoir for this spirochete, did not develop Lyme disease because
to offer significant protection they mounted an, effective humoral response to it. Careful studies revealed that mouse antibodies
against B. burgdorferi infection in were directed against two proteins found in the outer bacterial envelope of the bacteria, whereas
human beings.
humans made antibodies against the flagellar antigen and not against those two proteins.
15.7 PROTOZOAN DISEASES

Protozoa is a unicellular, eukaryotic organism that may cause several serious diseases in humans
such as malaria, sleeping sickness, leishmaniasis and ameobiasis. Protozoan infection stimulates
both defence mechanisms—humoral as well as cell-mediated. Humoral responses are elicited when
a protozoan parasite is outside the cell; while the same pathogen if it has an intracellular part of the
life cycle will induce a cell-mediated immune response.
15.7.1 MALARIA
» Globally, every 30 seconds a Malaria is a serious and sometimes fatal disease that affects about 500 million people worldwide;
patient dies due to malaria! about 1 million die in Africa alone.
» The name malaria is derived from About 1,000 cases of malaria are diagnosed in USA each year. Malaria is caused by four spe-
the Italian word mal’aria which im- cies of protozoa plasmodium—P. falciparum, P. vivax, P. ovale and P. malariae. P. falciparum is
plies “bad air” because this disease the most virulent and most prevalent of these protozoan species. Symptoms of malaria include
was prevalent near swamps where
malodorous vapours of swampy shaking chills, fever, headache, muscle aches, tiredness and nausea. Vomiting and diarrhoea may
waters were present. also occur. Jaundice and anaemia can also occur because of loss of blood cells.
PAT H O G E N E S I S O F M A L A R I A « Female anopheles mosquitoes

Humans get malaria from the bite of the malaria-infested female anopheles mosquitoes which feed feed on blood, while male
anopheles mosquitoes feed on
on human blood. plant juices.
The female anopheles mosquito serves as the biological vector for malaria and a part of the para-
site’s life cycle occurs in it. When plasmodium-infected mosquito bites a healthy human, sporozoites « During the 1890s, Patrick Manson
migrate from the mosquito’s mouth into the subcutaneous tissue and from there into the blood. suggested that malaria was trans-
mitted by mosquitoes.
Once sporozoites leave the bloodstream, they enter the liver within half an hour. Sporozoites
are a part of the plasmodium life cycle and are equipped with a specialized adhesion protein, cir- « The circumsporozoite has two
cumsporozoite, that binds liver cells. domains—the thrombospondin
Once inside the liver cells, they multiply and result in the formation of the next stage in the domain and the thrombospondin-
related adhesive protein do-
life cycle, merozoites. During the time in which the parasite is in the liver, the infected person does main—that bind specifically to
not feel ill. heparin sulphate proteoglycans on
The merozoites then leave the liver cells and enter into the red blood cells. Merozoites then hepatocytes.
transform to a large uninucleate, trophozoite. This trophozoite then divides asexually to produce a
« One single sporozoite can
large number of merozoites again, causing red blood cells to burst. This frees the parasite to attack generate tens of thousands of
other red blood cells. merozoites.
Every 48 hours, merozoites, together with merozoite toxins (which are believed to be cyto-
kines), are released. These merozoite toxins, erythrocyte debris released from lysed red blood cells,
« P. falciparum and P. vivax produce
cause the characteristic recurrent chills, shivering, fever and sweating associated with malaria. around 20 merozoites per mature
Eventually some (asexual) merozoites differentiate into male and female gametocytes which parasite in a red blood cell.
are ingested by the female anopheles mosquito during the mosquito bite. Within the mosquito
« The chills associated with malaria
gut, male and female gametocytes form gametes which fuse to form a zygote. This differentiates are caused by the action of toxins
into a sporozoite within the salivary gland. The infected female anopheles is ready for yet another on phagocytes (macrophages).
round of infection. A schematic representation of the life cycle of the malarial parasite is shown in These phagocytes release TNF
which induces malarial paroxysm
Figure 15.14. (fever with chills).
Mosquito Human
Sporozoites
Salivary
glands Liver
Sporozoites
Oocyst Merozoites
Asexual
cycle
Midgut Zygote
Gametes
Gametocytes
Transmission to Figure 15.14

Red blood Schematic diagram the
mosquito
cell showing life cycle of
malaria parasite.
HOST IMMUNE RESPONSE TO PLASMODIUM

Infants and children less than 14 years have poorly developed immune systems, consequently they
are most likely to develop this disease. Unless malaria is properly treated with chemotherapy, mor-
tality rate of children can reach up to 50 per cent. Both TH cells and Tcyt cells render some protection
against different stages of plasmodium. Studies on mice have led us to believe that TH cells mediate
immunity against malarial parasite when they are in the blood, while Tcyt cells inhibit multiplica-
tion of parasite in heptocytes. TH cells do not act on heptocytes as they do not express class II MHC
molecules. The low host immune response to malarial parasite is due to several reasons:
• The humoral and cell-mediated immune responses to sporozoites does not occur because
sporozoites remain for only about 30 minutes in blood. Such a short duration is ineffective
in inducing immune response.
• P. falciparum present in blood releases a soluble parasite antigen that binds to the antibody
generated and hence protects the parasite from host antibodies. This process is called im-
mune distraction, and involves the shedding of cell-surface antigen. Immune distraction is
mediated by endogenous phospholipase(s). Table 15.3 provides some interesting insights
into the mechanisms used by Plasmodium and other parasites in evading immune response.
• Macrophages in malaria endocytose hemozone—a breakdown product of malaria which
makes them less responsive.
• The surface molecules of the parasite undergo several changes of life cycle within the host
cell. The immune system cannot cope fully with this continuous changes of antigen.
• The parasite hides from the immune system by multiplying and living mostly inside the
body cells.
A N T I M A L A R I A L D R U G S A N D VACC I N E S
The common drugs used in malarial treatment include quinolines (that include quinine, chloro-
quine, amodiaquine, halofantrine), artemisinins (artemether, arteether, artesunate), antifolates
(sulphadoxine, pyrimethamine) and Atovaquone-proguanil, apart from common antibiotics such
as tetracycline and doxycycline.
The studies on vaccine development against malarial parasite have revealed theoretically ex-
cellent but impractical results. This is because different stages of the parasite express different an-
tigens and a vaccine effective, for example, in killing the liver-stage parasite may not inhibit the
growth of the blood-stage parasite. The sheer number of parasite proteins, estimated to be at least
Host Response
Affected Parasite Parasite Factor Mechanism
Antibody binding Plasmodium falciparum S antigen Soluble antigens are
Trypanosoma brucei VSG antigen shed to “distract”
antibody response
Oxidative burst of Schistosomes Glutathione-S- Resists and neutralize
phagocytes transferase oxidative burst
Filarial worms Glutathione peroxidase
Antibody and cell- Trichinella spiralis Lymphotoxic factor Lysis of lymphoid cells
mediated response and tissues
Antibody response Trypanosoma brucei Surface antigen varia- Parasite changes
tion surface antigen so
that previous antibody
becomes ineffective
Immune response Filarial worms Immunosuppression Clonal anergy against
parasites, generalized
Schistosomes Cytokines produced
immunosuppression
Complement and Schistosomes Thick tegument of Thick layer of tegument
Table 15.3 cytolysis by Tcyt parasite can resist Tcyt cell and
Selected parasite products that interfere
complement onslaught
with host immune response.
5,000–6,000, as well as extensive antigenic polymorphism, potentially limits the effectiveness of

any vaccine. For example, a single parasite clone contains roughly 50 different copies of the gene
for the variable surface antigen PFEMP1 (P. falciparum erythrocyte membrane protein 1).
The current approaches to malaria vaccine include vaccine directed against sporozoites or
asexual blood stages. One of the few P. falciparum vaccines that is undergoing field trials includes
synthetic peptide vaccine SPf66 directed at blood-stage parasites. A combination of three asexual
blood stage antigens (fragments of MSP1, MSP2 and RESA) have also been significant in decreas-
ing the frequency of parasite episodes. Although the MuStDO 9 vaccine that contains five plasmids
encoding nine different malarial antigens has generated antibody response in animal models, it has
not been not tested in humans as yet.
15.7.2 AFRICAN SLEEPING SICKNESS

There are two types of African sleeping sickness (African trypanosomiasis) depending on the « T. cruzi causes the American
area in Africa in which they are prevalent. East African trypanosomiasis (EAT) and West African sleeping sickness (Chagas’ disease)
Trypanosomiasis (WAT). EAT is caused by Trypanosoma brucei rhodesiense while WAT is caused which is prevalent in the tropics of
continental America. This disease is
by T. brucei gambiense. Trypanosoma brucei rhodesiense is found mainly in the savannas of East transmitted by the triatomid bug,
Africa while T. brucei gambiense is found in the rain forests of west Africa. which discharges pathogen-infested
African sleeping sickness is transmitted to humans by the bite of tsetse fly. The painful bite by faeces while feeding on human
blood. These faeces can enter the
the tsetse fly often develops into a red sore called chancre. The general symptoms of sleeping sickness host body through wounds.
include extreme fatigue, swollen lymph nodes, severe headache, fever, aching muscles and joints.
Sleeping for long periods during the day (and hence the name) and insomnia at night are most common « Domestic cattle and wild animals
symptoms. If left untreated, death can occur within weeks as the parasite invades the central nervous system. are the reservoir for trypanosomes.
Arthropod bite transmits the patho-
gen from animals to humans.
PAT H O G E N E S I S O F T R Y PA N O S O M I A S I S
The pathogen enters the host blood stream by the bite of the tsetse fly. Once in the blood, trypano-
somes differentiate and rapidly multiply, doubling every three to six hours. Through a series of « The flagellated protozoa, such
as trypanosomes and leishmania
stages, the parasite infects the central nervous system and causes many symptoms like seizure, parasite, that are transmitted by
slurred speech, difficulty in walking, and meningo-encephalitis, and loss of consciousness. the bites of infected arthropods are
called hemoflagellates
HOST IMMUNE RESPONSE
As the parasites enter the blood stream, they multiply and increase in number which triggers an « Sleeping sickness derives its
extracellular immune response—humoral response. Antibodies are formed against the surface pro- name from the symptoms of the
disease manifested by the patient.
tein of trypanosomes, called variant surface glycoprotein (VSG). The patient usually lies prostrate,
Antibodies bind to the parasites and the parasites are eliminated either by complement-medi- insensitive to pain, drools from the
ated cell lysis or by phagocytosis by macrophages which act easily due to opsonization. The para- mouth and appears to be lethargic
or in a sleep-like state. There are
sites escape this onslaught of antibodies by two main ways (see Figure 15.15). about 15 to 20 million cases each
year worldwide.
• VSG of the parasites are shed from its cell membrane by a phosphatidylinositol-specific
phospholipase. VSG is anchored to the plasma membrane by phosphatidylinositol (GPI)
« Trypanosome can change its
anchor which is cleaved by phospholipase. These antigens “mop up” the circulating anti- surface antigen (VSG) to evade
bodies that are formed against the parasite. humoral response.
• Some of the parasites change their surface VSG antigens and hence the antibodies formed
against a previous variant of the parasite are ineffective.
This change in the surface antigen is brought about by the fact that there are more than 1,000
VSG genes in the genome of T. brucei. However, a trypanosome expresses only one VSG at a time.
Through some unknown mechanism, under pressure from humoral response, a new VSG gene is
expressed resulting in the appearance of new variants of VSG protein that can escape immunological
detection and cause the next wave of parasitemia.
15.7.3 LEISHMANIASIS
Leishmaniasis is a protozoan disease that is spread by the bite of infected sand flies (of genera
Lutzomyia and Phlebotomus). There are two forms of this disease—cutaneous and visceral. The
symptoms of the cutaneous form of the disease are sores on the skin which are usually covered by
a scab. The visceral form of leishmaniasis is exhibited by enlarged liver and spleen (in some cases
spleen becomes larger than the liver), low RBC and platelet count and continual fever.
Complement-mediated
VSG antigen cell lysis
Immune
Response
Trypanosoma
Phagocytosis
Immune response against Trypanosoma Phagocyte

(macrophage/neutrophil)
Antibody ineffective against

new antigen
Shedding of antigen
neutralizes antibodies
Change of surface VSG antigen
Figure 15.15
Immune response and its evasion by
Trypanosoma. Evasion of immune response by Trypanosoma
There are about 1–2 million cases of the cutaneous form, while the visceral form of the disease
» Visceral leishmaniasis is known as
kala-azar in India. affects 500,000 individuals per year. Cutaneous leishmaniasis is mainly caused by L. tropica, while
the visceral disease is caused by L. donovani.
» Rodents and canines form the
reservoir of Leishmania spp. Liesh- H O S T R E S P O N S E T O L E I S H M A N I A S P P.
mania is transmitted from animals The resistance of individuals to leishmaniasis infection varies and may be controlled by number
to humans or from one individual to
another by infected sand flies. Sand of factors. Once inside the blood stream, the leishmania parasite, which is a flagellated protozoa,
flies bite humans to take their blood enters the macrophage. Figure 15.16 shows a leishmania parasite getting endocytosed by a macro-
meal and inadvertently introduce phage. Since it spends most of the time inside the macrophage, the main human defence mecha-
parasites.
nisms are generation of reactive oxygen species, reactive nitrogen species and lysosomal enzymes
within the macrophages so that intracellular pathogens are killed. Studies in mice have shown that
IFN-γ produced by activated TH- (TH1) cells activates macrophages to kill the protozoa (L. major)
that live within them. IL-4, if produced by TH2 cells, inhibits the production of IFN-γ and makes
mice more susceptible to leishmania parasite.
Parasites can resist host defences in naïve individuals (individuals not immunized) in a num-
» Leishmania can protect itself from
ber of ways, as follows:
reactive oxygen species (particu-
larly the superoxide anion) by syn- • Certain species of leishmania parasite (for example, L. donovani) have a membrane that
thesizing the enzyme superoxide
dismutase that neutralizes them. can resist complement-mediated cell lysis.
• Certain species can protect themselves from killer oxidative burst within the macrophage
by synthesizing the enzyme superoxide dismutase that neutralizes injurious oxidative free
radicals.
• Certain species have specialized lipophosphoglycan coat and glycoproteinins (eggp 63)
that provide protection against lysosomal enzymes as well reactive oxygen species generated
within the cell. They also downregulate class II MHC molecules making the cells immune
to T-cell surveillance.
These protozoan diseases clearly point to one thing. Prevention (through vaccine) is better than
cure, as the pathogen, even though unicellular, has evolved complex defence mechanisms over the
years. Chemotherapy is the last and perhaps the only alternative for the several of these diseases.
Figure 15.16
Scanning electron micrograph of a
leishmania parasite in the process of
being phagocytosed. (From Encyclopedia
of Life Sciences (2006). Copyright John
Wiley & Sons Limited. Reproduced with
permission.)
15.8 DISEASES C AUSED BY PAR ASITIC

WORMS
Parasitic worms are responsible for a wide variety of diseases in humans. Parasitic worms that
infect humans include trematodes or flukes (Schistosoma) roundworm (Ascaris), filarial worms
(Wuchereria spp.) and several other helminths that can enter the human body via food, such as
tapeworm (Ancyclostoma spp.) round worm (Trichinella spiralis), and hookworm. Hookworms and
schistoma larva enter via skin; tapeworm, pinworm and roundworm via contaminated food; and
filarial worm through the bite of intermediate insect vector. These parasitic worms reside in the hu-
man body, outside the cell and usually do not multiply in the host. Some of the important parasites
that infect human beings are shown in Figure 15.17.
15.8.1 HOST IMMUNE RESPONSE

Helminths, being extracellular parasites, are easily accessible to immune surveillance. However,
since the number of parasitic worms that enter the human body is quite small, a low level of im-
Schistosoma mansoni Leishmania donovani Trypanosoma brucei
Figure 15.17
False colour representation of important
Ascaris lumbricoides Trichinella spiralis Taenia saginata parasites of humans.
mune response to helminths occurs. These parasites are too large to be phagocytosed by neutrophils
» The major basic proteins secreted and macrophages which help clear extracellular pathogens. The major defence against worms are
by eosinophils are toxic to
helminths. IgE and eosinophils. Eosinophils react to IgE- or IgG-coated worms causing degranulation onto the
surface of the worms, killing them.
It is believed that TH2 subsets of TH cells play an important role in helminth infection. Cyto-
kines released by TH2 cells such as IL-4 induces class switching to IgE, IL-5 induces eosinophilia
(increase in the number of eosinophils) and IL-3 activates mast cells to degranulate. IgE antibodies
bind to the surface of the parasite. The free Fc region of helminth-bound antibody attaches to the Fc
receptor present on eosinophils. The cross-linking of the Fc receptor causes the secretion of gran-
ules from eosinophils (and mast cells) that destroys the parasite. Eosinophils may be more effective
at killing helminth parasites than other leukocytes because the major basic protein of eosinophils
is more toxic to helminths, than reactive oxygen and reactive nitrogen species and proteolytic en-
zymes produced by macrophages and neutrophils. Major basic protein is a non-specific protein,
but still does more harm to the parasite because it is secreted in close proximity to the parasite,
doing little damage to nearby host cells.
Sometimes, the immune system cannot completely eliminate the parasite, so it “isolates” the
organism (or its eggs) by “walling off” the parasite. This walling off effect is a chronic cell-mediated
response, that leads to the formation of fibrosis and granulomatous lesion. Schistoma mansoni eggs
in the liver stimulate TH cells that activate macrophages and induce delayed type hypersensitivity
reactions. Chronic delayed type hypersensitivity results in the formation of granuloma around
the eggs. Though the granuloma localize the Schistoma eggs and prevents their dispersal to other
places such as the intestine or bladder, fibrosis associated with granuloma disrupts blood flow in
the liver inducing hypertension and cirrhosis.
Schistosoma parasites can elicit antibody formation, resulting in the formation of an immune
complex. These complexes can be deposited in the kidneys, and blood vessels producing vasculitis
and nephrites.
Certain helminths may be expelled from the body (an IL-4 dependent response) by a mechanism
that is still not defined and does not require IgE. This mechanism, which probably involves
T cells, eosinophils and mast cell, is induced by changes in gut permeability, the coating of the
parasite with mucous and the shedding of a few layers of gut epithelium that helps shed to the
worms. Systematic contraction of intestinal muscle brought about by mast cell mediations facili-
tate expulsion by peristalsis.
15.8.2 E VA S I O N O F I M M U N E M E C H A N I S M BY H E L M I N T H S
Heminths evade the immune mechanism in the following ways:
• Antigenic disguise: Some helminths such as schistosomes acquire a surface layer of host
antigen (such as blood group A, B, H determinants and MHC molecules) which allows
them to remain in the host body, as the host is unable to distinguish them from self.
• Many parasitic worms develop thick integuments that make them resistant to complement-
mediated cell lysis, cytolytic action of Tcyt and cytocidal mechanisms of neutrophils and
macrophages. The best example is Schistosoma larvae which develop teguments during
their migration to lungs. Many nematodes shed their surface coat when they come under
immune attack, so surface antigens and antibodies are sloughed off.
• Some parasitic worms induce generalized immunosupression as well as specific anergy to
parasite antigens. Filarial worms infect and disrupt lymph nodes contributing to deficient
immunity. Clonal anergy against specific parasite antigens have been observed in severe
cases of schistosomiasis where immunosuppression occurs because of the production of
immunosuppressive cytokines by activated T cells and macrophages.
• Some parasitic worms express or secrete certain antioxidant enzymes/molecules that
resist the oxidative burst of phagocytes. Schistosomes have the antioxidant enzyme
glutathione-S-transferase, while filarial worms can secrete glutathione peroxidase.
Infection and disease in humans can be caused by a plethora of pathogens that include bacteria,
virus, protozoa, helminths and several others. The human body is equipped with a wide variety of
defence mechanisms that control, combat and most of the time destroy the invading pathogen.
The defence against these infectious agents is mediated both by non-specific innate immunity and
specific adaptive immunity. Antiviral immune responses include innate defences such as inter-
feron and natural killer cells, while specific defence includes Tcell- mediated immunity. Innate
bacterial defence comprises complement activation and phagocytosis, and the sensing of patho-
gen by TLRs and Nod molecules. Adaptive immune response comprises Tcyt lymphocytes and TH
cells. Helminths being extracellular parasites are easily accessible to immune surveillance and are
taken care of by eosinophils and IgE antibodies. Pathogens, in general, have evolved a variety of
strategies to evade these immune attacks. A constant struggle between pathogens and the immune
system that occurs in our body, in which, thankfully, the immune system wins, most of the time.
ELISA—Double Antibody Sandwich Assay

Double antibody sandwich assay of ELISA is another vari-
ation of ELISA that is used to detect/assay the presence of
Antibody adsorbed
a specific antigen. It is called sandwich assay because the on to microtiter plate
antigen to be detected is sandwiched between two layers
of antibody. In this assay, microtitre plates are first coated
with the specific antibody (concentration~10 μg/ml). This
antibody is adsorbed onto the wells by incubating it over-
night. This antibody which is bound to the wells of the mi-
crotitre plates is called capture antibody as it captures and
retains the specific antigen onto the microtitre plates.The
test antigen (usually 100 μl ) is then added in each well. Test antigen is
The capture antibody retains the specific antigen, and un- added. Antigen binds to
bound antigen, and non-specific antigens are then washed the adsorbed antibody
off. Finally, an enzyme-conjugated antibody specific for the
antigen (called detection antibody) is added. The microti-
tre plates are then washed to remove the non-specifically
bound antibodies. The complex that is finally formed has
the antigen sandwiched between the capture antibody
and the detection antibody. A substrate that gives a col-
oured product (chromogenic substrate) is then added and
the coloured product formed is detected by measuring
absorbance (see Figure 15.18). If antigen has been bound Antibody that is specific
for antigen and conjugated
to the capture antibody in the first step, the ELISA test will with enzyme is added. This
be positive. If the antigen is not recognized by the capture forms a sandwich of antibody
antibody, no binding of detection the antibody occurs, no with antigen in middle
coloured product is formed and the ELISA test is negative.
As in indirect ELISA, the enzymes that are conjugated with
detection antibody are alkaline phosphates (substrate
Substrate
used is p-nitrophenyl phosphate) or horse radish per-
oxidase (substrate used is hydrogen peroxide and ABTS
Product
as chromogen). The advantage of sandwich assay is that
there is no requirement for purity of antigen as the cap-
ture antibody only binds to the specific antigen. Moreover, Chromogenic substrate is added.
The reaction product is coloured
sandwich ELISA is three to five times more sensitive than
and is quantitated.
indirect ELISA. As with indirect ELISA, sandwich assay can
be used to give a qualitative result (presence or absence of
Figure 15.18
antigen) or a quantitative result (measuring concentration Double Antibody Sandwich ELISA.
of antigen). As a thumb rule, it should be remembered
that indirect ELISA detects antibodies while sandwich ELISA detects agents such as Helicobacter pylori (causative agent for stomach
antigen in the sample. This assay is routinely used to detect causative ulcers), cholera bacilli and Salmonella.
S U M M A R Y
• The defence against infectious agents is mediated both by non- • Bacteria have evolved a number of strategies that avoid
specific innate immunity and specific adaptive immunity. bacterial killing. These include anti-phagocytic mechanism,
antigenic disguise and changing surface antigens.
• Anti-viral immune response include innate defences such as
interferon, natural killer cells and macrophages. Specific defence • Protozoan infection stimulates both defence mechanisms—
include humoral response, mediated by antibody together with humoral as well as cell-mediated immunity. A humoral response is
complement. T-cell mediated immunity eradicates intracellular elicited when protozoan parasites are blood-borne, while the same
viral infections. pathogen will induce a cell-mediated response if it has an intracel-
lular part of the life cycle.
• Some viruses can avoid immune response by downregulating
• Helminths, being extracellular parasites, are easily accessible to
cellular proteins, mutating surface antigens or hiding in immune-
immune surveillance. Since these parasites are too large to be
privileged sites. phagocytosed by neutrophils and macrophages, the major defence
• Innate immunity to bacterial infection includes physical barriers against these worms are IgE and eosinophils.
(skin) and chemical barriers (fatty acids, lysozyme) of the body. • Helminths have developed a diverse array of mechanisms for
Innate bacterial defence comprises complement activation and evading the immune system. These include antigenic disguise (by
phagocytosis, and the sensing of pathogen by TLRs and schistosome), thick integuments (nematode), immunosuppression
Nod molecules. Adaptive immune response comprises Tcyt and TH (filarial worm) as well as secretion of antioxidant enzymes/
cells. molecules.
K E Y W O R D S
• antigenic drift 323 • Cornyebacterium diphtheriae 329 • major basic protein 338
• African sleeping sickness 335 • evasion of immunity 336 • malaria 332
• antigenic disguise 327 • helminths 337 • mycobacterium tuberculosis 330
• antigenic shift 323 • immune response 316 • NK cell 317
• antiphagocytic mechanism 327 • influenza 322 • PAMP 324
• bacterial immunity 324 • interferon 316 • plasmodium 332
• Borrelia burgdorferi 331 • leishmania 335 • trypanosomiasis 335
• chancre 335 • Lyme disease 331 • tuberculosis 330
1. How is the innate antiviral defence mechanism different from the 4. Can you suggest some reasons for low immune response to ma-
innate antibacterial defence mechanism? What role do NK cells larial parasite? Why has no successful vaccine against malaria been
play in these defence mechanisms? developed till date?
2. What is antigenic drift? Why is it different from antigenic shift? 5. Viruses are obligate intracellular parasites. What roles do antibody
What advantage does it give to the pathogen? and complement play in controlling viral infections? What role do
3. What are the various antiphagocytic mechanisms used by bacteria T cells play in combating viral infection?
to evade immune response? What are the other bacterial strategies
that allow them to resist host defence?

1. Which of the gene products is not induced by interferon? 3. Cell-mediated defence against virus is mediated by:
(a) Protein kinase R (a) Macrophages
(b) Oligoadenylate synthase (b) CD8+ Tcyt cells
(c) Cytosine deaminase (c) B cells
(d) RNase L (d) All of the above
4. Large changes in HA and NA antigens of influenza virus occur
2. IFN-γ is primarily produced by: because of all, except:
(a) NK cells (a) Mutation in genome
(b) B cells (b) Gene reassortment
(c) Resting T cells (c) Changes in surface antigen
(d) Basophils (d) Gene conversion
5. Which of the following is a part of the intracellular innate 8. The causative organisms of leishmaniasis are:
defence against bacteria? (a) Bacteria
(a) Complement system (b) Viruses
(b) Toll-like receptor (c) Nematodes
(c) Nod molecules (d) None of the above
(d) IgG
9. One of them is not a substage of the malaria parasite’s life cycle:
6. Borrelia burgdorferi causes: (a) Sporozoite
(a) Tuberculosis (b) Circumsporozoite
(b) Diphtheria (c) Merozoite
(c) Lyme disease (d) Gametocyte
(d) Leishmaniasis
10. In which of the following diseases is hypersensitivity evoked?
7. The major defences against helminths are all, except – (a) Lyme disease
(a) IgE (b) Malaria
(b) Eosinophils (c) Influenza
(c) Basophils (d) Leishmaniasis
(d) Th cells

1. CD4+ T cell protects host body against Mycobacterium onslaught. 4. Antitoxin formed against diphtheria toxoid binds and neutralize
it’s A chain.
2. Diphtheria toxin is produced by tox gene present in bacterio-
phage β in C.diphtheriae. 5. Tubercle bacilli evokes type IV hypersensitivity.
3. IFN-γ-induced RNase L edits amino acid substitution which
results in the formation of inactive protein.
Banchereau, J. and R. M. Steinmann (1998). “Dendritic Cells and Mims, C. A. (1987). Pathogenesis of Infectious Disease, 2nd ed.
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“The Pathogenic Basis of Malaria”, Nature, 415: 673–79.
Probably the first attempt to vaccinate individuals was thought of by
the Romans around CE 23 when they explored the use of liver extracts
from rabid dogs for protection against rabies. The term vaccine owes
its origin to the Latin word vacca, meaning cow. The term was coined
by Pasteur, when he found out that attenuated bacteria of fowl cholera
could be administered into healthy chickens to make them immune to
this disease. Though his work focused on fowls, he named attenuated “Let no one ever
bacteria as vaccine in honour of Jenner’s work on cowpox. Edward come to you
Jenner, a country doctor of Berkley, Gloucestershire, England made without leaving
a pioneering effort in 1798. He extracted the contents of cowpox better and
pustules and inoculated them into the arm of an eight-year-old boy, happier.”
—MOTHER TERESA
Joseph Meister. When he subsequently injected the smallpox virus in
the boy, the boy did not develop the disease. It was a brave attempt to
prevent an infectious disease, and it proved to be the most successful
application of immunological principles to human health. This
vaccination trial against small pox gave humankind’s first vaccine. The
benefits of vaccination were dramatically evident as smallpox, a deadly
disfiguring disease, was completely wiped out. In 1890, Von Behring should be able to:
and Kitasato discovered that immunity to certain diseases such as • Define the term vaccine and
explain the underlying concept
diphtheria and tetanus is due to the presence of antibodies. Moreover, • Describe natural live vaccine,
live attenuated vaccine,
the transfer of immune serum to a naïve recipient would protect the inactivated vaccine, toxoid
vaccine and polysaccharide
recipient from diphtheria. Finally, with advancement of the clonal vaccine
• Give the merits and demerits
selection theory (in 1957) and the discovery of lymphocytes, a of attenuated and inactivated
vaccine
profound influence upon immunological thought and immunological
• Describe live viral and bacterial
vector vaccines
practice was exerted. In the middle of the 19th century (1860
• Explain recombinant antigen
onwards), the mechanism behind vaccination was understood. It vaccine and DNA vaccine
• Demonstrate knowledge of live
became clear that vaccines induce specific immunity against pathogen vector vaccine
(bacteria, viruses and parasites) by inducing clonal expansion or • Give an account of some new
vaccine strategies
proliferation of specific T and/or B cells and, most importantly, the for- • List the characteristics of an
ideal vaccine
mation of specific memory T cells and B cells (see Figure 16.1). These
memory cells are triggered rapidly on subsequent encounter with the
pathogen (secondary response), unleashing a battery of antibodies
and effector T cells to counter the pathogen.
Vaccines
16.1 INTRODUCTION
A vaccine can be defined as a preparation of bacterial, viral or other pathogenic agents or of their
isolated antigens which is administered with the objective of stimulating a recipient’s protective
16
immunity. After primary exposure of antigen to immunocompetent lymphocytes, there occurs an
initial but slightly delayed immune response called primary immune response. Primary immune
response peaks on approximately the 14th day of antigen exposure. There are two main outcomes
of primary response: first, specific immunocompetent cells (B and T cells) are activated; and sec-
ond, and the more important one, memory cells are formed. Subsequent exposure to the same anti-
gen stimulates these memory cells which results in a rapid and more heightened immune response.
Secondary response usually occurs within two to three days. It must also be mentioned that a
primary immune response elicits the formation of IgM antibodies while a secondary response pro-
tects the host with higher-affinity IgG antibodies. It is the rapidity with which secondary response
occurs upon secondary exposure to antigen that protects the host against any potential threat by
repeated exposure to pathogen (see Figure 16.2). « Vaccine owes its origin to the Latin
Thus, a vaccine is basically an antigen or its component that can induce secondary or adap- word, vacca, meaning cow. Edward
Jenner did not introduce the term
tive immunity in the host. It aims to prevent severe complications of infections by reinforcing or vaccine, it was Pasteur who did
broadening the defences by introducing immunological memory. that.
Exposure to Infection and disease

pathogen prevented
Vaccination
Specific B and T cell Memory cells Re-expansion of specific

stimulated formed B/T Cells
Figure 16.1
What does a vaccine do? A vaccine forms
B and T memory cells before exposure
to the pathogen. The actual entry of
the pathogen evokes a strong and
quick defence from the host, thereby
preventing infection and disease.
Secondary response
Primary response
Antibody titer
First exposure of antigen,
memory cells formed
Second exposure of antigen,

stimulation of memory cells
IgG
IgM
7 14 3 7
Figure 16.2
Primary and secondary immune
responses. Time (days)
16.2 T YP E S O F VACC I N E S
» Vaccination is the deliberate There are several types of vaccines that are currently and conventionally used. These include natural
introduction of an antigen into a live vaccine, live attenuated vaccine, inactivated vaccine, toxoid vaccine, polysaccharide vaccine,
host with the intention of inducing
immunity. recombinant antigen vaccine, live vector vaccine and a more recent DNA vaccine. Figure 16.3
depicts some important types of vaccines. Table 16.1 lists some of the important types of vaccines.
Attenuated
(weakening)
Live Attenuated
pathogen non-pathogenic
form (vaccine)
Natural live vaccine Live attenuated vaccine
Chemical
Inactivation
modification
Live Killed pathogens
pathogens (vaccine) Toxin Toxoid
(vaccine)
Figure 16.3
Types of vaccines. Polysaccharide vaccine
VACCINES 345
Types Examples of Vaccines Used or Explored

Natural live vaccines Cowpox (vaccinia) virus used for immunization against
smallpox (historic importance, now redundant);
simian rotavirus is used in vaccinations against
gastroenteritis.
Live attenuated vaccines BCG; Sabin polio vaccine; MMR*; varicella- zoster
vaccine (for chickenpox); yellow fever vaccine
Inactivated vaccines Salk polio vaccine; rabies vaccine,; pertussis vaccine
Toxoid vaccines Diptheria vaccine; tetanus vaccine
Polysaccharide vaccines Hib† vaccine; Vaccine for pneumococcal pneumonia;
meningococcal meningitis
Recombinant antigen vaccines HBs AgΔ vaccine
Viral vector vaccines An HIV vaccine containing env, gag and, pol genes
in canary pox virus – vector (in clinical trials); animal
model is experimented with hepatitis B antigen; influ-
enza antigens inserted in vaccinia virus vector
Bacterial vector vaccines Attenuated strain of Salmonella typhimurium (Ty2la) is
explored as vector for cholera vaccine
DNA vaccines Rabies vaccine; Influenza vaccine; HIV vaccine—are
currently being explored
Table 16.1
Note: *Mumps – Measles – Rubella vaccine; †Haemophilus Influenza type b; ΔRecombinant hepatitis B virus.
Types of common vaccines for humans.
16.2.1 N AT U R A L L I V E VA C C I N E S
These preparations include natural non-pathogenic organisms, but which still induce specific
Rotavirus
immunity. Currently, live natural vaccines are rarely used. Apart from the historical cowpox virus Rotavirus belongs to the Reoviridae
vaccine, no other natural organism are usually used, though live simian and bovine rotaviruses family of viruses. This virus causes
have been used in vaccination against infant diarrhoea with moderate success. gastroenteritis among children
which manifests as severe diarrhoea
The problems of these vaccines reside in their ability (albeit hidden) to mutate and convert and vomiting and is responsible for
into forms that could be pathogenic to human hosts. about 600,000 deaths per annum in
developing countries.
16.2.2 L I V E AT T E N U AT E D VA C C I N E S
Attenuation (Latin: attenuare—to weaken) refers to the weakening of the pathogenic bacteria or « Natural live vaccine could turn
virus by making it less virulent without altering its immunogenicity. Microorganisms are attenu- pathogenic in a host.
ated or weakened so that they do not cause any disease. Attenuation can be achieved by growing
« Poliovirus infects only primates
pathogenic microorganisms (bacteria or virus) for a long period of time in a foreign host such as and other animal species are
embryonated eggs or tissue culture cells. The second and more famous attenuation was successfully usually not affected by it. This
carried out by two scientists Albert Calmette and I. Camille Guérin in 1921 (the first being carried species specificity is believed to
be due to the PVR protein found
out by Louis Pasteur himself in 1880 on bacteria of fowl cholera). Calmette and Guérin grew the on poliovirus. This PVR protein is
bovine strain of Mycobacterium tuberculosis (called M. bovis) for several years on a medium con- a member of the immunoglobulin
taining increasing concentration of bile. This in vitro cell culture of pathogenic M. bovis changed it superfamily.
into a less virulent and more suitable form of bacilli known as BCG (Bacillus Calmette–Guérin) a « The yellow fever virus is attenu-
commonly used vaccine against tuberculosis. Attenuated viruses are also used as vaccine, the vac- ated by passage through mice and
cines of polio, yellow fever and measles viruses being the most successful. chicken embryo cells, while Sabin
(type I) poliovirus is attenuated by
The process of attenuation involves growing microbes (bacteria or viruses) under abnormal growing the virus for prolonged
in vitro conditions, be it high bile concentration or passage through foreign cell/tissue such as periods in the epithelial cells of
embryonated eggs. These abnormal environmental conditions select those mutant cells that are kidney in monkeys.
able to survive and multiply under these conditions. These microbes are then harvested and used
as vaccine. In a normal host, these “pathogenic” microorganisms either fail to multiply or multiply
very slowly because these microbes are now “used to be grown” under abnormal conditions. The
reason for the loss of pathogenicity achieved by the “foreign cell passage” method is difficult to
determine. However, it is quite clear now that growing microbes in the unnatural host induces a
purely random series of mutations in their genome. This mutation can result in the change of sur-
face proteins of the virus (as in type 1 polio virus) or can make them temperature-sensitive (they
grow better at 320C than at 370C) or sometimes even cold-adapted (grow at 250C and not 370C).
» Polio was first described by Michel A D VA N TA G E S O F AT T E N U AT E D VA C C I N E S

Underwood in 1789. The first polio Because of the slow growth of attenuated viruses under normal body conditions, attenuated
outbreaks were reported around
the 1840s. vaccines provide for prolonged exposure of viral antigen to the immune system resulting in the pro-
duction of a large number of B and T cells and, more importantly, memory cells. As a consequence,
most of these vaccines are administered once in a lifetime and do not require repeated boosters.
The attenuated vaccines that are given orally to children (e.g. Sabin polio vaccine type I) is
» Sabin polio vaccine contains given orally on sugar cubes or drops. The attenuated virus enters the gastrointestinal tract and
about 1 million attenuated virus
particles of strains 1, 2 and 3. induces the production of secretory IgA as well as humoral IgG. These antibodies serve as an
important defence against naturally occurring poliovirus.
D I S A D VA N TA G E S O F AT T E N U AT E D VA C C I N E S
The principal limitation of these vaccines relate to concerns about their safety.
A major disadvantage of using live attenuated vaccine is the possibility of their reversion to
the virulent form. Type 2 and type 3 Sabin polio vaccine have been shown to revert frequently to
their wild type form. This has led to the outbreak of paralytic poliomyelitis and countries such as
Sweden have discontinued the use of Sabin polio vaccine.
Another disadvantage of live attenuated vaccines is that they cannot be given to persons hav-
ing immunodeficiency diseases as the immune system of these patients is severely compromised
» Albert Sabin, in 1961, developed
a vaccine containing attenuated and these pathogens, even though attenuated or weakened, can induce some damage.
poliovirus. This vaccine went on Since pathogenic microbes are attenuated by growing in tissue culture cells, sometimes
clinical trials in 1957 under the ae- these culture cells can become contaminated with other viruses. During the 1960s, SV40 virus-
gis of WHO. This attenuated vaccine
was administered to children in contaminated culture cells of kidney of monkey’s were inadvertently used in the production of
several countries including Russia, Sabin polio vaccine. Fortunately, no adverse reaction was reported but thereafter strict guidelines
Sweden and Holland. Following its were laid down to rule out the possibility of contaminating virus in the vaccine.
success, this vaccine was endorsed
by the United States Public Health Sometimes attenuated viruses shed from a recently vaccinated individual via faeces revert to vir-
Service in 1961. ulence and cause disease in other individuals. This has been reported for Sabin (type III) poliovirus.
16.2.3 I N A C T I VAT E D VA C C I N E S
» In 1955, Jonas Salk, an American Another commonly used method for vaccine production is to inactivate the whole pathogen and
physician, produced the first polio then use it for vaccination. The inactivation could be achieved by modifying the antigen (pathogen)
vaccine using inactivated virus.
He refused to patent the vaccine chemically by formaldehyde treatment or physically by heat treatment.
so that it is made and used as per The killed bacteria or viruses are then used in vaccines. Heat inactivation of microbes has one
its requirement for the benefit of potential problem. Heat inactivation of microbes causes the denaturation of surface proteins, that
humankind.
is, its antigenic determinants, and so their antigenic structure is altered. The antibody/immune
response induced by vaccinating these altered antigens may not be effective against natural patho-
gen. In contrast to heat inactivation, chemical inactivation gives better results. The bacteria or
viruses are treated with formaldehyde, phenol or propionolactone or other amino acid modifying
agents for a suitable period of time. This treatment usually results in the killing of the bacteria or
» Whooping cough vaccine contains
virus with almost no change in the antigenic structure. This approach has been successful for both
killed strains of the bacteria Borde- viral (Salk polio vaccine) and bacterial (whooping cough vaccine) pathogens.
tella pertussis, the causative agent However, chemically inactivated pathogens in vaccines are associated with some risks. The
of whooping cough. Whooping
cough is a contagious respiratory chemical agent sometimes does not kill all the pathogens present and, as a result, if these prepara-
disease characterized by violent tions (that contain chemically inactivated pathogen plus some live pathogen that has somehow
coughing spells due to clogging escaped inactivation) are administered, it might result in the disease in the vaccinee (person receiv-
of the respiratory passages by
thick mucous. Fatality is highest in ing vaccination). This happened with the early the batch of Salk polio vaccine when inactivation
infants under six months. procedures were still in their infancy. Formaldehyde failed to inactivate all of the polioviruses dur-
ing the preparation of Salk polio vaccine. The administration of this vaccine preparation caused
» Polio is the shortened form of large number of paralytic polio cases in the vaccinees.
poliomyelitis, derived from the
Greek word Polios meaning gray One of the greatest advantages of using inactivated/killed pathogen in a vaccine is that there is
and myelon meaning matter. no danger of mutation or reversion to the pathogenic form. Killed/inactivated vaccines induce suf-
Poliovirus is a single-stranded virus ficient humoral immunity (if boosters are given). However, they are less effective in inducing cell-
that belongs to the Picornaviridae
family, genus Enterovirus. mediated immunity or eliciting mucosal immunity. Since, these are killed pathogen inactivated
vaccine can safely be used in immunodeficient patients. The disadvantages include the need for
repeated boosters, higher cost, and sometimes, as mentioned previously, a failure in inactivation of
virus or bacteria resulting in immunization with virulent virus. The advantages and disadvantages
of inactivated vaccines are briefly summarized in Table 16.2.
VACCINES 347
Advantages Disadvantages
No mutation or reversion to wild type forms since Weak cell-mediated responses.
pathogenic organism is dead.
Provides sufficient humoral immunity. Requires booster stimulation since organism cannot
replicate inside host.
Heat-stable. Higher cost.
Can be used in immunodeficient patients Inadequate killing of virulent organism can result in Table 16.2
Advantages and disadvantages of an
occurrence of the disease.
inactivated vaccine.
Theoretically, live vaccines (natural or attenuated), are generally more effective than killed
ones. Live vaccines provide a large and continual antigenic challenge (slow-growing pathogen)
that replicates to give a large antigenic dose which lasts for days or weeks. Hence there is no need
for giving repeated booster doses. Moreover, antigens of live vaccine are presented by both class I
and class II MHC molecules inducing balanced response that includes Tcyt cell as well as TH cell and
antibody responses. Killed whole organism vaccine microbes are presented mainly by the class II
pathway and hence establish good humoral immunity. Dead microbe vaccines do not induce good
Tcyt-cell responses; the immunity is short lasting (requires booster) and often not detectable against Exotoxins
Exotoxins are toxic substances
all of the viral antigens (as inactivation may selectively modify or destroy the immunogenicity of secreted by living cells. They are
various virus proteins). highly potent and elicit major
damage to the host. Exotoxins are
proteinic in nature and hence heat-
16.2.4 TOXO I D VACC I N E S labile. Common toxin-producing
The virulence of some pathogenic bacteria depends primarily on the production of exotoxin. Exo- bacteria include Clostridium
botulinum and Pseudomonas
toxins are cytotoxic microbial poisons (proteinic in nature) produced by living cells. Some bacterial
aeruginosa.
pathogens such as diphtheria and tetanus bacilli produce exotoxins that induce several characteristic
symptoms associated with these infections.
In other words, the harmful part of these bacteria is their exotoxin, so if these exotoxins can Tetanus
be neutralized by the body the disease will not occur. These exotoxins are isolated and chemically Tetanus is a serious bacterial
disease. It is characterized by painful
modified (usually with formaldehyde) so that their toxicity (but not immunogenicity) is lost. These tightening of muscles all over the
non-toxic yet immunogenic derivatives of exotoxin, or toxoids, are commonly used in vaccines. body. It can lead to locking of the
Vaccination with a toxoid induces antitoxoid antibodies which are capable of binding the jaws of the victim so that the victim
cannot open the mouth or swallow
toxin and neutralizing it. Diphtheria and tetanus vaccines are among the most successful of all bac- anything. For this reason, it is also
terial vaccines produced from toxoids. The toxoids are generally mixed with aluminum hydroxide called lockjaw disease.
which acts as an adjuvant resulting in an increased production of specific antibodies, encouraging
its removal by phagocytic cells and hence stimulating T-cell response.
Toxoids
There are two major drawbacks. Not all pathogenic organisms produce exotoxin; secondly, Toxoids are modified toxins that
those who do produce them secrete them in minuscule quantities so it is difficult to purify them. have lost their toxicity yet are
Recently, the latter limitation was overcome by cloning and expressing the toxin gene in a different immunogenic. Toxoids are generated
either by a chemical modification
host. In this way, large quantities of toxins can then be harvested and toxoids formed. of the toxins by formaldehyde or by
heat treatment.
16.2.5 P O LY S A C C H A R I D E V A C C I N E S
The surface of bacteria is the first thing that is exposed to the immune system. Therefore, the surface « Botulinum toxin is one of the
antigens can serve as an excellent vaccine. Moreover, some bacteria have resistance to phagocytic most powerful naturally occurring
poisons in the world. Injections
invasion because these cells have capsular polysaccharides that resist phagocytosis (for example, of minuscule amounts of this
the polysaccharide capsules of S. pneumoniae, Haemophilus influenzae and Klebsiella pneumoniae). neurotoxin (as Botox) are used in
The coating of these capsular polysaccharides with antibodies and/or complement components the cosmetic industry for reducing
wrinkles on the face.
marks them for destruction by phagocytosis. These observations have paved the way for synthesiz-
ing vaccines against bacterial-purified polysaccharides.
The current vaccine against the bacteria Neisseria meningitidis which causes meningococcal
meningitis, contains purified capsular polysaccharide from types A and C. Another vaccine against
Haemophilus meningitis which is caused by bacteria Haemophilus influenzae type b consists of
purified capsular polysaccharide from H. influenzae type b (Hib). The vaccine for pneumococcal
pneumonia, (which is caused by about 80 different types of strains of Streptococcus pneumoniae) « Polysaccharide vaccines usually do
not stimulate T cells.
consists of 23 antigenically different types of capsular polysaccharides.
One of the major drawbacks of polysaccharide vaccines is their inability to stimulate TH cells « One out 20 people who contracts
or induce adequate memory response. Polysaccharide antigens can be made to stimulate TH cells pnuemococcal pneumonia dies
from it.
» Most healthy adults develop by conjugating them to some sort of a protein carrier. The polysaccharide–protein conjugate acti-
protection against pneumonia
within two to three weeks of
vates TH cells which in turn enables the formation of memory B cells. Even though memory T cells
vaccination. One dose of are not formed for some unknown reasons and very few memory B cells are formed, these poly-
pneumonia vaccine is needed in a saccharide vaccines can often provide long-lasting immunity probably because the antigens of the
lifetime.
capsules persist in the lymphoid tissue for a long time and are not easily degraded. This approach
has been tried for pnemococcal pneumonia (caused by S. pneumoniae) in which seven capsular
polysaccharides conjugated to protein are used in the vaccine. This vaccine unlike the previous one
that uses 23 capsular polysaccharides works better in infants.
A capsular polysaccharide covalently linked to a protein carrier is also used in the vaccine
for Haemophilus influenzae type b (Hib). To prevent an S. aureus infection, two types of capsular
polysaccharides linked to a carrier protein are usually given to the individual. The protein carriers
usually used are tetanus toxoid or diphtheria toxoid though the outer membrane protein of me-
ningococci is also considered as another potential carrier.
16.2.6 R E CO M B I N A N T A N T I G E N VACC I N E S
With the advent of recombinant DNA technology, virtually any gene-encoding immunogenic pro-
tein can be introduced and expressed in yeast, bacterial or even mammalian cells, using recom-
binant DNA technology. These cells are then cultured in the laboratory and the protein produced
endogenously is harvested. The genes that are selected for making recombinant antigen vaccine
are usually surface antigens (mostly glycoproteins). A number of genes coding for various surface
antigens have been successfully cloned in bacterial, yeast and mammalian cells cultures. Yeasts
have emerged as a better choice for making these surface antigens as they add and process carbo-
hydrate molecules on the protein in their Golgi bodies in a manner more similar to mammals, and
less care is needed in comparison with mammalian cells. One such vaccine approved for human
» Apart from a very popular recom- use is hepatitis B vaccine. A single gene for the major surface antigen of hepatitis B virus (HbsAg)
binant vaccine containing HBsAg, is cloned in yeast cells. The recombinant yeast cells are grown in fermenters. HbsAg, the surface
a recombinant vaccine has already
been approved and used for Lyme antigen, is expressed and accumulates inside the yeast cells. The yeast cells are then harvested and
disease. This vaccine, called Lymerix, then burst open by high pressure releasing the recombinant HbsAg (among other proteins). HbsAg
contains a recombinantly expressed is then purified by a standard biochemical technique such as affinity chromatography. The purified
surface protein of the causative
agent of Lyme disease, Borrelia antigen has been shown to induce humoral immunity. This approach has been used to make several
burgdoferi. potential HIV vaccines.
Recombinant antigen vaccines have both advantages and disadvantages. The advantages in-
clude the inexpensive production of a large amount of antigens as well as allowing space for genetic
manipulation of antigens. As a foreign gene is introduced from outside into the yeast cell, and usu-
ally the gene sequence of this gene is known, genetic manipulation of the gene is possible. Exotox-
ins of tetanus and diphtheria, which were previously modified chemically, can now be genetically
inactivated. Similarly, antigens can be made more immunogenic. The main limitation of this tech-
nique is the same as of inactivated vaccine. The recombinant antigens evoke humoral response,
stimulating B cells and TH cells (since antigen is processed by class II MHC pathway) but do not
generate potent Tcyt-cell response. Another limitation which is inherent to the technique itself is
that recombinant DNA technology cannot be used to synthesize carbohydrate antigen. Therefore,
this technique is ineffective in producing antigen if it is a carbohydrate.
16.2.7 L I V E V E C TO R VACC I N E S
In live vector vaccines, the desired gene coding for target antigens of the virulent pathogen is put
into a vector (attenuated bacteria or virus) and then this vector is infected (or administered orally)
to the vaccinee. This vector slowly replicates inside the inoculated individual and it serves as a
source of the antigen, delivering a large amount of antigen into the system and provoking a strong
immune response. A number of organisms have been used as vectors. The most commonly used
viral vectors are the vaccinia virus (the smallpox vaccine virus), adenovirus and canary pox virus
(that infects cells but does not replicate in humans). The bacterial vectors include attenuated Salmo-
nella typhi (Ty2la), BCG strain of Mycobacterium bovis, as well as the not-so-common but potential
vector Vibrio cholerae. In fact, all of the attenuated viral/bacterial vaccines have been suggested as
possible vectors for their use in live vector vaccines. The greatest advantage of using live vector vac-
cine is that there is complete immune response, that is, both humoral and cell-mediated protection
systems are activated.
VACCINES 349
V I R A L V E C TO R VACC I N E
The most commonly used viral vector is the vaccinia virus, though genetically engineered recom- « Vaccinia virus was the virus that
binant viral vectors such as pox viruses or adenovirus are also deliberated upon. was used for vaccination against
smallpox. Similar to the historical
Genes that have the potential to induce protective immunity (such as coat antigens) are in- cowpox virus used for smallpox
serted into attenuated live virus. Vaccinia, a commonly used virus has a large, double-stranded vaccination, it is a large, enveloped
genome (about 187,000 kb and approximately 200 genes). Because of its large size, a large number virus that belongs to the poxvirus
family. After the eradication of
of genes can be replaced with foreign (antigen) genes without causing any loss of their capacity to smallpox, this virus has been used
infect host cells. The disadvantage is, because of the large size, it becomes difficult to manipulate as a vehicle for delivering genes of
or insert the gene in vitro. So a simple but long method is used to prepare the vaccinia virus con- interest into biological tissues.
taining a foreign gene. The foreign gene (that encodes the antigen) is first inserted into a plasmid
vector. The plasmid vector is then transfected in tissue culture cells.
« Usually the genetic material is
These tissue culture cells are then infected with the vaccinia virus. Inside the tissue culture mixed with materials such as
cells, the vaccinia virus replicates in the cytosol where it undergo homologous recombination with calcium phosphate or calcium
plasmid containing antigen gene. This recombination event causes the antigen gene (which had chloride and then added to
eukaryotic cells. The DNA is taken
the vaccinia virus promoter associated with it) to get transferred to the vaccinia virus genome. This up by the eukaryotic cell by some
foreign gene is usually inserted into the viral tk (thymidine kinase) gene inactivating it. The viruses unknown mechanism.
that show tk-negative phenotype are selected (see Figure 16.4).
Foreign antigen gene

( Its protein product to be used as vaccine )
Vaccinia genome with

Plasmid vector Thymidine kinase
gene (tk)
Transfection
Vaccinia genome
Plasmid vector Vaccinia virus
infection
Mammalian cell culture
Homologous
recombination and assembly
Foreign antigen of virus particles
expressed
Foreign antigen gene integrated Foreign antigen gene not integrated

into vaccinia genome. Cell becomes wild type vaccinia virus showing
tk deficient because of this integration tk+phenotype Figure 16.4
Schematic representation of live viral
vector vaccine synthesis. The thymidine
kinase gene is used as a marker to check
Recombinant TK-deficient virus selected No foreign gene incorporated and hence the incorporation of an antigen gene
and used as vaccine antigen source of no use for vaccine production into the viral vector.
» Canary pox vector virus infects but This genetically manipulated vaccinia virus expresses a high level of gene product (for-
does not replicate in humans. How- eign antigen) and this serves as a potent source of immunogen inside the host. Effective live
ever, canary pox virus causes a fatal
disease in canaries and sparrows. vaccines based on this method have been developed for rabies in animals. Experimental viral
vector vaccines generated by this method include those that express influenza virus haema-
glutinin, malaria protein, hepatitis B surface antigen, herpes simplex virus proteins, among
others. An HIV vaccine that carries HIV env, gag, protease, part of pol genes in canary pox are
in clinical trials.
The advantages of viral vector vaccine include induction of both cell-mediated and humoral
immunity against the foreign antigen expressed by the vaccinia virus. Since a large chunk of for-
eign DNA can be inserted into the vaccinia virus, several antigens from different pathogens can be
inserted at the same time, introducing the possibility of a single vaccine for several diseases. The
major problem of viral vector vaccines is that since viral antigens are also expressed together with
foreign antigens, sometimes Tcyt-cell response occurs against virally infected cells, which results
in injury to the host cell. It is well known that live vaccinia virus can provoke a severe reaction in
a small percentage of people. Moreover, the risk created with the usage of attenuated live virus is
always there, that is, the danger of reversion of the virus to a pathogenic form. Some of the viruses
such as adenovirus which are also the candidate for carrying foreign antigens into the host cell, are
capable of transforming normal cells to cancerous one. Some of merits and demerits of viral vector
vaccines are given in Table 16.3.
» The full name of Salmonella B AC T E R I A L V E C TO R VACC I N E

typhimurium is Salmonella enterica Like the live viral vectors, some attenuated bacterial strains have been engineered to carry genes
serovar typhimurium. It is among the
most common Salmonella species
of virulent pathogens. The DNA encoding the antigenic determinants is inserted into the attenu-
that causes salmonellosis infections ated bacterial genome. The bacteria then express the antigen along with its own protein (see Figure
in USA. In humans, salmonellosis 16.5). The production and expression of antigen by the bacterial vector inside the host body stimu-
manifests as diarrhoea, fever and
abdominal cramps 12 to 72 hours
lates the immune system. An attenuated strain of Salmonella typhimurium (Ty2la), the causative
after infection and may last for agent of food poisoning, is being explored as a vector and is currently in human trials.
up to six to seven days. There are The advantage of this type of vaccine includes the fact that attenuated strains of
probably hundreds of millions of
cases every year in the world and
S. typhimurium, V. cholera and BCG are easily available and their genomes can easily be
Salmonella may kill twice as many manipulated. The use of such bacterial vaccine will produce immune response both against
people as typhoid. the vector as well as the inserted antigen (an inserted gene product). The use of some bacteria
such as Salmonella has an additional advantage: the bacteria not only induces cell-mediated
» Recently some vaccine antigens and humoral immunity but also mucosal immunity (IgA production) since these bacteria
have been expressed in plants!
Antigens from the plague pathogen
survive in the GI tract. Immunity against the pathogens of gonorrhoea and cholera are best
Yersinia pestis have been successful- provided by mucosal IgA. The disadvantages of bacterial vector vaccine include reversion and
ly expressed in the plant Nicotiana emergence of the pathogenic form of bacteria, rejection and elimination of bacterial vector
benthamiana.
before it can express the recombinant protein (as most of the population is already vaccinated
against the bacteria being used as vector). Moreover, antigens formed inside the bacteria may
be proteolysed by endogenous bacterial enzymes.
Strong humoral and cell-mediated im- Danger of reversion to virulence is always there.
mune responses.
Several segments of DNA encoding Some viruses have transforming capabilities making infected cells
antigens from different pathogens can be cancerous.
inserted in a single virus vector.
Can be targeted to specific tissues due to Immune response to virus-infected cells may cause damage to
viral tropism. vaccine.
No interference in protection produced
Table 16.3 by other types of vaccine.
Advantages and disadvantages of viral Inexpensive and easy to transport.
vector vaccine.
VACCINES 351
16.2.8 D N A VA C C I N E S Bacterial genome

