Introductory Textbook

@ NEW AGE INTERNATIONAL PUBLlSIIERS
IMMUNOLOGY
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As t ext books and jour nals of immunology gr ow in size and number and t he explosion of
infor mat ion floods t he libr ar ies, st udent s oft en find t hemselves over whelmed by t he volume of
r eading r equir ed t o under st and basic concept s. Assimilat ing a fast changing, ever expanding
subject like immunology can be a daunt ing pr ocess. As a t eacher of immunology and clinical
micr obiology for under gr aduat e medical st udent s, I have t r ied t o pr esent t he basic t enet s of t he
subject of immunology in a simple and under st andable manner , ver y much like t he many
lect ur es I have given t o st udent s over t he year s. I have t r ied t o evolve a st or y so t hat st udent s
may be inspir ed t o r ead on and exper ience t he wonder of scient ific discover y. To t his end I owe
my own enjoyment of t he subject t o t he many excellent ar t icles in ‘Scient ific Amer ican Medicine’,
t o t he pict or ial pr esent at ion of immunological concept s in Ivan Roit t ’s excellent t ext books of
Immunology and t o ‘Basic and Clinical Immunology’ of t he Lange Medical Publicat ion ser ies. I
gr at efully acknowledge t hese sour ces for t he many ideas and figur es t hat I have adapt ed for
t his book. The second edit ion has been car efully updat ed, wit h some chapt er s being r ewr it t en
incor por at ing sever al new illust r at ions, in keeping wit h scient ific advances. A new chapt er
dedicat ed t o t he immunology of HIV has been added in r ecognit ion of t he devast at ing pandemic
t hat has r e-defined global healt h. I hope t he book will be of use t o st udent s of medicine,
micr obiology, nur sing and for any one else in t he life sciences who feels t he need t o explor e t his
fascinat ing ar ea of science.
Lon d on Na n d i n i Sh e t t y
PREFACE TO THE SECOND EDITION
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PREFACE TO THE FIRST EDITION
As textbooks and journals of immunology grow in size and number and the explosion of
information floods the libraries, students often find themselves overwhelmed by the volume of
reading required to understand basic concepts. Assimilating a fast changing, ever expanding
subject like immunology can be a daunting process. As a teacher of immunology and clinical
microbiology for undergraduatemedical students, I have tried to present the basic tenets of the
subject of immunology in a simple and understandable manner, very much like the many
lectures I have given to students over the years. I have tried to evolve a story so that students
may be inspired to read on and experience the wonder of scientific discovery. To this end lowe
my own enjoyment of the subject to the many excellent articles in 'Scientific American Medicine',
to the pictorial presentation of immunological concepts in Ivan Roitt's excellent textbooks of
Immunology and to 'Basic and Clinical Immunology' of the Lange Medica! Publication series. I
gratefully acknowledge these sources for the many ideas and figures that I have adapted for
this book. I hope the book will be of use to students of medicine, microbiology, nursing and for
anyone else in the life· sciences who feels the need to explore this fascinating area of science.
London Nandini Shetty
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LIST OF ABBREVIATIONS
Chapter 1
BCG Bacille Calmet t e Guer in
C r egion Const ant r egion
Fab Fr act ion ant igen binding
Fc Fr act ion cr yst allizable
HLA Human leukocyt e ant igen
MHC Major hist ocompat ibilit y complex
T cell Thymus der ived cell or t hymocyt e
V r egion Var iable r egion
Chapter 2
CRP C-r eact ive pr ot ein
IL-1
IL-2
IL-3
IL-4 Int er leukins-1 t o 7
IL-5
IL-6
IL-7
NADPH Nicot inamide adenosine dinucleot ide phosphat e hydr ogen
Chapter 3
CD Clust er of differ ent iat ion
GALT Gut associat ed lymphoid t issue
MALT Mucosal associat ed lymphoid t issue
MBP Major basic pr ot ein
MPS Mononuclear phagocyt e syst em
NK Nat ur al killer
PMN Polymor phonuclear neut r ophil
Chapter 4
DNP Dinit r ophenyl
Chapter 5
CDR Complement ar it y det er mining r egions
C
H
Const ant heavy
C
L
Const ant light
H chain Heavy chain
L chanin Light chain
V
H
Var iable heavy
V
L
Var iable light
Chapter 6
D r egion Diver sit y
IVS Int er vening sequences
J r egion J oining r egion
Chapter 7
P Pr oper din
SLE Syst emic lupus er yt hemat osus
SRS-A Slow r eact ing subst ance-A
Chapter 8
CEA Car cinoembr yonic ant igen
CIE Count er immunoelect r ophor esis
ELISA Enzyme linked immunosor bent assay
HBsAg Hepat it is B sur face ant igen
HIV Human immunodeficiency vir us
IEP Immunoelect r ophor esis
IHA/PHA Indir ect /Passive haemagglut inat ion
RAST Radio aller gosor bent t est
RIA Radio immunoassay
RIST Radio immunosor bent t est
RPHA Rever se passive haemagglut inat ion
SDS-Page Sodium dodecyl sulphat e-Polyacr ylamide gel elect r ophor esis
Chapter 9
HAT Hypoxant hine aminopt er in t hymidine
HPRT Hypoxant hine phosphor ibosyl t r ansfer ase
PEG Polye t hylene glycol
Chapter 10
GBM Glomer ular basement membr ane
HTC Homozygous t yping cells
Ir genes Immune r esponse genes
Is genes Immune suppr essor genes
(xii)
MLR Mixed lymphocyt e r eact ion
PLT Pr imed lymphocyt e t yping
Chapter 11
TCR T cell r ecept or
Chapter 12
APC Ant igen pr esent ing cell
BCDF B cell differ ent iat ion fact or
BCGF B cell gr owt h fact or
ICAM Int er cellular adhesion molecule
IFN Int er fer on
IP3 Inosit ol t r iphosphat e
LFA Lymphocyt e funct ion associat ed ant igen
Tc T cyt ot oxic
Th T helper
Chapter 13
ADCC Ant ibody dependent cellular cyt ot oxicit y
BAF B cell act ivat ing fact or
LAF Lymphokine act ivat ing fact or
LPS Lipopolysacchar ide
MAF Macr ophage act ivat ing fact or
MDP Mur amyl dipept ide
MIF Migr at ion inhibit or y fact or
Mult i-CSF Mult i-Colony st imulat ing fact or
Chapter 14
CMI Cell mediat ed immunit y
EBV Epst ein Bar r vir us
ECF Eosinophil chemot act ic fact or
ESP Eosinphil st imulat or y pr omot er
FACS Fluor escence act ivat ed cell sor t er
GM-CSF Gr anulocyt e-monocyt e colony st imulat ing fact or
HTLV 1 Human T cell leukemia vir us 1
HTLV 2 Human T cell leukemia vir us 2
LIF Lymphocyt e inhibit or y fact or
LT Lymphot oxin
OAF Ost eoclast act ivat ing fact or
PHA Phyt o haemagglut inin
PPD Pur ified pr ot ein der ivat ive
PWM Pokeweed mit ogen
TNF Tumour necr osis fact or
(xiii)
Chapter 15
ECF-A Eosinophil chemot act ic fact or -anaphylaxis
NCF-A Neut r ophil chemot act ic fact or -anaphylaxis
PK Pr ausnit z küst ner
Chapter 16
Ab 1, 2, 3 Ant ibody 1, 2, 3
BSA Bovine ser um albumin
Id 1, 2, 3 Idiot ype 1, 2, 3
LATS Long act ing t hyr oid st imulat or
NZB New Zealand black
NZW New Zealand whit e
Chapter 17
LAK Lymphokine act ivat ed killer
Chapter 18
GVH Gr aft ver sus host
SCID Sever e combined immunodeficiency
Chapter 19
DNFB Dinit r oflur obenzene
FeLV Feline leukemia vir us
MCA Met hyl cholant hr ene
Chapter 21
HIG Human immunoglobulin
VZIG Var icella zost er immune globulin
Chapter 22
ADA Adenosine deaminase
AIDS Acquir ed immunodeficiency sundr ome
CVID Combined var iable immunodeficiency
(xiv)
CONTENTS
Preface to the S econd Edition .................................................................................... vii
Preface to the First Edition ........................................................................................ ix
List of Abbreviations ................................................................................................... xi
1. Milest ones in Immunology .......................................................................................... 1
2. Innat e Immunit y .......................................................................................................... 5
3. Immunobiology ........................................................................................................... 12
4. Ant igens and Immunogenicit y .................................................................................. 20
5. Immunoglobulins I: St r uct ur e and Funct ion ............................................................ 25
6. Immunoglobulins II: The Genet ics of Ant ibody Diver sit y ........................................ 33
7. The Complement Syst em .......................................................................................... 39
8. Det ect ion and Applicat ion of Ant igen-Ant ibody React ions ....................................... 50
9. Monoclonal Ant ibodies ............................................................................................... 70
10. The Major Hist ocompat ibilit y Complex .................................................................... 75
11. Immune Response Mechanismis I: B and T Lymphocyt es ....................................... 87
12. Immune Response Mechanismis II: Ant igen Pr esent at ion and
Pr ocessing; Mechanisms of Lymphocyt e Act ivat ion ................................................ 97
13. Cyt okines ................................................................................................................... 107
14. Cell-Mediat ed Immunit y ........................................................................................... 117
15. Hyper sensit ivit y ........................................................................................................ 126
16. Immunologic Toler ance and Aut oimmunit y ............................................................ 143
17. Immunopot ent iat ion and Immunosuppr ession ....................................................... 144
18 Tr ansplant at ion Immunology ................................................................................... 158
19. Tumour Immunology ................................................................................................ 166
20. Immunit y Against Infect ious Diseases ..................................................................... 174
21. Immunizat ion ............................................................................................................ 180
22. Immunodeficiency Diseases ..................................................................................... 186
23. Immunology of HIV Infect ion ................................................................................... 197
24. Immunit y and Malnut r it ion ..................................................................................... 200
Index .......................................................................................................................... 203
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W
it h t he evolut ion of t he ger m t heor y of disease, t he st udy of t he mechanisms of
i mmu n i t y a n d t h e pos s i bl e con qu es t of i n fect i ou s di s ea s es bega n a l mos t
simult aneously. Int er est in immunology gr ew out of t he ever yday evidence t hat t hose individuals
who sur vived: pocked and disfigur ed fr om t he dr ead disease, small pox never did cont r act t he
infect ion again. As ear ly as 1773, Volt air e r epor t ed on an ancient Chinese cust om wher e dr ied
and powder ed small pox scabs wer e inhaled, much like snuff, in at at t empt at pr event ing t he
disease. Alt hough it would t ake several years before bact eria and viruses were fully charact erized,
t heor ist s wer e alr eady elucidat ing t he mechanisms of host defence against infect ious diseases
and t heir possible pr event ion.
The p h a gocyt i c t h eor y was t he fir st t o be developed in t he 1880s a bold and imaginat ive
concept — t he fr uit of Eli e Met ch n i k off’s deep knowledge of biology. Met chnikoff hypot hesized
t hat t he basis of inflammat ion was t he cellular r eact ion and t hat vascular and ner vous r eact ions
wer e only of secondar y impor t ance. He post ulat ed fur t her t hat t hese migr at ing cells which
wer e able t o move in or der t o meet an enemy wer e t he major guar dians of healt h against
bact er ial infect ions. It was ar ound t his t ime t hat Lou i s P a st e u r r epor t ed on t he t r eat ment of
his fir st t wo pat ient s wit h r abies using ear ly vaccines. In 1888 Met chnikoff left Russia t o cont inue
his wor k on phagocyt osis in Past eur ’s Inst it ut e in Par is. In 1908 he shar ed t he Nobel Pr ize
wit h Paul Ehr lich for his ear ly cont r ibut ions t o t he under st anding of inflammat ion.
Aft er Met chnikoff died in 1916 ot her wor ker s who had long cher ished t he t heor y t hat
solu b le su b st a n ce s in blood wer e also bact er icidal, r epor t ed t heir obser vat ions. Be h r i n g,
Nu t t a ll and Ni sse n cont r ibut ed subst ant ially t o t he concept of soluble or h u mor a l fa ct or s
but found t hat t hese fact or s wer e not always bact er icidal. Finally in 1903, Almr ot h Wr i gh t
wit h his disciple St ewa r t Dou gla s obser ved t hat a humor al component which t hey designat ed
op son i n , could r ender bact er ia suscept ible t o phagocyt osis. They t hus for ged a link bet ween
t he t wo major t heor ies of immunit y. Since t he t ime humor al and cellular component s wer e
shown t o be int er woven in an int r icat e pat t er n, t he complex chemicals associat ed wit h immunit y
have been t he cont inuing subject of year s of r esear ch.
Mer e empir ical met hods of pr oducing or incr easing immunit y wer e not sufficient , t he
whole b a si s of i mmu n i t y needed quest ioning. Behr ing found t hat cer t ain diseases wer e t he
expr ession of t he act ion of t oxi n s which could be neut r alized by a n t i t oxi n s. And it could not
be ignor ed t hat t he blood of animals cont ained cer t ain pr efor med bact er icidal agent s. Emi l
von Be h r i n g and Ki t a sa t o in 1890 inoculat ed animals wit h t oxins of dipht her ia and t et anus,
t o pr oduce neut r alizing ant it oxin ser um. They int r oduced p a ssi ve i mmu n i za t i on int o moder n
medicine and for t his, von Behr ing was awar ded t he Fr ench legion of honour and t he Nobel
Pr ize in 1901. Behr ing maint ained cor dial t ies wit h Past eur - one of his gr eat her oes and wit h
Met chnikoff he est ablished a cont inuing fr iendship t hat began in 1888. This fr iendship was
unique because t he t wo men wer e at opposit e poles in explaining basic aspect s of immunit y -yet
enjoying t he st imulus of int ellect ual debat e.
MILESTONES IN IMMUNOLOGY
CHAPTER – 1
2 Immunology– Introductory Textbook
Though eminent ly successful in passive immunizat ion against dipht her ia and t et anus,
von Behr ing failed abysmally when he t r ied t o ext end t hese pr inciples t o passive immunizat ion
against t uber culosis. At t his t ime Rob e r t Koch t oo had t o t ast e failur e in his at t empt s at
pr oviding a successful vaccine against t uber culosis. The pr event ion of t uber culosis was not t o
be found by t he Ger mans. It was in Fr ance t hat Alb e r t Ca lme t t e and Ca mi lle Gu e r i n
developed an effect ive va cci n e for t u b e r cu losi s by t he met hods of at t enuat ion so dear t o
Past eur and disdained by Koch.
To est ablish a live but at t enuat ed st r ain of Mycobacterium tuberculosis t hey had t o
cont inually t r ansfer or sub cult ur e t he or ganism t ill vir ulence was lost . This odyssey t ook t hem
t hir t een year s and 230 t r ansfer s lat er , on J an. 5, 1921 it was complet ely avir ulent even at high
doses for all animal species. The Ba ci lle Ca lme t t e Gu e r i n (BCG) was t hus bor n - it did not
induce t he for mat ion of t uber cles by int r avenous, int r aper it oneal or subcut aneous inoculat ion
or even by ingest ion, but for med an effect ive pr ophylact ic against t uber culosis.
P fe i ffe r in 1894-95, discover ed t he phenomenon of in–vivo cyt olysis of Vibrio cholerae
when t he or ganism and immune ser um int er act ed int r aper it oneally in t he guinea pig. Fr om
t hese ear ly exper iment s t he nat ur e and funct ion of comp le me n t me d i a t e d cyt olysi s wer e
elucidat ed by P fei ffer and lat er by Bu ch n er and Bor d et . J ules Bor det was awar ded t he Nobel
Pr ize in 1920 for his pioneer ing wor k on complement , for his discover y of t he whooping cough
agent and for his enunciat ion of t he diver se aspect s of ant igen – ant ibody r eact ions and t he
blood coagulat ion syst em. Ar ound t his t ime Gr u b e r and Du r h a m descr ibed t he diagnost ic
value of t he a gglu t i n a t i on r ea ct i on , when agar cult ur es wer e mixed wit h t heir cor r esponding
immune ser a. The same year F e r d i n a n d Wi d a l t he Alger ian bor n son of a Fr ench ar my
sur geon descr ibed t he agglut inat ion r eact ion for t yphoid fever —a t est which is st ill widely used
and bear s his name.
In 1667, J ea n Ba p t i st e Den i s, physician t o Louis t he XIV, per for med what is consider ed
t o be t he fir st t r a n sfu si on of b lood in man. The exper iment was not well r eceived by his
fellow-physicians who wer e mor e accust omed t o blood-let t ing t han r ever sing t he flow. Till t he
ear ly 1900s t he pr act ice of t r ansfusion of blood fr om man t o man r emained a r isky, unpr edict able
business, when at t he t ur n of t he cent ur y, Ca r l La n d st e i n e r discover ed t he blood gr oups.
Landst einer r eceived t he Nobel Pr ize in 1930 for elucidat ing t he blood gr oups and for his wor k
on t he Rh fact or .
As ser ot her a py developed, it soon beca me evident t ha t a nt igen- a nt ibody r ea ct ions in
vivo could ha ve some ha r mful effect s a nd even pr oduce dea t h. An a p h yl a xi s , t he most
dr a ma t ic ma nifest a t ion of hyper sensit ivit y wa s fir st descr ibed by P .P or t i e r a nd Ch a r l e s
Ri c h e t in 1902. In 1913 Cha r les Richet r eceived t he Nobel Pr ize in r ecognit ion of his wor k
on anaphylaxis.
Two of t he most vit al immunological hypot heses made in t he 1800s went undet ect ed for
sever al decades. They wer e r ediscover ed only in t he 1940s and for m a basis of t he st udy of
immunology t oday. Elie Met chnikoff suggest ed t hat phagocyt es wer e t he pr ime det ect or s of
for eign mat er ial— we t r ace t he or igins of cellu la r i mmu n i t y t o t his hypot hesis. P a u l Eh r li ch
pr oposed t he pr e–exist ence of r ecept or s (which he called t oxophor es) on t he living cell, t hat
r eact ed wit h t oxins. Excess r ecept or s wer e liber at ed int o t he cir culat ion as ant ibodies–t he
molecular basis of h u mor a l i mmu n i t y, as we know it t oday, is r emar kably close t o t his novel
but or iginal concept . Thus, t he foundat ions of moder n immunology, as we r ecognize it t oday,
wer e laid by sever al Eur opean scient ist s over t he last hundr ed or mor e year s.
The t went iet h cent ur y saw phenomenal pr ogr ess in t he under st anding of immunological
concept s. How ant igens dir ect t he var ious immunological pr ocesses was t he chief point of
cont ent ion among ear ly t heor ist s who had t o choose bet ween t he inst r uct ive and t he select ive
Milestones in Immunology 3
t heor ies. In t he past 30 year s t he biological basis of t he immune r esponse was, in par t , clar ified
when t he t heor y of clon a l se le ct i on of a n t i b od y for ma t i on found accept ance. This t heor y
pr oposed, in essence, by Paul Ehr lich almost a 100 year s ago fell out of favour wit h t he
int r oduct ion of t he t emplat e t heor y. The fir st compr ehensive at t ack against t he t emplat e t heor y
was launched in 1955 by Ni els Ka j J er n e who r ecalled cer t ain cr it icisms made by Ma cfa r la n e
Bu r n e t against t he t emplat e t heor y. It , however , r emained for Bur net t o dr aw t oget her t he
new concept ualizat ion and in 1957, he asser t ed t hat “each cell and it s clones can pr oduce just
one kind of r ecept or ” t o explain t he specificit y of t he immune r esponse and t he exponent ial r ise
in ant ibody pr oduct ion following cont act wit h ant igen. The secondar y r esponse is mor e power ful
because ant igenic memor y leads t o r apid clonal expansion dur ing subsequent cont act wit h t he
same ant igen. We know t oday t hat dur ing t his second exposur e t he binding abilit y of ant ibody
impr oves a nd we a lso know t his t o be t he r esult of a ffinit y ma t ur a t ion a nd soma t ic
hyper mut a t ion. The clona l select ion t heor y a lso expla ined t oler a nce a s t he delet ion or
suppr ession of an ent ir e clone of cells which could occur befor e or soon aft er bir t h; or even
much lat er in some inst ances. Macfar lane Bur net was awar ded t he Nobel Pr ize in 1960 which
he shar ed wit h P e t e r Me d a wa r . In 1984 Niels J er ne was also awar ded t he Nobel Pr ize for his
t heor et ical cont r ibut ions t o immunology, t he most fundament al of which was his r ole in
developing he concept of clonalit y and for his descr ipt ion of t he idiot ype net wor k in t he r egulat ion
of immune r esponses.
The e lu ci d a t i on of a n t i b od y st r u ct u r e is cr edit ed t o Rod n e y R. P or t e r and Ge r a ld
M. Ed elma n who shar ed t he Nobel Pr ize for t heir discover y in 1972. In 1959 Edelman showed
t hat t he immunoglobulin molecule had 4 polypept ide chains: 2 of each kind and t hat each could
be separ at ed by chemical means. He called t hem light and heavy chains because of t heir size.
At t he same t ime Por t er showed t hat t he molecule could be cut int o 3 differ ent pieces ( Fab x
2; Fc x 1) by enzymes t hat cleave polypept ides. In 1970 Edelman showed t hat chemical differences
r esponsible for t he specificit y of ant igen binding wer e embodied in t he amino acid sequences of
t heir var iable r egions at t he upper , out er ar ms of t he ant ibody molecule. All subsequent wor k
has confir med his conclusions wit h some elabor at ions. In 1970 T.T. Wu and E.A Ka b a t
demonst r at ed t he pr esence of hyper var iable r egions.
Dr e ye r and Be n n e t in 1965, suggest ed t hat t he ger m line cont ains many var iable (V)
r egion genes and one const ant (C) r egion gene, which combine t o yield an immunoglobulin
molecule. As a cell mat ur es, it select s one V gene out of many and combines it wit h t he one C
r egion gene. The t heor y was at t r act ive; yet it r equir ed t hat t he cell have some means of
r ear r anging genes in somat ic cells. Evidence t hat immunoglobulin genes do under go soma t i c
r e comb i n a t i on , but in mor e complicat ed ways t han Dr eyer and Bennet suggest ed, was found
in 1970 by Hozu mi and Ton ega wa t hen at Basel, Swit zer land. They demonst r at ed t hat V and
C genes wer e far apar t in embr yonic cells but much closer t o each ot her in plasma cells. They
also delineat ed t he mechanism of shuffling of t he many gene segment s. Tonegawa was awar ded
t he Nobel Pr ize in 1987 for elucidat ing t he mechanism by which t he immune syst em gener at es
an almost limit less var iet y of ant ibodies. Somat ic hyper mut at ion, as anot her major sour ce of
t he t r emendous diver sit y of ant ibody specificit y gener at ed, was demonst r at ed by Wei ger t and
Coh n in 1970.
The pr oduct ion of mon oclon a l a n t i b od i e s by somat ic cell hybr idizat ion of ant ibody
for ming cells and cont inuously r eplicat ing cell lines was called t he t echnique of hybr idoma
for mat ion. This t echnique descr ibed by Ge or ge s Koh le r and Ce sa r Mi lst e i n in 1975, has
ena bled immunologist s t o pr epa r e vir t ua lly unlimit ed qua nt it ies of a nt ibodies t ha t a r e
chemically, physically and immunologically complet ely homogenous. For t his invaluable
cont r ibut ion t o immunology t hey wer e awar ded t he Nobel Pr ize in 1984.
4 Immunology– Introductory Textbook
Immunobiology of t r a n sp la n t a t i on and t ole r a n ce was an act ive field of r esear ch in t he
1940s and 50s. In 1953, Me d a wa r , Br e n t and Bi lli n gh a m per for med a ser ies of dazzling
exper iment s t o explain t he mechanisms t hat enable t he host t o r ecognize and dest r oy for eign
cells. Medawar showed conclusively t hat such a mechanism was immunological. He suggest ed
t hat specific individualit y mar ker s wer e associat ed wit h ever y cell of an or ganism and t hat an
animal can be immunized against for eign gr aft s by fir st inject ing it wit h cells der ived fr om
immunocompet ent t issues of t he donor . Ear lier Ra y Owen had shown, in his classic exper iment
wit h dizygot ic t win calves, t hat dissimilar blood gr oup ant igens of one calf wer e t oler at ed by t he
ot her t win calf. Dr awing fr om t his exper iment Medawar demonst r at ed t hat an inbr ed st r ain of
mice – st r ain A could be made t o t oler at e skin gr aft s fr om anot her inbr ed st r ain – st r ain B by
inject ing t he st r ain A animal soon aft er bir t h wit h st r ain B spleen cells. Wit h t his exper iment
he pr oved t hat r esist ance (or t he lack of it ) t o for eign t issue is an immunological phenomenon.
Pet er Medawar was awar ded t he Nobel Pr ize (wit h Bur net ) in 1960.
Exi s t en ce of ma r k er s of bi ol ogi ca l i n di vi du a l i t y – wh a t we n ow k n ow a s t h e
h i st ocomp a t i b i li t y a n t i ge n s was fir st suggest ed by Gor e r in 1937, who demonst r at ed t hat
loci det er mining blood gr oup ant igens and t hose cont r olling t umour r eject ion in mice wer e
dist inct . Wit h Sn e ll in 1948 he showed t hat t her e wer e mult iple alleles in t he mouse H-2 locus
and subsequent ly t hat t his locus was genet ically complex. The H-2 locus was shown t o cont r ol
t he phenot ype expr ession of cer t ain cell sur face mar ker s in mice. In 1950 Da u s s e t ident ified
t he HLA ( Human Leukocyt e Ant igen) locus or MHC (major hist ocompat ibilit y complex) as it is
somet imes called. Be n a ce r r a f showed t hat genes of t he HLA det er mining loci may cont r ol
immune r esponses. Sn ell , Da u sse t and Be n a ce r r a f wer e awar ded t he Nobel Pr ize in 1980.
Elucidat ion of t he st r uct ur es of some HLA molecules by Bjor k ma n and Wi le y in 1987, using
X-r ay cr yst allogr aphy, r evealed t hat t he molecules of t he major hist ocompat ibilit y complex
(MHC) bind t o ant igenic pept ides and pr esent t hese pept ides t o t he T cell r ecept or .
Fir st glimpsed in exper iment s by Alli son , and Ka p p ler & Rei n h er z, t he T cell r ecep t or
was st udied and char act er ized by Ta k W.Ma k and Ma r k M. Da vi s in 1984. These wor ker s
cloned and sequenced a gene expr essed and r ear r anged in T cells but not in B cells. Their
analysis showed t hat many of t he T cell r ecept or sequences wer e homologous t o t hose of
immunoglobulin genes. P e t e r Doh e r t y wit h Rolf M. Zi n k e r n a ge l, found t hat T cells fr om
mice infect ed wit h a meningit is vir us dest r oyed vir us-infect ed cells only fr om t he same st r ain
of mice, and t hey showed t hat T cells must r ecognize t wo signals on an infect ed cell—one fr om
t he vir us and one fr om t he cell’s own ant igens—t o dest r oy it . For t his new under st anding of
cellular immune mechanisms t hey shar ed t he Nobel Pr ize in 1996.
Immunology cont inues t o fascinat e and fr ust r at e r esear cher s t he wor ld over . A bet t er
under st anding of t umour immunology, aut oimmunit y and r esponses t o immunizat ion could
develop int o st r at egies for pr ot ect ion against many cr ippling diseases. Event ually, what has
evolved as a pr ecise and power ful t ool cr eat ed by nat ur e t o ensur e t he cont inued sur vival of t he
species could per haps be manipulat ed t o yield a bet t er qualit y of life.
Bi bli ogr a p h y
1. Lecheva lier HA a nd Solot or ovsky M. Thr ee cent ur ies of Micr obiology Dover
Publicat ions Inc; New Yor k, 1974.
2. Par ish, H.J . A. Hist or y of Immunizat ion, E. and S. Livingst one, Lt d, Edinbur gh,
1965.
™™™
T
aliafer r o said, “The host is an island invaded by st rangers wit h different needs, different
food r equir ement s, differ ent localit ies in which t o r aise t heir pr ogeny”. Ther e ar e a
for midable r ange of infect ious agent s t hat can use t he human body as a sanct uar y t o r aise t heir
offspr ing. The immune syst em faces t he t ask of pr oviding a defence mechanism t o est ablish a
st at e t hat is known as immunit y t o infect ion.
Th e con t e mp or a r y d e fi n i t i on of i mmu n i t y is t her efor e: “All t hose physiological
mechanisms t hat endow t he animal wit h t he capacit y t o r ecognize mat er ials as for eign t o it self
and t o neut r alize, eliminat e or met abolize t hem wit h or wit hout injur y t o it s own t issues”.
Immunological responses serve three broad functions:
• Defence against micr o or ganisms
• Homeost asis; r emoval of damaged or effet e cells
• Sur veillance; r ecognit ion and dest r uct ion of mut ant cells.
These r esponses have been widely classified as n on –sp e ci fi c and sp e ci fi c. Among t he
non-specific defence mechanisms t her e ar e t hose t hat for m a set of ill under st ood and per haps
gr ossly under -emphasized con st i t u t i on a l fact or s t hat make one species innat ely suscept ible
and anot her r esist ant t o cer t ain infect ions.
These con st i t u t i on a l fa ct or s can best be list ed as–
(a ) Ge n e t i c: bet ween species, for example:
• Mycobacterium leprae seems t o infect humans and ar madillos only.
• Bacillus anthracis is an infect ion of humans t hough not of chickens.
• Gonor r hoea is a disease of man and chimpanzees and not of any ot her species. Some
species seem t o be able t o har bour or ganisms wit hin t heir body t issues while t he
same or ganism may cause anot her species t o succumb t o such an infect ion. Wit hin
man, t her e ar e cer t ain well known r a ci a l d i ffe r e n ce s in disease suscept ibilit y:
• dar k skinned individuals have an incr eased suscept ibilit y t o coccidioidomycosis.
• cer t ain dar k skinned people lack t he r ed cell Duffy coat and ar e not suscept ible
t o vivax malar ia.
Genet ic cont r ol of disease has been shown t o be st r ongly associat ed wit h t he major
hist ocompat ibilit y complex.
(b) Age: The ver y young ar e mor e suscept ible t o many infect ions, in par t icular , Escherichia
coli meningit is; t his may be because bact er icidal IgM does not cr oss t he placent a.
At t he ot her end of t he spect r um r icket t sial infect ions and cer t ain vir al infect ions of
childr en ar e mor e sever e wit h age.
INNATE IMMUNITY
CHAPTER – 2
6 Immunology– Introductory Textbook
(c) Me t a b oli c: Hypoadr enal and hypot hyr oid st at es decr ease r esist ance t o infect ion. In
diseases such as diabet es mellit us wher e alt er ed met abolism causes incr ease in blood glucose,
decr ease in pH and a r educed influx of phagocyt es - infect ion can be a sever e complicat ion.
St er oid hor mones ar e known t o affect many modalit ies of t he immune r esponse.
(d ) Ther e is a gr owing body of evidence t hat suppor t s t he hypot hesis t hat immune pr ocesses
can be influenced by n e u r oe n d ocr i n e fa ct or s . Immunologic cells have r ecept or s for a whole
r ange of hor mones. Cor t icost er oids, andr ogens, oest r ogens and pr ogest er one depr ess immune
r esponses, wher eas gr owt h hor mone, insulin and t hyr oxine do t he opposit e. Immunologic or gans
ar e inner vat ed by aut onomic and pr imar y sensor ial neur ons; hor mone secr et ion is balanced by
neur al cont r ol. It t her efor e seems r easonable t o say t hat immune r esponses ar e finely t uned
by neur o endocr ine cir cuit s. At a mor e physiologic level, st r ess and cir cadian r hyt hms modify
t he funct ioning of t he immune syst em.
(e) En vi r on men t : Poor living condit ions, over cr owding and under nut r it ion also incr ease
suscept ibilit y t o infect ion.
Figure 2.1. Natural barriers to infectious agents.
Na t u r a l b a r r i e r s t o infect ious agent s ar e simple yet effect ive means of innat e defence
(Figur e 2.1). A major for m of defence in t his cont ext is t he i n t a ct sk i n which is imper meable
t o most infect ious agent s. Swe a t and se b a ce ou s gla n d s ar e pot ent ial point s of ent r y for t he
infect ious agent . However , most bact er ia fail t o ent er due t o t he low pH and dir ect inhibit or y
effect s of lysozymes , la ct i c a ci d and ot her fa t t y a ci d s of sweat and sebaceous secr et ions. An
except ion is S taphylococcus aureus which commonly infect s t he hair follicle and glands.
Mu cu s se cr e t i on s of t he t r act s t hat connect int er nal or gans t o ext er nal sur faces for m
an impor t ant for m of defence. They ent r ap and immobilize bact er ia and hence, pr event adher ence
and colonizat ion of epit helial sur faces. Ha i r s a t t h e e xt e r n a l n a r e s, t he cou gh r e fle x and
t he cilia t ed mu cu s membr a n e of t he respirat ory t ract help drive ent rapped organisms upwards
and out war ds. Ot her mechanical fact or s which help pr ot ect t hese t r act s ar e t he wa sh i n g
Innate Immunity 7
a ct i on of t e a r s, sa li va a n d u r i n e. Many secr et ions cont ain bact er icidal component s such as
a ci d i n ga st r i c ju i ce ; lysozyme in t ear s, nasal secr et ions and saliva; p r ot e olyt i c e n zyme s
in int est inal secr et ions; sp er mi n e a n d zi n c i n semen and la ct op er oxi d a se i n br ea st mi lk .
An impor t ant mechanism of defence is associat ed wit h n or ma l b a ct e r i a l flor a of t he
body. Nor mal flor a can suppr ess t he gr owt h of many pot ent ially pat hogenic bact er ia and fungi
by compet it ion for essent ial nut r ient s or by pr oduct ion of inhibit or y subst ances such as colicins
or acid. Pat hogenic invasion of t he vaginal flor a is inhibit ed by lact ic acid pr oduced by t he
commensal vaginal flor a. When nor mal human flor a is dest r oyed by br oad spect r um ant ibiot ics,
pat hogens such as Candida spp. and Clostridium difficile cause oppor t unist ic infect ions.
If micr oor ganisms do penet r at e t he body, t wo main defensive oper at ions come int o play –
p h a gocyt osi s and t he bact er icidal effect of solu b le ch e mi ca l fa ct or s (molecules such as
complement pr ot eins, acut e phase pr ot eins, and cyt okines). Ot her cells may also be involved:
cells t hat r elease inflammat or y mediat or s (basophils, mast cells, and eosinophils) and nat ur al
killer cells (NK cells).
Phagocytosis
The engulfment and digest ion of infect ious agent s is assigned t o t wo major cell populat ions :
t he polymor phonuclear neut r ophil (PMN) and t he macr ophage (M). Phagocyt osis is a mult iphasic
act (Figur e 2.2), r equir ing r ecognit ion, movement of PMNs out of blood vessels t owar ds t he
ir r it ant , at t achment t o micr oor ganisms, ingest ion and int r acellular killing.
Figure 2.2. Phagocytosis: a multiphasic act.
8
Immunology-Introductory Textbook
Pattern Recognition and Ingestion
Phagocytic cells are among the most important cells endowed with a variety of receptors
capable of recognizing molecular patterns expressed on the surface of pathogens or pathogen
.... sliOCiated molecular patterns (PAMPS); these are shared by a large group of infectious agents
and are clearly differentiated from 'selr patterns. Most body defence cells bave pattern-
recognition receptors (PRRs) for common pathogen-associated molecular patterns (Figure
2.3) and so there is an immediate response against the invading microorganism. Pathogen-
associated molecular patterns can also be rec;ognized by a series of soluble pattern-recognition
receptors in the blood that function as opsonins and initiate the complement pathways. In all,
the innate immune system is thought to recognize approximately lQ3 molecular patterns.
Most of these pattern recognition r eceptors (PRRa) are ledin.like and bind to externally
di Kplayed microbial sugars. They are glycoprotein in nature and are also known as toll-Uke
receptors and are found on the surface of various body defence cells_ They are so named
because they recognize and bind to pathogen-associated molecular patterns. molecular
components associated with microorganisms but not found as a part of eukaryotic cells. These
include bacterial molecules such as peptidoglycan, teichoic acids, lipopolysaccharide, mennans.
flagellin. pilin. and b.ltcterial DNA. There are also pattern-recognition molecules for viral double·
stranded RNA (dsRNA) and fungal cell wall components such as lipoteichoic acids, glycolipids.
manDans, and zymosan. Binding of the microbial molecule to the toll· like receptor sends a
signal through the cytoplasm to the nucleus of the cell where it activates genes coding for the
synthesis and secretion of cytokines.
There is evidence that an adherent particle may initiate ingestion by activating an actin-
myosin contractile system which extends pseudopods around the particle_ The particle is
eventually enclosed completely in a vacuole - the phagosome. Lysosomal granules come into
contact and finally fuse with the phagosome forming a pbagoly9080me. Several hydrolytic enzymes
are now released into the phagolysosome which act optimally at a low pH.
P'those"....noa.ted
molecular paltern$
FigUN 2.3. Palhogen-Associated Molecular Patterns Binding 10 Pattern-Recognition Rec.ptors on
Defence Cells.
Innate Immunity
Intracellular Killing
Intracellular killing utilizes two mechanisms:
(i) Oxygen dependent mechanisms and
(ii) Oxygen independent mechanisms (Table 2.1).
Table 2.1: Oxygen Dependent and Independent Mechanisms
Oxygen depenthllt 1tVr:.lIllnl.flftf
hexoslt
monophosphil le
phuspbate
Glucose + NADP'
..
+ NAOPlI
02 burrst + generation
shunt
of ::;uperoxide anion
cvtochmnHl
NADpt
NADPH+0
2

spontaneous




Spontaneous
Formation of further
.. OH+Olr+ IO!
microbicidlli agents
mye]operoxidllse
+ CI-
.. ocr + H
2
O Myel operoxidase
generation of
OCI-+

10
2
+ CI- + H
2
O
microbicidal
IIlQlecules
supcroxidc
Prot.ective
20
2
+2W

+
mechanisms used
di smut ilse
by host +
muny microbes

2H2.0:!
.. 2 H:lO+ (}.l
Oxygen independent mechanisms
Cationic proteins (incl. cathespin C)
Dflmage to microbial membranes
Lysozyme Splits mucopeptide in bacterial cell wcll
Lacwferrin
Deprives proliferating bacteria of iran
Prol.eolytic enzymes
nigest.ion of killed organisms
variety of other hydrolytic em:rmes
Oxygen-dependent mechanisms
9
With the formation of the phagolYBoBome, there is a dramatic increase in activity of the
hexose monophosphate shunt, increased glycolysis and incr eased oxygen consumption with an
exagger ated formation of hydrogen peroxide, lactic acid and a subsequent fall in pH - a
10
Immunology-Introductory Textbook
prerequisite for optimal functioning of hydrolytic enzymes. Collectively the stimulation of all
these pathways is called a "respiratory burst". The hexose monophosphate shunt generates
NADPH, which is ultimately utilized to reduce molecular oxygen hound tocytoebrome, cilusing
a burst of oxygen consumption. As a resul t OXYllen is converted to superoxide anion (OJ.
hydrogen peroxide, si nglet 02 (I0
2
) and hydroxyl radicals (Om - all of which are powerful
microbicidal agents. Furthermore the combination of peroxide, myeloperoxidase and halide
(en ions constitutes a potent halogenating system capable of kiUing both bacteria and viruses.
Killing by Nitric Oxide
Nitric oxide is known to be a physiologic mediator similar to factors that relax the
endothelium. It is formed within most cells particularly neutrophils and macrophages and
generates a powerful antimicrobial effect. It is thought to be particularly effective against
Salmonella and Leishmania spp., pathogens known to live comfortably within the cell and yet
escape phagocytic killing.
Oxygen independent mechanisms
As a r esult of t he oxygen dependent mechanisms the pH of the vacuole rises so as to allow
several cationic proteins which are microbicidal to act optimally. These molecules are known
as a.-defensins and act selectively on microbial lipid components. Other substances such as the
neutral proteinase (cathepsin G) are also powerful microbicidal agents. Lysozyme and lactoferrin
also constitute bactericidal or bacteriostatic factors which are oxygen independent and can
function under anaerobic conditions. Finally killed organisms are digested by hydrolytic enzymes
and degraded products released to the exterior.
Extra-cellular Killing
Natural killer cells arc large granular Iympocytes. Their main role is to kill virus
infected cells. this they do by secreting a cytolysin called perforin that attacks the membrane of
the infected cell.
Large parasites such as helminths cannot be physically phagocytosed. Extra cellular killing
by eosinophils (using the complement pathway) has evolved as a way of defence against these
parasites. Remember eosi nophilia can sometimes be an indirect clue that the patient has a
parasitic infestation.
Soluble (Humoral) Bactericidal Factors
Of the soluble bactericidal substances elaborated by the body. perhaps the most abundant
li nd widespread is the enzyme, lysozyme a muramidase which splits the peptidoglycan of the
bacterial cell wall. Human lHIefensins are proteins that play an important role in defending
against microbial invaders along mucosal tracts.
There are also a number of plasma proteins collectively called the "acute phase proteins"
which show a dramatic increase during infection. These include C-reactive protein (CRP).
serum amyloid A. a-I antitrypsin. mannose-binding protein. fibrinogen and
caeruloplasmin.
During infection , microbial substances such as endotoxins stimulate the r elease of
interle uki n-l (IL-l)-an endogenous pyrogen. IL· l in turn induces the liver to release more
CRP. The prime function of CRP is to bind to a number of micro organisms (in a calcium
dependant fashion) which contain phosphorylcholine. This then enhances activation of
Innate Immunity 11
complement and thereby induces the acute inflammatory response. CRP therefore acts as an
opsonin, coating organisms and triggering complement mediated lysis (see Chapter: 7).
Interferons are anti·viral agents synthesized by cells tha.t are infeded by viruses. They are
secreted into the extra-cellular fluid where they bind to receptors on uninfected cells. The
bound interferon exerts an antiviral effect and prevents the uninfected cell from becoming
infected.
However, many of these remarkable defence mechanisms are powerless in the face of
overwhelming infection. Experience with chronically ill or debilitated patients indicates that
many of these patients become "secondaril y" infected - a reflection of tbe waning innate or
natural defence mechanism in the host. Should innate immunity fail for some reason, all is not
lost; other strategies of defence, far more powerful and exquisitely precise are brought into
play, in the form of adaptive or acquired spe.::ific immunity,
T
he cellular or ganelles of defence in t he human body ar e found in, what is collect ively
t er med, t he lympho-r et icular syst em. This is br oadly classified int o
I n t er n a l Ext er n a l
Blood Respir at or y t r act
Tissues Gast r o-int est inal t r act
Thymus Genit o-ur inar y t r act
Lymph nodes
Spleen
The cellular const it uent s of t he lympho-r et icular syst em ar e:
• Phagocyt ic cells Polymor phonuclear neut r onphils
Mononuclear phagocyt es
Eosinophils
• Lymphocyt es
The Polymorphonuclear Neutrophil (PMN)
This cell has a common haemopoiet ic st em cell pr ecur sor and is t he dominant whit e cell
in cir culat ion. It is a non-dividing shor t lived cell wit h a mult ilobed nucleus and an ar r ay of
gr anules. The neut r ophil gr anules ar e of t hr ee t ypes, t he pr imar y azur ophilic gr anule cont ains
myeloper oxidase, some lysozyme and a family of cat ionic pr ot eins. The secondar y gr anules
hold lact ofer r in, lysozyme and a B
12
binding pr ot ein. The t er t iar y gr anules ar e t he convent ional
lysozymes wit h acid hydr olases. Met abolism by glycolysis enables t he cell t o funct ion under
anaer obic condit ions. The polymor phs pr ovide a major defence against pyogenic bact er ia- hence
t hey ar e oft en loosely called “pus cells”.
The Mononuclear Phagocyte
These cells ar e der ived fr om bone mar r ow pr omonocyt es. They ent er t he cir culat ion as
blood mon ocyt e s and finally set t le in t he t issues as mat ur e ma cr op h a ge s. They const it ut e
t he mon on u cle a r p h a gocyt e syst e m (MP S). They ar e pr esent t hr oughout t he connect ive
t issue and around t he basement membrane of small blood vessels. They are part icularly abundant
in t he lung as a lveola r ma cr op h a ges and in t he liver as Ku p ffer cells. They ar e st r at egically
placed in t he lining of spleen sinusoids and lymph node medullar y sinuses t o filt er for eign
IMMUNOBIOLOGY
CHAPTER – 3
Immunobiology 13
mat er ial. Me sa n gi a l ce lls in t he r enal glomer ulus, mi cr ogli a in t he br ain and ost e oscla st s
in bone ar e also par t of t he MPS. Unlike polymor phs, t hey have a long life span. The
macr ophages feat ur e pr edominant ly in combat ing int r acellular bact er ia, vir uses and pr ot ozoa.
Mononuclear phagocyt es expr ess a myeloid r ecept or (CD14) which ser ves as a r ecognit ion
molecule for a wide var iet y of bact er ial envelope molecules, such as LPS fr om Gr am negat ive
or ganisms and component s of Mycobact er ial and Gr am posit ive cell walls. Ligat ion of t his
r ecept or leads t o macr ophage act ivat ion.
Br oadly, t he macr ophage ser ves t wo major funct ions : t o ingest and dest r oy par t iculat e
mat t er – a funct ion gr eat ly enhanced when t he for eign mat t er is coat ed by complement or
ant ibody. The t er m opsonin is used t o descr ibe t his coat ing wit h bot h ant ibody and complement
t o facilit at e phagocyt osis. The ot her funct ion involves t he init ial r ecognit ion, pr ocessing and
pr esent at ion of ant igen t o t he T-cell t o elicit t he specific immune r esponse.
Eosinophils
Lar ge par asit es such as helmint hs cannot physically be phagocyt osed and ext r acellular
killing by eosinophils is lar gely t he mode of defence involved. These cells have dist inct ive
gr anules which st ain well wit h acid dyes. A ma jor b a si c p r ot e i n (MBP) is localized in t he
cor e of t he gr anules, while an eosinophilic cat ionic pr ot ein t oget her wit h a per oxidase have
been ident ified in t he gr a nule ma t r ix. Ot her enzymes found in t he eosinophil include
ar ylsulphat ase B, phospholipase D, and hist aminase. They have r ecept or s for complement
component C3b (see Chapt er 7) and when act ivat ed produce a “respirat ory burst ” and concomit ant
gener at ion of act ive oxygen met abolit es. Eosinophils also pr oduce a “per for in” like pr ot ein
which can pr oduce membr ane damage via t r ansmembr ane plugs.
Most helmint hs act ivat e t he alt er nat ive complement pat hway (see Chapt er 7) and hence
C3b is deposit ed all along t he helmint hic membr ane. This allows for adher ence of eosinophils
t hr ough t heir C3b r ecept or s. Upon act ivat ion, t he eosinophil t hen launches it s ext r a cellular
at t ack which includes r elease of MBP and cat ionic pr ot eins bot h of which damage par asit e
membr anes.
Lymphoid Organs, Lymphocytes and Lymphocyte Traffic
Any discussion on lymphocyt es t akes it s or igins fr om a det ailed st udy of t he lymphoid
or gans. The immune syst em consist s of a number of lymphoid or gans, classified commonly as
Pr imar y or cent r al and Secondar y or per ipher al or gans. The pr imar y or gans ar e t he t hymus
and t he bone mar r ow; t he secondar y or gans ar e t he spleen, lymph nodes and t he aggr egat es of
lymphoid t issue in t he r espir at or y, gast r o-int est inal and genit o-ur inar y t r act s.
Lymphocyt es der ive fr om st em cells. Init ially st em cells ar ise fr om t he yolk sac and t he
foet al liver , but lat er in per inat al development , some or iginat e in t he bone mar r ow. St em cells
differ ent iat e int o lymphocyt es in t he pr imar y lymphoid or gans, namely t he t hymus and t he
bone mar r ow (Figur e 3.1). Classificat ion of lymphocyt es on he basis of sur face mar ker s make
use of t wo impor t ant classes of char act er ist ics. One is known as clust er designat ion (CD) and
t he ot her t he ant igen r ecognit ion r ecept or s. CD ant igens r epr esent families of sur face ant igens
t hat can be r ecognized by specific ant ibodies pr oduced against t hem. Thus mat ur e T cells have
CD3, CD4 or CD8 mar ker s, and B cells have CDs 19-22. Ther e ar e mor e t han 200 dist inct CDs.
14 Immunology– Introductory Textbook
Figure 3.1. A simplistic overview of the immune system. * Bursal equivalent; since B cells were
known to originate in the Bursa of Fabricius in birds.
The Primary Lymphoid Organs
The Thymus
The t hymus is r esponsible for t he development of T-dependent lymphocyt es. It plays an
impor t ant r ole in immunogenesis in t he young and t he T cells der ived fr om it or chest r at e t he
immune r esponse t hr oughout life. This cent r al lymphoid or gan differ s fr om ot her lymphoid
t issues. All ot her lymphoid t issues, ar e st r at egically placed t o meet for eign par t icles, t he t hymus
is pr ot ect ed fr om ant igen cont act . Fur t her mor e, t he r at e of mit ot ic act ivit y is gr eat er t han in
any ot her lymphoid t issue and yet , t he number of cells leaving it ar e compar at ively less. The
assumpt ion is t hat a lar ge number of cells made in t he t hymus die wit hin it s subst ance. This
pr obably r epr esent s a syst em of homeost asis.
The t hymus consist s of t wo lobes sur r ounded by a t hin capsule which ext ends int o t he
subst ance of t he gland t o form sept a–wit h t he result ant format ion of lobes and lobules. Peripheral
por t ions of t he lobule ar e heavily infilt r at ed wit h lymphocyt es. Cent r al por t ions have fewer
lymphocyt es and mor e epit helial cells.
The t hymus is believed t o per for m t wo main funct ions, pr oduct ion of lymphocyt es in t he
cor t ex and pr oduct ion of humor al subst ances in t he medulla. These humor al subst ances (one of
which is called t hymosin) may induce differ ent iat ion of lymphocyt es dir ect ly wit hin t he t hymus
or may cont r ol differ ent iat ion in t he per ipher y. The t hymus does not ont ain any plasma cells.
Unlike ot her lymphoid or gans it cont ains t wo t issue t ypes: lymphoid and epit helial. Some
int er digit at ing cells, der ived fr om pr ecur sor s in t he bone mar r ow, ar e also found in t he t hymus.
These cells ar e r ich in major hist ocompat ibilit y ant igens (MHC), which t hey display as sur face
mar ker s. Thymic epit helial and int er digit at ing cells also influence T cell differ ent iat ion.
Following infilt r at ion of t he t hymus wit h plur ipot ent ial st em cells fr om yolk sac, foet al liver or
Immunobiology 15
spleen, these cells acquire new surface antigens as they undergo differentiation. During intra
thymic maturation thymocytes lose or retain certain surface markers_
Stage I thymocytes express a specific antigen termed CD2 on their surface. They also
ontain an activated gene known as the T gene, which forms part of an antigen binding receptor
on the T cell. As the thymocytes pass to Stage II they exhibit CD5 CD4 and COB surface
markers, in addition, the gene that encodes for the Il-chain of the T cell receptor becomes
activated. Stage 11 cells then lose the CD5 marker and differentiate further into one of two
types of a Stage In cell. Those that lose the COB surface marker become mature CD4+ T
cells, whereas those that lose the CD4 marker become COB T cells (Figure 3.2).
Stage ~
stage II
Stage III
C02
o
1
COAD4
Vb"
/\
~
CD2U '
Flgu ... 3.2. Stages of T cell differentilltion.
T cell receptor genes become activated in both types of stage III cells, allowing for the
expression of the complete T cell antigen binding receptor complex (CD3-a ~ ) . During the
process of maturation the T cells become immuno-competent : they learn to bind to specific
antigens, when these antigens are presented in association with MHC molecules. Hence they
arc schooled to recognize MHC antigens during thymic maturation. In addition, T cells learn to
differentiate between self and non self antigens. The T cells go through a selection process in
the thymus based upon the T cell receptor that they posses. T cells that recognize selfantigens
are destined to die by apoptosis 01" programmed cell death. Finally, there are two separate
populations of Stage III thymocytes released into the blood stream. These two populations are
called; T.helper inducer and T-cytotoxic suppressor T cells. In the blood stream they are
16
Immunology-Introductory Textbook
carried to the lymphoid system's peripheral organs where they reside in thymus dependent
regions of the peripheral lymphoid system.
The Bursal Equivalent and Bone Marrow
Birds have another primary lymphoid organ in addition to the thymus, situated near the
cloaca, it is called the Bursa of Fabricius. The bursa is derived from gut epithelium in the
embryo. Stem cells enter the Bursa of Fabricius where they differentiate into Beells capable of
producing antibody. Burscctomized birds are completely B cell deficient though T cell functional
activity is unaffected. There does not appear to be an organ in mammals that is equivalent to
th+! Bursa of Fabricius. Stem cells differentiate into B cells in the bone-marrow and in the
peripheral lymphoid organs themselves.
Secondary Lymphoid Organs
Lymph nodes
Cells and molecules are brought to the lymph nodes via afferent lymphatics (Figure 3.3).
Lymphocytes move through the sinuses and leave the lymph nodes through efferent lymphatics.
A great majority of lymphocytes that traverse through the lymph nodes come from the blood
stream. A sman percentage is generated in the lymph node from precursor cells. Most of the
blood lymphocytes arc T cells which migrate in and out of lymph nodes. A few B cells also come
from the blood stream and many localize in the germi nal centers of the eortex. The germinal
centers are hence peripheral bursal equivalents. When lymph nodes have been stimulated by
antigen, germinal centers increase in size and thereafter contain many lymphoblasts and is the
site of memory B cell proliferation.
Afferent
lymphiltic1
Outar Cortex (8 cell arCiI)
-""' .... _ Primary tOllicl,
""",,-,, +f-- Paril cortic.! (T cel!) Iru
c:::' -.Y.+fl-- Madu!IIrY , inusa,

Medullary cords
centre or
secondary tolUd,
Figure 3. 3. Cut section of lymph mode.
Cells of the dendritic cell lineage involved in T cell stimulation are bone marrow
derived. In the skin they are known as Langerhans cells . These cells efficiently process
antigen but cannot present it to T cellR. Langerhans cells pick up antigen in skin and carry it
Immunobiology 17
via a ffer en t lymp h a t i c vessels t o lymph nodes. Dendr it ic cells in lymph ar e known as “veiled”
cells. In lymph nodes t he cells, now known as t i ssu e d e n d r i t i c ce lls or i n t e r d i gi t a t i n g
ce lls , may efficient ly pr esent ant igen. The par a cor t ical ar eas cont ain t hese int er digit at ing
cells bear ing MHC class II molecules complexed t o for eign ant igen. They pr esent ant igen t o T
cells which abound in t he par acor t ical and sub cor t ical ar eas of t he lymph mode. T cell funct ion
is closely associat ed and r est r ict ed by t he pr esence of MHC ant igens on ant igen pr esent ing
cells.
Follicular d en d r i t i c cells ar e found in ger minal cent r es. They ar e called dendr it ic because
of t heir mor phology r at her t han any lineage r elat ionship wit h dendr it ic cells. In fact , t her e is
consider able uncer t aint y about t heir development al or igin. They display C3 complement and
IgG (Fc) r ecept or s. Their funct ion appear s t o ent r ap ant igen as an ant igen- ant ibody- C3 complex,
and pr esent it t o B cells, as a st imulus t o ant ibody pr oduct ion. They cannot pr esent ant igen t o
T cells but ar e impor t ant in developing r esponses by B cells.
The lymph nodes hence appear t o be compar t ment alized - ger minal cent er s wit h immobile
B cells, par a cor t ical ar eas wit h migr ant T cells. In addit ion, t her e ar e sinuses full of macr ophages
and a r et icular net wor k of dendr it ic cells t hat t ends t o hold ant igen for a long t ime. All t his
appear s t o facilit at e int er act ions bet ween t he differ ent t ypes of cells t hat ar e r equir ed for
gener at ing an immune r esponse.
Spleen
In immunological t er ms t he whit e pulp of t he spleen is t he bur sal equivalent and is t he
st or e house for B cells. The r ed pulp which sur r ounds t he whit e is a t hymic dependant ar ea
housing T-cells (Figur e 3.4).
Figure 3.4. Cut section of spleen.
Other Lymphoid Tissue
The r espir at or y, aliment ar y and genit o-ur inar y t r act s ar e guar ded immunologically by
sub epit helial accumulat ions of lymphoid t issue which ar e not const r ained by a connect ive
18 Immunology– Introductory Textbook
t issue capsule. These may occur as diffuse collect ions of lymphocyt es, plasma cells and phagocyt es
in t he lung and in t he lamina pr opr ia of t he int est inal wall, or as clear ly or ganized lymphoid
follicles. This includes t he t onsils, t he Payer s pat ches and t he appendix, lymphoid t issue found
in t he r espir at or y and ur inar y t r act s: collect ively t hey ar e t er med, mu cos a l a s s oci a t e d
lymp h oi d t i ssu e (MALT). It is believed t hat MALT for ms a separ at e int er connect ed secr et or y
syst em wit hin which cells commit t ed t o IgA or IgE synt hesis may cir culat e.
Lymphocyte traffic
Ther e ar e t hr ee major t ypes of lymphocyt e cir culat ion :
(a) The seeding of st em cells fr om t he foet al liver or bone mar r ow in t he pr imar y lymphoid
or gans and t he subsequent differ ent iat ion and dist r ibut ion of t hese cells t o t he
per ipher al lymphoid syst em,
(b) The r ecir culat ion of lymphocyt es fr om blood t o lymph t o blood and
(c) The dist r ibut ion of effect or cells t o par t icular par t s of t he body.
Immunocompetent cells involved in the immune response
The pr imar y cells involved in t he immune syst em ar e t he lymphocyt es. Funct ionally,
lymphocyt es ar e divided int o
(a) T cells: These cell t ypes r egulat e t he immune r esponse, ar e involved in cell mediat ed
immune r eact ions and induce B cells t o pr oduce ant ibody.
(b) B ce lls: These cells differ ent iat e int o ant ibody pr oducing p la sma ce lls .
In addit ion t her e ar e a het er ogenous gr oup or gr oups of lymphocyt es t hat ar e neit her T or
B cells. Morphologically t hese cell t ypes cannot be different iat ed. Binding of monoclonal ant ibodies
is cur r ent ly t he most specific t echnique used t o ident ify specific sub set s.
Sub sets of T cells
Depending on t he pr esence of sur face mar ker s (CD4 and CD8) T cells ar e br oadly and
funct ionally divided int o h e lp e r T ce lls bear ing t he mar ker CD4, cyt ot oxi c T cells (bear ing
t he CD8 mar ker ).
Large granular lymphocytes
A por t ion of t he lymphocyt es cir culat ing in blood lack t he sur face ant igens of T or B cells.
These cells ar e somet imes called n u ll ce lls. A major it y of null cells ar e n a t u r a l k i lle r (NK)
ce lls. They ar e lar ge gr anular lymphocyt es t hat , in vit r o, can kill a number of t umour cell
lines in a non specific manner . For a compr ehensive classificat ion of lymphocyt es, t heir subset s
and funct ions see Table 3.1.
Phylogenet ic evidence suggest s t hat t he development of immunologic compet ence coincides
wit h t he development of t he lymphocyt e. Using convent ional st ains t he lymphocyt e is a
mor phologically feat ur eless cell. Funct ionally however , t hese cells play pivot al r oles in t he
immunological r esponse t o for eign ant igen.
Immunobiology 19
Table 3.1: Classification of lymphocytes
Lymphocytes and Main cell surface Restrictions Functions
their sub-sets markers
T cells
T helper cell CD4+; CD3- T-cell MHC- Class II Stimulate B cell to produce
receptor; CD2+ antibody; Induce CD8+ T-
cell cytokine secretion;
macrophage activation
T cytotoxic cell CD8+; CD3- T-cell MHC- Class I Lyse antigen bearing target
receptor; CD2+ cell
B cells
Plasma cell and Ig+; CD19; CD20; Antigen specific Differentiate into antibody
Memory B-cells CD21; CD23; producing and memory
cells
Large granular lymphocytes
Null/ NK cells CD 56+ Not MHC Kill tumour cells and show
restricted antimicrobial activity
(mainly antiviral)
CD: Clust er differ ent iat ion designat ion
™™™
T
he t er ms a n t i ge n and i mmu n oge n ar e oft en used synonymously. However , t hese
t er ms ant igen and immunogen, imply t wo closely r elat ed ent it ies. One which descr ibes
a molecule t hat pr ovokes an immune r esponse is called an immunogen and t he ot her descr ibes
a molecule which r eact s wit h t he ant ibody pr oduced or wit h t he act ivat ed cellular const it uent s
of cell mediat ed immunit y, is r efer r ed t o as an ant igen.
In cont r ast t o t his is t he hapt en. Ha p t en s ar e small well defined chemical gr oupings such
as dinit r ophenyl (DNP) which ar e not immunogenic on t heir own but will r eact wit h pr efor med
ant ibodies. To make a hapt en immunogenic it must be linked t o a car r ier molecule which is
it self immunogenic.
Ant igens ar e r ecognised not only by ant ibodies but also by ant igen specific T cell r ecept or s.
In cont r ast t o immunoglobulins, which usually r ecognize int act ant igen, T cell sur face r ecept or s
r ecognize p r oce sse d a n t i ge n on t he sur face of ant igen pr esent ing cells, t oget her wit h t he
major hist ocompat ibilit y complex (MHC) Class I or Class II sur face pr ot eins (r efer chapt er 10).
Antigenic Determinants and Epitopes
The par t of t he ant ibody molecule which cont act s t he ant igen is t er med t he p a r a t op e.
Consequent ly, t hat par t of t he ant igen molecule t hat makes cont act wit h t he par at ope is called
t he e p i t op e. As most ant igens ar e pr ot ein in nat ur e t hey exist in a folded, t hr ee dimensional,
t er t ia r y st r uct ur e. Hence t her e ma y be a clust er of a mino a cid sequences on t he t hr ee
dimensional st r uct ur e const it ut ing a ser ies of epit opes. Each of t hese epit ope clust er s is what is
meant by an a n t igen ic d et er min a n t . An illust r at ion of t he ant igenic det er minant s on bact er ial
cells is shown in Figur e 4.1 a and b.
Figure 4.1. Antigenic determinants on a single bacterial cell.
ANTIGENS AND IMMUNOGENICITY
CHAPTER – 4
Antigens and Immunogenicity 21
Requirements for immunogenicity
The fir st and pr imar y r equir ement for any molecule t o qualify as an immunogen is t hat
t he subst ance be gen et i ca lly for ei gn t o t he host . Somet imes body const it uent s ar e r ecognized
as for eign leading t o aut oimmune disease. Nor mally t he body discr iminat es self fr om non self.
Cer t ain par t icles do not elicit an immune r esponse – t hey mer ely under go phagocyt osis, for
example, car bon as coal dust .
Mole cu la r si ze det er mines immunogenicit y t o some ext ent . The gener al r ule is t hat
par t icles wit h a molecular weight less t han 10,000 ar e only weakly immunogenic or not
immunogenic at all. The most pot ent immunogens ar e macr omolecular pr ot eins wit h molecular
weight s of 10,000 and mor e.
An immunogenic molecule needs t o possess a cer t ain degr ee of ch e mi ca l comp le xi t y. It
is difficult t o est ablish a definit e t hr eshold, however , t he gener al r ule holds. Only pur e lipids
ar e non immunogenic. A solut ion of monomer ic pr ot eins may act ually induce t oler ance, but is
highly immunogenic in t he polymer ic st at e. Sever al immunogens t hat do not induce an immune
r esponse in t he pur e for m, do so when t hey ar e par t of a lar ger par t icle. Hence, adjuvant s ar e
used t o enhance immunogenicit y.
Exper iment s have shown t hat con for ma t i on of a molecule could be impor t ant t o it s
ant igenicit y. For example, t he lysozyme molecule is a good ant igen in it s nat ive for m which
consist s of sever al amino acids folded int o a loop wit h t he aid of a disulphide bond. If t he
disulphide bond was dist ur bed, so t hat t he loop confor mat ion was no longer pr esent , t he
ant igenicit y of t he molecule would be dr amat ically r educed.
St udies have also shown t hat t he a mi n o a ci d se q u e n ce is impor t ant t o ant igenicit y.
Cer t ain molecules t hat have no appar ent complexit y in st r uct ur e, can ser ve as ant igens pr ovided
cer t ain shor t , but cr ucial st r et ches of amino acids ar e pr esent in an undist ur bed fashion.
Ot her wor ker s have shown t hat t he mob i li t y of a se gme n t of t he ant igen molecule
influences it s ant igenicit y. Using t he t obacco mosaic vir us, r esear cher s have found t hat cer t ain
segment s wit h high mobilit y (movement of 1 angst r om fr om t he pr ot ein back bone posit ion)
wer e mor e ant igenic t han non mobile segment s.
Par t s of t he pept ide chains which p r ot r u d e si gn i fi ca n t ly fr om t he globular sur face t end
t o be sit es of high epit ope densit y. Hence a cce ssi b i li t y of t hese high epit ope densit y ar eas t o
t he r ecognit ion syst em det er mines t he out come of t he immune r esponse (Figur e 4.2).
Figure 4.2. Accessibility of antigenic determinants.
22 Immunology– Introductory Textbook
Ant igens and ant ibodies int er act by sp a t i a l comp le me n t a r i t y and not by co-valent
bonding, and like enzyme – subst r at e int er act ions, can be r eadily r ever sed. The idea t hat
ant ibody r ecognizes ant igen t hr ough complement ar y shapes on par at ope and epit ope is
simplist ically illust r at ed using t he “lock and key” analogy (Figur e 4.3).
Figure 4.3. Antigen and antibody interact by spatial complementarity; the lock and key analogy.
The Forces that Bind Antigen to Antibody
The for ces t hat bind ant igen t o ant ibody become lar ger as int er cellular dist ances decr ease.
These for ces ar e, in essence, no differ ent fr om t he ‘non-specific’ int er act ions which occur bet ween
any t wo unr elat ed pr ot eins. These int er molecular for ces ar e:
• elect r ost at ic (at t r act ion bet ween opposit ely char ged ionic gr oups)
• hydr ogen bonding (r ever sible hydr ogen br idges bet ween hydr ophilic gr oups)
• hydr ophobic bonding (similar t o t he manner in which oil dr oplet s in wat er mer ge t o
for m a single lar ge dr op, side chains of valine, leucine and phenylalanine t end t o
associat e in an aqueous envir onment . This may account for over 50% of t he st r engt h
in an ant igen ant ibody bond).
• Van der Waals for ces : int er act ion bet ween t he elect r ons in t he ext er nal or bit s of t he
t wo differ ent macr o molecules.
At t his point of t he discussion, t her e ar e t wo commonly used t er ms t hat need definit ion:
(i) a ffi n i t y or st r engt h of binding of ant igen t o ant ibody. The t er m affinit y alludes t o t he
st r engt h of t he bond bet ween ant igenic det er minant and monovalent ant ibody. Hence t hose
ant ibodies t hat fit closely t o an ant igenic det er minant and ar e not easily dissociable ar e known
as high affinit y ant ibodies.
Antigens and Immunogenicity 23
(ii) a vi d i t y of ant iser um for ant igen r efer s t o t he over all st r engt h of int er act ion bet ween
a lar ge ant igen wit h mult iple epit opes and a polyvalent ant ibody molecule such as t he IgM.
Each one of t he many int er act ions is by it self a low affinit y one. This is a funct ion of mult ivalency
i.e. an ant igen wit h sever al det er minant s will elicit a var iet y of ant ibodies as illust r at ed in
Figur e 4.4. Ant iser um in Figur e 4.4b is mult ivalent , binding is gr eat er , dissociat ion is not easy
and it is of higher avidit y t han ant iser um 4.4a.
Figure 4.4. Avidity of antiserum for antigen a. monovalent and easily dissociated antiserum
b. multivalent antiserum has a higher avidity.
Antigen-antibody Specificity is not Absolute
It is widely known t hat ant igen-ant ibody r eact ions ar e exquisit ely specific. An ant ibody
r aised t o one det er minant will not r eact wit h anot her det er minant . However , in pr act ice, t he
t er m cr oss r e a ct i vi t y is widely used. An ant iser um r aised against a given ant igen can cr oss
r ea ct wit h a pa r t ia lly r ela t ed a nt igen which bea r s a n ident ica l or simila r det er mina nt
(Figur e 4.5).
Figure 4.5. Cross reactivity of antibodies.
24 Immunology– Introductory Textbook
Ant iser um r aised t o Ag1, will r eact wit h Ag2 due t o t he ident ical det er minant (hat ched)
shar ed by t he t wo ant igens. Ant iser um t o Ag1 will also r eact weakly t o Ag3 because, t he
det er minant s t hough not ident ical, ar e similar in shape.
Thus it is possible t hat each ant ibody will r eact not only wit h t he ant igen which st imulat ed
it s product ion, but also wit h some quit e unrelat ed molecule bearing similar looking det erminant s.
The Heterophile Antibody Response
A number of similar ant igen molecules ar e found in phylogenet ically unr elat ed species.
The cr oss r eact ing ant igens of human hear t t issue and t he gr oup A β-haemolyt ic S treptococcus
is an example.
The For ssma n a n t i gen is anot her example which is found in t he t issues of many species.
This ant igen is it self not found in man, however a gr eat var iet y of ot her cells and t issues could
sensit ize man t o t his ant igen. Following an at t ack of infect ious mononucleosis, an infect ion by
t he Epst ein-Bar r vir us, ant ibodies ar e pr oduced which r eact not only t o t he vir us but also t o a
complet ely unr elat ed ant igen-t he sheep r ed cell. This is known as t he het er ophile ant ibody
r esponse and for ms t he basis of t he Paul-Bunnel t est for infect ious mononucleosis.
™™™
T
he fir st r eal chemical infor mat ion r egar ding t he st r uct ur e of ant ibodies was pr ovided
by Ti se li u s a n d Ka b a t in t he ear ly 1940s. These wor ker s demonst r at ed t hat t he
fr act ion of ser um pr ot eins t hat migr at ed most slowly in elect r ophor esis cont ained most of t he
ser um ant ibodies. In t he 1950s P or t e r used pr ot eolyt ic enzymes such as papain t o cleave t he
ant ibody molecule in an at t empt at defining it s st r uct ur e and funct ion. Amino acid sequence of
t he IgG molecule was det er mined by Ed e lma n et a l in 1969.
In all t hese st udies Bence J ones pr ot eins which ar e monoclonal immunoglobulin light
chains shed in plasma and ur ine fr om pat ient s wit h plasmacyt omas or mult iple myelomas have
been an invaluable sour ce of lar ge amount s of immunoglobulin molecules.
Basic Structure of the Immunoglobulin Molecule
Immunoglobulins ar e glycopr ot eins (Figur e 5.1). The basic unit of t he molecule is a four
chain monomer . These four polypept ide chains consist of t wo ident ical heavy chains and t wo
ident ical light chains, designat ed H a n d L ch a in s r espect ively. Each polypept ide chain cont ains
an amino t er minal and a car boxy t er minal. The amino t er minal por t ion of t he molecule is par t
of t he va r i a b le or ‘V’ r e gi on s and t he car boxy t er minal por t ion t he con st a n t or ‘C’ r e gi on s.
Hence t he light chains have a var iable (V
L
) and a const ant (C
L
) r egion and so do t he heavy
chains have a var iable r egion (V
H
)