The term DNA vaccine is misno- Foreign gene
mer. It wrongly implies that the desired as
vaccine
+
DNA is used as an antigen and
antibodies are formed against Attenuated bacteria
DNA. This is not the case. The DNA Plasmid vector
vaccine (or more properly DNA-
based vaccine) represents a new Vector introduced
class of vaccines in which there is a into bacterial cell
deliberate introduction of a DNA
plasmid usually into the muscle
cell of the recipient. The plasmid
contains a protein-coding gene (of
antigen) that gets expressed in the Attenuated bacteria
cell, leading to both humoral and harbouring plasmid,
cell-mediated immune responses. containing foreign
The plasmid DNA can be intro- antigen
duced into the muscle cell either Recombination
by infection or by bombarding
the skin with DNA-coated gold Expressed
particles with a fine airgun (gene- foreign
Bacterial genome,
gun). Currently, attempts are also antigen
harbouring
underway to introduce DNA into foreign antigen
the nasal tissue via nasal drops. It Attenuated but foreign-
should be noted that once inside antigen- expressing
the cells of the recipient, the plas- bacterial cell
mid does not replicate, but only
expresses itself, that is, protein is
produced. Usually bacterial plas- Figure 16.5
Diagram showing how live (bacterial)
mid is used and a gene coding the Bacterial cells used as vector vaccines are made using
antigen is inserted into the control live vector vaccine attenuated salmonella strain.
of a mammalian promoter and
this chimeric plasmid is then in- « In the DNA vaccine, the DNA is not
troduced into the recipient. The recipient cell then expresses the foreign antigenic protein coded by used as the antigen. It codes for the
the introduced DNA in the host body. The immune system then responds to the antigen as to any antigen that is expressed inside the
host cell/tissue.
other antigen entering the body. Figure 16.6 shows the mode of action of a DNA vaccine.
The DNA vaccines have several advantages. They are heat-stable as the DNA is a stable mole- « The DNA vaccine is very stable and
cule and so the storage and transport of these vaccine are easy. can be stored at room tempera-
ture in dried form or in solution.
The chimeric plasmids can be easily made in a laboratory in large amounts and, if the need Conventional vaccines have to be
arises, slight modifications in the DNA sequence (and hence the antigens’ amino acid sequence) can stored in specialized refrigerators
easily be introduced. Moreover, since proteins, are produced inside the muscle (or any other cell) in called “cold chain”.
the same way as normal proteins they are processed post-translationally the same way as proteins of « Muscle cells take up DNA more
the host cell. Memory cells against the antigen are synthesized, giving lasting immunity. easily than any other cell and hence
This makes them better antigens that can mimic proteins produced by real virus. As plasmids are used as target cells in DNA
vaccines.
do not replicate, the DNA vaccines are non-virulent. The DNA vaccines stimulate both cell-medi-
ated and humoral responses. Since, there is an expression of antigen for a long period of time there
« Scientists have attempted to
is also the induction of immunological memory. The foreign DNA can be manipulated to express
create DNA vaccines for a number
diverse antigens and other protein costimulatory molecules such as cytokines to get optimum im- of diseases affecting humans but
mune response. their attempts have not met with
One of the major drawbacks of the DNA vaccine is the concern over potential integration success till now.
of plasmids into the DNA of cells. Such an integration could lead to insertional mutagenesis and
cause the cell to become cancerous. In addition, there is a danger of induction of anti-DNA anti-
bodies causing pathological autoimmune reactions. Another drawback of the DNA vaccine is the
fact that it can only be formed against protein antigens. Vaccines against polysaccharide antigens
cannot be formed, as expected.
A brief summary of the advantages and disadvantages of DNA vaccines is depicted in Table 16.4.
Gene gun
Gold particle-foreign DNA

adduct introduced into target cell
Gold particle
Foreign DNA coding

for antigen
(DNA vaccine)
Foreign DNA
Transcription
mRNA
Transport
Nucleus
Translation
Foreign antigen expressd

Antigen-processing
pathway
Muscle cell
Foreign antigenic peptides presented

MHC on host-cell MHC, evoke cell-mediated
Figure 16.6 and humoral response
Mode of action of DNA vaccine. Antigenic peptide
Easy to manufacture in large amounts. Insertion of foreign DNA into host genome may cause cell to
become cancerous
Stable and easy to transport
DNA sequence and hence antigen can eas- Danger of autoimmune response due to formation anti DNA
ily be changed. antibodies
Mixture of plasmids could be used to form May induce immunologic tolerance by antigen(s) expressed
broad-spectrum vaccine. inside host body
Absence of protein components ensures Cannot be formed against polysaccharide antigens.
Table 16.4 there is no strong immune response
Advantages and disadvantages of DNA
against vaccine
vaccine.
In animal studies conducted so far, DNA vaccines have shown promising results. The im-
mune response is long-lasting and mimics the situation seen in normal infection by homologous
pathogen. Protection by DNA vaccines has been demonstrated with rabies, Plasmodium yoelli,
Mycoplasma and influenza. Currently, anti-HIV vaccines are also being explored.
VACCINES 353
16.3 N E W VA C C I N E S T R AT E G I E S
Diverse types of antigen delivery systems are being developed and experimented for optimal
antigen delivery into the recipient. Figure 16.7 lists some of the new vaccine strategies. These can
be categorized into several classes as is given in Table 16.5.
Amino acids +
Antigen
Introduced
Chemical synthesis
in capsule
Antigen
Antigenic peptide Biodegradable capsule
synthesised
Used as antigen Leaching
Synthetic vaccine
Antigen slowly
Capsule degraded
leaches out
Antigen
released
Microencapsulation delivery system
Artificial lipid
bilayer
Aqueous
environment
Hydrophobic antigen
anchored in bilayer
Hydrophilic antigen
enclosed in aqueous environment
Liposomes as vehicle for antigen
Lipid bilayer + glycoside Matrix

Quil-A
Monclonal
Antigen antibody
Figure 16.7
Some new vaccine strategies. Quil A of
Antigen ISCOMs allow liposome-like structures
to survive in the hostile environment of
the GI tract. SMAA complexes are easily
phagocytosed because of their large
ISCOMS - Immune stimulating Solid matrix-antibody-antigen
size and hence invoke a strong immune
complexes (SMAA) complexues response.
Vaccine Strategy Disease(s) against which it is currently directed or

explored
Synthetic Vaccine Currently been experimented against diseases such as AIDS, malaria,
schistosomiasis, hepatitis(B). Use of multiple antigenic peptides increas-
es immune response
Micro encapsulation delivery system Influenza (inactivated virus), tetanus (toxoid)
SMAA Clinical trials ongoing for several infectious diseases
Liposomes and Micelles Hepatitis A
ISCOMS Measles
Anti-idiotype Vaccine Hepatitis B
Antigen Cochelate Currently been explored for number of infectious diseases
Table 16.5
New vaccine strategies. SMAA – Solid matrix antibody-antigen cochelate; ISCOMS – Immune stimulating complexes.
16.3.1 S YN T H E T I C VACC I N E S
If we know the peptide sequence of protein antigens, they can be chemically synthesized in a labo-
ratory. A protein antigen is a large molecule and there are technical difficulties in synthesizing such
large molecules and hence small parts, or antigenic determinants, are synthesized. These synthetic
antigens have one major problem. They are poorly immunogenic (and do not elicit a strong im-
mune response, in this particular case cell-mediated response). This could be due to the fact that
synthetic antigens may not fold (that is, they don’t have secondary and tertiary structures) as the
» The first human synthetic vaccine
was developed in 1999 against the native antigenic determinants because the 3D conformation of antigenic determinants is deter-
Heamophilus influenzae type b (Hib) mined not only by the linear amino acid sequence but also by the conformation of the intact pro-
bacteria that causes meningitis and tein, as well as that of the virus/bacteria as a whole.
pneumonia in children.
A synthetic vaccine containing repeats of ~10 amino acids (of HbsAg) are currently in trial as
a potential hepatitis B vaccine.
16.3.2 M I C R O E N C A P S U L AT I O N D E L I V E R Y S Y S T E M
» The polymer of lactic acid and
In microencapsulation delivery system antigens are placed inside a biodegradable capsule (micro-
glycolic acid is biodegradable and
has been used in resorbable sutures spheres, nanospheres) and introduced inside the body of the recipient. The antigens are released
and bone plates for some years. So from a capsule either when the capsule is phagocytosed by macrophages and dendritic cells or
their safety is documented. via slow leaching. The polymer capsule is made up of lactic and glycolic acid (specifically poly-
Lactide–co–Glycolide).
These capsules (~ 5μm in diameter) enclose the antigen and are delivered orally or nasally.
» Microspheres and nanospheres
induce not only humoral immunity Animal studies have shown that these polymer capsules release antigens in controlled phases due
but also T-cell immunity as they to slow polymer degradation, eliminating the need for booster immunization. Moreover, these
stimulate Tcyt and TH cells as well. capsules can be phagocytosed and hence degraded inside antigen-presenting cells targeting anti-
gens to the lymphoid system.
Animal models are currently being experimented by microsphere-encapsulated influenza vac-
cine as well as with tetanus toxoid and Bortedella pertussis. The major drawbacks of this delivery
system is the lack of cell-mediated immune response to enclosed antigens. In addition, the po-
lymerization of the capsule around the antigen involves treatment with organic solvents that may
denature some of the protein antigens.
16.3.3 LIPOSOMES AND MICELLES

Liposomes are tiny spheres that contain a phospholipid bilayer enclosing an aqueous environ-
ment, while micelles are amphipathic molecules (such as detergents) that are arranged with their
polar head outwards and non-polar hydrophobic tail inwards the globular structure. The liposome
interior is aqueous and that can be used to enclose soluble antigens while non-polar/hydrophobic
» The idea that a liposome can be antigens can be inserted in the membrane. When introduced into the recipient, the membrane of
used as a carrier of vaccine was sug-
gested by Prof. Junzo Sunamoto of liposome fuses with the cells, releasing the antigen into the cytosol of the recipient cells. Liposomes
Kyoto University in 1992. containing hepatitis A antigens were found to induce high titres of antibody in the recipient.
VACCINES 355
16.3.4 I S C O M S — I M M U N E - S T I M U L AT I N G
COMPLEXES
ISCOMS are liposome-like structures having an outer coat of naturally occurring lipids mixed
with a detergent, and a glycoside such as Quil A (derived from the bark of tree Quillaia saponaria
Molina). These substances form an outer lipid membrane that encages the antigens inside. ISCOMS
can survive the hostile environment of the GI tract as well as up to several years. ISCOMS have
been shown to stimulate both humoral immunity and Tcyt/TH cells. This property makes ISCOMS
suitable for oral vaccines. HIV antigens incorporated in ISCOMS are being investigated as potent
HIV vaccines in animal studies.
16.3.5 S O L I D M AT R I X – A N T I B O D Y – A N T I G E N
COMPLEXES (SMAA)
Monoclonal antibodies are first formed against antigenic determinants of pathogens. These anti-
bodies are then covalently linked to the solid matrices. The antibody–matrix complex is then satu-
rated with antigen which binds monoclonal antibodies. A variety of antigens can be presented on
the same matrix by the using mixture of monoclonal antibodies. The complex which contains solid
matrix–antibody–antigen is then introduced into the recipient. Because of their large size, complex-
es are easily phagocytosed, and humoral and cell-mediated immunity elicited. Animal studies have
shown positive results after the administration of SMAA in mice having paramyxovirus infection.
Tcyt cells were primarily stimulated by SMAA.
16.3.6 A N T I - I D I OT YP E VACC I N E S
The unique amino acid structure of an antigen-binding site is referred to as idiotype. The antigen-
Anti-idiotype antibody
binding site is complementary to the structure of the antigen. So antibodies raised against the Anti-idiotype antibody an antibody
idiotype of the antibody will mimic the three-dimensional structure of the antigen. This can be directed against the antigen-binding
site of an antibody molecule.
used as a vaccine. When the anti-idiotype antibody (anti-ids) are injected into an individual, the
antibodies formed (anti-anti-ids) recognize the original antigen (virus) and can potentially neu-
tralize the antigen. Efforts are on to develop an anti-idiotype vaccine against hepatitis B antigen.
Clinical trials of anti-idiotypic vaccine developed against metastatic melanoma (I-Mel-2) are in the
phase II stage of testing in human subjects.
16.3.7 A N T I G E N - C O C H E L AT E
Antigen-cochelate consists of a continuous sheet of phospholipid bilayer—Ca2+ complex—within
which antigens are dispersed. The entire sheet is rolled up like a carpet roll. Because of the folding
of the lipid bilayer, few antigens are exposed, most of them are hidden and protected from degrada-
tion. When this complex is introduced into the recipient, it is slowly degraded exposing the hidden
antigen. It generates both humoral and cell-mediated immunity. This technique is relatively new
and is still in its infancy.
16.4 W H AT S H O U L D A N I D E A L VA C C I N E
H AV E ?
A vaccine should be able to generate immunological memory. Both memory B cells and T cells
should be formed. Upon subsequent exposure to the pathogen, memory cells specific for that par-
ticular pathogen will provide an accelerated response and immediate protection to the host. A
vaccine should provide lifelong immunity with a single dose. It should be preferably introduced
into the recipient by non-invasive methods such as oral administration or nasal spray. It should not
have any side effects. Vaccines should stimulate both arms of the immune system—humoral and
cell-mediated immunity.
Vaccines should be inexpensive, easily manufactured and stable in extremes of temperature or
humidity. Figure 16.8 gives some important characteristics of a vaccine. The immunization sched-
ule recommended by Indian academy of pediatrics is given in Table 16.6.
To conclude, a vaccine can be defined as a preparation of killed or inactivated pathogens
(bacteria, viruses, etc.) or their isolated antigens or a significantly similar molecule which can be
B/T- memory
cell formed
Vaccine
Life-long immunity
Cheap, easy to store
in single dose
and transport
Figure 16.8 Should stimulate both cell-mediated

The features of an ideal vaccine. and humoral immune response
Vaccine Age Recommended

BCG Birth–2 weeks
Polio vaccine Birth, 6, 10 weeks, 14 weeks, 16–18 months, 5 years
DPT 6 weeks, 10 weeks, 14 weeks, 16–18 months, 5 years
Hepatitis B Birth, 6 weeks, 14 weeks, 10 weeks, 14 weeks
Haemophilus influenza conjugate 6 weeks, 10 weeks, 14 weeks, 16–18 months
Measles 9 months plus
MMR 15 months
Typhoid Above 2 years
Additional vaccines
Varicella 1 year onward
Hepatitis A 2 years onward
Table 16.6 Note: To prevent prenatal transmission, birth dose of Hepatitis B vaccine within 12 hours is essential. In India, in addition to routine polio
Immunization timetable recommended vaccine, recommended “Pulse doses of Polio Vaccine” are also mandatory. Apart from the earliest age indicated, MMR, typhoid, Varicella,
by the Indian Academy of Paediatrics. Hepatitis A can be given at any age relevant to local epidemiology. (DPT—diphtheria pertussis tetanus;MMR—measles–mumps–rubella).
administered with the objective of stimulating a recipient’s protective immunity. The host is delib-
erately exposed to inactivated/attenuated/subunits of pathogen to induce immunologic memory so
that when the “real” pathogen tries to invade the host body, the host is ready with his/her defence.
Conventional vaccines include natural live vaccine, live attenuated vaccine, inactivated vaccine and
toxoid vaccine, to name a few. A battery of new vaccine strategies such as ISCOMS, SMAA, liposome-
enclosed vaccine and DNA vaccine and anti-idiotypic vaccine are also currently being explored.
VACCINES 357
Ion Exchange Chromatography

Ion exchange chromatography is an extremely
useful method for the separation of proteins
Negatively
and the isolation of important proteins of the
charged
body, including antibodies. Ion exchange Binding of proteins
proteins to ion
makes use of the fact that proteins are inher-
Anion exchange exchange column
ently charged molecules that are capable of
column
binding to an oppositely charged matrix. If a
protein has a net positive charge, say at pH 7.4, Addition of increasing
it will bind to a negatively charged matrix concentration of positive
Change of pH Elution of
(such as carboxymethyl cellulose); while and negative ions (Na+Cl-)
bound proteins
negatively charged proteins will not bind to
this matrix. Similarly a negatively charged pro-
tein will bind to a positively charged matrix
(such as diethylaminoethyl cellulose) while
positively charged proteins will remain un-
bound. Once the protein is bound it can be Cl- Cl-
eluted by two different means. Cl- Cl-
The first is by increasing the ionic strength Cl- Column
Cl- Column
of elution buffer: for example, if a negatively Cl-
Cl- Cl-
charged protein is bound to a positively Cl- #L
charged column, the addition of increasing #L Cl- Eluted
concentration of salt such as NaCl to the col- Na+ Eluted protein
Na +
umn will elute (detach) the protein from the protein
Na+ Na+
column. This is because Na+ ions of the salt will Na+
compete with the proteins for the charged Oppositely charged ions bind Protein no longer
groups present on the ion exchanger. Hence, column and protein, and charged, hence, eluted
ion exchanger groups will bind the Na⫹ ions hence, protein is eluted
(because they are present in high concentra- Figure 16.9
tion) and release the protein which will be Principle of ion exchange chromatography.
eluted.
The second is by altering the pH of the eluting buffer. When the pH negatively charged proteins and vice versa. DEAE cellulose is com-
of the eluting buffer approaches the isoelectric point of the bound monly used for the fractionation of serum proteins. Typically, anion
protein its net charge becomes zero, and hence the protein no exchanger column (such as DEAE cellulose) is packed and the pro-
longer remains bound to the matrix and is eluted. tein is allowed to bind to the column. After loading, the protein is
eluted by slowly increasing the salt concentration of the elution
Both cationic (positively charged) and anionic (negatively charged) buffer (see Figure 16.9). The protein that has weakest interaction
exchangers are commonly used. Cationic exchangers are used for with the charged matrix will be eluted first.
S U M M A R Y
• Α vaccine is a preparation of pathogenic agents or their constituent • Attenuated vaccines use weakened pathogen to stimulate the
parts, which can be administered with the objective of stimulating immune system of the vaccinee. Since pathogens are attenuated or
protective immunity. weakened they rarely cause damage.
• Vaccines that are commonly used include attenuated vaccine, inac- • Inactivated vaccines use killed pathogen while toxoid vaccines use
tivated vaccine, toxoid vaccine and polysaccharide vaccine. a chemically modified toxin derivative that has lost its toxicity but
still retains immunogenicity.
• Capsular polysaccharides of bacteria have also been used as • DNA vaccine involves the direct introduction of an antigen gene
vaccine with excellent results. into the muscle cell or antigen-presenting cell. The target gene as a
gene–gold particle adduct is bombarded on the cell by a gene gun.
• In making recombinant antigen vaccine, gene coding for the anti- Once inside, the gene is expressed and the antigen displayed on the
gen is introduced into the host cell (yeast, bacteria) using recombi- host cell MHC that stimulates immune system.
nant DNA technology. The protein is then expressed and harvested,
and used as a vaccine. • An ideal vaccine should (a) provide lifelong immunity with a single
dose, (b) be non-invasive (c) stimulate both humoral and cell-
• Live vector vaccine (bacterial or viral) serves as a source of anti- mediated immunity (d) be cheap and easy to store and transport.
gen inside the vaccinee. Antigen genes are introduced in attenuated
bacteria or harmless viruses and are infected into the host system.
The antigens formed and released by these vectors stimulate both
B-cell- and T-cell-mediated immunity.
K E Y W O R D S
• active immunity 345 • live attenuated vaccine 345 • solid matrix–antigen–antibody

• antigen-cochelate 355 • live vector vaccine 348 complexes 355
• attenuation 345 • natural live vaccine 345 • toxoid vaccine 347
• bacterial vector vaccine 350 • polysaccharide vaccine 347 • vaccination 344
• DNA vaccine 351 • recombinant antigen vaccine 348 • vaccine 343
• inactivated vaccine 346 • synthetic vaccine 354 • viral vector vaccine 350
• ISCOMS 355
1. Why do you think that a DNA vaccine cannot be prepared for all H INT — All known diseases include infectious and non-infectious diseases,
antigens? Think and answer including deficiency diseases.
2. Tourists travelling to remote areas of South Africa are given a few
5. Give the advantages and disadvantages of attenuated and inacti-
days’ course of anti-amoebic drug. Do you think that this is a
vated vaccines. Which do you think will stimulate both humoral
vaccination? Comment.
and cell-mediated immunity? Which of them is better suited for an
3. Can inactivated AIDS virus be used as a vaccine? If no, give
immunocompromised individual ?
reasons.
4. Can we make vaccines against all the known diseases? Is it even
theoretically possible?

1. In DNA vaccine, DNA is used as: (c) Can replicate inside the host cell.
(a) An antigen (d) Stimulates both humoral and cell mediated immunity
(b) An adjuvant
(c) Genetic material to express antigen 5. An immunogenic yet non-toxic derivative of toxin is called:
(d) As a non-specific B-cell stimulator (a) Vaccine
(b) Toxoid
2. Polysaccharide vaccine usually does not stimulate: (c) Endotoxin
(a) B cells (d) Exotoxin
(b) T cells
(c) Production of antibodies 6. One of the advantages of using a live vector vaccine is:
(d) None of the above (a) Antigen delivery
(b) Stimulation of both B- and T-cell response
3. A vaccine will not be effective in inducing long-term immunity (c) Easy to manufacture
if it is: (d) Easy to store and transport
(a) Not stored properly
(b) Introduced by invasive method 7. An inactivated vaccine does not stimulate:
(c) Does not generate any memory cells (a) B-cell response
(d) Not cost effective (b) Tcyt-cell response
(c) NK-cell response
4. Which of the following statement is not true for a DNA (d) All of the above
vaccine?
(a) Can be transcribed in the host cell
(b) Can be translated in the host cell
VACCINES 359
8. DNA vaccine cannot be prepared for: (c) Subunit vaccine

(a) Toxin (d) Viral vector vaccine
(b) Surface antigen
(c) Capsular polysaccharide 10. Vaccination induces:
(d) Protein antigen (a) Naturally acquired active immunity
(b) Artificially acquired active immunity
9. A vaccine that will never lead to host cell injury is: (c) Naturally acquired passive immunity
(a) Natural live vaccine (d) Artificially acquired passive immunity
(b) Attenuated vaccine

1. Sabin polio vaccine can be safely administered to an immuno- 4. Natural live vaccines are natural non-pathogenic organisms that
compromised individual. induce specific immunity.
2. DNA vaccine contains nucleic acid with adjuvant. 5. Even a single nucleotide change in an inactivated vaccine can
make it virulent.
3. An ideal vaccine should stimulate cell-mediated, humoral and
mucosal immunity.
Arnon, R. and M. H. V. Van Regenmortel (1992). “Structural Fields, B. N. and R. M Chanock (1989). “What Biotechnology
Basis of Antigenic Specificity and Design of New Vaccines”, Has to Offer Vaccine Development”, Review of Infectious
FASEB Journal, 6: 3265–74. Disease, 11: 519–23.
Autran, B., G. Carcelain, B. Combadiere and P. Debre (2004). Garrison, F. H. (1917). An Introduction to the History of
“Therapeutic Vaccines for Chronic Infections”, Science, 305: 205–08. Medicine.
Bell, R. and G. Torrigiani (eds) (1986). Progress Towards Better Moss, B. (1985). “Vaccinia Virus Expression Vector:
Vaccines. Oxford: Oxford University Press. A New Tool for Immunologists”, Immunology Today
6: 243–45.
Clark, I. A. and K. A. Rockett (1996). “Nitric Oxide and Parasitic
Disease”, Advances in Parasitology, 37: 1–58. Roitt, I. M. (ed) (1984). New Trends in Vaccines in Immune
Intervention, 1. London: Academic Press.
Corral, R. S. and P. B. Petray (2001). “CpG DNA as a Thl-
promoting Adjuvant in Immunization Against Trypanosoma
cruzi”, Vaccine, 19: 234.
During ancient times, medicine was a curious concoction of primitive
science, intelligence, boldness and superstition. As time passed, medi- “He conquers
cal science slowly replaced superstition and surgeons began dreaming who endures.”
—PERSIUS
of replacing the missing or defective parts of the human body. Prob-
ably the first attempted transplantation was recorded by
G. Tagliacozzi in 1597. He successfully repaired the lost nose of a patient
using skin from the patient’s arm. Corneal transplantation in an
animal (gazelle)was attempted by Samuel Bigger in 1897. It is believed
that the first human corneal transplantation was done in 1906 by
Dr Edward Zirm while Dr Reverdin performed the first skin graft trans-
plantation. The technique of vascular anastomosis (joining of blood
vessels) was perfected by a French surgeon, Alexis Carrel, who made
the transplantation of organs and limbs feasible. He suggested, for After studying this chapter, you
should be able to:
the first time, that some unknown interactions are responsible for the
• Define autograft, isograft,
rejection of grafted organs. In 1944, P. B. Medawer, a British zoologist allograft, xenograft
(studying skin transplantation), who was interested in the immuno- • Describe transplantation
antigens
logical aspects of tissue transplantation established conclusively that • Differentiate between direct
and indirect presentation of
the rejection of foreign tissue has immunological specificity and is alloantigens
• Explain and illustrate mixed
based on the same mechanism that provides protection against invad- lymphocyte reaction
ing pathogens such as bacteria or virus. In 1949, Burnet introduced • Give an account of hyperacute,
acute and chronic rejection
the concept of self and non-self antigens. He suggested that since • Briefly summarize the
mode of action of different
self-antigens are present during embryonic life, they somehow cause specific and non-specific
immunosuppressive agents
destruction of self-reactive clones (a mechanism called clonal dele- • Describe hyperacute and
delayed xenograft rejection
tion) of antibody-forming cells. The deletion of these anti-self clones
• Explain graft vs. host disease
ensure that the body will not mount an immune response against self-
antigens. This thesis was tested and validated by Medawer, for which
Burnet and Medawer won the Nobel Prize in 1960. With the discovery
of classical transplantation antigens—the major histocompatibility
complex, the intricate mechanisms of graft acceptance and rejection
were finally deconstructed and understood. Figure 17.1 shows the role
of histocompatibility antigens in tissue transplantation reaction.
Transplantation
Immunology 17
17.1 INTRODUCTION
Transplantation is the process of taking tissues or organs (or even cells) and placing them into the
same or different individual. The tissues, organs or cells that are transferred from one individual
to another are called grafts. The individual who donates the graft is called donor and individual
who receives the graft is referred to as recipient. Clinical transplantation is usually performed to
overcome a functional or anatomical deficit in an individual. The transplantation of kidney, heart,
liver, cornea, lungs, pancreas and bone marrow is now performed worldwide.
Transplantation of a graft from donor to a recipient has one major drawback. When a damaged « Corneas have to be processed and
tissue of one individual is replaced by the healthy tissue of a genetically non-identical individual, transplanted within six days.
an inflammatory reaction sets in which leads to rejection of the graft by recipient. Immunologists
have classified grafts according to their origin and the identity of the recipient.
• Autologous graft (autograft): These are graft transplants from one region to another on
the same individual.
• Syngenic graft (syngraft)/Isograft: Isograft involves the transfer of graft between genetically
identical (syngenic) individuals of the same species. This is possible between genetically
identical twins or genetically identical mice.
Graft
Recipient (expressing same Graft accepted

histocompatibility antigen as
donor)
Histocompatibility
antigen
Donor
Graft
Recipient (expressing different Graft rejected

histocompatibility antigen as Figure 17.1
Diagram showing the role of
from donor)
histocompatibility antigens in
acceptance and rejection of grafts.
• Allogeneic graft (or allograft): A graft transplanted between two genetically non-
identical individuals of the same species is called allograft.
• Xenogeneic graft (or xenograft): These are graft transplants between individuals of dif-
ferent species. Figure 17.2 illustrates different types of tissue transplantations.
» Siblings have a one-in-four chance
of sharing all MHC antigens and It has been shown that graft is permanently accepted only when essentially all of its transplanta-
being ideal donors in an allograft. tion antigens (such as histocompatibility antigens) are present in the recipient. If the graft has
Relatives (genetically related)
usually share more MHC antigens transplantation antigens that are different from the transplantation antigens of the recipient, an
than other unrelated people, while immune response that leads to the rejection of the graft takes place.The molecules that are recog-
populations from different parts nized as foreign (antigen) on an allograft are referred to as alloantigens and those present on a
of the world are less likely to share
MHC antigens than people of xenograft are called xenoantigens. Usually autograft and isograft survive because they evoke little
similar origins. or no immune response whereas allograft and xenograft are rejected under normal conditions.
17.1.1 T R A N S P L A N TAT I O N A N T I G E N
Transplantation antigens (synonymously used with histocompatibility antigens) are those antigens
Transplantation antigen
Transplantation antigen is an (proteins) that are present on cells (or tissue surface) and are responsible for either the acceptance
antigen that is responsible for graft or the rejection of the graft. These antigens induce the immune response in the host that may cause
acceptance and rejection. In practice, the rejection of the transplanted tissue. The transplantation antigens are products of genes called
major transplantation antigens
are the major histocompatibility histocompatibility genes. One set of histocompatibility genes, called major histocompatibility com-
complex, the H-Y antigen; and, plex (MHC) genes, specify the cell surface molecules that elicit the most rapid allograft rejection.
to a lesser extent, the minor The MHC antigens are termed as human leukocyte antigen or HLA complex in humans (the genes
histocompatibility antigens. The
H-Y antigen is defined as a male of which reside on chromosome 6) and H-2 complex in mice (located on chromosome 17).
histocompatibility antigen. These The structural details of MHC antigens in humans and mice have already been discussed in Chap-
male-specific antigens cause ter 6. These MHC proteins are cell surface transmembrane glycoproteins that are present on all host
rejection of the male skin grafts by
female recipients of the same inbred cells and are involved in interaction with the cells of immune system. MHC genes are inherited from
strain of mice. both parents and are expressed codominantly. Figure 17.1 shows the role of MHC in transplantation.
Co-dominant expression means that animals of F1 generation of A and B (A × B) will ex-
press both A-strain and B-strain alleles (see Figure 17.3). This conclusion is based on the result of
experimental transplantation between inbred strains of mice. Grafts between individuals of the
Autograft Isograft
(Accepted) (Accepted)
Figure 17.2
Diagram showing difference between
autograft , isograft, allograft and Allograft Xenograft
xenograft. (rejected) (rejected)
TRANSPLANTATION IMMUNOLOGY 363
same inbred strain of species are

always accepted. In contrast, grafts
of tissues or organs between indi- HLA-A
HLA-B
viduals of genetically different inbred HLA-C
strain of same species of mice are HLA-D
always rejected. Moreover, inbred
strain (A × B) will never reject grafts
from either parents (A or B). This
is because A × B animals see both « Dissimilar MHC elicit the most
Maternal set of Paternal set of
A and B tissues as self. rapid allograft rejection.
MHC Alleles MHC alleles
17.1.2 IM M UNOLOGY
OF A L LO G E N E I C
TRANSPLANTATION
The immune response to alloantigens
of a graft can be both cell-mediated
and antibody-mediated. However, Progeny
T-cell responses are more impor-
tant for the rejection of transplanted
grafts but in some cases, as we shall
see, antibodies may also contribute.
This chapter focuses on allogeneic « The foetus is an almost perfect
allograft that is somehow never
transplantation because it is more rejected!
common and better understood.
P R E S E N TAT I O N O F
ALLOGENEIC MHC
TO T CELLS
Allogeneic MHC molecules (MHC, Figure 17.3
Line diagram explaining inheritance of
which are expressed on allogeneic
MHC antigens through simple Mendelian
tissues or cells) are presented to host inheritance.
T cells in two different ways.
• Direct presentation involves the recognition of intact MHC molecules (together with as-
sociated peptides) displayed on allogeneic donor cells.
• Indirect presentation involves the processing of a donor’s MHC protein molecules by the
recipient’s antigen-presenting cells and their subsequent display with self-MHC. This self-
MHC + foreign peptides (derived from donor MHC) present the antigen to self-T cells.
DIRECT PRESENTATION OF ALLOANTIGENS. Self-T cells are selected in such a way that they
recognize and respond to self-MHC molecule plus foreign peptide. Some of these T cells, can
cross-react with foreign MHC plus foreign peptide displayed on donor cells and elicit a T-cell
response.
Thus, the same TCR which recognizes self-MHC plus foreign peptide may also recognize one « Intact allogeneic MHC molecule
or more (foreign) allogeneic MHC molecules. Several experimental evidences point to this fact. plus foreign peptides are directly
recognized by self-T cells in direct
• Monoclonal antibodies against the antigenic binding site TCR inhibits the recognition of presentation.
both self-MHC–foreign peptide complex and allogeneic MHC–peptide molecules.
• Transfection of rearranged TCR genes into another T cells confers specificity of both for
self-MHC plus foreign peptide and for allogeneic MHC molecules.
A T-cell receptor normally recognizes self-MHC plus foreign peptide. What it actually “sees”
are particular amino acids arranged in a specific 3-D conformation that fits into the crevices of the
TCR. Sometimes what happens is that allogeneic MHC molecules alone provide specific amino
acids arranged in a 3-D conformation in such a way that the TCR confuses it with foreign peptide +
self-MHC and binds to it and elicits an effector response. Sometimes, allogeneic MHC molecules
together with bound peptides mimic the structure of self-MHC + foreign peptide. In either case,
the host T cells elicit a strong immune response leading to graft rejection. This recognition of al-
logeneic MHC–peptide complex by T cells, results from the fact that T cells that can respond to
self-peptide + allogeneic MHC are not entirely removed from the T-cell repertoire and hence re-
spond to the allograft. In fact, it has also been shown that each allogeneic MHC molecule may be
recognized by and responded to by many different TCR molecules on different T cells in vivo. It is
believed that around 1 per cent of the T cells can directly recognize and respond to a single alloge-
neic MHC (foreign) molecule. The direct presentation of alloantigen is illustrated in Figure 17.4.
INDIRECT PRESENTATION OF ALLOANTIGEN. Alloantigens, of which MHC molecules comprises

the major part, may also be endocytosed by host antigen-presenting cells. These MHC molecules
are processed like any other foreign peptide (through the endosomal pathway) and presented on
the antigen-presenting cell surface together with class II MHC molecules.
Indirect presentation of alloantigens occurs usually by class II MHC molecules and hence TH
cells of the host are stimulated, though cross-priming of Tcyt cells sometimes occurs as some al-
loantigens can enter the class I MHC pathway in antigen-presenting cells. Because an MHC mol-
ecule is one of the most variable/polymorphic proteins in the body, each allogeneic MHC molecule
can give rise to multiple antigenic determinants, each recognized by different T cells. Experiments
» Processed allogeneic MHC is
recognized by host T cells in an in class II MHC knockout mice have confirmed the indirect presentation of alloantigens. These
indirect presentation. mice express only class I MHC molecules on their cells. Skin grafts from mice that express only
class I MHC molecules have activated
the recipient’s TH cells and B cells, apart
T-cell response
from Tcyt cells. These results imply that
class I MHC molecules are taken up by
the recipient’s antigen-presenting cells,
which process and present them to TH
cells, apart from other cells. Figure 17.5
summarizes the indirect presentation of
alloantigen.
Effector T cell Cell exhibiting self-MHC
and foreign peptide
T-CELL RESPONSE TO
Normal T-cell response
ALLOANTIGENS IN VITRO:
MIXED LEUKOCYTE REACTION
T-cell response Graft rejection is often a T-cell mediated
response and we can actually see if the
donor cells can activate recipient T cells
to divide. The mixed leukocyte reac-
tion (MLR) is a useful in vitro model of
T-cell recognition of allogeneic MHC
gene products and is used as a predic-
Foreign cell exhibiting
foreign (allogeneic) MHC
tive test of cell-mediated graft rejection
Self-T cell or acceptance (that is, is used in tissue
that resembles self-MHC
+foreign peptide typing). The MLR is induced by incu-
bating cultured mononuclear leukocytes
(which include T cells, B cells, NK cells,
T-cell response
phagocytes and dendritic cells) from
the donor with mononuclear leukocytes
derived from the recipient. If two in-
dividuals have a difference in alleles of
MHC and hence are incompatible for
transplants, a large proportion of mono-
nuclear cells will be stimulated to divide
Foreign cell exhibiting
allogeneic MHC and peptide over a period of three to seven days. This
Self-T cell proliferative response indicates that the
that resmbles self-MHC
Figure 17.4 and peptide recipient will react to allogeneic antigen
Diagram showing the direct presentation
of alloantigens. Direct presentation and is not suitable for transplantation.
Class I MHC
Phagocytosis of
allogeneic cells
Processing by
antigen-presenting (?)
Class II MHC cells
Foreign cell expressing

allogeneic MHC
Peptide from allogeneic

MHC presented on self-MHC
Effector T cells
(Tcyt)
Activate cell-mediated
and humoral response
Antigen-presenting cell Effector T cell

with allogeneic (TH) Figure 17.5
MHC peptide on self-MHC Indirect presentation of alloantigens.
Histocompatibility varies inversely with the number of mitotic cells produced. This response is also « Histocompatibility is inversely
sometimes called allogeneic MLR. proportional to the clonal expan-
sion of lymphocytes in the MLR.
If mononuclear leukocytes from a donor and a recipient are mixed in culture and both sets of
leukocytes proliferate, it is called two-way MLR. However, in most cases, to simplify the analysis,
donor leukocytes are prevented from proliferating by γ-radiation/treatment with an antimitotic
drug such as mitomycin C. These donor cells which now cannot undergo mitosis are then incubated
with recipient leukocytes. In this one-way MLR, treated donor cells serve exclusively as stimulator
cells and recipient cells as responder cells. Figure 17.6 depicts a simplified diagrammatic represen-
tation of one-way and two-way MLRs. If histo-incompatible recipient cells respond, CD8+ T cells
differentiate into Tcyt cells specific for allogeneic MHC, and CD4+ T cells differentiate into TH cells
of both TH1/TH2 types that secrete cytokines.
The responding T cells recognize specific MHC molecules on the stimulator cells. Tcyt cells
recognize class I MHC molecules on stimulator cells namely HLA-A, HLA-B and HLA-C in hu-
mans and H-2k, H-D or HL in mice. TH cells, stimulated in allogeneic MLR are specific for class
II MHC molecules I-A and I-E in mice, and HLA-DP, HLA-DR and HLA-DQ in humans. Several
evidences suggest that molecular targets recognized by T cells in MLR are MHC molecules. This
was shown by the ability of Tcyt cells formed against stimulator cells (say A) to lyse target cells (say B)
from another donor if these targets share class I MHC molecule (or alleles).
The stimulation of T cells in an MLR reaction requires both MHC–peptide complex and its
« Though, most alloreacting CD8+
costimulating signal. The full differentiation of Tcyt cells requires stimulation by allogeneic class I T cells differentiate into Tcyt cells,
molecules, as well as costimulators of antigen-presenting cells or cytokines by TH cells. Similarly, some CD8+ T cells produce
full activation, proliferation and differentiation of CD4+ T cells can be induced only by profes- IL-2, IFN-γ and TNF similar CD4+
T cells. Similarly some CD4+ T cells
sional antigen-presenting cells that express allogeneic class II MHC molecules and also provide differentiate into alloreactive Tcyt.
costimulatory signals.
C E L L - M E D I AT E D R E S P O N S E T O A L L O G R A F T S I N V I V O
MHC molecules play a central role in initiating graft rejection (or acceptance) in vivo, as in MLR.
MHC molecules are presented by both direct and indirect pathways of allogeneic presentation. The
Donor leukocytes Recepient leukocytes Both donor and recepient

leukocytes multiply
Two-way MLR
γ-radiation
+
Mitomycin-c
Figure 17.6
Mixed leukocyte reaction: Line diagram Donor leukocytes Donor Recepient Only recepient leukocytes
explaining one-way MLR and two-way leukocytes leukocytes multiply
MLR. (mitosis inhibited)
importance of MHC molecules in allograft rejection was established by the fact that grafts between
congeneic strains of inbred mice were rejected when strains differed only in class I or class II MHC
alleles but not other genes.
Both donor and recipient antigen-presenting cells are likely to be involved in graft rejections.
The most important antigen-presenting cells are likely to be dendritic cells of either donor or recipi-
ent origin. Donor antigen-presenting cells may stimulate T cells entering the graft through the blood
supply. Alternatively, a donor’s antigen-presenting cells may migrate from the graft into the lymph
and reaching lymph nodes, where they activate naïve alloreactive T cells by direct presentation of
» Antigen-presenting cells of the
graft that pass into the host are allogeneic MHC. These motile antigen-presenting cells that move from graft to host are also called
called passenger leukocytes. passenger leukocytes. The role of passenger leukocytes in graft rejection is shown in Figure 17.7.
Passenger leukocytes
(dendritic cells) Direct presentation
of allogeneic MHC
IL-2,γIFN
TH
Tcyt IL-2,IL-4,IL-5
Effector Tcyt B cell

(Receipient)
Damage to
Recipient’s Antibodies
the graft
antigen-presenting
cell
Figure 17.7
Role of passenger leukocytes in graft
rejection. Donor organ Recipient’s lymph node
Similarly a recipient’s antigen-presenting cells may enter the graft and pick up donor al-
loantigens or alternatively the recipient or host antigen-presenting cells may also pick alloanti-
gens entering lymphoid tissues, process them and present them to alloreactive T cells. However,
the donor antigen-presenting cells may or may not play a vital role in graft rejection. Studies
on knockout mice suggest that allografts (of solid organs) that contain antigen-presenting cells
but lack costimulators B7-1 and B7-2 necessary for antigen-presenting function are still rejected
suggesting that donor antigen-presenting cells may not be essential to stimulate the rejection of
solid-organ allograft.
Antibodies are formed against alloantigens; however, the exact mechanism of B-cell activation
by foreign MHC molecules is still not known and probably involves stimulation of B cells with
other foreign antigens (apart from stimulation provided by cytokines).
17.2 TYPES OF GRAFT REJECTION

Depending on the time taken to reject the graft and the nature of immune response (humoral or
cell-mediated) to the graft, graft rejection has been classified into three histopathologic patterns:
hyperacute, acute and chronic rejections.
17.2.1 HYPERACUTE REJECTION « Hyperacute graft rejection occurs

The hyperacute rejection mechanism is initiated by pre-existing antibodies. It begins within a few almost immediately after transplan-
minutes to hours after a graft is transplanted. Once the blood vessels of the host are anastomosed tation, usually within 24 hours. This
rejection is either due to preformed
to graft vessels, pre-existing antibodies against the donor’s endothelial antigens enter the graft. anti-MHC antibodies, natural anti-
These antibodies bind endothelial antigen and fix the complement component damaging the bodies to blood type antigens, or
endothelial cell lining of the blood vessels. The damaged endothelial cells secrete the Von-Wil- those antibodies that are formed in
response to previous blood transfu-
lebrand factor which mediates platelet adhesion and aggregation. Moreover, endothelial cells sions or previous transplants or
lose from their surface an an- developed during pregnancy to the
ticoagulant, heparin sulphate, baby’s paternal MHC antigens.
which prevents blood coagu-
Inside the graft
lation under normal circum-
stances. The process of plate-
let aggregation and the loss of
anticoagulating factor from Preformed Blood vessel
endothelial cells contributes to antibodies Solid organ
thrombosis and vascular oc- in the host Complement activation
clusion, depriving the graft of and damage to
endothelial cells
blood supply and leading to
irreversible ischaemic damage
Von Willebrand Heparin
to the graft (see Figure 17.8).
factor Endothelial
Why are these pre-existing secreted antigen
antibodies against a graft
present in a supposedly nor- von Willebrand
mal individual? (a) Antibodies factor-mediated
platelet adhesion
against a graft (particularly
and aggregation
against donor blood cells that
invariably accompany any
graft) could be induced during Platelets
prior blood transfusion, previ-
ous rejection of another trans- Occlusion of blood vessel
plant or even during multiple
pregnancies. (b) Humans also
have pre-existing “natural
antibodies” against alloantigen
(of IgG or IgM type) at
high titre before any exposure
Figure 17.8
to alloantigens. These natural Line diagram showing the steps in
antibodies are believed to Damage to the grafted organ hyperacute rejection of graft.
» Natural antibodies against the arise because of carbohydrate antigens expressed by bacteria that normally colonize the gut (nor-
normal flora that circulate in the
body cause hyperacute allograft
mal flora) and other body parts. These antibodies, though formed against carbohydrate antigen of
rejection. bacteria, can cross-react with ABO antigen present on red blood cells (and vascular endothelial
cells on which they are also expressed) and elicit hyperacute rejection.
17.2.2 ACUTE REJECTION

» Acute rejection is mediated by Acute rejection usually begins after the first week of transplantation. It is mediated primarily by
T cells. effector T cells, though macrophage activation and antibodies also play a role. Within the T-cell
response, both TH and Tcyt cells may contribute to acute rejection.
The T-cell response occurs to responding alloantigens, including MHC molecules, alloanti-
gens on vascular endothelium cell as well as on other cells of the graft. Since, it takes a few days to
generate effector T cells (as well as B cells), acute rejection starts after a week of transplantation.
The activated Tcyt cells cause direct lysis of graft cells, and TH-cell-generated cytokines recruit and
activate inflammatory cells such as macrophages which cause necrosis. An acute rejection shows
histologically different patterns as compared to a hyperacute rejection. Acute rejection is charac-
terized by necrosis of graft vessel walls, with acute inflammation (see Figure 17.9), which is distinct
from the thrombotic blocking of blood vessels without necrosis of blood vessel wall observed in
hyperacute rejection.
Both types of T cells participate in
acute rejection reactions and experimen-
tal evidences suggest that both Tcyt and TH
cells play an important role in an acute
Solid organ rejection. Histological evidences reveal
(kidney) that grafts undergoing acute rejection
are markedly enriched for Tcyt cells spe-
cific for graft alloantigens. Tcyt cells can
be used to transfer acute cellular graft re-
» Alloreactive Tcyt cells directly lyse
jection to a naïve unexposed individual.
graft endothelial cells. Alloreactive Inside the graft However, other cells apart from Tcyt cells
TH cells summon and activate mac- also contribute, as knockout mice lack-
rophages and initiate grafted tissue
injury by delayed-type hypersensi- Tcyt ing Tcyt cells or perforin still elicit acute
tivity Alloreactive antibodies bind IL-2, IL-4, rejection of allograft, suggesting other
to the endothelium, activate the IL5, rIFN cells or mechanisms also contribute to
complement pathway and injure TH
graft blood vessels.
acute rejection.
TH cells participate in acute graft
T-cell response against allogeneic MHC
reaction by secreting cytokines and
inducing delayed-type hypersensitivity
reactions in grafts. The importance of TH
cells in acute rejection reaction can be as-
sessed from the fact that the transfer of al-
loreactive TH cells into naïve mouse cause
the rejection of an allograft.
Antibodies are formed by activated
B cells (stimulated by TH cells) against
Tcyt cells, antibodies and macrophages
damage the graft (necrosis)
antigens present on vascular endothelium
cells, leading to complement-mediated
cell lysis and acute rejection necrosis.
17.2.3 CHRONIC
REJECTION
Chronic rejection of an allograft is a
slow process taking months or years
Necrosis of Inflammed vessel (usually because of immunosuppressive
graft walls wall
treatment). Chronic rejection may be
Figure 17.9
Line diagram showing the steps in acute cell-mediated or antibody-mediated.
rejection of graft. Rejection of the graft When mediated by antibodies, antibodies
alone or an antigen–antibody complex

may cause damage to grafted tissue lead-
ing to graft rejection. The pathogenesis
of chronic rejection is not very well
understood. The main feature of chronic
rejection is the thickening of blood ves- Macrophage
sel walls which eventually get blocked.
Inside the graft γIFN
This arterial occlusion occurs as a result
of a proliferation of smooth muscle cells
which migrate (from the intima) in the Alloantigen TH
vessel wall and deposit matrix proteins
Graft arteriosclerosis
on the side of blood vessels leading to Graft arteriosclerosis is the blocking
vascular occlusion and blocking of arter- of an artery due to smooth muscle
ies in the graft. This is called graft Intimal smooth muscle
proliferation and production of
collagen by fibroblasts. This process
arteriosclerosis (see Figure 17.10). cell surrounding the vessel wall results in fibrosis which can cause
The smooth muscle proliferation of ischeamia and cell death.
the intima of blood vessels, is induced
by cytokines secreted by lymphocytes TGF-β
which are activated by alloantigens
present in the graft vessels. These cy-
tokines, particularly IFN-γ, stimulate
« Chronic rejection is characterized
macrophages that secrete platelet- by fibrosis with loss of normal organ
derived growth factor (PDGF) and structures. Some scientists suggest
TGF-β that chronic rejection is a form of
transforming growth factor-β (TGF-β). Secretion of growth factors delayed-type hypersensitivity.
TGF-β induces smooth muscle prolif- by macrophage
eration (which causes graft arterioscle-
rosis) while PDGF induces fibroblast
proliferation. Fibrosis is the result of
fibroblast proliferation and secretion
of collagen throughout the graft. The
formation of scar tissue or fibrosis
throughout the grafted organ or tissue
is another feature of chronic rejection.
It is because of chronic rejection that Blocking of blood vessel due to proliferation of
most of the kidney transplants have a smooth muscle cells (Graft arteriosclerosis)
half-life of about 10 years, though ad- Figure 17.10
Line diagram showing the steps in
vances in clinical science are seeking to Graft rejection chronic rejection of graft.
control the chronic rejection process.
17.3 IMMUNOSUPPRESSIVE THERAPY

OF ALLOGRAFT RE JECTION
Allogeneic transplantation requires some degree of immunosuppression to accompany it, other-
wise the transplantation invariably results in some form of rejection. Th ere are two main types of
immunosuppressive treatments: antigen-non-specific and antigen-specific.
17.3.1 ANTIGEN-NON-SPECIFIC IMMUNOSUPPRESSIVE

AGENTS
C YCLOSPORIN A
Cyclosporin A (Cs A) is a cyclic peptide made by a species of fungus. Cs A blocks the activation « Cyclosporin A is derived from
of resting T cells (TH cells) by inhibiting the transcription of IL-2 genes. Cs A binds a cellular the soil fungus Tolypodcladium
inflatum.
protein, cyclophilin, and this complex binds and inhibits calcineurin (Ca2/calmodulin-activated
phosphatase).
» Cyclosporin A is the most com- Calcineurin activates transcription factor NF-AT (nuclear factor of activated T cells) that is
mon immunosuppressive drug
administered in heart and lung
involved in the transcription of IL-2.The inhibition of calcineurin inhibits the transcription and
transplantation. The most adverse expression of IL-2, thus inhibiting IL-2-dependent growth and differentiation of T cells.
effect of this drug is nephrotoxicity. CsA also suppresses cytokine production by T cells by reducing the expression of the receptor
» Cyclosporin A inhibits the tran-
of IL-2 on lymphocytes. Moreover, it also induces the synthesis of immunosuppressive cytokine
scription of IL-2 gene. TGF-β. In addition, it blocks the synthesis of IFN-γ (which has antigraft properties).
CsA is one of the agents that has been shown to prolong the graft survival of kidney and liver.
Before the use of CsA, the majority of heart and liver transplantations were rejected within a short
period of time. Now a majority of these grafts survive for about five to eight years.
F K - 5 0 6 ( TA C R O L I M U S )
» FK-506 comes from the Japanese Another potent metabolite that has a potent immunosuppressive effect is FK-506. It functions like
filamentous bacterium Streptomyces cyclosporin, and binds a binding protein in the cell. This protein is called FKBP (FK-binding pro-
tsukabaensis. FK-506 is most effec-
tive in liver transplantation. tein) also binds and inhibits calcineurin’s activity. FK-506 is most effective in liver transplantation
and in those cases where kidney rejection is not adequately controlled by CsA. FK-506 blocks the
transcription of IL-2, IL-3, IL-4, IFN-γ and TNF.
R A PA M YC I N
» Rapamycin is produced by a fun- Another immunosuppressive agent is rapamycin, a fungal product whose principal action is to in-
gus found on Easter Island (which hibit T-cell proliferation. Rapamycin also binds to FKBP. This rapamycin–FKBP complex does not
the inhabitants call Rapa Nui from
which it derives its name). inhibit calcineurin but binds to another cellular protein, MTOR (mammalian target of rapamycin),
and this ultimately interferes with the intracellular signalling pathways associated with IL-2 recep-
tor and prevents IL-2-dependent T-cell activation and proliferation.
M YC O P H E N O L AT E M O F E T I L ( M M F )
» A common adverse effect of MMF MMF is a relatively new drug that kills proliferating mature T cells activated by alloantigen and also
is leukopaenia. inhibits the maturation of T cells (in fact it is a lymphocyte and not T-cell-specifi c) from immature
precursors. MMF blocks the synthesis of guanine nucleotide by the de novo pathway by inhibiting
lymphocyte-specific isoforms of inosine monophosphate dehydroxygenase. Since MMF has nar-
row toxicity as it inhibits only lymphocyte-specifi c isoform of this enzyme, it has few toxic effects
on other cells of the host.
CORTICOSTEROIDS
» Steroid hormones are lipid- Corticosteroids are one of the most potent anti-infl ammatory agents. The proposed mechanism
soluble hormones that pass of action of these “natural hormones” is to suppress activated macrophages. Steroids block the
through the plasma membrane to
bind cytosolic steroid receptors. synthesis and secretion of inflammatory cytokines such as IL-1, IL-6 and TNF. Th ese cytokines
They then move to the nucleus and are essential for lymphocyte–antigen-presenting cell communication and a decrease in cytokine
bind DNA. It is believed that steroid production effectively obstructs the capacity of an antigen-presenting cell to activate allograft-
receptors regulate expression of
about 1 per cent of human genes. specific lymphocytes. Corticosteroids also suppresses various effector mechanisms of macro-
phages such as generation of reactive oxygen and nitrogen species, prostaglandins. High doses
of corticosteroids inhibit the secretion of cytokines by T cells, reduce MHC expression and even
lyse T cells. The corticosteroids commonly used in preventing graft rejection are dexamethasone
and prednisone.
A Z AT H I O P R I N E / C YC L O P H O S P H A M I D E
» Azathioprine and cyclophos- Azathioprine is an antiproliferative drug, often administered both before and after transplantation
phamide are the most commonly to prevent T-cell proliferation. Azathioprine, an analogue of 6-mercaptopurine, blocks the synthe-
used cytotoxic drugs for immune
suppression. These drugs block sis of inosinic acid, a precursor of purines—adenine and guanine. Moreover, its incorporation into
DNA synthesis and affect rapidly the DNA of dividing cells prevents their further replication. Azathioprine is toxic to proliferating B
dividing cells. and T cells as well as to the enterocytes in the gut.
Cyclophosphamide, cyclized nitrogen mustard, is an alkylating agent that inserts in DNA and
cross-links it, leading to the disruption of the DNA chain. It is especially active against prolif-
erating T cells which rapidly divide in response to an allograft.
However, MMF has rapidly replaced azathioprine/cyclophosphamide and is currently being
used with cyclosporine to prevent allograft rejection. The mode of action of some non-specific im-
munosuppressive agents is shown in Figure 17.11.
IL-2
IL-2 receptor
expression inhibited γIFN
γIFN IL-2
TNF IL-3
Cyclosporin Tcyt
IL-4
FK-506
(Obtained from the fungi (Obtained from the bacterium

Tolypocladium inflatum) Streptomyces tsukubaensis)
IL-1
Blocks cell
proliferation
ROS, RNS Figure 17.11
Synthesis Line diagram showing the mode of action
blocked of non-specific immunosuppressant on
B or T cell transplantation. Cyclosporin blocks the
synthesis of IFN-γ, IL-2 and IL-receptor;
FK-506 blocks IFN-γ, TNF, IL-4 and
Azathioprine IL-3. Azathioprine blocks cell division.
TNF
Corticosteroid blocks IL-1, TNF, ROS and
(Analogue of 6-mercaptopurine) Corticosteroid
RNS synthesis.
17.3.2 SPECIFIC IMMUNOSUPPRESSIVE AGENTS

Non-specific immunosuppressive agents abolish or diminish the activity of the immune system
regardless of the provoking alloantigen. These usually leave recipients vulnerable to infections. The
best approach would be to inactivate only those clones of T or B lymphocytes that are activated by
alloantigens, leaving the rest of the immune system intact. Such highly specific immunosuppres-
sion still remains a dream though some efforts made in this direction are detailed below.
MONOCLONAL ANTIBODIES TO T-CELL ANTIGENS

« Monoclonal antibodies are now
Monoclonal antibodies directed against various surface molecules of the cells of the immune being used and tested on humans.
system have been used to suppress T-cell activity. The most widely used reagent is OKT3, a mouse Monoclonal antibodies being used
monoclonal antibody specific for human CD3. Once injected in vivo OKT3 binds CD3 on T cells, include anti-CD3 and those that are
tested include anti-CD4, anti-CD40L
activating the complement pathway to induce cell lysis or activating phagocytes by acting as an and a fusion protein of CTLA-4
opsonin. This clears T cells from circulation which aids in graft acceptance. and the Fc region of the human
Another antibody in common use is specific against CD25 (α subunit of IL-2 receptor) mol- antibody.
ecule present on the surface of T cells. Anti-CD25 antibodies which are usually administered at the « The most recent FDA-approved
time of transplantation bind IL-2 receptors thus blocking IL-2 binding to T cells (hence T cells are monoclonal antibodies are the
not activated). Additionally, they delete CD25-expressing T cells by a mechanism similar to OKT3. IL-2 receptor antagonists that are
genetically engineered chimeric
The major drawback experienced is that in both cases mouse monoclonal antibody, being an an- antibodies possessing both human
tigen, is rapidly cleared from the human system. For this reason antibodies is either humanized/ and murine antibody sequences.
chimerized. A chimeric (human–mouse) antibody, Basiliximab, is also currently used to prevent
acute graft rejection. This antibody acts as an IL-2 receptor antagonist as it binds a subunit of IL-2 Chimeric and humanized
receptor (CD25) and prevents the binding of IL-2 to its receptor. antibodies
Cell surface adhesion molecules another target for monoclonal antibody therapy. The simul- Chimeric antibodies have constant
taneous treatment of cardiac grafts with monoclonal antibodies to adhesion molecules such as domains of the human IgG molecule
that are combined with the murine
ICAM-1 and LFA-1 for a week after transplantation has resulted in a successful life-long survival variable regions by transgenic fusion
of graft. However, antibodies against both adhesion molecules should be administered simultane- of the immunoglobulin genes,
ously as the administration of as of antibody alone leads to the rejection of transplantation. while humanized antibodies were
developed in such a way that 6 CDRs
of the heavy and light chains of the
B L O C K I N G C O S T I M U L AT O R Y S I G N A L S murine monoclonal antibody were
The method of blocking costimulatory signals has successfully induced tolerance to kidney and grafted by recombinant technology
to the CDR-depleted human IgG.
pancreatic allografts in monkeys but is still in clinical trials on human subjects. The blocking of B7
Antigen-presenting
CD3 cell
CD25 B7 Soluble CTLA-4