and a const ant r egion (C
H
). The C
H
r egion is fur t her divided
int o t hr ee ar eas and t hese ar e designat ed CH
1
, CH
2
and CH
3
in or der fr om t he amino end t o t he
car boxy end of t he heavy chain.
The par t of t he ant ibody molecule t hat binds ant igen is for med only by small number s of
amino acids in t he V r egions of t he H and L chains. The h i n ge r e gi on is for med in t he t wo
heavy chains bet ween t he fir st and second C r egions. It is flexible and mor e exposed t o enzymes
and chemicals due t o it s high pr oline cont ent , hence papain act s at t he hinge r egion t o fr agment
t he immunoglobulin molecule. Digest ion of t he molecule by papain pr oduces t wo Fab (ant igen
binding) fr agment s and one Fc (cr yst allizable) fr agment , which lacks t he abilit y t o bind ant igen.
The enzyme pepsin st r ikes at a differ ent point . It cleaves t he Fc fr agment fr om t he r emainder
of t he molecule, leaving t he hinge r egion int act . It leaves behind a lar ge fr agment designat ed
F(ab)
2
since it is st ill divalent wit h r espect t o ant igen binding just like t he par ent ant ibody. The
immunoglobulin molecule is linked t oget her by sever al d i su lp h i d e bon d s fr om H t o H chains,
H t o L chains and L t o L chains.
Int r achain links also exist . However , t he disulphide bonds ar e not r eally r esponsible for
holding t he chains of an immunoglobulin molecule t oget her because r emoval of t hese bonds
r et ains t he int act ness of t he molecule. Besides, a few subt ypes of immunoglobulin molecules
do not have disulphide links bet ween chains, yet r et ain t he basic four chain st r uct ur e. It has
been shown t hat noncovalent for ces (elect r ost at ic, hydr ogen bonding and Van der Waal’s for ces)
IMMUNOGLOBULINS I:
STRUCTURE AND FUNCTION
CHAPTER – 5
26 Immunology– Introductory Textbook
ar e far mor e impor t ant in maint aining t he int egr it y of t he molecule t han t he co-valent disulphide
bonds. All immunoglobulins ar e measur ed using a sed i men t a t i on coeffi ci en t (mea su r ed by
Sve d b e r g) or S va lu e . Higher t he molecular weight of immunoglobulin, lar ger is t he S value.
Figure 5.1. Structure of an immunoglobulin molecule.
Isotypes
Based upon t he st r uct ur e of t heir heavy chain const ant r egions, immunoglobulins ar e
classed int o major gr oups t er med classes. These classes ar e designat ed IgG, IgM, IgA, IgE, and
IgD. Since t hese classes ar e all var iant s of t he immunoglobulin molecule t hey ar e t er med
isot ypic var iant s or i sot yp e s . The 5 differ ent isot ypes ar e defined by differ ences in t he amino
acid sequences of t heir const ant r egions and t her efor e by ant igenic differ ences in t he C
H
r egions
of t he molecule. The C
H
r egion of t he r espect ive immunoglobulins ar e designat ed by gr eek
alphabet s. Hence IgG has γ C
H
r egion; IgM a µ; IgA an α; IgE an ε and IgD a δ t ype heavy chain
const ant r egion. IgG, IgA and IgM have been fur t her divided int o subclasses by r elat ively
minor differ ences in t heir C
H
r egions. Ther efor e, an IgG1, IgG2, IgG3 and IgG4 have been
descr ibed and similar ly an IgA1 and IgA2 as well.
Since t he immunoglobulin molecule is a pr ot ein st r uct ur e wit h ant igenic differ ences in
t he var ious isot ypes, it is possible t o r aise an ant iser um t o any one isot ype (say IgG) which can
be absor bed t o r emove any cr oss r eact ing ant ibodies. This ant iser um will t hen be capable of
r eact ing wit h IgG but not wit h IgM, IgA, IgE or IgD.
Likewise t he light chain const ant r egions also exist in isot ypic for ms known as κ and λ.
Immunoglobulins possess eit her κ or λ light chains, never mixed, on a single molecule. Thus
IgG exist s as IgG κ or IgG λ , IgM as IgM κ or IgM λ and so on.
Immunoglobulins I: Structure and Function 27
The Variable Regions and Heterogeneity of Immunoglobulins
The ant ibody populat ion in any individual is incr edibly het er ogenous. In effect , t his means
t hat t he nor mal immune syst em can gener at e an immunoglobulin for ever y possible immunogen
t hat t he syst em may encount er dur ing it s life t ime. It does t his by const ant ly changing t he
amino acid sequences in t he var iable r egions of t he immunoglobulin molecule. Thus inst ead of
car r ying t he enor mous load of having t o pr oduce millions of billions of immunoglobulins t o
encount er ever y for eign agent , t he immune syst em is equipped wit h t he pr ovision of r apidly
changing and modifying t he var iable r egions of immunoglobulins t o suit a par t icular ant igen.
The t er minal 110 amino acid sequences of t he L and H chains const it ut e t he var iable or
het er ogenous r egions of t he immunoglobulin molecule. Cer t ain sequences in t he var iable r egion
show quit e r emar kable diver sit y and ar e t er med h yp e r va r i a b l e r e gi on s , hot spot s or
comp le me n t a r i t y d e t e r mi n i n g r e gi on s (CDR). These hyper var iable sequences have been
localized t o t hr ee segment s each on t he H and L chains. Not sur pr isingly, t hese ar eas of
hyper var iabilit y ar e int imat ely involved in t he for mat ion of t he ant igen binding sit e.
Idiotypes
J ust as t he C r egions of t he var ious isot ypes have ant igenic det er minant s differ ent fr om
each ot her , idiot ypes ar e for med based on ant igenic det er minant s in t he var iable r egion t hat
dist inguish one V domain fr om t he ot her . And just as an ant iser a can be r aised t o t he differ ent
isot ypes an ant i idiot ypic ser um can be r aised t o t he var ious idiot ypes of immunoglobulin
molecules. Such ant i idiot ypic ser a ar e useful in det ect ion and t yping of t he var ious idiot ypes
pr oduced by t he syst em. A st ar t ling r ealizat ion r esult ing fr om r esear ch in t his ar ea was t hat an
individual could make ant ibodies t o his own idiot ypes (aut o ant i idiot ypic sera). The consequences
of t his will be discussed lat er in t he J er ne net wor k t heor y. (Chapt er 16)
Immunoglobulins in their natural state
In t heir nat ur al st at e immunoglobulins ar e folded int o a t hr ee dimensional st r uct ur e.
Significant ly t he hyper var iable, ant igen binding sequences all appear as loops at one end of t he
var iable domain, clust er ed close t o each ot her (Figur e: 5.2). They ar e t her efor e in an ideal
Figure 5.2. Characteristic folding pattern of the immunoglobulin molecule. An antibody molecule is
a Y-shaped protein made of four polypeptide chains. Two heavy chains extend from the stem
(blue), two light chains are confined to the arms (grey).
28 Immunology– Introductory Textbook
posit ion t o ser ve t he funct ion of ant igen r ecognit ion and t his has been confir med by X-r ay
cr yst allogr aphic analysis. Besides sequence het er ogeneit y, t he hyper var iable loops ensur e
t r emendous diver sit y in combining specificit y for ant igen t hr ough var iat ion in t he shape and
nat ur e of t he sur face t hey cr eat e. (Figur e 5.3)
Biological Role of the Constant Regions
If t he var iable r egions funct ion as ant igen binding sit es, what biological funct ions do t he
const ant r egions ser ve? The classes of ant ibody differ fr om each ot her in t heir abilit y t o car r y
out cer t ain secondar y biological funct ions. For example, t he CH
2
r egions of IgG and IgM fix
complement most efficient ly; t he CH
3
domain of IgG binds t o Fc r ecept or s on phagocyt ic cells,
NK cells (nat ur al killer ) and placent al syncyt iot r ophoblast s; also t o St aphylococcal pr ot ein A,
and t he CH
3
r egion of IgE binds t o Fc r ecept or s on homologous mast cells and basophils.
Figure 5.3. An analogy for the antigen binding site. The three fingers of each hand constitute the
six hypervarible regions which form the antigen binding site. The apple is the antigen!
Immunoglobulin Classes and Subclasses
The following sect ion descr ibes t he physical and biological char act er ist ics of t he 5 major
immunoglobulin classes: IgG, IgD and IgE exist in t he basic four chain st r uct ur al for m, IgM
and IgA occur as complexes of t his basic four chain unit .
Immunoglobulin G
IgG const it ut es 75% of t he t ot al ser um immunoglobulins in humans. Dur ing t he secondar y
immune r esponse it is t he major immunoglobulin t o be synt hesized. Hence it plays a vit al r ole
in t he defence against infect ion. Being t he immunoglobulin t hat can cr oss t he placent a in
humans it is r esponsible for t he pr ot ect ion of t he neonat e in t he fir st mont hs of life. This is
r einfor ced by t he pr esence of colost r al IgG in br east fed infant s. IgG diffuses r eadily int o
ext r avascular spaces and hence pr ovides a major defence against bact er ial t oxins and ot her
blood bor ne infect ious agent s. Or ganisms coat ed wit h immunoglobulin G at t r act macr ophages
via t heir Fc r ecept or s: t hus enhancing phagocyt osis. Fur t her , complexes of ant ibody and bact er ia
also act ivat e complement t her eby chemot act ically at t r act ing polymor phonuclear phagocyt ic
cells and pr omot ing phagocyt osis, since “polymor phs” also possess r ecept or s for t he Fc por t ion
of immunoglobulin and for complement component s. The complement binding sit e on t he IgG
molecule appear s t o be in t he CH
2
domain.
Immunoglobulins I: Structure and Function 29
In a similar way t he ext r a cellular killing of t ar get cells coat ed wit h IgG is mediat ed
lar gely t hr ough r ecognit ion of t he Fc por t ion of t he IgG by sur face r ecept or s on NK cells. Such
Fc r ecept or s ar e also pr esent on plat elet s and when complexed may lead t o r elease of vasoact ive
amines. IgG is unable t o bind fir mly ont o most cells, but has t he abilit y t o bind t o guinea pig
skin - t he significance of which r emains unclear . As will be discussed lat er t he pr oper t y of t he
Fc por t ion of IgG t o bind t o pr ot ein A on t he sur face of S taphylococcus aureus has been gr eat ly
exploit ed for use in diagnosis and r esear ch.
Immunoglobulin M
IgM is t he lar gest immunoglobulin in size (Figur e 5.4). It exist s as a pent amer of t he basic
four chain subunit s held t oget her by disulphide bonds. A r elat ively small molecule; t he J chain
par t icipat es in t he polymer izat ion of IgM via a sulphydr yl r esidue near t he car boxy t er minal.
The heavy chains of IgM ar e designat ed ‘µ’ chains.
Figure 5.4. Structure of the IgM pentamer.
Elect r on micr oscopy st udies r eveal t hat it is shaped like a st ar , but when it is at t ached t o
a bact er ium, it s ant igen binding sit es ar e bound t o t he bact er ial sur face. This changes t he
appear ance of IgM t o a cr ab like for m and causes cr oss-linking of t he differ ent ant igenic
det er mina nt s or epit opes on t he ba ct er ia l cell sur fa ce by t he polyva lent I gM molecule
(Figur e 5.5).
The µ chains bear an ext r a CH r egion; t he IgM heavy chain t her efor e consist s of r egions
CH
1
t o CH
4
. IgM ant ibodies t end t o be of r elat ively low affinit y but because of t heir valency
t hey bind wit h high avidit y t o ant igens wit h mult iple epit opes.
IgM ant ibodies appear ear ly in t he r esponse t o infect ion and because of t heir size, ar e
lar gely confined t o t he blood st r eam. They ar e an impor t ant defence mechanism against bact er ia.
The size and valency of IgM makes it a ver y effect ive agglut inat ing and cyt olyt ic agent . This is
so because it is t he most efficient complement fixing immunoglobulin. IgM pr edominat es in
cer t ain ant ibody r esponses such as t he “nat ur al” isohaemagglut inins (ant i-A, ant i-B) and t o t he
t yphoid ‘O’ ant igen. Since IgM does not cr oss t he placent a it s pr esence in cor d blood indicat es
act ive foet al infect ion, fur t her mor e, since t he IgM r esponse is shor t lived it s pr esence may be
30 Immunology– Introductory Textbook
helpful in est ablishing an acut e infect ion. Monomer ic IgM has a hydr ophobic sequence st it ched
int o t he ‘C’ t er minal of t he heavy chain t o anchor t he molecule int o t he cell membr ane of t he
B-lymphocyt e. The anchor ed IgM is t he major ant ibody r ecept or used by B-lymphocyt es t o
r ecognize ant igen.
Figure 5.5. (a) IgM free and (b) bound to the bacterial surface.
Immunoglobulin A
IgA is act ively secr et ed by, what is now known as, mucosal associat ed lymphoid t issue
(MALT). IgA appear s select ively in ser o-mucus secr et ions such as saliva, t ear s, nasal fluids,
colost r um and in secr et ions of t he lung, genit o-ur inar y and gast r o-int est inal t r act s. It is pr esent
in t hese fluids as a dimer , st abilized against pr ot eolysis by combinat ion wit h anot her pr ot ein,
t he secr et or y component , which is synt hesized by local epit helial cells and has a single pept ide
chain of molecular weight 60,000 (Figur e 5.6). The IgA is synt hesized locally by plasma cells
and dimer ized int r a cellular ly befor e secr et ion, wit h t he help of a cyst eine r ich polypept ide
called t he J chain which has a molecular weight of 15,000. The dimer ic IgA is r eleased fr om t he
plasma cell and binds st r ongly t o a secr et or y component pr ecur sor pr esent on t he sur face of
t he glandular epit helial cell. It is act ively endocyt osed and t r anspor t ed wit hin t he endocyt ic
vacuole t o t he mucosal sur face. Cleavage of t he r ecept or r eleases t he IgA, st ill at t ached t o par t
of t he r ecept or : t er med t he secr et or y piece, int o ser o-mucus secr et ions (Figur e 5.7).
Figure 5.6. Structure of the IgA dimer.
Immunoglobulins I: Structure and Function 31
Figure 5.7. Formation of secretory IgA. The glandular cell on the mucosal surface synthesizes an
immunoglobulin receptor inserted into the basal membrane. Dimeric IgA binds to this receptor, is
endocytosed in a vacuole and transported to the mucosal surface. Cleavage of the receptor yields
secretory IgA into the lumen with part of the receptor (as the secretory piece) still in place.
Since IgA is most abundant in body secr et ions it per for ms t he r ole of defending t he exposed
ext er nal sur faces of t he body against at t ack by micr o or ganisms. IgA funct ions by inhibit ing t he
adher ence of coat ed micr o or ganisms t o t he sur face of mucosal cells t her eby pr event ing ent r y
int o t he body t issues. IgA act ivat es complement via t he alt er nat ive pat hway but is unable t o do
so via t he classical pat hway. IgA does not cr oss t he placent a, but cont r ibut es t o t he pr ot ect ion
of t he newbor n by being in abundance in colost r um. IgA is t he pr ime funct ional unit of t he
mucosal associat ed lymphoid t issue (MALT).
Immunoglobulin E
IgE is pr esent in ver y low concent r at ions in ser um. IgE ant ibodies have a high affinit y for
mast cells and binding occur s via t he Fc por t ion of t he immunoglobulin molecule. On cont act
wit h specific ant igens called aller gens, t he mast cells under go degr anulat ion wit h r elease of
vasoact ive amines. This pr ocess is r esponsible for t he wheal and flar e skin r eact ion in aller gy,
for t he sympt oms of hay fever and ext r insic ast hma. IgE also has t he abilit y t o at t ach t o human
skin wher e t hey ar e pr obably bound t o mast cells. IgE is found mainly in t he linings of t he
r espir at or y and gast r o int est inal t r act s, wher e t hey for m const it uent s of MALT.
The main physiological r ole of IgE would appear t o be pr ot ect ion of ext er nal mucosal
sur faces wher e r elease of vasoact ive amines could per pet uat e t he acut e inflammat or y r esponse.
Infect ious agent s penet r at ing t he IgA defences would combine wit h specific IgE on t he mast
cell sur face t o t r igger t he r elease of vasoact ive agent s and ot her fact or s chemot act ic for
gr anulocyt es. This would lead t o an influx of plasma IgG, complement , polymor phs and
eosinophils causing an effect ive inflammat or y r esponse. It is possible t hat IgE act s in t his way
as a defence against helmint hic infect ions; which ar e char act er ized by an exagger at ed IgE
r esponse.
32 Immunology– Introductory Textbook
Immunoglobulin D
This immunoglobulin has t he basic four pept ide st r uct ur e. It is pr esent in ser um in t r ace
amount s. Because of an ext ended hinge r egion it is r elat ively liable t o degr adat ion by pr ot eolyt ic
enzymes. The main funct ion of IgD has not yet been det er mined; wit h IgM it is found abundant ly
on t he sur face of B-lymphocyt es. It has been suggest ed t hat t hey may oper at e as ant igen
r ecept or s and in t he cont r ol of lymphocyt ic act ivat ion and suppr ession.
Immunoglobulin Sub-classes
IgG has four isot ypic sub classes t er med IgG1, IgG2, IgG3 and IgG4. The differ ences lie in
t he chains of t he molecule. Cer t ain funct ional and biological differ ences exist bet ween t he
classes for eg: IgG3 does not combine wit h st aphylococcal pr ot ein A, IgG2 does not cr oss t he
placent a r eadily, IgG4 does not bind t o complement in t he classical pat hway nor does it bind t o
monocyt es.
IgA exist s as IgA1 (const it ut es 80-90% of t ot al IgA), IgA2 is unusual because it lacks H t o
L disulphide bonds.
Major char act er ist ics of t he immunoglobulin classes have been summar ised in Table 5.1.
Table 5.1: Characteristics of the immunoglobulin classes
Characteristics IgG IgM IgA IgE IgD
Sedimentation 7S 19S 11S 8S 7S
coefficient
Molecular weight 150,000 900,000 160,000 200,000 185,000
Functions
Complement
fixation
classical ++ +++ – – –
alternative – – + – –
Cross placenta + – – – –
Fix to homologous – – – + –
mast cells /
basophils
Bind to + – + – –
macrophages and
polymorphs
™™™
Most abundant
in extra-
vascular
spaces;
combats micro-
organisms and
their toxins
Early in immune
response.
Effective in
agglutination;
first line defence
in bacteraemia
Major Ig in
sero-mucus
secretions,
defends
external body
surfaces
Mostly
present on
surface of B
lymphocytes
Protects
external body
surface,
responsible
for
symptoms of
allergy.
Raised in
parasitic
infections.
T
he immune syst em is clear ly essent ial for sur vival; wit hout it deat h fr om infect ion
would be inevit a ble. The molecules of t he immune syst em ma int a in const a nt
sur veillance against invading micr o or ganisms. They r ecognize an almost limit less var iet y of
for eign subst ances, t hey can dist inguish and bind t o ever y possible ant igen t hat t he host is
likely t o meet dur ing it s lifet ime and some t hat it is not . Fur t her mor e, t hey r emember each
infect ion so t hat a second exposur e t o t he same or ganism is dealt wit h mor e efficient ly. This
implies t hat millions of species of ant ibody molecules need t o be synt hesized. Since an ant ibody
is an assembly of pr ot ein chains, it s st r uct ur e is specified by a gene. It would appear t hat t he
genome must cont ain millions of ant ibody genes, since each ant ibody is differ ent depending on
t he ant igen t hat pr ovokes it s for mat ion. Yet t he t ot al genet ic complement of a mammal amount s
t o per haps a million genes. How t hen does t he immune syst em funct ion on such a small defence
budget demanding t hat only a small shar e of t he genome and t he body’s r esour ces be ear
mar ked for t he pr oduct ion of ant ibodies?
The par adox of a limit ed number of genes and an appar ent ly limit less capacit y t o gener at e
differ ent ant ibodies has been a major puzzle for immunologist s,and essent ially t wo compet ing
t heor ies wer e put for war d in explanat ion - t he ger m line and somat ic r ecombinat ion t heor ies.
The Dreyer-Bennett Hypothesis
William Dr eyer and Claude Bennet t in 1965 wor king at t he Calt ech labor at or ies made a
r adical pr oposal. Inst ead of assuming t hat t he genet ic infor mat ion for an ant ibody light chain is
specified by a cont inuous ar r ay of codons (a codon is a t r iplet of nucleot ides which codes for an
amino acid), t hey pr oposed t hat t her e wer e sever al hundr ed st r et ches of DNA t hat coded
separ at ely for differ ent kinds of var iable r egions and one gene coded for t he const ant r egion in
t he ger m line DNA (DNA in t he sper m cell and ovum). They also implied t hat t hese separ at e
list s of infor mat ion must somehow come t oget her t o for m a cont iguous, coher ent message.
This pr oposal init ially at t r act ed consider able cr it icism since it opposed t he cent r al dogma of
pr ot ein biosynt hesis; t he one gene, one polypept ide t heor y. Dr eyer and Bennet t claimed t hat
t wo or mor e genes could cont r ol t he pr oduct ion of one polypept ide chain, at a t ime when no
means of r ear r angement of genes in ger m line cells was known of. Yet t heir idea pr oved t o be
essent ially cor r ect .
The Generation of Antibody Diversity by Somatic Recombination
The DNA in t he ger m cells (in t he male sper m and t he female egg) or in t he ear ly embr yo
cont ains sever al bit s and pieces of t he gene t hat dict at es ant ibody for mat ion. These bit s and
pieces ar e shuffled in t he DNA of t he B-lymphocyt e t o yield t he r equir ed ant ibody. The
mechanisms r esponsible for shuffling of immunoglobulin DNA became clear when it was possible
IMMUNOGLOBULINS II:
THE GENETICS OF ANTIBODY DIVERSITY
CHAPTER – 6
34 Immunology– Introductory Textbook
t o sequence fr agment s of DNA aft er t hey wer e cloned in bact er ia. These exper iment s by
Ton ega wa , Hozu mi and ot her s showed t hat i) V and C genes wer e far t her apar t in embr yonic
cells t han in ant ibody pr oducing cells and ii) a par t icular light chain had a small segment of
DNA coding for a par t of t he V r egion: t his fr agment was called t he V segment . The r est of t he
V region was coded for by a st ret ch of DNA locat ed t housands of base - pairs away. This int erposed
segment was called J for “joining”. The C segment was also locat ed about 1300 base pair s
“upst r eam” and was found t o code for t he C r egion. Each light chain was assembled by combining
t he scat t er ed V,J and C segment s.
Ther eaft er it was discover ed t hat t her e wer e a few hundr ed V segment s in ger m line
DNA, each differ ing slight ly in amino acid sequence, and four dist inct J segment s.
The pot ent ial diver sit y of t he heavy chains is even gr eat er . In addit ion t o V and J segment s
t he genes for t he heavy chain var iable segment include a t hir d fr agment designat ed D (for
diver sit y). Mouse ger m line cells have about 20 D segment s. In pr inciple, t hese segment s can
be br ought t oget her in over 10,000 combinat ions. Combining a light wit h a heavy chain can
yield over 10 million dist inct ant igen binding sit es.
The Assembly of a Functioning Immunoglobulin Gene
The assembling of a funct ional immunoglobulin gene t akes place in t wo st ages of a pr ocess
called Soma t i c Recomb i n a t i on . As shown in Figur es 6.1 and 6.2 t he ger m line DNA cont ains
Figure 6.1. Organization of the human light chain gene; illustrates the process of kappa light chain
secretion. The same principle holds for the lambda light chain. Multiple variable (Vk) region bits exist
in the germ line DNA, each accompanied by a leader (L) sequence. There are 5 joining (J) segments
coding for amino acids 96-108. There is only one constant (C) gene. DNA rearrangement joins 1 V
region to 1 J region to the C region. The intervening sequences (IVS) are removed by RNA splicing.
This yields one kind of high chain. Many other permutations and combinations are possible.
Immunoglobulins II: The Genetics of Antibody Diversity 35
an ar r ay of segment s fr om which per mut at ions and combinat ions occur . Each B- cell DNA
cont ains r andomly select ed V and J segment s for a light chain or V,D and J segment s for a
heavy chain. These segment s ar e fused by enzymes t hat delet e all t he int er vening DNA,
somet imes t er med i n t e r ve n i n g se q u e n ce s (I VS) or i n t r on s. Each of t he V segment s is
pr eceded by a shor t coding sequence known as t he “leader ” sequence and t his is t hought t o
play a par t in t he t r anspor t of t he ant ibody molecule t hr ough t he cell membr ane, it is t hen
cleaved away as t he ant ibody molecule passes acr oss t he membr ane.
The second st age in t his pr ocess r elies on mechanisms of RNA sp licin g. An RNA t r anscr ipt
is obt ained as shown in Figur e 6.1, wit h t he select ion of a V, coding for amino acids(1-95), a J
coding for amino acids 96-108 and t he C gene separ at ed fr om t he J gene by an int r on of 1250
nucleot ides. Aft er RNA splicing t he mRNA cont ains a single V gene in combinat ion wit h a
single J which in t ur n t ags on t o a single C gene. All int r ons have been excised. This is t hen
t r anslat ed t o for m t he pr ot ein molecule which is t he ant ibody. In case of t he heavy chain gene
or der t he fusion or der yields a V,D, J and C segment s (See figur e 6.2).
Figure 6.2. Organization of the human heavy chain gene. Similar to the k light chain V fragments,
there are multiple variable (V
H
)regions with preceding leader (L) sequences. There are the 6
functional joining (J
H
) segments and in addition, a family of diversity (D
H
) segments increase the
variety of possible combinations between segments. Single VH, DH and JH regions recombine with
each other at the DNA level. RNA splicing removes the intervening sequences (IVS) to join the
constant (C
µ
) to the rest yielding a secreted IgM protein - among the first immunoglobulins to be
secreted by a stimulated plasma cell.
Fusion of gene segment s in B- cell DNA is unusual and may even be unique t o t he immune
syst em. It employs a set of enzymes t hat can br ing t oget her dist ant V,D and J segment s delet ing
all t he DNA t hat separ at es t hem. Cer t ain signal sequences t hat guide t he sit e of act ion of t hese
splicing enzymes have been discover ed. As shown in Figur e 6.3, just down st r eam fr om t he V
gene of a kappa chain t her e is a dist inct ive pat t er n composed of a hept amer or seven nucleot ide
unit , followed by a spacer and a nonamer or nine nucleot ide unit . J ust upst r eam of t he J
segment t her e is a complement ar y nonamer -spacer -hept amer pat t er n. These unit s could pr ovide
a t emplat e for t he enzymes t o cut and r ejoin t he double helix. Similar signal sequences ar e
found in t he heavy chain genes.
36 Immunology– Introductory Textbook
Humans ut ilize lambda light chains in one t hir d of t heir immunoglobulins.In cont r ast t o
kappa light chains, lambda light chains have mult iple const ant r egion genes ar r anged in t andem.
Figure 6.3. Sequences that facilitate V-J, V-D and D-J joining. The characteristic sequences form
a base paired heptamer, a spacer of 12 bases (at the V end) and 23 bases (at the J end) and then
a base paired nonamer. These characteristic sequences facilitate splicing by DNA repair enzymes
bringing distant V-J regions into proximity. Similar mechanisms exist for joining V-D and D-J segments.
Ther e ar e at least t wo a d d i t i on a l sou r ce s of va r i e t y t hat cont r ibut e t o ant ibody
diver sit y. One of t hese is a lack of pr ecision in t he DNA splicing machiner y t hat fuses V,D and
J segment s. The sit e of t he junct ion can var y by sever al base pair s. Fur t her mor e, in some
cases, addit ional base pair s ar e inser t ed in t he pr ocess of combining segment s. Bot h kinds of
change can obviously alt er t he amino acid sequence of t he polypept ide. As a r esult , even if t wo
ant ibodies ar e specified by t he same set of gene segment s, t hey may st ill have slight ly differ ent
ant igen binding sit es.
Anot her major sour ce of diver sit y is s oma t i c h yp e r mu t a t i on . Spont aneous genet ic
changes in t he developing cells ar e known t o occur . Mut at ions ar e seen in t he var iable domain
genes and in t he immediat ely adjacent r egions, but not in t he const ant domains. Est imat es of
t he r at e of mut at ions suggest t hat t her e should be one change in t he V r egion for ever y t hr ee
t o 30 cell divisions, a r at e sever al or der s of magnit ude gr eat er t han t he aver age mut at ion r at e
for eukar yot ic genes. It seems likely t hat B cells or t heir pr edecessor s car r y enzymes t hat
induce mut at ions in just t hese set s of genes.
The pr ocess of somat ic r ecombinat ion cr eat es a populat ion of cells t hat var y widely in
t heir ant igen specificit y; fr om which few cells ar e select ed by any given ant igen. The mut at ional
mechanism is called int o act ion dur ing pr olifer at ion of t he select ed B-cell clones. By alt er ing
individua l nucleot ide ba ses t he mut a t ions “fine-t une” t he immune r esponse cr ea t ing
immunoglobulin genes whose pr oduct s bet t er mat ch t he ant igen.
The effect s of DNA - joining inaccur acy and somat ic mut at ion, in addit ion, incr ease t he
number and var iet y of ant igen binding sit es and t oget her wit h somat ic r ecombinat ion, t he t ot al
number is est imat ed at well over a billion differ ent ant igen binding t ypes.
Immunoglobulin Class Switching in Individual B-cells, and the Heavy Chain Gene
Order
An immat ur e B cell bear ing only sur face IgM, develops int o a cell t hat simult aneously
pr oduces IgM and IgD and is subsequent ly capable of swit ching t o t he pr oduct ion of IgG, IgA or
Immunoglobulins II: The Genetics of Antibody Diversity 37
IgE. It is impor t ant t o not e t hat each of t hese heavy chain classes is associat ed wit h t he same
var iable heavy chain r egion (V
H
) in a given cell and hence bear s t he same ant igen binding
specificit y.
The exact mechanism by which t he differ ent classes ar e pr oduced became clear er when
t he heavy chain gene or der was sequenced. As descr ibed ear lier , t his gene or der was similar in
pr inciple t o t he V, J and C segment s of t he light chain gene. As can be seen fr om Figur e 6.4,
t her e ar e sever al V
H
segment s followed by t he D
H
and J
H
segment s. The µ const ant (C µ) r egion
is closely associat ed wit h t he δ const ant r egion (C δ). Consider able space separ at es t hese t wo
genes fr om t he γ const ant (C γ) clust er of genes which appear in t he or der of Cγ3, Cγ1, Cγ2b and
Cγ2a. This is followed by t he ε C r egion and last ly t he α C r egion. The init ial r ear r angement of
t he V-D-J segment s in t he B cell DNA is similar t o t he light chain genes and has been descr ibed
ear lier . St udies on t he var ious ‘C’ r egion segment s showed t he pr esence of highly homologous
Figure 6.4. Gene assembly for immunoglobulin class switching. Following the initial DNA re
arrangement recombining VH, DH and JH region a B cell can utilize alternative sites of RNA splicing
to simultaneously produce IgM and IgD. Alternatively such a B cell can further differentiate and
switch to producing another class of immunoglobulins. For example a DNA recombination can occur
at the switch site (Sα) and bring about a V-D-J-C joining leading to IgA production. Similar homologous
switch sites are found (not shown in this figure) in front of each of the ‘C’ regions.
swit ch sit es (S-sit es) in fr ont of t he C µ and C α segment s (Figur e 6.4). Similar swit ch sit es (not
shown in diagr am) ar e found in fr ont of each of t he ot her C r egions. If RNA splicing occur s at
t he S µ sit e, t hen mRNAs can be pr oduced simult aneously for IgM and IgD by ut ilizing a
t r anscr ipt wit h t he C µ r egion for IgM pr oduct ion and t he C δ r egion for IgD pr oduct ion. Such a
B cell would be equipped t o secr et e bot h IgM and IgD. Alt er nat ively, t he same B cell can
fur t her differ ent iat e and swit ch t o pr oducing anot her heavy chain class. This would necessit at e
a second DNA r ear r angement at t he highly homologous swit ch sit es of S µ and S α in fr ont of
t he C µ and C α r egions. Wit h t r anscr ipt ion and RNA splicing all t he int er vening sequences ar e
38 Immunology– Introductory Textbook
r emoved yielding an mRNA consist ing of V-V-D-J -C α (see Figur e 6.4). Such a B cell would
effect ively swit ch t o producing IgA wit h t he same ant igen specificit y. Because similar homologous
swit ch sit es ar e found in fr ont of t he γ and ε r egions, alt er nat ive pr oduct ion of immunoglobulins
of all t he above classes is possible.
It is essent ially cor r ect , however , t o st at e t hat one B cell pr oduces one kind of ant ibody,
since what ever be t he heavy chain class of t he immunoglobulin pr oduced, t hey will all r eact
only t o one ant igenic det er minant , since t hey all have similar var iable r egions and t her efor e
similar ant igen binding specificit y.
™™™
T
he complement syst em is t he pr imar y humor al mediat or of ant igen ant ibody r eact ions.
The syst em consist s of at least 20 chemically and immunologically dist inct plasma
pr ot eins, capable of int er act ing wit h each ot her , wit h ant ibody and wit h cell membr anes.
Following act ivat ion of t his syst em, t hese int er act ions lead t o t he gener at ion of a wide r ange of
biological act ivit y fr om lysis of differ ent kinds of cells, bact er ia and vir uses t o dir ect mediat ion
of t he inflammat ory process. In addit ion, complement is able t o recruit and enlist t he part icipat ion
of ot her humor al and cellular effect or syst ems and induce hist amine r elease fr om mast cells,
migr at ion of leucocyt es, phagocyt osis and r elease of lysosomes fr om phagocyt es.
The individual pr ot eins of t he complement syst em ar e nor mally found in t he ser um as
funct ionally inact ive molecules, belonging t o t he plasma globulin fr act ion. Component s ar e
designat ed by numer als C
1
, C
2
, C
3
et c and by names such as Fact or B and Fact or D. Each
component is act ivat ed sequent ially (similar t o t he t r igger ed enzyme cascade phenomenon in
coagulat ion), r esult ing in t he complement r eact ion. Once act ivat ed t he complement component s
become enzymes and are designat ed wit h a bar such as
B Fact or or C1
. A complement component
can somet imes be cleaved by an enzyme leading t o t he for mat ion of cleavage pr oduct s such as
C4a and C4b.
Ther e ar e t wo par allel but ent ir ely independent pat hways leading t o t he t er minal most
act ive par t of t he complement syst em (Figur e 7.1). The t wo pat hways ar e called t he cla ssi ca l
and t he a lt e r n a t i ve pat hways. Each pat hway is t r igger ed by differ ent subst ances. The t wo
pat hways conver ge at a point fr om which t he fi n a l common p a t h wa y ensues, t o t er minat e at
t he end point of complement act ivat ion which is cyt olysi s or cyt ot oxi ci t y. Ther e ar e ot her
non complement enzymes of ser um or cellular or igin which can act ivat e t he complement syst em
midway for example : t r ypsin - like enzymes such as plasmin.
Table 7.1: Activators of the complement system
Classical Alternative
Immunologic Antigen-antibody complexes
Non-immunologic Trypsin-like enzymes DNA
C-reactive protein
Staphylococcal protein A.
THE COMPLEMENT SYSTEM
CHAPTER – 7
IgA, IgG
Lipopolysaccharide, plant and
bacterial polysaccharides,
cobra venom;
Trypsin-like enzymes,
40 Immunology– Introductory Textbook
F
i
g
u
r
e