ICAM-1
added
Monoclonal
B7 binding blocked
antibodies
CD28
LFA-1
Monoclonal
antibodies
Figure 17.12 T cell CD40 Ligand
Diagram explaining various specific (blocked)
immunosuppressive agents that Monoclonal antibodies T cell
suppress graft rejection. against T-cell antigen Blocking costimulatory signals
molecules on antigen-presenting cells is performed by a soluble form of CTLA-4. Soluble CTLA-4

binds B7 and prevents it to bind CD28 antigen on T cells, suppressing T-cell activation. Similarly
monoclonal antibody that binds CD40 ligand on T cells prevents its interaction with CD40 on
antigen-presenting cells. We know that T-cell activation requires both ligand binding at T-cell
receptor as well as costimulatory signals. In the absence of costimulatory signals, T cells become
anergic and usually lead to a state of immunosuppression which increases the life of the graft. Some
specific immunosuppressive therapies are shown in Figure 17.12.
17.4 IMMUNOLOGY OF XENOGENEIC

T R A N S P L A N TAT I O N
One of the major obstacle in the transplantation of tissues or organs is the lack of availability of
donor organs. The need for an alternative source of donor organs has focused the attention on other
mammals such as non-human primates, baboons, chimpanzee, monkeys and pigs.
The major immunologic barrier with xenotransplantation is that immune rejection is quite
vigorous even in the presence of immunosuppressive drugs. The immune reaction that results
in xenograft rejection are hyperacute xenograft rejection, delayed xenograft rejection and acute
T-cell-mediated rejection.
17.4.1 HYPERACUTE XENOGRAFT REJECTION

» Karl Landsteiner in 1901 was the Hyperacute reaction to xenograft transplantation occurs because humans have “natural antibodies”
first to describe the existence of that can react with xenogeneic determinants. As discussed previously, natural antibodies are IgM
separate blood groups in humans.
The naming A, B, AB and O of the antibodies directed towards non-self carbohydrate determinants of the ABO blood group system.
blood groups was done by Von Over 95 per cent of humans have such natural antibodies that react strongly with the corre-
Dungren and Hirszfield in 1910. sponding antigens manifested on evolutionary distant species. Natural antibodies are rarely produced
» A vast majority of pig organ xeno- against carbohydrate determinants of closely related species such as humans and chimpanzees. Thus
grafts are rejected because humans the chimpanzee monkey can technically be used as an organs donor to human beings, but is not done
have natural anti-pig antibody for both logistic and ethical reasons. Xenograft from pigs is preferred due to several reasons. They
against a cell surface antigen that
is analogous to the human blood breed rapidly, have large litter, and have several physiologic and anatomic compatibility.
group H antigen, but has galactose Hyperacute reactions to pig xenograft induce rejection reactions similar to human allograft
(pig) instead of fucose (human). rejections. The reactions include the loss of the anticoagulant heparin sulphate from endothelial
» Xenograft rejection occurs mainly cells, that inhibits complement activation and generation of endothelial cell pro-coagulants and
by hyperacute mechanism. platelets (aggregating substances). The activation of the complement pathway both by human an-
tibody binding to pig cells and loss of heparin sulphate leads to a severe reaction to xenogeneic pig
cells. This complement-induced damage is more severe than natural antibody-induced pathoge-
nicity because the complement regulatory protein—decay accelerating factor (DAF)—synthesized
by pig cells cannot interact with human proteins of the complement pathway and hence limit its
damage. DAF normally dissociates C3 convertase and prevents the activation of C3 and C5. Efforts
are underway to construct and breed transgenic pigs that have cells that can overexpress human
blood group H antigen and human complement regulatory proteins such as DAF, thus overcoming
two main reasons that generate hyperacute reactions to xenografts.
17.4.2 D E L AY E D X E N O G R A F T R E J E C T I O N
Even when hyperacute xenograft reaction is prevented by immunosuppressive therapy, xenograft « Delayed xenograft rejection
are still rejected within four to five days of transplantation. This form of rejection is called delayed involves antibodies but not
complement activation.
xenograft rejection.
The mechanism of delayed xenograft rejection is still not clear but it involves antibodies
not complement activation. It is believed that damage to graft tissue is caused by the production
of antibodies that damage xenogeneic endothelial tissues. NK cells and macrophages have
also been implicated in the reaction. Delayed xenograft rejection is characterized by intravascular
thrombosis and necrosis of blood vessel walls. In experimental animals, the prevention of delayed
xenograft rejection is done by reducing antibody-response by the use of cytotoxic drugs such as
cyclophosphamide and methotrexate.
17.4.3 T - C E L L - M E D I AT E D X E N O G R A F T R E J E C T I O N
Grafts from xenogeneic species such as pig induces a similar T-cell response as human allogeneic « Pig tissues might contain retro-
transplants. However, the xenoresponse is too vigorous or strong to be controlled by immuno- viruses called porcine endogenous
retrovirus and there is a fear that
suppressive drugs. Human T cells are activated by pig MHC molecules presented both by direct these might infect the human
and indirect pathways. T-cell activation leads to cell-mediated rejection of xenografts. Methods for recipient.
inducing tolerance to xenografts include the administration of MHC peptide from xenogeneic spe-
cies and blocking of costimulation when the recipient is first exposed to xenograft.
In addition to the problem of rejection, xenotransplantation has the potential of spreading
pathogen from xenogeneic donor to human recipient, for example, HIV-2 and herpesvirus B
which infect several species of monkey can lead to deadly infection in humans. Apart from intro-
ducing new viruses to humans, they can create new viruses or a new agent of disease: for example,
theoretically SIV (simian immunodeficiency virus) may combine with human HIV to create a new,
more dangerous virus that could infect both the species.
17.5 TRANSPLANTS TO PRIVILEGED SITES « The cheek pouch of hamsters is

also a privileged site.
Certain areas such as brain, anterior chamber of the eye, cornea, uterus and testis are regarded as
immunologically privileged sites because these sites can tolerate grafting without eliciting a rejec-
tion reaction.
These sites fail to induce an immune response because of the dearth or complete lack of lym-
phatic vessels and in some cases even blood vessels. Consequently, the alloantigens of the graft are
not able to sensitize the recipient lymphocyte, increasing the likelihood of acceptance even when
HLA antigens are not matched. Cornea, being a non-vascular tissue is also a privileged location
and hence corneal transplants are highly successful. If, however, the cornea is placed on a vascular-
ized tissue or if trauma at the time of operation causes inflammation and vascularization occurs,
the grafted cornea is rejected.
17.6 O R G A N T R A N S P L A N TAT I O N
Ever since the first kidney transplant between identical twins in the 1950s, millions of organs or « Before organ transplantation, HLA
tissue transplantations have been performed. About 400,000 kidney transplants, 80,000 bone mar- (MHC antigens) and ABO blood
groups of the donor and the recipi-
row transplants, 60,000 liver transplants, 45,000 heart transplants, lung transplants 6,500 and 2,000 ent are matched. ABO blood groups
pancreas transplants have been performed worldwide. Organs transplants are usually performed and HLA compatibility are essential
when the organ is damaged or dysfunctional and there is threat to the patient’s life and there is for the survival of the graft.
always a sense of urgency associated with transplantation.
17.6.1 K I D N E Y T R A N S P L A N TAT I O N
Kidney transplantation is usually done in patients who have two failing or dysfunctional kidneys. « The kidney was the first solid
Usually a person with a single functional kidney lives a normal life without any clinical problems. organ to be transplanted.
Kidney failure that requires transplantation of a new kidney is usually elicited by a wide variety of « Kidney transplantation is per-
diseases such as glomerulonephritis, drug-induced nephritis and various other types of kidney dis- formed usually within 48 hours as a
ability diseases such as diabetes. donor kidney cannot be stored for
more than two days. About half of
As can be predicted from studies, there are two main problems associated with kidney transplant. all the kidneys transplanted come
First, kidneys are always in short supply because of lack of donors. The second problem is the large from brain-dead individuals.
number of sensitized recipients. An average kidney transplant lasts about five to eight years. Rejec-
tion of the first transplant sensitizes the patient, activating the immune response (both humoral and
cell-mediated) directed against kidney antigen. Any subsequent kidney transplant is hence quickly
rejected. Therefore, a detailed tissue-typing procedure is performed to ascertain that the patient has
no antibodies or active cellular mechanism directed against the potential donor kidney. Tissue typing
is a procedure that determines the type of histocompatibility antigens present on the donor (and re-
cipient) cells or tissues. This procedure is typically used prior to transplantation of tissues or organs to
ensure as close a match as possible between the donor and the recipient. Tissue-typing can be done by
several ways, which include HLA typing (see Figure 17.13 and also Experimental Insight of Chapter 6)
and functional assays such as MLR (discussed previously).
» Lung transplantation is performed
on patients with respiratory insuf- 17.6.2 L U N G T R A N S P L A N TAT I O N
ficiency or failure. All the donated Conditions such as cystic fibrosis and acute damage due to smoking, irreversibly damage the lungs
lungs are from brain-dead, heart- necessitating grafting of lungs. Lung transplants are either performed alone or together with heart
beating donors. Donors must be
non-smokers of less than 65 years transplant. No systematic study has been performed except that it has a survival rate of 60 per cent
of age. during the first year.
» Heart transplantation is an option
for patients suffering from the end 17.6.3 H E A R T T R A N S P L A N TAT I O N
stage of coronary heart disease, Heart damage due to various types of diseases, or congenital or acquired defect in heart circuitry
congenital heart disease or arrhyth- or valves necessitates heart transplantation. Accident victims who are brain-dead but have an intact
mia. The donor heart is preserved
in special freezers and transplanted
within four to six hours.
Microtiter wells coated

HLA-B HLA-B with donor cells
Variant 1 Variant 2
Antibodies against
HLA-B variant 2 added
Antibodies bind cells bearing

HLA-B variant 2
Complement
proteins added
Cells bearing HLA-B

variant 2 lysed
Coloured dye, eosin or

trypan blue added
Dye permeates cell,

Figure 17.13 Dye excludes cell implies cell displays HLA-B
Simplified view of tissue typing. variant 2 molecule on its surface
circulatory system and functioning heart are usually the source of donated heart. There is a big scarcity
of donors. Though tissue HLA matching is desirable, it is not often possible due to lack of donors. A
donor’s heart is kept viable for a short period of time in an ice-cold buffer that short-circuits the pace-
maker. The release of electric impulse from the pacemaker could damage the isolated heart. The recipi-
ent’s heart is removed and patient is kept alive wholly artificially by a heart–lung machine that circulates
and aerates the patient’s blood till the donor heart is transplanted. The one-year survival rate for the
transplantation of the heart is more than 80 per cent. Heart transplant rejection is probably antibody-
mediated, leading to a atherosclerotic lesion in the coronary arteries of the transplanted heart.
17.6.4 L I V E R T R A N S P L A N TAT I O N
Liver damage can occur due to variety of reasons such as cirrhosis, exposure to virus as in viral « After kidney, liver transplantation
hepatitis or ingestion of harmful chemicals, chronic alcoholism, hepatocellular carcinoma and is the second most common solid
organ transplantation. In this case,
fulminant hepatic necrosis. A damaged liver usually regenerates itself once the cause or causative agent the ABO group and size of the liver
has been cleared. If the liver fails to regenerate, it has to be transplanted. However, the majority of liver of the donor and the recipient are
transplants are used as therapy for congenital abnormalities of liver. A liver donor is a cadaver. matched.
Liver transplantation is not an easy task as the liver is so extensively vascularized; it immuno-
logically resists re-implantation. Moreover, the paucity of donors has necessitated that a liver from
a single brain-dead donor be split and successfully transplanted to two different individuals. The « Liver transplantation undergoes
rejection of liver transplant is effected by antibody-mediated hyperacute rejection. Donor blood acute rejection in 50 per cent of the
cases.
cells carried within the transplanted liver elicit humoral response in recipients. Anti-blood group
antibodies against donor red blood cells or antibodies formed against passenger leukocytes initiate
damaging hyperacute reactions.
17.6.5 PA N C R E A S T R A N S P L A N TAT I O N
Pancreas is an insulin producing organ that controls the blood sugar level and hence the occurrence
of diabetes. The pancreas, or more specifically its islet of Langerhans that produces insulin, can be
damaged due to a variety of reasons, varying from viral infections to generation of anti-islet antibod-
ies. This response leads to pancreas dysfunction and consequent damage (pancreatitis) with resultant
diabetes. Pancreas transplantation can be performed by whole organs or better by transplanting in-
sulin producing islet cells. Pancreas transplantation usually restores the body’s function of producing
insulin. Kidney and pancreas transplantations are usually performed simultaneously as damage to the
pancreas results in diabetes which in turn leads to kidney damage. A pancreas donor is a cadaver.
17.6.6 S K I N T R A N S P L A N TAT I O N
Skin grafts in humans are usually performed with autologous skin, that is, skin transplanted from « Skin cells may be grown in culture
one region to another from the same individual. If skin damage is more widespread, frozen skins to form artificial skin. Skin, by the
virtue of its rich vascular bed of
are taken from tissue banks and used. The grafted skin does not grow on the recipient but acts as blood capillaries and lymphatics, is
a biological dressing till the body grows its own skin tissues. Skin grafts are replaced from time to highly immunogenic.
time as their cellular contents become no longer viable.
17.6.7 B O N E M A R R O W T R A N S P L A N TAT I O N
Bone marrow transplantation is used as a remedy for a number of malignant and non-malignant
conditions which lead to defects in the haematopoietic system, such as leukemia, thalassemia,
aplastic anemia, lymphoma, as well as several immunodeficiency diseases. It has also been pro-
posed as a means of correcting inherited deficiencies or abnormalities of enzymes by providing a
self-renewing source of normal stem cells.
For the purpose of bone marrow transplantation, bone marrow inoculum is obtained from a liv-
ing donor (usually allogeneic donor) by aspiration through a hypodermic syringe. Bone marrow in-
oculum contains haematopoietic stem cells that can give rise to a variety of lineages such as erythroid,
myeloid, megakaryocytic and lymphocytic lineages. Transplanted allogeneic stem cells will be readily
rejected by even a minimally immunocompetent host. So, the usual procedure is for recipients of
a bone marrow transplant to be immunologically ablated or suppressed to permit successful bone
marrow transplants. Such ablation is accompanied by total body irradiation or chemotherapy by cy-
totoxic drugs such as cyclophosphamide. The bone marrow inoculum is then injected intravenously
into the host whose T cells have been ablated by radiation or by chemotherapy. The intention of this
bone marrow transplantation is to reconstitute the recipient’s immune system using an infusion of
haematopoietic stem cells of the donor. The transplanted donor cells then reconstitute the entire
haematopoietic system of the recipient. The reconstitution depends on variety of factors, including
histocompatibility, between donor and host and the number of bone marrow cells inoculated.
These transplanted stem cells must establish themselves in the appropriate niches inside the
» About 108–1010 cells/kg body bone marrow cavities. If these niches are somehow occupied at the time of transplantation, bone
weight of the recipient are injected marrow grafting is usually not successful. Even after successful transplantation, one major problem
into host body during bone marrow
transplantation. frequently associated with bone marrow transplantation is graft-versus-host disease (GVHD).
It may be noted that allogeneic transplantation (as well as xenogeneic transplantation) can be
considered as chimera formation since the donor cells or tissues carry different DNA sequences from
those of a recipient. From the viewpoint of genetic constitution, the recipient carries two types of
DNA sequences—one derived from germ-line DNA and other from the transplanted DNA; hence the
recipient can be considered as a chimera. Thus, as very aptly pointed out by Chigra (1997), chimera
is a genetic concept while transplantation is an immunological term. In general these two terms can
be considered identical. In successful bone marrow transplantation, the “self ” dictated by germ-line
DNA is “cancelled”(by irradiating the bone marrow of the recipient that is the source of immune
» Chimera (Greek: khimaira— self) and a new “self ” is reconstituted from allogeneic/grafted bone marrow cells that recognize both
she-goat) is a Greek mythological host cells and donor cells as the “new self ”. Such a bone marrow chimera has been constructed for
fire-breathing monster with the
head of a lion, the body of a goat several animal models such as mouse (recipient )–human (donor), sheep (recipient)–human (donor),
and the tail of a serpent. and among mice/rats of different strains. Such animal models are useful in various studies related
to organ transplantation such as the
development of tolerance-induction
Bone marrow inoculum
protocols and testing of immunosup-
containing T cells (Donor cells) pressive drugs, and their success has
been translated into success in humans.
Transplant
17.7 GRAFT
Immunocompromised VERSUS HOST
host (recipient)
DISEASE (GVHD)
GVHD reactions are an expression
Inside the graft
of T-cell function. When an immu-
Donor
nologically competent bone marrow
TH cell inoculum is transplanted into an im-
munologically compromised host,
Minor histocompability Cytokines the graft tissue can mount an immu-
antigen IL-1,IL-2,TNF-b nologic attack on the alloantigens of a
host. This occurs when the host is so
immunocompromised that it is una-
Minor histocompatibility ble to reject the allogeneic cells in the
antigens graft. Since both host and graft share
Polymorphic alloantigens other Recipient’s antigen-presenting cell similar MHC molecules (without
than MHC which produce weak or
which bone marrow transplantation
slow rejection reaction are called
minor histocompatibility antigens. is not possible), GVHD reactions are
Examples include H-Y antigen, usually directed against minor histo-
an antigen encoded on the Y
Activates compatibility antigens (see Figure
chromosome of male mice and HA-2,
an antigen derived from myosin. 17.14). GVHD can also occur when
other organs such as lungs, alimen-
Donor
cells
tary tract and liver (which contains a
number of T cells) are transplanted.
Tcyt NK cell Macrophage GVDH could be acute or chronic.
Acute GVDH is manifested as early
as 8 to 10 days after transplantation
by a measles-like skin rash, diarrhoea,
gastrointestinal tract haemorrhage
Figure 17.14 Mediates tissue destruction of
immuno-compromised recepient and jaundice. The target organs are
Graft vs host disease.
liver (causing hepatitis), skin(dermatitis) or gastrointestinal tract (enteritis) where GVDH causes
necrosis. Chronic GVHD,which usually manifests three months after transplantation, is character-
ized by fibrosis and atropy of one or more organs (liver, gastrointestinal tract, skin, lungs) without ne-
crosis. Chronic GVHD if it appears in a severe form, leads to destruction of organs and can be fatal.
GVHD is initiated by T-cell recognition of host alloantigens. Donor T cells recognize the re-
cipient peptide–MHC complex displayed on antigen-presenting cells. Activated TH cells secrete a
number of cytokines (one of them being IL-2). Cytokines activate a variety of cells, including effec-
tor cells such as NK cells, Tcyt cells and macrophages. Since, NK cells cannot recognize alloantigens,
it is proposed that NK cells are activated by cytokines to differentiate into lymphokine-activated
killer cells that can lyse normal allogeneic cells of the host. Tcyt can directly lyse host cells and elicit
tissue damage. TNF-β and perhaps IL-1 also appear to be important mediators of GVHD.
Both acute and chronic GVDH reactions in bone marrow transplantation are treated with intense
immunosuppression. CsA and metabolic toxin methotrexate are usually administered prophylactically
to the recipient. Agents that suppress cytokine production such as thalidomide have been effective in
treating GVHD. HLA-typing is very important for preventing the occurrence of GVHD. Most human
bone marrow transplants are performed between siblings who are completely identical at all HLA loci.
Immunofluorescence
Fluorophore-labelled
antibody
Slide
Antigens fixed Antibody binds antigen.
on a slide Fluorescence detected,
presence of antigen confirmed
Direct immunofluorescence
Antibody reacted Fluorophore-labelled

with antigen anti-IgG antibody added
Antigen fixed on a slide Antibody to be detected Labelled anti-IgG

binds target antigen detects bound antibody
Indirect immunofluorescence
Figure 17.15 Principle of direct and indirect immunofluorescence assay.
Immunofluorescence is a process in which fluorophores are used to pathogenic microbes and even detect antipathogen antibodies in
label antibodies (or antigen) to make them fluoresce or emit visible the serum. Labelled antibodies are observed by fluorescence micro-
light. Fluorescent dyes such as fluorescein isothiocyanate (FITC) are scope or by confocal microscope.
coupled to antibody molecules without changing antibody specifi-
city. The fluorescent antibody molecule can then be used to study There are two main types of immunofluorescence assays—
the distribution pattern of target antigens in cells or tissues, identify direct immunofluorescence and indirect immunofluorescence.
Direct immunofluorescence is a simple two-step technique for antibodies against that pathogen in the serum. These antibodies are
detecting antigen (protein, cell or microbes). Antigen is first fixed on identified with the help of indirect immunofluorescence. In this assay,
to the slide. Primary antibodies labelled with FITC are then added the antigen is first coated onto the slide. This slide is then incubated
and the non-specific antibodies bound are then washed off. The with the (suspected) antiserum that contains its specific antibodies.
slide containing antigen – FITC-labelled specific antibody is then These antibodies are unlabelled. This antigen–unlabelled antibody
examined with fluorescence microscope (see Figure 17.15). Direct complex is then washed off and the location of the first or primary
immunofluorescence has been used to identify a variety of antigens antibody is then detected by FITC-labelled anti-IgG antibodies and
including E. coli and Salmonella typhi. examined under a fluorescence microscope. Indirect immunofluo-
rescence is routinely used in the diagnosis of syphilis in which the
Indirect immunofluorescence assay is used to detect the pres-
patient’s serum is analysed for the presence of Treponema pallidum
ence of antibodies in the serum. If an individual is infected with a
(causative organism of syphilis) antibodies.
particular pathogen, they have a detectable amount of specific
S U M M A R Y
• Transplantation is the process of taking tissues or organs (or even • Chronic rejection may take months or years and can be cell-medi-
cells) and placing them onto the same or different ated or antibody-mediated. The main feature of chronic rejection is
individuals. the thickening of blood vessel walls, which eventually get blocked.
• Transplantation antigens are those proteins that are present on cells • Allogeneic transplantation endures if it is complemented with some
or tissue surfaces and are responsible for either the acceptance or degree of immunosupression. The two main types of immunosup-
the rejection of a graft. pressive treatment are antigen-non-specific or antigen-specific.
• MHC genes specify the cell surface molecules that elicit the most • Non-specific immunosuppressants include cyclosporin A, FK-506,
rapid allograft rejection. rapamycin and MMF that have been shown to prolong graft sur-
• Host’s T cells recognize either intact allogeneic MHC molecules vival by diminishing the activity of the immune system regardless
(direct presentation) or processed allogeneic MHC molecules of the provoking alloantigen.
(indirect presentation). • Specific immunosuppressive agents are theoretically designed to
• Graft rejection has been classified as hyperacute, acute or chronic inactivate only those clones of T or B lymphocytes that are acti-
depending on the time taken to reject the graft and the nature of vated by alloantigens, leaving the rest of the immune system intact.
immune response. These include blocking costimulatory signals on antigen-presenting
cells by soluble receptors and the use of monoclonal antibodies
• Hyperacute rejection is initiated by pre-existing antibodies against directed against T cells to suppress T-cell activity.
the donor’s endothelial antigen, leading to platelet aggregation,
thrombosis and irreversible ischaemic damage. • When an immunologically competent bone marrow inoculum is
transplanted into an immunocompromised host, the graft tissue can
• Acute rejection involves CD4+ and CD8+ T-cell response against mount an immune attack on the host tissue. This is called graft ver-
the responding alloantigen. Tcyt cells induce lysis of graft cells sus host disease (GVHD) and is mediated primarily by donor T cells.
while TH cells invoke inflammation. GVHD can be acute or chronic and can affect more than one organ.
K E Y W O R D S
• acute rejection 368 • direct presentation 363 • hyperacute • passenger

• allograft 362 • delayed xenograft rejection 367 leukocyte 366
• arteriosclerosis 369 rejection 373 • isograft 361 • rapamycin 370
• autograft 361 • FK-506 370 • indirect • privileged site 373
• azathioprine 370 • graft rejection 364 presentation 363 • organ transplantation 373
• corticosteroid 370 • graft vs host • mixed leukocyte • sensitization 373
• chronic rejection 367 reaction 376 reaction 364 • transplantation
• cyclosporine 370 • histocompatibility • mycophenolate antigen 362
• cyclophosphamide 370 antigen 362 mofetil 370 • xenograft 362
1. Bone marrow transplantation is frequently associated with GVHD. against graft present in a supposedly normal individual? How are
How can this pathological condition be prevented? Why can’t we they generated?
take immunocompetent recipients to avoid GVHD? 4. Anti-CD25 antibodies are usually administered into a recipient at
2. How is direct presentation of allogeneic MHC different from indi- the time of solid organ transplantation. What does this antibody
rect presentation? Can both presentations occur at the same time in do? What strategies should be adopted to make this antibody more
an individual?. Give some experimental evidences to prove direct effective?
and indirect presentation of allogeneic MHC. 5. What are the different immunosuppressive treatments that accom-
3. Hyperacute rejection is mediated by preformed antibodies already pany solid organ transplantation? Give an account of some non-
present in an individual. Why are these pre-existing antibodies specific and specific treatments currently practised?
Choose the appropriate option. 6. A drug that inhibits T-cell maturation is:
(a) FK-506
1. For a successful allogeneic transplantation, one of the follow- (b) Rapamycin
ing should match between donor and recipient: (c) Cyclosporin
(a) Class III MHC antigen (d) Mycophenolate mofetil
(b) Antigens and antibodies
(c) Classes I and II MHC antigens 7. Passenger leukocytes are:
(d) TH and Tcyt cells (a) Antigen-presenting cells
2. In graft vs host disease: (b) T cells
(a) Host tissue mounts an immune response on the graft (c) B cells
(b) Donor T cells recognize and react with host antigen- (d) NK cells
presenting cells
(c) Immune reactions are directed against MHC 8. In mixed leukocyte reaction:
(d) Disease can occur in immunocompetent host (a) Histocompatibility is directly proportional to clonal
expansion of cells
3. During direct presentation of allogeneic MHC, self-T cells (b) Histocompatibility is inversely proportional to mitotic cell
recognize: produced
(a) Self-MHC + foreign peptide (c) Histocompatibility cannot be judged by clonal expansion
(b) Foreign MHC + foreign peptide (d) None of the above
(c) Foreign MHC + self-peptide
(d) Self-MHC + self-peptide 9. In allograft rejection, the most important antigen-presenting
cells are:
4. Antibodies are involved in pathogenesis of all, except: (a) Dendritic cells
(a) Allogeneic hyperacute rejection (b) Macrophages
(b) Allogeneic acute rejection (c) B cells
(c) Allogeneic chronic rejection (d) Endothelial cells
(d) Hyperacute xenograft rejection
10. In which of the following disease is hypersensitivity evoked?
5. Graft arteriosclerosis occurs in: (a) Lyme disese
(a) Acute graft rejection (b) Malaria
(b) Chronic graft rejection (c) Influenza
(c) Hyperacute graft rejection (d) Leishmaniasis

1. In indirect presentation of alloantigens, peptides derived from 4. Hyperacute allograft rejection is mediated by preformed antibodies
allogeneic MHC are presented to T cells on self-MHC. and Tcyt cells present in the recipient.
2. Cyclophosphamide and cyclosporin exert their immunosuppres- 5. In GVHD, donor T cells mount an immune response against host
sive effect by inhibiting DNA replication. molecules.
3. Transplantation between genetically identical members of the
same species is an autograft.
Chapman, L. E. (2003). “Xenotransplantation: Public Health Singh, N. (2003). “Impact of Current Transplantation
Risks—Patient vs Society in an Emerging Field”, Current Topics in Practices on the Changing Epidemiology of Infections in
Microbiology and Immunology, 278: 23–45. Transplant Recipients”, Lancet Infectious Diseases,
3: 156–61.
Fox, A. and I. C. Harrison (2000). “Innate Immunity and Graft
Rejection”, Immunological Review, 173: 141. Thomson, A.W. (1994). “Immunosuppressive Drugs and the
Induction of Transplantation Tolerance”, Transplant Immunology,
Gould, D. S. and H. Auchincloss (1999). “Direct and Indirect 2: 247–51.
Recognition: The Role of MHC Antigens in Graft Rejection.
Immunosuppressive Strategies in Transplantation”, Physiological Waldman, H. and S. Cobbold (2004). “Exploiting Tolerance
Review, 79: 99–141. Process in Transplantation”, Science, 305: 209–12.
Von Seventer, G. A., R. T. Semnani, E. M. Palmer, B. L. McRae Woo, S. B., S. J. Lee and M. M. Schubert (1997).
and J. M. Van Seventer (1998). “Integrins and T-helper Cell Acti- “Graft vs Host Disease”, Critical Review in Oral Biology
vation”, Transplantation Proceedings, 30: 4270–74. and Medicine, 8: 201.
The word cancer came from the father of medicine, Hippocrates. He “Life is a school
used the Greek words, carcinos and carcinoma to describe tumours we never
and called cancer karkinos. The Greek term karkinos, which means crab, graduate from.”
—NINA YOMEROWSKA
probably comes from the appearance of the surface of a solid tumour
with a roundish hard centre surrounded by pointed projections,
vaguely resembling a crab. He later added the suffix oma, Greek for
swelling, giving the name carcinoma. Although Hippocrates came up
with the name cancer, he was not the first one to record these diseases.
The oldest record of human cancer was found in Egyptian papyri writ-
ten between 3000–1500 BCE. The oldest specimen of a human cancer
was found in the remains of a skull dating back to 1900–1600 BCE. The
treatment of cancer during ancient times involved cauterization, a
method to destroy tissue (tumour) with a hot instrument or treating a you should be able to:
patient’s four humors with diet and laxatives. The first modern attempt • Define benign and malignant
tumours
to treat various forms of tumour was made in the mid-1890s by • Differentiate between
oncogenes and tumour
Hericourt and Richet (France), and Salvati and de Gaetano (Italy) who suppressor genes
attempted to cure cancer by anti-tumour antibodies. Their efforts, • Explain the difference between
tumour-specific and tumour-
associated antigens
though unsuccessful at that time, showed other scientists the way to
• Given an account of protective
modern immunotherapy against cancer. With the advent of modern immunity rendered by T cells
and NK cells against tumour
techniques, the structural and functional differences between a • Briefly summarize how a
tumour evades immune
normal cell and a tumour cell became clear; and techniques to counter response
• Describe how active and
the onset and propagation of cancer became more accurate and passive immunotherapies can
fight tumour cells
fine-tuned. Figure 18.1 highlights the structural differences between a
• Explain, giving examples,
normal cell and a cancerous cell. adoptive cellular
immunotherapy
• List the potential uses and
drawbacks of humoral
immunotherapy
Cancer and the Immune
System 18
18.1 INTRODUCTION
Cancer (Greek: karkinos—crab) is a large class of diverse diseases, all of which exhibit uncontrolled
cell growth and division. Under normal conditions, the production of new cells is regulated in such
a way that the number of any type of cell remains constant. Occasionally, variants of normal cells
arise that have lost their usual growth control. They acquire the ability to grow in inappropriate
locations or to propagate indefinitely making them lethal for the host’s body. In a non-circulating « Normal cells can divide only about
tissue, such uncontrolled cell growth produces clones of the cell that manifest as cell masses called 70 times before their certain death.
tumour or neoplasm (Greek: neoplasm—new growth).
The tumours or neoplasms that are capable of indefinite growth and do not invade other « The term metastasis was coined by
the French physician Joseph Claude
body parts are said to be benign or non-cancerous. Cancerous or malignant tumours are those Recamier in 1829 in his treatise Re-
in which cells grow indefinitely, and detach and migrate into healthy surrounding tissue. Me- cherches du Cancer. He was the first
tastasis (Greek for transition) is the most damaging feature of cancer. It is the ability or process scientist to provide evidence that
metastasis is caused by cancer cells
of migration of cancer cells into the normal tissue so that they can move away from their origin moving from one place to another.
and establish new colonies of cancerous cell (secondary tumours) elsewhere in the body. A
schematic representation of the process of conversion of benign to malignant tumour is shown
in Figure 18.2.
Cancers have been classified according to the tissue of its origin. Carcinoma (of endodermal
or ectodermal tissue) is a malignant tumour of epithelial origin such as skin and epithelial
lining of internal organs, and glands such as lung, breast, prostrate and colon. Sarcoma is a
malignant tumour of connective tissues or other tissue of mesodermal origin such as bone, fat
or cartilage.
Leukaemia and Lymphoma are malignancies derived from haematopoietic cells. Leukae- « About 90 per cent of all human
mia is a malignant disease due to the abnormal proliferation of white blood cells. Depending on cancers are carcinomas.
Figure 18.1
Schematic diagram explaining the
difference between normal and tumour
cells. Normal cells adhere to the surface
of the culture dish, while the transformed
cells tend to overgrow on one another,
forming clusters.
Cell becomes cancerous
Tissue Immortalization and

transformation
Blood vessel
Benign tumour
Benign tumour grows
Blood vessel
Tumour becomes
malignant and spreads
to other tissues through
Metastasis the blood stream
Blood vessel
Figure 18.2
Benign and malignant tumours.
lineage, leukaemia could be myeloid leukaemia or lymphoid leukaemia. Lymphomas are cancers
involving secondary lymphoid organs such as spleen and lymph nodes. Leukaemia generally pro-
liferates as a single cell while lymphoma arises as solid tumour masses.
» In 2005, out of 58 million deaths
occurring worldwide, 7.6 million
people died of cancer. 18.1.1 M A L I G N A N T T R A N S F O R M AT I O N O F C E L L S
Normal cells are judged by:
• anchorage dependence;
• serum/growth factor dependence;
• density-dependent inhibition of cell growth; and
• cytoskeleton organization.
When normal cells are converted into tumour cells, the process is called transformation. Trans-
formation describes the failure of normal cells to observe the normal constraints of growth.
Carcinogens Transformed cells have an altered morphology and growth properties. They also have a rounded
Carcinogens are those substances appearance, do not require anchorage dependence (that is, they do not need a solid surface to attach
that are capable of inducing or
propagating cancer in humans or to), have a reduced serum growth factor dependence, grow in density in an independent fashion
animals. The first virus that was and most importantly may induce tumours when injected into appropriate test animals.
shown to induce cancer in animals Certain events or agents (physical or chemical) convert normal cells into transformed cells.
was the Rous sarcoma virus.
Agents that convert normal cells into cancerous ones are called carcinogenic agents or carcinogens.
CANCER AND THE IMMUNE SYSTEM 383
A carcinogen may initiate epigenetic changes in the cell that leads to transformation of cells, or it may
not initiate but promote transformation induced by some other carcinogens. Carcinogenic agents
can be physical (such as UV light and ionizing radiation), chemical (such as DNA-alkylating agents)
or biological (DNA and RNA viruses).
There are number of ways cells can be transformed and cancer induced in the cells, but broadly
Oncogene
speaking the activation of two main events leads to the induction of tumour/cancer. One is muta- An oncogene is dominant-
tion in host genome. The other is the activation of tumour-inducing genes present or integrated in acting gene that stimulates the
formation of tumours. Oncogene
the host cells. Such genes are divided into two classes: genes that have gain of function (activating)
can potentially induce cancerous
mutation leading to cancer-known as oncogenes (Greek: onkos—tumour) and genes which have transformation in the cell where they
loss of function (inactivating) mutation in cancer known as tumour suppressor genes (see Figure occur or are introduced. Galen was
the first person to use word oncos to
18.3). More than a 100 different oncogenes and more than 30 different tumour suppressor genes
describe all sorts of tumours.
have been identified.
The importance of mutation can easily be seen in xeroderma pigmentosum, a type of skin cancer. « An important difference between
This cancer is caused by mutation in the gene that encodes DNA-repair enzyme, UV-specific oncogenes and tumour suppressor
endonuclease. The skin cells of such individuals exposed to UV light from the sun leads to mutation genes is that oncogenes result from
and consequently skin cancer. The inactive tumour-inducing genes present in normal host cells, the activation (turning on) of proto-
oncogenes, but tumour suppressor
including humans, are called proto-oncogenes. These normal cellular proto-oncogenes can be genes cause cancer when they are
converted into cellular oncogenes by mutation or by association with new regulatory sequences inactivated (turned off).
through recombination. Additional transformation may also result from infection by number of « The oncogenes that occur in
tumour inducing virus such as polyoma, adenovirus, Epstein–Barr virus and SV40 virus (simian viruses are named viral oncogenes
virus 40). The viruses also contain oncogenes called viral oncogenes that are able to induce uncon- while closely related oncogenic
sequences in eukaryotes are
trolled or cancerous growth in the host cells in culture. referred to as proto-oncogenes.
Viral oncogenes are prefixed by v
18.1.2 ONCOGENES AND CANCER INDUCTION (for example, v-myc) while proto-
oncogenes are prefixed by c (for
Considering that oncogenes induce uncontrolled cell division resulting in tumours, one might an- example, c-myc).
ticipate these genes must play an important role in the regulation of cell division. Oncogenes were
initially identified as genes carried by viruses that cause transformation of their target cells. « The link between cancer and
chromosome was first suggested
by the German cytologist Theodor
Boveri in 1914.
Proto-oncogene Oncogene (mutated)
Mutuation in
either allele
Homozygous Heterozygous
(+/+) (+/-)
Wild type Dominant mutation
Normal cell
Oncogene Cell proliferation
division
Mutuation in
both alleles
Figure 18.3
Line diagram showing the role of
Homozygous Tumour suppressor Tumour suppressor Homozygous oncogenes and tumour suppressor
genes in inducing cancer. Mutation in
(+/+) gene gene (mutated) (-/-)
proto-oncogene is dominant and hence
Wild type Recessive mutation mutation of only one allele results in the
cell becoming cancerous. The mutated
tumour suppressor gene is recessive and
Normal cell hence both alleles have to be mutated or
Tumour suppressor gene Cell proliferation
division deleted for the development of cancer.
Virus Host V-oncogenes Functions of V-oncogene

Products
Abelson murine leukaemia virus Mouse abl Tyrosine kinase
Avian erythroblastosis virus Chicken erbA Thyroid hormone-receptor analogue
Simian sarcoma virus Monkey sis Platelet-derived growth factor
Harvey-murine sarcoma virus Rat H-ras GTP-binding protein
Table 18.1
Some common viral oncogenes.
Kirsten-murine sarcoma virus Rat K-ras GTP-binding protein
These viral oncogenes (v-onc) are different from their cellular counterpart called proto-
oncogenes or cellular oncogenes (c-onc). Proto-oncogenes contain introns while viral oncogene
are single exons. This suggests that v-oncogenes are derived from c-oncogenes and not vice-versa,
as v-oncogenes lack introns.
Apparently, the mRNA copy of a proto-oncogene is ligated into an RNA genome of a ret-
rovirus by a recombination mechanism. What could be of greater value to a virus than to have a
new gene that stimulates an increased growth of its host which in turn helps in virus sustenance.
Table 18.1 lists some of the common viral oncogenes that affect animals.
I N D U C T I O N O F C E L L U L A R P R O L I F E R AT I O N
Proto-oncogenes code for various cellular proteins that control cell division and proliferation.
These include four main groups:
• those that encode growth factor receptors (c-erbB) or growth factor (c-sis);
• those that encode GTP-binding protein (c-ras);
» The sis oncogene leads to the • those that encode protein kinases (c-src), tyrosine specific protein kinase (c-abl) or serine/
overproduction of platelet-derived threonine specific kinases (c-mil); and
growth factor which stimulates cells
to grow uncontrollably. • those that encode transcriptional regulators (c-jun, c-myc).
» It has been suggested that the The differences between viral oncogenes and proto-oncogenes and the functions of the four
fifth class of oncogenes includes main groups of proto-oncogenes are shown in Figure 18.4. Some important representative groups
regulators of programmed cell of proto-oncogenes together with their function are given in Table 18.2.
death. An example of this fifth
class of oncogenes is bcl-2, which
prevents the cell from committing Exon
apoptosis. The normal functions
of the bcl-2 gene include rescuing
cells during the selection of B and T Oncogene
cells, and haematopoiesis, in which No introns
a large number of cell deaths occur.
Viral genome
The activation of this gene during Growth-factor
cancer results in the encoding of receptor
Viral oncogenes
a protein that gives immortality to
cells which then become cancerous.
Growth
factor
Oncogene
DNA
Protein
GTP kinase
ATP ADP + P
Phosphorylated
GTP-binding
protein protein
Transcriptional
regulator
Functions of some proto-oncogenes

Exon Intron
Human DNA
Figure 18.4
Proto-oncogenes and their functions. Proto-oncogene
Group Proto- Functions of proto- Mechanism of

oncogenes oncogene Products Activation
Growth-factor c-erbB Epidermal-growth-factor receptor Amplification
receptor kinase
c-fms Colony-stimulating factor-1 Mutation
receptor kinase
Growth factor c-sis Platelets-derived growth factor— Mutation
B chain
Int2 Growth factor Insertion mutation
GTP-binding c-ras GTP-binding protein Point mutation
proteins
gsp Gα-stimulating protein Point mutation
gip Gα-inhibitory protein Point mutation
Tyrosine kinase c-src Membrane-associated tyrosine Mutation
kinase
c-abl Cytosolic tyrosine kinase Translocation
Serine/threonine c-raf Cytosolic serine/threonine kinases Insertion mutation
kinase
c-mos Cytosolic serine/threonine kinase Insertion mutation
c-mil Cytosolic serine/threonine kinase Mutation
Transcription c-jun Leucine zipper protein Activates transcription from
factors enhancer/promoter
c-fos Leucine zipper protein Activates transcription
c-myc Transcriptional regulator Amplification/translocation Table 18.2
Some representative groups of proto-
c-erbA Thyroid hormone receptor Mutation
oncogenes.
A C T I VAT I O N O F P R O T O - O N C O G E N E T O O N C O G E N E
« The genomes of all vertebrates
A variety of genomic changes can activate proto-oncogene to become oncogenic. They include contain three distinct, but related,
mutations, amplification, translocation and insertion of proto-oncogenes, which causes them to ras proto-oncogenes: C- H-ras, C-K-
become active cellular oncogenes. ras and N-ras. All three of these ras
proto-oncogenes have been shown
Some proto-oncogenes are frequently amplified in a particular type of cancer. Such an ampli- to undergo mutation to oncogenic
fication event plays a causative role in the oncogenic process that gives rise to the cancer cells. This derivatives. Oncogenic variants of
amplification of proto-oncogenes and it the subsequent overexpression of the proto-oncogene the three c-ras proto-oncogenes
have been detected in promy-
product have been demonstrated in lower eukaryotes such as Leishmania tropica and has been elocytic leukaemia, fibrosarcoma,
recently confirmed in some human cancers. The proto-oncogene c-myc is found to be amplified and colon, mammary and bladder
in small cell carcinoma of the lung and colon in which multiple copies of c-myc were detected on tumours.
abnormal X-chromosomes. Two more proto-oncogenes are frequently amplified namely, L-myc « The alteration/mutation of just
and N-myc. one allele of a proto-oncogene is
The translocation (which is breakage and transfer of parts of chromosomes to non-homologous enough to transform a normal cell
into a cancerous cell.
chromosome) of proto-oncogenes was repeatedly observed with certain types of cancer cells. This
is best illustrated in the Philadelphia chromosome found in cells with chronic myelogenous « Philadelphia chromosome was the
leukaemia. In this type of cancer, the proto-oncogene c-abl is translocated from chromosome 9 to first chromosomal abnormality ever
linked to a specific cancer. It was
chromosome 22, resulting in the activation of the oncogenic gene. discovered by Tough and his co-
Proto-oncogenes have been known to be activated by insertion of a virus into a cellular genome. workers in 1961.They named it the
The virus may contain v-oncogenes such as v-src or may not, like slow-transforming viruses Philadelphia chromosome after the
city in which it was discovered.
carry any oncogene yet still induce a neoplastic state in the cell. Slow-transforming viruses most
frequently induce cancer by integrating as proviruses adjacent to proto-oncogenes and resulting Tumour suppressor genes
in their activation. The different mechanisms for activation of proto-oncogenes are depicted in Tumour suppressor genes are
Figure 18.5. normal cellular genes that slow
down cell division, repair DNA
mistakes and tell cells when to die.
TUMOUR SUPPRESSOR GENES When tumour suppressor genes
Apart from oncogenes, tumour suppressor genes represent another kind of cell division regulating don’t work properly, cells grow out
of control, leading to cancer.
genes. Certain tumours are caused by loss of both alleles of these tumour suppressor genes in the
Proto-oncogene
Amplification Translocation to Insertion

and activation other chromosome
Activation
Becomes oncogene
(activated) Insertion of virus
(with or without
oncogene)
Figure 18.5
The different mechanisms for activation Oncogene Oncogene amplified Oncogene Activation to
of proto-oncogenes. and activated oncogene
individual. Tumours results from loss of function. This led to the identification of tumour suppres-
sor genes whose products are needed for normal cell function and whose loss of function causes
tumour. About 30 tumour suppressor genes have been identified, including p53, BRCA1, NF-1,
APC, and RB. A well-characterized tumour suppressor gene that codes for the protein RB, and is
responsible for retinoblastoma is taken as an example.
» The retinoblastoma gene was iso- Retinoblastoma (RB) is a human childhood disease involving a tumour on the retina. It has
lated by Stephen H. Friend working been associated with the deletion of band 914 on human chromosome 13. A normal individual has
in the Weinberg Laboratory in 1986.
two RB+ alleles. Retinoblastoma arises when both copies of these alleles are inactivated. RB is nuclear
protein of 105 kDa, that influences the cell cycle. RB can exist as the normal dephosphorylated form
or the phosphorylated form. The normal dephosphorylated RB prevents cell proliferation. The RB
presumably associates with transcription factors such as E2F group of proteins, which blocks the abil-
ity to transcribe genes required for DNA synthesis. The deletion of this “brake” protein RB, results in
uncontrolled cell proliferation. Some important tumour suppressor genes are linked in Table 18.3.
I M M O R TA L I Z AT I O N A N D T R A N S F O R M AT I O N
Most cancerous or tumour cells arise as a result of multiple events in the normal cells. The require-
ment for multiple events reflects that normal cells have multiple mechanisms to regulate their growth
and differentiation, and several separate changes may be required to bypass these controls. These
multiple events may be done by a single virus such as adenovirus or by several mutational events
occurring over a period of time. An adenovirus transforms a human cell in culture by two ways:
Tumour Function of Tumour Suppressor Associated Tumour

Suppressor Genes Gene Product
p53 Transcription factor, regulates cell Several types of tumours,
proliferation Li Fraumeni syndrome
RB Transcription factor regulator Retinoblastoma, lung tumours
NF-1 GTPase activator Neurofibromatosis,
Nerve tumour
Table 18.3 WT -1 Transcription factor Wilms tumour
Some representative tumour suppressor
genes. DCC/APC ? Colon tumour
it carries the ElA gene which allows cells to grow indefinitely and the ElB region which causes mor-
phological changes characteristic of the transformed state.
Immortalization and cancer induction is also a multistage process. The cancer of the gastroin-
testinal tract begins as small adenoma of epithelial cells, growing gradually and amplifying to acquire
a malignant phenotype. It involves a multistep sequence of gene changes involving the proliferation
of K-ras, a proto-oncogene and loss of the three tumour repressor genes—p53, APC, DCC.
18.2 TUMOURS OF THE IMMUNE SYSTEM

The tumours of the immune system are classified as leukaemia or lymphoma. Leukaemia is uncon-
trolled proliferation of white blood cells or leukocytes. Leukaemia tends to proliferate as single cells
and can develop in cells of lymphoid or myeloid lineages. The disorder may progress rapidly, often
fatally (acute leukaemia), or may progress slowly (chronic leukaemia).
Lymphoma is a malignant disorder involving the secondary lymphoid organs such as the spleen
and lymph nodes. It usually arises as a solid outgrowth in lymphoid tissues. Lymphomas include
Hodgkins, and non-Hodgkin malignancies, as well as B- and T-cell lymphomas. One of the best char-
acterized B-cell lymphoma is Burkitt lymphoma. This lymphoma, usually develops when the proto-
oncogene c-myc located on chromosome 8 is translocated to chromosome 14, next to the antibody
genes. This c-myc is expressed abnormally in a new location, because the c-myc gene is placed next to
a heavy-chain gene cluster that is expressed constitutively in these cells. This abnormal expression of
proto-oncogene contributes to the oncogenic transformation of Burkitt lymphoma cells.
18.2.1 TUMOUR ANTIGENS

A variety of tumour antigens are recognized by T and B lymphocytes in animal and human cancers.
Antigens that are expressed on tumour cells but not on normal cells are called tumour-specific Tumour-specific antigens
antigen (TSA). Tumour-specific antigens are
antigens that are expressed on
Some TSA are even specific for particular tumour while other are shared among tumours of tumour cells and not on normal
same type. Some of tumour antigens that are also present on normal cells are called as tumour- cells. The tumour antigens that are
associated antigens (TAAs). TAA usually are those proteins that are normal cellular proteins also expressed on normal cells are
referred to as tumour-associated
but which are expressed aberrantly on adult cells (such as expression of fetal proteins on adult antigen.
tumour). TAAs may also be those proteins that are normally expressed at low level in normal cells
but are expressed at higher levels in tumour cells.
18.2.2 TUMOUR-SPECIFIC ANTIGENS

Tumour-specific antigens are those antigens which are derived from tumour-causing genes and are
expressed exclusively in tumour tissue. The most important tumour-specific antigens include anti-
gens that are expressed at the wrong place in cancer, for example, the expression of MAGE; proteins « Tumours induced by physical
from tumour-inducing viruses, for example, HPV’s E6 and E7 proteins; and protein variants that or chemical carcinogens usually
express tumour-specific antigens.
are created by somatic mutation within tumour cells, for example, CDK-4, CDKN2A proteins.
EXPRESSION OF ANTIGEN IN THE WRONG PLACE

Certain tumour-specific antigens may be derived from genes that are not expressed in a normal
tissue. The transformation may result in an inappropriate expression of normal genes in the wrong
tissue or at a wrong time. The expression of melanoma antigen (MAGE) under normal conditions is
restricted to the testes and placenta. MAGE genes are silent in other tissue. However, MAGE proteins
(MAGE-1 and MAGE- 2) are expressed on tumours, carcinomas of the bladder, lung, breast and, skin,
melanoma and sarcomas. The tumour-specific antigens induce a response from memory Tcyt cells.
VIRUS-INDUCED TUMOUR-SPECIFIC ANTIGENS

The products of oncogenic viruses function as tumour-specific antigen. In most virus-induced tumours,
virus-encoded protein antigens are found almost anywhere in the cell—nucleus, cytosol, plasma mem-
brane of tumour cells. The endogenously synthesized proteins are processed in the cellular machinery and
hence can be displayed together with class I MHC molecules on a tumour cell surface. Viruses implicated
in the development of cancer in humans include Epstein–Barr virus (associated with naso-pharyngeal
carcinoma, and B-cell lymphoma), and human papilloma virus associated with cervical cancer. These
virus-induced TSAs are subject to immune surveillance and, hence, attack by Tcyt cells.
The RNA tumour virus can also give rise to TSA upon infection to host cells, for example, an
RNA tumour virus, Human T-cell lymphotropic virus-1 (HTLV-1), that causes adult T-cell leu-
kaemia/lymphoma (cancer of TH-cells) induces tumour-specific antigens on tumour cells, though
it is not clear whether they are any good in generating protective immunity. Similarly, host cells
infected with human papilloma virus display peptides E6 and E7 on their surface.
M U TAT E D C E L L U L A R G E N E P R O D U C T A S T U M O U R - S P E C I F I C A N T I G E N
There are several well-characterized examples of tumour-specific antigens that are derived from the
mutated version of normal cellular products. Examples include cyclin-dependent kinase-4 (CDK-4),
which is involved in cell cycle progression in humans. A specific mutation in the 24th codon of
CDK-4 (changing arginine to cysteine) is seen in many cases of sporadic and familial melanomas.
This mutated gene product shows aberrant interactions with another gene product (p16) that re-
sults in unregulated tumour cell growth. Such mutated cellular gene products expressed/displayed/
on tumour cells are tumour-specific antigens. A schematic representation of different types of tu-
mour-specific antigens is shown in Figure 18.6.
18.2.3 T U M O U R - A S S O C I AT E D A N T I G E N S
A large number of tumour antigens are not restricted to tumour cells, but are also present on nor-
mal cells. These include proteins expressed only on foetal cells but not on normal adult cells (onco-
foetal or embryonic tumour antigens),
Tumour-inducing virus products of mutated proto-oncogenes,
overexpressed or aberrantly expressed
Viral genome
proteins, altered glycolipid and glyco-
protein antigens and differentiation
Viral genome antigen. Some important tumour-as-
sociated antigens are listed in Table
Viral peptide Host cell 18.4.
Processing and
display of peptide O N CO F O E TA L T U M O U R
ANTIGEN
Tumour-specific viral Class I MHC Oncofoetal tumour antigens are anti-
peptide displayed
gens that are found on oncogenic or
cancerous cells as well as normal foe-
Virus-induced tumour-specific
tal cells but not in normal adult tissue.
antigen (e.g. proteins from HPV-such as E7)
These antigens appear early in embry-
Oncogenic onic development before the develop-
mutation ment of a competent immune system.
Altered It is believed that the genes en-
self-antigen coding these proteins are silenced
Genome during development. These genes, if
Normal expression expressed upon malignant transfor-
mation, will be recognized as non-self
Self-antigen and will induce an immunologic re-
Tumour cell expressing sponse. Two most thoroughly char-
mutated self-antigen (e.g. CDK4) acterized oncofoetal antigens include
» The normal range for CEA in an carcinoembryonic antigens (CEA)
adult is 2.5–5.0 ng/ml in blood. Normally and alpha-foetal proteins (AF).
The presence of both benign and silent gene CEA (CD66) is an oncofoetal an-
malignant tumours in the human
body increases the concentration tigen membrane glycoprotein of the
of CEA.
Genome
immunoglobulin superfamily. It func-
Oncogenesis tions in binding tumour cells to one
another. CEA expression is increased
Antigen expressed in carcinomas of colon, breast, stom-
on wrong cell ach, lung, and bladder.
Figure 18.6
Schematic diagram showing an overview Expression of antigen at Alpha-foetal protein (AFP) is a
of various tumour-specific antigens. wrong place (e.g. MAGE) circulating glycoprotein synthesized
Class of Antigen Tumour-associated Antigen Associated Cancer

Oncofoetal tumour antigen Carcinoembryonic antigen (CEA) Carcinoma of colon, lungs
Alpha-foetal protein Germ cell tumour, pancreatic cancer,
liver cancer
Mutated protein p53 Colorectum, and liver cancer
Aberrantly expressed Her2/Neu, Ovary and breast cancer
normal cell protein †
PSA Prostate cancer
Differentiation antigen MART-1, Melanoma
gp-100 Melanoma
Table 18.4
Ganglioside* antigen GM2/GD2/GD3 Neuroblastoma, melanoma, sarcoma Tumour-associated antigens.
Note: *A lipid antigen. †PSA—prostate-specific antigen
and secreted in foetal life by the yolk sac and liver. Foetal AFP concentration is around 2–3 mg/ml « Increase in AFP in pregnant moth-
which drops to nanogram levels in a normal adult. AFP concentration in the serum can increase ers could also be due to spinabifida
significantly in germ cell tumour, hepato-cellular carcinoma, and pancreatic and gastric cancers. (an opening in the spine), Down
syndrome and trisomy18 (chromo-
AFP elevation serves as a useful indicator of germ cell tumour or liver cancer. some abnormalities).
ONCOGENIC PRODUCT AS TUMOUR ANTIGEN

A number of tumours have been shown to express tumour-associated antigens encoded by cellular
oncogenes. These antigens are also present in normal cells encoded by proto-oncogenes. Often
oncogenes are produced by point mutation, deletions, chromosomal translocation or viral gene in- « Ras was the first human oncogene
sertion of cellular proto-oncogenes. The product of these altered proto-oncogenes are synthesized to be cloned (in 1982).
in the cytosol of tumour cells and like any other cytosolic proteins enter the class I MHC antigen
processing pathway. The altered peptides when displayed on the surface of tumour cells elicit host
T-cell response. Some cancer patients have activated TH cells and Tcyt cells that respond to products
of mutated oncogenes, such as ras, p53.
A B E R R A N T LY E X P R E S S E D N O R M A L C E L L P R O T E I N
Some tumour-antigens are normal cellular proteins that are overexpressed or aberrantly expressed
proteins in tumour cells. These include proteins such as NY-ESO-1 and tyrosinase. NY-ESO-1 is a
protein found specifically in the testes and ovaries but aberrantly expressed in a number of malig-
nancies. Tyrosinase, an enzyme involved in melanin biosynthesis, is expressed in melanocytes and
melanomas. The surprising fact is that tyrosinase is a normal self-protein, yet in melanoma TH cells
and Tcyt cells recognize and respond against it. Apparently, tyrosinase is expressed in such a small « In the USA, melanoma or skin
cancer is the seventh most com-
amount and in a few cells in a normal state that it fails to be recognized by the immune system as mon type of cancer, affecting about
self and hence fails to induce tolerance. 300,000 people till date.
A LT E R E D G LY C O L I P I D A N D / O R G LY C O P R O T E I N A N T I G E N S « Differentiation antigens are

Tumours often have a disregulated expression of normally detected only at a
enzymes. The enzymes synthesize carbohydrate Oncofoetal antigen Altered particular phase of differentiation
(e.g. AFP) glycolipid of a particular cell type. It should
side chains which leads to the appearance of glycoprotein be made clear that these antigens
tumour-associated altered glycolipid/glycoprotein. Oncogen antigen may or may not play any role in
product differentiation of that cell.
These altered molecules include blood group (e.g. GM-2)
as tumour
antigens and ganglioside antigens such as mono- antigen
sialoganglioside GM2 expressed in melanomas
and sarcomas, and disialoganglioside GD2 and
Figure 18.7
GD3 antigens expressed in melanomas, neurob-
Schematic diagram showing an
lastomas and sarcomas. These altered antigens overview of various tumour-associated
induce both antibody and T-cell response in antigens. All antigens are shown as
surface molecules for the sake of
cancer patients. Being strongly immunogenic, Aberrantly expressed Differentiation clarity. AFP—alpha foetal protein;
the altered glycoproteins are considered as normal cell proteins antigen PSA—prostate-specific antigen; GM—
potential candidates for cancer vaccine. (e.g. PSA) (e.g. MART) monosialoganglioside.
One tumour-associated antigen that does not raise any immune response is tissue-specific
differentiation antigen. This is a differentiation antigen because it is specific for a particular lineage
or differentiation stage. Differentiation antigens are normal self-antigens and therefore do not in-
duce immune response in a tumour-bearing host: for example, CD10 surface marker expressed on
B-cell lineage cells, MART-1 and gp-100 are also differentiation antigens expressed in melanoma.
Some representative tumour-associated antigens are shown in Figure 18.7.
18.3 IMMUNE RESPONSE TO TUMOURS