7
.
1
.


T
h
e

C
o
m
p
l
e
m
e
n
t

C
a
s
c
a
d
e
.
The Complement System 41
The Classical Complement Pathway
The classical complement pat hway is act ivat ed as descr ibed above (r efer Table 7.1). Among
t he IgG sub classes, IgG
3
is t he most act ive, followed by IgG
1
and IgG
2
. IgM is t he most efficient
immunoglobulin act ivat or of t he classical complement pat hway. Act ivat ion occur s by binding of
t he fir st complement component C1, t o a sit e locat ed in t he CH
2
r egion of t he immunoglobulin
molecule. Hence ant igen ant ibody complexes or aggr egat ed immunoglobulins ar e a must for
act ivat ion of t he classical complement syst em. As list ed, non immunologic subst ances also
act ivat e complement . Her e, act ivat ion occur s by dir ect binding of C1 or in t he case of enzymes,
by dir ect pr ot eolyt ic at t ack of C1.
C1
The st eps involved in t he act ivat ion of C1, following at t achment or pr ot eolyt ic at t ack,
compr ise t he fir st funct ional unit of t he complement pat hway. C1 consist s of 3 dist inct pr ot ein
molecules: C1q, C1r and C1s held t oget her by calcium bonds. C1 is nor mally pr esent as a
complet e ent it y; individual pr ot eins of C1 ar e found only in disease st at es. C1q is unique because
it s st r uct ur e is ver y similar , chemically, t o t hat of collagen or basement membr ane.
C1q cont ains a t ot al of 18 polypept ide chains of 3 dist inct t ypes, t hat ar e or ganized int o a
st r uct ur e consist ing of 6 per ipher al globular por t ions connect ed by fibr illar st r ands t o a cent r al
st r uct ur e. The polypept ide chains have been visualized as for ming 6 sub unit s, each in t he for m
of a t r iple helix (Figur e 7.2).
Figure 7.2. Schematic representation of C1q.
The C1q molecule bear s t he sit es which enable t he C1 molecule t o bind t o t he Fc r egion of
IgG and IgM ant ibodies. It is t her efor e able t o bind t o 6 IgG molecules (Figur e 7.2).
Once C1q binds t o t he immunoglobulin molecule, C1r , a β globulin molecule, acquir es t he
abilit y t o enzymat ically act ivat e C1s.Int egr it y of t he C1 macr omolecule and calcium ions ar e
r equir ed for t his pr ocess. C1s, once act ivat ed,
) C1s (
acquir es pr ot eolyt ic act ivit y. Once C1s is
act ivat ed t he init ial phase of t he classical complement pat hway is complet ed; t he earlier react ant s
including ant igen, ant ibody and C1q or C1r ar e not necessar y for pr ogr ession of t he complement
r eact ion. Act ivat ed C1s mediat es t he next phase of t he complement cascade.
C4 and C2
C1s cleaves C4, yielding C4a and C4b (Fig 7.3); it also cleaves C2 and init iat es t he for mat ion
of t he key enzyme
C4b2a
on t he act ivat or sur face. C4b cont ains a labile binding sit e which
42 Immunology– Introductory Textbook
binds t o it s act ivat or in a t r ansient manner . C2a also has a labile binding sit e which allows it t o
bind t o C4b . Magnesium ions ar e r equir ed for t he for mat ion of t he
C4b2a
complex. Act ivat ed
C4b2a
is a pr ot eolyt ic enzyme t hat assumes t he r ole of cont inuing t he classical complement
r eact ion. Once it has been for med, ear lier r eact ant s ar e no longer necessar y. C4b2a is also
t er med C3 conver t ase as it cleaves and t her eby act ivat es C3. The enzymat ic sit e r esides in t he
C2 moiet y of t he complex.
Figure 7.3. Schematic model of C4: disulphide linkages between the three chains go into
forming the C4 molecule.
C3
Cleavage of C3 r esult s in t he for mat ion of C3a and C3b (Figur e 7.4). C3a is a biologically
pot ent pept ide and will be discussed lat er . The at t achment of C3b t o membr anes in t he vicinit y
of
C4b2a
molecules leads t o t he gener at ion of t he last enzyme of t he classical pat hway:
C4b2a3b
.
This enzyme act s t o cleave C5.
Figure 7.4. Schematic structure of C3 and its cleavage products. The B chain together with
the disulphide linked fragments remaining after Factor I cleavage is termed C3c.
The Complement System 43
The classical complement pat hway t hus consist s of a ser ies of enzyme subst r at e r eact ions
in sequence, which lead t o t he for mat ion of sever al mor e complement enzymes. The r eact ions
involved ar e highly specific and t her e is consider able t ur nover of t he molecules involved i.e.
C2, C3, C4 and C5. In addit ion, sever al biologically act ive molecules ar e gener at ed and it is
evident t hat a r elat ively small st imulus t o complement act ivat ion may lead t o consider able
gener at ion of t hese molecules wit h t he r esult t hat sever e t issue r eact ion can ensue.
The Alternative Complement Pathway
The alt er nat ive complement pat hway const it ut es t he humor al component of nat ur al
defence against infect ions which can oper at e wit hout ant ibody par t icipat ion. Six pr ot eins, C3,
B, D, H, I, and P, by t hemselves per for m t he funct ions of init iat ion, r ecognit ion, and amplificat ion
of t he pat hway. A var iet y of act ivat or s of t his pat hway have been descr ibed such as cer t ain
par t iculat e polysacchar ides, for example, bact er ial (LPS), yeast (zymosan), or plant (inulin)
polysa ccha r ides, fungi, ba ct er ia , vir uses, cer t a in ma mma lia n cells, a nd a ggr ega t es of
immunoglobulins, for example, t he Fab por t ions of IgA or IgE (Table: 7.1).
It is not yet known which st r uct ur es t hese act ivat or s have in common as far as r ecognit ion
by t he pat hway is concer ned. The mechanisms by which t hese het er ogeneous gr oups of
subst ances can init iat e t he act ivat ion of t his pat hway, ar e also not fully under st ood. It is clear ,
however , t hat r ecognit ion involves C3b.
Proteins of the alternative complement pathway
• P (pr oper din) – a γ2 globulin
• fact or B – a β2 pr ot ein
• fact or D – an α globulin
• fact or s I and H – β globulins
The alt ernat ive complement pat hway, because of it s major role in t he resist ance t o infect ion,
is consider ed t o be t he pat hway t hat is mor e impor t ant t o man. The alt er nat ive pat hway was so
named because act ivat ion pr oceeds in a manner differ ent fr om t hat of t he classical pat hway,
and bypasses t he need for C1, C4 and C2.
An init ial r equir ement for act ivat ion is t he pr esence of C3b, which is cont inuously
gener at ed. This occur s following cle a va ge of a t h i o e st e r b on d i n C3, t hus for ming C3*,
which r eact s wit h fact or s B and D t o gener at e an enzyme able t o cleave C3 int o C3a and C3b
(t he C-3 cleaving enzyme, also known as t he pr iming C3 conver t ase). Wat er can hydr olize C3
and for m C3i, a molecule t hat funct ions in a manner similar t o C3b. It is possible t hat C3 in
cir culat ion is also cleaved by enzymes of t he coagulat ion and fibr inolyt ic syst ems; t he enzyme
zymosan : a yeast polysacchar ide also causes C3 act ivat ion in t he pr esence of magnesium ions.
Most of t he newly gener at ed C3b r emains in t he fluid phase, some binds t o var ious cellular
sur faces. In eit her case t his C3b is r apidly inact ivat ed by cont r ol pr ot eins, fact or s I and H,
which cleave it . This st eady level of C3b in t he body is gr eat ly modified by par t iculat e act ivat or s
of t he alt er nat ive pat hway, such as lipopolysacchar ides and cer t ain cells. The C3b deposit ed on
such act ivat or s is “pr ot ect ed” fr om dest r uct ion by fact or s I and H. This sur face bound, pr ot ect ed
C3b int er act s wit h fact or B in t he pr esence of magnesium ions t o for m C3bB. Wit h t he addit ion
of fact or D, which act s on bound B t o cleave it yielding Bb, an enzyme
C3bBb
is gener at ed.
This sur face bound enzyme is also called t he amplifying C3 conver t ase and is able t o cleave
ver y lar ge amount s of C3. This r esult s in t he accumulat ion of C3b and as shown in Figur e
7.5, t her e is a cyclical amplificat ion of t he cr it ical C3b component . This is also called a posit ive
44 Immunology– Introductory Textbook
feed back mechanism which enhances C3b for mat ion. Fur t her mor e, if pr oper din (P) wer e t o
complex wit h
C3bBb
yielding
C3bPBb
, t he enzyme becomes funct ionally mor e efficient .
Figure 7.5. The positive feed back mechanism for C3b.
Many of t he C3b molecules t hus gener at ed, eit her by
C3bBb
or
C3bPBb
, bind t o t he
sur face of t he act ivat or par t icle in close pr oximit y t o each ot her . This r esult s in t he for mat ion
of modified enzymes,
Bb C3b
n
or
) 1 n ( PBb C3b
n
>
, which ar e able t o cleave C5 and init iat e t he
final common membr ane at t ack pat hway (see Figur e 7.1). Hence t he cyclic amplifying syst em,
in conjunct ion wit h t he cr ucial “pr ot ect ed” sur face r epr esent s t he key event s in act ivat ion of
t he alt er nat ive pat hway.
Cell sur face sialic acid, which is pr esent in glycopr ot eins, glycolipids and polysacchar ides
incr eases t he affinit y of membr ane associat ed C3b for H. Thus cells t hat have abundant sialic
acid ar e non act ivat or s of t he alt er nat ive pat hway. It has been hypot hesized t hat some bact er ial
species t hat car r y capsular sialic acid ar e mor e pat hogenic for neonat es t han or ganisms t hat
lack t his subst ance. Bact er ia wit h capsular sialic acid include t ype III gr oup B S treptococcus,
gr oups B and C Neisseria meningitidis and K1 Escherichia coli. Human par asit es such as
Leishmania major and Trypanosoma cruzi have evolved several st rat egies for evading recognit ion
by t he alt er nat ive pat hway, including synt hesis of glycopr ot eins t hat cont ain sialic acid, r apid
degr adat ion of bound C3b and synt hesis of a pr ot ein t hat compet es wit h B for binding t o C3b.
Host cells ar e pr ot ect ed fr om t he effect s of complement act ivat ion by membr ane pr ot eins
which pr event act ivat ion of classical and alt er nat ive pat hways. The alt er nat ive pat hway may
be act ivat ed by an isolat ed pr ot ein obt ained fr om cobr a venom. This pr ot ein appear s t o r epr esent
cobr a C3b.
The Membrane Attack Mechanism: C5 - C9
The t er minal por t ion of t he complement sequence is t er med t he membr ane at t ack syst em,
since C5b-9 must become membr ane bound in or der for membr ane changes or damage t o
occur .
The complement at t ack mechanism is init iat ed on cleavage of C5 by
Bb b 3 C , C4b2a3b
n
or
PBb C3b
n
or cer t ain enzymes such as plasmin (see Figur e 7.1). The act ivat ion r eact ion r esult s
The Complement System 45
in gener at ion of a biologically act ive pept ide: C5a and a lar ger C5b. C5b has t he abilit y t o bind
C6 and C7 for ming a complex of
67 b 5 C
. This complex has only t r ansient abilit y t o bind t o
membr anes. However , t his pr ocess is modulat ed by S pr ot ein which is a nor mal ser um pr ot ein.
The S pr ot ein act s by binding t o t he membr ane at t he same binding sit e as t he
67 b 5 C
complex,
t hus nat ur ally inhibit ing it . Each
67 b 5 C
complex possesses a binding sit e for t he molecule C8.
Membr ane leakage begins at t his st age. The cyt olyt ic pr ocess is,however ,gr eat ly enhanced by
t he at t achment of t he last complement component : C9, t o t he membr ane bound
678 b 5 C
complex. The r esult ing C5b6789 complex, cont aining one molecule of C5b,C6,C7,C8 and sever al
molecules of C9, r epr esent s t he fully assembled cyt olyt ic mechanism of t he complement syst em.
The Molecular Mechanism of Cytolysis
Binding of ant ibody t o a t ar get cell t r igger s t he complement cascade in which successive
pr ot eins of t he complement syst em ar e act ivat ed as descr ibed in t he pr evious sect ion. Event ually
C5b binds t o C6 and C7 and is inser t ed int o t he t ar get cell membr ane. C8 and sever al C9
pr ot eins t hen aggr egat e ar ound t he C5b67 complex binding t oget her t o for m a cylindr ical channel
or a por e like st r uct ur e. This per mit s r apid influx of ions because of a leaky membr ane and
ult imat ely leads t o cell lysis (Figur e 7.6). J ust one such membr ane - at t ack complex ( or por e
channel) of t he complement syst em need be inser t ed int o t he t ar get cell t o cause cell lysis. This
is called t he “one-hit ” mechanism of complement mediat ed cell lysis.
Figure 7.6. The mechanism of complement mediated membrane leakage.
46 Immunology– Introductory Textbook
Control Mechanisms of the Complement System
Uncont r olled act ivat ion of t he complement syst em is pr event ed by t he fact t hat act ive
sit es of t he complement component s a r e la bile. In a ddit ion, sever a l complexes such a s
b 3 a 2 b 4 C and a 2 b 4 C , bBb 3 C
dissociat e in a t ime and t emper at ur e dependant fashion.
Ot her pr ot eins limit r unaway complement act ivat ion by binding t o or enzymat ically
at t acking cer t ain specific complement component s. C1 i n h i b i t or is one such pr ot ein which
inhibit s enzymat ic act ivit y of C1 and it s C1r and C1s subunit s by rapidly forming firm, irreversible
complexes.
Anot her key cont r ol pr ot ein of t he complement syst em is F a ct or I . Fact or I at t acks C3b
fr ee, in solut ion or on t he sur face of cells and cleaves t he molecule. Anot her r egulat or t hat
act s on C3b is F a ct or H, a ser um pr ot ein t hat binds t o C3b and acceler at es t he dest r uct ion of
C3b by fact or I. It also binds t o C3b on int er mediat e complexes and inhibit s t he enzyme
bPBb 3 C
. A C4 b i n d i n g p r ot e i n also exist s which binds t o C4b and facilit at es it s dest r uct ion
by Fact or I. A C8 bin d in g p r ot ein inhibit s binding of C9 t o
678 b 5 C
and prevent s t he membrane
a t t a ck mecha nism fr om going int o oper a t ion. Huma n ser um cont a ins a n enzyme t he
a n a p h yla t oxi n inact ivat or , an α globulin, which dest r oys t he biological act ivit ies of C3a, C4a
and C5a. The S pr ot ein binds t o t he
67 b 5 C
complex and pr event s membr ane leakage.
Biological Consequences of Complement Activation
Besides t he labile binding sit es involved in t he pat hways, sit es ar e gener at ed or uncover ed
in t he fr agment s of C3 and C4 as a consequence of pr ot eolyt ic cleavage of t he molecules dur ing
complement act ivat ion. Pr ot eolyt ic cleavage also r esult s in fur t her br eakdown pr oduct s of t he
molecules C3 and C4. Ther e ar e t her efor e sever al r eact ive sit es gener at ed in C3b, C4b and in
t heir br eakdown fr agment s. These r eact ive sit es ar e r ecognised by var ious cells having specific
r ecept or s for t hese fr agment s. Table 7.2 list s t he var ious complement fr agment s, t heir r ecept or
specificit y and t he cells bear ing t hese r ecept or s.
TABLE 7.2: SPECIFICITY AND CELLULAR DISTRIBUTION OF COMPLEMENT RECEPTORS
Receptor Complement fragment Cell type
CR1 C3b Erythrocytes,PMNs; B-
lymphocytes; subsets of T-
lymphocytes; monocytes;
macrophages; dendritic cells
CR2 C3d B-lymphocytes
CR3 C3bi PMNs, monocytes, macrophages
CR4 C3d PMNs, monocytes
C3a and C5a C3a, 4a, 5a PMNs, monocytes,
macrophages, mast cells
The Complement System 47
Results of Complement–Receptor Interactions
The major complement r ecept or s ar e:
(i) CR1 pr efer ent ially binds t he init ial C3 act ivat ion pr oduct C3b. These r ecept or s ar e
found on many cell t ypes as list ed in Table 7.3. The act ivat or par t icle bear ing t he
ant ibody-C3b complex is t her efor e at t ached t o var ious cell t ypes by t hese CR1 r ecept or
molecules. The immune complex for ms a br idge bet ween t he act ivat or and t he cell
t ype. When t he cell t ype involved is t he er yt hr ocyt e (see Figur e 7.7), t her e is an
immune r ed cell adher ence ar ound t he act ivat or -ant ibody-C3b complex. This helps t o
r egulat e complement act ivat ion and aids in t he clear ance of immune complexes, as
r ed cells “car r y” t hese complexes t o t he liver for degr adat ion.
On cer t ain lymphoid cells and phagocyt ic cells t he r ecept or s help t o link t he act ivat or
par t icle via t he ant ibody-C3b link t o phagocyt ic cells. This helps t o augment phagocyt ic
dest r uct ion of an act ivat or par t icle t hat is coat ed (opsonized) by t he ant ibody- C3b
complex (Figur e 7.7).
Figure 7.7. Biologic consequences of complement-receptor interactions
(ii) The r ecept or known as CR2, binds t he t er minal C3 cleavage pr oduct s i.e. C3d.This
r ecept or is confined t o B lymphocyt es. CR2 has also been shown t o be t he st r uct ur e
used by t he Epst ein-Bar r vir us t o at t ach t o and infect B cells. CR2 plays an impor t ant
r ole in t he pr esent at ion of ant igen t o specific B and T cells and in t he cont r ol of B-cell
pr olifer at ion. Evidence clear ly suggest s t hat CR2 is involved in t he induct ion of a
pr imar y humor al r esponse.
(iii) The CR3 r ecept or binds t he int er mediat e complement C3 cleavage pr oduct , C3bi.
Int er act ions wit h C3bi and r ecept or s on phagolyt ic cells enhance ant ibody dependant
phagocyt ic r esponses, leading t o dest r uct ion of t he act ivat or . Individuals genet ically
lacking CR3 ar e pr edisposed t o r ecur r ent life t hr eat ening bact er ial infect ions.
(iv) CR4 is a C3 r ecept or t hat binds C3bi and C3d. CR4 has an impor t ant r ole in host
r esist ance t o infect ion. C3bi-coat ed immune complexes have a high affinit y for t he
48 Immunology– Introductory Textbook
CR4 r ecept or s on phagocyt ic cells of t he liver and spleen, t o wher e t hey ar e t r anspor t ed
and degr aded.
(v) Recept or s for C3a and C5a bind t o anaphylat oxins (see below).
Biologic Actions of Complement Cleavage Products
The low molecular weight fr agment s of C3, C4 and C5 - C3a, C4a and C5a r espect ively ar e
known as anaphylat oxins. These hor mone like pept ides induce smoot h muscle cont r act ion,
enhance vascular per meabilit y, r elease vaso act ive amines fr om mast cells and basophils and
induce lysosomal enzyme r elease fr om gr anulocyt es.
In addit ion, C5a is also chemot act ic ie: it is able t o induce t he migr at ion of leukocyt es int o
an ar ea of complement act ivat ion. The C5a molecule has a number of ot her pr oper t ies, which
include gr anulocyt e aggr egat ion, and act ivat ion of int r acellular pr ocesses in cer t ain cells, leading
t o var ious effect s such as r elease of oxygen met abolit es and SRS-A (slow r eact ing subst ance of
anaphylaxis). Hist amine induced effect s of C3a, C4a and C5a ar e blocked by ant i hist amines.
Ther e ar e ot her biologic consequences of t he complement act ivat ion syst em. These include
t he gener at ion of a kinin, possibly a fr agment of C2, which causes smoot h muscle cont r act ion
and incr eased vascular per meabilit y. This kinin does not funct ion t hr ough r elease of hist amine.
Biologic Significance of the Complement System
The sum t ot al effect of t he int egr at ed complement syst em is t hat it is able t o pr oduce
inflammat ion and facilit at e t he localizat ion of an infect ive agent . (Figur e 7.8 ). The C3b, C4b
r ecept or s on phagocyt ic cells help link phagocyt ic cells t o act ivat or par t icles t hat ar e coat ed
wit h ant ibody and C3b or C3b alone. This coat ing and facilit at ion of phagocyt osis has been
called opsonizat ion. The t er m opsonin is der ived fr om t he gr eek wor d “opsons” meaning t o
pr epar e food for : (phagocyt es).
Figure 7.8. Biological effects of complement mediated reactions
The Complement System 49
A classic descr ipt ion of opsonin was given in 1906, by Geor ge Ber nar d Shaw, in his play
“The Doct or s Dilemma”. The lead char act er Sir Colenso Ridgeon was closely pat t er ned aft er
t he Br it ish scient ist Almr ot h Wr ight . “The phagocyt es” Ridgeon says in t he play, “won’t eat t he
micr obes unless t he micr obes ar e nicely but t er ed for t hem. Well t he pat ient manufact ur es t he
but t er for himself alr ight , t hat but t er , I call opsonin .” Ther e ar e t wo pr incipal classes of opsonin:
specific ser um ant ibody and complement component fr agment s.
Ot her biologic effect s such as smoot h muscle cont r act ion, vascular per meabilit y and
chemot axis of leukocyt es facilit at e t he acut e inflammat or y pr ocess.
Evidence for t he biologic impor t ance of t his syst em can be obt ained fr om human and
animal complement deficiency st at es. Ther e is a mar ked incr ease in suscept ibilit y t o infect ion.
Infect ions and ot her human disor der s associat ed wit h congenit al complement deficiencies ar e
given in Table 7.3.
Table 7.3: Human disorders associated with congenital complement deficiencies
C1q Systemic lupus erythematosus(SLE) or similar
syndrome, hypogammaglobulinaemia, nephritis
C1r Renal disease, SLE or similar syndrome, recurrent
infections, rheumatoid disease
C1s & C4 SLE
C2 Arthralgia, SLE or similar syndrome, nephritis,
susceptibility to infection
C3 Recurrent infections with pyogenic bacteria
C5 SLE, recurrent infections, recurrent gonococccal
infections
C6 Recurrent gonococcal and meningococcal infections
and Raynaud’s phenomenon
C7 Recurrent gonococccal and meningococcal infections,
SLE
C1 inhibitor Hereditary angioedema
C3b inhibitor Repeated infections, recurrent infections with
pyogenic bacteria
™™™
DETECTION AND APPLICATION OF
ANTIGEN–ANTIBODY REACTIONS
CHAPTER – 8
B
asic knowledge r egar ding t he molecular basis of ant igen and ant ibody st r uct ur e and
funct ion, will event ually influence diagnosis and t her apeut ics in clinical medicine.
Much of t his chapt er will be devot ed t o t he dynamics and clinical labor at or y applicat ions of
ant igen-ant ibody int er act ions.
For ease of descr ipt ion t hese ant igen ant ibody r eact ions have been br oadly classified int o:
• Pr ecipit at ion
• Agglut inat ion
• Complement fixat ion
• Immuno assay using labelled r eagent s
• Immuno hist ochemist r y (Immunofluor escence)
• Cyt okine immunoassays (ELISPOTR )
• DNA innunoassays
Precipitation
The classical pr ecipit at ion r eact ion was fir st demonst r at ed by i mmu n od i ffu si on in gel.
It det ect s t he r eact ion bet ween solu b le a n t i ge n and a pot ent ant iser um mixed in t he cor r ect
pr opor t ions. The ant igen-ant ibody pr ecipit at e t hat is so for med in any semi solid medium such
as agar or agar ose is also dependent on buffer elect r olyt es, pH and t emper at ur e. The for mat ion
of pr ecipit a t ion lines in a ny immunodiffusion syst em is highly dependent on r ela t ive
concent r at ions of ant igen and ant ibody.
Th i s r el a t i on s h i p i s depi ct ed
schemat ically in Figur e 8.1, maximal
pr ecipit at ion is for med in t he zone of
equivalence; not in t he zones of ant igen
or a n t i body exces s . Th e p r o z o n e
p h e n ome n on (Figur e 8.2), is a t er m
which is used t o descr ibe subopt imal
pr eci pi t a t i on or i n compl et e l a t t i ce
for mat ion which occur s in t he r egion of
ant ibody excess. A similar phenomenon
occur s in t he r egion of ant igen excess
and is somet imes called t he post zone
effect . Figure 8.1. The zone of equivalence in
antigen antibody reactions: where maximal
precipitate is formed.
Detection and Application of Antigen–Antibody Reactions 51
Figure 8.2. The optimal ratio of antigen to antibody (middle) yields an insoluble precipitate in the
classic precipitation test. Extreme antibody excess (left) or antigen excess (right), however, does
not lead to lattice formation.
Immunodiffusion or Agar Gel Diffusion
Agar gel diffusion is t he simplest and most dir ect means of demonst r at ing soluble ant igen-
ant ibody r eact ions in t he labor at or y. In 1946 Ou d i n descr ibed a syst em of single diffusion of
ant igen and ant ibody in agar filled t ubes. This impor t ant advance was soon followed by
Ou ch t e r lon y’s classic descr ipt ion of double diffusion in agar layer ed on slides. This met hod is
st ill widely used t oday t o det ect and analyze ant igen-ant ibody r eact ions.
Agar gel diffusion r eact ions may be classified as single or double. In single agar gel diffusion
eit her ant igen or ant ibody r emains fixed and t he ot her r eact ant is allowed t o diffuse fr eely in
t he gel. In double immuno diffusion bot h r eact ant s ar e fr ee t o move t owar ds each ot her and
pr ecipit at e. Movement wit hin t he gel may be linear or r adial. The most impor t ant clinical
applicat ion of immunodiffusion is t he quant ificat ion of ser um immunoglobulins.
(i) Ouchterlony’s Double Diffusion
This t est is per for med by pour ing molt en agar ont o glass slides or int o Pet r i dishes and
allowing it t o har den. Small wells ar e punched out of t he agar , a few millimet r es apar t . Samples
cont aining ant igen and ant ibody ar e placed in opposing wells and allowed t o diffuse t owar d one
anot her in a moist chamber for 18- 24 hour s. Ant igen and ant ibody diffuse out of t he wells in a
r a dia l fa shion. Two a r cs fr om ea ch well a ppr oxima t e wit h ea ch ot her t o for m a line of
pr ecipit at ion, which can be viewed by indir ect light or wit h a magnifying lens (Figur e 8.3). Gels
can be dehydrat ed and st ained by prot ein binding dyes like Coomassie brilliant blue and preserved
indefinit ely.
Figure 8.3. Agar-gel diffusion.
Double diffusion can also be per for med bet ween one or mor e ant igens and a single ant ibody
for compar at ive pur poses. The t hr ee basic char act er ist ic pat t er ns of such r eact ions ar e shown
in Figur e 8.4. Double diffusion in agar can also be used for semi-quant ificat ion of ant igen or
ant ibody r eact ant s as shown in Figur e 8.5. It is st ill an impor t ant init ial t est for t he det ect ion
of ser ologic r eact ions t o var ious human, animal and plant ant igens.
52 Immunology– Introductory Textbook
Figure 8.4. Reaction patterns with double diffusion in agar R=antigen-R, S=antigen-S, R1=antigen-
R1, αR = antibody to R; αS = antibody to S
Reaction of identity : Precisely similar precipitin lines have formed in the reaction between R and anti
R. The lines meet at a point, showing that the two antigens are similar. Reaction of non-identity.
Precipitin lines completely cross owing to two separate interactions: R with αR and S with αS;
showing that R and S are dissimilar and non cross- reacting antigens. Reaction of partial identity: αR
reacts with both R and R1; the precipitin lines do not cross, a characteristic spur is formed,
indicating that antigenic determinants are partially shared R and R1.
Figure 8.5. Semi-quantitative analysis using double immunodiffusion; semiquantitative analysis of
antigen. Antigen x is serially diluted and placed in a ring of wells surrounding a central well containing
antibody to x. Precipitin lines form with decreasing thickness until no longer visible at an antigen
dilution of 1:32. The whole process can be reversed to quantitate antibody.
Figure 8.6. Single radial immuno diffusion. Antibody is added to the agar which is then poured onto
a slide or petri dish. Wells are punched in the agar and dilutions of test antigen-S are put in the
wells. The plates are left for 24 hours during which time antigen diffuses out of the wells and binds