In vitro, both cell-mediated immunity and humoral immunity have been shown to kill tumour
cells. In general, cell-mediated response is believed to provide more protection in vivo. It is still a
challenge for tumour immunologists to determine whether humoral or cell-mediated immunity
provides protection against tumours. Here we discuss various effector mechanisms which are most
likely to be relevant to human tumours.
18.3.1 T - C E L L - M E D I AT E D I M M U N I T Y
In general, Tcyt-cell-mediated killing of tumour cells appear to a play major role in killing tumour
cells. The ability of Tcyt cells to provide effective anti-tumour immunity in vivo has been experi-
mentally demonstrated in animals. In DNA-virus-induced carcinoma and carcinogen-induced tu-
mours, Tcyt cells recognize and kill tumour cells that display oncogenic peptides together with class I
MHC molecules. The mechanism by which tumours stimulate Tcyt cells is also very interesting.
Tumours mostly arise from body cells other than professional antigen-presenting cells. Cells other
than professional antigen-presenting cells do not express costimulators needed to initiate Tcyt-cells
effector functions. The stimulation of TH cells is necessary for the activation and differentiation of
Tcyt-cells. It is likely that the tumour cells or their antigens are taken up by the host antigen-presenting
cells. This antigen can then be processed and displayed on the antigen-presenting cell surface
together with class I MHC molecules for recognition by Tcyt cells. The host antigen-presenting
Cross-priming or cell also display costimulatory molecules that differentiate Tcyt cells into anti-tumour Tcyt. This
Cross-presentation process is called cross-priming or cross-presentation to indicate that one type of cell (professional
The ability of antigen-presenting
cells to acquire extracellular antigens antigen-presenting cell) may prime T cells for antigen of another cell (tumour cell). The role of T
by phagocytosis and present cells in inducing immunity against tumour cells is shown in Figure 18.8.
processed material by means of Moreover, professional antigen-presenting cells also have class II MHC molecules that can
class I MHC molecules to Tcyt cells is
termed as cross-priming or cross- present internalized tumour antigens to TH cells. Once stimulated, effector Tcyt cells can recognize
presentation. Cross-priming is a and kill the tumour cells without the requirement of costimulation.
defence against pathogens such as Recent studies on animal models have identified new antigens on tumour cells that are
viruses, bacteria, tissue grafts and
tumours. recognized by TH cells. Tumour eradication, however, was dependent on the induction of tumour-
specific Tcyt cells, demonstrating the principle that TH cell and Tcyt-cell responses must act in
» Cross-priming was reported by concert to fight cancer.
M. J. Bevan in 1976 for the minor The activation of an antigen-specific Tcyt cell response by a TH cell is dependent on the highly
histocompatibility antigen. specialized and efficient antigen-presenting cells—dendritic cells. The interaction between CD40
» The role of immune surveillance expressed on the dendritic cell and its ligand, CD40L, expressed on TH cells can lead to an amplifi-
by T cells has recently been chal- cation of the immune response and tumour destruction. Cytokines secreted by tumour specific TH
lenged. It has been reported that cells may also lead to tumour destruction by activating an assault by eosinophils and macrophages
nude mice that lack functional T
cells do not show more frequent to produce toxic superoxide and nitric oxide radicals.
development of non-viral-induced It was believed right from the 1900s that, when tumour cells arise, the cells of the body rec-
tumours. This implies that T cells are ognize them as foreign and are eliminated. This theory which was called immune surveillance
not solely responsible for keeping
non-viral-induced tumours in check theory, was later specified for Tcyt cells as they were believed to be the cells involved in surveillance
by immune surveillance. functions by recognizing and killing potentially malignant cells.
18.3.2 N K - C E L L - A N D M A C R O P H A G E - M E D I AT E D
IMMUNITY
NK cells can lyse many types of tumour cells, especially those tumour cells that have reduced class I
MHC molecules and can escape detection by Tcyt cells. This is because the recognition of tumour
cells by NK cells is not MHC-restricted. In vitro studies have shown that NK cells lyse virally
infected cells as well tumour cell lines, especially tumours of haematopoietic origin. NK cells also
kill those cells that have decreased class I MHC molecules on their surface, a mechanism called
Class I MHC
No costimulators expressed
on tumour cell, no stimulation
Tumour cell
of T-cell response
Class II MHC
Processed tumour cell antigen
Tumour cell
Tumour antigens
Internalization of
Endocytosis of tumour
tumour cell and
antigen and processing
processing by
by class II MHC pathway
class I MHC
pathway
Antigen-presenting
cell
B7
B7 co-stimulator
CD28 CD28 Figure 18.8

T-cell-mediated immunity against cancer.
Antigen-presenting cells, apart from
stimulating CD4+ T-cell response against
Tcyt cell TCR THcell tumour antigen, may also cross-prime
cytotoxic CD8+ T cell against tumour
antigens. The ability of APC to present
cell remnants/antigen by class I MHC
Activation of CD8+ Tcyt cell Activation of CD4+ TH cell pathway is called cross-penetration or
(Cross presentation) cross-priming.
missing self hypothesis. This is because some viruses that induce tumours have developed mecha-
nisms that inhibit the expression of class I MHC so that they escape scrutiny by Tcyt cells. NK cells
are natural killer of such MHC-depleted cells.
NK cells can also bind to antibody-coated tumour cells via their Fc receptor and mediate cell
lysis by ADCC mechanism. In vitro studies have shown that the tumourcidal activity of NK cells
is increased by IL-12. In fact, IL-2-activated NK cells, called lymphokine-activated cells (LAK) are
used in adoptive tumour immunotherapy.
The importance of NK cells in tumour immunity in humans can be gauged by the Chediak– « Strictly speaking, LAK cells com-
Higashi syndrome, in which there is marked decrease in NK cells with the resultant increase in the prise both IL-2-activated NK cells
and Tcyt cells.
incidence of certain types of cancer.
Various experimental evidences in vitro have demonstrated that activated macrophages also play « Some scientists believe that the
a significant role in anti-tumour immunity. Activated macrophages can kill tumour cells in a non- reason T-cell-deficient mice do not
have a high incidence of spontane-
MHC-restricted manner. This anti-tumour activity is probably mediated by the repertoire of lytic ous tumours is because NK cells
enzymes and ROS/RNS expressed in macrophages. Activated macrophages also produces tumour serve as immune surveillance cells.
necrosis factor (TNF), which, as the name implies, induce haemorrhage and necrosis of the tumour
and not the normal cells. Tumour necrosis can be induced by thrombosis in tumour blood vessels,
resulting in the ischaemic degradation of tumour or direct apoptosis of tumour cells mediated by the
binding of TNF to its receptor on the tumour cells. Macrophages also express the Fc receptor that helps
them bind antibody-coated tumours cell and mediate either phagocytosis or ADCC. The role of NK
cells and macrophages in generating an immune response against tumours is shown in Figure 18.9.
NK cell
KIR
Perforin and
granzyme
Decreased expression
of class I MHC
Tumour cell
Tumour antigen
ADCC
Fc receptor
NK cell
NK cell-mediated attack on tumour cell
Tumour cell TNF receptor
Tumour
antigen
ADCC
Fc receptor
Phagocytosis
-
O2
NO
Lytic enzymes
ROS,RNS
Figure 18.9
Schematic diagram showing how NK Macrophage
cells and macrophages are involved in
the killing of tumour cells. Macrophage-mediated attack on tumour cell
18.4 E VA S I O N O F I M M U N E R E S P O N S E
BY TUMOURS
Under ideal conditions, cells of the innate immune system detect the “danger signals” provided by
growing tumours. These signals induce inflammation, activate the innate effector cells with anti-
tumour activity and stimulate the antigen-presenting cells to endocytose tumour cells or antigens,
and then travel via lymph nodes to inform adaptive (T and B) lymphocytes. Despite this excellent
scrutiny operation, the presence of tumour indicates that the progressing tumour somehow avoids
detection or overwhelms the immune response.
Developing cancer cells have evolved a number of strategies that help them evade or resist the
Tumour escape host immune response (see Figure 18.10). By understanding the mechanism by which these cells
The evasion of the immune response evade the immune response, scientists hope to craft techniques to increase the immunogenicity of
by tumours is called tumour escape. tumours and the success of their treatment. The process of evasion of the immune response often
called tumour escape can result from the manifestation of one or more of the following mechanisms.
18.4.1 D O W N R E G U L AT I O N O F C L A S S I M H C M O L E C U L E S
Tumour-inducing viruses have evolved ways to decrease class I MHC expression and assembly
with peptides thereby blocking the presentation of the viral antigen to Tcyt cells. The viruses that
Class I MHC
(expression decreased) Tumour antigens
(expression decreased)
Tumour cell
Downregulation of class I MHC Disappearance of surface

tumour antigens
Glycocalyx
Tumour antigen
Antigen masking
Lymphocyte/
macrophage
inhibited
TGFβ
Fas ligand (recognizes

Fas on leukocyte
Costimulator molecules causing their apoptosis)
(expression decreased)
Figure 18.10
Decreased expression of Expression of immunosuppresive Line diagram explaining evasion of
costimulators tumour products immune response by tumour cells.
downregulate the presentation of peptides to Tcyt cells, include adenovirus that downregulates the
transcription of class I MHC molecules, herpes simplex virus inhibits peptide transporter, TAP,
that is associated with antigen processing. These strategies are operative in a normal viral infec-
tion as well as a virally induced tumour. Even when tumours are not virally induced, tumour cells
show downregulation of class I MHC molecules or β2-microglobulin or TAP, or some component « Human melanoma cells use down-
of proteasomes. All these changes result in a decreased presentation of peptide to Tcyt cells and regulation of class I MHC to escape
immune recognition.
the resulting tumour becomes resistant to Tcyt cells. In vitro systems have shown that an increased
expression of class I MHC molecules on tumour cells (by IFN-γ) results in an increased susceptibility
of these cells to Tcyt cells in vitro.
18.4.2 B L O C K I N G O F T CYT R E S P O N S E B Y A N T I B O D I E S
In some cases, antibodies formed against a tumour-bearing host may bind to tumour antigens,
effectively blocking epitopes from Tcyt cells. This blocking effect can be mediated by anti-tumour
antibodies alone or by antigen–antibody complex. These complexes may also bind NK cells or
macrophages and may inhibit ADCC.
18.4.3 M O D U L AT I O N O F T U M O U R A N T I G E N S
Certain tumour-specific antigens have been shown to disappear from the surface of tumour cells in
the presence of serum antibody. Such “antigen loss variants” are usually detected in rapidly dividing
tumours. Because of the high mitotic rate of tumour cells and their genetic instability, these antigen
» The loss of the antigen MAGE loss variants arise. If these antigens are not essential for the growth or survival of the transformed
is detected in some variants of
melanoma.
phenotype, these antigen loss variants have growth advantage in the host, and hence survive and
proliferate.
18.4.4 L A C K O F C O S T I M U L AT O R S
T-cell activation requires an activating signal-triggered binding of the peptide–MHC molecule by
TCR and a costimulatory signal-triggered binding by the interaction of B7 on the antigen-presenting
cell with CD28 on T cells. Both signals are needed to induce activation and proliferation of
T cells. If tumour cells are B7-negative, that is, they lack costimulator molecules, then the maximal
anti-tumour Tcyt-cell differentiation and hence effector T-cell response does not occur. B7-negative
tumour cells when transfected with genes for B7-1 and B7-2 molecules, are able to elicit strong cell
» Lack of costimulators leads to mediated immune response. Once stimulated, these Tcyt cells can act on B7-negative tumour cells,
tolerization of T cells. as the effector phase of Tcyt killing does not require costimulation.
18.4.5 SUPPRESSION OF ANTI-TUMOUR IMMUNE

RESPONSE
Some tumour cells express/produce immunosuppressive tumour products such as transforming
growth factor β (TGF-β) in a large quantity, to inhibit cell division and the effector function of
lymphocytes and macrophages. Other tumour cells express Fas ligand (FasL) that recognize the Fas
molecule on leukocytes. When FasL binds Fas, it results in the apoptotic death of the leukocytes.
18.4.6 ANTIGEN MASKING

The cell surface tumour antigen may be hidden from the immune system by glycocalyx molecules
such as sialic acid. This phenomenon is called antigen masking and and occurs because tumour
cells exhibit more glycocalyx (surface carbohydrate) molecules than normal cells. Similarly a tu-
mour cell may shield itself from immune system by activating coagulation cascade and coating
itself with fibrin. The anti-tumour antibodies or the specific Tcyt cells which are formed fail to react
with the concealed tumour cells, making the tumour resistant to immune attack.
18.4.7 P R E V E N T I N G I N F L A M M AT O R Y R E S P O N S E
Some tumours prevent the triggering of an inflammatory response by secreting cytokines/growth
factors such as IL-10, or the vascular endothelial growth factor (VEGF) that interferes with
dendritic-cell activation and differentiation or blocking the production of pro-inflammatory
molecules by the tumour cells.
18.5 IMMUNOTHERAPY FOR CANCER

Immunotherapy which is also sometimes called biologic therapy uses the host’s own immune sys-
tem to fight tumour or cancerous cells or their side effects. Tumour usually elicit an immune re-
sponse in the host in some form or the other. However, this response does not provide sufficient
protective immunity to the host and as a result cancer develops. Theoretically, there are two main
immunologic ways to treat cancer patients. The first, called active immunotherapy, is to support
or augment the host’s immune response towards tumours (that is, provide active immunity) and
this can occur only if host immunity is not significantly weakened. However, if the host’s immune
system is eroded or weakened by the developing cancer, tumour can controlled by administering
tumour-specific antibodies or specific Tcyt cells, which amounts to a type of passive immunity and is
» More than $50 billion have already
called passive immunotherapy. (The non-immunologic ways to treat cancer cells include radia-
been spent on cancer research. tion therapy and chemotherapy which are well beyond the scope of this book.)
18.5.1 S T I M U L AT I O N O F A C T I V E I M M U N I T Y A G A I N S T
TUMOUR
The immunization of the tumour-bearing host with killed tumour cells or tumour peptide antigens
may result in an enhanced immune response against tumours. Since tumour cells are damaged or
controlled by T cells, the generation of an activated population of T cells that recognize oncogenic
antigen is a key element in the generation of active anti-tumour immunity. A patient’s own killed
tumour cells mixed with a bacterial adjuvant such as BCG was first used as a vaccine to immunize
cancer patients. This patient–specific vaccine is usually impractical for common use. The use of « Nine out of 10 cancer deaths result
from metastasis of tumour.
common tumour antigens such as mutated p53, as well as MAGE and tyrosinase as immunogens
has the potential for a broad spectrum of cancers.
Nowadays, efforts are being made to identify tumour antigens that strongly activate T cells.
The MHC–peptide complex is isolated from particular tumour cells and bound peptides eluted.
The peptides are then tested on target human T cells. Those peptides that are able to sensitize
T cells the most, towards the tumour cells, are isolated and used in vaccination. Presenting the
antigen is the next big obstacle. A simple injection of tumour antigen may not provide long-last-
ing sustained protection against tumours. Recent researches focus on the introduction of the tu-
mour antigen gene via recombinant viral or bacterial agents. Infection with these vaccine vec-
tors and the subsequent expression of tumour antigen may provide an effective way of displaying
oncogenic peptides on MHC molecules of tumour cells. The approach was effective in reducing
feline leukaemia in cats and preventing herpesvirus-induced lymphoma in chickens. Another ap-
proach to elicit active immunization against a tumour is either to isolate and incubate a patient’s
« Dendritic cells, professional
professional antigen-presenting cells or transfect them with gene-encoding tumour antigens and
antigen-presenting cells, are found
then re-infuse these cells back into the patient to provide proper stimulation of tumour specific everywhere except the brain and
Tcyt and TH cells. testes.
A U G M E N TAT I O N O F I M M U N E R E S P O N S E B Y C O S T I M U L AT O R S
Several research groups have demonstrated that cell-mediated immunity to tumours may be en-
hanced by expressing costimulators and cytokines that stimulate the proliferation and differentia-
tion of T cells and NK cells. We know that tumour cells induce a weak immune response because
they lack costimulators and do not express class II MHC molecules so they do not induce the
activation of TH cells. Thus, there are two loose ends—proper expression of costimulators and pro-
duction/presence of specific cytokines for the activation of TH cells. Several cytokines themselves
have anti-tumour activities.
The efficacy of enhancing costimulation for anti-tumour immunotherapy has been demon-
strated by experiments in mice. Mice tumour cells were transfected with a gene that encodes B7
costimulatory molecules and is used in vaccinating animals. These B7-expressing tumour cells
induce protective immunity against unmodified tumour cells, possibly by the activity of specific
Tcyt cells against tumour cells.
S T I M U L AT I O N B Y C Y T O K I N E S
Cytokines are also used to enhance the immune response against tumours. Tumour cells may be
transfected with cytokine genes to localize the effect of cytokines where they are needed: for exam-
ple, mice transfected with IL-2, IL-4, IFN-γ or GM-CSF (granulocyte-macrophage colony stimu-
lating factor) genes are injected into animals. The tumours are usually rejected or regressed by the
host. The local production of cytokines augments T-cell response to the tumour antigen.
Since cytokines are low molecular weight molecules, and can be obtained commercially in
high yield, systemic cytokine therapy for various tumours has recently been introduced. Systemic
cytokine therapy involves systemic administration (injection) of pure cytokine along or with che-
« IL-2 has been effective in tumour
motherapy or adoptive cellular therapy for the treatment of tumours. An alternate strategy is to regression in 10–15 per cent of
expand and activate NK cells in vitro by culture with IL-2, followed by an infusion of a large num- patients with renal cell cancer and
ber of activated NK cells back into the patient, alone or with a high dose of IL-2. advanced melanoma.
IFN-α has an anti-proliferative effect on tumours, increases cytolytic activity of NK cells and « Tumour necrosis factor, as the
augments class I MHC expression, thereby increasing Tcyt-cell activity against tumours. In addi- name implies, has been shown
tion, the interferon has been shown to inhibit cell division of both normal and malignantly trans- to exhibit direct antitumour
activity—killing some tumour
formed cells in vitro. IFN-α therapy is effective against Kaposi sarcoma, various lymphoma, renal cells and reducing the proliferation
carcinoma and melanoma. The toxic side effects of cytokine therapy include fever, pulmonary of tumour cells while sparing the
oedema and vascular shock. normal cells.
The TNF-α or TNF-β induces characteristic tumour haemorrhage, necrosis and regression.
TNF-α inhibits tumour-induced angiogenesis (vascularization) by damaging the vascular en-
dothelial cells in the vicinity of a tumour, thereby reducing blood flow and inhibiting tumour
growth. Active immunotherapy against tumour administered by transfecting genes of cytokines or
costimulators is shown in Figure 18.11(a).
TCR
No stimulation
of Tcyt cell
CD28 Tcyt cell

Tumour cell
(Lacking costimulators)
Costimulator B7 CD28 Tcyt cell Tcyt cell

gene transfected
Tumour cell expressing Tumour-specific Tcyt cell
costimulator proliferate
Augmentation of immune reponse by costimulators
No stimulation
of Tcyt cell
Tumour cell Tcyt cell
Figure 18.11 (a)

Tcyt cell Tcyt cell Tcyt cell
Diagram showing the various ways
Cytokine Tumour cell Cytokines Cytokines augment differentation
to stimulate active immunity against
tumours. Transfection of tumour cells gene expressing and expansion of Tumour-specific
by costimulator or cytokine genes lead transfected cytokines
to “more visibility” of tumour cells and
hence a stronger immune reaction. Augmentation of immune reponse by costimulators
A U G M E N TAT I O N O F A N T I G E N P R E S E N TAT I O N
Antigen-presentation and antigen-presenting cells play a central role in specific immune response
to tumour cells. This has led to a number of approaches aimed at expanding the population of
antigen-presenting cells so these cells can activate TH or Tcyt cells specific for tumour antigens.
One approach is to isolate dendritic cells from peripheral blood progenitor cells and culture
them in the presence of GM-CSF, TNF-α and IL-4. This leads to cell proliferation and formation
of a massive dendritic cell population. These antigen-presenting cells are then primed with tumour
antigens and then reintroduced into the patient where they can activate TH and Tcyt cells specific
for the tumour antigen.
Another approach is to transfect tumour cells with the gene-encoding GM-CSF. These tumour
cells, when re-introduced in the patient will divide massively (being cancerous cells) but will also
secrete GM-CSF. This secreted GM-CSF will enhance the differentiation and activation of antigen-
presenting cells, particularly dendritic cells, which present tumour antigens to TH cells and Tcyt cells.
Active immunotherapy against cancer using antigen-presenting cells is shown in Figure 18.11(b).
18.5.2 N O N - S P E C I F I C S T I M U L AT I O N
OF THE IMMUNE SYSTEM
If the immune system is activated either by the local administration of inflammatory substances
or agents that act as polyclonal activator of lymphocytes, it results in a better immune response
Tumour-bearing
host
Isolate Isolate
Dendritic Tumour cell

cell
GM-CSF
Transfect
gene
GM-CSF,
TNF-α,IL-4
Cell culture
Tumour cell
transfected with
cytokine gene
Proliferation of Re-introduced
dendritic cells into host cell
Tumour
antigen Secretion of GM-CSF from
Priming with expanded tumour cell
tumour antigen
Figure 18.11 (b)

Dendritic cells re-introduced into Activation and differentation Diagram explaining how immune
tumour-bearing host-specific Tcyt of dendritic cell which present antigen response to tumour cells may be
and TH cell stimulated to Tcyt and TH cells enhanced by antigen-presenting cells
« Non-specific stimulation of the

to tumours. Non-specific stimulation of tumour patients by injecting attenuated strains of Mycobac- immune system can also be
terium bovis for example, BCG, or CpG-containing oligodeoxynucleotides (recognized by Toll-like achieved by Staphylococcus
receptor on innate immune cells), results in better humoral and cell-mediated (innate and adaptive) pyogenes and Corynebacterium
parvum.
immune response against tumour cells.
BCG mycobacteria activates macrophages by increasing the expression of cytokines (secretion),
class II MHC molecules and B7 costimulatory molecules, thereby promoting the macrophage-mediated
killing of tumour cells. These activated macrophages which are better activators of TH cells may
stimulate T-cell response to tumour cells. CpG-containing oligodeoxynucleotides stimulate innate
immune cells, leading to the production of pro-inflammatory cytokines and facilitating the interac-
tion between the adaptive and innate immune systems. Another approach that has been tested on
animal models is the treatment of tumour-bearing animals with anti-CD3 antibodies. Anti-CD3 anti-
bodies result in polyclonal activation of T cells and the concomitant regression of tumour growth.
18.5.3 PASSIVE IMMUNOTHER APY FOR TUMOURS

Passive immunotherapy involves the transfer of immune effector agents such as tumour-specific
« The term immunotherapy was
T cells and antibodies in tumour-bearing patients. Passive immunotherapy does not provide probably employed for the first
long-term immunity. time by Paul Ehrlich in 1906.
18.5.4 ADOPTIVE CELLULAR IMMUNOTHERAPY

Adoptive cellular immunotherapy is the transfer of cultured immune cells that have anti-tumour
activity into a tumour-bearing host. Animal studies have shown that lymphocytes can be activated
against the tumour antigen in vitro by expanding them against X-ray killed tumour cells in the
» A majority of LAK cells are NK cells
that have been stimulated by IL-2. presence of IL-2 and then re-infusing the activated lymphocytes back into the patient.
LAK cells are functionally different In 1980, Rosenberg discovered that NK cells that are spontaneously reactive against tumour
from NK cells because they can lyse cells can be cultured in the presence of IL-2 (without the tumour antigen) that could kill fresh tu-
tumour cells that were previously
shown to be resistant to NK cell mour cells but not normal cells. These cells, which Rosenberg referred to as lymphokine-activated
lysis. cells or LAK cells are then injected into the tumour-bearing host.
This adoptive cellular therapy with autologous LAK cells together with chemotherapy and ad-
ministration of IL-2 results in regression of tumours in mice. Human LAK cell therapy for humans
is still under clinical trials. One of the main side effects of LAK cell therapy is the side effect of IL-2
that is co-infected with LAK cells. It leads to vascular leak syndrome characterized by migration
of lymphoid cells and plasma from the peripheral blood vessels into the tissue. Another variation
» TILs are different from LAK cells. of this technique is to isolate tumour infiltrating lymphocytes (TIL) from the inflammatory infil-
A majority of cells in TILs are T lym- tration present in and around the solid tumour. These TILs are isolated by biopsy and their clone
phocytes. TILs are at least 100 times
more sensitive to the presence of expanded by incubating with IL-2. With this approach, it has been possible to expand TILs (which
IL-2 than LAK cells. are tumour-reactive T cells) to enormous numbers in vitro and infuse billions of specific cells, oc-
casionally completely eliminating tumour masses. Renal cell carcinoma and malignant tumours
show a partial regression in patients after injection with autologous TILs.
Adoptive cell therapy for cytomegalovirus and EBV infection in an immunosuppressed indi-
vidual has demonstrated that increased in vivo immune response occurs when Tcyt cells are present
with TH cells. Moreover, the infusion of T-cell clones rather than T-cell lines represent a sophistica-
tion of T-cell therapy because of the precisely defined specificity, avidity and effector functions of
infused cells. A schematic representation of adoptive cellular therapy is shown in Figure 18.12(a).
18.5.5 HUMORAL IMMUNOTHERAPY

The use of tumour-specific monoclonal antibodies for specific immunotherapy for cancer is allur-
ing for scientists and is a very active area of research. Anticancer monoclonal antibodies eliminate
tumours cells by the same effector mechanism that is used for killing microbes, namely, the activa-
tion of complement and the activation of phagocytosis with or without opsonization.
The first humoral immunotherapy against cancer involved the transfer of monoclonal antibod-
ies, which were therapeutically tested in humans, targeting the IL-2 receptor expressed by many
cancerous T cells. Subsequently, a monoclonal antibody specific for oncogenic product HER2/neu
has shown success in breast cancer patients. Monoclonal antibodies are also successful in treating
B-cell lymphoma and T-cell laeukemia in some patients.
Monoclonal antibodies exhibit their anti-tumour activity by a variety of ways depending on
» The chimeric antibody Herceptin the onco-antigen towards which they are directed. Anti-Her2/neu antibody interferes with the
(trastuzumab) has been approved growth-signalling function of HER2/neu molecules, apart from the usual effector function against
for treating breast cancer. However tumour cells. Monoclonal antibodies targeted against B-cell lineage antigen, CD20, is used in anti-
patients receiving this antibody
treatment are at increased risk for body therapy for B-cell lymphoma. Antibodies induce B-cell-specific cell destruction by comple-
congestive heart failure. ment activation or phagocytosis.
Monoclonal antibodies are also targeted against growth factor receptors, which are an excel-
lent target for anti-tumour antibodies. A variety of tumours express significantly increased level
of growth factor receptors such as HER2/neu, an epidermal growth-factor-like receptor which is
» Immunotoxins are proteins that
overproduced and implicated in about one-third of all breast cancers. The preparation of a chime-
contain a toxin along with an anti- ric antibody, Herceptin, directed against HER-2 that has mouse Fab and human Fc region has been
body (or growth factor) that binds approved by the FDA for treatment of breast cancer. The most widely used monoclonal antibody
specifically to target cells and kills
the target cell.
is rituximab, which binds CD20 and if given alone or with chemotherapy can induce remission in
patients with B-cell lymphomas possibly by ADCC mechanism.
Many variations of anti-tumour monoclonal antibodies have been tried in order to improve their
» The suggestion that an antibody
might be linked with a toxic mol- effectiveness. Tumour-specific antibodies have been variously linked to radioisotopes, antitumour
ecule and targeted to tumour cells drugs and toxic molecules so that tumour cells can be specifically targeted. Toxins such as ricin and dip-
was first made in 1946 by David theria toxin are potent inhibitors of protein synthesis and can be cytotoxic if they enter tumour cells.
Pressman, who later established the
Sloan–Kettering Institute for cancer One approach utilizes the best of both the worlds. It conjugates monoclonal antibodies directed
research. against tumours to the inhibitory chain of toxin such as ricin or diptheria. These “immunotoxins”
Isolate TILs Isolate NK cells reactive

reactive against tumour cells against tumour cell
Tumour-bearing
host
TILs NK cells
Clonal expansion in
presence of IL-2
Expanded population of TILs Lymphokine-activated

cells (LAK)
Transfer TILs into Transfer LAK cells into

tumour-bearing host tumour-bearing host together
together with IL-2 with IL-2
Figure 18.12 (a)

Tumour regression Schematic diagram explaining passive
immunotherapy (via adoptive cellular
Adoptive cellular Immunotherapy therapy).
are delivered specifically to tumour cells which are then eliminated. Immunotoxins have been
effective in several cancers such as metastatic breast carcinoma and colorectal carcinoma and various
lymphomas and leukaemias. A schematic representation of humoral immunity in assaulting tumour
cells is shown in Figure 18.12(b).
However the following precautionary measures should be kept in mind.
• The antibody must not bind normal healthy cells.
• A sufficient amount of antibody should be injected so that a significant amount reaches
tumour cells before being cleared by Fc-receptor-bearing phagocytes in the blood.
• Toxic molecules such as radioisotopes or toxins may have their own systemic effect when
circulating through normal tissues so the minimal required amount should be administered.
• The administration of immunotoxins may result in antibody response against the toxins
and the injected heterologous antibodies.
Anti-tumour antibodies are also used to treat cancer cells from the bone marrow before autologous
marrow transplantation. In this procedure, the patient’s bone marrow is removed and treated with
monoclonal antibodies specific for the tumour antigen that kill the tumour cells. This treated bone
marrow is then re-transplanted but not before the patient is given doses by radiation and chemo-
therapy to destroy all cells (normal as well as tumour cells). The re-transplanted bone marrow then
reconstitutes the haematopoietic system destroyed by irradiation and chemotherapy.
Passively Toxin Radioisotope

mAb
transfered mAb mAb (Rituximab)
Complement Growth-factor
Tumour receptor CD20
antigen
MAC
Figure 18.12 (b) Membrane lysis Cell-cycle
Diagram explaining how humoral
retardation Metabolic
immunotherapy is used for DNA
inhibition ADCC
assaulting tumour cells. Passively damage
transferred mAbs can be linked Cell death
to a variety of effector molecules
that ultimately kill tumour cells. Overview of Humoral immunotherapy
Immunoblotting
Transfer of proteins
from SDS-gel to
nitrocellulose sheet
SDS–PAGE of proteins Proteins transferred to

nitrocellulose sheet
Addition of primary antibodies

against proteins
Addition of chromogenic
substrate of enzyme
makes specific
proteins visible
Addition of enzyme-conjugated
secondary antibody,
that binds primary antibody already
bound to antigen
Figure 18.13
Western blotting/Immunoblotting.
Immunoblotting allows the detection of specific protein in a sample groups that non-specifically bind proteins, these binding sites on
of a variety of proteins. For immunoblotting, proteins are first sepa- the nitrocellulose membrance are blocked by incubating it with
rated on the basis of size on denaturing SDS–PAGE. Once this elec- a blocking agent such as bovine serum albumin or skimmed milk
trophoretic separation is achieved, the wet gel is then placed against powder. These blocking materials adsorb to sites other than the
the nitrocellulose paper in a special electrophoresis chamber. The gel blotted proteins and hence block the highly charged surface of the
is then subjected to electrophoresis which transfers proteins from nitrocellulose membrane. Primary antibody (such as murine anti-
the polyacrylamide gel onto the nitrocellulose membrane where the body) is then added which binds the target protein. Secondary anti-
proteins adhere irreversibly. These proteins which have been “blotted” body (such as goat antimurine IgG) conjugated with an enzyme
onto nitrocellulose can be detected by a protein stain or probed by such as alkaline phosphatase or horseradish peroxidase is then
antibodies. This transfer of proteins from the polyacrylamide gel to added which binds the primary antibody. The addition of chro-
the nitrocellulose paper is referred to as Western blotting. The pro- mogenic substrates for these enzymes (such as X-phos for alkaline
teins on the nitrocellulose sheet can be detected by a reversible red phosphatase) allows the detection of bands of target protein. Im-
stain such as Ponceau S (0.02 per cent solution). Once the proteins munoblotting is routinely used for identification of single protein
are visualized, this stain can be completely removed by washing the
from a mixture of proteins separated by PAGE. It is also used for
stained nitrocellulose membrane in tap water.
screening expression libraries. Since it is a rather long procedure,
For immunoblotting, a specific antibody against the target pro- gels can be probed by up to eight to nine antibodies at a time to
tein must be available. Since, the nitrocellulose membrane carries increase the protein identification rate.
S U M M A R Y
• Cancer comprises a large class of diverse diseases all of which • In general, a cell-mediated immune response is believed to provide
exhibit uncontrolled cell growth and division. more protection to the host than humoral response against cancer-
ous cells. In general Tcyt cells, and NK cells, appear to play a major
• Transformation describes the failure to observe the normal role in killing tumour cells.
constraints of growth such as serum/growth factor dependence,
anchorage dependence, density-dependent inhibition of cell • Developing tumour cells have evolved a number of strategies that
growth, etc. help them evade or resist host immune responses. These include
downregulation of class I MHC molecules, modulation of tumour
• Cancer can be induced by activating mutation in oncogenes and/or antigens, deficient expression of costimulators, antigen masking, etc.
inactivating the mutation of tumour suppressor genes.
• Immunotherapy for cancer uses the host’s own immune system to
• Cellular proto-oncogene can become oncogenic by a variety of fight tumour cells or its side effects. It can be (a) active immuno-
genomic changes such as mutation, amplification, translocation
therapy if the host is not significantly weakened; (b) passive im-
and gene insertion.
munotherapy when tumour-specific antibodies or specific Tcyt cells
• The loss of function by inactivating mutation in both the alleles are administered into cancer patients because the host’s immune
of tumour suppressor gene results in oncogenesis. Two well- system is too weak to respond.
characterized tumour suppressor genes are p53 and RB-gene.
• Active immunotherapy augments the host immune response
• Antigens that are expressed on tumour cells but not on normal against tumours. It includes immunization of the tumour-bearing
cells are tumour-specific antigens (TSAs). The tumour antigens host with killed tumour cells, augmentation of immune response by
that are also present on normal cells are called tumour associated costimulators and stimulation by cytokines.
antigens (TAAs).
• Passive immunotherapy for the tumour-bearing host includes
• Common examples of TSAs include MAGE proteins, while TAAs transfer of immune effector agents such as tumour-specific T cells,
include oncofoetal tumour antigens, and aberrantly expressed NK cells (adoptive cellular immunotherapy) or tumour-specific
normal cell proteins, among others. monoclonal antibodies (humoral immunotherapy).
K E Y W O R D S
• alpha foetal protein 388 • humoral • lymphoma 381 • tumour

• antigen masking 394 immunotherapy 398 • lymphokine-activated suppressor gene 383
• active • passive killer cell 398 • tumour-specific
immunotherapy 394 immunotherapy 393 • oncofoetal antigen 387
• adoptive cellular • immune surveillance antigen 388 • tumour-associated
immunotherapy 398 theory 390 • oncogenes 383 antigens 388
• benign tumour 382 • retinoblastoma 386 • p53 386 • tumour infiltrating
• carcinogens 382 • specific differentiation • proto-oncogenes 383 lymphocyte 398
• carcinoembryonic antigen 390 • Philadelphia • tumour escape 392
antigen 388 • MAGE 387 chromosome 385 • tumour
• cytokine therapy 395 • malignant tumour 381 • tumour 381 immunotherapy 391
1. Why are a large number of tumours curable if detected early? What 4. Telomerase inhibitor was one hailed as a “cure” for cancer. Why?
happens if their detection is delayed? Why did this attempt fail?
H INT —Repeated damage to chromosome ends usually terminate cell
2. Why is tumour-specific immune response incapable of controlling life. Cancer cells rebuilt broken ends by telomerase. The magic bullet failed
progression? What role does Tcyt and TH cells play in anti-tumour because it also eliminated “immortal” but much-needed stem cells.
immunity?
5. How is tumour induction by oncogenes different from tumour
3. How are active and passive immunotherapies used to modulate induction by tumour suppressor genes? Citing examples, support
specific immune response against tumours? your point of view.

1. Anti-tumour monoclonal antibodies targeted against which 6. Proto-oncogenes can code for all of the following, except:
surface antigen will be most effective? (a) Growth factor receptor
(a) Growth factor receptors (b) Calcium-binding protein
(b) Class I MHC (c) GTP-binding protein
(c) CD3 molecule (d) Protein kinases
(d) CD22 molecule
7. Chronic myelogenous leukaemia which expresses Philadelphia
2. Which cytokine is used for clonal expansion of LAK and TIL chromosome exhibits:
cells to be used for adoptive cellular therapy? (a) Activation of tumour suppressor gene
(a) IL-1 (b) Activation of oncogene
(b) IL-2 (c) Amplification of proto-oncogene
(c) IFN-γ (d) Insertion of virus into cellular genome
(d) TNF
8. Lymphokine-activated cells are primarily:
3. Which one of the modes is NOT used by tumour cells to evade (a) NK cells
host immunity? (b) CD8+ T cells
(a) Downregulation of class I MHC (c) CD4+ T cells
(b) Downregulation of costimulatory molecules (d) Dendritic cells
(c) Antigen masking
(d) Molecular mimicry 9. Sarcoma is a malignant tumour of the tissues of:
(a) Ectodermal origin
4. The stimulation of immune response in tumour patients can (b) Secondary lymphoid organs
be achieved by injecting all of the following, except: (c) Endodermal origin
(a) Mycobacterium bovis (d) Mesodermal origin
(b) CpG-containing oligodexynucleotides
(c) Streptococcus aureus 10. Which of the following is a cancer of circulating tissue:
(d) Anti-CD3 antibodies (a) Carcinoma
(b) Lymphoma
5. Which of the following cell is likely to be least relevant in anti- (c) Leukaemia
tumour immunity? (d) Sarcoma
(a) CD8+ T cells
(b) NK cells
(c) Macrophages
(d) Eosinophils
State true or false against each statement. If false give reason(s).

1. Viral oncogenes contain introns while proto-oncogenes are 4. Tumour-reactive T cells have been named as TILs and LAK
single exons. cells.
2. Genomes of all vertebrates contain three ras proto-oncogenes— 5. Monoclonal antibodies directed against tumours conjugated with
C-H-ras, C-K-ras, and N-ras. toxin molecules are referred to as immunotoxins.
3. Generation of B7-negative tumour cells results in maximal Tcyt
effector response.
F U R T H E R R E A D I N G S
Bishop, J. M. (1983). “Cellular Oncogenes and Retrovines”, Marshall, C. J. (1991). “Tumour Suppressor Genes”, Cell, 64:
Annual Review of Biochemistry, 52: 301–54. 313–26.
Blattman, J. N. and P. D. Greenberg, (2004). “Cancer Immuno- Ockert, D., M. Schmitz, I. M. Hamp and E. P. Rieber (1999).
therapy: A Treatment for the Masses”, Nature, 305: 200–05. “Advances in Cancer Immunotherapy”, Immunology
Today, 20: 63.
Cerundolo, V. (1999). “Tumour Immunology: T-cells Work
together to Fight Cancer”, Current Biology, 9: R695–97. Old, L. J. and Y. T. Chen, (1998). “New Paths in Human Cancer
Serology”, Journal of Experimental Medicine, 187: 1163–67.
De Pinho, R. A. (2000). “The Age of Cancer”, Nature, 408:
248–54. Schar, P. (2001). “Spontaneous DNA Damage Genome Instability
and Cancer When DNA Replication Escapes Control”, Cell, 104:
Fearon, E. R. and C.V. Dang, (1999). “Cancer Genetics: Tumour 329–32.
Suppressor Meets Oncogene”, Current Biology, 9: R62–65.
It was probably in 1950 that the first immunodeficiency disease was “It is healthy
reported. This immunodeficiency disease involved defects in the to be reminded
functioning of both B cells and T cells and was referred to as essential that the strongest
lymphocytophthisis by E. Glanzmann and P. Riniker. In 1952 Bruton might weaken
described, for the first time, sex-linked hypogammaglobulinemia and the wisest
which later came to be known as X-linked agammaglobulinemia. IgA might err.”
— M A HAT M A G A N D H I
deficiency was first reported by J. H. Rockey and his associates in 1964.
Immunodeficiency related to defects in T-cell functions came to be rec-
ognized in 1965 by DiGeorge. In 1967, IgM deficiency was reported by
Hobbs , Milner and Watts in two brothers whose father also manifested
low IgM concentration in the serum. With increased awareness and
advancement of technologies, the etiology and symptoms of other
immunodeficiency diseases came to be reported and studied. you should be able to:
Figure 19.1 shows a photograph of a child affected by one of the well- • Differentiate between
primary and secondary
characterized immunodeficiency diseases—the DiGeorge syndrome. immunodeficiencies
• Briefly describe
immunodeficiency diseases
affecting the myeloid cell
lineage
• Explain X-linked and autosome-
linked severe combined
immunodeficiencies
• Give an account of
immunodeficiency diseases
affecting B- and T-cell lineages
• Describe the animal models
of primary immunodeficiency
diseases
• Give an account of the AIDS
epidemic
• Explain the structure and life
cycle of HIV
• Describe the mechanism of
immunosuppression by HIV
• Describe the currents
treatments and potential
vaccines that are currently
being explored
Primary and Secondary
Immunodeficiencies 19
19.1 INTRODUCTION
The immune system is a complex defence system essential for combating infectious organisms and
their toxic products. Defects or deficiency in one or more components in the vast network of the
immune system can lead to serious consequences or disorders. These immune disorders that mani-
fest as a result of one or more deficiencies in the immune system are termed as immunodeficiency
disorders. The main fallout of immunodeficiency is the increased susceptibility to infectious
organisms. A deficiency of humoral immunity increases the chances of bacterial infection as an-
tibodies are the primary defence against bacteria. Defects in the effector arm of cell-mediated
immunity lead to an increased incidence of virus infection. An immunodeficient state makes a
person more prone to oncogenic viruses such as Epstein–Barr virus (EBV) and cytomegalovirus.
Immune disorders are broadly divided in two main groups—primary or congenital immunodefi-
ciency and secondary or acquired immunodeficiency
19.1.1 PRIMARY IMMUNODEFICIENCY

Primary or congenital immunodeficiency is the result of genetic defect(s) in either the innate or
adaptive immune system. This defect leads to increased susceptibility to infection that is frequently
manifested in infancy and childhood.
19.1.2 SECONDARY OR ACQUIRED

IMMUNODEFICIENCY
Secondary immunodeficiency is a result of defects in the immune system acquired during the
life time of an individual. This form of immunodeficiency can develop as a result of infection of
immune cells as in AIDS, or due to malnutrition, disseminated cancer or during the course of treat-
ment with immunosuppressive drugs.
This chapter focuses on the main types of primary and secondary immunodeficiencies
and their pathogenesis, and the components of the immune system that are involved in dealing
with them.
Figure 19.1
Photograph showing a boy with
DiGeorge syndrome. A child with this
anomaly has (a) low-set ears; and a
(b) fish-shaped mouth. Reproduced with
permission from DeBerardinis,
R. J., L. Medne, N. B. Spinner and
E. H. Zackai (2005). “DiGeorge Anomaly in
a Patient with Isochromosome 18p Born
to a Diabetic Mother”, American Journal
of Medical Genetics, 138A: 155–159.
19.2 PRIMARY IMMUNODEFICIENCIES

Probably the first case of primary immunodeficiency involving defects in both B- and T-cell func-
tion was reported by Glanzmann and Riniker in 1950. They called it essential lymphocytopthisis.
Primary immunodeficiencies may result from a defect or impairment of mediators of innate
immunity such as neutrophils (Job syndrome), phagocytes (Chediak–Higashi syndrome), comple-
ment components (complement-deficiency disorders) as well as deficient class II MHC molecules
» Primary immunodeficiencies are
the result of genetic defects in (bare lymphocyte syndrome). A deficiency in the specific components of adaptive immunity re-
either the innate or the adaptive sults from the defects in B cell and T cells.
immune system. Abnormalities in B-lymphocyte development and function leads to increased susceptibility to
infection by extracellular pathogens. It is usually diagnosed by deficient antibody production,
reduced level of serum antibodies, poor antibody response to vaccination, and in rare cases, reduced
B cells in circulation and reduced plasma cells in the tissues. Abnormalities in T-cell development
and function lead to deficient cell-mediated immunity and increased susceptibility to intracellular
pathogens. T-cell immunodeficiency is diagnosed by decreased peripheral blood T cells, low pro-
» Congenital or surgical asplenia
leads to Tuftism deficiency, an liferative response to T-cell activator and deficient delayed-type-hypersensitivity response towards
immunodeficiency disease. allergens. In addition, there are immunodeficiencies that stem from developmental defects that
impair the proper functioning of an organ of the immune system.
Table 19.1 describes the major primary immunodeficiencies that are commonly found. In the
following section we will describe immunodeficiencies caused by defects in the various compo-
nents of acquired and innate immunity and conclude with a brief discussion of therapeutic strate-
gies to overcome these diseases.
19.2.1 LY M P H O I D C E L L D I S O R D E R
As we have seen in Chapter 2, there are two main cell lineages that are important to immune func-
tions. These include the lymphoid cell lineage that includes B or T cells and the myeloid cell lineage
that gives rise to macrophages, neutrophils and other phagocytes.
The process of lymphocyte maturation from the stem cells to functionally competent mature
lymphocytes is a multi-step process. It involves cell proliferation, expression of antigen receptors,
positive and negative selection of cells and, of course, changes in the expression of diverse genes.
Genetic defect(s) at one or more steps in these stages lead to abnormalities in lymphocyte develop-
ment. Figure 19.2 highlights the location of defects in lymphocyte development that give rise to vari-
ous primary immunodeficiencies. Disorders that affect both B and T lymphocytes result in defects
in both humoral and cell-mediated immunity and are called severe combined immunodeficiencies
(SCIDs). Other diseases that affect either the B- or T- cell lineage are given a more specific name.
SEVERE COMBINED IMMUNODEFICIENCY (SCID)

SCID
SCID, which is the acronym for SCID is a family of disorders which are X-linked recessive or autosomal recessive, and affect the
severe combined Immunodeficiency, development of either T cells or T and B cells. About 50 per cent of SCID cases are X-linked and 50
represents a family of rare,
per cent are autosomal-linked.
sometimes fatal, congenital
disorders characterized by little or no
immune response. X-LINKED SCID. X-linked SCID cases arise due to mutations in genes encoding the γc chain
(common γ chain) which is shared by several cytokine receptors such as receptors for IL-2, IL-4,
IL-7, IL-9 and IL-15. The genes are located on the X chromosome and are recessive. Heterozygous
» Essential lymphocytophthisis is
now known as severe combined females with two X chromosomes are phenotypic normal carriers. Males who inherit the defective
immunodeficiency. X chromosome manifest the disease. Since developing cells in females randomly switch off one X
chromosome, the γ protein will not be expressed in half of the cell population of female carriers.
» X-linked SCID results from a muta-
tion in the interleukin receptor γ X-linked SCID has normal levels of B cells, but greatly reduced numbers of mature T cells.
gene which produces the common In spite of having a normal concentration of B cells, X-linked SCID animals or humans show a poor
γ chain subunit, a component of humoral response. The humoral immunodeficiency in this disease is attributed to a lack of T-cell help
several interleukin receptors. Defec-
tive interleukin receptors prevent for antibody production. Common γ chain (γ c chain) is also a part of the IL-7 receptor and X-linked
the proper development of T SCID is due to the inability of IL-7 (not IL-2) to stimulate the growth of immature thymocytes.
lymphocytes that play a major role
in identifying invading pathogens,
as well as activating and regulating AUTOSOME-LINKED SCIDS. Of the 50 per cent of autosome-linked SCID, half are due to the
other cells of the immune system. deficiency of an enzyme called adenosine deaminase (ADA). The ADA form of SCID is inherited
PRIMARY AND SECONDARY IMMUNODEFICIENCIES 407
Mode of
Disorder Cells Affected Defective Functions Inheritance
B-cell deficiencies
• X-linked agammaglobulinemia B cells Defect in Bruton’s tyrosine kinase; No maturation beyond XL
pre-B cells
• Common variable immunode- B cells Block in differentiation pathway that transforms B cells to AD/AR
ficiency plasma cells; plasma cells absent
Selective immunoglobulin isotype
deficiencies
¾ IgA B cells Low serum IgA; defect in the inability to synthesize or secrete IgA Variable
¾ IgG B cells Low serum IgG (IgG3 or IgG2); abnormal terminal B-cell differ- XL
entiation/homozygous deletion of constant region gene of IgG.
• X-linked hyper IgM syndrome B cells T cells, CD40 ligand deficient; no class switching, no memory cells XL
macrophages formed, large number of IgM secreting plasma cells present
• Transient ypogammaglobu-
h B cells IgG synthesis in infants is delayed; B cells do not get help from Not sex-linked
linemia of infancy TH cells
T-cell deficiencies
• DiGeorge yndrome
s T cells T-cell deficiency, defective development of thymus (and AD (not hereditary)
parathyroid gland) XL
• X-linked ympho-proliferative
l B cells, T cells Development of B-cell tumours; mutation in adaptor molecule AR
disease involved in signalling pathway that regulates B-cell and T-cell
proliferation
• Bare lymphocyte syndrome Antigen-presenting Deficiency of CD4+ T cell, decreased antibody production; class
cells, T cells II MHC absent or deficient
• Defective class I MHC expression T cells Deficiency of class I MHC molecule; defective TAP molecules, Autosomal
defective pumping of peptide molecules into ER. inheritance
Combined B-cell and T-cell deficiencies
• Wiskot–Aldrich syndrome Platelets, B cells, Defect in Wiskot–Aldrich syndrome protein (WASP); B and T cells XL
T cells are functionally abnormal; thrombocytopenia
• Ataxia–telangiectasia B cells and T cells Uncoordinated muscle movement, abnormal dilation of blood AR
vessels, lymphopenia, decreased level of IgG, IgA
Phagocytic cell deficiencies
• Chronic Granulomatous Disease Neutrophils, Defect in one or more proteins of phagocytic, oxidase, namely, XL/AR
macrophages cyt b558 or p67Phox or p47Phox
• Leukocyte adhesion deficiency-1 Leukocytes Defective β2-integrins, impaired extravasation of leukocytes, AR
(LAD=1) adhesion-dependent functions of leukocyte are impaired.
• Chediak–Higashi yndrome
s Macrophages, Defective cytosolic protein LYST which is involved in intracel- AR
neutrophils, platelets, lular trafficking; defective degranulation, defective fusion of
dendritic cells, NK cells lysosome with phagosome and formation of giant lysosomes
SCID
• X-linked CID
S T cells, B cells Mutation in γc chain of cytokine receptor, reduction in mature XL
T cells, poor humoral response
• Autosome linked SCID
¾ ADA deficiency T cells, B cells Deficiency of ADA enzyme, reduced number of B and T cells AR
¾ PNP deficiency T cells, B cells Deficiency of PNP enzyme, accumulation of toxic metabolites in cell AR
¾ ZAP-70 deficiency T cells Defective tyrosine kinase, ZAP-70-, low number of Tcyt cells, AR
normal level of non-functional TH cells
¾ Reticular dysgenesis B cells, T cells Mutation in gene coding for RAG-1 and RAG-2, defective Unknown
antigen-receptor gene rearrangement, absence of B and T cells
AR—autosomal recessive; AD—Autosomal dominant;XL—X-linked.
Table 19.1
Major primary immunodeficiency syndromes
Stem cell
Lymphoid precursor
Myeloid precursor
Severe
Neutropenia
combined
immundeficiency
Monocyte
Neutrophils
Pre-B cell Pre-T cell
Chronic Leukocyte
granulomatous adhesion
disease disease DiGeorge
X-linked syndrome
agammaglobulinemia
Bare
lymphocyte
syndrome
B cell T cell
Wiskot–Aldrich
syndrome
Common variable
immunodeficiency disease
Selective
IgA
deficiency
Selective
X-linked IgG
syndrome deficiency
IgM IgE IgG IgA
Figure 19.2 Common

Line diagram depicting points at which a variable
lymphopoietic development block may immunodeficiency
lead to primary immunodeficiencies. disease
The sites of defects in the lymphopoietic
development of immune cells that
result in primary immunodeficiencies
Plasma cells
are shown.
» In X-linked SCID, there is a

as an autosomal recessive form. ADA functions in the salvage pathway of purine degradation where
defective production of NK cells. it catalyses the conversion of deoxyadenosine to inosine.
A deficiency of this enzyme leads to the accumulation of deoxyadenosine and its precusor de-
» The enzyme adenosine deami-
nase (ADA) is coded by a gene on oxyadenosine triphosphate and S-adenosylhomocysteine. The accumulation of these toxic products
chromosome 20. leads to the inhibition of DNA synthesis. ADA deficiency leads to reduced numbers of B and T cells.
Infants with ADA-deficient SCID, Guanine Decrease Deoxyinosine

« SCIDs with ADA deficiency
constitute about 15 per cent of all
have near-normal lymphocyte count SCIDs and 50 per cent of autosomal
at birth which falls off exponentially in PNP ADA SCIDs.
the first year. Those lymphocytes that deficiency deficiency
remain fail to proliferate upon antigen Deoxyguanosine Deoxyadenosine
stimulation.
In about half of the cases, little or
no specific information regarding the
genetic defect is known. It is known
dGTP Increase dATP
that another autosomal SCID, though
rare, is caused by deficiency of purine
nucleoside phosphorylase, PNP, that
is also involved in purine catabolism.
PNP catalyses the conversion of inosine Ribonucleotide reductase
to hypoxanthine and guanosine to inhibition
guanine. A deficiency of PNP leads
to the accumulation of deoxyguanosine
and deoxyguanosine triphosphate « Apart from the forms of SCIDs
that has toxic effects on immature T Toxic mentioned here, there are a num-
lymphocytes. The toxic effect of both for ber of other forms of autosomal
SCIDs (involving deficiency of either B/T cells SCIDs that are universally found.
These include diseases caused by
ADA or PNP) is the inhibition of the mutation in the Jak-3 gene, the CD3
enzyme ribonucleotide reductase, gene, the Artemis gene and the CD
45 gene.
which is needed for DNA synthesis/
replication. Since ADA and PNP are SCID
found in all mammalian cells, why do
these defective genes exert their effect Autosome-linked SCID
on lymphocytes? The possible reason Multichain cytokine receptor
is that other cells have another enzyme
5’ nucleotidase that compensates for
defective ADA and PNP. Lymphoid Mutation in
cells either lack or are deficient in common γ chain Figure 19.3
Schematic diagram showing points of
this enzyme. Another kind of SCID defects in autosome and X-linked SCID.
which is neither inherited nor caused α β γ Flow chart showing accumulation of
deoxyadenosine and deoxyguanosine
by a specific genetic defect is known as
X-linked SCID and its resulting toxicity.
reticular dysgenesis. A mutation
in genes RAG-1 and RAG-2 that code for specific recombinases, causes a defect in the antigen
receptor gene rearrangement. This form of SCID is characterized by an absence of B and T lym-
phocytes, but NK cells are present at almost normal concentration.
Another type of SCID is characterized by the depletion of Tcyt cells and non-functional TH
cells. This form of SCID has a defective tyrosine kinase (ZAP-70) that plays an important role in
T-cell signal transduction. Infants with this form of SCID have normal level of antibodies, normal
level of non-functional TH cells and a very low level of Tcyt cells. Figure 19.3 shows the defects that
occur in autosome-and X-linked SCIDs.
D E F E C T S I N B - C E L L M AT U R AT I O N
X-LINKED AGAMMAGLOBULINEMIA. This disease which is also called Bruton’s agammaglo- « O. C. Bruton described X-linked
bulinaemia is characterized by the absence of gamma globulin in blood and hence the name. O. C. agammaglobulinemia as
hypogammaglobulinemia in 1952.
Bruton was the scientist who first described this disease as sex-linked hypogammaglobulinae.
It is the most common X-linked congenital immunodeficiency disease. This disease is char-
acterized by the failure of B cells to mature beyond the pre-B-cell stage in the bone marrow. Pre-B
cells have a rearranged heavy chain but the light chain genes are in their germ-line configuration.
This occurs because of mutations or deletions in the gene encoding an enzyme, Bruton’s tyrosine
kinase (BtK) in B cells. It is believed that BtK, which is a cytosolic enzyme is involved in transduc-
ing signals from the pre-B-cell receptor to the cellular machinery which is required for continued
» The BtK gene was discovered

maturation of the cells. The exact function or pathway of action of this protein is still not clear. In
in 1993. the female carrier, which has one normal and a mutant allele of BtK, only those B cells that have an
inactivated X-chromosome-carrying mutant allele, mature.
For unknown reasons, in X-linked agammaglobulinemia, a non-random X-chromosome in-
activation is not observed in T cells. (In SCID, both B and T cells show random X-chromosome
inactivation.) Patients with X-linked agammaglobulinemia usually have no IgA, IgD, IgE or IgM.
» Patients with X-Linked agam- IgG is detected in very low concentration. They have absent or reduced B cells in the lymphoid
maglobulinemia are susceptible organs or peripheral blood circulation. They also show no detectable germinal centre, absent or
to infections because they lack hyplastic Peyers’ patches and tonsils, and no plasma cells in tissues.
antibodies. The common infec-
tions that occur in this disease are However, the number and function of T cells in patients are normal. For the first 6–10 months
sinusitis, conjunctivitis, bronchitis, of life, infants are protected from infection by the maternal IgG that has crossed placenta and en-
pneumonia, gastroenteritis and tered foetal circulation. As this maternal defence molecule is exhausted, the affected male infant de-
nasal infections.
velops repeated bacterial infection. The infectious complications are greatly reduced if the patient is
infused intravenously with periodic (weekly or monthly) injections of a large dose of gammaglobulin.
Such antibody preparations contain preformed antibodies that can provide effective protective
immunity against common pathogens.
COMMON VARIABLE IMMUNODEFICIENCY (CVID). This group of heterogenous and variable

diseases is defined by the presence of hypogammaglobulinemia, a decreased number of
» CVID generally manifests after antibody-producing plasma cells, poor response to pyogenic organisms, protozoa (Giardia
20–30 years of age. Individuals with lamblia) or vaccine. Many patients also develop other autoimmune diseases including pernicious
CVID have enlarged lymph nodes in
the neck, chest or abdomen. anaemia, haemolytic anaemia and rheumatoid arthritis. CVID is generally acquired after 20 to
30 years of age and both males and females are equally affected. The genetic basis of the disease
is not known. Both sporadic and familial cases occur. Because of its inheritance mode, CVID
is considered to be a primary immunodeficiency and it shows both autosomal dominant and
recessive inheritance pattern.
Mature B cells are present in CVID but fully differentiated B cells that is, plasma cells are absent
» Plasma cells are absent in CVID in the lymphoid tissue. This suggests that CVID patients have a block at a differentiation pathway
and hence most individuals with that transforms mature B cells to antibody-producing plasma cells. Since the exact cause of this
CVID are prone to recurrent bacte-
rial infections abnormality is not known, a number of possibilities are suggested. These include intrinsic B-cell de-
fect, failure of proper signals from T cells and excessive “suppressor cell” action on mature B cells.
SELECTIVE IMMUNOGLOBULIN ISOTYPE DEFICIENCIES. A number of immunodeficiency