to antibody; when the point of equivalence is reached, antigen and antibody precipitate to form a
ring. The area within the precipitin ring is proportional to the antigen concentration.
(ii) S ingle radial immunodiffusion
The double immunodiffusion is only semi quant it at ive. In 1965, Mancini int r oduced a
single diffusion t echnique for quant it at ive est imat ion of ant igens. Radial diffusion is based on
t he pr inciple t hat a quant it at ive r elat ionship exist s bet ween t he amount of ant igen placed in a
well (wher e t he ant ibody is incor por at ed int o t he gel) and t he r esult ant r ing of pr ecipit at ion
(Figur e 8.6). An impor t ant applicat ion of t his t est is in t he det ect ion and semi-quant ificat ion of
many ant igens.
Detection and Application of Antigen–Antibody Reactions 53
Electrophoresis
Simple zone elect r ophor esis uses t he pr inciple of separ at ion of individual pr ot eins ( in a
complex pr ot ein mixt ur e such as human ser um) in an elect r ical field.
The medium in which t his occur s is gener ally one t hat does not impede or enhance t he
flow of molecules in an elect r ical field. Gener ally, paper , agar ose or cellulose acet at e st r ips ar e
used for t his pur pose. Technically, t he biologic fluid is
spot t ed at t he or igin and subject ed t o an elect r ical cur r ent ,
allowing for separ at ion of individual const it uent s. Lar ge
molecular weight subst ances move slowly and t r aver se a
s h or t er di s t a n ce i n t h e gi ven t i me per i od, s ma l l er
mol ecu l a r wei gh t fr a gmen t s t r a ver s e fu r t h er .
Densit omet er scanning conver t s t he separ at ed bands int o
peaks. Figur e 8.7, shows separ at ion of nor mal human
ser um int o 5 major elect r ophor et ic bands: albumin, α1
globulin, α 2 globulin, β globulin and γ globulin. Human
immunoglobulins fall int o t he γ globulin fr act ion of human
ser um.
Zone elect r ophor esis is ext r emely valuable in t he diagnosis of human par apr ot ein disor der s
and disor der s of t he γ globulin fr act ion of ser um such as hypo or agammaglobulinemia.
Immunoelectrophoresis (IEP)
Immunoelect r ophor esis combines elect r ophor et ic separ at ion and immunopr ecipit at ion of
pr ot eins. Bot h ident ificat ion and appr oximat e quant ificat ion can t her eby be accomplished for
individual pr ot eins pr esent in ser um, ur ine or ot her biologic fluids. The t echnique is illust r at ed
in Figur e 8.8, using human ser um as ant igen and ant iser um r aised against whole human
s er u m a s a n t ibody. Th e a pplica t ion s of immu n oelect r oph or es is in clu de dia gn os is of
par apr ot einemias, hypo or agammaglobulinemia and ident ificat ion of L chains in t he ur ine of
pat ient s wit h plasma cell dyscr asias or aut oimmune disor der s. Thus wit h specific ant i-κ or
ant i-λ ant iser a, t he monoclonal nat ur e of Bence-J ones pr ot eins in myeloma can be confir med.
Immunoelect r ophor esis is also helpful in ident ifying incr eased amount s of pr ot eins pr esent in
t he cer ebr o spinal fluid in pat ient s wit h var ious neur ologic diseases.
Figure 8.8. Technique of immunoelectrophoresis : (1) Agar gel slab is prepared, a trough and well
are cut and the well is filled with antigen. (2) Antigens are separated by electrophoresis and the pH
of the gel is chosen so that positively charged proteins move to the negative electrode and negatively
charged proteins move to the positive electrode. (3) After antigen separation, the trough is filled with
antiserum which is left to diffuse. (4) The separated antigens and the antibodies in the trough
diffuse, interact and form precipitin arcs. Immunoelectrophoresis permits the comparison of
complicated mixtures of antigens found in human serum.
Figure 8.7. Electrophoretic
separation of normal human serum.
54 Immunology– Introductory Textbook
Electroimmunodiffusion
In imm unodiffusion t echniques descr ibed t his far , ant igen and ant ibody ar e allowed t o
come int o cont act and t o pr ecipit at e in agar pur ely by diffusion. However , t he chance of an
ant igen and ant ibody meet ing and t he speed of development of a pr ecipit at ion line can be
gr ea t l y en h a n ced by el ect r i ca l l y dr i vi n g t h e t wo t oget h er . Two va r i a t i on s of
el ect r oi mmu n odi ffu s i on h a ve r ecei ved cl i n i ca l a ppl i ca bi l i t y. Th ey a r e (i ) Cou n t er
immunoelect r ophor esis (CIE) and (ii) Laur ell’s r ocket elect r ophor esis.
(i) Counter immunoelectrophoresis
The basic pr inciple involves, as befor e, pour ing molt en agar on a slide and punching wells
int o it a few millimet r es apar t , once t he gel has solidified. Ant igen and ant ibody ar e placed in
t he opposing wells. In a clinical condit ion t he biologic fluid is placed in one well and known
ant ibody in t he ot her . This det ect s ant igen in t he clinical mat er ial. The syst em can be r ever sed
t o det ect ant ibody in t he clinical sample. The slide is t hen placed in an elect r ophor esis t ank and
bat hed in buffer solut ions of opt imal pH and connect ed t o a power pack supplying t he elect r ical
cur r ent . As shown in Figur e 8.9, t he ant igen solut ion being a pr ot ein moves t owar ds t he posit ive
pole. The pr ot eins in t he given ant iser um well also move t owar ds t he posit ive pole. However ,
t he γ globulin fr act ion which cont ains all t he immunoglobulins is dr iven in t he opposit e dir ect ion
by elect r o endosmosis. This dr ives ant ibody t owar ds t he ant igen and pr ecipit in lines may be
seen t o be for med a few hour s aft er beginning of elect r ophor esis. Elect r o endosmosis is a
phenomenon in which molecules wit hin t he agar gel t end t o move in t he dir ect ion opposit e t o
t he flow of cur r ent when in an elect r ical field. γ globulins being r elat ively low molecular weight
subst ances ar e hence car r ied along by elect r o endosmosis t owar ds t he ant igen, facilit at ing t he
ant igen- ant ibody r eact ion.
Figure 8.9. Counter immuno electrophoresis. Antigen and antibody are placed in wells and driven
towards each other by electric current, a precipitin line forms in the zone of equivalence, where
antigen and antibody meet.
This t echnique can pr oduce visible pr ecipit in lines wit hin 30 minut es and is 10 t imes
mor e sensit ive t han agar gel diffusion. However , it is only semi quant it at ive. CIE is widely
used in many plant , animal and human immunology labor at or ies for t he det ect ion of nor mal
and abnor mal pr ot eins.
(ii) Laurell’s rocket electrophoresis
In t his t echnique ant iser um t o t he par t icular ant igen or ant igens is incor por at ed int o an
agar ose medium and pour ed over a glass slide. This ensur es t hat ant ibody does not migr at e.
The specimen cont aining t he unknown ant igen is placed in a small well. Elect r ophor esis of t he
ant igen int o t he ant ibody cont aining agar ose is t hen per for med. The r esult ant pat t er n of
immuno-pr ecipit at ion r esembles a spike or r ocket – hence t he t er m r ocket elect r ophor esis
(Figur e 8.10).
Detection and Application of Antigen–Antibody Reactions 55
Figure 8.10. Rocket electrophoresis (Laurells’ technique). Varying concentrations of the antigen
are placed in wells 1-4. Electrophoresis is performed and antigen is driven into the antibody -
containing gel. Precipitin pattern forms in the shape of a “rocket.” Amount of antigen is directly
proportional to height of the rocket.
The pr incipal applicat ion of t his t echnique is t o quant ify ant igens in biological fluids. The
r ocket pat t er n occur s because pr ecipit at ion occur s along t he lat er al mar gins of t he moving
boundar y of ant igen, as t he ant igen is dr iven int o t he agar cont aining t he ant ibody. Gr adually
as t he ant igen is lost t hr ough pr ecipit at ion it s concent r at ion at t he leading edge diminishes and
t he lat er al mar gins conver ge t o for m a shar p point . The t ot al dist ance of ant igen migr at ion
(lengt h of spike) for a given ant iser um concent r at ion is linear ly pr opor t ionat e t o t he ant igen
concent r at ion. This syst em cannot be used t o quant ify immunoglobulins because t heir weak
negat ive char ge pr event s t heir elect r ophor et ic mobilit y in t his syst em.
(iii) Crossed immunoelectrophoresis
One power ful var iant of Laur ell’s r ocket syst em, t he cr ossed immunoelect r ophor esis
involves a pr elimina r y elect r ophor et ic sepa r a t ion of a n a nt igen mixt ur e in a dir ect ion
per pendicular t o t hat of t he final “r ocket st age”. In t his way each of sever al ant igens in a
mixt ur e can be quant ified (Figur e 8.11). The t echnique is used in many immunology labor at or ies
for t he det ect ion of ant igens in human and animal biological fluids.
Figure 8.11. (a) Antigens are separated on the basis of electrophoretic mobility in agar gel (dotted
vertical bands). (b) A narrow longitudinal strip (dashed lines) containing the separated antigens is
cut out as shown, laid over another gel, which contains antibody and electrophoresis carried out in
a direction at right angles to the first run to drive the antigen bands through the antibody containing
gel. Antigen and antibody complex to form precipitin peaks. The area under the peak is related to the
concentration of antigen.
56 Immunology– Introductory Textbook
Agglutination
Wher eas cr oss linking of mult ivalent pr ot ein ant igens of a soluble nat ur e leads t o
precipit at ion, cross-linking of cells or large part icles by ant ibody direct ed against surface ant igens
leads t o agglut inat ion. Since most cells ar e elect r ically char ged, a r easonable number of ant ibody
links bet ween t wo cells is r equir ed befor e t he mut ual r epulsion is over come. The agglut inat ion
of cells bear ing only a small number of sur face det er minant s may t her efor e be difficult t o
achieve. Similar ly t he higher avidit y of mult ivalent IgM ant ibody r elat ive t o IgG, makes it a far
mor e effect ive agglut inat ing agent . Impor t ant advant ages of agglut inat ion ar e t hat r eact ions
can be r ead visually, have a high degr ee of sensit ivit y and an enor mous var iet y of subst ances
ar e det ect able t hr ough t his met hod.
Agglut inat ion r eact ions may be classified as eit her dir ect or indir ect (passive). Besides
t hese t her e ar e sever al modificat ions of t he agglut inat ion pr inciple, which have been used
successfully in clinical, labor at or y medicine.
Agglutination techniques
(i) Direct agglutination
In t his simple dir ect t echnique, a cell or insoluble par t icle is agglut inat ed dir ect ly by
ant ibody. An example is t he agglut inat ion of gr oup A r ed cells by ant i-A ser a in a simple slide
agglut inat ion t est . In t his way sever al species of bact er ia (gr own on cult ur e) can be dir ect ly
agglut inat ed and t hus definit ively diagnosed by specific ant ibody using a slide agglut inat ion
r eact ion.
Tube agglutination r eact ions ar e used t o det ect specific ant ibody (in human or animal
ser um) in t he pr esence of a const ant amount of ant igen. Classically,t he ser um t o be t est ed is
init ially, ser ially dilut ed in t est t ubes, using t wo fold dilut ions such as 1 in 2, 1 in 4 or 1 in 8 et c.,
alt er nat ively dilut ions such as 1 in 10, 1 in 20, 1 in 40 et c., may be used. Essent ially, what t his
means is t hat 1 par t of ser um is mixed wit h 1 par t of a diluent in a 1 in 2 dilut ion, or one par t
of ser um is mixed wit h 3 par t s of diluent in a 1 in 4 dilut ion and so on. The diluent s used ar e
usually nor mal saline or any ot her suit able buffer solut ion. St andar dized amount s of known
ant igen ar e t hen added t o all t he t ubes. The t ubes ar e incubat ed at suit able t emper at ur e
(usually 37 C), for most t est s except for cold r eact ing ant ibodies or cold agglut inins which need
r efr iger at ion. Aft er a few hour s of incubat ion, agglut inat ion is complet e and t ubes ar e examined
for clumping. The r esult s ar e usually expr essed as a t it er i.e., t he highest dilut ion of t he t est
ser um at which a posit ive agglut inat ion r eact ion occur s. Impor t ant clinical applicat ions of t his
t echnique ar e t he Wi d a l t e st for diagnosis of t yphoid fever , t he Br u ce lla agglut inat ion t est
for Br ucellosis and t he We i l F e li x t est for Ricket t siosis, t o name just t hr ee.
(ii) Indirect/ passive agglutination
When a soluble ant igen needs t o be used in an agglut inat ion r eact ion, it is oft en coat ed
ont o a car r ier par t icle, t hus r ender ing it par t iculat e. Soluble ant igens can be passively adsor bed
or chemically coupled t o r ed blood cells of var ious species, in which case t he t est is called an
indir ect (passive) haemagglut inat ion t est (IHA or PHA). Many ant igens will spont aneously
couple wit h r ed cells and for m st able r eagent s for ant ibody det ect ion. Treponema pallidum
haemagglut inat ion (TPHA) for t he diagnosis of syphilis is a widely used t est t hat exploit s t his
pr inciple. Coupling agent s var y widely, however , a good example is t annic acid which incr eases
t he amount of most pr ot ein ant igens coupled ont o r ed cells.
Agglut inat ion t est s ar e per for med in t ubes or micr ot it er plat es which hold ser ially dilut ed
ser um samples. A const ant amount of coupled ant igen is t hen added t o each ser um dilut ion and
Detection and Application of Antigen–Antibody Reactions 57
incubat ed. Aft er a few hour s of incubat ion, a posit ive r eact ion is seen as clumping of r ed cells,
a negat ive r eact ion is evidenced by set t ling of r ed cells at t he bot t om of t he t ube or well as a
but t on (Figur e 8.12). Passive haemagglut inat ion wit h pr ot ein sensit ized cells can det ect ant ibody
at concent r at ions as low as 0.03 µgs/ml.
Figure 8.12. Indirect (passive) haemagglutination (a) red cells are coated with antigen (b)
specific antibody causes indirect (passive) haemagglutination.
A modificat ion of t he above t echnique t hat is commonly used involves coupling of ant ibody
ont o r ed cells so as t o d et ect a n t i gen in t he pat ient ’s sample. Since t he r eact ant s ar e r ever sed
t he t est is somet imes called r ever sed passive haemagglut inat ion (RPHA).
Passive car r ier s of ant igen, ot her t han r ed blood cells, have been widely used in ser ology
for t he demonst r at ion of agglut inat ing ant ibody. Among t he commonest ones used ar e iner t
part icles like lat ex in t he lat ex agglut inat ion (fixat ion) t est or st aphyloccal prot ien A (Figure 8.14).
The lat ex agglut inat ion t est is widely used for det ect ion of Rheumat oid fact or . Rheumat oid
fact or is a 19S IgM ant ibody (an aut o ant ibody) dir ect ed against t he pat ient ’s own 7S IgG. If
lat ex par t icles ar e adsor bed wit h 7S IgG, when mixed wit h ser um of pat ient wit h r heumat oid
ar t hr it is, t he 19S IgM in t hese ser a r eact wit h 7S IgG coat ed ont o lat ex par t icles and cause
visible clumping of t he par t icles.
A passive haemagglut inat ion t est is also used t o det ect r heumat oid fact or . This t est is
ca lled t he Ros e -Wa a l e r t e s t . Ant ibodies (I gG) a ga inst sheep r ed cells a r e r a ised in
r abbit s.Human r heumat oid fact or (IgM) has been shown t o r eact against t he r abbit IgG r aised
against sheep r ed cells. This ant iser um is also known as ambocept or . Tanned sheep r ed cells
ar e coat ed (sensit ized) wit h t he ambocept or in sub agglut inat ing t it er s. These sensit ized sheep
cells ar e allowed t o r eact wit h ser ial dilut ions of human ser um. Rheumat oid fact or in human
ser um will r eact wit h r abbit ant i sheep IgG coat ed ont o t he sheep r ed cells, t o cause clumping
of r ed cells. This occur s by vir t ue of t he fact t hat r abbit IgG and human 7S IgG cr oss r eact
(Figur e 8.13). Befor e int er pr et ing t he t est r eading it is impor t ant t o ascer t ain t hat t he given
pat ient ’s ser um does not r eact wit h nor mal unsensit ized sheep cells, causing non specific
clumping.
(iii) Haemagglutination inhibition
Anot her cat egor y of agglut inat ion involves spont aneous agglut inat ion of r ed cells by cer t ain
vir uses. This vir al haemagglut inat ion r eact ion can be specifically inhibit ed in t he pr esence of
ant ivir al ant ibody. Thus vir al haemagglut inat ion can be used t o det ect pr esence of vir us;
alt er nat ively, t he haemagglut inat ion inhibit ion r eact ion can be used t o det ect t he pr esence of
58 Immunology– Introductory Textbook
specific ant ivir al ant ibody in pat ient ’s ser um. This happens because vir al ant igen r eact s wit h
ant ivir al ant ibody in pat ient ’s ser um; t he haemagglut inat ing sit es on t he vir al par t icle t hus lie
complexed t o ant ibody and ar e no longer available for r eact ion wit h r ed cells.
Figure 8.13. Principle of the Rose-Waaler test a) Sheep red cells sensitized with rabbit IgG raised
against the sheep red cells (sub agglutinating doses) b) Human IgM (rheumatoid factor) reacts with
rabbit IgG causing agglutination of red cells.
Figure 8.14. Co-agglutination (a) Protein A containing S.aureus binds avidly to the Fc portion of the
IgG molecule. (b) The Fab portion of the bound IgG complexes with antigen resulting in clumping of
S.aureus cells.
Coomb’s Test
In some cases a shor t ant ibody molecule dir ect ed against a deeply locat ed membr ane
ant igenic det er minant cannot agglut inat e (Figur e 8.15 a),as t he t wo Fab ar ms ar e not long
enough t o br idge det er minant s on apposing r ed cells. These ant ibodies ar e somet imes called
“incomplet e” or “blocking” ant ibodies. A good example of such a det er minant is t he Rh ant igenic
det er minant on r ed cells. To det ect t he pr esence of ant i Rh ant ibodies, alr eady fixed t o r ed
cells, an ant i human immunoglobulin is added which is direct ed against t he “incomplet e” ant ibody.
The addit ion of t his second ant ibody leads t o agglut inat ion (Figur e 8.15 b).
Detection and Application of Antigen–Antibody Reactions 59
Figure 8.15. The Coomb’s test (a) red cells are sensitized with “incomplete antibodies” (b) Anti-
antibody reacts with the “incomplete antibody” causing agglutination of red cells.
The Coomb’s t est is per for med in t wo ways:
(i) The d i r e ct Coomb ’s t e st det ect s immunoglobulin alr eady pr esent on t he r ed cell by
addit ion of t he ant iglobulin (as descr ibed above). The dir ect Coomb’s t est is posit ive
in an alr eady sensit ized individual, such as t he Rh posit ive infant bor n t o an Rh
negat ive mot her . Such an infant ’s r ed cells ar e coat ed wit h ant i Rh ant ibody pr oduced
by t he Rh negat ive mot her against her own baby’s Rh posit ive r ed cells. The infant is
t hus at r isk of developing haemolyt ic disease of t he newbor n.
(ii) The in d ir ect Coomb’s t est is a 2 st age r eact ion for det ect ion of cir culat ing incomplet e
ant ibodies against t he Rh det er minant , which ar e usually pr esent in t he mot her ’s
ser um aft er t he bir t h of an Rh posit ive infant . The pat ient s ser um is fir st incubat ed
wit h r ed cells possessing t he Rh det er minant . The incomplet e ant ibodies r eact wit h
t he Rh det er minant s and coat t he r ed cells. Once coat ing has t aken place, t he
ant iglobulin is added and t he appear ance of agglut inat ion indicat es t he pr esence of
ant i Rh ant ibodies in mat er nal ser um. The Coomb’s t est is also used in t he diagnosis
of aut o immune haemolyt ic anaemia.
Complement Fixation Test
The fixat ion of complement occur s dur ing t he int er act ion of ant igen and ant ibody. Thus
t he consumpt ion of complement in vit r o, can be used as a t est t o det ect and measur e ant ibodies,
ant igens or bot h. The t est depends on t he use of a haemolyt ic indicat or syst em, consist ing of
sheep r ed cells, ambocept or (ant ibody t o sheep r ed cells) and complement . When added t oget her ,
sheep r ed cell ant igen complexes wit h ant ibody, ut ilizes complement and r esult ant r ed cell
lysis occur s (Figur e 8.16). The act ual t est is done in t wo st ages: t he fir st st age consist s of
adding t he t est r eagent s which ar e ant igen and ant ibody (nor mally t he biologic fluid such as
human ser um) and a st andar dized amount complement . If ant igen and ant ibody ar e specific for
each ot her t hey will complex and ut ilise t he complement as well. The second st age of t he t est ,
is done by adding t he sheep cell-ambocept or mixt ur e (also known as sensit ised sheep r ed cells).
Aft er a per iod of incubat ion t he t est is r ead looking specifically for t he pr esence and degr ee of
r ed cell lysis.
60 Immunology– Introductory Textbook
Figure 8.16. The Complement fixation test.
Interpretation
Occur r ence of lysis indicat es: t est ant igen and ant ibody in st age one did not complex,
t her efor e did not ut ilise complement . Complement was hence fr eely available for ut ilisat ion by
t he haemolyt ic indicat or syst em, wit h r esult ant r ed cell lysis. Lysis t her efor e indicat es a negat ive
t est r eact ion since t he pat ient ’s ser um did not cont ain t he ant ibody in quest ion. The a b se n ce
of lysi s indicat es: a posit ive t est r eact ion since ant igen was complexed t o ant ibody (pr esent in
pat ient ’s ser um) and ut ilised most or all of complement . Complement was hence not available
t o t he haemolyt ic indicat or syst em and sheep r ed cell lysis did not occur (Figur e 8.16). Result s
ar e shown as t he highest ser um dilut ion showing a posit ive r eact ion.
Complement fixat ion t est s have r eceived widespr ead use in bot h clinical and r esear ch
labor at or ies, t hough t hey have now been r eplaced by less cumber some immunoassay t est s.
Immunoassay Using Labelled Reagents
One of t he most impor t ant analyt ic met hods developed in r ecent year s is t he immunoassay
of ant igen and ant ibody using labelled r eagent s, also known as binder -ligand assays.
Detection and Application of Antigen–Antibody Reactions 61
The fir st ligand assay met hod t o be developed was t he r adio immunoassay (RIA), which
was int r oduced t o det ect human insulin using ant i insulin ant ibodies. The met hod was descr ibed
by Ber son & Yalow and in 1977; Yalow was awar ded t he Nobel pr ize for her cont r ibut ions t o t he
field of binder -ligand assays. In t he past t wo decades ligand assays have r evolut ionized t he
quant ificat ion of hor mones, dr ugs, t umour mar ker s, aller gens and a var iet y of ant igens and
ant ibodies associat ed wit h infect ious agent s. The chief goal of an assay is t o det er mine t he
concent r at ion of some molecule of int er est - t he analyt e, be it an ant igen or an ant ibody.
Radio immunoassay
Radio immunoassays has been used t o quant ify hor mones such as insulin, t hyr oxine et c,
in plasma. This is done by using ant ibodies r aised in r abbit s or guinea pigs t o t he concer ned
hor mones. An init ial st ep in t he use of t he RIA is t o plot a st andar d gr aph for each hor mone
t est ed. This st andar dizat ion pr ocedur e is descr ibed below:
(i) For est imat ion of t he hor mone X (for e.g.,t hyr oxine) ant iser um t o X is r aised in
r abbit or guinea pig.
(ii) Pur e hor mone X is obt ained and r adio labelled t o yield X*.
(iii) The concent r at ion of labelled hor mone i.e., X* is t it r at ed so as t o r eact wit h 70% of
binding sit es when added t o ant i X.
(iv) A r ange of known concent r at ions of unlabelled pur e X is added simult aneously wit h
t he t it r at ed X* t o t he ant ibody and t he mixt ur e is incubat ed.
(v) Labelled X* + ant ibody is separ at ed fr om fr ee X by t he addit ion of an ant i-ant ibody.
(vi) Fr om t he amount of bound X* measur ed out at var ious X concent r at ions a cur ve is
const r uct ed. The cur ve is linear over a r elat ively limit ed r ange, hence t he sample
con t a in in g X n eeds t o be dilu t ed so a s t o give a r ea din g wit h in t h is r a n ge
(Figur e 8.17).
Figure 8.17. Standard curve for hormone assays by RIA.
Aft er t he st andar dizat ion is achieved, t he exper iment is r epeat ed r eplacing X in t he above
st ep no.(iv) wit h t he pat ient ’s sample. The per cent age of X bound t o ant ibody is est imat ed in
t he pr esence of t he clinical specimen. Using t he gr aph (Figur e 8.18), t he concent r at ion of t he
hor mone X in t he pat ient s sample can be ascer t ained.
62 Immunology– Introductory Textbook
Figure 8.18. The radio-immunosorbent test (RIST) detects total IgE in patients serum.
Solid phase radio immunoassay
Solid phase r adio immunoassays may be used t o det ect bot h ant igen and ant ibody. A good
example t o illust r at e t his is t he r a d i o i mmu n osor b e n t t e st (RI ST) which det ect s t ot al IgE
in a pat ient ’s ser um. The t est is usually done in a micr ot it er poly - car bonat e plat e wit h mult iple
wells. Ant i human IgE r aised in r abbit s in used t o coat t he well (Figur e 8.18), or is coupled via
cyanogen br omide t o micr o-cellulose discs. The solid phase is blocked wit h non-specific pr ot ein
t o decr ease non-specific binding. Dilut ions of t he pat ient ’s ser um ar e added t o t he coat ed well.
Excess ser um is washed off ensur ing t hat human IgE alone, is in t he well coupled t o ant i IgE.
The bound IgE is t hen measur ed by addit ion of labelled ant i human IgE r aised in yet anot her
animal. Measur ement s of r adioact ivit y in t he well ar e t hen r ecor ded and quant ified using a
geiger count er .
The r a d i o a ller go sor ben t t est (RAST) measur es t he amount of IgE t o specific ant igens
(aller gens) in t he pat ient ’s ser um. The specific aller gen (eg: pollen ext r act ) is coupled t o a paper
disc in a micr ot it er well. This is t r eat ed wit h t he pat ient s ser um, washed and bound human IgE
det ect ed wit h labelled ant ihuman IgE similar t o t he pr ocedur e used for t he RIST (Figur e 8.19).
Figure 8.19. The radio allergosorbent (RAST) test to detect specific IgE.
Detection and Application of Antigen–Antibody Reactions 63
Enzyme labelled immunosorbent assays (ELISA)
The use of r adio labelled r eagent s car r ies wit h it t he hazar ds of r adioact ivit y, t he pr oblems
of safe disposal and t he pr ohibit ive cost of r eagent s and equipment .
Radiolabelled r eagent s have now been lar gely r eplaced by enzyme- labelled ant ibodies,
which when exposed t o colour ed subst r at e complexes r elease t he colour . Fur t her mor e, t he
r eact ion can be quant ified by measur ing t he densit y of colour using a simple colour imet er . The
basic pr inciple is ver y similar t o t he solid phase r adi o immunoassay (Figur e 8.20 a and b).
Figure 8.20. (a) The enzyme linked immunosorbent assay (ELISA) to detect antibody (b) The
ELISA to detect antigen.
a.
Coat plate with
Solid phase
antigen
<J> Antigen
Wash

Add test seru m
ant i body
(contains antibody)
Wash
A,',hom" "libod,
Add enzyme labelled
antihuman antibody
<J>: conjugat ed t o an enzyme
Wash

00 substrat e _ chromogen
Add substrate-
compl ex is cleaved by
chromogen complex
<J> enzyme
••
Measure degree

of colour change
Colour changes
b.
Sensitize plate with
Solid phase
capture antibody to a
"libo', specific antigen
Wash
Ant igen in quest ion
Add patients sample
(contains antigen)
Wash

Add enzyme - labelled
L<
2nd antibody
Wash

2nd antibody conjugat ed
Add substrate-
-----./-- to an enzyme
chromogen complex
Measure degree of

Colour change In
colour change
chromogen
64 Immunology– Introductory Textbook
Commonly used enzyme labelled ant ibodies and t heir r espect ive colour subst r at e complexes
ar e given in Table 8.1.
Table 8.1: Enzyme labelled antibodies with respective substrate complexes
Antibodies conjugated to Substrates
Horse radish peroxidase hydrogen peroxide + ortho phenylene diamine
Alkaline phophatase p–nitrophenyl phosphate
β– galactosidase o–nitro–phenyl–β–d– gal actopyranoside
Biotin / avidin enhanced immuno assays
A var iat ion of t he ELISA is a syst em which uses t he st r ong affinit y of compounds like
biot in and avidin for each ot her . These assays have been shown t o be sever al hundr edfold mor e
sensit ive t han t he RIA, and can t her efor e be used t o det ect ver y small quant it ies of ant igen or
ant ibody. The var iat ion involves biot inylat ing t he second ant ibody aft er which avidin conjugat ed
t o an enzyme is added, t his for ms a fir m complex which is det ect ed using a subst r at e-colour
compound as in convent ional ELISAs.
The ELISA and it s many var iat ions ar e widely used in clinical immunology (Table 8.2).
Class specific ant ibodies (IgG, IgM, IgA or IgE) can also be det ect ed using t he appr opr iat e
second ant ibody.
Table 8.2: Applications of the ELISA
For antigen detection
Hepatitis B surface antigen (HBsAg)
HBeAg
For antibody detection
Anti Hbs
Anti Hbc
Antibodies to toxoplasma,
Antibodies rubella and other viruses
Antibodies to HIV 1 and HIV 2
Immunochromatography (Immunocard tests)
Immunoassays have been used in a var iet y of commer cially available immunocar d t est s.
These t est s use t he pr inciple of immunochr omat ogr aphy. The disposable device consist s of
chr omat ogr aphic membr ane wit h a liquid-phase

ant ibody conjugat ed t o a colour ed subst r at e
(eg: conjugat ed mouse monoclonal ant ibody)

t o t he ant igen t o be det ect ed. This is called t he
signal ant ibody; while t he t wo solid-phase

ant ibodies ar e a polyclonal ant ibody t o ant igen and a
polyclonal

ant ibody t o mouse immunoglobulin in t wo st at ionar y ant ibody zones (Figur e:8.21).
Pat ient sample (st ool ext r act for example) is int r oduced int o t he device and incubat ed at r oom
Detection and Application of Antigen–Antibody Reactions 65
t emper at ur e. The specimen migr at es via capillar y act ion along t he membr ane unt il it r eaches
t he fir st st at ionar y specific monoclonal ant ibody t o t he ant igen in quest ion. The specimen, as it
migr at es, also dissolves t he embedded signal ant ibody. Ant igen binds t o t he st at ionar y ant ibody,
t he signal ant ibody also binds t o ant igen. React ion t akes place and a colour ed line appear s. The
excess

signal ant ibody which does not bind t o ant igen migr at es fur t her

unt il it r eact s wit h t he
polyclonal ant ibody t o mouse immunoglobulin,

pr oducing a separ at e, second colour ed line.
Thus, t wo colour ed lines

on t he t est st ick indicat e t he pr esence of ant igen.

In t he absence of
ant igen in t he pat ient ’s sample, only one colour ed line develops,

as a r esult of t he r eact ion
bet ween t he signal ant ibody and t he

ant ibody t o mouse

immunoglobulin. Colour must always
be seen at t he cont r ol line. If no colour is seen, t he t est has failed and must be r epeat ed.
Result s ar e r ead by compar ison of t he int ensit y of colour at t he t est line wit h t he cont r ol line –
equivalent colour is posit ive, dar ker colour is st r ongly posit ive. These t est s ar e now widely
available for diagnosis of Clostridium difficile toxins A and B, Helicobacter pylori faecal ant igen
t est s, pr egnancy t est s and many ot her ant igen det ect ion t est s.
Figure 8.21. Immunochromatography.
DNA immunoassays
DNA enzyme immunoassays have been developed t o det ect a var iet y of micr o or ganisms.
Commer cial kit s oft en use st r ept avidin

coat ed micr owell plat es t o which is added a biot inylat ed
specific

pr obe based on t he gene t o be det ect ed (for eg you may want t o det ect t he Ur eC gene of
H. pylori). The amplified gene pr oduct

(by pr ior PCR) is added t o t he plat e and t he duplex DNA
det ect ed wit h an enzyme

linked ant ibody against double st r anded DNA. These PCR-ELISAs ar e
becoming ver y popular diagnost ic t ools for a var iet y of micr o or ganisms wher e it is impor t ant
t o det ect t he specific pat hogenic or ant ibiot ic r esist ant gene in t he or ganism.
Immuno blotting (Western blots)
West er n blot s ar e used t o det ect ant ibody t ar get ed t o individual ant igenic det er minant s
in a cr ude whole cell a nt igen mixt ur e. I nit ia lly, t he a nt igen mixt ur e is subject ed t o
elect r ophor et ic separ at ion in a gel using, for example, sodium dodecyl sulphat e (SDS) poly
66 Immunology– Introductory Textbook
acr ylamide gel elect r ophor esis (SDS -PAGE). The separ at ed pr ot ein bands ar e t hen t r ansfer r ed
or blot t ed ont o nit r o-cellulose sheet s by t r a nsver se elect r ophor esis, wher e t hey bind
nonspecifically. These nit r o-cellulose sheet s ar e t hen incubat ed wit h t he pat ient ’s ser um, when
ant ibodies ar e allowed t o bind t o individual pr ot ein ant igens. Bound human ant ibody is t hen
det ect ed using enzyme labelled or r adio labelled ant ihuman immunoglobulin, much like t he
syst em used in an ELISA or RIA (Figur e 8.22).
Figure 8.22. Immunoblotting (Western blots).
The r ecombinant immunoblot assay (RIBA) used for t he diagnosis of Hepat it is C uses t he
West er n blot pr inciple; ser um is incubat ed on nit r ocellulose st r ips on which four r ecombinant
vir al pr ot eins ar e blot t ed. Colour changes indicat e t hat ant ibodies have adher ed t o t he pr ot eins.
An immunoblot is consider ed posit ive if t wo or mor e pr ot eins r eact .
Immunohistochemical techniques
I mmu n ofl u or e s ce n ce is essent ially a hist ochemical or cyt ochemical t echnique for
det ect ion and localizat ion of ant igens. Fluor escent dyes such as fluor escein and r hodamine can
be coupled t o ant ibodies wit hout dest r oying t heir specificit y. Such conjugat es can combine wit h
ant igen pr esent in a t issue sect ion and t he bound ant ibody can be visualized by means of a
fluor escence micr oscope. In t his way t he dist r ibut ion of ant igen t hr oughout a t issue and wit hin
cells can be demonst r at ed.
Immunofluor escence t est s can be eit her dir ect or indir ect (Figur e 8.23 a and b). In t he
d i r e ct i mmu n oflu or e sce n t t est t he ant ibody t o t he t issue subst r at e is it self conjugat ed wit h
t he fluor escent dye and applied t o cells or t issues fixed on a slide. Alt er nat ively, ant igens can be
spot t ed ont o a slide and det ect ed by dir ect fluor escent labelled ant ibody (Figur e 8.23a).
Detection and Application of Antigen–Antibody Reactions 67
Figure 8.23. The basis of immunofluorescence (a) direct immunofluorescence (b) indirect
immunofluorescence.
The in d i r e ct i mmu n oflu or e sce n ce t est is a double layer ed t echnique (Figur e 8.22 b).
The unlabelled ant ibody is applied t o t he cell pr epar at ion,t issue subst r at e or ant igen spot t ed
ont o a slide. This ant ibody is t hen t r eat ed wit h an ant i human immunoglobulin labelled t o a
fluor escent dye. When visualized under a fluor escent micr oscope, bound, labelled ant i globulin
fluor esces, emit t ing a char act er ist ic colour .
Immunofluor escence t echniques can be used t o det ect bot h ant igen and ant ibody in clinical
mat er ial. Clinical applicat ions of immunofluor escence ar e given in Table 8.3.
Table 8.3: Clinical applications of Immunofluorescence
Identification of T and B cells in blood.
Detection of immunoglobulins in tissues.
Detection of complement components in tissues.
Detection of specific tissue fixed antibody.
Rapid identification of micro organisms in tissue or culture e.g.,
Chlamydia trachomatis
Identification of tumour specific antigens.
Identification of transplantation antigens.
Cytokine Immunoassays
Det ect ing an immune r esponse t o t he pat hogen has been a successful met hod of diagnosis
of infect ion for many year s. However , t her e ar e a number of limit at ions t o using ant ibody
det ect ion for t he diagnosis of acut e infect ion: it is difficult t o differ ent iat e past infect ion fr om
acut e cur r ent infect ion; and somet imes t he ant ibody r esponse is insufficient and cannot be
used as a diagnost ic met hod. A classical example is t uber culosis(TB) wher e ant ibody det ect ion
has not played a r ole in diagnosis.
68 Immunology– Introductory Textbook
Our under st anding of t he immune r esponse mechanisms t ells us t hat ant ibodies ar e
invar iably for med wit h T cell help. So wher e t her e ar e ant ibodies t her e must also be act ivat ed
T cells. T cells, in par t icular , cont r ol t he fight against int r a-cellular pat hogens such as M.
tuberculosis, vir uses and t umour ant igens. Unt il r ecent ly, T cells have been difficult t o det ect .
The T-spot  or ELISPOT t echnology is a simple met hod t hat can be used in rout ine laborat ories
t o det ect pat hogen specific T cells in blood.
When whit e cells fr om t he pat ient s blood ar e exposed t o a specific ant igen (for e.g, M.
tuberculosis ant igen), t he T lymphocyt es pr oduce γ int er fer on as a r esult of act ivat ion by ant igen.
This γ int er fer on is capt ur ed by ant ibody t o γ int er fer on which is pr e-coat ed in wells of a micr o-
t it r e plat e. By a modificat ion of t he ELISA met hod t he capt ur ed γ int er fer on is det ect ed by a
second ant i-γ int er fer on ant ibody conjugat ed t o a suit able subst r at e-indicat or complex (Figur e:
8.24). Cyt okine immunoassays ar e being developed for a number of ot her vir al and t umour
ant igens. The ELISPOT met hod for t uber culosis is sensit ive and specific, is able t o det ect
act ive and lat ent TB in immunocompet ent as well as immunosuppr essed individuals and does
not cr oss r eact wit h BCG.
HOW THE T SPOT
TM
TECHNOLOGY WORKS
Figure 8.24. Cytokine immunoassay– ELISPOT® techonology
Plasma
Whit e cells
Gel barri er
Cryt hnocyt es
and cosmophi ls
Col lect whit e cel ls using t he commercially
available t ube or wit h Ficoll ext ract ion
Add whit e cells and TB anti gens t o wells,
T cel ls release IFN–γ
IFN–γ capt ured by ant ibody coated on well
Add substrat e and count spot s by
eye or use a reader
Incubat e, wash and add conjugated
second ant ibody IFN–γ
Each spot is an individual T cell t hat has
released IFN–γ
Detection and Application of Antigen–Antibody Reactions 69
Predictive Theory and Immunologic Testing
When any t est is used t o make a decision t her e is some pr obabilit y of dr awing an er r oneous
conclusion. This divides r esult s int o four cat egor ies:
• t r ue posit ives (a+c)
• t r ue negat ives (b+d)
• false posit ives (b)
• false negat ives (c)
Gold Standard test
Positive Negative
New test Positive a b
Negative c d
Di a gn ost i c se n si t i vi t y of a new t est is t he pr opor t ion of t r ue posit ives (as defined by a
gold st andar d t est ) cor r ect ly ident ified by t he new t est . Sensit ivit y = a / a+c.
Di a gn ost i c sp eci fi ci t y is t he pr opor t ion of t r ue negat ives (as defined by a gold st andar d
t est ) cor r ect ly ident ified by t he new t est . Specificit y = d / b+d.
Th e p osi t i ve p r e d i ct i ve va lu e (P P V) of t he new t est is t he pr opor t ion of individuals
showing a posit ive r esult wit h t he new t est who act ually have t he disease. PPV = a / a+b.
Th e n ega t i ve p r ed i ct i ve (NP V) of t he new t est is t he pr opor t ion of individuals showing
a negat ive r esult wit h t he new t est who do not act ually have t he disease. NPV = d / c+d.
™™™
MONOCLONAL ANTIBODIES
CHAPTER – 9
F
or many year s ant ibodies have had a major r ole in t he diagnosis of a wide var iet y of
diseases. Immunologic assays are used ext ensively, as discussed in t he previous chapt er,
in diagnost ic labor at or ies t o det ect and quant ify dr ugs, bact er ial and vir al pr oduct s, t umour
ant igens and cir culat ing immunoglobulins. These assays have been complicat ed because of
ant ibodies of r est r ict ed r eact ivit y and t he het er ogeneit y of all ant iser a obt ained by convent ional
met hods. These met hods, most commonly involve inject ing whole ant igen, wit h or wit hout an
adjuvant int o animals such as r abbit s, mice, goat , sheep et c. Sever al B cell clones ar e involved,
each pr oducing a slight ly differ ent ant ibody molecule t o t he myr iad ant igenic det er minant s,
encompassing t he whole ant igen. This phenomenon is r efer r ed t o as het er ogeneit y of t he
ant iser um and occur s even when appar ent ly pur e ant igen and inbr ed st r ains of animals ar e
used. The affinit y and quant it y of t he ant ibody var ies even fr om one bleed t o anot her . Such an
ant iser um, secr et ed by many ant ibody pr oducing B cell clones, is t er med a polyclonal ant iser um.
The var iabilit y of sub specificit ies and cr oss r eact ivit ies in polyclonal ant iser a have plagued
immunologist s all along and for ced t hem t o r emove unnecessar y ant ibodies by r epeat ed
absor pt ion, t her eby deplet ing t he or iginal ant ibody cont ent and st r engt h of t he ser um. In ot her
cases, cer t ain ant iser a ar e r ar e t o come by and r efer ence labor at or ies face t he ar duous t ask of
keeping r ar e r efer ence ser a in const ant supply.
The Genesis of Monoclonal Antibodies
As it oft en happens in science, t he solut ion gr ew out of a ser ies of basic and complet ely
unr elat ed exper iment s. To examine t he genet ic cont r ol of immunoglobulin pr oduct ion, a number
of labor at or ies had been at t empt ing t o fuse mouse myeloma cells t o each ot her or ot her cell
lines. In t he cour se of such st udies Geor ges Kohler and Cesar Milst ein fr om Cambr idge, England
showed t hat cult ur ed mouse myeloma cells could be fused t o nor mal spleen cells of animals
immunized wit h an ant igen, in t heir case, sheep r ed cells. Since t hese wor ker s hybr idized
single cell mixt ur es, t hey concluded t hat subsequent ant ibody for ming cell lines wer e clones of
(pr ogeny of ) t his one cell (mouse myeloma) t o one cell (nor mal spleen cell) hybr idizat ion.
These clones wer e, in fact , pr oducing ant ibody t o a single det er minant by a single hybr idized B
cell, hence t he t er m monoclonal ant ibody. These cell lines gr ew cont inuously in cult ur e because
of t he cancer ous myeloma hybr id, for ming lit t le t umour like masses in vit r o - giving r ise t o t he
t er m hybr idoma. These hybr idomas cont inuously secr et ed ant ibody; t hey could be fr ozen away,
r ecover ed and inject ed int o t he per it oneal cavit y of mice, wher e t hey gr ew and pr oduced an
ascit es which yielded lar ge amount s of ant ibody. The yields ar e t o t he t une of upt o a 100 ugs of
ant ibody/ml of cult ur e, and 10 mgs/ml in ser um or ascit ic fluid of t umour bear ing mice . These
findings have exceeded t he wildest dr eams of immunologist s and have r evolut ionized ser ology.
For t his invaluable cont r ibut ion t o t he pr ogr ess of medicine, Kohler and Milst ein shar ed t he
Nobel Pr ize in 1984, wit h Niels J er ne who int r oduced t he t heor et ical concept of clonalit y of t he
immune r esponse.
Monoclonal Antibodies 71
The Principle of Monoclonal Antibody Production
Mice ar e immunized wit h t he whole ant igen of int er est (cont aining, let us say, t wo epit opes:
a and b), and given anot her inject ion t o obt ain a secondar y r esponse (Figur e 9.1). Two t o four
days lat er t he spleen is r emoved and t eased apar t t o for m a suspension of spleen cells. Some
of t hese spleen cells will be commit t ed t o pr oduce ant ibodies t o epit ope “a”, ot her s t o epit ope
“b”. The spleen cells ar e mixed wit h mouse myeloma cells t hat have been pr eviously adapt ed
t o gr ow in cont inuous cult ur e. Polyet hylene glycol (PEG) is added t o pr omot e t he fusion bet ween
cell membr anes and t he cells ar e suspended in t issue cult ur e medium. The mouse myeloma
cells used ar e var iant s, in t hat , t hey lack t he enzyme hypoxant hine phosphor ibosyl t r ansfer ase
(HPRT

) and ar e also non-secr et or s of immunoglobulin (Ig

). The spleen cells on t he ot her
hand, being nor mal, possess bot h t he enzyme (HPRT
+
) and t he abilit y t o secr et e immunoglobulin
(Ig
+
). Only one in ever y 2 x 10
5
spleen cells act ually for ms a viable hybr idoma; it is t her efor e
necessar y t o eliminat e t he unfused cells t o allow r ecover y of t he hybr id. This is done by gr owt h
in select ive medium cont aining: H – hypoxant hine; A – aminopt er in; T – t hymidine.
Because myeloma cells lack t he enzyme HPRT, t hey cannot use exogenous hypoxant hine
t o synt hesize pur ines. Aminopt er in blocks endogenous synt hesis of pur ines and pyr amidines,
hence myeloma cells die aft er a per iod of t ime. Spleen cells being nor mal, diploid cells do not
have a ver y long life span in cult ur e and will also die befor e long. However , a hybr id bet ween a
myeloma cell and a spleen cell which possesses t he enzyme HPRT, is able t o synt hesize pur ines
and pyr amidines,and sur vives. The hybr ids ar e t hus immor t alized because t he myeloma cell
line cont r ibut es t he pr oper t y of gr owt h in cont inuous cult ur e and t he spleen cell pr ovides t he
hybr id wit h HPRT. Non immunoglobulin pr oducing myeloma cells a r e essent ia l a s t he
immunoglobulin desir ed is t he one t hat t he spleen cell is commit t ed t o pr oduce - myeloma
immunoglobulin or hybr id immunoglobulins would pose many obvious pr oblems.
The mixt ur e of spleen and myeloma cells gr ows in wells of a micr ot it er dish. Two t o four
weeks aft er fusion, hybr idomas become visible on gr oss examinat ion and t he super nat ant of
t hese clones ar e examined for specific ant ibody t o var ious individual ant igenic det er minant s
usually by RIA or ELISA; since t hese ar e sensit ive, quick and r eliable assays. Clones pr oducing
ant ibody ar e gr own in mass cult ur e and r ecloned. These can eit her be fr ozen away or inject ed
t o induce t umour s in mice. The r esult ant ascit ic fluid for med in t he mice yields ver y high
concent r at ions of ant ibody.
Advantages of Monoclonal Antibodies
Once a hybr idoma has been est ablished, t he exact same ant ibody can be pr oduced by
differ ent gr oups of wor ker s indefinit ely. This elimina t es differ ences in r esult s bet ween
labor at or ies. This is impor t ant in t he case of diagnost ic r eagent s. Besides, lar ge amount s of
ant ibody can be pr oduced in t his way, pr oviding a st eady supply of r eagent s for diagnost ic
immunoassays, blood gr ouping and HLA t yping.
Drawbacks of Monoclonal Antibodies
Since monoclonal ant ibodies pr oduce ant ibodies t o a single det er minant , t hey do not for m
t he lat t ice necessar y for pr ecipit at ion and so cannot be used in pr ecipit at ion assays, r adial
immunodiffusion, immunoelect r ophor esis or agar gel diffusion. Some ant ibodies gener at ed
may not fix complement and hence ar e not useful in complement fixat ion t est s.
72 Immunology– Introductory Textbook
Ver y high t it er ant ibodies also pose cer t ain difficult ies. They may r ecognize ant igens of a
low r eact ivit y as in r ecipient s of mult iple t r ansfusions or mult ipar ous women. Int er pr et at ion of
t hese r esult s becomes confusing and difficult . This kind of cr oss r eact ivit y cannot be r emoved
by absor pt ion as in t he case of convent ional ant iser a.
Figure 9.1. The principle of monoclonal antibody production.
The Production of Hybrid Antibody Molecules
Wit h cur r ent t echnology it is possible t o int r oduce DNA int o cells and have it expr essed as
if it wer e par t of t he cells own genet ic appar at us. By t his pr ocess called DNA t r ansfect ion, t he
immunoglobulin gene DNA can be int r oduced int o myeloma cells. In t his manner not only can
Monoclonal Antibodies 73
myeloma cells pr oduce monoclonal ant ibodies, t he DNA can be cust om - alt er ed t o yield modified
monoclonal ant ibodies. For example, r ecombinant ant ibodies can be pr oduced wit h t he desir ed
ant igen binding capacit y and fused t o a por t ion of a molecule wit h enzymat ic funct ion. Such a
molecule could be used in immunoassays wit h no need for second ant ibody t echniques. The
gene for a specific ant igen binding r egion (say, a t umour ant igen) could be coupled t o t he gene
for a t oxin, so t hat t he final molecule could be used as a specific t oxin t o kill t umour cells.
Using similar met hodology, invest igat ors have generat ed monoclonal ant ibodies t hat are hybrids
of mouse and human immunoglobulin molecules. In t hese molecules, t he ant igen combining
por t ion is fr om t he mouse gene and t he C-r egion is fr om t he human gene. This met hodology
may be useful in cases wher e it is necessar y t o have human immunoglobulin molecules, and
wher e it is not possible t o eit her immunize humans or t o gener at e an in vit r o ant ibody r esponse
wit h human cells.
Some Applications of Monoclonal Antibodies
Besides a sour ce of pur e ant ibody for diagnosis of infect ious diseases, monoclonal ant ibodies
pr ovide a means of:
(i) Enumeration of human lymphocyte sub populations
Ant i-CD3 ident ifies all mat ur e T cells, ant i-CD4 ident ifies subset s cont aining T helper
cells and ant i-CD8 ident ifies cyt ot oxic T cells.
(ii) Analysis of viral antigens
Monoclonal ant ibodies ar e used in t he classificat ion and diagnosis of vir al diseases. The
best example of t his is t he dissect ion of t he ant igenic st r uct ur e of t he influenza vir us. Monoclonal
ant ibodies have been used against t he haemagglut inin glycopr ot ein t hat is exposed on t he
sur face of t he vir us and t hese ant ibodies have also been found t o neut r alize t he vir us. In t his
way, ever y ant igenic dr ift or shift can be ident ified. Monoclonal ant ibodies ar e used t o map
ar eas of ant igenic var iat ion on t he vir us par t icle. Such funct ional mapping has also been done
for polio vir us and cer t ain r eovir uses.
(iii) Analysis of putative protective antigens
Monoclonal ant ibodies can define ant igen st r uct ur e and help ident ify cer t ain ant igenic
det er minant s t hat evoke a pr ot ect ive immune r esponse. Non immunodominant det er minant s
can also be ident ified and t heir r eact ivit y analysed. Wit h t he help of monoclonal ant ibodies
synt het ic pept ides have been pr oduced as subunit vaccines, wit h configur at ions cor r esponding
t o t he pr ot ect ive ant igen.
(iv) Analysis of immunologically competent cell surface molecules
Mon oclon a l a n t ibodies ca n be u sed t o ma p a n d st u dy t h e va r iou s r egion s of a n
immunoglobulin molecule and t he basis for immunoglobulin var iabilit y. Ant i CD8 inhibit s killing
by cyt ot oxic T cells. The Ant i Ia molecule inhibit s T cell r esponses t o macr ophage pr ocessed
ant igen. A cockt ail of ant i CD3 + complement kills T cells in human bone mar r ow and can be
used t o pr event gr aft ver sus host r eact ion.
(v) Blood grouping
Ant i blood gr oup monoclonals pr ovide a mor e r eliable st andar d r eagent t han convent ional
ant iser a.
74 Immunology– Introductory Textbook
(vi) HLA typing
Monoclonal ant ibodies pr ovide a r eliable means of HLA ant igen det ect ion using individual
specificit ies t o t he A,B,C and DR loci.
(vii) Diagnosis of Cancer
Ant ibodies have been gener at ed t hat dist inguish bet ween malignant and nor mal cells.
Malignant melanoma cells and acut e lymphocyt ic leukemia cells ar e among t he few t hat can be
differ ent iat ed. Radioact ive ant i car cino-embr yonic ant igen is used t o localize colonic t umour s
or secondaries on scanning. It may be possible t o deliver cyt ot oxic drugs conjugat ed t o monoclonal
ant ibodies r ight int o t umour specific cell t ypes - t he new “magic bullet ” t her apy.
(viii) In autoimmunity and immune deficiency
Imbalances in t he r at io of T helper and T suppr essor subset s indicat e immunodeficiency
or aut oimmunit y. These st at es ar e accur at ely monit or ed by monoclonals against specific T cell
ant igens. Monoclonal ant ibodies t o t he TSH* and ACH* r ecept or s ar e used t o st udy Gr aves
disease and myast henia gr avis.
(ix) In the control of fertility
Using monoclonal ant ibodies against hor mones and r epr oduct ive t r act ant igens such as
HCG*, LH* and LHRH*, at t empt s ar e being made t o cont r ol fer t ilit y.
(x) Monoclonal mutants
Mut ant s lacking Fc st r uct ur es ar e used for defining biologic r oles of Fc domains and for in
vivo neut r alizat ion of t oxic dr ugs, for example in cases of digoxin over dose.
Hybr idoma t echnology has not only impr oved t he qualit y and discr iminat ing power of
diagnost ic and invest igat ive ser ology, it pr omises t o pr ovide new r eagent s t hat will be useful in
t he diagnosis and t r eat ment of many disease pr ocesses.
*TSH : Thyroid stimulating hormone
ACH: Adrenocortico hormone
HCG: Human chorionic gonadotrophin
LH: Luteinising hormone
LHRH: Luteinising hormone releasing hormone
™™™
THE MAJOR HISTOCOMPATIBILITY
COMPLEX
CHAPTER – 10
T
he discover y of t he major hist ocompat ibilit y complex in humans ar ose fr om st udies of
t r ansplant at ion of t umour s and gr aft s in mice. It was found t hat inbr ed mice of st r ain
A would r eject skin gr aft s fr om inbr ed mice of st r ain B and vice ver sa, by pr oducing ant ibodies
against membr ane ant igens on t he for eign gr aft ed t issue. These membr ane ant igens ar e encoded
by genes on chr omosome 17 of t he mouse, in a r egion designat ed H2.
In humans evidence for t he exist ence of similar loci(a locus is a posit ion on t he chr omosome
wher e a given gene may be found) dat es fr om t he mid 1950s when leuko agglut inat ing ant ibodies
(ant ibodies t hat agglut inat e leukocyt es), wer e found in r ecipient s of mult iple t r ansfusions and
in t he ser a of mult ipar ous women. Such ser a wer e found t o agglut inat e or lyse leukocyt es fr om
some per sons but not ot her s. The chr omosomal r egion analogous t o t he mouse H2 const it ut es
t he “major hist ocompat ibilit y complex” (MHC), in all species found t o possess it . In humans,
t his r egion and t he ant igens encoded by t his r egion ar e bot h known by t he acr onym HLA
(h u ma n leu kocyt e a n t igen ). Con s equ en t ly, t h e t er ms MHC a n d HLA a r e oft en u s ed
int er changeably.
The vit al role played by HLA ant igens in t issue and organ t ransplant s was soon appreciat ed.
However , in 1973 cer t ain HLA ant igens wer e found t o be associat ed wit h specific diseases in
high pr opor t ion. In addit ion, it was r ealized t hat t he major hist ocompat ibilit y complex r egulat es
sever al aspect s of t he human immune r esponse.
Nomenclature and Genetic Organization of the MHC
The nomenclat ure of t he MHC/HLA syst em is devised by t he HLA Nomenclat ure Commit t ee
under t he auspices of t he Wor ld Healt h Or ganizat ion. An Int er nat ional Wor kshop meet s ever y
year t o updat e t he descr ipt ion and nomenclat ur e of t he MHC.
The ent ir e hist ocompat ibilit y complex occupies a segment on t he shor t ar m of chr omosome
6. Though t he t er ms HLA and MHC ar e somet imes used int er changeably, t he acr onym HLA is
mor e oft en used t o pr ecede t he individual ant igens wit hin t he complex for example, HLA – A,
HLA–B, HLA – C, et c. Figur e 10.1 schemat ically depict s t he cur r ent concept of t he MHC,
showing t he genet ic r egions cont aining t he HLA loci in r elat ion t o each ot her . The officially
r ecognized genet ic loci ar e HLA–A, HLA-B, HLA–C, HLA – E, HLA – F, HLA–G, H, J , K, L;
t oget her wit h t he HLA–D r egion. The HLA-D r egion consist s of t he following loci : HLA–DR
(D-r elat ed) HLA –DQ and HLA–DP. Mor e HLA r elat ed loci ar e cont inually being and ident ified.
Sever al addit ional genet ic r egions have been linked t o t he HLA complex. The complement
r egion, which has been mapped bet ween t he HLA–B and HLA–DR r egions cont ains genes
det er mining t he complement component s C2 and C4 of t he classical complement pat hway and
pr oper din and fact or B F of t he alt er nat ive pat hway.
76 Immunology– Introductory Textbook
Figuere 10.1. Genetic organization of the HLA system
In t he upper par t of t he figur e t he posit ion of t he HLA complex, on t he shor t ar m of
chr omosome no: 6, is shown in r elat ion t o t he ot her mar ker s: PGM
3
= phosphoglucomut ase 3;
GLO = glyoxylase; Pg5 = ur inar y pepsinogen. The lower par t of t he figur e shows t he expanded
ver sion of t he HLA complex. Class I loci ar e t he HLA–A, HLA–B and HLA–C r egions. A clust er
of closely linked complement genes: C4, BF and C2 lies next t o t he HLA–B locus. The t hr ee
class II loci ar e t er med HLA–DP, HLA–DQ and HLA–DR fr om t he 5' t o t he 3' end.
At each locus, one of sever al alt er nat ive for ms (alleles) of a gene may be found. Officially
r ecognized alleles at each locus ar e designat ed by t he locus and a number , for example, HLA–
A1, HLA–A2 et c., ar e t he allelic (alt er nat ive for ms) of t he HLA–A locus t hat may exist . Alleles
t hat have been t ent at ively assigned t o a given locus, but ar e not yet officially r ecognized ar e
designat ed by a w (for wor kshop) placed befor e t he number for example, HLA–DR w1. The HLA
syst em is ext r emely polymor phic, having mult iple differ ent alleles at each known locus; for
inst ance t her e ar e 30 or mor e differ ent alleles at t he HLA–A locus and at least 60 at t he HLA–
B locus. Each allelic gene det er mines a differ ent gene pr oduct or cell sur face ant igen which is
also given t he same number . In sever al inst ances HLA ant igens init ially t hought t o be a single
ant igen, have been found t o be a gr oup of 2 or 3 closely r elat ed ant igens. They ar e ident ified
numer ically and in br acket s for eg., HLA– A25(10), 10 being closely r elat ed t o 25.
The combinat ion of alleles at each locus on a single chr omosome is usually inher it ed as a
unit . This unit is r efer r ed t o as t he haplot ype. Since we inher it one chr omosome fr om each
par ent we have t wo HLA haplot ypes. Because all HLA genes ar e co-dominant , bot h alleles, one
each fr om t he mat er nal and pat er nal chr omosomes, at a given HLA locus ar e expr essed, and 2
complet e set s of HLA ant igens can be det ect ed on cells. Hence an individual can be t yped:
HLA–A1, A11 and HLA–B27, B13 and so on.
Linkage Disequilibrium
This is a phenomenon t hat occur s when cer t ain combinat ions of alleles ar e found wit h a
fr equency far exceeding t hat expect ed fr om pur e r andom mat ing. As an example HLA–B8 and
HLA –A1 ar e found t oget her six t o 21 t imes mor e oft en in t he same individual in some populat ion
gr oups, t han would be possible by pur e co incident al associat ion. The clinical significance of
linkage disequilibr ium is as yet unclear . However , t he mechanism r esponsible for linkage
disequilibr ium r emains t he subject of many speculat ions. Cur r ent ly, t he most pr efer r ed
explanat ion post ulat es t hat cert ain combinat ions of alleles at different HLA loci are advant ageous
t o t he sur vival of an individual. The pr ecise nat ur e of t his advant age has not been defined, but
The Major Histocompatibility Complex 77
it could r epr esent an enhanced r esist ance t o cer t ain infect ions or diseases, especially t hose
t hat ar e pr evalent in t he envir onment of a given populat ion.
Antigens of the Major Histocompatibility Complex (MHC)
Based on t heir t issue dist r ibut ion and st r uct ur e, t he MHC ant igens have been divided
int o t wo classes. The Cla ss I MHC a n t igen s, also t er med t he classic hist ocompat ibilit y ant igens
include t he HLA – A; HLA – B; HLA – C ser ies.
Th e Cla ss I I MHC a n t i gen s, analogous t o t he Ia ant igens in t he mouse include t he HLA
– DR; HLA – DQ; HLA– DP ant igens.
HLA typing
Lymphocytotoxicity
Availabilit y of monoclonal ant ibodies t o a var iet y of HLA ant igens have made ser ological
t est ing a popular met hod of HLA t yping. Using t he lymphocyt ot oxicit y assay, lymphocyt es ar e
added t o ant i-ser a which may or may not have ant ibodies dir ect ed t o HLA ant igens (shown
schemat ically in Figur e 10.2).
Figure 10.2. Typing for HLA Class I antigens: Microcytotoxicity assay. The above reaction
patterns allow the cell to be typed as HLA B27 +; HLA B8