» J. H. Rockey reported the first IgA diseases have been characterized that have significantly lower amounts of a specific immunoglobulin
deficiency (IgA) in 1964.
isotype. The most common is selective IgA deficiency that affects 1 in every 700 causcasians but
occurs rarely in other ethnic groups.
IgA deficiency disease is sporadic, but familial cases with either an autosomal dominant or a
recessive pattern have ensured it a place in primary immunodeficiency. IgA deficiency is charac-
» IgA protects mucosal surfaces
from infections. Hence a common
terized by very low serum IgA, usually 30–50 μg/ml (the normal being 200 mg/dl) with normal
problem in IgA deficiency is suscep- levels of other isotypes. About 20 per cent of IgA deficiency cases also lack IgG2 and IgG4.
tibility to infections. Recurrent ear IgA deficiency arises because IgA-bearing B cells are unable to undergo normal differentia-
infections, sinusitis, bronchitis and
pneumonia are the most common
tion to the plasma-cell stage [see Figure 19.4(a)]. It is not clear whether the block in B-cell differ-
infections seen in patients with IgA entiation is due to some B-cell defect or an abnormality in T-cell help such as the production of
deficiency. cytokines (TGF-β, IL-5) that enhances IgA secretion, or no B-cell response to these cytokines, or
» The IgG circulating in the blood-
the presence of excessive suppressor T cells. IgA-bearing B cells are present in normal numbers
stream is: 60 per cent–70 per cent but they are defective in their ability to synthesize or release IgA. The clinical manifestation of this
IgG1, 20 per cent–30 per cent IgG2, disease is recurrent sino-pulmonary infection, gastrointestinal disease, autoimmune disease, ma-
5–8 per cent IgG3 and 1–3 per cent
IgG4.
lignancy as well as hypersensitivity.
Other isotype-IgG subclass deficiencies have also been reported. In this more specific IgG sub-
class deficiencies, the deficiency of IgG3 is most common. IgG2 deficiency is associated with IgA
deficiency and is most common in children. In humans, IgG2 subclass is responsible for binding
and neutralizing capsular polysaccharide of pyogenic bacteria. IgG2 deficiency results in recurrent
pyogenic bacterial infection. Selective IgG subclass deficiencies are usually due to abnormal termi-
nal B-cell differentiation and sometimes due to homozygous deletion that occurs in the constant
region genes of IgG.
IgG-secreting plasma IgA-secreting plasma
cells not formed cells not formed
a) Selective immunoglobulin isotype deficiencies
CD40L absent
TH cell
B cell
(Expresses IgM)
No class
switching
B cell
IgA IgE IgG
No IgA, IgE, IgG produced
Plasma cell
IgM
Figure 19.4
Line diagram showing
immunodeficiency caused by defects in
b) X-linked hyper IgM syndrome B-cell maturation.
X-LINKED HYPER IGM SYNDROME. X-linked hyper IgM (X-HIGM) syndrome is associated
with defective isotype switching of B cells to the IgG, IgE and IgA isotype. These antibodies are
therefore absent and there is a compensatory increase in IgM (to about 10 mg/ml from a normal of
1–2 mg/ml) in blood. It is an X-linked recessive disease that affects both men and women, although
acquired X-HIGM cases are also reported. The affected individuals have a large number of IgM-
secreting plasma cells in their peripheral blood and lymphoid tissues. The GI tract in particular
is infiltrated with IgM-producing cells. Patients tend to form IgM autoantibodies to neutrophils,
» In hyper IgM there is no class platelets and red blood cells, thereby adding the complexity of another disease—autoimmune
switching. disease to immunodeficiency. They are susceptible to pyogenic infections, particularly respiratory
pathogens such as Pneumocystis carinii.
In X-HIGM, B cells cannot make the switch from IgM to IgG, IgA or IgE that normally occurs
in B-cell maturation. Individuals with X-HIGM have normal B cells expressing IgM or IgD, but
they lack B cells expressing membrane-bound IgG, IgA or IgE. Though the defect is visible in B
cells which do not secrete IgG, IgA or IgE, it is actually instilled by mutated genes located in the X
chromosome of T cells. The defective gene codes for CD40 ligand (CD40L) on TH cell surface [see
» On account of the deficiency in Figure 19.4(b)]. For this reason, some workers place X-HIGM in the mixed-cell disease category
CD40 ligand on T cells, TH cells are that affect both B and T cells.
unable to instruct B cells to switch
their production of antibody from Since an interaction between CD40 on B cells and CD40L on TH cells is required for B-cell ac-
IgM to IgG , IgE or IgA. tivation and isotype switching, the absence of CD40–CD40L interaction leads to an absence of the
stimulus that causes B cells to undergo heavy-chain isotype switching. However, B-cell response
to T-independent antigen, which comprises mainly IgM antibodies, remains unaffected. Since the
formation of memory B cells also requires costimulation by the CD40–CD40L pathway, the ab-
sence of this interaction in X-HIGM leads to a failure to produce memory B cells. In addition,
X-HIGM individuals fail to generate germinal centre formation as T-cell help (via CD40–CD40L
interaction) is missing.
TRANSIENT HYPOGAMMAGLOBULINEMIA OF INFANCY. This immunodeficiency results when

the onset of immunoglobulin synthesis particularly that of IgG, is delayed beyond its normal time.
Infants are normally protected by the mother’s IgG. The maternal IgG is catabolized with a half-life
of approximately a month. Normal infants begin to synthesize their own IgG by the 3rd month and
hence in the 1st and 2nd months most normal babies experience recurrent respiratory infection.
In some infants, however, who experience transient hypogammaglobulinemia of infancy,
there is a delay in the ability to synthesize IgG. The IgG synthesis can delayed by 16–36 months
after which the situation cures itself. Infants with this disorder suffer from recurrent pyogenic
Gram-positive infections of skin, respiratory tract or even meningis of the brain. The B cells of
diseased infants appear to lack help from TH cells in synthesizing antibodies.
DEFECTS IN T-CELL DEVELOPMENT

DIGEORGE SYNDROME. DiGeorge syndrome is named after A. M. DiGeorge who reported it
» A. M. DiGeorge reported T-cell
deficiency in 1965. This syndrome is
for the first time in 1965.
caused by an abnormal develop- This syndrome is a selective T-cell deficiency that occurs due to the congenital defective de-
ment of certain cells and tissues velopment of the thymus and parathyroid gland. In fact, it involves the defective development
of the neck during growth and
differentiation of the foetus. As a
of structure, during foetal life, of the third and fourth pharyngeal pouches from which the thy-
result, the affected children have mus and parathyroid glands are derived. The basis for the developmental of the abnormality is
an upward bowing of their mouth, not known, though there appear to be an autosomal inheritance pattern in some. Thymic aplasia
an underdeveloped chin, eyes that
slant downward, low set ears and
(or hypoplasia) results in cellular immunodeficiency with profoundly impaired T-cell function.
defective upper portions of their Peripheral blood T cells are absent or greatly reduced in numbers. T cells do not respond to poly-
ear lobes. clonal T-cell activators or mixed leukocyte reactions. Antibody levels are usually normal but may
be reduced in severely affected individual.
The DiGeorge syndrome is manifested by deficient T-cell maturation causing recurrent bacte-
rial, viral, fungal and protozoan infections. The absence of the parathyroid gland leads to hypocal-
» Most patients with the DiGeorge
syndrome have a small deletion in
cemic tetany (muscle twitching due to decreased calcium concentration). The facial appearance is
a specific part of the 22nd chromo- abnormal with low-set ears and fish-shaped mouth in affected patients and their eyes are widely
some at position 22q11.2. It is for separated (hypertelorism). The patients also show impairment in delayed hypersensitivity reac-
this reason that this syndrome
is also called 22q11.2 deletion
tions and allograft rejection reactions. The immunodeficiency associated with DiGeorge syndrome
syndrome. can be corrected by transplantation of thymus from foetus or by HLA-identical bone marrow
transplants. Hypocalcemia can be controlled by administration of vitamin D and calcium. Most

patients improve with age even without a thymus transplant and are relatively normal by the age of
5 or 6. It is believed that some ectotopic sites (extra-thymic sites) are involved in T-cell maturation.
X-LINKED LYMPHOPROLIFERATIVE DISEASE. This X-linked disease is due to mutation that

occurs in the gene-coding adaptor molecule involved in the signalling pathway that regulates both
B- and T-cell proliferation. This disorder is characterized by the development of B-cell tumours
and a decrease in the level of gammaglobulin. This disorder imparts an inability to eliminate EBV,
leading to the development of infectious mononucleosis.
BARE LYMPHOCYTE SYNDROME. This autosomal recessive immunodeficiency is a rare disease

in which the expression of class II MHC gene products HLA-DP, HLA-DQ or HLA-DR on antigen-
« Class II MHC molecules are
presenting cells (macrophages, dendritic cells) and B cells is deficient or absent. Moreover, these deficient or absent on antigen-
cells fail to express class II MHC molecules in response to IFN-γ. presenting cells in individuals with
Since, the development of TH cells depends on positive selection by class II MHC molecules in the bare lymphocyte syndrome.
the thymus, class-II-MHC-deficient individuals have a deficiency of CD4+ T cells. The lack of TH
cells leads to a deficiency in antibody production. Affected infants are also deficient in delayed-type
hypersensitivity reactions. This disease which is characterized by recurrent infection, particularly of
the GI tract, appears within the first year of life and is usually fatal if left untreated. Affected individ-
uals do not express class II MHC molecules on antigen-presenting cells and B cells. This is because
of the fact that class II MHC molecules are not transcribed and hence not expressed. Class II MHC
molecules are not transcribed because transcription factor RFX5 or transcription co-activator CII
TA which binds and activates the transcription of class II MHC genes is inactive. This abnormality
results in reduced or nil transcription of class II MHC genes which leads to this syndrome.
DEFECTIVE CLASS I MHC EXPRESSION. This disease is an autosomal class I MHC deficiency
that is characterized by reduced T-cell numbers and function. Since the development of T cells in
the thymus depends on positive selection by class I MHC molecules, individuals lacking class I
MHC molecules lack functional T cells. The absence or reduction of class I MHC molecules occurs
because of mutation in the gene encoding TAP subunits. TAP is required for “normal pumping”
of peptides into the ER where they are used for class I MHC–peptide assembly. The TAP-deficient
individual expresses very few class I MHC molecules as class II MHC–peptide complex is expressed
on the cell surface and not class I MHC–peptide . These patients suffer from recurrent bacterial
infections of the respiratory tract. In spite of the fact that T cells are defective, viral infections in
these patients is surprisingly not common.
COMBINED B-CELL AND T-CELL DISORDERS

Variable degrees of B- and T-cell immunodeficiency diseases occur that are congenital and involve
both B and T cells. One such disease is SCID that has already been discussed. A few others are
discussed below.
WISKOTT–ALDRICH SYNDROME (WAS). Wiskott–Aldrich syndrome is an X-linked immuno-

deficiency disorder. It comprises several cardinal features: (a) thrombocytopenia—the presence
of few and small platelets (in this case lymphocytes also)—a symptom that is present right from
birth and can lead to fatal bleeding; (b) recurrent pyogenic and opportunistic infections starting
from 6 months of age; (c) eczema (skin irritation and rashes) of varying degrees starting at about
1 year of age; (d) normal serum level of IgA and IgE, but slightly increased IgG level and decreased « In 1937, Dr Wiskott described this
amount of IgM; (e) T-cells defective in function, worsening malfunction as the disease progresss; condition in three brothers who
(f) B cells normal in function but seem to be associated with the failure to make an antibody had low platelet counts, eczema
and recurrent ear infections. Seven-
response to polysaccharide antigens. As the disease progresses, symptom of WAS increases in teen years later, in 1954, Dr Aldrich
severity and usually results in fatal infection or lymphoid malignancy. demonstrated that this syndrome
WAS is caused by mutations in the gene which produces a protein, named after the disorder, showed an X-linked recessive in-
heritance and hence this syndrome
the Wiskott–Aldrich syndrome protein (WASP). The WASP gene is located on the short arm of came to be called the Wiskot–
the X chromosome. The function of this protein is not yet understood. Aldrich syndrome.
ATAXIA–TELANGIECTASIA (AT). Ataxia–telangiectasia is an autosomal recessive immunodeficiency

disease. It is characterized by uncoordinated muscle movements (ataxia), dilation of small blood
vessels (telangiectasis) that is readily observed in the eyes and on skin by 6 years of age. The afflicted
children have neurological problems, increased incidence of tumours and immunodeficiency.
Children with AT may sway when they stand or sit, and they wobble or stagger when they walk. This
unsteady posture and gait results from neurological abnormalities that affect the part of the brain
(the cerebellum) that controls balance.
This immunological defect affects both B and T cells. T-cell defect is associated with T-cell de-
» Ataxia means uncoordinated ficiency due to thymic hypoplasia. About 60-70 per cent of AT patients also have humoral defects
muscle movement and telangiec- manifested by IgA and IgG2 deficiency. This is mainly due to chromosomal aberration that occurs
tasia means dilation of small blood in chromosome 11 that encodes a kinase that is structurally related to phosphatidylinositol-
vessels.
3-kinase. The precise function of this kinase is still not clear but it is believed to be involved in
the repair of double-strand breaks in the DNA. One of the clinical features of AT is an increased
susceptibility to infections. Infections most commonly involve the lungs and sinuses, and are
usually caused by bacteria or viruses. The infections are, at least in part, due to the variable immuno-
deficiency seen in AT.
19.2.2 DEFECTS IN MYELOID LINEAGE

Defects in myeloid lineages leads to defects in the innate immune functions (see Figure 19.5). Since
» NADPH oxidase present on the
membrane of phagocytes is innate immunity constitutes the first line of defence against infectious organisms, defects in this de-
defective in CGD. fence system result in recurrent microbial infection of varying severity. These defects are congenital
disorders and hence are placed in the category of primary immunodeficiencies.
C H R O N I C G R A N U L O M AT O U S D I S E A S E ( C G D )
CGD is a rare disease that affects about 1 in 1 million individuals in the USA. It exists in two genetic
forms—about 70–75 per cent show the X-linked pattern of inheritance and the remainder exihibit
the autosomal recessive pattern. CGD is characterized by recurrent infections of various Gram-
negative (for example, Klebsiella, Escherichia, Serratia) and Gram-positive (for example, Staphylo-
coccus) bacteria and fungal infections.
Because the infections cannot be controlled by phagocytes, they stimulate a chronic cell-mediated
immune response that results in the formation of granuloma (made up of activated macrophages) in
many organs. The disease is usually fatal even when treated with antibiotic therapy.The most common
form of CGD is caused by mutation in a component of the phagocyte oxidase—91 kDa membrane
protein of cytochrome b558 located on the X chromosome. Cyt b558 is a part of NADPH oxidase
enzyme that catalyses the one electron reduction of oxygen (O2) to O2- anion by this reaction.
NADPH + 2O2 ⎯→ NADP+ + 2O2- + H+
Patients with CGD have a defective NADPH oxidase, and hence are incapable of forming
superoxide anions (and H2O2) by phagocytes following the ingestion of microbes. Therefore, they
cannot efficiently kill ingested pathogens. As a result, pathogens remain alive in phagocytes which
gives rise to persistent cell-mediated response to intracellular pathogen leading to the formation
» Patients with CGD have normal
of granuloma. Three other types of CGD (autosomal recessive) result from a defect in the 22 kDa
antibody production, normal T-cell chain of cyt b558 or mutation in one or the other protein, called p67phox or p47phox (phox being the
function and a normal complement abbreviation for phagocytic oxidase).
system. In other words, the rest
of their immune system is normal
except that the phagocytes are LEUKOCYTE ADHESION DEFICIENCY-1 (LAD-1)
unable to kill ingested pathogens. It is another rare immunodeficiency disorder. This disease is characterized by recurrent fungal
Hence, recurrent bacterial and viral
infections occur. Pneumonia is infection and infection from Gram-positive and Gram-negative bacteria. The viral immunity is de-
common in patients with CGD. pressed in LAD-1 patients. They also have impaired wound healing. In LAD, adhesion-dependent
» LAD is a group of diseases in functions of leukocytes are abnormal. These include impairment of adhesion to vascular endothe-
which the adhesion-dependent lium, phagocytosis, cytotoxic effect of neutrophils, NK cells and T cells, and neutrophil aggregation
functions of leukocytes are and chemotaxis. The defective leukocyte membrane proteins that are important in the adhesion of
abnormal.
leukocytes to other cells are absent or deficient.
The affected proteins include β2-integrins or CD11–CD18 family of proteins which includes
LFA-1, Mac-1, p150, 95. All these proteins share the same 95 kDa β chain (encoded in gene
gp91 Decreased
O2 O2
- rap 1 phagocytic ability
Figure 19.5
Leukocyte Extracellular Line diagram showing defects in myeloid
lineage. Impaired leukocyte adhesion
α-integrin β-integrin NADPH Cytosol Abnormal
disrupts the ability of phagocytes to bind
missing Oxidase giant lysosmes
the endothelium, resulting in defective
gp67 diapedesis in LAD-1. Mutant gp 91/ gp67
gp47 component of NADPH oxidase is unable
Impaired leukocyte Neutrophil to generate superoxide in CGD. Impaired
adhesion NADPH NADP phagocytic ability of neutrophils
and the presence of giant lysosomes
are hallmarks of Chediak–Hiagshi’s
Leukocyte adhesion Chronic granulomatous Chediak-Higashi syndrome that results in defective killing
deficiency-1(LAD-1) disease (CGD) syndrome of pathogens.
on chromosome 21), which is mutated or absent in defective leukocytes. This β chain is part of
complement receptor 3 (CR3), which binds to complement fragment C3b bound on opsonized
microbes, and is critical for ingestion of bacteria by phagocytes. The β chain is also part of LFA-1
that interacts with intercellular adhesion molecule-1 (ICAM-1) present on the endothelial cell
surface and other cell membranes. Because of these defects in CR3 and LFA-1, leukocytes show
defective phagocytosis and decreased adhesion to vascular endothelium which results in impair-
ment in resolution of infection.
CHEDIAK–HIGASHI SYNDROME
Chediak–Higashi syndrome is another rare autosomal recessive disorder. It is characterized by
recurrent infection by pyogenic bacteria, partial oculo-cutaneous albinism (lack of skin and eye
« Chediak–Higashi syndrome (CHS)
pigment), aggressive but non-neoplastic infiltration of various organs by lymphocytes. The gene is a rare, inherited, complex,
responsible for this disorder has been mapped to chromosome 1 that codes for a cytosolic protein immune disorder of childhood,
(LYST) which is involved in the regulation of intracellular trafficking. (usually) characterized by abnor-
mally pale skin and eyes.
Though the mechanism is still not clear, defects in this protein lead to abnormal granule
membrane fusion (which usually results in the formation of gaint lysosomes) in neutrophils and
macrophages, as well as in other cells of the body. Increased granule fusion decreases phagocytic
capabilities of macrophages and neutrophils so that they cannot kill ingested bacteria. It also
affects melanocytes (leading to albinism), neurons (causing neurological problems) and platelets
(leading to bleeding disorder). Defects in lysosomes of dendritic cells and macrophages leads to
impairment of antigen processing and presentation. Figure 19.5 shows the defects in Chediak–
Higashi syndrome, LAD-1 and CGD.
NEUTROPENIA OR GRANULOCYTOPENIA
Neutropenia is a decrease in the total number of circulating neutrophils. This decrease in neutro-
phil number (below 1500/mm3) could be due to decreased production of neutrophils or increased
destruction of neutrophils.
The decreased production of neutrophils arises because of several conditions, including leu-
kaemia and administration of bone marrow depressant drugs such as nitrogen mustard, as well as
genetic defects in the development of all bone marrow stem cells (for example, reticular dysgene-
sis) that affects myeloid progenitor cells. This genetic defect which is called congenital neutropenia
results from reduced production of neutrophils during haematopoeisis. Another genetic disease,
agranulocytosis, results from an almost complete absence of neutrophils from the circulation. In
agranulocytosis, myeloid stem cells are present in the bone marrow but rarely differentiate beyond
the promyelocyte stage. This is probably due to the decreased production of the granulocyte colony
stimulating factor (G-CSF) which results in the failure of myeloid stem cells to differentiate along
the granulocytic lineage.
An increased destruction of neutrophils can be caused by autoimmune diseases such as Sjo-
gren’s syndrome or SLE or by autoimmune reactions that occur following the administration of
certain drugs such as oxacillin and quinidine. Hypersplenism, which results in aggravation of de-
structive function of the spleen, also results in neutropenia.
19.3 DEFECTS IN THE COMPLEMENT

SYSTEM
Deficiencies of complement components or functions have been associated with increased suscep-
tibility to bacterial infection and a variety of other diseases. C1 esterase inhibitor (C1Inh) deficiency
causes transient but recurrent localized oedema, affecting skin, GI tract and respiratory tract.
Clq deficiency is associated with SCID, hypogammaglobulinemia and repeated bacterial infections.
C2 and C4 deficiencies cause the failure of complement-dependent mechanism and symptoms
» The first case of complement similar to SCID. C3 deficiency leads to life-threatening infections by Neisseria meningitidis and S.
deficiency was described by A. M. pneumoniae. Since there is no generation of C3b, phagocytosis is impaired and there is no production
Silverstein in 1960. of the chemotactic C5a fragment. C5 deficiency leads to increased bacterial infection due to im-
paired chemotaxis.
C6, C7 and C8 deficiencies can lead to increased susceptibility to meningococcal and gono-
coccal infection as complement-mediated lysis is a major control mechanism in these infections.
19.4 T R E AT M E N T A P P R O A C H E S F O R
IMMUNODEFICIENCY
In theory, the ideal way to cure an immunodeficiency is to supply or replace the defective gene or
protein or enzyme or cell lineage with a new self-renewing set.
The replacement of a single defective gene as in ADA-SCID or chronic granulomatous disease
with defective p67Phox has been successfully attempted. Clinical trials show disease remission up to
18 months in SCID and 7 months in CGD individuals. In both of these procedures, self-renewing
stem cells are isolated from patients and transfected with a normal copy of the defective gene.
These transfected stem cells are then reinfused into the patient where they multiply and produce
corrected stem cells.
Passive immunization with pooled gammaglobulin is the classic course for agammaglobulin-
aemic patients. Pooled human gammaglobulin is administered intravenously or subcutaneously to
successfully protect the patient with X-linked agammaglobulinemia. Antibodies against the par-
ticular pathogen with which the person is repeatedly infected is given and maintained at a higher-
than-normal (5 mg/ml) level in the serum. Bone marrow transplantation is the choice of treatment
for LAD, bare lymphocyte syndrome and SCID with ADA deficiency. It is successful only after
complete HLA-matching and T-cell depletion in the host, to avoid graft rejection by the host. En-
zyme replacement therapy is being attempted for ADA and PNP SCIDs which provides temporary
success for SCID patients.
Cell-replacement therapy, particularly replacement of self-renewing stem cells from HLA-
identical donor has reported high rates for success. The recent attempts on SCID infants were
successful though it is still not clear how long the cell transplantation will last. Donor T cells are
depleted and stem cells are enriched and then introduced into the recipient. These transplantations
are successful only when the haplotype, that is, HLA gene sets, are identical.
19.5 ANIMAL MODELS OF PRIMARY

IMMUNODEFICIENCY
Immunologists use several well-established animal models of primary immunodeficiency for
various experimental purposes. These include nude (athymic) mouse, SCID mouse, beige mouse,
CBA/N mouse.
» Nude mice lack the thymus and 19.5.1 NUDE MOUSE

are hairless, from which they derive Nude mouse exhibits a genetic trait which is designated nu. Mice homozygous for this trait nu/nu
their name.
are hairless and lack the thymus.
Heterozygous nu/+ have normal hair and normal thymus. It is thus clear that the nu gene is
recessive. This gene is found on chromosome 11. It is believed that the nu gene codes for the
transcription factor that is required for the normal development of epidermal cells (defective gene
make them hairless), and third and fourth pharyngeal pouches from which the thymus is derived
(hence when the gene is defective, there is no development of the thymus).
The nude (nu/nu) mice lack cell-mediated response and antibody-making capacity. They can-
not survive more than 25 weeks and half of them usually die within the first two weeks after birth.
Because of this complete immunodeficiency, nude mice are ideal for studies on allografts and
xenograft as these grafts are easily tolerated. They are also useful in studying tumour immunology
as solid tumour from any origin may be grown as ascites or as implanted tumours.
19.5.2 SCID MOUSE

A SCID mouse has virtually no B and T cells because of early blocks in differentiation of B and T
lymphocytes. Cells other than lymphocytes, such as red blood cells, monocytes and granulocytes
are normal and functional. This SCID trait in mouse is due to an autosomal recessive mutation
in its genome, because of which a component of protein kinase which is required for DNA break
repair is mutated. This defect results in the abnormal joining of Ig and T-cell receptor gene seg-
ments during lymphocyte development and hence the failure to express B-cell and T-cell receptors.
SCID mice can neither make antibody nor elicit delayed-type hypersensitivity nor graft rejection
response. Unless these immunodeficient animals are kept in an extremely clean environment, they
succumb to infections early in life.
SCID mice are extremely useful for studying cellular immunology. SCID mice lack their own
immune system. In these SCID models, the human immune precursor cell may be used to establish
the “human” immune system within a SCID mouse. This mouse could then be infected with differ- « Normal mice do not get HIV-1
ent viruses (for example, HIV) and various therapeutic strategies developed. infection.
19.5.3 CBA/N MOUSE

CBA/N mouse is an inbred mouse strain that has an X-linked defect in B-cell maturation due to a
point mutation in Btk gene. This mouse is used as the animal model for X-linked agammaglobu-
linemia. The problem however is that this mice has less severe defect than its human counterparts.
Though it shows a defective antibody response as in humans, unlike humans the number of B cells
are near normal.
19.5.4 BEIGE MOUSE

This mouse is an animal model for the Chediak–Higashi syndrome. It is characterized by defective
NK-cell function and giant lysosomes in the leukocytes. The mutation in beige mouse is located on « Beige mouse is an animal model
for the Chediak–Higashi syndrome.
the same gene as its homologue in the human Chediak–Higashi syndrome.
19.6 SECONDARY IMMUNODEFICIENCY

AND AIDS
As mentioned previously, secondary immunodeficiency diseases arise as the result of defects in the
immune system acquired during the lifetime of an individual. The acquired immunodeficiency could
be induced by steroid treatment (which induces lymphocytopaenia and monocytopaenia), treatment
with immunomodulatory drugs such as cyclophosphamide and chlorambudil (which decreases
lymphocyte numbers), treatments with immunosuppressive drugs to inhibit transplantation rejection
(such as cyclosporin A, rapamycin, FK506) or be acquired through viral infection (AIDS).
19.6.1 THE AIDS EPIDEMIC

In late 1970s and early 1980s, several patients in the USA and Europe sought advice and treatment
of symptoms of immunological dysfunction. These patients displayed generalized lymphodenopa-
thy, opportunistic infections (typically Pneumocystis carinii pneumonia, cryptococcal meningitis,
cytomegalovirus-associated retinitis) and several unusual cancers (such as Kaposi sarcoma). « AIDS was first called a gay-
related immune deficiency.
They also had marked depletion of TH lymphocytes. The medical community of the Center for
Disease Control and Prevention noticed this disease on 18 June 1981, after the Center for Disease « Gaeten Dugas is suspected to be
Control reported about five California men with severe immunodeficiency in their Morbidity and the first carrier of the AIDS virus.
Mortality Weekly Report.
This report was followed by several other reports describing homosexual men and intrave-
nous drug users with impaired immune systems exhibiting similar immunodeficiency diseases. It
was also reported from other groups including haemophiliacs, blood transfusion recipients and
promiscuous heterosexual individuals of either sex and their partners, and infants born to such an
immunodeficient mother. This acquired immunodeficiency syndrome or AIDS as the disease was
soon called, is caused by a new retrovirus with genetic characteristic of the lentivirus genus.This
virus was named human immunodeficiency virus or HIV (and was later named HIV-1). This virus
was associated with AIDS in the US, Europe and Central Africa. Another retrovirus that is related
to HIV-1 but is less pathogenic and immunologically distinct is HIV-2, which also causes AIDS.
» HIV is a member of the lentivirus HIV infects a variety of cells of the immune system including CD4-expressing TH cells, den-
genus of Retroviridae family. It is a
spherical virus with a diameter of dritic cells and macrophages. HIV causes profound immunosuppression with associated opportu-
110 nm. nistic infection, wasting, central nervous system degeneration and a variety of malignancies.
19.6.2 THE HIV VIRUS
HIV is a member of lentivirus family of animal retroviruses. Two closely related types of HIV have
been identified. HIV-1 is by far the most common cause of AIDS but HIV-2 which differs in ge-
nomic and immunological characteristics causes a similar syndrome.
HIV contains two identical strands of a single-stranded RNA genome (of positive polarity)
» The HIV envelope is studded with
about 72 knob-like proteins. Each
each approximately 9.2 kb long, packed within a core of viral proteins and surrounded by a lipid
knob is made up of two proteins bilayer envelope (see Figure 19.6). The virus phospholipid bilayer envelope is derived from the host
gp120 and gp41. HIV genome cell membrane but includes virally encoded membrane proteins.
resembles eukaryotic cellular mRNA
having both the 5’ cap and the THE GENETIC COMPOSITION OF HIV
poly-A tail.
HIV genome has the basic arrangement of nucleic acid sequences characteristic of retrovirus. Long
» A large variation in the sequence
terminal repeats (LTR) at each end of the genome regulate viral integration into the host genome,
of the env gene is responsible for viral replication and viral gene expression. The gag sequence encodes a 53 kDa protein that is the
classifying HIV into nine subtypes precursor of core structural proteins (p24 and p17). The env sequences encode 160 kDa precursor of
(from HIV-1A to HIV-1I).
envelope proteins gp41 and gp120, which is required for the fusion of virus with the cell.
» Vif protein removes those blocks The pol sequence encodes
that hinder viral replication inside Lipid bilayer reverse transcriptase, integrase
the host cell. Reverse
transcriptase gp 41 Envelope and viral protease enzymes
gp120 proteins needed for viral replication. In
addition to these typical ret-
p17 gag matrix protein
roviral genes, HIV-1 includes
Figure 19.6 p24 gag capsid number of genes such as vif,
ssRNA protein
Schematic diagram showing the vpr, tat, rev, nef and vpu. The
structure of HIV. HIV-1 is an enveloped
tat gene encodes a protein that
retrovirus. It has two ssRNA genomes p9 p7
associated with enzymes, including attaches to the viral RNA and
Host
reverse transcriptase, protease and
protein enhances transcription. Genes
integrase. The phospholipids bilayer of p32 integrase
the envelope contains 72 external spikes,
found in HIV-1 genome and
including two major envelope proteins their associated functions are
gp120 and gp41. p9 and p7 are structural p10 protease given in Figure 19.7.
proteins.
Enhances
transcription
Transcription
activator
Promotes
pol nuclear
LTR vpr tat tat LTR
export of
5’ gag 3’
vif RNA
rev rev
Binding site Reverse
env nef
for transcriptase,
Core, vpu
transcription protease,
Figure 19.7 matrix Virion Promotes
factor NFκB endonuclease
Schematic diagram showing structure proteins morphogenesis viral replication,
SP1
of the genome of HIV-1. The genome is (p24, p17, p7, p9)
a single-stranded RNA. However, several
and budding Envelope downregulates
genes overlap extensively. The functions glycoproteins class I MHC
of the gene products are shown. (gp120,gp41)
Gene/DNA Sequence Functions of Gene/Gene Products

LTR Viral integration into host genome; binds transcription factors, NFκ B,SP1
gag Precursor of nucleocapsid and matrix proteins
env Precursor of envelope proteins, required for fusion of virus to the cell
pol Encodes reverse transcriptase, integrase and viral protease, needed for viral
replication
vif Encodes protein that allows virus to survive host cell’s stress
vpr Encodes protein that is a weak transcription activator of viral DNA
vpu Encodes protein that promotes assembly and budding of virus, down/
regulates host CD4 molecules
tat Encodes protein that is a transcription activator of viral DNA
rev Encodes protein involved in nuclear export of incompletely spliced or
unspliced viral mRNAs.
nef Encodes protein that promotes viral replication, downregulates host cell
Table 19.2
CD4 expression
Functions of HIV-1 genes
The rev gene codes for regulatory protein that changes the way by which viral RNA transcripts
are processed, a day after infection. vif, an accessory protein, is needed by HIV-1 under certain
“stressful” conditions imposed on the virus by its cellular home. vpu, vpr products regulate viral
assembly and budding. The functions of HIV genes are briefly summarized in Table 19.2.
CD4 antigen is the receptor for the virus. This antigen is present on CD4+ TH lymphocytes and
cells of monocyte/macrophage lineage. Viral gp120 binds to CD4 but chemokine co-receptor is also
involved in viral gp41-mediated fusion and internalization of the virus.
THE LIFE C YCLE OF HIV

The viral particles that initiate infection are usually found in blood, semen and other body fluids of
« It is believed that apart from TH
the individual and are introduced into another individual by sexual contact or needle. HIV’s repli-
cells which have CD4, dendritic
cative cycle begins with adsorption of virus particles to CD4 molecules on the surface of susceptible cells also express CD4 molecules,
cells, followed by fusion of the virus envelope with the membrane of the target cell. the receptor for HIV, and thus are
The first step in the process is the binding of the gp120 protein of the virus to a CD4 molecule potentially capable of binding HIV,
and/or being infected by it.
of the target cell. This binding induces a conformational change that promotes the binding of gp120
to a co-receptor, a seven-membrane spanning CC or CXC family of chemokine receptor (specifi- « IThere are only nine genes in the
cally CDR5 and CXCR4).This co-receptor binding induces conformational change in another viral HIV RNA.
envelope protein gp41.
The conformational change exposes the hydrophobic region called fusion peptide that inserts
into the target cell membrane and induces viral fusion. This is a direct fusion of virus with the cell
and is not receptor-mediated endocytosis. Following entry, subviral particles become active and
begin the viral reproductive cycle. The nucleoprotein core of the subvirus is uncoated and the RNA
genome of the virus is transcribed into the DNA-stranded RNA form by viral reverse transcriptase.
The double-stranded viral DNA combines with a set of unknown proteins to form a nucleoprotein–
preintegration complex that is “actively” transported into the nucleus. Viral integrase also enters
the cell and catalyses the integration of viral DNA into the host cell genome. The integrated HIV
DNA, now called provirus may remain transcriptionally inactive for months or years with no or
little production of viral proteins, making the HIV infection latent.
The transcription of integrated proviral DNA is regulated intrinsically by the LTR (long ter-
minal repeats) upstream of viral structural genes, as well as by extrinsic factors such as cytokine
or other physiological stimuli to T cells, or other virus-harbouring cells. LTR contains polyad-
enylation signal sequences, TATA box, as well as the binding sites for two host cell transcrip-
tion factors NFκB and SP1. The initiation of gene transcription is linked to a variable factor that
acts on T cells or macrophages, and monocytes in which the virus resides. IL-2, TNF, lympho-
toxin and TCR-binding lectins all stimulate HIV gene expression in T cells. IL-1, IL-3, IL-6,
IFN-γ, TNF and lymphotoxins are stimulators of HIV gene expression and viral replication in
infected monocytes and macrophages. A high level of viral RNA is produced by the coordinated
action of HIV-encoded Tat protein and cellular NFκB and Spl proteins that activate the host RNA
pol II apparatus. Tat protein is required for HIV gene expression and acts by increasing the com-
pletion of mRNA transcription. Mammalian RNA pol II is very inefficient in transcribing HIV
genes and usually stops before the mRNA is completely transcribed. Tat proteins bind to the na-
scent mRNA (and not viral “DNA”) and allows the transcription of viral genes to be completed by
increasing the processing of RNA polymerase.
HIV gene expression may be divided into early stage and late stage. The early stage of HIV gene
expression is when regulatory genes such as rev, tat and nef are expressed, soon after infection. The
late stage includes the expression of env, gag and pol which encode structural components of the
virus. mRNA encoding the various HIV proteins are all derived from a single full-genome-length
transcript by differential splicing events. These spliced (or partially spliced) transcripts are then
exported to the cytoplasm by virus encoded shuttle, rev proteins. Subsequently, gp160 synthesis (coded
by env gene) occurs in the ER whereas gag/pol proteins are synthesized on cytosolic ribosomes.
A 160 kDa glycoprotein, gp160, is cleaved by cellular protease into gp120 and gp41 proteins. gag
gene proteins encode 55 kDa protein that is cleaved into p24, p17 and p15 polypeptides by the action
of the viral protease encoded by pol gene. Pol gene product is a precursor protein that is systemati-
cally cleaved to form reverse transcriptase, ribonuclease, protease and integrase enzymes.
After the synthesis of various viral proteins in the cytoplasm and cellular ER, viral particles
then begin packaging the full-length RNA transcript of the pro-viral genome within a nucleocapsid
that includes gag core proteins and pol-encoded enzymes needed for the next infectious cycle. The
nucleocapsid is then enclosed within a membrane envelope (that integrates viral proteins also) and
released from the cell by a process of budding from the membrane. The production and release of
new viruses is associated with lysis of the cell which is an important mechanism of the cytopathic
» HIV changes the surface antigen
even in the same individual at effect of HIV. A simplified representation of the HIV life cycle is shown in Figure 19.8.
different times.
19.6.3 HIV’S MECHANISM OF IMMUNOSUPPRESSION
HIV infection is combated both by humoral and cell-mediated immunity. Nonetheless it is clear
that these immune responses fail to eradicate all the viruses and in most cases infection eventually
overwhelms the immune system.
HIV
CD4
Chemokine CD4+ T cell

receptor
Viral RNA Reverse transcriptase
ssDNA copy synthesized

by reverse transcriptase
dsDNA formed Viral RNA degraded
Host Nucleus
Integration
genome
of viral
genome in HIV provirus
host DNA IL,TNF,activation
HIV-RNA
of HIV provises
transcript
Nucleocapsid
Cell activation RNA
HIV-RNA
Assembly
Viral RNA translated,
Figure 19.8 yielding viral enzymes
Viral budding of
Line diagram showing the life cycle and structural proteins HIV
of HIV. infectious virions
The initial adaptive response is similar to the immune response to other viruses. There is a
massive clonal expansion of CD8+ Tcyt cells specific for peptides derived from HIV proteins. More
than 10 per cent of the circulating Tcyt cells may be specific for HIV during the early stages of infec-
tion. Humoral response to HIV antigen such as gp 120, gp41 as well as reverse transcriptase, pol, gag
and p24 products have minimal effect in limiting this disease. HIV infection ultimately results in
a major impairment of the innate and adaptive arms of the immune system. This is due to various
direct cytopathic effects of HIV on infected CD4+ cells. These include the following:
• There is direct lysis of CD4+ TH cells by the virus. Recent studies have reported that HIV
preferentially infects memory CD4+ TH cells, which explains the collapse of anti-HIV
immune response and the consequent loss of immunological control of HIV replication.
• The expression of viral protein gp41 in the plasma membrane of the host cells results in an
influx of a large amount of Ca2+ into the cell with resultant osmotic lysis of the cell. It inflicts
a major CD4+ TH-cell loss.
• HIV-infected T cells fuse with uninfected TH cells. This fusion, mediated by the gp120–
CD4 interaction, leads to the formation of giant multinucleated cells or syncytia which
ultimately die within 48 hours.
• A toxic effect is induced by the large amount of viral DNA as several viruses fuse in a single
cell. Non-functional viral RNA may also be toxic to infected cells.
• Viral replication can interfere with normal cellular activities of TH cells, leading to cell
death: for example tat protein can interact with regulatory protein such as p300, coactivator
the of transcription involved in cytokine synthesis.
Ca2+
Ca2+ Ca2+
Ca2+
Ca2+ gp41
Infected
gp120
target cell
Direct lysis of Osmotic lysis
CD4+ T cell of CD4+ T cell
gp120
CD4
Infected Uninfected
CD4+ T cell CD4+ T cell
Formation of dysfunctional gaint cell
HIV virion ADCC-mediated

apoptosis
gp120
Viral Viral
RNA DNA CD4+ T cell
Death of CD4+ T cell due ADCC of HIV-infected cells Figure 19.9

Mechanisms of immunosupression used
to DNA/RNA load by HIV.
• The depletion of CD4+ TH cells displaying viral antigens can occur because of Tcyt-
cell-mediated cell lysis.
• The antibodies bound to gp120 displayed on an infected cell can trigger apoptosis via antibody-
dependent cell-mediated cytotoxicity (ADCC).
Apart from these, other postulated mechanisms that could account for loss of CD4+ TH cells may
involve loss of CD4+ stem cell population and induction and secretion of soluble factors with
cytotoxic effects on CD4+ cells. Some important mechanisms used by HIV to suppress the immune
system are shown in Figure 19.9.
19.6.4 M E C H A N I S M O F E VA S I O N U S E D BY H I V
The failure of the host immune response to control or eradicate HIV infections is probably due to
several factors. CD4+ TH cells that are required to initiate or promote cell-mediated and humoral
immunity are massively depleted by the virus. The immune system becomes too compromised to
eliminate the virus. The extremely high mutation rate because of the error-prone reverse transcrip-
» It is estimated that every possible tion of the viral genome generates a large number of antigenically variable components of the virus.
point of mutation in the viral
genome occurs everyday.
The V3 loop of protein gp20 varies in its antigen determinant composition and structure even if it
is taken from the same individual at a different time. Such a rapid change of antigen on the HIV
system tends to overwhelm the host immune system.
HIV-encoded protein Nef can downregulate class I MHC molecule expression particularly of
HLA-A and HLA-B. Thus, HIV infected cells may evade Tcyt-cell-response as Tcyt response is class-I-
MHC-restricted. The mechanisms of evasion used by HIV are given in Figure 19.10.
19.6.5 THE COURSE OF HIV INFECTION AND AIDS

The diagnosis of AIDS includes evidence for infection with HIV-1 (either the virus itself or its anti-
bodies), greatly diminished CD4+ TH cells (less than 200 cells/mm3), impaired delayed-type hyper-
sensitivity reaction and the occurrence of opportunistic infection by the fungus Candida albicans.
Patients usually succumb to multiple diseases that include pneumonia, tuberculosis, diarrhoea and
various malignancies.
Multinucleate syncytia
formation
Cell lysis due to CD4+ T cell
Accumulation viral budding
of RNA/DNA
load Nef
ADCC-induced (Downregulates
cell lysis class 1 MHC
expression)
Decreased class
I MHC expression
Tcyt-induced Diminished
lysis (a) (b) Tcyt response
Antibodies Antibodies
formed formed
against against
original variant
gp120 1 gp120
Previous Ineffective
antibody antibodies
ineffective
Figure 19.10
Line diagram explaining the various Virus mutates Virus mutates
mechanisms of evasion used by HIV.
gp120 again
(a) Depletion of CD4+ T-cell; (b) Down
regulation of class I MHC (c) Variation of HIV Variant 1 HIV Variant 2 HIV
envelope gp120. (c)
The course of HIV infection can be followed Acute Clinical

by measuring the amount of virus in the patient’s HIV latency Symptomatic
plasma and by the TH cell count. Within days of syndrome period AIDS
the first exposure of HIV, abundant viral repli-
cation is detected in the lymph nodes followed 1000 Death
by viraemia, during which large a number of
CD4+ T cells/mm3
« The HIV infection is termed as
HIV particles are present in the patient’s blood. Opportunistic AIDS when the TH cell count falls
This is accompanied by a variety of non-specific CD4+
diseases below 200 cells/mm3.
T cell
signs of viral disease such as fever, lymphade- 500
Severe
nopathy, rashes and sore throat with pharyngi- Fever,
Figure 19.11
immuno- Clinical course of HIV infection. HIV
tis, but symptoms do not persist for more than lymphoadenopathy, suppression infection causes progressive loss of CD4+
a few weeks. After the initial acute infection, a rashes T cells that results in a gradual decrease
second “chronic” phase of disease develops dur- of immunity. The fall of CD4+ T cells
0 to below 200 cells/mm3 increases the
ing which lymph nodes and spleen see viral rep- 0 6 12 5 6 7 8 9 10 11 12
susceptibility to opportunistic infections,
lications and tissues destruction. In this phase Weeks Years leading leads to full-blown AIDS.
there are few or no clinical manifestation of HIV
infection. This phase which is called clinical la-
tency phase shows only low levels of virus and the majority of peripheral blood T cells do not
harbour viruses. However, the destruction of CD4+ TH cells within the lymphoid tissues steadily
progresses during the latent period and the number of circulating blood TH cells
steadily declines. This is because more than 90 per cent of the body’s T cells are found
in lymphoid tissues and the destruction of these cells occurs when individuals are
asymptomatic. The last and final
phase of HIV infection is called AIDS Exposure to HIV-
when the destruction of the periph- containing fluids
eral lymphoid tissues is essentially
complete and the TH cell count falls
below 200 cells/mm3. The clinical
course of HIV infection is shown in
Figure 19.11.
AIDS patients suffer from opportu- Infection—viraemia
nistic infections, malignancies, cachex-
ia (AIDS wasting syndrome), kidney
failure (AIDS nephropathy) and CNS
degeneration (AIDS encephalopathy) Weeks or Months
that ultimately ends in death. Figure
19.12 shows how HIV infection slowly
evolves into AIDS. Table 19.3 shows
three important phases of HIV infec-
tion, together with associated symptoms Sore throat,Fever, Prodromal symptoms
and number of TH cells in each phase. Lymphoadenopathy,
Rashes,Night sweats (Non-specific symptoms)
19.6.6 T R E AT M E N T A N D
PREVENTION
OF AIDS Years
So far, the treatment of AIDS has proved
very difficult. Although now the course
of the infection is clearly understood, Opportunistic infection,lymphoma
there is still much to be accomplished of brain and lymphatic tissue,
Kaposi sarcoma, AIDS
as far as treatment and therapy are wasting syndrome, CNS
concerned. A two-pronged approach degeneration
is being worked on by scientists—new
effective drugs to treat AIDS and an
Months or Years
efficient vaccine that imparts immunity
Figure 19.12
to AIDS. Line diagram showing how HIV infection
Death
is established and causes AIDS.
Phase of Disease Clinical Course

Acute infection Some patients are asymptomatic, others show fever,
sore throat, general malaise, lymphoadenopathy.
Viraemia exhibited. Persists for few weeks. ( TH cells
more than 500 cells/mm3 ).
Clinical latency phase A relatively asymptomatic period, low level of viral
replication, but there is gradual destruction and
decline of TH cells. Lymph nodes are the predominant
location of HIV-infected cells ( TH cells between
200-499 cells/mm3).
AIDS TH cells count falls below 200 cells/mm3. Patients
suffer from opportunistic infection, such as candidiasis,
cryptococcosis, pneumocystis, mycobacteriosis,
toxoplasmosis. Other AIDS-related diseases include
cytomegalovirus infection, retinitis, Kaposi sarcoma,
Burkitt lymphoma, large B-cell lymphoma, CNS
lymphoma, HIV encephalopathy, multifocal
Table 19.3
The clinical course of HIV infection.
leukoencephalopathy, cachexia (wasting syndrome).
DEVELOPMENT OF EFFECTIVE DRUGS

Theoretically, there are several possible strategies for the development of effective anti-HIV drugs.
An ideal drug should be specific for HIV virus, should interfere minimally with normal cell pro-
cesses and should be cheap. The first success was with drugs that interfere with the reverse tran-
scription of viral RNA to cDNA. Several drugs are used that interfere with reverse transcription.
» The first group of drugs available These drugs, which are nucleotide analogues, include 3-azido-3'-deoxythymidine (AZT), 2'3'-
to treat HIV was the nucleoside/ dideoxyinosine (ddI) and 2', 3'dideoxycytidine (ddC). These drugs are effective in reducing plasma
nucleotide reverse transcriptase
inhibitors. It was introduced HIV RNA levels for several months or years. However they do not halt the progression of HIV-
in 1987. induced diseases mainly because of the evolution of viruses with mutated forms of reverse
transcriptase that are resistant to these drugs.
» The second group of antiretroviral More recently, drugs have been introduced which block the stage in which precursor proteins are
drugs that was approved in 1997 cleaved into units needed for making new viruses. This step which requires the conversion of precursor
was the non-nucleoside reverse
transcriptase inhibitors. These
proteins into native units, requires a specific viral protease which can be inhibited by a chemical agent.
drugs stop HIV from replicat- However, when these viral protease inhibitors are used, mutant viruses rapidly emerge. How-
ing within cells by inhibiting the ever, these protease inhibitors are now being used as part of a new triple-drug therapy commonly
reverse transcriptase protein.
called HAART (highly active antiretroviral therapy).
In most cases HAART uses two nucleoside analogues and one protease inhibitor. This strategy
» The third type of antiretroviral
drug comprised the protease
appears to overcome the ability of the virus to rapidly produce mutants that are drug-resistant.
inhibitors. In 1995, the first protease HAART has proved to be remarkably effective in reducing the viral load to almost undetectable
inhibitor was approved for HIV levels in most treated patients for up to three years. Whether resistance to this therapy will develop
treatment.
with its usage for longer periods is not yet known.
» The fourth group of antiretroviral

D E V E LO PM E N T O F A N E F F E C T I V E VACC I N E
drugs comprises entry inhibitors
which prevent HIV from entering The traditional approaches to AIDS treatment have involved immunization with either a killed virus
human cells. One such commonly or attenuated virus or recombinant HIV proteins using an immune response booster agent-adjuvant.
used entry inhibitor is called T-20. These, it was hoped, would stimulate the immune response that can block HIV infection of cells. Unfor-
A new type of entry inhibitor,
maraviroc (introduced in 2007) tunately, it was found that killed HIV does not retain antigenicity; recombinant HIV proteins provided
blocks the CCR5 co-receptor on hu- brief, if any, humoral immunity and the use of attenuated virus was discouraged because of a high muta-
man cells, preventing the AIDS virus tion rate; there was always a possibility that the virus could become virulent, an unacceptable result.
from attaching to the cell surface of
the host. An effective vaccine will have to stimulate both humoral and cell-mediated responses to the
viral antigen. Although an anti-HIV vaccine has yet to successfully prevent infection, several vac-
» An example of HAART: Two cines are currently evaluated as adjuncts to anti-retroviral therapy:
nucleoside/nucleotide reverse
transcriptase inhibitors zidovudine • Non-virulent hybrid viruses composed of a part HIV and a part SIV sequence or a virus that
and lamivudine are combined has been attenuated by deletion of part genome such as nef gene, are being tried. Such hybrids
with the non-nucleoside reverse
transcriptase inhibitor efavirenz or a stimulate a strong Tcyt response. One concern about such attenuated vaccines is their potential
protease inhibitor. to revert to the wild type after combining with the live virus present in the host system.
• Another approach is to use a non-viral vector such as canary pox vaccine expressing several
HIV-1 antigens, such as gp120. This recombinant vaccine elicits a strong Tcyt response to HIV
antigens and is presently undergoing phase III human trial.
• DNA vaccines containing one or more HIV genes or HIV peptides fragments of the env
protein are also undergoing preliminary trials.
• A cocktail of HIV peptides (or protein fragments) is also undergoing human trial with
modest success.
• Another canary pox vaccine, expressing antigens such as gp120 combined with lipopeptides
and IL-2, has given encouraging results.
Despite the prevailing uncertainty about the efficacy of an anti-HIV vaccine for prevention of
infection by the virus, the perseverance and determined effort of 140,000 scientists across the globe
is bound to pay off.
Radioimmunoassay
Ag/Radioactive labelled free Ag

Radioactive labelled bound
Radiolabelled antigen No free labelled
Mix and incubate antigen in
supernatant
Specific antibody Antigen-antibody

precipitate
Standard or blank Non-radioactive Ag

Standard plot
Labelled antigen Low concentration

Unlabelled antigen of labelled antigen Higher

in supernatant concentration
of labelled
antigen in the
Specific antibody supernatant
Low concentration of unlabelled antigen High concentration of unlabelled antigen
Figure 19.13
Principle of Radioimmunoassay.
Radioimmunoassay (RIA) was developed by Rosalyn Yalow and of radio labelled antigen with its specific antibodies. Unlabelled
Solomon A. Berson in 1960 for estimating insulin concentration (test) antigen, whose concentration is to be determined is then
in plasma. It was the first time that a technique was devised that added to this labelled antigen–antibody mixture, Unlabelled
measured hormone levels in blood by an in vitro method. There are antigen displaces labelled antigen from the antigen–antibody
four basic requirements for RIA—antibody against test antigen to complex. Displaced labelled antigen is then separated from the
be measured, availability of radioactively labelled test antigen, a mixture (by molecular sieving, precipitation by polyethylene glycol,
method which can separate antibody bound with antigen from test ammonium sulphate precipitation). Separated (displaced) labelled
antigen and a standard unlabelled test antigen. This technique in antigen is estimated to give the concentration of bound test antigen
its classical form (classical RIA) involves reacting known quantities (see Figure 19.13).
Radioisotopes (β or γ emitters) are used for labelling compounds to be compounds of various types have been subjected to quantitative
detected by RIA. Radioactive isotopes of Iodine I131 or I125 are commonly analysis by RIA and include the measurement of peptide hormones,
used since they can be easily attached to the tyrosine residues in a pro- non-peptide hormones, prescribed and abused drugs. This immuno-
tein, although isotopes of selenium, cobalt and inidium have also been logical technique has both the advantages and disadvatages of em-
used successfully. Radioactive iodine can be attached to antigen or an- ploying radioactivity. The major advantage includes detection of test
tibody with the help of oxidizing agents such as chloramines T, iodogen antigen with great sensitivity, while the disadvantages stem from the
or enzyme (lactoperoxidase). Radioactivity from these isotopes (β or γ use of radioactive isotopes whose effects are dangerous and cumuli-
emitters) can be counted using automated β or γ counters.
tative, and for which proper precautions need to be taken. However,
In theory, however, RIA maybe used for the measurement of any RIA is still a major tool for estimating plasma levels of almost all the
substance against which antibody can be raised. More than 300 hormones.
S U M M A RY
• The defects or deficiencies of one or more components of the im- • AIDS is caused by a retrovirus with the genetic characteristic of
mune system are termed as immunodeficiency disorders. the lentivirus genus named human immunodeficiency virus (HIV).
• Primary immunodeficiencies may result from genetic defects in • HIV infects a variety of cells, including CD4-expressing T cells,
either innate or adaptive immune systems. dendritic cells and macrophages, causing immunosuppression as-
sociated with opportunistic infection and a variety of malignancies.
• Primary immunodeficiencies may result from the defect or impair-
ment of innate immunity ( Chediak–Higashi syndrome) or adaptive • The viral particles that initiate infections are present in the body
immunity. fluids of the individual and are introduced into another individual
by sexual contact, infected needles or from an HIV positive mother
• Deficiency in adaptive immunity may result from defects in B cells
to the foetus.
(X-linked agammaglobulinemia) or T cells (DiGeorge syndrome).
• Direct cytopathic effects of HIV infection results in the loss of TH
• Disorders that result from the defects in both humoral and cell-
cells which causes a major impairment of the innate or adaptive
mediated immunity are called severe combined immunodeficiencies
arm of the immune system.
(SCID). SCID can be X-linked or autosome-linked.
• The best treatment of AIDS is prevention. At present there is no
• There are a number of animal models of primary immunodeficiency
cure for AIDS. However, AIDS, if it has occurred can be treated
that are used in the studies. These include nude mouse, SCID
with nucleotide analogues that inhibit reverse transcription (AZT,
mouse, beige mouse, CBA/N mouse.
ddI, ddc) or a cocktail of viral protease inhibitors (HAART) which
• Secondary immunodeficiency diseases result from the defects in delays the onset of the disease or its symptoms.
the immune system acquired during the lifetime of the individual.
• Effective anti-HIV vaccines have yet to successfully prevent
• Secondary immunodeficiency could be induced by steroid treatment infection. Through extensive effort and perseverance of scientists
(monocytopenia), treatment with immunomodulatory drugs, or im- across the globe we expect to see soon a light at the end of the
munosuppressive drugs, or acquired by viral infection (AIDS). tunnel.
K E Y W O R D S
• AIDS 405 • bare lymphocyte • Chediak–Higashi • nude Mice 416

• AIDS virus 417 syndrome 413 syndrome 406 • Kaposi sarcoma 417
• AIDS vaccine 423 • beige mouse 417 • CBA/N mouse 416 • SCID 406
• acquired immuno- • chronic granulomatous • HIV 418 • X-linked agammaglobulin-
deficiency 405 disease 416 • isotype deficiencies 407 emia 409
• AZT 424 • common variable • lymphoid cell disorder 406 • X-linked SCID 406
• autosome-linked immunodeficiency 410 • leukocyte adhesion • X-linked hyper IgM 412
SCID 406 • DiGeorge deficiency 414 • Wiskott–Aldrich
• ataxia–telangiectasia 414 syndrome 412 • neutropenia 415 syndrome 413
1. What are the major cytopathic effects of HIV that could account for vaccine? What are some current vaccine strategies that are being
loss of TH cells? evaluated?
2. What are the known primary immunodeficiencies of lymphoid cell 4. What are the differences between chronic granulomatous disease
lineage? How are they different from primary immunodeficiencies and Chediak–Higashi syndrome?
of myeloid cell lineage? Which of the two affects innate immunity? 5. What is X-linked agammaglobulinemia? How is it different from
3. Why does the host immune response not control HIV infection transient hypogammaglobulinemia of infancy?
in the body? What are your suggestions for an effective AIDS

1. Primary immunodeficiency diseases results from: 6. Mutation in phagocyte oxidase is observed in:
(a) Steroid treatment (a) Chronic granulomatous disease
(b) Viral infection (b) Ataxia telangiectasia
(c) Genetic defects (c) Bare lymphocyte syndrome
(d) Treatment with immunosuppressive drugs (d) Chediak–Higashi syndrome
2. Which disease is not manifested in common variable 7. Mouse that lacks the thymus is:
immunodeficiency? (a) Nude mouse
(a) Hyper IgM (b) SCID mouse
(b) Haemolytic anaemia (c) Beige mouse
(c) Rheumatoid arthritis (d) CBA/N mouse
(d) Pyogenic infections
8. Two proteins that play important role in attachment
3. DiGeorge syndrome is associated with the defective of HIV are:
development of: (a) gp120 and p24
(a) Thyroid and parathyroid glands (b) p17 and p24
(b) Thyroid and spleen (c) gp41 and gp120
(c) Thymus and parathyroid gland (d) gp41 and p24
(d) Spleen and thymus
9. Pick the odd one out:
4. In bare lymphocyte syndrome, the following gene product is (a) gp160
not expressed: (b) gp120
(a) Class I MHC (c) Gag protein
(b) Surface antibody (d) gp41
(c) T-cell receptor
(d) Class II MHC 10. Which one of the following is not expressed at early age of HIV
infection?
5. Wiskot–Aldrich syndrome is: (a) rev gene
(a) X-linked immunodeficiency disorder (b) env gene
(b) Autosome recessive disorder (c) tat gene
(c) Autosome dominant disorder (d) nef gene
(d) X-linked and autosomal recessive disorder