.
If t he ser um cont ains an ant ibody specific t o an HLA (Class I or Class II) ant igen on t he
lymphocyt es, t he ant ibody will bind t o t his HLA ant igen. Complement is t hen added. The
complement binds only t o posit ive cells (i.e., wher e t he ant ibody has bound) and in doing so,
78 Immunology– Introductory Textbook
causes membr ane damage. The damaged cells ar e not complet ely lysed but suffer sufficient
membr ane damage t o allow upt ake of vit al st ains such as eosin or fluor escent st ains such as
et hidium br omide. Micr oscopic ident ificat ion of t he st ained cells, indicat es t he pr esence of a
specific HLA t ype.
The cells used for t he t est ar e lymphocyt es because of t heir excellent expr ession of HLA
and ease of isolat ion compar ed t o most ot her t issues. The most impor t ant use of t his t est is t o
det ect specific donor -r eact ive ant ibodies pr esent in a pot ent ial r ecipient pr ior t o t r ansplant at ion.
Hist or ically, t his t est , using ant iser a of known specificit y has long been used t o t ype for
HLA Class I and Class II ant igens,. However, t he problems of non-availabilit y of cert ain ant ibodies
has led t o t he int r oduct ion of DNA based met hods. Cur r ent ly, many labor at or ies have changed
t o molecular genet ic met hods for HLA Class t yping.
Mixed Lymphocyte Reaction (MLR)
The HLA - DR and HLA - DQ ant igens ar e t yped using a ser ologic pr ocedur e similar t o t he
assay for Class I ant igens. Inst ead of using t he pat ient s mononuclear cells, t hese assays ar e
done on pur ified populat ions of B lymphocyt es. The t yping ser a ar e pr et est ed t o make cer t ain
t hat t hey do not det ect t he Class I ant igens. The HLA - DR ant igens also elicit a r eact ion called
t he mi xe d lymp h ocyt e r e a ct i on (MLR). This occur s when lymphocyt es fr om one individual
ar e cult ur ed wit h t hose fr om anot her . If t hese t wo lymphocyt e populat ions possess differ ing
(het er ozygous) HLA - DR ant igens, t he cells ar e st imulat ed t o divide. They become met abolically
ver y act ive in t he pr esence of a for eign HLA - DR ant igen and begin t o synt hesize excessive
a mou n t s of DNA. Th i s ph en omen on i s ca l l ed b l a s t t r a n s f o r m a t i o n . Du r i n g bl a s t
t r ansfor mat ion t her e is incr eased upt ake of t hymidine int o t he cells t o facilit at e incr eased
DNA synt hesis. If t he t hymidine can be labelled, as in 3H t hymidine, t he blast t r ansfor mat ion
can be measur ed by following t he r at e of upt ake of 3H t hymidine. In an MLR, a panel of known
HLA - DR cells called t he h omozygou s t yp i n g cells (HTC) is used. These st imulat or cells ar e
ir r adiat ed or t r eat ed wit h mit omycin C t o pr event t heir pr olifer at ion in r esponse t o t he unknown
cells. The r esponder cells of t he individual t o be t yped ar e cult ur ed wit h t he HTC (Figur e 10.3).
If t he responder cells, aft er incubat ion wit h t he st imulat or cells, display excessive DNA synt hesis,
t hen it is concluded t hat t he pat ient ’s cells ar e not of t he same t ype as t he HTC. If only base
line DNA synt hesis occur s, t han t he pat ient s cells and t he HTC ar e homozygous (similar ).
An impor t ant use of t he MLC is in it s use as a “cellular cr ossmat ch” pr ior t o t r ansplant at ion
especially for bone mar r ow t r ansplant s. By t est ing t he pr ospect ive donor and r ecipient , an
invit ro t ransplant model is est ablished which is an ext remely useful indicat or of possible reject ion
or Gr aft ver sus Host r eact ion.
Molecular typing techniques
RFLP
Rest r ict ion Fr agment Lengt h Polymor phism (RFLP) met hods r ely on t he abilit y of cer t ain
enzymes t o r ecognise exact DNA nucleot ide sequences and t o cut t he DNA at each of t hese
point s. Thus t he fr equency of a par t icular sequence will det er mine t he lengt hs of DNA pr oduced
by cut t ing wit h a par t icular enzyme.
The Major Histocompatibility Complex 79
Figure 10.3. Typing for HLA Class II antigens: Mixed lymphocyte reaction. The above reactions
allow the cell to be typed as HLA Dw3
+
; HLA-Dw2