1. CBA/N mouse has no B and T cells. 4. MHC molecules are deficient or absent in all nucleated cells in
bare lymphocyte syndrome.
2. Agranulocytosis disease results from the almost complete
absence of neutrophils from circulation. 5. Nude mice lack cell-mediated and humoral response.
3. X-linked hyper IgM is associated with defective switching in
B cells to IgD, IgE, IgA isotypes.
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Autoimmune disease is an immune reaction against self-molecules “Et tu Brutus.”
which evokes pathological consequences due to the involvement of —WILLIAM SHAKESPEARE
(JULIUS CAESAR)
humoral, cell-mediated or complement-mediated immunity. The possi-
bility that an individual’s immune system may react with self-antigens
and induce tissue damage was first pointed out by Alexandre Besredka
at the Pasteur Institute in 1901. This point was epitomized in P. Ehrlich’s
famous diction of horror autoxicus (also in 1901), meaning harmful
(toxic) immune reactions against self. With the discovery of the
first autoimmune disease—paroxysomal cold haemoglobulinuria by
Donath and Landsteiner in 1904, the concept of autoimmunity had
established its first scientific milestone in the speculative environment
of that time. Later, F. F. Krusius experimentally demonstrated that the
“self” lens protein could induce autoimmunity in experimental you should be able to:
animals. This statement corroborated Ehrlich’s concept of horror • Define autoimmune reaction
and autoimmune disease
autoxicus and the science of deconstructing autoimmunity never • Differentiate between
organ-specific and systemic
looked back. Figure 20.1 shows various organs and tissues that are autoimmune diseases
the target sites of autoimmune reactions. • Briefly summarize different
organ-specific autoimmune
diseases
• Explain and illustrate the
mechanism of induction of
single-organ autoimmune
disease
• Describe important systemic
autoimmune diseases such
as SLE, rheumatoid arthritis,
multiple sclerosis, scleroderma
• Give an account of different
animal models used to
understand autoimmune
diseases
• Explain the different
mechanisms for induction of
autoimmunity
• Briefly summarize various
strategies for treating
autoimmune diseases
Autoimmunity and
Autoimmune Diseases 20
20.1 INTRODUCTION
The immune system has the capacity to mount an immune response against virtually all molecules—
foreign as well as self. However, several mechanisms exist within the human system that prevent or
subdue response to self-antigens. The immune system has developed a series of checks and balances
that enable it to distinguish dangerous from harmless signals and allow it to respond to foreign and
not self-antigens. When these mechanisms undergo a breakdown or are overridden, a response
directed against self-antigens can occur, resulting in autoimmune reactions and autoimmune dis-
eases. The consequences of autoimmunity may vary from minimal to catastrophic depending on
the extent to which the integrity of self-tolerance has been affected. Autoimmunity is an important
cause of diseases in humans and is estimated to affect 5–6 per cent of the entire human population.
The autoimmune diseases often involve distinct anatomical regions. For example, the immune sys- « Autoimmune diseases affect
5 per cent of North Americans and
tem attacks the synovial lining of the joints in rheumatoid arthritis, the thyroid gland in thyroiditis, Europeans, a majority of them
the myelin sheath of brain cells and the spinal cord in multiple sclerosis. An autoimmune disease is being women.
Thyroid
(Hashimoto’s
thyroiditis)
Neuromuscular
junction
(Myasthenia Stomach
Gravis) (Pernicious
anemia)
Kidney (SLE)
Pancreas
(Insulin-dependent Nerves
diabetes (Multiple sclerosis)
mellitus)
Joints
(Rheumatoid arthritis)
Figure 20.1
Diagram showing target organs of
various autoimmune diseases.
classified as organ-specific when it affects one organ (for example, in diabetes mellitus pancreatic
β cells are the target) or systemic, when it affects multiple organs or glands (for example, systemic
lupus erythematosus—SLE).
Organ-specific diseases include thyroiditis, anaemias, Goodpasture syndrome, diabetes mellitus,
Graves’ disease, and myasthenia gravis among others. However, when antibodies are formed against
an antigen that is shared by multiple tissue sites, multiple organs or glands are affected. This leads to
systemic autoimmunity and autoimmune diseases such as rheumatoid arthritis (RA), SLE, multiple
sclerosis (MS) and scleroderma. Individuals may develop more than one type of autoimmune diseases
at a time (for example, individuals affected with gastric autoimmunity usually develop thyroditis).
Autoimmune disease may be mediated primarily by antibodies (Graves’ disease), cellular immu-
nity (multiple sclerosis) or a combination of humoral and cell-mediated immunity (RA). The most
» Rarely, non-autoimune diseases common autoimmune diseases are Graves’ disease, type I diabetes, pernicious anemia, RA, MS and
like cancer can provoke autoim-
mune conditions like paraneoplas- SLE, which account for about 90 per cent of all cases. In general, women are three times more likely
tic syndromes. than men to develop autoimmune diseases.
20.2 SINGLE-ORGAN AUTOIMMUNE

DISEASE
In organ-specific autoimmune diseases, the immune response is directed towards antigens specific
for a particular organ or gland so that tissue damage is largely limited to that organ. The common
target organs in organ-specific diseases include the thyroid, pancreas, adrenals and stomach. The
cells of these target organs may be damaged directly either by humoral or cell-mediated immune re-
sponse; or antibodies binding to the target organs may induce malfunctioning of the target organ.
20.2.1 AUTOIMMUNE DISEASES DUE TO TISSUE

DESTRUCTION OF ORGANS
CHRONIC THYROIDITIS (HASHIMOTO’S THYROIDITIS)

Hashimoto’s thyroiditis is a disease of the thyroid that affects mainly women in the age group
of 30–50 years. The clinicopathological features include an enlarged thyroid, ensuing goitre and
» Immune targets in Hashimoto’s eventual atrophy of the thyroid gland which results in hypothyroidism. Histological examination
thyroiditis are thyroid peroxidase, reveals fibrosis of thyroid follicles and the presence of lymphocytes, macrophages and plasma-cell
thyroglobulin and second colloid infiltrate. The individual produces autoantibodies and sensitized TDTH (TH cells involved in delayed-
antigen. It is believed to affect
about 0.1–5 per cent of all adults in type hypersensitivity) cells specific for thyroid antigens, such as microsomal antigen (thyroid per-
Western countries. oxidase) from thyroid epithelial cells, second colloid antigen and thyroglobulin.
Antibody-dependent cell-mediated cytotoxicity may also be involved. The binding of anti-
bodies to these cell proteins interferes with the iodine uptake and leads to decreased production
» One of the diagnostic tests of of thyroid hormones leading to hypothyroidism symptoms such as dry skin, puffy face, brittle
Hashimoto’s thyroiditis is the detec- hair and nails and feeling of being cold. The destruction of the thyroid results in the gland getting
tion of high levels of antibodies
against thyroglobulin and thyroid enlarged as it attempts to regenerate. The factors contributing to the development of Hashimoto’s
peroxidase in the patient’s blood. thyroiditis are shown in Figure 20.2.
TDTH cell
Antibodies against
Antibodies against microsomal antigen
second colloid
antigen
Antibodies against
thyroglobulin
Figure 20.2
Factors contributing to the induction of Iodine uptake
Hashimoto’s thyroiditis. Thyroid gland
AUTOIMMUNITY AND AUTOIMMUNE DISEASES 431
PERNICIOUS ANAEMIA
Pernicious anaemia results from defective red blood cell maturation due to faulty absorption of Pernicious anaemia
vitamin B12. Under normal conditions, dietery vitamin B12 is transported across the small intestine Pernicious anaemia is a clinical
condition in which the human body
into the body as a complex with the intrinsic factor. The intrinsic factor is synthesized by parietal does not make enough red blood
cells in the gastric mucosa. cells due to lack of vitamin B12 in the
In pernicious anaemia, the patient produces antibodies against the intrinsic factor (as well as body. This form of anaemia usually
occurs in individuals who have lost
three other parietal-cell antigens). The binding of autoantibodies to the intrinsic factor blocks the the ability to absorb vitamin B12 from
attachment of vitamin B12 to the intrinsic factor. This blocks the absorption of vitamin B12 from the food as autoantibodies are formed
small intestine (see Figure 20.3), suppressing or adversely affecting proper haematopoieisis which against the intrinsic factor. Under
normal conditions, the intrinsic factor
results in a decrease in red blood cell count below normal. Defective red blood cell maturation leads binds and transports vitamin B12.
to anaemia with accompanying weakness, loss of appetite, pallor, fatigue and weight loss. Pernicious
anaemia is treated with injections of vitamin B12. « This anaemic condition was
named pernicious anaemia because
A U T O I M M U N E H A E M O LY T I C A N A E M I A it was often fatal during ancient
times when its cause (and hence
An individual with autoimmune haemolytic anaemia makes autoantibodies (IgG or IgM) against cure) had not been discovered.
a variety of red-blood-cell antigens such as Rh determinants, antigens I or i, or antigen P. These
« Autoimmune haemolytic anaemia
antibodies react with self RBC, destroying or removing the blood cells. There are two proposed is characterized by the production
mechanisms of red blood cell destruction: of autoantibodies that attack red
blood cells as if they were foreign to
• Complement-mediated cell lysis of antibody-coated red blood cells—the haemoglobin the body.
released to appears in the urine (haemoglobinuria); and
• phagocytic clearance of antibody-/C3b-coated red blood cells.
The antibodies that are formed in autoimmune haemolytic anemia can be divided into two groups de-
pending on their physical properties.
• Warm antibodies—They are so called since they react with red blood cells under warm » In patients with advanced autoim-
(37°C) conditions. These antibodies are of IgG class and usually directed against Rh mune haemolytic anaemia, a corti-
antigens. Because Rh antigens are located far from each other, the IgG bound to Rh antigen costeroid drug such as prednisone
is usually recommended. If the
cannot activate the complement system (complement activation requires a close alignment patient does not respond to corti-
of at least two molecules of IgG), but is effective in inducing phagocytosis. costeroids or if the corticosteroid
• Cold agglutinins—These antibodies attach to red blood cells only when the temperature causes side effects, then splenec-
tomy is often the best option.
is below 37°C. These antibodies are of IgM class and are specific for I or i antigens present
on the surface of the red blood cells. IgM is highly efficient in activating the complement
system and causing lysis of the red blood cells to which they attach.
The role of IgG and IgM antibodies in inducing autoimmune haemolytic anaemia is shown
in Figure 20.4.
» Drug-induced haemolytic anaemia
D R U G - I N D U C E D H A E M O LY T I C A N A E M I A occurs when a drug causes the
body’s immune system to react
Certain drugs such as penicillin or α-methyldopa (an anti-hypertensive drug) interact with red blood against red blood cells. Drugs that
cell antigens to generate neoantigens. These neoantigens are recognized as foreign and the body are known to trigger drug-
generates autoantibodies directed against these antigens. The drug-induced immune reactions induced haemolytic anaemia
include cephalosporins, quinidine,
(complement-mediated cell lysis or phagocytosis) are mediated by autoantibodies leading to haemolytic levodopa, apart from penicillin and
anaemia (see Figure 20.5). The disease is self-limiting and disappears when the drug use is discontinued. methyldopa.
Food Food
Vit B12
Vit B12
Intrinsic Vit B12

factor
Vit B12 Autoantibodies
absorbed Vit B12 not
bound to intrinsic
absorbed
factor Figure 20.3
Diagram showing the reasons for
No pernicious anaemia Pernicious anaemia develops pernicious anaemia.
RBC
P
I
Rh
Generates
antibody
Antibodies
(IgG or IgM)
IgM-mediated IgG-mediated
Activates complement Phagocyte
Figure 20.4
Diagram showing the mechanism of Haemolysis Phagocytosis
autoimmune haemolytic anaemia. of RBC
Antigen
RBC Penicillin
Neoantigen formed
Generates antibody.
Antibody binds RBC
Complement activation
Complement-mediated
haemolysis Phagocytes
Figure 20.5
Diagram showing the possible
mechanisms for drug-induced anaemia.
THROMBOCYTOPENIC PURPURA
It is an acute or chronic autoimmune disease that re- +
sults from the destruction of platelets by autoantibod-
ies. Patients demonstrate a variety of bleeding problems Drugs
Platelets
including petechia (skin spots due to ruptured blood (e.g. antihistamine)
vessels), gingivitis (bleeding gums), bleeding in the
GI and genitourinary tracts. Antibody-coated plate-
lets are endocytosed and destroyed by phagocytes of
the spleen and liver. This decreases both the half-life Neoantigen
» Thrombocytopenic purpura is a
on platelets
of platelets in the blood as well as their number (to life-threatening disorder that was
less than 1 × 105 cells/ml). Certain drugs such as anti- first described by Moschcowitz in
Generation 1924. For untreated patients, the
histamines and quinine can bind to the surface of the mortality rate of this disease is
platelets generating neoantigens. Neoantigens can elic- and binding approximately 95 per cent.
it antibody formation that binds and destructs plate- of antibodies
lets by complement activation (see Figure 20.6). The against neoantigen » This syndrome is named after
Goodpasture who first described
treatment of such drug-induced thrombocytopenic this disorder in 1919. He reported
purpura consists of withholding the drug. Splenectomy this as a case of pulmonary haemor-
is also sometimes recommended in advanced cases. rhage and glomerulonephritis
Anti-neoantigen during an influenza epidemic. There
antibodies is strong evidence to suggest that
G O O D PA S T U R E S YN D R O M E
individuals with a specific HLA type
It is a rare autoimmune disease of the lungs and kid- (HLA-DR) are more susceptible to
neys. This disease affects all age groups. The mechanism this condition.
of this disease involves the synthesis of autoantibodies
specific for the basement membrane of alveoli and
glomeruli. The binding of these autoantibodies to
basement-membrane antigens leads to complement ac- Complement-mediated Figure 20.6
Line diagram showing the induction of
tivation with resultant cellular damage. Damage to the platelet lysis
thrombocytopenic purpura.
glomerular and alveolar basement membrane causes
kidney and pulmonary damage, leading to glomerulonephritis, pulmonary haemorrhage, haemol-
ysis and haematourea. Death often ensues within several months of initiation of this disease.
INSULIN-DEPENDENT DIABETES MELLITUS (TYPE I DIABETES)

This disease, which affects about 1 person out of every 500 in a population, is caused by the immu- « In type I diabetes, the immune
nological destruction of the pancreas. The attack is directed against specialized insulin-producing system targets insulin, hsp60, and
glutamic acid decarboxylase of the
β cells of the islet of Langerhans in the pancreas. The autoimmune onslaught de- islets of Langerhans.
stroys β cells resulting in decreased production of insulin. The inability to synthesize insu-
« Patients of type I diabetes may
lin makes the patient susceptible to wide fluctuations in blood glucose levels. This destruction
lose as much as 6.8 kg weight
of β cells by the autoimmune response may involve Tcyt cells, followed by antibody plus complement lysis within two weeks.
or lysis by antibody-dependent cell-mediated cytotoxicity (see Figure 20.7). Genetic factors include
the presence of several susceptible genes such as genes in the region of class II MHC region, insulin
gene (chromosome 11) and at least a dozen other non-HLA-linked diabetes susceptibility genes.
The acute manifestation of insulin insufficiency includes ketoacidosis, polyuria, polydipsia and
associated abnormal metabolic events that lead to cardiovascular diseases, kidney problems, cata-
ract and neuropathies. Patients with type I diabetes require daily insulin injections.
20.2.2 AU TO I M M U N E D I S E A S E S INDUCED BY ANTIBODY

BINDING
We have seen in the previous section that a large number of autoimmune diseases result from tis-
sue destruction of specific glands or organs. However, there are a number of autoimmune disorders
that could be induced by a simple binding of antibodies.
« Graves’ disease is named after
G R AV E S’ D I S E A S E ( HYP E R T HYR O I D I S M )
Robert Graves, the physician who
This autoimmune disease results from the overproduction of thyroid hormones (thyroxine). For first described this form of hyper-
reasons not understood, a patient with Graves’ disease develops autoantibodies to the receptor for thyroidism.
thyroid stimulating hormone (TSH). The binding of these autoantibodies to TSH receptors mimics
« Graves’ disease is often associated
the action of pituitary hormone or TSH. The result of this interaction is the overproduction of the with the inflammation, swelling and
thyroid hormone (see Figure 20.8) and hyperthyroidism. bulging of the eyes.
Foreign
antigen
IL-4,IL-5
B cell
Phagocyte
IL-2
TH cell
TH cell
TH cell
Normal immune response
IL-2
Tcyt cell
Antibody
Complement
ADCC
Native autoantigen
resembling foreign B cells
antigen NK cell
Perforin
Figure 20.7
Line diagram showing how damage to
β cells in type I diabetes is induced. Tcyt cell
Anti-TSH This disease mostly affects women in their 30s

antibodies and 40s. Patients demonstrate fatigue, nervousness,
Stimulation increased sweating, palpitation, heat intolerance and
weight loss. Anti-thyroid drugs such as propylthio-
Thyroid follicle uracil or methimazole may be used. Surgical or radio-
TSH receptor
active (I131) ablation of the gland is also effective. A
Graves’ susceptibility gene has recently been localized
on chromosome 20 (20q 11.2).
Figure 20.8
Line diagram explaining the mechanism Overproduction of M YA S T H E N I A G R AV I S
of Graves’ disease. thyroid hormones It is chronic autoimmune disease resulting from
faulty neuromuscular transmission. Patients with
« The name myasthenia gravis liter-
this disease generate autoantibodies (frequently of
ally means grave muscle weakness IgG3 isotype) against the acetylcholine receptor. It appears that antibodies bind acetylcholine re-
in Greek. In myasthenia gravis, ceptors located at the myoneural junction (neuromuscular junction). Antibody-binding blocks the
antibodies are produced against
the acetylcholine receptors at the
receptors, making them immune to the presence of acetylcholine. In addition, antibodies bound to
neuromuscular junction. These an- receptors either cross-link them to a non-functional state or induce endocytosis of receptors.
tibodies block, alter or destroy the Antibody-binding also mediates complement-mediated degradation of cell-bearing recep-
receptors for acetylcholine at the
neuromuscular junction thereby
tors, resulting in a decrease in the number of acetylcholine receptors. The different ways by
preventing muscle contraction. which autoantibodies block the binding of acetylcholine to its receptor are shown in Figure
20.9. The disease is characterized by muscle weakness and fatigueability particularly of the
» Myasthenia gravis may be
ocular, facial, laryngeal and skeletal muscles. This results in difficulty in chewing, swallow-
transmitted to the foetus from the
mother through the passage of ing and breathing, and eventually death from respiratory failure. The female to male ratio is
autoantibodies across the placenta. reported to be 3:2.
Nerve cell
Acetylcholine
Antibodies block the No binding of Neuromuscular
binding of acetylcholine junction
acetylcholine Acetylcholine
to its receptor Cross-linking and
receptor
blocking of receptor
Muscle cell
Removal of receptor Figure 20.9
from cell surface Receptor Line diagram explaining the mechanism
degradation of myasthenia gravis.
20.3 SYSTEMIC AUTOIMMUNE DISEASES

In systemic autoimmune diseases, the immune system is directed towards multiple organs or
glands. The widespread tissue damage is both from cell-mediated and humoral responses as well as
the deposition of immune complexes.
20.3.1 S Y S T E M I C L U P U S E R Y T H E M AT O S U S ( S L E )
This is a chronic, inflammatory multi-organ autoimmune disorder that predominantly affects
young women between 20 to 40 years. The ratio of female to male patients is 10:1.
SLE (which takes its name, red wolf, from the reddish facial rash on the cheeks, an early symp- « Lupus is a condition characterized
by chronic inflammation of body
tom) patients may produce antibodies against a variety of antigens, including ssDNA, dsDNA, tissues. Systemic lupus erythema-
RNA, red blood cells, platelets, mitochondria, lysosomes, ribosomes and thrombin. Patients of SLE tosus, as can be inferred from the
may have 10 to 15 autoantibodies of different specificities, including that against DNA, RNA, red name, is characterized by chronic
systemic inflammation that affects
blood cells, platelets, thrombin, mitochondria, lysosomes and ribosomes. many parts of the body.
The interaction of autoantibody with platelets and red blood cells results in thrombocytopenia
and haemolytic anaemia. Antibodies against thrombin leads to a decreased clotting reaction. When « Butterfly rash on the cheeks , the
characteristic feature of SLE, is
antibodies react with different self-antigens, an antigen–antibody complex is formed that is deposited called Malar rash.
along the walls of small blood vessels (including blood vessels or renal glomerulus). This deposited
complex activates the complement system that forms pore complexes and anaphylatoxins that
ultimately damage the blood vessels, resulting in vasculits and glomerulonephritis (see Figure 20.10).
The main organs affected include skin, kidney, brain, mucosa and the cardiovascular system. The gen-
eral symptoms include erythematous skin rashes, lupus nephritis (deposition of immune complex in
the glomerulus) as well as malaise, fever, lethargy and weight loss. The most characteristic feature is a
skin lesion, particularly the butterfly rash (see Figure 20.11).
This erythematous rash, which occurs over the nose and cheeks, resembles the wings of a but-
terfly. Other lesions include discoid skin rashes and kidney lesions which cause the most mortality
from SLE. Double-stranded DNA can become trapped in the glomerular basement membrane due
to its electrostatic interactions with membrane constituents such as collagen and laminin. The ds-
DNA binds circulating antibodies and forms immune complexes. These immune complexes may
activate the complement cascade resulting in damage to kidneys (glomerulonephritis), leakage of
protein (proteinuria) and haemorrhage (haematuria). The antigen that initiates SLE is still un-
known. Infectious agents, environmental factors and genetic predisposition are still being explored
as possible triggers for this autoimmunity. Non-steroidal anti-inflammatory drugs (aspirin), steroi-
dal anti-inflammatory drugs (cortisone) are useful in treating the symptoms of SLE.
20.3.2 R H E U M AT O I D A R T H R I T I S
Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease affecting mainly women
(usually with MHC of HLA-DR4 type) from the 4th to the 5th decades of life.
RA is characterized by chronically inflamed synovial membranes, densely surrounded by in-
» About 2 million people in the
flammatory cells in the synovial fluid which results in destruction of the cartilage and bone of the USA are believed to be affected by
joints. General symptoms include weight loss, malaise, fever, fatigue and weakness; the cardiovascular, rheumatoid arthritis.
SLE patient
Produces autoantibodies against
ssDNA dsDNA ssRNA RBC Platelets Mitochondria Lysosome Ribosome
Complement activation
Damages blood vessel Damages kidney
Figure 20.10
Diagram showing the mechanism and
consequences of SLE. Erythematous skin rashes Lupus nephrites
haematological and respiratory systems are also

affected. The hallmark of RA is the presence of
rheumatoid factor—an immunoglobulin (IgM
Rheumatoid factor
Rheumatoid factor is an IgG or IgM or IgG type) that reacts with determinants in the
antibody. Fc region of the circulating IgG.
The Fc region of the IgG is modified by ROS
» Rheumatoid arthritis most often
affects the smaller joints, such as
and appears as non-self, resulting in the gen-
those of the hands, feet, wrists, eration of the rheumatoid factor. This complex
elbows, knees, and/or ankles. Indi- of IgG rheumatoid factors is deposited in the
viduals affected with this disease
have difficulty carrying out normal Butterfly rash
synovial membrane of the joint which triggers an
activities such as standing, walking, on cheeks inflammatory response. The erosion of cartilage
dressing, washing, etc. and bone in RA are mediated by TH cells, Tcyt cells,
Figure 20.11
activated macrophages and NK cells which become
Schematic representation of butterfly stimulated by cytokines secreted from activated
rash of SLE. TH cells. These cells release cytokines (TNF-α,
IL-1), degradative enzymes and mediators that
set the inflammation). It is not clear what triggers the initial response. It could be ROS-modified
IgG rheumatoid factor, bacteria, heat shock proteins (γδ T cells of RA patients recognize hsp moi-
eties), Epstein–Barr virus (specific B cells against it are detected in some cases). Inflammation de-
stroys cartilage and bone, leading to joint deformity.
20.3.3 M U LT I P L E S C L E R O S I S
It is an autoimmune neuromuscular disease that affects the central nervous system. The symptoms » The name multiple sclerosis
include motor weakness leading to numbness of limbs or paralysis, ataxia (uncontrolled movement refers to the occurrence of several
scleroses ( plaques or lesions) in the
of limbs), impaired vision, urinary bladder dysfunction and mental aberration. Usually people be- white matter of the central nervous
tween the ages of 20 and 40 are affected. Patients of this disease produce autoreactive T cells that system.
participate in the formation of inflammatory lesions (sclerotic plaques) along the myelin sheath of
» Multiple sclerosis affects the
nerve fibres of the central nervous system. neurons in the white matter of the
Most patients show activated T lymphocytes in the spinal fluid that infiltrate the brain tissue brain and spinal cord.
and cause characteristic sclerotic lesions. These lesions along the myelin sheath lead to numerous
» The immune system targets
mental aberrations. It is assumed that this disease shows some genetic predisposition suggested by a multiple myelin antigens, including
close association with HLA-DR2 and HLA-DOW1. myelin basic protein, proteolipid
The cause of multiple sclerosis, like other autoimmune diseases, is not fully understood. It is protein and myelin oligodendro-
cyte glycoprotein in multiple
believed that infection by certain viruses tend to predispose an individual to multiple sclerosis. sclerosis.
The failure of clonal deletion of cells active against the myelin sheath and the sensitization of the
immune system by neuroantigens have also been suggested as possible reasons. Though MS pa-
tients tend to have elevated levels of antibodies to the measles virus, no definitive proof at present
is available for the involvement of any virus.
20.3.4 SCLERODERMA
Scleroderma is a rare autoimmune disease that affects mainly women between the ages of 35 and « The three major forms of sclero-
54. The common symptoms of this disease include gradual lightening of the skin in regions such derma are diffuse type, limited
type and morphea/linear type.
as hands, feet and face. It results in the deposition of excess collagen in the connecting tissue of Diffuse and limited scleroderma
these regions which results in the thickening of the skin and hence the name (sclero—hard and are systemic forms of the disease,
derma—skin). An individual with scleroderma develops the CREST syndrome. CREST is an affecting multiple organs, whereas
the linear/morphea form is local-
acronym for five symptoms: calcinosis—calcium depositions in the skin; reynauld’s phenomenon— ized to the skin.
abnormal blood flow in response to stress or cold; esophageal dysfunction involving difficulty in
swallowing; scelerodactyl—tightening or scaling of the skin; telangiectasia—formation of red spots
on the face, lips, palms and forearms. The systemic form of this disease affects multiple organs such
as kidney, heart, lungs, GI tract and joints and skin. The cause of this disease remains unknown.
20.3.5 GUILLIAN–BARRÉ SYNDROME

Guillian–Barré Syndrome is an autoimmune disease that commonly occurs after an infectious disease « Guillian–Barré syndrome is named
(for example, measles) or after a vaccination (for example, influenza). The common symptoms include after two French physicians Guillain
and Barré who, for the first time
fever, malaise and progressive weakness, leading to paralysis. The cause of this disease involves anti- in 1916, recognized and reported
body- and T-cell mediated damage to nerve tissues, leading to demyelination and excessive cytokine the nature of this paralytic illness.
production by sensitized lymphocytes. Most patients recover normal function in 6–10 months. Gullian–Barré syndrome is also
known as acute idiopathic poly-
Plasmapheresis or plasma exchange therapy which exchanges the offending antibodies and neuritis.
immune complex containing plasma with normal serum or frozen plasma, is beneficial in Guil-
« In USA alone, the incidence of the
lian–Barré syndrome.
Guillian-Barré Syndrome is 1–3 per
100,000 inhabitants, making it the
most common cause of paralysis.
20.4 ANIMAL MODELS OF AUTOIMMUNE
DISEASES
Animal model for autoimmune diseases helped in understanding the mechanism of and potential
treatments for autoimmune diseases found in humans. These genetically determined animal mod-
els spontaneously develop autoimmune disease(s).
20.4.1 M O D E L S O F S P O N TA N E O U S AU TO I M M U N I T Y
One example is obese strain (OS) chicken. The OS chicken in which thyroid autoantibodies occur
spontaneously and thyroid undergoes progressive destruction resembles what is seen in Hashimoto’s
thyroiditis. The sera of this bird contains thyroglobulin autoantibodies. OS chicken resembles human
Hashimoto’s thyroid disease in terms of lesion of the gland, generation of antibodies against thyro-
globulin and the simultaneous generation of autoimmunity against stomach cells. The role of B and
T cell in thyroiditis have been elucidated from the experiments on OS chicken. The removal of thy-
mus at birth aggravates thyroiditis, which suggests that the thymus exerts a controlling effect on the
disease. Ironically, T-cell depletion in adult chicken by anti-T-cell antibodies inhibits both autoanti-
body production and destruction of thyroid. Similarly the removal of the bursa of Fabricus (in which
B cells mature) after the hatching of the chicken strongly diminishes the pathogenesis of the disease.
This suggests that both B and T cells play an important role in the development of thyroiditis.
« At least 14 genes contribute to the Another important animal model is non-obese diabetic mouse (NOD) which shares many
development of diabetes in NOD
mice.
key features with human insulin-dependent diabetes mellitus (IDDM). Like its human counterpart,
this NOD mouse exhibits the destruction of pancreatic β-islets of Langerhans by lymphocytes.
Various studies in NOD mice suggest that it is T cells that play the decisive role in the development
of IDDM. For example, if the immune system of a healthy NOD mouse is destroyed by X-radiation
and then reconstituted with normal bone marrow cells, the NOD mouse does not develop diabetes.
On the other hand, if the immune system of a normal mouse is destroyed by lethal doses of X-ray and
reconstituted with bone marrow of a NOD mouse, the reconstituted mouse develops diabetes.
The animal model for multiple sclerosis is experimental autoimmune encephalomyelitis
(EAE) rat or mouse. These animals when injected with myelin basic protein (MBP) or proteolipid
antigen develop lesions in the myelin sheaths of the central nervous system, leading to demyelin-
» Obese chicken is the animal model
of Hashimoto’s thyroiditis. ation and eventual paralysis. These symptoms resemble those observed in MS patients.
Similarly New Zealand black (NZB) mouse is the animal model for autoimmune haemolytic anae-
mia while the F1 progeny of the cross between NZB mouse and NZW (New Zealand white) mouse
which does not develop autoimmune disease, develop a high titer of anti-nuclear antibodies and is
used as a model for SLE.
20.4.2 MECHANISM FOR INDUCTION OF AUTOIMMUNITY

The potential for provoking immunity against self-antigen exists in all individuals. However, a
mechanism exists within the body that inactivates or deletes the self-reactive system and induces
self-tolerance. Autoimmunity results from a failure or breakdown of the mechanism which is nor-
mally responsible for maintaining self-tolerance. Immunologic abnormalities may result from de-
fects in T cells, B cells, or both.
FA I LU R E O F B - C E L L TO L E R A N C E
Normal individuals fail to produce autoantibodies against self-antigens because of clonal deletion
of self-reactive cells or tolerance of TH cells or B cells to self-antigen or clonal ignorance (refers to
state whereby autoreactive lymphocytes are neither anergized or deleted but remain in an inacti-
vated state because of low antigen concentration or low affinity for antigen).
A number of viruses (such as Epstein–Barr virus and cytomegalovirus) and bacterial anti-
gens (such as LPS) can act as polyclonal B-cell activators. The polyclonal B-cell activator induces
the proliferation of a large number of clones of B cells, including some that are specific for self-antigens.
This results in the formation of autoantibodies which leads to cellular or tissue destruction. The infec-
tion by EBV leads to infectious mononucleosis that results in the production of a variety of autoan-
tibodies including those that react with self T and B cells (see Figure 20.12). Certain diseases such as
Graves’ disease and myasthenia gravis that are autoantibody-mediated actually result from defects in
the tolerance of T cells. Direct evidence for the involvement of T cells comes from animal models of
autoimmune diseases. However such evidence is more difficult to obtain in human disorders.
MOLECULAR MIMICRY BY CROSS-REACTIVE MICROBIAL ANTIGENS

Autoimmunity may also arise when an immune assault specific for microbial antigen cross-reacts
with self-antigens because epitopes in the microbes resemble self-antigenic determinants. This is
called as molecular mimicry.
Molecular mimicry
Molecular mimicry is the condition
The target antigens for autoimmune disorders can be cell surface proteins, as well as cytoplasmic,
when surface molecules on microbes nuclear or even secreted molecules of the host cells. Some proteins or enzymes are highly conserved
resemble self-antigens. The concept and some conserved sequences or conformations are found in almost all organisms—from microbes
of molecular mimicry as one of the
causes of autoimmune diseases was
to humans. These include heat shock proteins, enzymes (such as tyrosinase in vitiligo, tyrosinase in
first proposed in 1985. celiac disease) or their substrates. The primary immune response to these microbe-specific epitopes
may induce a cross-reactive response to the homologous human protein as these epitopes show a
strong conserved sequence. Such an “unintentional” autoimmune response which is directed to-
» In the early 1970s, rheumatic fever
was first linked to α-haemolytic wards the foreign antigen that shows molecular mimicry results in the autoimmune disease. The
Streptococcus. classic example of such molecular mimicry is evident in rheumatic fever. Antibodies are formed
B cell B cell B cell

Reactive against
self-antigen (switched off)
EBV, Bacterial LPS

(Polyclonal B-cell activator)
B cell Auto-reactive B cell

activated B cell activated activated
Figure 20.12
Diagram showing the failure of
B-cell tolerance in inducing
autoimmunity. Polyclonal B-cell
activator induces proliferation of a large
Antibodies Auto-reactive Antibodies number of B-cell clones, including some
antibodies autoreactive B cells.
against carbohydrate antigens on Streptococci that infect the throat. These antibodies cross-react with
self-cardiac myosin on heart valves resulting in damage to the heart valves (see Figure 20.13).
Another compelling example of molecular mimicry is between epitopes of certain animal » HIV-1 has been shown to cause
diseases of the central nervous
viruses such as hepatitis B virus, Epstein-Barr virus, adenovirus that mimics antigenic determinants system in humans through mo-
of encephalitogenic myelin basic protein (MBP): for example, the P3 peptide of measles virus mimics lecular mimicry. The host produces
MBP peptide (residue 61–69) epitope. Similarly polymerase of hepatitis B virus exhibits sequence antibodies against gp41 proteins
present on HIV-1 These antibodies
homology with MBP peptide. These peptides from the viruses initiate both humoral and cell-mediated can cross-react with surface anti-
responses (mainly T-cell mediated) in the infected individuals, that affects the central nervous gens present on astrocytes within
system, leading to the symptoms of encephalitis such as ataxia and flaccid paralysis. Similarly in the human central nervous system
and act as autoantibodies.
autoimmune diabetes, T cells active against Coxsackievirus were found to coss-react with glutamic
acid decarboxylase (an antigen in β cells of islets of Langerhans).
Animal studies have pointed out another interesting case of virus-induced autoimmunity.
Infection of mice with herpes simplex type 1 leads to destruction of the central nervous system tis-
sue. However, it also leads to a disease called herpes stromal keratinitis (HSK), an autoimmune-like
disease in which T cells specific for a particular peptide attack corneal cells, leading to blindness
in mice. Mutant HSV-1 lacking a particular epitope affects only the central nervous system (cns)
without causing blindness.
Carbohydrate antigen
Streptococci
Throat infection
Antibodies specific for

carbohydrate antigen
Control of infection Autoimmunity
Figure 20.13
Molecular mimicry. Antibodies formed
against carbohydrate antigens of
Streptococci cross-react with self-
cardiac-mysosin leading to Resolution of infection Antibodies cross-react with cardiac
autoimmunity. antigen
AVA I L A B I L I T Y O F S E Q U E S T E R E D S E L F - A N T I G E N S
« Lens protein, a self-antigen is Tolerance is induced during the embryonic stage of development. Antigens that are absent or ana-
never exposed to the immune tomically separated or sequestered from the immune system during this period are not recognized
system. as self. If in later life, these sequestered antigens are released from the protective or concealed place
due to injury, trauma, inflammation or anatomic alteration in tissue, they can stimulate immune
cells that have escaped tolerance and induce the development of an autoimmune disease. Antigens
that fit within this category include antigens of the lens of eye, heart muscle, myelin basic protein
(a CNS antigen) and sperm antigen.
The release of lens protein due to injury or trauma to one eye leads to production of autoanti-
bodies that damages the non-traumatized eye. Similarly autoreactivity to cardiac antigens develops
as a consequence of the exposure of sequestered antigens upon heart damage (see Figure 20.14). In
animal models (mice), the infection of myelin basic protein (MBP), a CNS antigen that is normally
sequestered from the immune system, leads to an autoimmune attack on the myelin sheath antigen
of CNS neuron leading to demyelination and paralysis.
ABERRANT EXPRESSION OF CLASS II MHC MOLECULES

Class II MHC molecules are expressed on the cells of the immune system, and these are involved in
presenting antigens to TH cells. If these class II MHC molecules are aberrantly expressed on non-
immune cells of the body, antigens presented by them will activate TH cells and can hence lead to
Heart
Myocardial infarction
Sequestered antigens released due to

heart damage
Antibodies formed against

sequestered cardiac antigens
Autoimmune reaction
against cardiac antigen
Heart damage
Figure 20.14
Line diagram showing how availability
of sequestered self-antigen can cause
autoimmunity.
activation of B cells, Tcyt cells or TDTH cells against the presented self-antigens. This happens in case
of pancreatic β cells of individuals with insulin-dependent diabetes mellitus (IDDM). These diabet-
ic patients express class II MHC antigens and express only a few class I MHC molecules. Similarly
acinar cells of the thyroid of patients of Graves’ disease express class II MHC molecules, which they do
not express normally. These inappropriately expressed class II MHC molecules present self-antigens
to TH cells. TH cells get sensitized which activates B cells or Tcyt against self antigens, leading to an
autoimmune disease and the consequent damage.
Experimental evidences suggest that dysregulation of cytokine network can also lead to aber-
rant expression of class II MHC molecules which activates the autoimmune response. IFN-γ induces
the expression of class II MHC molecules on non-antigen-presenting cells such as pancreatic β cells,
melanoma cells, thyroid acinar cells and intestinal epithelial cells. Viral infection or injury/trauma
in the organ may increase the localized concentration of IFN-γ in the affected organ. This may lead
to the expression of aberrant class II MHC molecules in non-antigen presenting molecules on the
cells, which activate TH cells leading to autoimmune response. It is worth mentioning that SLE
patients with a large variety of autoreactive antibodies also have a high level of IFN-γ in their blood
as compared to a normal individual. This suggests that an increase in IFN-γ in these patients may
lead to an inappropriate expression of class II MHC molecules and thus to T-cell activation against
a variety of autoantigen. This self-reactivity is not simply a result of non-specific IFN-γ-induced
local inflammatory reaction, since normal islet cells grafted at a different site are specifically rejected
suggesting autoimmune response is quite specific for islet cells and not for any other self-cells.
20.5 THERAPEUTIC APPROACHES

TO AUTOIMMUNE DISEASES
The strategies for treating immune-mediated diseases are aimed at removing problematic symp-
toms so that the patient leads a near-normal life. The ideal treatment of an autoimmune disease
will be to reinstate specific immune tolerance to the “culprit” self-antigen. However, the induction
of such a specific tolerance is difficult to achieve during an ongoing immune response. The treat-
ments currently available do not distinguish between protective immune response of the host and
pathological autoimmunity. Current treatments aimed at suppressing the autoimmune response
include treatment with immunosuppressive drugs, the administration of cytotoxic drugs, organ
ablation when autoimmunity is against a particular organ, plasmapheresis or plasma exchange
process in which the patient’s plasma which contains autoantibodies is replaced by normal
plasma from a matching individual. Non-steroidal anti-inflammatory drugs such as aspirin, or
corticosteroids such as glucocorticoids, dampen the inflammatory reaction by slowing the mi-
totic activity of lymphocytes provide encouraging results in SLE. Cytotoxic drugs—cyclosporin
A or FK506 inhibit the signal transduction mediated by T-cell receptor. These drugs inhibit only
antigen-activated T cells and are reported in the treatment of type I diabetes mellitus. When
an autoimmune response is directed towards antigens of a particular organ, the removal of that
organ, if it is dispensable, gives positive results. Thymectomy of patients having myasthenia gravis
gives relief from symptoms of the disease. A soluble TNF receptor that binds and neutralizes TNF
(a pro-inflammatory cytokine) is now being used to treat rheumatoid arthritis. The removal of
» β interferon is used in the immune complexes and autoantibodies by plasmapheresis benefits patients with SLE, RA, myas-
non-specific therapy of multiple
sclerosis.
thenia gravis and Graves’ disease.
Plasmapheresis involves the removal of blood from the body of the patient. Plasma is removed
from the patient’s blood and the cells, particularly red blood cells, are resuspended in a normal
suitable medium and re-injected back into the patient. This results in the removal of autoantibod-
ies and immune complexes, leading to a temporary relief of symptoms. Similarly, the replacement
of damaged cells such as platelets in autoimmune thrombocytopenia, chemicals such as vitamin
B12 in pernicious anaemia and cholinesterase inhibitors in myasthenia gravis also provides a tem-
porary relief of symptoms.
20.6 O T H E R S T R AT E G I E S
A number of strategies are underway that aim at either inducing tolerance towards specific antigens
or eliminating self-reactive T and B cells. These approaches are being tested on experimental auto-
immune animal models and are likely to be experimented in human subjects.
20.6.1 TOLERANCE INDUCTION

The administration of tolerance towards antigens that elicit autoimmunity, involves the introduc-
tion of antigen via the oral route (mucosal immunity) in an attempt to reintroduce specific immu-
nity toward self-antigens. Mice fed with myelin basic protein do not develop encephalitis even after
the injection of MBP. The injection of MBP in mice that had not been previously exposed to oral
MBP caused encephalitis. In similar experiments in humans, men and women undergoing clini-
cal trials for human MS were fed with bovine MBP. Male recipients (but not females) underwent
significant reduction in T cells specific for MBP. Several more advanced and more specific clinical
trials with tolerance induction with oral antigen are going on for RA as well as MS.
20.6.2 MONOCLONAL ANTIBODY AGAINST

AUTOANTIGENS
Monoclonal antibodies can be raised against some components specifically involved in an autoim-
mune reaction and injected in the experimental animal. These antibodies bind specifically to the
pathological antigen causing either its blocking or destruction of the cells bearing it. The removal
of the irritating antigen does not activate the host system to form autoantibodies. Class II MHC
molecules are aberrantly expressed in some autoimmune diseases; monoclonal antibodies directed
against class II MHC molecules retard autoimmune reactions though they also impede normal
immunity. The blocking of TNF by administration of chimerized anti-TNF antibody has led to a « Monoclonal antibodies against
remarkable improvement of functions in RA patients. CD52 show some promise in the

treatment of multiple sclerosis.
Similarly antibodies directed towards TCR could the block development of autoimmunity
if it is T-cell mediated. The binding of monoclonal antibody specific for the Vβ region of TCR « Treatment with humanized anti-
prevented the induction of encephalitis in mice after injection with MBP. In fact, these antibodies integrin monoclonal antibodies
could reverse the symptoms of encephalitis (in mice) after its induction. The role of monoclonal resulted in short-term reduction
in the number of active lesions in
antibodies in blocking autoantigens is shown in Figure 20.15. multiple sclerosis.
Encouraging results were obtained in non-obese diabetic mice after treatment with anti-CD4
monoclonal antibodies. This therapeutic treatment led to remission in symptoms of diabetes. As
this anti-CD4 treatment was non-specific, it led to an overall blocking of TH cells with a resultant
decrease in total immune response. A better strategy involves blocking only activated and not
resting TH cells. Activated TH cells are likely to be involved in autoimmune reactions. One such
antigen that is specifically expressed only in activated TH cells is IL-2 receptor. IL-2 receptor’s
α subunit is expressed at high levels on autoimmune T cells. Monoclonal antibodies directed
towards the α subunit of IL-2R block the binding of IL-2 and hence preferentially inactivate
activated (that includes autoreactive) TH cells. This was evident in the animal system in which
rats were co-injected activated MBP specific T cells and anti-IL-2 antibodies. Almost 60–80 per
cent of rats survived, while all control rats, which were injected with only MBP-specific T cells,
succumbed to encephalitis.
Monoclonal antibodies
Blocking
peptide
Antigenic
peptide
Class II TCR
CD4 entry
MHC
blocked
Class II
MHC
Antigen-presenting T cell T cell
cell Figure 20.15
Line diagram explaining the role of
Monoclonal antibodies against monoclonal antibodies in blocking
Blockage of MHC molecules
some autoantigens autoantigens.
20.6.3 BLOCKAGE OF MHC MOLECULES

« Statins, the inhibitor of HMG-CoA MHC molecules, particularly class II MHC molecules, are involved in TH-cell activation and hence
reductase, reduce the expression of play a major role in T-cell-mediated autoimmunity. Blocking peptides that have a different amino
class II MHC and are used to treat
multiple sclerosis. acid sequence from the antigenic peptides have been developed in animal models. By binding to
the antigen-binding cleft on MHC these blocking peptides prevent the binding of antigen to MHC
and thereby antoimmune responses (see Figure 20.15).
It has been shown that in encephalitis-prone mice (EAE mice), when synthetic peptides
differing by only one amino acid (from their MBP counterpart) were administered along with
MBP peptide, the development of encephalitis was blocked. Apparently synthetic peptide acts as a
competitor for MBP, occupies the antigen-binding site of MHC and prevents the binding of
autoimmunogenic peptides to MHC. Whether such a mechanism is successful in human trials
remains to be seen.
20.6.4 INDUCTION OF T-CELL SUPPRESSION

Mouse encephalitis has been shown to be suppressed by the administration of low doses of MBP-
specific T cells into the mice. Moreover, when these T cells immunized the mice and the mice were
injected with by a lethal dose of MBP, they did not develop encephalitis. This was observed when
either low-dose MBP-specific T cells were used or cross-linked (by glutaraldehyde) T cells were
injected in the EAE mouse. This is probably due to the fact that injected T cells activated the
T-“suppressor” cells specific for the autoimmune reaction. These T-suppressor cells suppress
the action of autoimmune T cells that mediates mouse encephalitis. The vaccination of animals by
T cells has proved successful in animal models and is being considered for human trials.
20.7 ROLE OF MHC, TH CELLS AND TCR

IN AUTOIMMUNITY
20.7.1 ROLE OF MHC
It has been identified that the occurrence of certain HLA haplotypes appears to predispose indi-
viduals to increased susceptibility to certain autoimmune diseases. The strongest such association
has been observed between a HLA-B27, a class I allele and ankylosing spondylitis, an inflamma-
tory autoimmune disorder of the joints. Individuals with B27 HLA allele stand a 100 times greater
chance of developing this disorder than individuals that lack B27. Class II HLA-D/DR3 is associ-
» Rhematoid arthritis shows a ated with SLE. D/DRS is associated with Hashimoto’s thyroiditis, pernicious anaemia and rheu-
strong genetic component in the matoid arthritis. As can be seen, class II MHC molecules are more associated with autoimmunity
etiology with its association with mainly because these MHC molecules are involved in the selection and activation of TH cells. These
MHC alleles, such as HLA-DR4 and
HLA-DR1. TH cells regulate both humoral and cell-mediated immune response under conditions of normal
and autoimmune response. It should be remembered that presence of particular disease-associated
HLA alleles is not by itself the cause of any autoimmune disease but may be one of the several fac-
tors that contribute to autoimmunity.
20.7.2 TH CELLS IN AUTOIMMUNITY

TH cells are the key regulators of all immune responses, particularly to protein antigens. Inap-
propriate response to self-antigens can involve both the cell-mediated and humoral types. TH-cell
abnormalities may also lead to autoantibody production because TH cells are necessary for the
production of antibodies. The role of TH-cell in autoimmunity is established by experiments
on animal models. The introduction of only specific TH-cell clones for thyroglobulin in normal
syngenic mice leads to the development of thyroditis. Similarly, the introduction of MBP-specific
TH-cell clone into normal syngenic mice, results in the development of mice encephalitis and
demylination of nerve fibre. This implies that TH cells play a significant role in the development
of autoimmunity. Experimental evidences suggest that balance of TH1/TH2 plays a pivotal role in
autoimmunity. TH1 plays a role in the development of autoimmunity while TH2 stops the progres-
sion of autoimmune response.
It has been experimentally proved that TH1-cell clones, specific for the MBP protein, transfer » TH2 cytokine production restrains
encephalitis in mice while TH2 cells do not. In fact the administration of TH2 cells actually protects the autoreactive response.
mice against encephalitis induced by subsequent injection of MBP plus adjuvant.
20.7.3 ROLE OF T-CELL RECEPTORS IN AUTOIMMUNITY

It has been observed that T cells obtained from patients of multiple sclerosis and myasthenia
gravis show a preferential expression of the TCR variable gene in T cells involved in the autoim-
mune response. It is reported that the T-cell receptor containing particular Vα and Vβ domains
are preferentially clonally expanded probably by a single autoimmune epitope which result in
autoimmunity.
Another T-cell receptor (inhibitory) is CTLA-4 that binds B7-1 or B7-2 present on target cells.
The binding of B7-1 or B7-2 by CTLA-4 receptor inhibits T-cell response and induces T-cell an-
ergy. Thus CTLA-4 normally functions to prevent immune response against self-antigens. Knock-
out mice of gene encoding CTLA-4 suffer from fatal autoimmunity and massive tissue destruction
involving the pancreas, heart, liver and several other organs. This points to the central role that T
cells play in autoimmune diseases.
ELISPOT
ELISPOT stands for enzyme-linked immunosorbent
Antigen is coated on
spot assay. This technique was developed by Cecil
microtiter plate
Czerkinsky in 1983. Classically, ELISPOT was used for
enumerating antibody-forming cells. Now this labo-
ratory technique is routinely employed for detect-
ing biological cells that generate (or secrete) various
substances such as cytokines or antibodies. When
this technique is used to enumerate antibody-secret-
Antibody-secreting cells
ing cells, the antigen is first coated on the wells of layered on antigen-coated plate
microtitre plate. Antibody-secreting cells (prepared
from the spleen) are then layered on the wells of this
antigen-coated plate. These antibody-secreting cells
secrete antibodies which bind to the coated anti- Primary antibody
gen molecules. Cells are then washed away, leaving Cells are washed away,
antibodies bound to antigens. The bound antibodies leaving specific antibodies
bound to the antigen
are revealed by the addition of secondary anti-IgG
antibody that has been conjugated with enzyme
such as peroxidase. A chromogenic substrate is then
added together with agarose over this antigen–an- Secondary antibody
tibody–anti-IgG–enzyme complex. The colourless
chromogenic substrate is cleaved by the conjugated Enzyme-linked secondary
enzyme to form a coloured product. The coloured antibody detects primary antibody
product is formed in those regions where the plasma
cell had originally bound (see Figure 20.16). Aga-
rose, which has a gel-like consistency is mixed with
the substrate and added to prevent diffusion of the
coloured product. Each developed spot represents a
single antibody-secreting cell. These coloured spots
Addition of chromogenic substrate
are then counted and the number of plasma cells together with agarose reveal coloured
enumerated. spots corresponding to plasma cells
Figure 20.16
The principle of ELISPOT.
S U M M A R Y
• The immune response against self-components, that evokes • The mechanisms that provoke immunity against self-antigens
pathological consequences, is called autoimmunity. include the failure of B-cell tolerance, molecular mimicry by
• Autoimmune diseases can be organ-specific when one organ is af- cross-reactive microbial antigens, availability of sequestered self-
fected, or systemic when multiple organs or glands are affected. antigens and aberrant expression of class II MHC molecules.
• Organ-specific diseases may be mediated by antibody-binding • The strategies for treating autoimmune diseases are aimed at
(Graves’ disease) and antibody-binding and complement activa- removing problematic symptoms so that patients lead a near-
tion (Goodpasture syndrome), or may= involve both cell-mediated
normal life. These therapeutic approaches include the administra-
and humoral response (Hashimoto’s thyroiditis, type I diabetes).
Systemic autoimmune diseases may result from cell-mediated and tion of cytotoxic drugs, immunosuppressive drugs, organ ablation,
humoral responses as well as the deposition of immune complexes plasma exchange process as well as treatment with non-steroidal
(SLE, rheumatoid arthritis, multiple sclerosis). anti-inflammatory drugs such as aspirin.
• A number of animal models have been developed to understand • Other approaches that aim at either inducing tolerance, prevent-
the mechanism and potential treatments of autoimmune diseases. ing MHC molecules from presenting autoantigens to T cells or the
These include obese chicken that spontaneously develops Hashi- induction of T-cell suppression are being tested on animals and are
moto’s thyroiditis, NOD mouse which exhibits insulin-dependent
likely to be explored on human subjects
diabetes mellitus, New Zealand black mice (NZB) which manifests
autoimmune haemolytic anaemia.
K E Y W O R D S
• autoantibodies 430 • Goodpasture • multiple sclerosis 436 • pernicious

• autoimmune haemolytic syndrome 433 • myasthenia gravis 434 anaemia 431
anaemia 431 • Hashimoto’s • organ-specific autoim- • systemic lupus
• chronic thyroiditis 430 thyroiditis 430 mune disease 430 erythematosus 435
• drug-induced haemolytic • horror autoxicus 428 • New Zealand black • thrombocytopenic
anaemia 431 • insulin-dependent diabetes mice 445 purpura 433
• Graves’ disease 433 mellitus 437 • NOD mouse 437 • scleroderma 447
• Gullian–Barré • obese chicken 437 • rheumatoid arthritis 435 • tolerance induction 442
syndrome 437 • molecular mimicry 438 • rheumatoid factor 436 • T-cell suppression 444
1. Lens protein is a self-antigen, yet exposure of an individual’s own 4. What purpose do animal models for autoimmune disease serve?
lens protein (in some trauma) to (self) immune system leads to the Why do we need them?
production of autoantibodies. Why?
5. What is single-organ autoimmune disease? What happens in sys-
2. What is known about the various therapeutic strategies for autoim- temic autoimmune disease?
mune diseases?
3. Certain viruses and bacteria elicit autoimmune response instead of
the normal expected immune response. What is happening?

1. Hashimoto thyroiditis is characterized by all of the following, 3. Thrombocytopenic purpura results from destruction of:
except: (a) Red blood cell
(a) Atrophy of thyroid (b) Platelets
(b) Stimulation of antibodies against thyroglobulin (c) Leukocytes
(c) Enlargement of thyroid (d) ds DNA
(d) Complement-mediated red blood cell lysis
4. Which of the following is not correct about Graves’ disease?
2. “Warm” antibodies are generated in: (a) Autoantibodies formed against TSH
(a) Pernicious anaemia (b) Overproduction of thyroid hormone
(b) Autoimmune haemolytic anemia (c) Susceptibility gene localized on chromosome 20
(c) Drug-induced haemolytic anemia (d) Usually affects women in their 30s and 40s
(d) Thrombocytopenic purpura
5. Antibodies are formed against acetycholine receptors in: (a) NOD mouse
(a) Myasthenia gravis (b) EAE rat
(b) SLE (c) NZB mouse
(c) Rheumatoid arthritis (d) Obese chicken
(d) Multiple sclerosis
9. Autoimmunity can be induced by all of the following
6. Reynaulds phenomenon is associated with this disease: mechanisms, except:
(a) Guillain–Barré syndrome (a) Molecular mimicry
(b) Scleroderma (b) Failure of B-cell tolerance
(c) Multiple sclerosis (c) Release of sequestered self antigens
(d) Chronic thyroiditis (d) Reintroduction of self antigens
7. Which statement is not true for SLE? 10. In pernicious anemia, antibodies are produced against:
(a) Affects mainly women between the 5th to 6th decade of life (a) Intrinsic factor
(b) Exhibits haemolytic anaemia (b) Rh antigens
(c) Exhibits thrombocytopenia (c) Neoantigens
(d) Exhibits butterfly rash on cheeks (d) Erythrocytes
8. Animal model that will exhibit multiple sclerosis on injection

of myelin basic protein is

1. In Goodpasture syndrome, autoantibodies specific for basement 4. B- and T-cell mediated damage to nerve tissue occurs in
membrane of alveoli and glomeruli are generated. Guillain-Barré syndrome.
2. Rheumatoid factor is an antibody of IgE and IgM type that is 5. Immune response against Epstein–Barr virus may affect the
formed against circulating IgG. central nervous system.
3. Pernicious anaemia can be treated with injections of vitamin B12.
Davidson, A. and B. Diamond (2001). “Autoimmune Diseases”, King, C. and N. Sarvetnick (1997). “Organ-specific Autoimmunity”,
New England Journal of Medicine, 345: 340. Current Opinion in Immunology, 9: 863–71.
Diebold, S. S., T. Kaisho, H. Hemmi, S. Akira and R. Souza Liblan, R. S., S. M. Singer and H. O. McDevitt (1995). “TH1 and
(2004). “Innate Antiviral Responses by Means of TLR7-mediated TH2 cells in the Pathogenesis of Organ-specific Autoimmune
Recognition of Single Stranded RNA”, Science, 303: 1529–31. Disease”, Immunology Today, 16: 34–38.
Harris, E. D. (1990). “Rheumatoid Arthritis: Pathophysiology and Rich, R. R., T. A. Fleisher and W. T Shearer (eds:) (2001). Clinical
Implications for Therapy”, New England Journal of Medicine, 322: Immunology: Principles and Practice. Vol. I and II, 2nd edn. St.
1277–89. Louis, Mosby.
Holmdahl, R. (1999). “Autoimmunity: Another Pathway Towards Steinman, L. (1993). “Autoimmune Disease”, Scientific American,
Arthritis”, Current Biology, 9: R528–30. 269: 107–114.
Janeway, C. (1982). “Beneficial Autoimmunity”, Nature, 299: Steinman, L. (2004). “Immune Therapy for Autoimmune
396–97. Diseases”, Science, 305: 212–16.
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GLOSSARY
A Affinity Measure of strength with which an

antigen-combining site interacts with cognate antigenic
determinant.
ABO-blood-group antigen Polymorphic
antigens expressed on red blood cells, used for typing Affinity maturation Increase in average affinity
human blood for transfusion, first blood group system to of antibody for an antigen with time, without any change
be identified. in its specificity.
Acquired immunity Immunity acquired during Agammaglobulinemia see X-linked

the lifetime of an individual. agammaglobulinemia
Acquired immuno deficiency syndrome Agglutination Clumping of particulate molecules

(AIDS) An immunodeficiency disease caused by HIV such as beads or cells, usually induced by antibodies,
infection, characterized by the functional depletion of lectins or other cross-linking ligands.
T cells of CD4 class, leading to increased susceptibility and Agretope Classical term for the region of processed
occurrence of a variety of diseases. antigenic determinant that binds MHC molecule.
Accessory molecules Surface molecules of T cells AIDS See acquired immunodeficiency syndrome.
other than T-cell receptors (and MHC) that are involved in
T-cell adhesion to antigen-presenting cells; also involved
Alleles Alternative forms of the same gene that confer
alternative characteristics.
in signal transduction and activation of T cells.
Active immunity Immunity rendered by the host
Allelic exclusion Situation in heterozygous
lymphoid cells in which only one of the pair of alleles of an
as a result of intentional and unintentional administration
antigen receptor (Ig or TCR) is expressed in a diploid cell.
of antigen.
Acute inflammation Inflammatory reaction Allergen An antigen that induces a state of
hypersensitivity or allergy.
characterized by a rapid onset and sharp peak, followed
by a distinct decline. Allergy A hyperimmune response to an apparently
innocuous antigen; condition in which contact with
Acute phase proteins Group of proteins found
allergen provokes type I reaction.
in blood and synthesized by liver cells, whose levels
are elevated in response to infection, inflammation or Alloantigen Antigen from a genetically different
trauma. These proteins form an important component of individual of the same species.
the innate defence against microbes. Allogeneic Derived from genetically different
Adaptive immune response See acquired individual of the same species; describes genetic
immune response. variations among the members of the same species.
Adaptor proteins Intracellular proteins that act as Allograft Tissue graft between two genetically non-
a link between receptors and other members of signalling identical individuals of the same species.
pathways. Allotype Any one of the serologically
ADCC (Antibody-dependent cell-mediated distinguishable protein product (or variants) of an Ig
cytotoxicity) Non-specific cytotoxic process in which molecule produced as a consequence of allelic variation
the effector cell, bearing Fc receptor, binds to the Fc in Ig-coding genes.
region of the antibody attached to the target antigen, Alpha foetal protein (AFP) Circulating
subsequently lysing the target cell. glycoprotein synthesized normally during foetal life but
Adenosine deaminase (ADA) not in an adult, whose concentration in serum increases
deficiency Autosomal recessive form of severe during certain types of cancer.
combined immunodeficiency, in which affected Altered receptor model A model for T-cell
individuals lack ADA enzyme which catalyses the recognition that suggests that TCR recognizes self-MHC
conversion of deoxyadenosine to inosine. that is altered by the binding of non-self-peptide.
Adhesion molecules Protein molecules present Alternative complement pathway Pathway
on cell surface that mediate the binding of one cell to of complement activation initiated by the spontaneous
other cells or to extracellular matrix proteins. hydrolysis of C3. This pathway is activated without the
Adjuvant A substance usually administered with need for antigen–antibody complexes.
antigen that enhances immune response against that Alveolar macrophage A type of macrophage
antigen. found in lung alveoli.
Adoptive cellular Anamnestic response Heightened immune
immunotherapy Transplantation of cultured response to secondary or subsequent administration of a
immune cells in the host so that the recipient is able to particular antigen; another name for secondary immune
elicit or participate in an immune response. response which involves immunological memory.
Addressin Protein molecules present on lymphocyte Anaphylatoxin Small cationic peptides generated
surface that bind to receptors on endothelial cells and by the complement cascade that induces the release of
hence help in their transmigration. pharmacologically active mediators from mast cells.
450 GLOSSARY
Anaphylaxis A systemic type I hypersensitivity reaction occurring as a Autoantigen Self-molecules that can act as antigen.
result of IgE-mediated mast cell degranulation.
Anchor residues Amino acid residues of processed antigenic peptide
Autoimmune disease Disease characterized by immune reaction
against self-tissues and organs.
that bind to the pockets in the binding cleft of MHC.
Anergy A state of unresponsiveness to antigens induced in B or T cells. Autograft Transplantation of cells/tissue from one part of the body to
These cells, though present, are unable to respond to antigen. another in the same individual.
Antibody Specific glycoproteins synthesized by vertebrates in response to an Avidity Overall strength of binding between multiple antigenic
antigen that can combine specifically with the antigen which elicited its formation. determinants and polyvalent antibody molecules.
Antibody-dependent cell-mediated cytotoxicity (ADCC) Azathioprine A deoxyribonucleoside analogue that inhibits the activity
Non-specific cytotoxic process in which the effector cell, bearing Fc receptor, of retroviral enzyme—reverse transcriptase, used in the chemotherapy of AIDS.
binds to the Fc region of the antibody attached to the target antigen,
subsequently lysing the target cell.
Antigen Any foreign substance that can bind B-cell receptor/antibody/
B
T-cell receptor and induce an immune response. B cells A subset of lymphocytes which are produced and which mature in
Antigen presentation The display of antigenic peptides bound to the bone marrow, and are the precursors of antibody-synthesizing cells.
MHC molecules on the surface of cells. B-cell receptor(BCR) Cell surface receptor of B cell comprising surface
Antigen-presenting cell (APC) Cell bearing class II MHC antibody and non-covalently–associated, signal-transducing invariant Igα and
molecules, capable of presenting antigen to TH cell. Igβ chains.
Antigen processing Shredding of antigens into small peptides so that B-cell co-receptor complex Group of three cell surface proteins
they can be displayed on MHC. comprising CR2 (CD21), CD81 (TAPA-1) and CD19 associated with B-cell
receptor. The co-receptor complex enhances or amplifies B-cell response.
Antigenic determinant Small restricted portion on the antigen that
determines its antigenicity. This region binds an antibody or T-cell receptor. B7-1 (CD80) and B7-2 (CD86) Costimulatory molecules expressed
This is also called epitope. on antigen-presenting cells that interact with T lymphocytes. They are members
Antigenic drift Minor changes in a pathogen’s surface antigen resulting of the Ig superfamily and their ligands include CD28 and CTLA-4.
from point mutations of genes. Such changes generate antigenic variations that Bacillus Calmette Guérin (BCG) An attenuated strain of
give rise to different strains in a given species.
Mycobacterium bovis, developed by Calmette and Guérin and used as a vaccine
Antigenic shift Extensive or major changes in antigenic specificity that against tuberculosis. It is also used as an adjuvant.
occurs suddenly, leading to the appearance of new subtypes of the pathogen in
a given species. Bronchus-associated lymphoid tissue (BALT) Secondary
lymphoid tissue comprising mainly B cells, situated along the bronchi of the lungs.
Anti-idiotypic antibodies Antibodies formed against antigen-
binding site (variable region domain) of another antibody. The binding site of Bare lymphocyte syndrome An autosomal recessive
anti-idiotypic antibodies mimics the original antigenic determinant. immunodeficiency characterized by failure to express class II MHC molecules.
Antiserum Serum containing high concentration of antibodies, against Basophils Non-phagocytic granulocytes, containing granules that stain
one or more antigens. with basic dyes. These cells express high affinity receptors for IgE antibodies.
Antitoxin General name given to antibodies formed against toxin; also β2-microglobulin Non-polymorphic chain that is associated with
antiserum, containing antibodies to one or more toxins. polymorphic α chain to form class I MHC molecules.
Antiviral proteins Proteins that inhibit the replication of viruses in bcl-2 Product of bcl-2 gene located on chromosome 18 (human).
cells, tissues or organs, usually induced by the action of interferon.
Expression of bcl-2 prevents apoptosis.
Apoptosis A regulated form of eukaryotic cell death brought about by the
activation of caspases. It involves DNA fragmentation, nuclear blebbing and Bence-Jones protein Dimers of light chains of antibody molecules
the formation of apoptotic bodies. It is a normal process that occurs during found in the urine of patients with multiple myeloma. It is named after the
embryogenesis in vertebrates and metamorphosis in invertebrates. physician Henry Bence-Jones who first described its occurrence.
Appendix Worm-like pouch containing lymphoid tissues located at the Benign tumour Non-malignant form of tumour.
beginning of the colon.
Bone marrow Living connective tissue that fills the cavities of bone; site
Arthus reaction A severe localized type III hypersensitivity reaction of haematopoietic activity.
seen in skin after intradermal injection of antigen. The reaction becomes
maximal 3–12 hours after intradermal injection and involves erythema, oedema Booster Supplementary injection of antigen given to stimulate
and local necrosis. immunologic memory provided by an earlier dose.
Asthma A type of pathophysiological type I allergic reaction characterized Bradykinin A vasoactive peptide (C50H73N15O11) produced as a result
by reversible blockage of the airway passage, bronchial inflammation with the of tissue damage, that dilates blood vessels and causes contraction of smooth
participation of mast cells, eosinophils and neutrophils. Some cases of asthma muscles.
are not allergic reactions. Bruton’s agammaglobulinemia X-linked agammaglobulinemia
Ataxia–Telangiectasia Autosomal recessive immunodeficiency characterized by the absence of gammaglobulin in the blood; named after the
characterized by the lack of muscle coordination (ataxia), permanent dilation of scientist O.C. Bruton.
small blood vessels (telangiectasis) in the eyes and on skin. Btk Bruton’s tyrosine kinase, an enzyme that is defective in Bruton’s
Atopy Allergy to environmental allergens such as pollens, usually agammaglobulinemia.
exihibiting a genetic predisposition.
Bursa of Fabricus A lympho-epithelial organ (primary a lymphoid
Attenuation Process through which pathogen loses its virulence and organ) that is found at the junction of the hind gut and cloaca in birds; site of
hence becomes incapable of causing a disease. B-cell maturation in birds.
GLOSSARY 451
C Chemokinesis Overall increase in the motility of cells in response to

chemical stimuli. The increase in motility is not in a particular direction.
Chemotaxis Directional movement of cells along the concentration

C9 Complement component composed of a single polypeptide chain coded gradient of chemical stimuli. It could be towards the chemical stimuli or away
by the gene located on chromosome 5. Several C9 molecules associate with the from the stimuli.
C5b678 complex present on pathogen surface and form a osmolytic pore.
Chimera A mythical animal having the head of a lion, the middle portion
C9 deficiency A rare autosomal disorder characterized by the defective of a goat and the tail of a snake; refers to an individual (or embryo) containing
activation of the terminal pathway and formation of a membrane-attack cells or tissue of two or more genetically different individuals.
complex. The patient exhibits recurrent bacterial infections.
Chromosome translocation A mutational event in which a small
Calcineurin A calmodulin/calcium-dependent serine-threonine portion of chromosome is transferred from one chromosome to the other.
phosphatase that plays a key role in signalling through TCR.
Chronic granulomatous disease (CGD) A disorder associated
Calnexin Chaperone protein that binds and assists in the proper folding of with a defect in NADPH oxidase enzyme that generates bactericidal superoxide
the α chain of class I MHC. radical. It shows X-linked inheritance in a majority of cases.
Capping Aggregation of cell surface proteins at one end of the cell. Chronic myeloid leukaemia Malignant tumour in which cells of
Carcinoembryonic antigen (CEA) Oncofoetal membrane glycoprotein the myeloid lineage (granulocyte, monocyte, platelets and erythrocytes) are
of Ig superfamily. It is involved in the binding of tumour cells to one another. overproduced.
Carcinoma Tumour arising from cells of epithelial origin. Class I MHC molecules Class I MHC molecules are membrane
glycoproteins composed of two polypeptides—polymorphic α chain associated
Carrier An immunogenic macromolecule that is linked to a hapten molecule with invariant β2-microglobulin chain. Expressed by all nucleated cells, and they
to make the hapten immunogenic. A carrier molecule also contains its own present antigen to Tcyt cells.
antigenic determinants.
Class II MHC molecules Heterodimeric glycoproteins composed of
Caspases A family of cysteine proteases that cleave after aspartate residues two non-covalently associated membrane polypeptides—α and β chains. They
of polypeptides. These proteases play an important role in apoptosis. are expressed by cells of the immune system and present antigens to TH cells.
CD molecule System of naming cell surface molecules of human Class III MHC molecules Several proteins that are coded within
leukocyte recognized by (a group of) monoclonal antibodies. Numbers after the MHC locus but are not involved in antigen presentation. These include
CD have a chronological basis. complement components, tumour necrosis factors, heat shock proteins.
CD3 A complex of non-polymorphic polypeptides that is associated with Class switching An intrachromosomal recombination process that
T-cell receptor. It consists of three transmembrane polypeptides γ, δ and ε. CD3 changes the class of antibodies being produced by the B cells.
is a part of the T-cell receptor complex.
Classical complement pathway Mechanism of activation of the
CD4 Cell surface glycoprotein that is expressed on a subset of T cells
complement pathway initiated by the antigen–antibody complex. It involves
(TH cells). It acts as a co-receptor and binds to class II MHC molecules during
complement components C1 to C9 and terminates after the formation of a
antigen presentation to the T cell. CD4 also acts as a receptor for HIV.
membrane-attack complex.
CD8 Cell surface glycoprotein that is expressed on a subset of T cells (Tcyt
Clonal anergy State of functional irresponsiveness of B or T cells when
cells). This molecule binds class I MHC molecules during antigen presentation
these cells are unable to respond to an antigen.
to the T cell.
CDR Parts of the variable region of antibodies that bind antigen. CDRs make Clonal deletion Elimination of self-reactive immature lymphocytes after
contact with antigen and determine specificity of antibodies. coming in contact with an antigen.
CEA See carcinoembryonic antigen. Clonal selection theory Currently accepted theory of antibody
formation. This theory proposes that an antigen selects a specifically reactive
Cell adhesion molecules (CAM) Cell surface proteins that are clone from pre-existing clones of a lymphocyte, initiating an immune
involved in recognition and adhesion of one cell to another. response.
Cell-mediated cytotoxicity Killing of target cells by effector Clone Identical copies (of cells, organisms) arising from a single progenitor.
lymphocytes or macrophages.
Cluster of differentiation (CD) See CD molecules.
Cell-mediated immunity Immune response mediated by T cells and
various non-specific immune cells. This form of immunity can be transferred Cold antibody Agglutinating antibodies of IgM class, optimally active at
to a non-immune individual by the transfer of cells, but not by cell-free less than 37ºC.
fraction.
Colony-stimulating factors Glycoproteins that govern the growth
Centroblast Large, rapidly dividing B cells, found in the germinal centre and differentiation of different haematopoietic progenitor cells in vivo and
of the dark zone, that do not express surface Ig. in vitro.
Centrocyte Small, rapidly dividing B cells that express surface Ig and are Combinatorial diversity Diversity of Ig and TCR that arises from a
found in the light zone. diverse combination of V, (D), J gene segments during gene rearrangement.
Chediak–Higashi syndrome Autosomal recessive Common variable immunodeficiency Heterogeneous group of
immunodeficiency manifested due to defective expression of cytosolic protein, diseases characterized by deficiency of mature antibody-secreting plasma cells.
LYST. Characterized by abnormal lysosomes and diminished phagocytic ability
of neutrophils and phagocytes. Complement Collective term for the large number of enzymes,
proenzymes and proteins present in normal plasma and tissue fluids that
Chemokine Family of low molecular weight cytokines (8–10 kDa) participate in enzymatic cascade which provides extracellular immunity.
that mediates chemotaxis and inflammatory reaction; functions primarily as
chemoattractants for leukocytes. Complementarity determining regions See CDR.
452 GLOSSARY
Conformational determinants Antigen determinants found on Diapedesis Trans-endothelial migration of blood cells across blood vessel
the surface of proteins, formed by amino acids far from each other in a primary wall, from the blood into the tissue.
sequence. These determinants are destroyed upon denaturation of the antigen.
DiGeorge syndrome Genetic defect characterized by the congenital
Congenic Individuals/animals that are different at only one locus. absence of the thymus and parathyroid glands. In this syndrome, T cells are
Constant region Almost invariant carboxyl terminal portion of an diminished in number.
antibody. It does not have antigen-binding domains. Diphtheria toxoid A non-toxic preparation of diptheria toxin generated
Contact dermatitis Cell-mediated delayed-type hypersensitivity that (classically) by formaldehyde modification of toxin; commonly combined with
develops on the skin in response to an allergen contact. tetanus toxoid and pertussis vaccine as DPT vaccine.
Costimulatory signal Signals other than antigen-specific signals, that Diversity gene segment See D gene segment.
are required for the activation of lymphocytes: for example, CD28 (for T cells)
and CD40 (for B cells) coming in contact with B7 and CD40L on an appropriate DM molecules Non-classical class II MHC molecule which functions in
stimulating cell, constitute costimulatory signals. loading antigenic peptides to class II MHC molecules.
Complement receptor Cell surface receptors for complement DNA vaccination A vaccination procedure in which DNA coding for
components or their fragments expressed on a variety of cells. antigen is injected or introduced in antigen-presenting cell or muscle cell to
initiate immune response.
Complement receptor 1 Single chain polymorphic transmembrane
receptor for complement components; expressed on red blood cells, and various Domain Structurally/functionally discrete portions of a protein, usually
myeloid and lymphoid cells; responsible for clearing circulating immune occurring as a compact segment.
complexes.
Double-negative thymocyte An intermediate stage of T-cell
Complement receptor 2 Single chain cell surface complement development in the thymus in which they lack T-cell co-receptors—CD4 and
receptor present on epithelial cells, B cells and dendritic cells; also functions as CD8 proteins.
receptor for Epstein–Barr virus.
Double-positive thymocyte An intermediate stage of T-cell
Cromolyn sodium A drug that blocks the release of pharmacological development in the thymus that is characterized by pre T-cell receptor, and
active mediators from the mast cells; Relieves type I hypersensitivity reaction presence of both co-receptors—CD4 and CD8 proteins.
symptoms.
Cytotoxic T Lymphocyte (CTL) See cytotoxic T cells. DP, DQ, DR molecules Human class II MHC molecules expressed
constitutively on the immune cells.
Cyclosporin A A fungal peptide used as an immunosuppressive drug in
preventing allograft rejection. It interferes with signal transduction in
T cells, preventing their activation. E
Cytokines Diverse array of glycoproteins secreted by cells that act on other
cells. These proteins form a dynamic network of intercellular messengers that Effector cells Cells that are capable of or involved in immune response
regulate various aspects of health including immune response. without any further differentiation: for example, mature T cells, mature B cells
and macrophages.
Cytotoxic T cells A effector T cell that can kill host cells which are
infected with viruses or other pathogens. T cells make direct physical contact Eicosanoids Polyunsaturated 20-carbon fatty acids. Some eicosanoids
with the target cell in an antigen-specific manner; killing could be mediated by or their derivatives can act as local chemical mediators, for example,
perforin or contact of T cells (FasL) with target cell. prostaglandins.
Electrophoresis Separation of molecules such as ions, proteins, DNA
and RNA, under the influence of electric field.
D
Enzyme-linked immunosorbent assay (ELISA) An assay for
Death domain Domains in proteins encoded by genes involved in quantitation of antigen or antibody that uses antibody tagged with enzyme
apoptosis. capable of generating a coloured product.
Decay accelerating factor (DAF) Integral membrane glycoprotein Endocytosis Process used by eukaryotic cell for ingesting
present on most blood cells, particularly red blood cells. DAF facilitates macromolecules, fluids nutrients, etc.
dissociation of C3 convertases (that is, C4b2a; C3bBb).
Endogenous antigen Foreign antigen synthesized in cytosol, for
D gene segment One of the three gene segments involved in the example, viral proteins.
assembly of variable regions of Ig heavy chain and T-cell receptor. This segment
Endosome Small membranous structure containing ingested
codes for the third CDR of most receptors.
macromolecules formed by infolding of the cell membrane.
D–J joining Ligation of a diversity (D) gene segment to a joining (J) Endothelium Cells of mesodermal origin that line the blood vessels and
segment during rearrangement at the Ig heavy chain and T-cell receptor DNA. lymphatics.
Degranulation Release of contents stored in the granules of mast cells Endotoxin Lipopolysaccharide present in the outer membrane of a Gram-
and basophils. negative bacteria that is released as toxin upon bacterial disintegration.
Delayed-type hypersensitivity Cell-mediated hypersensitivity Eosinophil Leukocytes containing cytoplasmic granule that stain with red
response, mediated primarily by CD4+ TH1 cells. The reaction appears to take acidic dye, eosin; main effector cell of ADCC against helminths.
24 to 48 hours to reach maximum intensity after antigen exposure. Epitopes Surface motifs of antigen that interact with antibody or T-cell
receptor (see also antigenic determinant).
Dendritic cells Very efficient antigen-presenting cells that are potent
stimulators of T cells; found primarily in secondary lymphoid organs. Epstein–Barr virus Virus isolated by Epstein and Barr in 1964 which is
the casual agent of Burkitt’s lymphoma and infectious mononucleosis.
Desensitization Prevention of allergic reactions in an individual
through exposure (injections) of multiple doses of antigen. Erythema Redness of skin observed in hypersensitivity reaction.
GLOSSARY 453
Erythroblastosis foetalis Haemolytic type II hypersensitivity reaction G

in which maternal antibodies against foetal Rh antigen cross the placenta and
induce haemolysis of foetal red blood cells, usually with disastrous effect.
Erythroid lineage Lineage of cell arising from haematopoietic stem cell,
G protein Heterotrimeric family of GTP-binding protein that acts as a
signal transducer in eukaryotic cells.
that gives rise to red blood cells.
Erythropoiesis Formation of red blood cells.
Gammaglobulin Serum proteins that migrate with gamma mobility
in electrophoresis. Gamma globulin fraction was found to contain most of the
Erythropoietin Cytokine that regulates the rate of erythrocyte formation immunoglobulins.
from committed erythroid precursors.
Gene Basic unit of heredity coding for protein and/or RNA molecule.
Exocytosis Process by which intracellular contents of a cell are released outside.
Gene complex Cluster of related genes that are located adjacent to each
Exogenous antigen Antigen entering the cell from an outside source. other on the chromosome.
Exotoxin Toxic protein molecules secreted by a living cell. Gene conversion Somatic rearrangements of DNA occurring in genes
Extravasation Trans-endothelial migration of blood cells across the of some species such as chicken, involving the transfer or exchange of DNA
blood vessel wall. between different gene segments.
Exon Region of DNA coding for a protein or part of a protein. Gene knockout Targeted disruption of a gene with resultant loss of
function.
Germinal centre A structural characteristic of lymph nodes and spleen
F where rapidly dividing B cells undergo activation and differentiation in plasma
and memory cells. They occur in secondary follicles.
Fab (fragment antigen binding) Fragment of antibody containing
one antigen binding site, originally generated by treating antibody with the Globulin A general term for any globular protein, soluble or sparingly
enzyme papain which cleaves antibody at the hinge region. soluble in water.
F(ab’)2 V-shaped fragment of an antibody containing two antigen binding Glomerulonephritis Group of diseases in which pathological lesions of
sites, generated by treating antibody with the enzyme pepsin. glomeruli occur, resulting in kidney damage (nephritis)
Factor B An alternative complement pathway component, that is cleaved Graft Cell or tissue or organ, that is taken from one individual and placed/
by factor D to produce C3bBb in the presence of C3. transplanted in another individual.
Factor D Serine protease that acts on C3bB complex to form C3bBb— Graft arteriosclerosis Blocking of artery caused by intense
alternative C3 convertase. proliferation of smooth muscles surrounding blood vessels.
Factor H Regulatory component of alternative complement pathway, that if Graft vs Host disease Pathological immune response initiated by cells
bound to alternative C3 convertase facilitates its dissociation. of tissue graft against tissues of immunocompromised host.
Factor I Serine protease responsible for breakdown of C3b to iC3b and Granulocyte Leukocytes of myeloid cell lineage containing prominent
finally to C3c and C3dg in the alternative pathway. cytoplasmic granules.
Factor P See Properdin. Granulocyte colony-stimulating factor A cytokine that
Fas (CD95) A cell surface molecule of the receptor family of the tumour stimulates the growth of leukocytes in particular cells of granulocyte lineage.
necrosis factor, expressed on a variety of cells, which binds FasL present on Tcyt Granuloma A nodule of inflammatory tissue containing activated TDTH
cell. Fas–FasL interaction triggers cytolysis of Fas-bearing cell. cells, macrophages and multinucleated giant cell.
Fas Ligand (CD178) A cell surface molecule of the tumor necrosis Granzyme Group of enzymes, mainly serine proteases, found in granules
factor family expressed on a number of cells, including Tcyt cells.
of Tcyt and NK cells.
Fc fragment (fragment crystallizable) A crystallizable
Graves’ disease An autoimmune disease resulting from overproduction
protein fragment of antibody obtained by treatment with enzyme papain.
of thyroid hormones due to formation and binding of antibodies against TSH
It is c-terminal region (stem) of antibody that binds the Fc receptor on a
receptor.
variety of cells. This fragment lacks an antigen-binding site.
Fc receptor Cell surface receptor that recognizes and binds the Fc region
of the antibody. H
FK-506 Immunosuppressive drug that inhibits signalling pathways from H-2 MHC region of the mouse. It is located on chromosome 17 and contains
T-cell receptor.
K, I, D, L sub-regions.
Follicular dendritic cell Cells within lymphoid follicles that do not Hapten Non-immunogenic low molecular weight compound.
express class II MHC molecule and hence do not present antigen to TH cells.
They bear Fc receptors/complement receptor that can hold and present immune Hashimoto’s thyroiditis Autoimmune disease characterized by the
complexes to B cells. production of auto-antibodies and sensitized TDTH cells specific for thyroid
antigens.
Framework region A relatively invariant sequence of peptide within
the variable region of Ig light and heavy chains. They constitute about Heat shock Exposure of cells/tissues/organisms to high but non-lethal
70 per cent of the total variable region. This region acts as a scaffold holding temperature.
CDRs in place.
Heat shock proteins Family of conserved proteins which are either
Freund’s complete adjuvant An oil in water emulsion containing killed induced or whose concentration increases in response to heat shock.
Mycobacteria that enhances the immune response when injected with antigen.
Heavy chains Larger of two kinds of polypeptide chains that constitute
Freund’s incomplete adjuvant Freund’s complete adjuvant lacking an antibody molecule.
killed Mycobacteria, that is injected together with antigen in booster doses to
Helper T cells A CD4+ T cell that “helps” other lymphocytes trigger an
enhance the already stimulated immune response.
immune response. These T cells trigger B cells and other T cells to make an
Fyn One of the Src family of tyrosine kinases that is involved in B-cell activation. immune response.
454 GLOSSARY
Haematopoiesis The process of formation and differentiation of blood Immune complex Antigen–antibody complex.
cells (red blood cells, leukocytes and platelets).
Immune response Defence response characterized by the tendency of
Haematopoietic organ An organ such as the bone marrow where the host to eliminate, reject or counteract foreign organisms
blood cells are produced. or cells within its tissues.
Haematopoietic stem cells Cells, which upon division, produce Immune response genes (Ir genes) Class II MHC genes that
haematopoietic stem cells and a population of progenitor cells that give rise to regulate the ability to mount immune response against protein antigens.
different haematopoietic lineages. Immune stimulant Any compound or organism (usually bacteria) that
Haemolysis Lysis or destruction of red blood cells. non-specifically stimulates the immune system.
Haemolytic disease of the newborn See erythroblastosis foetalis. Immune surveillance Surveillance system of the body in which
lymphocytes search for cancerous or virus-infected cells and then kill them.
Hereditary angioedema Disorder caused by decrease or absence of
C1 inhibitor, characterized by recurrent oedema attacks. Immunity A state characterized by a defence response to the onset of
disease after infection by viruses, bacteria or a variety of other pathogens.
Heterophilic antigen Similar (cross-reacting) antigens that occur in
different species such as humans and bacteria. Immunization Deliberate introduction of antigen into the host body to
produce a state of immunity.
High endothelial venules Specialized blood vessels found in
lymphoid organs, from where lymphocytes migrate into lymphoid tissues. Immunocompetent Immune cells or system capable of recognizing
and responding to an antigen.
Hinge region Flexible and open structure of antibody molecule occurring
between the first and second “constant” domains of an antibody molecule that can Immunodeficiency Clinical state in which the immune function is
act as a hinge when the need arises. defective and there is a deficiency of immune responsiveness. Such a state could
be induced by a congenital defect or could be acquired during ones lifetime.
Histamine A basic vasoactive amine produced by decarboxylation of
histidine; found mainly in mast cells and basophils. It causes vasodilation and Immunodominant Antigenic determinants that produce a stronger
smooth muscle contraction. immune response than others.
Histocompatibility antigen Antigens present on cells/tissues that Immunoelectrophoresis A procedure in which protein
determine whether a graft will be accepted or rejected. antigens are first separated by gel electrophoresis and then identified by
immunoprecipitation.
Human immunodeficiency virus (HIV) Causative retrovirus that
infects TH cells and causes AIDS. Immunofluorescence Method that uses antibodies tagged with
fluorescent dyes to visualize the distribution of particular antigen.
Human leukocyte antigen (HLA) MHC in humans are designated
as HLA. Products of these linked genetic loci occur on cell surfaces and serve as Immunogen Any macromolecular substance capable of eliciting a
markers which distinguish self-cells, tissues from non-self cells/tissues. protective immune response.
Humoral immunity Host defence system that involves antibodies; can Immunogenicity The ability of a molecule to evoke an immune
be transferred from one individual to another by using plasma/serum or other response.
antibody containing body fluids. Immunoglobulin A glycoprotein produced by vertebrates that
Hybridoma The product or progeny of hybrid cells formed by the fusion specifically binds antigen (See also antibody).
of a normal antibody-producing cell (plasma cell) with a cell that has the Immunoglobulin domain Domain or folded globular structure
capacity to divide indefinitely (tumour cell); used as a source of antibodies. containing an immunoglobulin fold. Found in members of the immunoglobulin
Hyperacute graft rejection Graft rejection mediated by preformed superfamily.
natural antibodies present in the recipient against allogeneic graft. Immunoglobulin fold Folding motif found in variable and constant
Hypergammaglobulinemia Increased antibody level in plasma or regions of an immunoglobulin. It consists of domains containing two β sheets
serum caused by chronic infection, autoimmune disease or other linked by a conserved disulphide bond.
pathologies. Immunoglobulin superfamily Family of proteins that exhibit
Hypersensitivity Heightened immune response to an apparent characteristic immunoglobulin domains and have less than 50 per cent
innocuous antigen. sequence similarity. Proteins that share more than 50 per cent sequence
similarity are said to belong to one family.
Hypervariable region One of three portions of light and heavy chains
that exhibits high sequence variability from one antibody to another.
Immunosuppression Inhibition of the immune system either by drugs
or as a result of some disease.
Immunotoxin Antibodies coupled to toxin (toxic proteins from bacteria,
plant or animals).These antibodies are directed against tumour antigens.
I Immunotoxins are used as anticancer agents as they kill target cancer cells to
which they bind.
Ia antigen Murine class II MHC molecule.
Iccosomes Small membranous bodies released by follicular dendritic cells
Inflammation A non-specific defence response to a tissue injury or infection
that involves trans-endothelial migration of leukocytes, plasma proteins and fluid.
that are coated with immune complexes.
Idiotope An epitope formed by variable domains (VL or VH) of an antibody. Innate immunity Non-specific defence mechanism that hinders
the early phase of infection. It involves physical and chemical barriers,
Idiotype Sum total of idiotopes that characterize an antibody. phagocytosis, acute phase proteins, inflammation, and fever.
Idiotypic network A regulatory network of reactions exhibiting Insulin-dependent diabetes mellitus Autoimmune disease
idiotype, anti-idiotype and anti-anti-idiotypic interactions that help control an characterized by destruction of the β cells of islets of Langerhans in the pancreas
immune response. during infancy. Autoimmune attack is mediated by specific T cells and antibodies.
Immediate hypersensitivity Hypersensitivity reactions mediated by Integrins A large family of cell surface adhesion molecules that play an
IgE, mast cells and basophils (type I) or an antigen–antibody complex (type II) important role in cell adhesion and leukocyte migration. These proteins are
that occur within a short span of time (minutes to hours) after exposure to antigen. heterodimers of α and β subunits.
GLOSSARY 455
Interdigitating dendritic cells Dendritic cells found in lymphoid LAV Lymphoadenopathy-associated virus, now known as HIV-1.
tissue as well as in blood that play a key role in the negative selection of T cells.
Leader peptide Hydrophobic sequence of amino acids located at the
Interferon (IFN) Family of anti-viral cytokines that are produced by n terminus of immunoglobulin chains as well as at other secretory or
virus-infected cells. These proteins induce an antiviral state in the neighbouring membrane proteins.
cells as well as in IFN-producing cells.
Lectins Carbohydrate-binding proteins.
Interleukins Group of proteins (cytokines) secreted by a variety of
leukocytes that act as an intercellular signal for other leukocytes.
Leukaemia Malignant proliferation of white blood cells/leukocytes.
Intraepidermal lymphocyte γδ subtype of T cells found in the epidermis. Leukocyte White blood cells.
Intraepithelial lymphocyte γδ subtype of T cells found in the Leukocyte-adhesion deficiency (LAD) Rare autosomal recessive
mucosal epithelia of various organs; usually recognizes non-peptide antigens. immunodeficiency characterized by the defective expression of β integrins,
CD11–CD18 group of proteins, which causes impairment in adhesive functions
Intron Intervening sequence of DNA between coding exons, that does not of leukocytes.
code for any protein.
Leukocytosis An increase in the number of circulating leukocytes.
ISCOMS Liposome-like structure having a lipid bilayer encaging antigens
inside. These vesicles are designed for delivering antigens to be used as vaccine, Leukopenia Decrease in the count of circulating leukocytes.
into the host body. Ligand Generic term for any molecule that binds to a receptor.
Invariant chain Invariant or non-polymorphic polypeptide that binds Light chain The smaller chain of an antibody molecule, λ or κ chain, that
class II MHC molecule before it binds antigenic peptide. joins with a heavy chain to form an antibody molecule
Isograft Transplantation or graft between genetically identical individuals. Lipopolysaccharide Compound containing lipid and polysaccharide,
Isotype Each of five classes of immunoglobulins (IgG, IgA, IgM, IgD, and that is the component of a Gram-negative bacterial cell wall.
IgE); another name for antibody class. Isotypes differ from each other in the Locus Location of a specific gene on a chromosome, plasmid or other
amino-acid sequence in the constant region of the heavy chain genetic material.
Isotype switching Genetic rearrangements that occur when B cells Lyme diseases Infectious disease caused by the Gram-negative bacteria
stop secreting antibodies of a particular isotype or class, and start producing Borrelia burgdorferi.
antibodies of another class. (see also class switching).
Lymph Extracellular fluid that surrounds the cells of tissues. It is derived
Isotypic determinant Antigenic determinants located on the constant from plasma and contains proteins, tissue products, antibodies and very few, if
region of the heavy chain, that determine antibody class or isotype. All members of any, cells.
one species carry the same isotypic determinant on the same class of antibodies.
Lymph node Secondary lymphoid organ that contains lymphocyte-
rich tissue in which B and T lymphocytes respond to antigens brought by the
J lymphatic system.
J (joining) chain A small peptide that joins monomers (heavy chains of Lymphatic system System of interconnected vessels found in the body
antibody monomers) in polymeric IgM and IgA. through which lymph travels. It also includes lymphnodes which filter antigens
and present them to lymphocytes.
J (joining) segments A short DNA segment that is located 3' to
the variable gene segment and codes for about 12–20 amino acid residues in Lymphoblast Large and rapidly dividing lymphocytes.
immunoglobulin and TCR genes. This region joins the variable gene segment to Lymphocyte Small mononuclear leukocytes that expresses antigen-
the constant gene segment in Ig and TCR genes. specific receptors; found in circulation and lymphoid tissue; includes B and
Junctional diversity The diversity in immunoglobulin and T-cell T lymphocytes.
receptor gene repertoire generated due to imprecise joining of V, (D,) J gene Lymphokine Cytokines produced by lymphoid cells.
segments during the assembly of immunoglobulin and T-cell receptor genes.
Lymphokine-activated killer cells (LAK) Lymphocytes activated
with IL-2 in vitro to generate a large population of activated anti-tumour cells.
K The majority of these cells are NK cells, although a small number of T cells are
also believed to be present.
Kappa (␬) light chain One of the two mutually exclusive types of light
Lymphoma Malignant tumour of the spleen and lymph nodes.
chains found in antibodies.
Kinin Group of vasoactive peptides released during inflammation. Lymphotoxin Cytokine produced by activated T cells, that activates
neutrophils and endothelial cells; also referred to as tumor necrosis factor β.
Killer activatory receptor (KAR) Receptor expressed on NK cells,
which upon activation induces the killing of the target cell.
Lysosome Cytosolic organelle that contains about 40–50 acid hydrolases.
Killer inhibitory receptor (KIR) Receptor expressed on NK cells Lysozyme An enzyme present in external secretions such as tears and saliva
that binds class I MHC molecule on the target cell. The binding of KIR to class I that digests mucopeptides in the bacterial cell wall. It functions as a non-specific
chemical barrier of the human body.
MHC molecules prevents the killing of target cell by NK cell.
Killer T cell See cytotoxic T cells.
Kupffer cell Type of macrophage found in the sinusoids of liver. M
M cells Specialized cells of the intestinal mucosa that have microvilli
L on their surface. These cells deliver antigen from the apical surface to
lymphocytes and antigen-presenting cells present in the pocket of basolateral
Lambda (λ) light chain One of the two light chains found in antibodies. surface.
Langerhans cell Cells of the dendritic cell family found in the skin and Macrophage Large mononuclear phagocyte found in tissues. It plays an
parts of the GI tract. These are very effective antigen-presenting cells. important role in immune response.
456 GLOSSARY
Major histocompatibility complex (MHC) A cluster of genes results in decreased synaptic transmission at the neuromuscular junction and a
that codes three different classes of polypeptide products (classes I, II and III). patient experiences lethargy.
Class I and class II MHC molecules are polymorphic cell surface molecules Myeloid lineage Lineage of blood cells that gives rise to granulocytes.
involved in antigen presentation to T cells. Class III genes encode complement
components, factor B and heat shock proteins, and are not involved in antigen Myeloma Malignant tumour of plasma cells usually associated with the
presentation. secretion of monoclonal antibodies.
Mucosal-associated lymphoid tissue (MALT) Lymphoid tissue Myeloma proteins Monoclonal antibodies secreted by myeloma cells.
that lines the digestive, respiratory and urogenital systems; protects the body
against microbial invasion of the vulnerable membrane surface.
Mannose receptor A carbohydrate-binding receptor (mannose, fucose) N

expressed by macrophages, that binds carbohydrate residues present on the
pathogen surface and initiates phagocytosis. N nucleotides Non-template (n) nucleotides added by terminal
deoxynucleotidyl transferase at 3' end of V, (D), J segments during genetic
Mannan-binding lectin Serum proteins that binds cell surface rearrangement.
mannose residues of pathogens and activate the complement system.
Naïve Lymphocyte Lymphocytes that have not yet encountered antigen;
Mast cell Bone-marrow-derived cell containing stored granules having virgin lymphocytes.
chemical mediators. It is a non-circulating cell found in tissues, expresses IgE
Natural antibody Antibodies against foreign antigen usually of IgM
receptor and plays a key role in inflammation and hypersensitivity.
type that are found in the serum of a normal or un-immunized individual.
Megakaryocyte Parent cell of leukocytic origin that produces platelets. Natural killer (NK) cell A lymphocyte that is neither a B nor a T cell,
found in normal individuals. They are natural “killers” of tumour or virus-
Membrane attack complex (MAC) Complex of C5–C9 complement
infected cells.
components, that is formed both in the classical and alternate pathways. This
complex is embedded in the target cell membrane creating a pore and lysing the cell. Necrosis Cell death due to infection or other pathological causes that
releases intracellular components to the environment.
Memory cell A lymphocyte that is generated after initial contact with
antigen. This cell expresses an antigen receptor and has a relatively longer life as Negative selection Elimination of B or T cells that are reactive against
compared to other immune cells. Upon stimulation, these cells can give rise to a self-antigen.
rapid and heightened immune response. Neutropenia Clinical stage characterized by a low number of
neutrophils in the blood.
MHC See major histocompatibility complex.
Neutrophil Circulating phagocytic granulocytes, containing cytoplasmic
MHC class I molecule Polymorphic glycoprotein coded by MHC granules that stain with neutral dyes.
complex that is involved in presenting antigen to Tcyt cells.
Neutrophilia Increased number of neutrophils in blood.
MHC class II molecules Polymorphic glycoprotein coded by MHC
complex that is involved in presenting antigens to TH cells. NKT cell A subset of T cell that express α/β T-cell receptor as well as NK
cell markers.
MHC class III molecules Non-polymorphic proteins such as tumor
necrosis factors and complement components that are encoded by the MHC Non-sequential epitopes Epitopes that are formed by amino acids
complex but are not involved in antigen-presentation process. that are located far apart in a primary sequence.
MHC restriction Property of T cell to respond to antigen, only when it is Nude mouse Animal model (mouse) that has no thymus and is hairless
presented together with an MHC molecule. (hence the name).
Minor histocompatibility antigen Antigen encoded outside the Null cell Lymphocytes that lack markers characteristic of T and B cells;
MHC complex that contributes to allograft rejection. Unlike MHC, they do not another name for NK cell.
elicit strong graft rejection.
Mitogens Any substance that under appropriate conditions can O
non-specifically promote mitosis.
Mixed Lymphocyte Reaction (MLR) In vitro proliferative Oncofoetal tumour antigen An antigen that is expressed during the
response of lymphocytes that occurs when lymphocytes are activated by MHC foetal stage and is aberrantly expressed in tumour cells. It is not expressed on
antigens of an allogeneic cell. normal adult cells.
Molecular mimicry One of the hypotheses of autoimmunity which Oncogene A gene that can potentially induce cancer/tumour in the cell in
predicts that autoimmunity results because some of the antigens of the which it occurs or is introduced.
pathogen mimic/resemble antigens expressed on self-molecules.
Monoclonal antibody Population of identical antibody molecules
Oncogenic virus Virus that can induce neoplastic transformation in the
cell which it infects.
derived from a clone of B cells. These antibodies show the same specificity, class
and affinity and have the same structure. Opsonin A molecule such as an antibody or a complement fragment that
Monocyte Large mononuclear phagocytic leukocyte that resides briefly in binds on an antigen surface and promotes phagocytosis.
blood; finally settles in tissue to become a macrophage. Opsonization Coating of opsonin on the antigen which leads to
Mucin Mucin is a vague term for a group of serine-and threonine-rich enhanced phagocytosis.
glycoproteins, that functions as adhesion molecules. These proteins bind selectin.
Multiple sclerosis Autoimmune disease in which immune response P
occurs against the myelin sheath of a neuron, resulting in demyelination and
consequent degeneration of the central nervous system.
P addition Addition of nucleotides to form a palindromic sequence at the
Myasthenia gravis Autoimmune disease in which antibodies are junction of V–D or D–J gene segments during antibody or T-cell receptor gene
formed against acetylcholine receptor at the neuromuscular junction. This rearrangement.
GLOSSARY 457
Paracortex A T-cell rich area of the lymph node between cortex and Primary immune response Immune response that occurs in the
medulla. host’s body after the first exposure to antigen.
Paratope Antigen-binding site of an antibody or T-cell receptor that binds Primary lymphoid organs Organs in which B and/or T lymphocytes
to an epitope of an antigen. mature for example, bone marrow and thymus.
Passive immunization Administration of preformed antibodies in an Priming Activation of virgin or naïve lymphocytes by exposure to antigen.
individual.
Privileged site Anatomical location within the body where allograft can
Pathogen Organism that causes disease. be accepted.
Pax-5 A B-cell-specific protein whose synthesis ensures commitment to Progenitor cell Undifferentiated derivative of stem cell that has lost its
B-cell lineage. self-renewal capacity and is committed to a particular cell lineage.
Programmed cell death See apoptosis.
Perforin A protein synthesized by T cells and natural killer cells, that
polymerizes on target cell membrane to form a pore, causing cell lysis. Properdin Regulator of the alternative pathway of complement, stabilizes
alternative C3 convertase, – C3bBb; also called as factor P.
Periarteriolar lymphoid sheath (PALS) T-cell rich, innermost
region of the white pulp. Prostaglandins Local chemical mediators derived from arachidonic acid
via the activity of cyclo-oxygenase. Prostaglandins can induce inflammation,
Peripheral blood lymphocytes Mature immunocompetent B and stimulate smooth muscle contraction, and also act as vasodilators.
T lymphocytes.
Protein A Protein derived from Staphylococcus aureus which binds IgG.
Peyers’ patches Cluster of lymphoid follicles that are distributed along
Proto-oncogenes A normal cellular gene that regulates growth
the lining of gastrointestinal tract.
control and whose alteration by mutation/recombination can result in the cell
Phagocytosis Process of engulfment of particulate matter such as cell becoming malignant.
debris or bacteria by phagocytes. Provirus Viral DNA integrated into the genome of host DNA.
Phagolysosome An endocytic vesicle formed by the fusion of lysosome Pseudogene DNA sequence that resembles a normal gene but differs in
with phagosome. carrying deleterious mutation and hence has no apparent function.
Phenotype The physical traits expressed by an individual’s genotype. Pus A mixture of dead bacteria, leukocytes and fluid exudates that is present
in wounds, formed as a result of inflammatory response.
Phylogeny Evolutionary history of a plant or animal species.
Pyrogen A chemical substance that stimulates the hypothalamus, inducing
Pili Hair-like protein filaments that project from some bacterial cells. They fever.
can function in bacterial attachment and conjugation.
Pinocytosis Fluid phase endocytosis. Q
Plasma Fluid phase of unclotted blood.
Quaternary structure Three-dimensional structure of protein
Platelets Bone-marrow-derived cells that play a crucial role in blood represented by the number and arrangements of protein subunits.
clotting and inflammation.
Polyclonal Arising from two or more clone of cells. R
Polyclonal activator A chemical substance that activates many Radioallergosorbent test (RAST) Radioimmunoassay that detects
different clones of B and/or T lymphocytes. a specific IgE formed against a particular antigen
Polyclonal antibodies Antibodies produced against a single antigen Radioimmunoassay An immunological test for quantitating antigen
but directed against different antigenic determinants. These antibodies are or antibody, using radioactively labelled antibody.
produced by a several B-cell clones. Recombination activating genes (RAG) Two genes RAG-1 and
Poly-Ig receptor Receptor for IgA or IgM expressed on the basolateral RAG-2 whose products are essential for the V(D)J rearrangements occurring in
surface of mucosal cells of the gut and salivary glands. After the binding B and T cells.
of antibody to this receptor, the antibody is transported from one face to Reactive nitrogen species Highly reactive chemical species (such as
another. nitric oxide, peroxynitrite) containing nitrogen and oxygen that are generated
by phagocytes: These species help in killing extracellular and intracellular
Polymorphism Existence of a character in two or more forms as a result
pathogens.
of multiple alternative alleles at a particular genetic locus.
Reactive oxygen species Highly reactive chemical species containing
Polymorphonuclear leukocytes (PMN) White blood cells oxygen, such as hydroxyl radical and superoxide anion that are formed by
containing multi-lobed nucleus. There are three types of PMNs—eosinophils,
phagocyte. These cytotoxic species have a potent cytotoxic and antimicrobial
basophils and neutrophils.
activity.
Positive selection The process by which B and T cells, that can respond Receptor cell surface A cell surface transmembrane protein that has a
to foreign antigens, are allowed to survive and proliferate within the primary high affinity for ligands.
lymphoid organs.
Receptor editing Process by which antigen receptor genes of
Pre-B cells Bone-marrow-derived cells of B-cell lineage that have rearranged B cells undergo secondary rearrangements to generate different antigenic
heavy chain genes. Light chain genes have not yet rearranged in pre-B cells. specificity.
Pre-T cells Cells committed to T-cell lineage that have rearranged β chain Recombination signal sequence (RSS) Heptamer–nonamer
genes of T-cell receptor and express both CD4 and CD8. β-chain polypeptide is sequence found in antibodies and T-cell receptor genes that plays a critical role
expressed with pre-Tα to form pre-TCR. in somatic recombination events.
Pre-T-cell receptor Receptors expressed by pre-T cell. Comprises Repertoire Diverse array of different antigenic specificities present in
β chain of TCR associated with pre-Tα protein and CD3 proteins. B- and T-cell populations.
458 GLOSSARY
Reticulo-endothelial system (RES) A collective term for non- Signal transduction Transmission of a signal received through a cell
lymphoid cells in the body including phagocytes. surface receptor into the nucleus of the cell via a cascade of reactions.
Reverse transcriptase An enzyme that “reverse” transcribes RNA Slow-reacting substance of anaphylaxis (SRS-A) Group of
genome of a retrovirus into DNA. leukotrienes B, C and D released by mast cells during anaphylaxis that induces
smooth muscle contraction.
Rheumatoid arthritis An autoimmune disease characterized by
inflammation of joints. Smallpox A now-eradicated disfiguring infectious disease caused by the
variola virus.
Rheumatoid factor Antibodies, usually of IgM class, that are found in
patients of rheumatoid arthritis. These antibodies are directed towards antigenic Somatic hypermutation Increased level of somatic mutations
determinants present on IgG molecules. (103–104 times higher) that occurs in the variable region of an antibody,
Rhogam Antibodies directed against Rh antigen of blood cells, produced by B cells. It results in increased affinity of antibodies.
administered to prevent erythroblastosis foetalis. Spleen Largest and major secondary lymphoid organ that is the major site
of immune response to blood-borne antigens.
Splice Joining of two fragments whether of DNA, RNA or even protein
S segments.
Sarcoma Tumour of cells or tissues of mesodermal origin such as Stem cell Those cells that retain the capacity to self-renew as well as to
connective tissue. produce progeny that can generate cells of multiple lineages.
Scavenger receptor Receptor present on macrophages that was Superantigen Antigen that can bind and stimulate certain T-cell
classically shown to remove damaged lipoproteins. It also mediates the (receptors) that express a particular β-chain region. Superantigen show
phagocytosis of various pathogens. specificity to stimulate certain types of T cells. Mitogens, on the other hand,
SCID mouse A mouse strain that is homozygous for scid mutation on stimulate all T cells to divide.
chromosome 16. This strain is unable to rearrange antibody and T-cell genes Superfamily A group of proteins that share less than 50 per cent
and hence lacks T and B cells. sequence similarities among themselves. Proteins that share more than 50 per cent
Secondary follicle Primary follicles that have been antigenically sequence similarities are said to belong to one family.
stimulated. It carries the germinal centre where memory cells and plasma cells Suppressor cell An ill-defined class of T lymphocytes that is claimed to
are formed. suppress the response of other cells to antigen.
Secondary immune response A more rapid, enhanced immune Surrogate light chains Two non-polymorphic Vλ5, Vpre polypeptides
response that occurs after a second or subsequent exposure to an antigen (see that associate with μ heavy chain in pre-B cell.
also anamnestic immune response).
Switch region Region within the heavy chain of B cells at which
Secondary lymphoid organs Organs such as lymph nodes and recombination occurs during isotype switching.
spleen where mature B and T cells encounter antigens and where these
lymphocytes proliferate and get activated. Syngeneic Genetically identical strains of animal or genetically identical
monozygotic twins.
Secondary immunodeficiency A non-genetic immunodeficiency
disease that is acquired during the lifetime of an individual. Syngraft Graft of tissue or organs transplanted between genetically
identical individual.
Secretory component A fragment of poly-Ig receptor that remains
bound to the multimeric form of IgA and IgM. It mediates transcytosis of the Systemic lupus eryethematosus (SLE) A systemic autoimmune
antibody across the epithelial membrane. disease characterized by the presence of high levels of anti-DNA antibodies and
the presence of soluble circulating antigen–antibody complexes. These immune
Secretory IgA Dimer of IgA linked by a J chain, with an attached complexes get deposited in joints and various other tissues and organs.
secretory component.
Selectin Family of cell adhesion molecules (of lectin type) present on T
leukocyte (L-selectin) or endothelium (E-selectin) or platelets (P-selectins).
Their ligands include GlyCAMs.
T cells A class of lymphocyte whose maturation and differentiation requires
Selective immunoglobulin deficiency A condition in which the thymus. They express T-cell receptor, CD3 complex, CD4 or CD8 co-
there is a low level or absence of one or more antibody classes. IgA deficiency is receptor. They are involved in cell-mediated immune response.
the most common form.
T-cell receptor Structurally diverse antigen receptors expressed on every
Self-tolerance Unresponsiveness of the host immune system to self-antigens. T cell. It consists of either α/β chains or γ/δ chains heterodimers.
Sensitization Intentional or unintentional immunization by allergens. TDTH cell T cells, usually of CD4+ type, that are involved in delayed-type
hypersensitivity.
Sepsis Condition in which pathogens (usually bacteria) live and multiply in
the blood stream. TH1 cell Subset of CD4+ TH cells that secrete IL-2 and IFN-γ which induce
inflammation and help cell-mediated immune response.
Serology Study of antigen and antibody reaction under laboratory
conditions. TH2 cell Subset of CD4+ TH cells that secrete IL-4, IL-5 and IL-13 that
support humoral immune response.
Serum Yellow residual fluid derived from clotted blood. It is free from cells
and differs from plasma in that it does not contain fibrinogen. T-suppressor A disputed subset of T cells that is claimed to suppress
Serum sickness A type III hypersensitivity response induced by the humoral and cell-mediated immunity.
administration of large doses of antiserum from a foreign source. It is Transporter associated with antigen processing
characterized by the formation of circulating, soluble immune complexes in the (TAP) Proteins associated with the membrane of endoplasmic reticulum
blood and their deposition in tissues. that are involved in transporting antigenic peptides from the cytoplasm to
endoplasmic reticulum for antigenic processing and presentation.
Severe combined immunodeficiency (SCID) An immuno-
deficiency disease characterized by lack of adaptive immune response due to Target cell Cells that express class I MHC molecules on their surface and
the absence of B and T lymphocytes. hence are potential target for Tcyt cells.
GLOSSARY 459
Tapasin TAP-associated protein that forms a bridge between class I MHC V

molecules and TAP protein during the cytosolic pathway of antigen processing.
Tcyt See T cytotoxic cell. V region (variable region) n-terminal part of antibody or T-cell
TdT (terminal deoxynucleotidyl transferase) DNA receptor that varies in amino acid sequence.
polymerase that inserts non-template nucleotides at the V, (D), J junction
during antibody and T-cell receptor gene rearrangement. These additions V–(D)–J joining Process of controlled somatic rearrangements that
generate additional diversity into antigen receptors genes. occurs in B or T cells that can join V–(D)–J gene segments.
Tertiary lymphoid tissue Loose aggregates of lymphocytes often Vaccine A preparation of attenuated or inactivated pathogens or their isolated
found in connective tissue, for example cutaneous-associated lymphoid tissue.
fragments that are used as antigen to induce immunity in the host.
Thoracic duct Largest lymphatic vessel that collects lymph from the lower
portion of the body and empties it into left sub-clavian vein near the heart. Vaccination Prophylactic administration of an antigen (vaccine) to
Thy-1 Earliest differentiation antigen expressed on T cells. stimulate a protective immune response.
Thymocyte A developing lymphocyte that matures into a T cell in the Variable region See V region.
thymus.
Thyroglobulin Globular protein that binds and stores iodine in the
Variolation Primitive method of vaccination with small doses of live
thyroid gland. small pox virus, variola, to protect an individual against smallpox.
Tumour infiltrating lymphocyte (TIL) Lymphocyte derived from Variant surface glycoprotein (VSG) Variable surface proteins that
inflammatory infiltration present in and around solid tumours.
are found on the African parasite, trypanosoma.
Thymus-dependent antigen An antigen that requires assistance
from TH cells to induce an immune response. Virion A complete virus particle.
Thymus-independent antigen An antigen that can elicit antibody

synthesis in the absence of T cells or their products.
W
Titre The reciprocal of the highest dilution of a serum that gives a detectable
immunological (precipitin) test. Wheal and erythema Characteristic symptom of type I
Tolerance Inability of an antigen to provoke a B-or T-cell response. hypersensitivity appearing as a raised area due to oedema (wheal) and redness
Toll-like receptor Family of pattern-recognition receptors that bind of affected region (erythema).
microorganisms, present on cells of the immune system such as macrophages.
White pulp White portion of the spleen that surrounds an artery.
Toxoid Non-toxic yet immunogenic derivative of toxin.
Transcytosis Transfer of polymeric antibody molecule across the
epithelial and endothelial cells in membrane-bound vesicles.
X
Transfection Process of genetic modification of animal cell by the uptake
of naked DNA.
Xenogeneic Originating from another species.
Transplantation Process of grafting cells, tissues or organs from one
individual to another. Xenograft Graft or trans-plantation of cells/tissues/organ(s) from an
Tuberculin An extract of Mycobacterium tuberculosis that is used in the individual of one species to another.
diagnostic test for tuberculosis.
X-linked agammaglobulinemia A congenital immunodeficiency
Tuberculin test Subcutaneous injection of tuberculin to detect whether characterized by the absence of gammaglobulin in the blood. This disease is
an individual is exposed to tubercle bacilli. DTH response develops at the
injection site 24–48 hours later. characterized by the failure of B cells to mature beyond the pre-B-cell stage, due
to the absence of or defect in Bruton’s tyrosine kinase (Btk).
Tumour antigens Cell surface antigens that are expressed on tumour cells.
Tumour associated antigens (TAA) Proteins that are normal X-linked hyper IgM X-linked recessive immunodeficiency disease
cellular proteins but are aberrantly expressed on tumour cells: examples include characterized by increased IgM and IgM-secreting plasma cells in blood and
the expression of foetal proteins on adult (tumour) cells.
lymphoid tissues. This disease is due to a defect in the expression of CD40
Tumour-specific antigen (TSA) Antigens that are expressed only on
ligand on the T-cell surface.
tumour cells.
Tumour necrosis factor (TNF) Two cytokines TNF-α (produced by X-linked lymphoproliferative disease X-linked immunodeficiency
macrophage, NK cells) and TNF-β (produced by T cells) that are cytostatic or disease characterized by the development of B-cell tumours and a decreased level
cytotoxic to tumour cells.
of gammaglobulin.
Tumour suppressor genes Class of genes involved in the normal
control of cellular growth and whose inactivation causes formation of
tumour. Z
U ZAP-70 (Zeta associated protein-70) A 70 kDa tyrosine kinase
that binds the zeta chain and is involved in T-cell activation.
Ubiquitin A conserved polypeptide with which proteins destined for
degradation are tagged. Zymogen Inactive precursor of enzymes.
ICONS USED IN THE BOOK
Antigenic T-cell
CD4 Class I MHC Cytokine
peptide receptor
Cytokine
Antibody CD3 CD8 Class II MHC
receptor
Immature THcell Tcytcell Natural killer cell

thymocyte
B cell Plasma cell Bone marrow Erythrocyte

stromal cell
Neutrophil Basophil Eosinophil Mast cell
Dendritic cell Monocyte Macrophage Antigen-presenting cell

INDEX
Page numbers in italic type refer to adenosine deaminase , 406 Ancyclostoma spp., 337
pages containing tables or illustrations. adenoviruses, 4, 383, 438 antibodies, 5, 317–18. See also
adjuvants, 65–68, 66 immunoglobulins
# adoptive cellular immunotherapy, 398

adult stem cell, multipotent, 26
affinity maturation of, 109
as antigens, 86–87
α chain, 120 vs embryonic cells, 27 association of light and
αβ TCR, 139 affinity chromatography, 133, 133 heavy chains, 110
β chain allelic exclusion, 145 affinity matrix, 133 classes of, 78–83
β sheets, 75, 87, 122, 75 affinity maturation defined, 71
δ chain of CD3 complex, 151–52 of antibodies, 109 effector functions, 83–85
ε chain of CD3 complex, 152 of B cells, 200 elucidation of, 73
γ chain of CD3 complex, 152 African sleeping sickness (African enzymatic cleavage of, 72
γ−interferon, 131,133 trypanosomiasis), 335 pockets in, 72
γδ TCR, 139–40 agglutination reaction, 262, 263 role in mucosal immunity, 85
η chain of CD3 complex, 152 agglutinins, 20, 81 role in neonatal immunity, 85
ζ chain of CD3 complex, 152 AID protein, see activation-induced structure, 71–77
12/23 rule or one-turn/two-turn cytosine deaminase protein superfamily, 87–88
rule, 102 AIDS, 20. See also HIV virus antibody-dependent cell-mediated
2,4 dinitriphenol epidemic, 417–18 cytotoxicity, 37–38, 84, 85, 256
p-aminobenzenearsonate, 65 treatment and prevention, 423–25 antibody-mediated immunity, 16
4-hydroxy-3-nitrophenylacetyl-chicken alexin, 210 antibody repertoire, 106
gammaglobulin, 202 alkylamines, 241 antibody specificity, 71
5-hydroxytryptamine, 285 Allegra, 280 anti-CD40 monoclonal antibodies, 194
alleles, 86 anti-CAM antibodies, 309
A allelic exclusion, 105–106, 145

T-cell receptors, 146–47
antifolates, 334
antigen–antibody interactions, 89
ABC, see ATP-binding cassette proteins allergen, 267 antigen cochelate, 355
aberrantly expressed proteins, 389 allergic rhinitis, 277 antigenic determinants, 13, 60–61, 61, 88
acidic gastric secretions, 5 alloantigens, 155, 362 antigenic drift, 323
acid proteases, 14 allogeneic graft (or allograft), 362 antigenic shift, 323
acquired complement deficiency allogeneic MLR, 365 antigen masking, 394
diseases, 222 allotopes, 86 antigen processing and
acquired immuno deficiency allotypic determinants, see allotopes presentation, 229
syndrome, see AIDS allotypic markers, 86 antigen presentation, 13
activation-induced cytosine deaminase alpha-foetal protein (AFP), 389 antigen-presenting
protein, 109, 176, 202 altered glycolipid/glycoprotein, 389 cells, 10, 12, 12, 230–32
active death-promoting signals, 169 altered-self model, of T-cell endogenous, 232–37
acute inflammation, 296 recognition, 138, 138 enxogenous, 237–40
acute-phase proteins, 9 alternative C3 convertase evidences, 229–30
ADA, see adenosine deaminase (C3bBb), 221, 221 pre-requisite properties of, 230
ADA-deficient SCID, 406–409 alternative pathway, of complement presentation of non-peptide
adaptive defence, against intracellular system, 213–14, 214 bacterial antigen, 240–41
bacteria, 326 alum, 65 antigen recognition, 13
adaptive enzyme theory, 93 aluminium hydroxide, 65 antigen recognition activation
adaptive immune response, to aluminium phosphate, 65 motif (ARAM), 151
extracellular bacteria, 325 alveolar macrophages, 7, 38 antigens
adaptive immunity, 10 anaphylatoxins, 211, 217–18 adjuvants, 65–68, 66
ADCC, see antibody-dependent anaphylaxis, 20, 276–77 chemical complexity, 63
cell-mediated cytotoxicity anchor residues, 124, 125 defined, 13, 59
462 INDEX
antigens (Continued) autosomal recessive form, 408 somatic hypermutation and, 188 C3b receptor (CR1), 284
distinction between self and avidity theory, 169 stages in development, 186–87 C3b, 6, 213, 219, 237, 324
non-self, 63 azathioprine, 370 tolerance failure of, 438 C3bBb complex, 213
haptens, 65 B1B subset, of B cells, 188 C3bBb3b complex, 213
macromolecules as, 61
molecular size, 62–63
B B7 molecules, 176
B7 family, 249
C3b-coated immune complexes, 286
C3dg, 220
properties, 60 B lymphocytes, 11, 13, 26, 33, 34, 55 B-cell epitopes, 64 C3d-opsonized microbial surface, 190
purified protein derivative B cells, 52, 63, 230–32 B-cell receptors, 137, 190 C4a, 217
(PPD), 178 activation of, 189–93 and NK cells, 252 C4a, 211
response to epitomes, 63–64 affinity maturation of, 200 Bacillus anthracis, 44 C4b molecules, 211
route of entry, 63 antibody-mediated bacterial infections, 329–32 C4b, 218
superantigens, 64–65, 177 regulation, 204–205 immunity to, 324–29 C4b2a, 209
antihistamines, 280 antigen-mediated regulation, 204 bacterial vector vaccine, 350 C5 convertase, 211, 213
anti-idiotype vaccine, 355 antigen recognition, 189–90 Bare lymphocyte syndrome, 407, 413 C5 serum protein, 215, 217
anti-inflammatory agents, 308–309 B1 subset of, 188–89 basolateral (pocket) membrane, 52 C5a receptor, 218
anti-inflammatory cytokines, 306 cross-linking with Ig and basophils, 10, 13, 41, 218, 270–71, 275 C5a, 209
antimicrobial peptides, 6 Fc receptors, 204–205 activation of, 273 C5b67 complex, 215
antimicrobial proteins, 20 deficiencies, 407, 409–410 bcl-2, 197 C5b67 protein complex, 303
antiphogocytic mechanisms, 327–28 development, 185–89 Behcet’s disease, 162 C5b678 complex (C5b-8), 215
AP-1 transcription factor, 174 differentiation into effector Bence Jones proteins, 72 C8 molecule, 215
Apaf-1(apoptotic protease- plasma cells, 195–96 BiP, 235 C9 complement component, 251
activating factor-1), 30 differentiation into memory blast transformation, 32 C9 molecules, 216
apoptosis, 8, 29–30, 31 cells, 197 blebbing, 30 c-abl, 384
arachidonic acid, 303–304 early immature stage of, 186 blood cells, formation and development c-erbB, 384
artemisinins, 334 electron micrograph of, 185 of. See haematopoiesis c-jun, 384
arthropod vectors, 4 germinal centre reactions, 200–203 blood-clotting system, 8, 42 c–NH2–terminal kinase
Arthus reaction, 284, 287 and idiotypic network theory, 205 blood transfusion, 20 (JNK), 174
arylsulphatase, 274 IgE and IgG (IgG4) bone marrow, 46 c-kit, 186
arylsulphatase B, 271 production in humans, 196 booster response, 198 c-mil, 384
Ascaris, 337 IgM-expressing, 186–88 Borrelia burgdorferi, 331, 332 c-myc, 384
asparagine-linked carbohydrate immunoglobulin genes of, 202 bovine serum albumin (BSA), 65 C-reactive complement protein (CRP), 9
moieties, 235 interaction with TH cells, 203 bradykinin, 224, 274 C-reactive protein, 306
associative or linked recognition, 199 lag phase duration, 198 bronchopulmonary aspergillosis, 271 c-sis, 384
asthma, 277 lymphocyte-mediated bronchus-associated lymphoid c-src, 384
astrocytes, 218 regulation, 205 tissue (BALT), 45 C-type lectin receptors, 36
ataxia–telangiectasia (AT), 414 maturation of, 185 Bruton’s tyrosine kinase (BtK), 409 CIIA, see class II transcription activator
“at least one” hypothesis, 254 memory, 189 bubble boy disease, 105 CAD, see caspase activatable DNase
atopic dermatitis, 278 mitosis phases, 191 Burnet’s indirect template theory, 94 cadherins, 298
atopy, 20, 277 naïve, 220 bursa of Fabricus, 45–46, 45 calcineurin, 173, 370
Atovaquone-proguanil, 334 negative selection, 187–88 butterfly rash, 435 calcinosis, 437
ATP-binding cassette proteins , 235 negative selection of, 187–88 BZLF2, 321 calnexin, 235, 238
attenuated vaccines, 345–46 pathway of maturation, 187 calreticulum, 235
attenuated viruses, 345
autoimmune diseases, 19, 429
primary immune response, 198
proliferating, in germinal centre, 201
C CAMs, see cell-surface adhesion
molecules
animal models, 437–42 proliferation phase, 191 C1 component (C1qr2s2), 210 cAMP-dependent protein kinase, 273
role of MHC, 444 regulation of immune C1 inhibitor, 224 cancer. See also tumours
role of T cells, 444 responses, 203–205 C1 regulation, 220 cell division and proliferation, 384
sequestered self-antigen, 441 role in humoral response, 199 C1Inh deficiency, 224 classification, 381–82
single-organ, 430–34 role of TH cells in activation C1q subunit, 210 and costimulator molecules, 394
systemic, 435–37 of, 193–97 C2a, 209 defined, 381
treatment, 442–44 secondary immune C2b, 209 immunotherapy, 394–400
autoimmune haemolytic response, 198 C3 convertase, 211, 213–14 malignant transformation
anaemia, 431–32 signalling through co-receptor classical, 220–21 of cells, 382–83
autoimmune reactions, 429 complex, 190–91 regulation of, 220–22 oncogenes and, 383–87
autologous graft (autograft ), 361 sites for induction of humoral C3a, 217 cancerous or malignant tumours, 381
autosomal recessive disease, 223 response, 199–200 C3b generation, 218 Candida albicans, 216
INDEX 463
carbohydrate residues, 140 CD81, 190 CIIV, 239 constant (C) region of light (CL)
carboxypeptidase A, 275 CD86, 34, 191 circulating dendritic cells, 41 chain, 75
carboxypeptidase N, 218 CD94, 36 cis-acting elements, 132 Cornynebacterium diphtheriae, 329
carcinoembryonic antigens (CEA), 388 CD94/NKG2 receptor, 254 classical pathway, of complement Coronavirus, 5
carcinogenic agents/carcinogens, 382 CDR, see complementarity- system, 210–12, 212 cortical macrophage, 165
carcinoma, 381 determining regions class III MHC molecules, 122–23 corticosteroids, 309, 370
carrier effect phenomenon, 199 Cecropin A, 20 class III MHC proteins, 12 Coxsackievirus, 438
caspase 8, 30 cell-free fluid (lymph), 54 class II MHC molecules, 12, 14, CR2 (CD21), 190
caspase activatable DNase, 252 cell-mediated immunity, 17–19 121–22, 122, 232, 237–40, 440 CREST syndrome, 437
caspases, 30 to allografts, 365–67 class II MHC molecules CRP, see C-reactive complement protein
catalytic function, of C1, 210 antibody-dependent, 256 class II transcription activator, 133 cromolyn sodium, 273
cathepsin G, 275 cells and effector molecules class I MHC molecules, 14, 120–21, CSF, see colony-stimulating factors
cathepsin L, 238 involved in, 248 122, 232–37, 255, 392–93 CTMC, see connective tissue mast cells
CD1 family, 240 delayed-type hypersensitivity class switching phenomenon, 112–14 cyanogen-bromide-activated
CD1, 127 (DTH), 257–62 clonal deletion, 168 matrix, 133
CD14, 10, 38 lymphocytes and, 248 clonal selection, 17 cyclophosphamide, 370
CD154, 34 meaning, 247 theory, 94–95 cyclosporin A, 369–70, 442
CD16 (FcγRIII), 36 non-specific arm of, 247, 248 clones, of lymphocytes, 17 cytocidal membrane-attack
CD19, 34, 190 role of cytokines, 260–61 clonotypic antibodies, 139 complex, 215–16
phosphorylation of, 191 Tcyt cells and, 248–52 Clostridium tetani, 324 cytokines, 7, 9, 46, 53, 57, 276, 395
CD1b-presented antigens, 241 cell migration, 295 clotting system, 302, 303, 305 and DTH, 260–61
CD1c-presented antigen, 241 cells, of immune response, 10–13 C8 molecule, 215 cytokines, role in regulating heavy-
CD20, 34 blood platelets, 42 C9 molecules, 216 chain class switching, 196
CD22, 191 dendritic cells (DC), 41–42 cognate interaction, 193 cytokine-secreting TH cells, 32
CD24, 34 granulocytes, 39–41 cold agglutinins, 432 cytolysis, 216–17
CD27 marker, 34 lymphocytes, 32–38 collectin, 210 cytomegaloviruses, 39
CD28, 35 mononuclear phagocytes, 38–39 collectin (C1q)-like structure, 214 cytopathic effect, of the virus, 316
CD3 complex, 150–52 cell-surface adhesion molecules (CAMs) colony-stimulating factors (CSFs), 27 cytoplasmic viral proteins, 258
accessory molecules, 153–54 cadherins of, 298 common variable immunodeficiency cytosol, 19, 232–34
functions of, 152–53 defined, 296 (CVID), 410 cytotoxic T cells, see Tcyt cells
CD3 proteins, 150, 154 immunoglobulins of, 297 complementarity-determining cytotoxic T lymphocyte-associated
CD31 ligand, 44 integrins of, 297 regions, 76 molecule-4 (CTLA-4), 175
CD32, 38 mucin family of, 297–98 of T-cell receptors, 149 cytotoxic T lymphocytes,
CD4 molecules, 122 selectins of, 297 complement fixation test, 224–25, 225 see Tcyt cells.
CD4 proteins, 156 cell-surface receptors, 10 complement proteins, 6, 209
CD4+ cells, 163 cellular composition, of adult complement system, 83–84, 84, D
CD4+ T cells, 174–75, 179, 199 human blood, 31 303, 318
CD40, 163 centroblast, 200, 220 activation of, 209 Dalton, defined, 63
CD40–CD40 ligand interaction, centrocytes, 200 alternative pathway, 213–14, 214 dark zone, 200
194, 195, 200 chancre, 335 biological functions of dead microbe vaccines, 347
CD45RA, 35 Chediak–Higashi syndrome, 415 complement proteins, 216–20 decay accelerating factor (DAF),
CD45RO marker, 34 chematactic factors, 286 classical pathway, 210–12, 212 213, 221
CD46, 216 chemical/physiological barriers, of cleavage of complement defence collagen, 214
CD56, 36 immunity, 5–6 components, 209 defence mechanisms
CD58 (LFA-3), 176 chemokine CXCL-13, 199 deficiencies, 222–24 adaptive immunity, 10
CD59, 217 chemokines, 300–301 defined, 209 chemical barriers, 5–6
CD64, 38 chemotactic molecules, 299 effector functions of, 209 fever, 7
CD66, 388 chemotaxis, 44 formation of a cytocidal inflammation, 7–9
CD72, 34 chimeric receptors, 153 membrane-attack mechanical barriers, 4–5
CD8 accessory molecules, 154 Chlamydiae, 327 complex, 215–16 phagocytosis, 6
CD8 antigen, 121 chondroitin sulphate, 271 mannan-binding pathway, 214–15 role of acute-phase proteins, 9–10
CD8 marker, 35 chronic granulomatous regulation of, 220–22 defensins, 6, 44
CD8 proteins, 156 disease (CGD), 414 conformational determinants, 61, 61 delayed haemolytic transfusion, 283
CD8+ cells, 163 chronic inflammation, 296 congestin, 267 delayed-type hypersensitivity, 19
CD8+ T cells, 174–75, 179 Churg–Strauss syndrome, 271 conjunctiva, 4 delayed-type hypersensitivity (DTH)
CD80, 34, 191 chymase, 271 connective tissue mast cells, 270 cytokines and, 260–61
464 INDEX
delayed-type hypersensitivity
(Continued)
dsRNA, 316
dual receptor model, of T-cell
erythropoietin (EPO), 27
E-selectin, 259, 297, 305
G
defined, 257 recognition, 138, 138 ES cells, see embryonic stem cells Galectin-1, 186
detection of, 261 Duffy blood group antigen, 300 Escherichia coli, 44, 88, 217, 247 gastrointestinal tract, 4
effector phase of, 258–60 exogenous antigens, 14–15 GATA-1 gene-coded transcription
sensitization phase of, 257–58
significance of, 261–62
E Exotoxin A, 328
exotoxins, 347
factor, 29
GATA-2 transcription factor, 29
delayed xenograft rejection, 373 EAE, see experimental autoimmune experimental autoimmune gel electrophoresis, 242, 242
dendritic cells, 38, 165, 231 encephalomyelitis encephalomyelitis, 437 gel filtration chromatography, 67, 67
follicular, 199–200 EBV, see Epstein–Barr virus extracellular bacteria, 324 Gell and Coombs classification, of
dendrocytes, see dendritic cells ECF-A, 275 extracellular signal-regulated kinase allergic reactions, 268–81
deoxyadenosine triphosphate, 408 echinoderms, 20 enzyme, 173 gene conversion, 108–109
deoxynucleotidyl transferase Echovirus, 216 genetic complement
enzyme, 148
dermal dendritic cells, 41
eczema, see atopic dermatitis
effector response, of DTH, 258–60
F deficiencies, 222
germinal centre
dermis, 4 eicosanoids, 218 factor P, 213 affinity maturation, of B cells, 200
des-Arg forms, 218 electron microscopes, 180 factors B and D, 213 defined, 199
desensitization (or hypo- electrophoresis, 242 FADD, see Fas-associated protein proliferating B cells in, 201
sensitization), 280 ELISA, see enzyme-linked with death domain reactions, 200–203
diabetes mellitus, 19 immunosorbent assay farmer’s lung, 284 selection of high-affinity
diacylglycerol (DAG), 173, 190 double antibody sandwich assay Fas-associated protein with death B cells, 203
diapedesis, 8, 259 of, 339, 339 domain, 251–52 somatic hypermutation,
diapedesis, of leukocytes, 299 ELISPOT, 445, 445 Fas ligand(Fas-L), 30, 251, 254 200–203, 201
differentiation-specific genes embryonic stem cells, pluripotent, 26 Fas pathway, 251–52 germ-line theory, 106
expression of, 27 vs adult stem cells, 27 Fc receptor for neonatal (FcRN), 85 globulins, 71
soluble factors, 29 embryonic stem cell, 25 Fc receptors, 34, 82, 85 gluteraldehyde-treated antigen-
DiGeorge syndrome, 20, 405, 407, 412 endocytosis, of proteins, 237–38 Fc region, of antibody, 13 presenting cells, 229
dinitrophenol (DNP), 65 endogenous antigens, 14, 232–37 FcεRI, 272 GlyCAM-1, 297
diphtheria, 329–30, 347 endogenous pyrogens, see endotoxins FcεRII, 272 glycosyl residues, 235
disulphide bonds, 75, 120–21 endothelial cells, 218 fever, 7 glycosyl transferases, 282
disulphide bonds140 endothelial venules, high, 47 fibrin, 303 Gm markers, 86
disulphide-linked heterodimeric endotoxin, 324 fibrinolysis, 305 Goodpasture syndrome, 432–33
plasma protein, 211 endotoxins, 7 fibrinolytic system, 303 graft arteriosclerosis, 369
disulphide-linked polypeptides, 35 enhancer A, 132 fibroblast, 26 graft rejection, 20, 366
disulphide (-S-S-) bonds, 77 enhancer B, 132 fibrosis, 260 graft versus host disease, 376–77
divalent, 72 enhancers, 114 fimbriae, 44 Gram-negative bacteria, 7, 217,
DNA-cleaving enzymes, 251 exogenous antigens, 237–40 fixation, 157 324, 326, 331
DNA-dependent protein kinase , 104 enzyme-linked immunosorbent FK-506, 370, 442 Gram-positive bacteria, 5, 217
DNA-dependent protein kinase, assay, 310, 310 FK-binding protein (FKBP), 370 granule-associated enzymes, 250
defects, 105 eosin, 271 flagellins, 9, 192 granulocyte-CSF (G-CSF), 27
DNA methylation, 29 eosinophil chemotactic factor, Flaviviruses, 4 granulocyte–macrophage-CSF
DNA-PK, see DNA-dependent 274, 278 flow cytometry, 54, 54 (GM-CSF), 27
protein kinase eosinophil-derived proteins, 271 Flt-3 ligand, 27 granulocytes, 26
DNA vaccine, 351 eosinophilic chemotactic factor of fluid shear stress, 299 basophils, 41
double antibody sandwich assay, of anaphylaxis (ECF-A), 271 fluorescence-activated cell sorter, eosinophils, 40–41
ELISA, 339 eosinophils, 10, 12, 40–41, 47, see flow cytometry mast cells, 41
double diffusion agar assay 218, 271, 271 fluorescent light, 54 neutrophils, 39–40
technique, 206 epitopes, 60, 64 focal adhesin kinase, 30 granulocytopenia, 415
double-positive (DP) thymocytes, Epstein–Barr virus, 216, 383, follicular dendritic cells, 42, 199–200 granuloma, 307
163, 165 405, 438 food allergies, 278 granulomas, 260
DP region, 129 Epstein–Barr Virus protein, 321 foreign antigens, 165 granzymes, 30
DQ region, 129 ERK, see extracellular signal- forward scatter, 54 Graves’ disease, 433, 440
Drosophila, 29 regulated kinase framework regions (FRs), 76 Group A Streptococci, 44
Toll protein, 247 Erp 57, 236 Freund’s complete adjuvants, 65 GTP-/GDP-binding protein, 173
DR region, 129 erythema, 8 frustrated phagocytosis, 286 guanidium-hydrochloride, 133
drug allergies, 282 erythroblastosis foetalis, 283 functional competence, 161 Guillian–Barré syndrome, 437
INDEX 465
gut-associated lymphoid tissue Hepatitis A virus, 5 Rhesus (Rh) antigen disorders, 19–20
(GALT), 45, 51–52 Hepatitis B virus, 438 incompatibility, 283–84 evolution of, 20–21
GVHD reactions, see graft versus Hepatitis B virus (HbsAg) antigen, 348 role of basophils, mast cells and host defence against
host disease hereditary angioneurotic edema eosinophils, 270–71 viruses, 319–22
(HANE), 223–24 role of IgE receptors in, 272 to influenza virus, 323–24
H herpes simplex virus, 44, 320

herpes stromal keratinitis (HSK), 438
therapeutic measures, 280–81
transfusion reactions, 282–83
innate, 4–10
and lymphatic systems, 53–55
H2020 strain, 95 herpes virus, 37, 216 tuberculin reaction, 290 origin of cells, 28
H-2K antigen, 126 herpes-virus protein, 178 type I, 268–76 role of lymphoid tissues or
H2L2 molecule, 112 high-mannose type β chain, 141 type II, 281–84 organs, 44–53
haematopoeisis, 10 hinge region, 76–77 type III, 284–89 role of M cells, 52, 53
cellular composition, of adult histamine, 275, 218, 271, 274 type IV, 289–90 types of response, 15–17
human blood, 31 histocytes, 7 hyperthyroidism, see Graves’ disease against viruses, 316–19
cytokines involved in, 27 HIV, see human immuno hypervariable regions, 75 immunoblotting, 400–401, 400
differentiation of HSC, 27 deficiency virus hypochlorite (HOCl), 43 immunodeficiency, 20
homeotic genes in, 29 HIV virus, 418 hypothyroidism, 19, 430 immunodeficiency disorders, 405
role of apoptosis, 29–30 and AIDS, 422–23 animal models, 416–17
role of progenitor and stromal
cells, 27, 29
genetic composition, 418–19
life cycle, 419–20
I deficiencies of complement
components, 416
haematopoiesis, 25–29 mechanism of evasion, 422 Iκ B, 173, 190 primary/congenital
in the bone marrow, 27 mechanism of immunosuppression, ICAM-1, 52 immunodeficiency, 405–416
comparison of embryonic 420–22 ICAM-2, 52. See also intercellular secondary immunodeficiency,
and adult stem cells, 27 HLA-DM, 240 adhesion molecules 405, 417–25
regulation, 29–31 HLA typing, 134 ICE, see interleukin-1 converting severe combined
site of, 26 homeotic genes, 27 enzyme immunodeficiencies,
in spleen, 26–27 homologous restriction factor, 222 iC3b, 218 406–409
haematopoietic-inducing HRF, see homologous restriction factor iC3b-dependent phagocytosis, 224 treatment approaches, 416
microenvironment (HIM), 27 HSC, see haematopoietic stem cell iccosomes, 200 immunoelectrophoresis, 291, 291
haematopoietic stem cell, 26 human immunodeficiency virus, 20, 42 ICP-47, 236 immunogen, defined, 59
haemocyanin, 138 human leukocyte antigen (HLA), 130 idiotypes, 86–87 immunogenic carrier molecule, 65
haemoglobinuria, 283 human papilloma virus (HPV), 236 idiotypic network theory, 205 immunoglobulin A (IgA), 5, 80
haemolytic anaemia, 432 humoral immunity, 13, 317. See also Igα/Ig β of BCR, 191 immunoglobulin D, 81–82, 111
haemolytic antibody, 86 antibody-mediated immunity I genes immunoglobulin domain, 75
Haemophilus influenzae, 347 humoral immunotherapy, 398 IgE-mediated type-I hypersensitivity immunoglobulin E, 82
Haemophilus influenzae type b humoral responses, 17–18 reactions, 41 128 immunoglobulin fold, 75
(Hib), 348 B-cell activation and, 18 IgE receptors, 272 immunoglobulin G, 78–79, 79
Haemophilus meningitis, 347 hydrogen peroxide, 43 IL-1 cytokines, 38, 218, 276, 305, 332 C1 complement-binding site on, 210
Hageman factor, 303 hydrogen peroxide (H2O2), 43 IL-12 cytokine, 261, 326 immunoglobulin (Ig) class, 34
haplotypes, 126 hydrophobic amino-acid sequences, 110 IL-2 cytokine, 174, 194 immunoglobulin-like domains, 151
hapten–carrier conjugates, 199, 202 hydroxyl radicals (OH), 43 IL-3 cytokine, 276 immunoglobulin M, 80–81, 81, 111
haptens, 59, 65, 68, 71 hyperacute reaction, to xenograft IL-4 cytokine, 59, 194, 276, 336 expressing B cells, 186–88
Hashimoto’s thyroiditis, 430 transplantation, 372–73 IL-5 cytokine, 194, 276 immunoglobulins. See also
hay fever, see allergic rhinitis hypermutation, 109 IL-6 cytokine, 218, 270, 305 antibodies
H box, 132 hypersensitivity, 20 IL-7 cytokine, 186 allelic exclusion, 105–106, 105
heat shock proteins, 178 activation of mast cells and IL-8 cytokine, 261 assembly and secretion of, 112
μ-heavy-chain genes, 110–11 basophils, 273 immature B lymphocytes, 187 cell-surface adhesion molecules
heavy-chain rearrangement biological mediators of, 273–76 immune stimulating complex, 65 of, 297
allelic exclusion, 106 clinical consequences of immune surveillance theory, 390 class switching phenomenon,
B cells, 195–96 type I, 276–78 immunity 112–14
CDR1, 107 contact, 289 activation of, 17–19 comparison with TCR, 140
immunoglobulins, 98–99 defined, 267 adaptive, 10 complementary-determining
helminths, 337–39 diagnosis of type I, 278–80 to bacterial infections, 324–29 regions, 140
hen egg lysozyme, 61, 62 drug-induced, 282 cells of immune response, 10–13 generation of antibody
heparin, 271, 274 granulomatous, 290 defence against infectious diversity, 106–110
heparin sulphate, 186 late-phase reaction, 278 agents, 315 gene rearrangement, 98–105
hepatitis, 284 reactions, 267–68 defined, 3 genetic organization, 96–98
466 INDEX
immunoglobulins. See also interleukins, 59 lck, 172 lymphoid progenitor cells, 27

antibodies (Continued) interstitial dendritic cells, see tissue- LCMV, see lymphocyte lymphoid tissues/organs
heavy-chain rearrangement, 98–99 resident dendritic cells choriomeningitis virus in birds, 45
light-chain rearrangement, 100–101 intracellular bacteria, 324 lectin, 210, 324 major, 45
membrane-bound, 110–11, 193, 195 intraepidermal lymphocytes, 53, 178 leishmaniasis, 335–36, 337 primary, 45–48
rearrangement of V, D or intraepithelial lymphocytes, 178 Leishmania tropica, 289, 385 secondary, 45, 48–53
J gene segments, 101–105 intrapulmonary Arthus-type Lentiviruses, 39 lymphokine-activated killer
and regulation of gene reaction, 287 lepromin, 257 cells, 255, 398
transcription, 114 intron, 96 Leptospira, 4 lymphoma, 381
secreted, 110–11 invariant chain (Ii), 238 leukaemia, 381 lyn, 194
selective immunoglobulin ion exchange chromatography, 357, 357 leukocyte adhesion deficiency, lys, 189
isotype deficiencies, 407 IP-10, 301 224, 414–15 lysed cells, 134
immunoglobulin superfamily IRF-I, see interferon response factor leukocyte migration lysing virus-infected cells, 19
(IgSF), 87–88 Ir genes, 128 activation, 298 lyso-glyceryl ether
immunology of allogeneic ischemia, 284 adherence to endothelium, 298–99 phosphorylcholine, 275
transplantation, 363–67 ISCOMS, 355. See also immune chemokines, 300–301 lysosomal enzymes, 20
origin, 3 stimulating complex chemotactic molecules, 298, 302 lysosomal hydrolases, 271
immunopathological response, 316 isoheamagglutinins, 282 tethering, 298 lysosomes, 6
immunoprecipitation, 89, 89 isotype, 86 leukocytes, transmigration of, 295 lysozymes, 5, 20
immunoreceptor tyrosine-based I-TAC, 301 leukotrienes, 273–75, 303 lytic virus, 316
activation motifs, 151), ITAM, see immunoreceptor tyrosine- leupeptin, 238 Lyt markers, 162
253–54, 271 based activation motifs LGL, see large granular lymphocytes Lyt protein, 162
immunotoxins, 398–99 ligand analogue, 133
inactivated vaccines, 346–47
incomplete adjuvants, 65
J ligand isopentenyl
pyrophosphate, 178
M
indirect immunosorbent jellyfish toxins, 267 light-chain rearrangement M cells, 52
assay, 310–11, 310 J (joining) chains, 77 allelic exclusion, 106 M-protein, 44
infection, development of, 315 junctional diversity, 107–108 immunoglobulins, 100–101 macroglobulin, 78
inflammatory cytokines, 303 linear/ sequential determinants, 61 macrophage-CSF (M-CSF), 27
inflammatory responses, 295–96
anti-inflammatory agents,
K lipid antigens, 241
lipid mediators, inflammatory,
macrophages, 7–8, 43, 218, 237
major histocompatibility complex
308–310 KAR, see killer activation receptors 303–304 antigens, 119
mediators of, 302–303 K antigen, 44, 284 lipopolysaccharide, 324 major histocompatibility complex
process, 303–308 kallidin, 303 lipopolysaccharide (LPS), 9, 63, 192 (MHC), 11–16, 35
inflammatory responses, to kallikrein, 224, 303 liposomes, 354 class I MHC molecules, 120–21,
anaphylatoxins, 217–18 keyhole limpet haemocyanin, 138 Listeria monocytogenes, 249, 257, 324 232–37, 255, 392–93
influenza, 322–24 killer activation receptors, 36, 253 live attenuated vaccines, 345–46 class II MHC molecules,
influenza viruses, 322–23 killer inhibitory receptor, 36, 253 live vector vaccines, 348–50 121–22, 232, 237–40, 440
inhibitors, of viral infections, 5 kinetic proofreading model, 169–70 LMP2, 234 class III MHC molecules,
innate defence, against intracellular kinin system, 303, 305 LMP7, 234 122–23
bacteria, 325–26 KIR, see killer inhibitory receptor LMP10, 234 codominant expressions of, 131
innate immune defence, against Klebsiella pneumoniae, 347 LTB4, 271 distribution of class I and class II
virus, 316–17 Kupffer cells, 7, 38, 284 luetin, 257 molecules, 130–31
innate immunity, 4–10 Lutzomyia, 335 gene map, 125–28
innocent bystander effect, 215
inosine monophosphate
L lyme disease, 331–32
lymphatic vessels, 55
haplotypes, 126
HLA typing, 134
dehydroxygenase, 370 L-selectin, 297 lymph nodes, 48–49 human loci of, 128–30
inositol 1, 4, 5 triphosphate L. donovani, 336 lymphoblasts, 32 I genes, 128
(IP3), 173, 190 L. tropica, 336 lymphocyte choriomeningitis murine, 126–28, 126–127
integrins, 297 LAD, see leukocyte adhesion virus, 204 peptide-binding clefts of, 123–24
intercellular adhesion molecules deficiency lymphocytes, 9–11, 324 peptide interactions with, 124–25
(ICAM), 87 LAK cells, see lymphokine-activated B cells, 34. peptides from, 120
interdigitating dendritic cells, 41 killer cells in brain, 85 polymorphism, 130
interferon response factor, 132 lamina propria, 45 development, 32–34 role of, 119
interferons, 5, 132 Langerhans cells, 26, 41, 53, 63 natural killer (NK) cells, 36–38 on thymic antigen-presenting
interleukin-1 converting enzyme, 251 large granular lymphocytes, 36, 252 T cells, 34–36. cells, 166–67
INDEX 467
transcriptional regulation of molluscs, 20 cytotoxicity, 254–56 oedema, 8

molecules, 132–33 Molluscum contagiosum virus function of, 37 oncofoetal tumour antigens, 388–89
major immunogene complex (pox virus), 321 mediated immunity, 390–91 oncogenes, 383
(MIC), 128 monoclonal antibodies, 139, 398 receptors, 253 and cancer induction, 383–87
malaria, 4, 284, 332–35 monocyte-/macrophage-based surface markers, 252–53 one gene–one polypeptide
Malar rash, 435 surface markers, 38 surface molecules, 37 principle, 94, 96
MALT, see mucosa-associated monocytes, 218 natural live vaccines, 345 opsonins, 9
lymphoid tissue mononuclear phagocytes, 13 NCF-A, 275 opsonization, 85
mammalian TLR, 247 mononuclear phagocyte system necrosis, 8, 30 Orthomyxoviridae, 216, 317, 322
mannan-binding pathway, of cells of, 38 negative selection, of immature Ouchterlony technique, 205, 205
complement system, 214–15, 215 mononuclear phagocytic cells, 231 B cells, 187–88 oxygen-dependent killing
mannan (mannose)-binding lectin, 214 monosaccharides, 65 negative selection, of T cells, mechanism, 43
mannosyl–fucosyl receptors, 38 MOPC321, 95 168–71, 168 oxygen-independent killing
MAPK, see mitogen-activated protein mouse TCR genes locus, 141–43 Neisseria infections, 223 mechanism, 43–44
kinase mucin, 297 Neisseria meningitidis, 223, 328,
margination, 8
MAS, see Mycoplasma arthritidis
mucosa-associated lymphoid tissue,
45, 51
347, 416
neoantigenic determinants, 62
P
mast cell, 218, 270–71, 271 mucosal immunity, 85 neonatal immunity, 85 p50 homodimer, 132
activation of, 273 mucosal mast cells (MMC), 270 neoplasm, see cancer p53 protein, 236–37
mast cells, 13, 41 mucous membrane, 4–5 neutralization, of viral infection, 220 P. falciparum, 334–35
MBL, see mannan (mannose)- multi-lineage-CSF (multi-CSF or neutropenia, 20, 415 P-selectin, 297
binding lectin IL-3), 27 neutrophil chemotaxis, 44 PAF, see platelet-activation factor
MBP, see myelin basic protein multiple germ-line gene segments, 107 neutrophils, 10, 13, 39–40, 43, PAMP, see pathogen-associated
MC159, 321 multiple sclerosis, 19, 436–37 218, 278, 324 molecular patterns
measles virus, 216 mutated cellular gene products, 388 New Zealand black (NZB) mouse, 438 papain cleavage site, 121
mechanical/physical barrier, of myasthenia gravis, 433–34 nitric oxide, 43 Papillomoviruses, 4
immunity, 4–5 mycobacteria, 178, 204, 324 nitrogen species, 20 Paramyxoviridae, 317
medullary macrophages, 165 Mycobacterium avium, 240 NK-cell surface molecules, 37 Paramyxoviruses, 216
membrane-bound immunoglobulin, Mycobacterium bovis, 348 NKG2 proteins, 36 paraoxysmal nocturnal
110–11 Mycobacterium leprae, 240, 257 NOD mice, see non-obese diabetic haemoglobulinuria, 224
membrane immunoglobulin (mIg), 193 Mycobacterium tuberculosis, 85, 240, mouse parapoptosis, 30
membrane-spanning proteins, 120 257, 261, 289, 327, 330–31, 345 Nod proteins, 247 parasitic worms, diseases from, 337–38
memory cells, 17–18, 33, 34 Mycoplasma arthritidis, 64 non-Hodgkin B-cell lymphoma cells, 241 passive immunity, 16
B cells, 197, 97 myelin basic protein, 438–39 non-lysozyme bactericidins, 20 passive immunotherapy, 397
meningitis, 284 myeloid progenitor cells, 27, 38 non-obese diabetic mouse, 437 patch test, for hypersensitivity, 280
mesangial cells, 38 myeloperoxidase enzyme, 39, 43 non-overlapping determinants, 61 pathogen-associated molecular
metastasis, 381 Myxomavirus, 4 non-peptide bacterial patterns, 9, 44, 247, 248, 324–25
micelles, 354 antigens, 240–41 pathogens, 4
microencapsulation delivery system, 354
microglia, 8
N non-polymorphic amino acid
residues, 124
pattern-recognition receptors , 9
Pauling’s direct template theory, 94
β2-microglobulin, 14, 120, N nucleotides, 103–104, 108, 148, 186 non-productive rearrangement, 107 Pax-5, 185
131, 240 N. gonorrhoeae, 223 non-sequential determinants, 61 PDGF, see platelet-derived growth
microlymphocytotoxicity test , 134 N-linked carbohydrate chains, 151 non-steroidal anti-inflammatory factor
microthrombi, 284 N-linked oligosaccharides, 121, 141 drugs, 309–310 penicillin, 65
migration inhibition factor, 289 NADPH oxidase complex, 43 NSAID, see non-steroidal anti- peptide antigens, 192
MIIC, 239 naïve B cells, 188, 220 inflammatory drugs peptide-binding clefts, 120, 121,
minor histocompatibility antigens mature, 188 nuclear factor-Y (NF-Y), 132 123–24, 123
(mH), 119 vs memory B cells, 197 nucleotides, 60. peptide-binding unit, 121
missing self hypothesis, 37 naïve cells, 32, 111 null cells, see natural killer (NK) cells peptides, role in positive selection, 167
mitochondria, 29 nasal-associated lymphoid tissue nurse cells, 47 peptide transportation, from
mitogen-activated protein (NALT), 45, 51 NY-ESO-1, 389 cytplasm to ER, 234–35
kinases, 173, 190 natural antibodies, 189 peptidoglycans, 9
Mls protein, 176
MLCT, see microlymphocytotoxicity
natural killer (NK) cells, 10, 12, 27,
36–38, 88, 179–80, 247,
O perforin, 30, 32
perforin and granzyme pathway, 250–51
test 252–56, 317 O-linked carbohydrate side chains, 297 perforin-mediated osmotic lysis, 37
molecular mimicry, 438 activatory receptors, 253–54 obese strain (OS) chicken, 437 perforin monomer, 251
468 INDEX
perforin monomers, 254 P-region nucleotides, 102, 104, 148 RAG-1, 103, 105 scavenger receptors, 10
peripheral blood lymphocytes, 34 prenylpyrophosphates, 241 RAG-2, 103, 105 schistosoma parasites, 338
pernicious anaemia, 19, 431, 431 pre-T-cell stage, 162 RANTES, 301 SCID, see severe combined
peroxynitrite, 43 primary/congenital rapamycin, 370 immunodeficiency.
Peyers’ patches, 49, 52, 45, 52 immunodeficiency, 405 rapamycin–FKBP complex, 370 scleroderma, 437
PFEMP1, 335 cause of, 406 Ras-associated binding protein, 251 sebaceous glands, 4
pH, of skin, 5 major, 406–414 Ras pathway, 172 secondary immune response, 17
phagocytes, 6, 8 primary immune response, 343 Ras protein, 173 secondary immunodeficiency, 405,
and apoptic cells, 30 productive rearrangement, 107 RB, see retinoblastoma 417–25
phagocytic cell deficiencies, 407 professional antigen-presenting RCA, see regulators of complement secreted immunoglobulin, 110–11
phagocytosis, 38, 218, 231 cells, 230 activation gene selectins, 297
bacterial killing mechanism, progenitor B cell (pro-B cell), 186, 205 reactive nitrogen species , 326 selective IgA deficiency, 410
43–44 programmed cell death, see reactive oxygen species , 20, 218, 326 self-MHC molecules, 149,
defined, 42 apoptosis receptor editing, 187 155, 255
strategies to overcome phagocytic promoters, 114 receptor occupancy model, of sensitization phase, in DTH response,
defences, as 44 properdin, 213 T cells, 169 257–58
phagocytosis, of invading PRR, see pattern-recognition receptor of complement (CR1), 219 sensitized allergens, 267
microorganisms, 6 receptors recombinant antigen vaccines, 348 sensitized indicator cells, 224
phagosome, 6 prostaglandins, 273–74 recombination signal sequences, 101 septae, 47–48, 47
phagosomes, 42 prostaglandins (PGD2), 275 red blood cells, 26 serine proteases, 274
Philadelphia chromosome, 385 pro-T cells, 162 red marrow, 46 serosal macrophages, 38
Phlebotomus, 335 proteasome complex, 233, 234 regulators of complement activation serum sickness, 20, 288
phospholipase-C-initiated pathway, 172 protectin, 222 gene, 221 severe combined immunodeficiencies,
phosphophatidylinositol-specific proteinase inhibitors, 238 regulatory factor X , 132 105, 406–409
phospholipase Cγ 1, 190 protein tyrosine kinases, 172 regulatory Treg cells, 36 Shigella flexineri, 247
phosphorylation, 62 proteoglycans, 271 respiratory burst, 43, 218 side scatter, 54
phylogenetic distance, 63 proteolysis, of protein antigen, 62 respiratory tract, 4–5 silencers, 114
Picornaviruses, 4–5 proto-oncogenes, 383–85, 384, 385 retinoblastoma, 386 single radial immunodiffusion, 115, 115
pigeon fancier lung, 284 protozoan infections, 332–37 retrograde translocation, 235–36 skin test, for hypersensitivity, 278–80
plague epidemic, 3 pseudogenes, 143 Retroviruses, 20, 216 SLE, see systemic lupus erythematosus
plasma cells, 11 Pseudomonas aeruginosa, 44, 247, 328 rhesus (Rh) antigen compatibility, SLP-76, 172
plasmapheresis, 437, 442 pTαβ heterodimers, 145 283–84 small lymphocytes, see naïve cells
plasmoblasts, 200 PTK, see protein tyrosine kinases rheumatoid arthritis , 19, 284, 436 small pox vaccination, 3
Plasmodium, 4 purified protein derivative rheumatoid factor, 436 smooth muscle cells, 218
platelet-activation factor , 271, 275 antigen, 178, 261, 290 Rhinoviruses, 4–5 Snell’s experiment, 119
platelet-derived growth factor, 261 pus formation, 8, 296 rhogam, 283 sodium cromolyn, 280
platelets, blood, 42 pyrogenesis, 296 Rickettsia, 44 sodium dodecyl sulphate (SDS), 242
PNH, see paraoxysmal nocturnal pyrogenic exotoxins, 64 RMA-S cells, 235 solid-matrix–antibody–antigen
haemoglobulinuria RSS, see recombination signal complexes (SMAA), 355
Pneumococci, 4, 9
polacrylamide gel, 242
Q sequences
rubor, 304
somatic diversification theory, 106
somatic hypermutation, 109–110
polyadenylation, 110–11 Quil A, 65 somatic rearrangements, 141
polyclonal B-cell activators, 192
polyisoprenoid phoshoglycolipids, 241
quinolines, 334 S Sos catalyse, 173
specific immunity, 10
polymorphism, 126, 130–31
polyoma, 383
R S box, 132
S protein, 222
specimen preparation, of TEM, 157
sperm whale myoglobin, 59
polysaccharide antigens, 60 RA, see rheumatoid arthritis S region, 110 spleen, 49–51
polysaccharides, 63 Rab protein, see Ras-associated binding Sabin polio vaccine, 346 src, 190
polysaccharide vaccines, 347–48 protein S-adenosylhomocysteine, 408 Src family tyrosine kinase, 162
polyvalent polysaccharides, 204 Rac pathway, 172, 174 Salk polio vaccine, 346 SRS-A, 303
positive selection, of T cells, radioallergosorbent test (RAST), Salmonella, 217 Staphylococcal enterotoxin, 176
165–68, 170–71 for hypersensitivity, 280 Salmonella typhi, 44, 327 Staphylococcal enterotoxin B (SEB), 64
PPD, see purified protein derivative radioimmunoassay (RIA), Salmonella typhi (Ty2la), 348 Staphylococcal exfoliative toxin, 64, 176
pre-B cells, 186 425–26, 425 scanning electron microscopy (SEM), staphylococcal infective endocarditis, 284
precipitin, 206 radioimmunosorbent test (RIST), for 180–81, 180 Staphylococci, 4
pre-cytotoxic T lymphocytes, 249 hypersensitivity, 280 scavenger cells, 38 Staphylococcus aureus, 44, 65
INDEX 469
stem cell, defined, 25 complex, 150–53 mediated graft rejection, 364–65 thymocytes, 47
stem-cell differentiation, 27 cross-reactivity of, 155–56, 156 mediated immunity against thymotaxin, 162
stem-cell factor (SCF), 27, 186 DNA-level gene rearrangement, 144 cancer, 390 thymus, 45, 47, 45
stop codon, 148 enhancers, 149–50 mediated immunity against thymus-dependent (TD) antigens,
streptococcal streptolysin, 44 genetic arrangements, 141–43 tuberculosis, 261 192–93, 193
Streptococci, 327 glycosylation of, 141 mediated xenograft rejection, 373 thymus-independent (TI) antigens,
Streptococcus enterotoxin, 176 human genes locus, 142–43 memory, 175 192–93, 193
Streptococcus pneumoniae, 44, 247, immunoglobulin genes monoclonal antibodies and, 371 thyroiditis, 19
328, 347, 416 rearrangements, 147 naïve, 174 thyroid-stimulating hormone (TSH)
Streptococcus pyogenes, 329 imprecise recombinations, 147–48 natural killer (NK), 179–80 receptors, 19
Streptococcus pyrogens, 64 junctional diversity, 147 negative selection, 168–71 TI-1 antigens, 192
stromal cells, 27 and MHC molecules, 137, 137 positive selection, 165–68, 170–71 tingible body macrophages, 200
suicide (apoptosis), of the target cell, 37 models for T-cell recognition, 138 precursors, 162 tissue-resident dendritic cells, 41
superantigen cross-links Vβ chain, of mouse genes locus, 141 proliferation, 174 tissue-specific (TS) stem cells, 25
TCR, 169 and NK cells, 252 specificity of γδ, 178 TNF receptor associated factor
superantigen-encoded protein, 168 P- and N-nucleotide superantigen-induced activation, (TRAFs), 194
superantigens, 64–65, 177, 177 additions, 148 176–77 Togaviridae, 317
superoxide anion, 43 pairing of α- and β-chain tolerance of, 168 tolerance induction, in lymphocytes, 63
surface molecules, on a genes, 148 T suppressor cells (Ts), 36 tolerance mechanism, 19
macrophage, 39 peptide–MHC complex, 171 TAP heterodimers, 235 Toll-like receptors, 324
surrogate light chains, 186 promoters, 149–50 TAP-associated protein, see tapasin toxins, 86
SV40 virus, 346, 383 RAG-1/RAG-2 expression, 146 TAPA-1 (transmembrane- toxoids, 347
switch recombination, 113 rearrangement of α-chain genes, protein-1), 190 toxoid vaccines, 347
switch regions (S), 113 145–46 Tcyt cells, 12, 17–18, 35–36, 140 transcription factor-CREB, 190
syk, 190, 194 relation with immunoglobulins, antigen-specific, 247–52 transcytosis, 52
syngenic graft (syngraft )/isograft, 361 139–41 apoptosis in, 30 trans-endothelial migration, of
synthetic homopolymers, 63 role of k chain enhancer, 147 CD8+, 249 leukocytes, 259
synthetic vaccine, 354 silencers, 149–50 cytolytic function of, 250–52, 252 transfusion reactions, 282–83
systemic lupus erythematosus, splicing and maturation of the effector function of, 171 transient hypogammaglobulinemia of
19, 284, 435 primary transcript, 144–45 Fas ligands on, 250 infancy, 412
structural diversity, 147–48 pre-, 249 transmembrane segment, 121
T structure of, 139–41

superantigen cross-links Vβ chain
proliferation and differentiation
of, 249–50
transmission electron microscope
(TEM), 157, 157
tapasin, 235 of, 169 tartrazine, 273 transplantation antigens, 362–63
T lymphocytes, 11–13, 26, 33, surface expression of, 152 teichoic acid, 9 transplantation immunology
34–36, 55 TCR–antigen–MHC telangiectasis, 414 acute rejection, of an allograft, 368
T regulatory (Treg) cells, 175 ternary complex, 154–55 terminal deoxynucleotidyl allegeneic transplantation, 363–67
T-cell differentiation, 46 V, (D), J combinations, 147, 149 transferase (TdT), 103 bone marrow transplantation,
T-cell epitopes, 64, 68 T-cell rich paracortex, 199 terminal deoxyribonucleotidyl 375–76
T-cell marker, 35–36 T cells, 34–36, 63 transferase (TdT), 186 chronic rejection, of an allograft,
T-cell maturation, 17, 161 activation of, 171–76 tetanus bacilli, 347 368–69
T-cell receptors, 162 CD4+ cells, 174–75, 179 tetrapeptides, 271 donor, defined, 361
α and β chain genes, 144–46 CD8+ cells, 174–75, 179 Tfa, 190 GVHD reactions, 376–77
α-chain enhancers, 149 and costimulation, 175–76 thalassin, 267 grafts, defined, 361
α-chain gene rearrangement, 163 deficiencies, 407, 412–13 TH cell-derived cytokines, 194 heart transplantation, 374–75
β-chain enhancers, 149–50 and dendritic cells, 231 T helper cells, 12, 17–18 histocompatibility, 365
δ- silencer, 150 derived IFN-γ, 260 and antigen-presenting cells, 229 hyperacute rejection mechanism,
γ and δ genes, 146 development, 162–64, 164 cytokine-secreting, 247 367–68
γ-chain enhancers, 150 differentiation, 174 TH1 cells, 36 immunosuppressive treatments,
γ- silencer, 150 effector, 175 TH2 cells, 36 369–72
accessory molecules of, 153–54 fall of response, 176 thiolester bond, 210 kidney transplantation, 373–74
allelic exclusion of, 146–47 functional competence of, 161 thrombocytopenic purpura, 432 leukocyte reactions, 365, 366
CD3 complex of, 151 functions of γδ, 178–79 thromboxanes, 304 liver transplantation, 375
clones, 139 maturation, 161, 163, 164 thymic corpuscles, 47 lung transplantation, 374
complementarity determining mediated antiviral mechanism, thymic education, 47–48, 165, 166 method of blocking costimulatory
regions, 149 318–19 thymic epithelial cells, 165 signals, 371–72
470 INDEX
transplantation immunology
(Continued)
tumour-specific antigens,
387–88
vascular cell adhesion molecule
(VCAM), 87–88
X
organ transplantation, 373–76 tumour suppressor genes, 383, 383, vasoactive amines, 286 X1 box, 132
pancreas transplantation, 375 385–86, 386 vasodilation, of blood vessels, 8 X2 box, 132
recipient, defined, 361 type I diabetes, 433 Vav, 174 X2 box, 132
skin transplantation, 375 type-I IFN functions, 316 VCAM, 52, 259 xenoantigens, 362
transplantation antigens, 362 typhoid bacilli, 5 V(D)J recombinase, 102 xenogeneic graft (or xenograft),
transplants to priveleged sites, 373 tyrosine residues, of ITAM, 154 defects in, 104–105 362
types of graft rejection, 367–69 veiled cells, see circulating dendritic xeroderma pigmentosum, 383
xenotransplantation, 372–73
Treponema pallidum, 44
U cells
vertebrate immune system, 20
X-linked aggamaglobulinemia,
409–410
Trichinella spiralis, 337 ubiquitin, 233 very late antigens (VLAs), 299 X-linked disease, 223, 413
tuberculin, 257 ubiquitination process, 233 Vibrio cholerae, 324 X-linked hyper IgM (X-HIGM)
tuberculin reaction, 290 urogenital tract, 4 viral avoidance, of immune syndrome, 412
tuberculin-type hypersensitivity, response, 320–22 X-linked SCID, 406
30, 289
V viral double-stranded RNA, 9
tuberculosis, 257, 330–31
tumour cell lysis, 178 V, (D), J gene recombination, 107
viral infections, 322–24
immunity to, 316–19
Y
tumour necrosis factor (TNF), 256 V, D or J gene segments, viral tk, 348 Y box, 132
tumour, 381 rearrangement of, 101–105 viral vector vaccine, 348–50 yellow marrow, 46
tumour cells, 19 vaccination, 3 virus-encoded protein
tumour infiltrating lymphocytes
(TIL), 398
vaccines
defined, 343
antigens, 387
V–J recombination, 187
Z
tumours, of the immune system. developments, 353–55 VLA-4, 186 ZAP-70, 172–73, 409
See also cancer function of, 343 zymogens, 220
antigen masking, 393
evasion of immune response,
ideal, 355–56
types of, 344–52
W
392–93 Vaccinia, 348 Waldeyer’s ring, 51
immune response to, 390–92 val, 189 warm antibodies, 432
tumour antigens, 387 variable (V) region of light (VL), 75 whooping cough vaccine, 346
tumour-associated antigens, variant surface glycoprotein Wiskott–Aldrich syndrome, 413
388–90 (VSG), 335 Wuchereria spp., 337
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The Elements of

Delhi • Chennai • Chandigarh

About the Cover

Library of Congress Cataloging-in-Publication Data

Khan, Fahim Halim.

Copyright © 2009 Dorling Kindersley (India) Pvt. Ltd.

Head Office: 482 FIE, Patparganj, Delhi 110 092, India

Layout designed by Satwinder Singh Channey

Pearson Education Inc., Upper Saddle River, NJ

1 An Introduction to the Immune System

1.2 THE COMPONENTS OF IMMUNITY 4

1.3 TYPES OF IMMUNE RESPONSE 15

1.4 ACTIVATION OF THE IMMUNE RESPONSE 17

1.5 IMMUNE DISORDERS 19

1.6 EVOLUTION OF IMMUNITY 20

2 Cells and Organs of the Immune System

2.2 REGULATION OF HAEMATOPOIESIS 29

2.3 CELLS OF THE IMMUNE SYSTEM 31

2.5 LYMPHOID TISSUES 44

2.6 LYMPHATIC SYSTEM 53

3.2 GENERAL PROPERTIES OF ANTIGENS 62

3.3 B-CELL AND T-CELL EPITOPES 63

4.2 LANDMARKS IN THE ELUCIDATION OF ANTIBODY STRUCTURE 71

4.3 ANTIBODY STRUCTURE 74

4.4 CLASSES OF IMMUNOGLOBULIN 78

4.5 ANTIBODY-MEDIATED EFFECTOR FUNCTIONS 84

4.6 MUCOSAL IMMUNITY 85

4.7 NEONATAL IMMUNITY 85

4.8 ANTIBODIES CAN BE ANTIGENS TOO 86

4.9 IMMUNOGLOBULIN SUPERFAMILY 87

5 Generation of Antibody Diversity

5.2 GENETIC ORGANIZATION OF IMMUNOGLOBULIN GENES 96

5.3 REARRANGEMENT OF GENES 98

5.4 ALLELIC EXCLUSION 105

5.5 THE GENERATION OF DIVERSITY IN IMMUNOGLOBULINS 106

5.5.3 JUNCTIONAL DIVERSITY 107

5.6 MEMBRANE-BOUND AND SECRETED IMMUNOGLOBULINS 110

5.7 ASSEMBLY AND SECRETION OF IMMUNOGLOBULINS 112

5.8 CLASS SWITCHING 112

5.9 REGULATION OF IMMUNOGLOBULIN GENE TRANSCRIPTION 114

6 Major Histocompatibility Complex

6.2 CLASS I MHC MOLECULES 120

6.3 CLASS II MHC MOLECULES 121

6.4 CLASS III MHC MOLECULES 122

6.5 STRUCTURE OF PEPTIDE-BINDING CLEFT 123

6.6 PEPTIDE–MHC INTERACTION 124

6.7 GENE MAP OF THE MAJOR HISTOCOMPABILITY COMPLEX 125

6.8 HUMAN MHC LOCI 128

6.9 MHC POLYMORPHISM 130

6.10 DISTRIBUTION OF CLASS I AND CLASS II MHC MOLECULES 130

6.11 TRANSCRIPTIONAL REGULATION OF MHC MOLECULES 132

7.2 STRUCTURE OF T-CELL RECEPTOR 139

7.3 ORGANIZATION OF T-CELL RECEPTOR GENES IN THE GERM LINE 141

7.4 REARRANGEMENT OF GENES TO FORM MATURE AND β GENES 144

7.5 REARRANGEMENT OF AND δ GENES 146

7.6 ALLELIC EXCLUSION OF TCR GENES 146

7.7 INHIBITION OF IMMUNOGLOBULIN GENE REARRANGEMENT IN T CELLS 147

7.8 GENERATION OF STRUCTURAL T-CELL RECEPTOR DIVERSITY 147

7.9 COMPLEMENTARITY DETERMINING REGIONS (CDR) OF T-CELL RECEPTOR 149

7.10 TCR GENES DO NOT UNDERGO SOMATIC HYPERMUTATION 149

7.11 PROMOTERS, ENHANCERS AND SILENCERS OF T-CELL RECEPTORS 149

7.12 T-CELL RECEPTOR COMPLEX 150

7.13 ACCESSORY MOLECULES ON T CELLS 153