.
The DNA for one HLA (Class II) ant igen, e.g., DR15, will have t hese par t icular enzyme
cut t ing sit es (or “r est r ict ion sit es”) at differ ent posit ions t o anot her ant igen, e.g., DR17. So t he
lengt hs of DNA seen when DR15 is cut by a par t icular enzyme, ar e char act er ist ic of DR15 and
differ ent t o t he sizes of t he fr agment s seen when DR17 is cut by t he same enzyme.
Polymerase Chain Reaction
The Polymer ase Chain React ion (PCR) is a r ecent ly developed and r evolut ionar y new
syst em for amplifying t he DNA nucleot ide sequence of a par t icular r egion of int er est in any
individual. Ver y small amount s of DNA can be used as a st ar t ing point . Sequencing DNA is now
available using comput er ised and aut omat ed met hodology.
The fir st st ep in t his t echnique is t o obt ain DNA fr om cells of an individual. The double
st r anded DNA is t hen denat ur ed by heat int o single st r anded DNA. Oligonucleot ide pr imer
sequences ar e t hen chosen t o flank a r egion of int er est . The oligo- nucleot ide pr imer is a shor t
segment of complement ar y DNA which will associat e wit h t he single st r anded DNA t o act as a
st ar t ing point for r econst r uct ion of double st r anded DNA at t hat sit e.
If t he oligonucleot ide is chosen t o be close t o a r egion of special int er est like a hyper var iable
r egion of HLA-DR t hen t he par t of t he DNA, will be amplified when DNA polymer ase and
deoxy-r ibonucleot ide t r iphosphat es ar e added. Fr om one copy of DNA it is t hus possible t o
make t wo. Those t wo copies can t hen, in t ur n, be denat ur ed, r eassociat e wit h pr imer s and
pr oduce four copies. This cycle can t hen be r epeat ed unt il t her e is sufficient of t he select ed
por t ion of DNA t o isolat e on a gel and t hen sequence or t ype.
80 Immunology– Introductory Textbook
Ther e ar e a number of PCR based met hods in use. For example:
S equence S pecific Priming (S S P)
I n t h is t est , t h e oligon u cleot ide pr imer s u sed t o st a r t t h e PCR h a ve sequ en ces
compliment ar y t o known sequences which ar e char act er ist ic t o cer t ain HLA specificit ies.
The pr imer s which ar e specific t o HLA-DR15, for example, will not be able t o inst igat e t he PCR
for HLA-DR17. Typing is done by using a set of differ ent PCRs, each wit h pr imer s specific for
differ ent HLA ant igens.
S equence S pecific Oligonucleotide (S S O) Typing
By t his met hod, t he DNA for a whole r egion (e.g., t he HLA DR gene r egion) is amplified in
t he PCR. The amplified DNA is t hen t est ed by adding labelled (e.g., Radioact ive) oligonucleot ide
pr obes, which ar e complement ar y for DNA sequences, char act er ist ic for cer t ain HLA ant igens.
These pr obes will t hen “t ype” for t he pr esence of specific DNA sequences of HLA genes.
In gener al, because we possess 2 haplot ypes each and because HLA expr ession is co-
dominant it possible t o t ype 2 ant igens fr om each locus. Occasionally only one ant igen can be
t yped when it is r epor t ed as HLA-B27; B- for example. Such a r epor t could mean t hat t he ot her
HLA-B ant igen was homozygous wit h t he t yped ant igen or t hat t yping was not possible by t he
met hods available.
Uses of HLA Typing
HLA t yping is used primarily for det erminat ion of HLA compat ibilit y prior t o t ransplant at ion,
for pat er nit y t est ing, for ant hr opologic st udies and t o est ablish HLA-disease associat ions.
Structure and function of the MHC antigens
MHC Class I Antigens
The MHC Class I ant igens consist of t wo polypept ide chains held t oget her non-covalent ly
(Figur e 10.4). One chain (t he alpha chain), is heavy and glycosylat ed (44,000 dalt ons).The ext r a
cellular por t ion of t his Class I heavy chain is
divided int o t hr ee domains, designat ed α1, α2
and α3 looped t oget her by disulphide bonds
(Figur e 10.4). The ext r acellular por t ion has an
N t er mi n a l a n d i s h ydr oph i l i c, t h e
t r a nsmembr a ne por t ion is hydr ophobic, t he
int r a cellular por t ion is hydr ophilic and has t he
car boxy t er minal. The ot her chain is a small
pr ot ei n (11, 500 da l t on s ) k n own a s β2–
micr oglobulin which is encoded by a gene on
chr omosome 15. Bot h t he β2 micr oglobulin and
t he α3 domain of t he heavy chain of t he MHC
Class I ant igen ar e r elat ively non- polymor phic
a n d demon s t r a t e con s i der a bl e a mi n o a ci d
homology wit h t he CH3 por t ion of t he const ant
r egion of t he IgG molecule. X-r ay diffr act ion
st udies of t he HLA-A2 molecule have shown t hat
t he heavy chain domains α 1 and α 2 ar e most
Figure 10.4. Structure of the MHC Class I antigen.
The Major Histocompatibility Complex 81
dist al fr om t he cell membr ane and for m a gr oove along t he t op sur face of t he molecule. The
sides of t he gr oove ar e for med by t he α1 and α2 domains, t he st r et ches of pr ot ein t hat for m t he
walls of t he gr oove lie in an alpha helix configur at ion. The base of t he gr oove also cont ains
par t s of t hese α1 and α 2 domains, t he pr ot ein st r et ches of t he base ar e found in a β pleat ed
configur at ion(Figur e 10.5). Analysis of t he pr ot ein st r et ches for ming t he gr oove show t hat
t her e ar e hyper var iable r egions along t he sides of t he gr oove and t o a lesser ext ent in t he β
pleat ed base. In all st udies concer ning t he MHC Class I ant igen, wor ker s found t hat t he gr oove
always cont ained an unident ified pept ide molecule, pr esumably r epr esent ing pr ocessed ant igen.
These findings ar e consist ent wit h t he idea t hat MHC molecules bind and pr esent pr ocessed
ant igens t o r esponding T cells and t hat t he T cell r ecept or co r ecognizes for eign ant igen only if
pr esent ed wit h t he MHC Class I ant igen. The genes t hat det er mine a Class I ant igen consist of
sever al exons int er spaced by int r ons. A separ at e exon codes for each of t he t hr ee α domains
and gene polymor phism exist s wit hin each of t hese exons, mor e so in t he α 1 and α 2 r egions.
Figure 10.5. A schematic representation of the α2 and α3 domains of the MHC Class I antigen
reveals a groove thought to be involved in antigen binding and presentation to T cells. The view is
looking down on the top of the molecule, showing the surface that faces away from the cell
membrane. The groove has a base composed of eight β strands, shown as thick arrows pointing in
the amino to carboxyl direction. The helices, which form the sides, are shown as helical ribbons.
The two connected spheres represent a disulfide bond, and the N terminus is indicated. Polymorphism
is found both along the edges of the helices and in the base.
MHC Class I ant igens ar e expr essed on all cell t ypes except er yt hr ocyt es and t r ophoblast s.
St r iat ed muscle cells and liver par enchymal cells ar e nor mally negat ive for Class I ant igens or
t hey may expr ess only a low densit y of Class I molecules. However , in inflammat or y st at es
t hese cells begin t o expr ess lar ge number s of Class I molecules.
By a pr ocess called capping, it can be shown t hat t he MHC Class I ant igens ar e separ at e
ent it ies on t he cell membr ane. When exposed t o specific ant ibody, t he MHC ant igen on t he cell
sur face will move fr om it s usual, even dist r ibut ion, t o a single small ar ea or cap on t he cell. If
a cell is exposed t o ant ibody t o HLA–A ant igen, only t hese ant igens will for m a cap; t he HLA–
B and HLA–C ant igens will r emain evenly dist r ibut ed. If ant ibody t o t he β2 micr oglobulin is
used, a ll a nt igens will ca p a s t his molecule is common t o a ll Cla ss I a nt igens. The β2
micr oglobulin helps t o keep t he configur a t ion of t he MHC Cla ss I a nt igens. I f t he β2
micr oglobulin is select ively r emoved, t he st r uct ur al configur at ion of t he ant igen is lost ;
par t icular ly t hat of t he gr oove bet ween t he α1 and α 2 domains.
82 Immunology– Introductory Textbook
Functions of the MHC Class I antigens
In cell mediat ed cyt olysis t he Class I ant igens ar e t he t ar get ant igens r ecognized by t he
killer / cyt ot oxic T lymphocyt es. MHC class I molecules specifically bind CD8 molecules expr essed
on cyt ot oxic T lymphocyt es. Class I ant igens ar e t he pr incipal ant igens r ecognized by t he host
dur ing t issue gr aft r eject ion.
The t r ue physiologic r ole of t he MHC Class I ant igens lies in t he cell mediat ed lysis of
vir us infect ed cells. When T lymphocyt es ar e exposed t o a vir al ant igen, t hey will r ecognize it
only if associat ed wit h a Class I ant igen; i.e., t hese vir al ant igens ar e pr esumably t he pept ides
found wit hin t he gr oove for med bet ween t he α1 and α2 domains. The cyt ot oxic T lymphocyt es
elicit ed by such an exposur e ar e r est r ict ed in t heir killing t o t hose t ar get cells which bear bot h
t he same vir al ant igen and t he same Class I ant igen as wer e pr esent on t he cell t hat fir st
st imulat ed t heir pr olifer at ion. These T lymphocyt es will not kill t ar get cells bear ing t he same
vir al ant igen and a differ ent Class I ant igen, nor will t hey kill cells bear ing t he cor r ect Class I
ant igen and a differ ent vir al ant igen.
In summar y MHC class I expr ession is widespr ead on vir t ually ever y cell of t he body. This
is consist ent wit h t he pr ot ect ive funct ion of cyt ot oxic T lymphocyt es which cont inuously sur vey
cell sur faces and kill cells har bour ing met abolically act ive micr oor ganisms. MHC class I
molecules bind pept ide fr agment s der ived fr om pr ot eolyt ically degr aded pr ot eins endogenously
synt hesized by a cell. Small pept ides ar e t r anspor t ed int o t he endoplasmic r et iculum wher e
t hey associat e wit h nascent MHC class I molecules befor e being r out ed t hr ough t he Golgi
appar at us and displayed on t he sur face for r ecognit ion by cyt ot oxic T lymphocyt es.
MHC Class II antigens
Class II ant igens ar e found chiefly on sur faces of immunocompet ent cells, including
monocyt es/macr ophages, act ivat ed T cells, dendr it ic cells and most not ably on B cells. As wit h
Class I ant igens, inflammat or y st at es cause many ot her t issues t o expr ess Class II ant igens. In
humans t he Class II ant igens ar e encoded by t he HLA–D gene r egion, which is divided int o at
least t hr ee sub r egions : HLA–DP,HLA–DQ and HLA–DR. The det ailed st r uct ur e of an HLA–
DR molecule has been elucidat ed and ser ves as a pr ot ot ype for t he Class II molecule. The MHC
Class II ant igen, like t he Class I ant igen, consist s of an α chain (34,000 dalt ons) and a β chain
(29,000 dalt ons) in non covalent associat ion
(Figur e 10.6). Bot h chains ar e anchor ed in t he
cell membr ane and display an ext r acellular
h ydr oph i l i c r egi on , a t r a n s membr a n e
h ydr oph obi c r egi on a n d a n i n t r a cel l u l a r
hydr ophilic r egion. The α chain int r acellular
region can be phosphorylat ed. As shown in figure
10.7 bot h chains cont ain ext r acellular domains
t er med α1, α2 and β1, β2. Posit ions of disulphide
bon ds a n d ca r boh ydr a t e lin ka ges a r e a ls o
illust r at ed in t he figur e. Like in t he MHC Class
I molecule, t he sides of t he α1 and β1 domains
for m a gr oove in which t he sides ar e in an α
helix configur at ion and t he base cont ains β
pleat s. Again, hyper var iable r egions ar e locat ed
pr imar ily along t he gr oove which suggest s t hat
for eign ant igen r est s wit hin t his gr oove and t hat
T cells r ecognize for eign ant igen in conjunct ion
wit h t he MHC ant igen.
Fig. 10.6. Structure of the MHC Class II antigen.
The Major Histocompatibility Complex 83
The genet ic or ganizat ion of t he MHC Class II ant igens is a lit t le mor e complex (Figur e
10.7). α genes encode t he α chains and β genes code for β chains. As shown in Figur e 10.7, t he
HLA –DR gene r egion has one locus for t he α chain and t hr ee loci for t he β chain; alt hough one
of t he lat t er may be a pseudogene (ie: it does not det er mine a pr oduct ). The DR α chain can
combine wit h any of t he ß chains t o pr oduce a DR molecule. The same happens for t he DP and
DQ molecules.
Figure 10.7. Genetic organization of the MHC Class II antigen showing the DP, DQ and DR
regions and their constituent α and ß chain genes.
Functions of the MHC Class II antigens
The MHC Class II ant igens ar e pr incipally r esponsible for an in vivo cor r elat e of t he
mixed lymphocyt e r eact ion (MLR) –t he gr aft ver sus host r eact ion. In t he physiological sit uat ion
for eign ant igen is r ecognised in conjunct ion wit h Class II molecules by cer t ain gr oups of T
cells: t he CD4+ T cells or T–helper cells. Hence MHC Class II molecules play an impor t ant par t
in pr esent ing pr ocessed ant igen t o T cells and aid collabor at ion bet ween T and B cells. Whet her
T cells r ecognize ant igen in conjunct ion wit h Class I or Class II molecules, it follows t hat T cells
must be equipped wit h r ecept or s bot h for t he MHC ant igens as well as for eign ant igens. This
phenomenon, when T cell act ivit y is r est r ict ed t o t hose cells which bear ant igen in conjunct ion
wit h eit her t he MHC Cla ss I or Cla ss II a nt igens, is known a s t he MHC Re s t r i c t i on
P h en omen on .
In summar y, MHC class II expr ession is r est r ict ed t o “ant igen pr esent ing cells.” This is
consist ent wit h t he funct ions of helper T
H
lymphocyt es which ar e locally act ivat ed wher ever
t hese cells encount er macrophages, dendrit ic cells, or B cells t hat have int ernalized and processed
ant igens pr oduced by pat hogenic or ganisms. MHC class II molecules bind pept ide fr agment s
der ived fr om pr ot eolyt ically degr aded pr ot eins exogenously int er nalized by “ant igen pr esent ing
cells,” including macr ophages, dendr it ic cells, and B cells. The r esult ing pept ide fr agment s ar e
compar t ment alized in t he endosome wher e t hey associat e wit h MHC class II molecules befor e
being r out ed t o t he cell sur face for r ecognit ion by helper T lymphocyt es.
Disease and the Major Histocompatibility Complex
Diseases associat ed wit h HLA ant igens have sever al char act er ist ics : (a) t hey ar e of
unknown cause and unknown pat hophysiologic mechanism, wit h a her edit ar y pat t er n of
dist r ibut ion, (b) t hey ar e associat ed wit h immunologic abnor malit ies and (c) t hey have lit t le or
no effect on r epr oduct ion. Table 10.1 list s t he diseases showing posit ive HLA - disease
associat ions.
84 Immunology– Introductory Textbook
Table 10.1: Diseases with positive HLA associations
Disease HLA Antigen Relative Risk
Rheumatic Ankylosing spondylitis B27 69.1
Reiter’s syndrome B27 37.0
Acute anterior uveitis B27 8.2
Reactive arthritis (yersinia, B27 18.0
salmonella, campylobacter)
Psoriatic arthritis (cenral) B27 10.7
B28 9.1
Psoariatic arthritis (peripheral) B 27 2.0
B38 6.5
Juvenile rheumatoid arthritis B27 3.9
Juvenile rheumatoid arthritis DR5 3.3
pauciarticular
Rheumatoid arthritis DR4 3.8
Sjogren’s sundrome DR3 5.7
Systemic lupus erythematosus DR2 / DR3 2.6
Gastro-intestinal Gluten sensitive enteropathy DR3 11.6
Chronic active hepatitis DR3 6.8
Ulcerative colitis B5 3.8
Haematologic Idiopathic haemochromatosis A3 6.7
B14 26.7
A3, B14 90.0
Pernicious anaemia DR5 5.4
Skin Dermatitis herpetiformis DR3 17.3
Psoriasis vulgaris B13, B17, 7.5
Bw57, Cw6
Behcet’s disease B5 3.8
Endocrine Insulin dependent diabetes DR4 3.6
mellitus DR3 4.8
DR2 0.2
Graves disease B8 2.5
DR3 3.7
Addison’s disease Dw3 10.5
Subacute thyroiditis (deQuervain) B35 13.7
Hashimoto’s throiditis DR5 3.2
Congenital adrenal hyperplasia Bw47 15.4
The Major Histocompatibility Complex 85
Disease HLA Antigen Relative Risk
Neurologic Myasthenia gravis (without thymoma) B8 3.3
Multiple sclerosis DR2 2.7
Bipolar affective disorder B16 2.3
Narcolepsy DR2 130.0
Schizophrenia A28 2.3
Renal Idiopathic membranous DR3 5.7
glomerulonephritis
Goodpasture’s syndrome DR2 15.9
Minimal change nephrotic syndrome DR7 4.2
IgA nephropathy DR4 3.1
Polycystic kidney disease B5 2.6
Infectious Tuberculoid leprosy (Indians) B8 6.8
Paralytic polio B16 4.3
Low vs high response to Cw3 12.7
vaccinia virus
The pr ot ot ype HLA –disease associat ion is t hat of ankylosing spondylit is wit h HLA–B27.
Ninet y per cent of Amer ican Caucasian pat ient s wit h ankylosing spondylit is possess HLA–B27.
In some cases a disease may be associat ed wit h ant igens det er mined by 2 differ ent HLA loci
(r efer Table 10.1).
Sever al hypot heses have been advanced t o explain HLA disease associat ions. In t he 1960’s,
it was discover ed t hat t he mouse MHC (called H2) played a par t in t he genet ic suscept ibilit y of
mice t o cer t ain leukaemias and in t heir immune r esponse t o cer t ain ant igens. Since t hen
numer ous r epor t s have been published descr ibing t he r ole of t he human MHC in t he cont r ol of
immune r esponsiveness and disease suscept ibilit y.
Ther e ar e t wo gener al explanat ions for HLA and disease associat ions. Fir st ly, t her e may
be linkage disequilibr ium bet ween alleles at a par t icular disease associat ed locus and t he HLA
ant igen associat ed wit h t hat disease: t his is so for HLA–A3 and idiopat hic haemochr omat osis.
Anot her possible explanat ion is t hat t he HLA ant igen it self plays a r ole in disease; as
explained by one of t he following models:
(a) by being a poor pr esent er of a cer t ain vir al or bact er ial ant igens
(b) by pr oviding a binding sit e on t he sur face of t he cell for a disease pr ovoking vir us or
bact er ium
(c) by pr oviding a possible means of t r anspor t for t he vir us t o ent er t he cell
(d) by having close molecular similar it y t o t he pat hogen, t he immune syst em fails t o
r ecognise t he pat hogen as for eign and fails t o mount an immune r esponse against it .
86 Immunology– Introductory Textbook
It is most likely mor e t han one mechanism is involved, but t o var ying ext ent s in differ ent
diseases. In mult iple scler osis and ankylosing spondylit is, cell mediat ed immunit y is oft en
depr essed, not only in t he pat ient s but also in t heir par ent s and siblings. Complement (C2)
levels ar e known t o be low in syst emic lupus er yt hemat osus, a disease associat ed wit h HLA
DR2 and DR3. In glut en ent er opat hy, which shows a high associat ion wit h HLA DR3, a specific
gene pr oduct is t hought t o act as an abnor mal r ecept or for gliadin, t he wheat pr ot ein, and
pr esent it as an imunogen t o t he body.
What ever t he explanat ion for HLA and disease associat ion, it is clear t hat t he HLA syst em
and ot her non-linked genes oper at e concur r ent ly t o pr oduce disease.
™™™
IMMUNE RESPONSE MECHANISMS I:
B AND T LYMPHOCYTES
CHAPTER – 11
The Origin of B Lymphocytes
T
he B lymphocyt e and it s secr et ed end pr oduct , t he ant ibody molecule has been
r ecognized as a power ful immunologic t ool for over a cent ur y. St udies in bir ds showed
t hat t he bur sa of Fabr icius, a hindgut lymphoid or gan was t he sit e of ear ly development of
ant ibody pr oducing cells. This lineage of cells was t her efor e t er med B cells. In mammals, cells
of B lineage ar e init ially gener at ed in t he foet al liver , when haemopoiet ic st em cells migr at e t o
t he liver fr om t he yolk sac. This pr ocess begins dur ing t he 8t h week of human gest at ion. The
foet al liver cont inues t o be a major sit e for pr oduct ion of t he er yt hr oid/ myeloid ser ies including
B cells, unt il well int o t he second t r imest er . St em cells t hen populat e t he bone mar r ow, t her eaft er
B cells ar e cont inuously pr oduced in t he bone mar r ow t hr oughout life. B cells develop fr om a
plur ipot ent ial st em cell t hat can give r ise t o all of t he differ ent t ypes of haemopoiet ic cell t ypes.
The Biology of B Lymphocytes
B lymphocyt es display immunoglobulins as int egr al pr ot eins of t heir cell membr anes.
These membr ane bound immunoglobulins ar e t he B cell r ecept or s for ant igen and t hey differ
fr om secr et ed immunoglobulins in sever al ways. They ar e anchor ed in t he cell membr ane wit h
t he car boxy t er minal embedded int o t he membr ane. The t r ans membr ane r egion is hydr ophobic
in nat ur e. The amino or ant igen binding end is ext r acellular and st r at egically placed t o r eceive
for eign ant igen. Immat ur e B cells init ially display membr ane bound IgM monomer s. IgD
molecules ar e not found on newly for med B cells. Immat ur e B cells r espond negat ively t o cr oss
linkage of t heir IgM molecules by mult ivalent ant igen. Thus ear ly exposur e t o ant igen eliminat es
B cell r esponsiveness leading t o t oler ance t owar ds t hat ant igen. As B cell development pr oceeds,
IgD molecules appear on t heir sur face membr anes along wit h IgM and t he IgD becomes t he
pr edominant membr ane bound isot ype found. Rest ing B cells display higher levels of sur face
IgD; as act ivat ion of B cells commences IgD is lost and ot her r ecept or s t hat aid B cell act ivat ion
appear on t he cell sur face.
Immat ur e B cells r apidly acquir e r ecept or s for C3d, Epst ein - Bar r vir us, C3b and Fc
por t ions of IgG molecules. Hist ocompat ibilit y Class I ant igens ar e pr esent on immat ur e B
lymphocyt es. Act ivat ed B cells expr ess incr eased amount s of HLA-D r egion encoded molecules.
Recept or s for Fc(gamma) and C3b also show an incr ease. The lat t er t wo t hen decr ease dur ing
t he t er minal st ages of B cell differ ent iat ion. Act ivat ed B cells also display r ecept or s for
int er leukin-2 (IL-2) and ot her fact or s needed for full differ ent iat ion of B cells. Figur e 11.1
shows t he full complement of r ecept or molecules on t he B cell sur face. Monoclonal ant ibodies
ar e now available which can ident ify cell sur face mar ker s and t her eby accur at ely st age t he B
cell in it s development .
88 Immunology– Introductory Textbook
Figure 11.1. Receptor molecules on the B Cell surface.
Aft er ant igen or mit ogen st imulat ion B cells can pr oceed along one of t wo pat hways. They
can differ ent iat e int o plasma cells and secr et e lar ge amount s of immunoglobulin or t hey can
divide and r et ur n t o a r est ing st age. These lat t er cells ar e called memor y B cells and can
r apidly differ ent iat e int o plasma cells following second exposur e t o t he same ant igen. Plasma
cells ar e t he t er minally differ ent iat ed st at e of B lymphocyt es. The ant ibody for ming machiner y
of t hese cells ar e t ur ned up full for ce. Over 40% of t he t ot al pr ot eins pr oduced by plasma cells
ar e immunoglobulins.They r elease t housands of ant ibody molecules ever y second. The plasma
cell seldom divides and has an aver age life span of less t han 4 days.
The Clonal Selection Theory
How do cells make ant ibodies in such enor mous var iet y? One ant ibody can neut r alize just
one t ype of ant igen - and ant igens come in an incr edible diver sit y of shapes, sizes and chemical
composit ions. Yet , t he human syst em is capable of manufact ur ing a pr act ically unlimit ed r ange
of ant ibodies against bact er ia and t heir t oxins, vir uses, pollen gr ains, incompat ible blood cells
and even some novel man made molecules.
Over 30 year s of int ensive r esear ch has led t o t he doct r ine t hat is most widely accept ed
t oday and is called t he Clon a l Select i on Th eor y of ant ibody for mat ion. The concept ual fat her
of t he clonal select ion t heor y was P a u l Eh r li ch . Over a hundr ed year s ago he post ulat ed t hat
a whit e blood cell’s sur face bor e r ecept or s wit h side chains t o which for eign subst ances became
chemically linked. This binding pr ompt ed t he cell t o pr oduce and secr et e copies of t his bound
r ecept or - t he ant ibody -int o t he cir culat ion (Figur e 11.2). For decades aft er Paul Ehr lich
pr oposed his t heor y, he failed t o win validat ion in scient ific cir cles. It was only in t he 1960s t hat
immunologist s r esumed t heir sear ch and br ought t his t heor y int o it s own.
It was Ni els J er n e who int r oduced t he concept of clonalit y and for mulat ed a not ion similar
t o Ehr lich’s side chain t heor y. He suggest ed t hat any animal possesses in it s ar mament ar ium,
small number s of pr efor med ant ibodies against all ant igens. An ant igen–ant ibody complex
int er act s wit h whit e cells and mor e ant ibody t o t he same ant igen is pr oduced. Da vi d Ta lma ge
Immune Response Mechanisms I: B and T lymphocytes 89
in 1957 went a bit fur t her , t o say t hat whit e cells ar e select ed for pr olifer at ion when t he
ant ibody t hey synt hesize mat ches t he invading ant igen.
Fig.ure 11.2. Ehrlich’s side chain theory (a) Combination of antigen with preformed receptors
(b) Cell is triggered to produce and secrete more of these receptors.
In r et r ospect it is obvious t hat J er ne and Talmage laid t he foundat ions of t he clonal
select ion t heor y , it r emained for Ma cfa r la n e Bu r n e t t o cr yst allize t he concept of clonal
select ion. Cent r al t o Bur net ’s t hinking was t he t enet t hat one cell pr oduced just one kind of
ant ibody. He pr oposed t hat binding of an ant igen wit h an ant ibody -cum-r ecept or on t he cell
sur face, t r igger s t he cell t o mult iply and manufact ur e mor e of t he same r ecept or . He asser t ed
t hat each cell and it s clones, or offspr ing, can pr oduce just one kind of ant ibody and he coined
t he t er m clonal select ion t o descr ibe his t heor y. Figur e 11.3 illust r at es t he clonal select ion
concept .
Figure 11.3. The Clonal Selection Theory.
90 Immunology– Introductory Textbook
The human syst em is endowed wit h ant ibody pr oducing cells bear ing an ent ir e libr ar y of
ant igen binding r ecept or s (t he immunoglobulin molecule). This occur s dur ing development ;
ea ch B-lymph ocyt e becomes gen et ica lly pr ogr a mmed, t h r ou gh a pr ocess ca lled g e n e
t r a n sloca t i on , t o make a unique B-cell r ecept or . Molecules of t hat B-cell r ecept or ar e placed
on it s sur face wher e it can r eact wit h epit opes of an ant igen. If an ant igen ent er s t he syst em it
mat ches wit h one of t he r ecept or s and binds t o it . This induces t he cell t o pr olifer at e, t o pr oduce
sever al clones of it s kind, each capable of secr et ing mor e of just t hat one par t icular t ype of
r ecept or (ant ibody) mat ched for t he invading ant igen. Once an ent ir e cadr e of cells has been
gener at ed, t he second r esponse t o t he same ant igen is mor e int ense since t her e ar e many
mor e cells await ing t o bind ant igen. Each cell will t hen gener at e mor e clones secr et ing t he
same ant ibody. Once clones of ant ibody pr oducing cells have been gener at ed, pr olonged exposur e
t o ant igen is not necessar y t o maint ain ant ibody pr oduct ion. The clonal select ion t heor y t oget her
wit h an underst anding of t he genet ics of ant ibody diversit y has provided many answers regarding
t he mechanisms which cont r ol t he pr oduct ion of t he vast r eper t oir e of ant ibodies t hat t he
immune syst em is endowed wit h.
Finally, clonal select ion explained immunologic t oler ance as t he complet e delet ion of an
ent ir e clone of cells, which would occur befor e or aft er bir t h, if an ant igen over whelmed t he
met abolic capabilit ies of t he cells. In 1960, Macfar lane Bur net shar ed t he Nobel Pr ize wit h
Pet er Medawar for his accomplishment s in under st anding immunologic t oler ance. In 1984,
Niels J er ne also r eceived t he Nobel Pr ize for his t heor et ical cont r ibut ions in concept ualizing
t he idea of clonalit y in immunology.
T-Lymphocytes
The t hymus der ived lymphocyt es, or T cells mediat e 2 gener al t ypes of immunologic
funct ions: effect or and r egulat or y. The effect or funct ions ar e mediat ed by (i) secr et ion of soluble
subst ances called lymphokines or cyt okines and (ii) by t heir abilit y t o kill ot her cells (cyt ot oxicit y).
The r egulat or y funct ions ar e r epr esent ed by t heir abilit y t o amplify cell mediat ed cyt ot oxicit y
by ot her T cells and by t he “help” t hey r ender in t he pr oduct ion of immunoglobulins by B cells.
These funct ions also r equir e t he synt hesis of lymphokines (cyt okines).
St em cells migr at e t o t he t hymus and move fr om cor t ex t o medulla and out int o t he
per ipher y - a jour ney t hat t akes t hr ee days. As T cells gain mat ur it y, cer t ain sur face molecules
begin t o be expr essed. An under st anding of T cell funct ion has been possible because of t he
ident ificat ion and char act er izat ion of t hese sur face molecules. The most impor t ant of t hese
sur face molecules is list ed in Table 11.1.
Table 11.1: Molecules on the T cell surface
Main cell surface Restrictions Functions
markers
T helper cell or T4 cell CD4+; CD3–T–cell MHC–Class II Stimulate B cell to produce
receptor; CD2+ antibody; Induce CD8+
T–cell cytokine secretion;
macrophage activation
T cytotoxic cell or CD8+; CD3–T–cell MHC–Class I Lyse antigen bearing target
T8 cell receptor; CD2+ cell
T cells subsets
Immune Response Mechanisms I: B and T lymphocytes 91
Each of t he molecules list ed is det ect able by monoclonal ant ibodies and plays an impor t ant
r ole in T cell differ ent iat ion and funct ion. As shown in Figur e 3.2, st em cells do not display any
sur face molecules. As T cells move int o t he t hymic cor t ex, t he CD2 molecule is t he fir st t o
appear , followed by CD3, CD4 and CD8. In t he t hymic medulla, t wo dist inct lineages become
evident : one bear ing CD2, CD3 and CD4 molecules and t he ot her bear ing CD2, CD3 and CD8
molecules. In per ipher al lymphoid t issue, 65% of t he cells possess CD2, CD3 and CD4 molecules
on t heir sur face and 35% display CD2, CD3 and CD8 ant igens.
CD4+ T cells or T4 cells const it ut e t he helper T cells and CD8+ T cells or T8 cells for m t he
cyt ot oxic/suppr essor T cells. This demar cat ion, however , is not wat er t ight and it has been
shown t hat T4 cells t ake par t in cyt ot oxicit y t ar get ed at cells bear ing MHC Class II molecules.
Br oadly, t he T helper or T4 cells, r ender B cell help, t hey secr et e cyt okines t o pr opagat e t his
funct ion and also t o amplify cell mediat ed immunologic r eact ions. The T cyt ot oxic (T8) cells act
as specific killer cells.
In t he t hymus all T cells lear n t o r ecognize self MHC gene pr oduct s and t his helps t hem
t o r eact wit h ant igen in conjunct ion wit h MHC gene pr oduct s in t he per ipher y.
T4-Lymphocytes (T4-Helper Cells, CD4
+
Cells)
Funct ionally, t her e ar e differ ent t ypes of T4-lymphocyt es based on t he cyt okines t hey
pr oduce. The t wo pr imar y t ypes ar e T
h
1 cells and T
h
2 cells. (Bot h T4-lymphocyt es and T8–
lymphocyt es can exhibit T
h
1 or T
h
2 cyt okine pr ofiles).
T
h
1 lymphocytes
T
h
1–lymphocyt es r ecognize ant igens pr esent ed by macr ophages and funct ion pr imar ily t o
act ivat e and height en cell-mediat ed immunit y by pr oducing cyt okines such as int er leukin-2
(IL-2), int er fer on-gamma (IFN-gamma) and t umour necr osis fact or -bet a (TNF-bet a). Collect ively
t hese cyt okines enable T8-lymphocyt es t o pr olifer at e and differ ent iat e int o cyt ot oxic T–
lymphocyt es capable of dest r oying infect ed host cells and mut ant cells; act ivat e cyt ot oxic T–
lymphocyt es and NK cells; act ivat e macr ophages enabling t hem t o dest r oy int r acellular
pat hogens; st imulat e t he pr oduct ion of opsonizing and complement -act ivat ing ant ibodies for
enhanced at t achment dur ing phagocyt osis; act ivat e neut r ophils; st imulat e incr eased pr oduct ion
of monocyt es in t he bone mar r ow; and funct ion as chemoat t r act ant s for phagocyt es.
T
h
2 lymphocytes
T
h
2-lymphocyt es r ecognize ant igens pr esent ed by B-lymphocyt es. They pr oduce cyt okines
such as int er leukins 2, 4, 5, 10, and 13 t hat pr omot e ant ibody pr oduct ion. Collect ively t hese
cyt okines enable act ivat ed B-lymphocyt es t o pr olifer at e, st imulat e act ivat ed B–lymphocyt es t o
synt hesize and secr et e ant ibodies, pr omot e t he differ ent iat ion of B-lymphocyt es int o ant ibody–
secr et ing plasma cells, and enable ant ibody pr oducing cells t o swit ch t he class of ant ibodies
being pr oduced. Anot her major funct ion of t he cyt okines pr oduced by T
h
2 cells is t o enable B–
lymphocyt es t o act ivat e eosinophils and pr oduce incr eased amount s of IgE against helmint hs.
IgE act s as an opsonizing ant ibody enabling eosinophils t o at t ach t o helmint hs for ext r acellular
killing.
T8-Lymphocytes (T8-Cells; CD8
+
Cells; Cytotoxic T-Lymphocytes)
Cyt ot oxic T cells consit it ut e one of t he body’s major defences against vir uses, int r acellular
bact er ia, and t umour cells. These cyt ot oxic T cells ar e effect or cells der ived fr om T8-lymphocyt es
92 Immunology– Introductory Textbook
dur ing cell-mediat ed immunit y. However , in or der t o become cyt ot oxic, naive T8-lymphocyt es
must become a ct i va t e d by cyt okines pr oduced by ant igen-pr esent ing cells (APCs). This
int er act ion bet ween APCs and naive T8-lymphocyt es occur s pr imar ily in t he lymph nodes, t he
lymph nodules, and t he spleen.
The T Cell Receptor for Antigen
Few pr oblems in immunology have been as difficult t o solve and as cont r over sial as t hat
of char act er izing t he T cell r ecept or for ant igen. The B cell r ecept or for ant igen has long been
ident ified as t he IgM monomer bound on it s sur face. Fur t her , t he B cell r ecept or is capable of
r ecognizing and r eact ing against fr ee, nat ive ant igen. St udies in t he 1960s and ear ly 1970s
clear ly demonst r at ed t hat T cells t oo, wer e ant igen specific. Ther efor e it followed t hat T cells
must have a r ecept or capable of r ecognizing ant igen. The molecules compr ising t he T cell
r ecept or and t he genes t hat code for t hem have been char act er ized.
The T cell r ecept or is a het er odimer composed of 2 chains, each of molecular weight 40-
50,000 dalt ons, linked by a disulphide bond on t he T cell sur face (Figur e 11.4).The t wo chains
ar e t er med α and β chains and ar e int egr al membr ane pr ot eins ext ending 4-5 amino acids int o
t he cyt oplasm. Each chain is folded int o t wo domains, one having a r elat ively const ant st r uct ur e,
much like t he const ant r egion of t he immunoglobulin molecule. The ot her domain exhibit s far
mor e var iabilit y t han t he immunoglobulin molecules and is analogous t o t he immunoglobulin
var iable r egion. It st ands t o r eason, t her efor e, t hat t he var iable r egions ar e st r at egically
placed and const r uct ed t o bind wit h for eign ant igen.
Figure 11.4. The T cell receptor for antibgen.
Immune Response Mechanisms I: B and T lymphocytes 93
Genetic Organization of the T Cell Receptor
A st udy of t he genet ic or ganizat ion of t he T cell r ecept or (TCR) led t o t he under st anding
t hat T cells t oo, ar e capable of r ecognizing an enor mous r ange of ant igens. Separ at e set s of
genes encode t he α and β chains. The genet ic locus cont aining t he α chain genes is pr esent on
chr omosome no:14, while t he genes coding for t he β chain ar e pr esent on t he 7t h chr omosome.
These genet ic loci can be fur t her divided int o r egions similar t o t he gene ar r angement for t he
immunoglobulin molecule (r efer Chapt er 6). The r egions have been named: t he var iable r egion
genes (V), diver sit y segment genes (D), joining r egion r egions (J ) and const ant r egion r egions
(C). Each gene r egion is compr ised of gene clust er s, for example, t her e ar e 60 V r egion genes
for t he α chain and 21 for t he β chain. Ot her r egions also, ar e highly polymor phic and have
sever al gene clust er s. The polymor phic genet ic or ganizat ion of t he T cell r ecept or is shown in
Table 11.2.
Table 11.2: Genetic organization of the T cell receptor
Gene regions α αα αα chain β ββ ββ chain
V region 60 21
D region – 2
J region 40 12
C region 1 2
Rear r angement s occur bet ween gene segment s, analogous t o t hat descr ibed for t he
immunoglobulin molecule (r efer Chapt er :6). This pr ovides t he diver sit y t o account for t he
exist ence of over a million dist inct T cell clones per individual, each having a differ ent ant igen
specificit y. Ant igen diver sit y is account ed for not only by r ear r angement of gene segment s as
wit h t he immunoglobulin molecule, but also by int er act ions bet ween t he α and β chains of t he
T cell r ecept or molecule. Ther efor e, differ ent ant igen specificit ies ar e cr eat ed when a single α
chain int er act s wit h differ ent β chains.
The CD3 Complex
In all immunocompet ent T cells t he T cell ant igen r ecept or is non covalent ly but int imat ely
linked wit h closely r elat ed molecules called t he CD3 complex. The t hr ee pept ide molecules of
t he CD3 complex have been called CD3/γ CD3/δ and CD3/ε (Figur e: 11.4). Each of t he CD3
molecules is an int egr al par t of t he T cell membr ane and ext ends int o t he cyt oplasm far t her
t han eit her t he α or β chains of t he r ecept or . The CD3 molecules t r ansduce t he ant igen
r ecognit ion signal r eceived by t he α – β het er odimer t o t he inside of t he cell.
Surface CD4 and CD8 Molecules
The CD4 and CD8 molecules play an impor t ant r ole in T cell act ivat ion. Bot h t he CD4 and
CD8 molecules and t he genes t hat encode t hem have been ident ified. Ther e is no evidence of
polymor phism or gene r ear r angement in t he case of t hese molecules and t hey appear ident ical
in cells having differ ent ant igen specificit ies. Ther efor e t hey do not seem t o par t icipat e in
ant igen r ecognit ion.
94 Immunology– Introductory Textbook
The CD4 and CD8 molecules have been shown t o funct ion as r ecognit ion mar ker s for t he
MHC gene pr oduct s. The pr esence of CD4 on T cells indicat es t hat t he T cell is pr ogr ammed t o
r ecognize and int er act wit h cells bear ing MHC class II molecules; while T cells bear ing CD8
molecules r ecognize cells displaying MHC Class I molecules as par t of t heir ant igen specificit y.
It has been hypot hesized t hat t he CD4 and CD8 molecules ar e not involved in act ivat ion of T
cells, but can incr ease int er act ion bet ween T cells and t ar get cells. They seem t o behave like
ext r a “glue”, so t hat act ivat ion can occur . For cells seeing ant igen for t he fir st t ime t his “glue”
may play an impor t ant r ole. It is also t hought t hat since t hey ar e st r uct ur ally const ant , t hey
play a par t in r ecognizing t he const ant r egions of t he MHC Class I and II molecules. This is
bor ne out by t he finding t hat t he CD4 and CD8 molecules ar e closely associat ed wit h t he T cell
r ecept or and act ually bind t o t he const ant r egion of t he MHC Class I and II molecules, t hus
guiding t he T cell r ecept or ont o t he ant igen (Figur e 12.2, r efer Chapt er 12).
The CD2 Molecule
The CD2 molecule on T lymphocyt es is a t r ansmembr ane sur face glycopr ot ein which
facilit at es cell-cell cont act . The amino t er minal domain of CD2 (domain 1) mediat es it s adhesion
funct ion by binding t o LFA-3 (CD58), anot her cell sur face glycopr ot ein widely expr essed on
var ious cell t ypes including hemat opoiet ic and epit helial cells. Bot h CD2 and CD58 ar e member s
of t he I mmu n oglob u li n ge n e su p e r fa mi ly ( se e b e low). The impor t ance of CD2 funct ion in
t he nor mal human immune r esponse has been well document ed: (i) r ecognit ion involving
helper T cells and ant igen pr esent ing cells; and (ii) for t he cyt olyt ic effect or funct ion of nat ur al
killer (NK) cells and cyt ot oxic T lymphocyt es (CTL).
The Immunoglobulin Gene Superfamily
All t he molecules involved in ant igen r ecognit ion descr ibed so far ar e member s of, what is
now known as, a “gene super family”. These ar e t he heavy and light chains of t he immunoglobulin
molecule, t he α and β chains of t he T cell r ecept or , t he α and β 2 micr oglobulin chain of t he
MHC Class I ant igen and t he α and β chains of t he MHC Class II ant igen (Figur e 11.5). It is
obvious t hat all of t hese molecules have cer t ain common char act er ist ics ; t hey all r ecognize
and bind for eign ant igen and t o do t his t hey ar e equipped wit h var iable r egions as par t of t heir
st r uct ur es. This is consequent t o t he polymor phic gene st r uct ur e and char act er ist ic gene
r ear r angement s seen in all of t hese molecules. It has been found t hat cer t ain sequences (about
110 amino acids long) ar e common t o all t hr ee st r uct ur es. Besides t hese, homologous sequences
have been conser ved ar ound disulphide bonds and in t he char act er ist ic ant ipar allel β pleat ed
st r ands. This char act er ist ic β pleat ed folding of shor t pr ot ein st r et ches is a shar ed feat ur e
bet ween all t hr ee molecules. An addit ional feat ur e, common t o t hese st r uct ur es, ar e t hat
many ar e 2 chain molecules wit h st r ong int er domain non covalent int er act ions. It is believed
t hat all t hr ee molecules evolved fr om a single pr imor dial ancest r al gene. The success of t hese
molecules indicat e t hat t he for ces of evolut ion will make sur e t hat t hey ar e widely exploit ed in
nat ur e. Since molecules unr elat ed t o t he immune syst em also fulfil cr it er ia for inclusion int o
t he “super gene family”, it has been suggest ed t hat r ecognit ion of cell sur face molecules (immune
or ot her wise), is t he pr imar y r ole of t hese molecules.
Immune Response Mechanisms I: B and T lymphocytes 95
Figure 11.5. The Immunoglobulin gene superfamily
Regulatory (Suppressor) T cells
The exist ence of specific suppressor T cells and t he phenomenon of immunologic suppression
has fallen int o disfavour in t he pr esent decade. However , most immunologist s believe t hat
some for m of suppr ession exist s. The r eason for t he suppr essor cell cont r over sy is t hat no one
has been able t o clone a unique suppr essor T cell. Of t he or iginal suppr essor T cells (Ts cells),
t he most pr omising r ecent candidat es have been given ot her names. Examples of t hese ar e:
• Tr cells
• Tr 1 cells
• Th3 cells
Tr Cells
Most CD4+ T cells belong t o eit her t he T
h
1 or T
h
2 subset s. However some 5–10% of t hem
do not . These have been t er med T-r egulat or y (Tr ) cells. Like ot her T cells t heir cell sur face
molecules have been char act er ised. They expr ess a t r ansmembr ane pr ot ein called CD25,
which is t he α chain of t he r ecept or for int er leukin-2 (IL-2). Like ot her T cells, t hey also
expr ess t he α β (alpha-bet a) T-cell r ecept or for ant igen (TCR) and can only be act ivat ed if it
binds t o t he pept ide-class II MHC molecule. If act ivat ed, t hey secr et e lar ge amount s of
i n t er leu k i n 10 (I L-10) and oft en some t r ansfor ming gr owt h fact or -bet a (TGF-β ββ ββ) as well. Bot h
t hese lymphokines ar e power ful i mmu n osu p p r e ssa n t s inhibit ing bot h T
h
1 help for cell-
mediat ed immunit y (including gr aft -ver sus-host disease) and inflammat ion, and T
h
2 help for
ant ibody pr oduct ion, and, possibly, t he act ion of CD8+ cyt olyt ic T lymphocyt es (CTL).
The ant igenic pept ides r ecognized by t heir TCRs t end t o be self-pept ides and per haps t he
major funct ion of Tr cells is t o inhibit ot her T cells fr om mount ing an immune at t ack against
self component s; t hat is, t o pr ot ect t he body against a u t oi mmu n i t y.
Tr1 Cells
Tr 1 cells r esemble Tr cells in sever al ways, alt hough t hey do not expr ess lar ge amount s of
CD25 on t heir sur face. They r equir e IL-10 for t heir for mat ion and once mat ur e, secr et e lar ge
amount s of it . Tr 1 cells ar e abundant in t he int est ine, and t heir chief funct ion may be t o make
96 Immunology– Introductory Textbook
t he host t oler ant t o t he many ant igens t hat ar e par t of it s diet . It is believed t hat pat ient s who
develop Cr ohn’s disease lack t he down r egulat ion char act er ised by IL-10 via t he Tr 1 cells
making t hem int oler ant t o a number of diet ar y and ot her ant igens.
Th3 Cells
Th3 cells ar e also pr evalent in t he int est ine, but unlike Tr 1 cells, t heir main lymphokine
is TGF-β. Also like Tr 1 cells, t hey suppr ess immune r esponses t o ingest ed ant igens. Fur t her
r esear ch is needed t o elabor at e t he r elat ionships bet ween t hese and ot her T cells t hat suppr ess
immune r esponses. This wor k is r elevant for pat ient s who r isk r eject ion of or gan t r ansplant s;
and for t he management of pat ient s wit h aut oimmune disor der s like lupus er yt hemat osus,
insulin-dependent diabet es mellit us (IDDM-t ype I diabet es) and Cr ohn’s disease.
™™™
IMMUNE RESPONSE MECHANISMS II:
ANTIGEN PRESENTATION AND PROCESSING;
MECHANISMS OF LYMPHOCYTE ACTIVATION
CHAPTER – 12
W
hen cells of t he immune syst em encount er an ant igen, a humor al immune r esponse
or cellular immune r esponse, or bot h may occur . Humor al immunit y is mediat ed by
B cells, which aft er st imulat ion pr olifer at e and differ ent iat e int o ant ibody pr oducing plasma
cells. Cellular immunit y is mediat ed by T cells, which become act ivat ed t o secr et e a number of
subst ances impor t ant in t he immune r esponse; t hey also kill vir us infect ed and malignant cells
dir ect ly, fulfilling t he r ole of T cyt ot oxic lymphocyt e(Tc/CTL) or killer T cells. Enhancing and
supplement ing t he act ivit y of B cells, ar e anot her set of T cells. These T cells help B cells t o
pr olifer at e and secr et e ant ibodies and ar e popular ly known as T helper (Th) cells. It is t her efor e
obvious t hat T cells and B cells need t o communicat e and int er act wit h each ot her and wit h
ot her ant igen pr esent ing cells. This t hey do t hr ough r ecept or s, soluble fact or s and var ious cell
adhesion molecules.
Antigen Presentation
Ver y ear ly st udies have shown t hat T cells and B cells r ecognize differ ent par t s of an
ant igen. If a hapt en is coupled wit h a car r ier par t icle t o yield an immunogen, B cells r eact wit h
t he hapt en t o pr oduce hapt en specific ant ibodies wher eas T cells r espond pr edominant ly t o
par t s of t he car r ier molecule. Pr esent at ion of ant igen t o T cells differ s fr om pr esent at ion of
ant igen t o B cells. B cells can r ecognize nat ive, unmodified ant igen and t his ant igen need not
be pr esent ed in conjunct ion wit h cellular MHC ant igens. Though most B cells do r equir e T cell
help for an adequat e ant ibody r esponse and for memor y cells t o be gener at ed.
In cont r ast , T cells do not r ecognize nat ive ant igen; ant igen must fir st be p r oce sse d by
an ant igen pr esent ing cell and t hen p r e se n t e d in associat ion wit h MHC Class I or Class II
molecules. In ot her wor ds, T cells ar e blind t o nat ive ant igen and will “see” ant igen only when
pr ocessed and pr esent ed t oget her wit h an MHC molecule. To r eit er at e a gener al r ule of t humb
t hat has been discussed in pr evious chapt er s: t he CD8+ T cell r ecognizes ant igen in associat ion
wit h an MHC Class I molecule and a CD4+ T cell r ecognizes ant igen in conjunct ion wit h an
MHC Class II molecule. Mor e oft en t han not , CD4+ T cells act as helper cells and CD8+ T cells
act as cyt ot oxic cells, t hough t his is not a r igid r ule as we shall see in t he following sect ions.
The Pathways of Antigen Processing
Macr ophages wer e t he fir st accessor y or ant igen pr esent ing cells t o be ident ified. Lat er
dendr it ic cells (specialized cells found in t he lymph nodes and spleen) and B cells t hemselves
wer e added t o t he pr incipal list of pot ent ial ant igen pr esent ing cells. Sever al ant igen pr esent ing
cells (APCs) do mor e t han simply capt ur e ant igen and display it on t heir sur face, t hey br eak
98
Immunology-Introductory Textbook
down antigen into peptide fragments before presenting it, obscuring its shape and leaving only
its distinctive amino acid sequence as the target. a process that requires both time and energy.
This distinctive target peptide fragment processed within the APe, is then transported to the
l'iurface of the cell and displayed in the antigen binding cleft or groove thatis an integral part of
both MHC Class I and Clagg II mulecules (Refer chapter 10). This explains why investigators
found that T cells have no interest in native antigen shape.
A scheme that describes the antigen processing pathway takes into account all of the
above data and is illustrated in Figure 12.1. Pathway A, describes the events that take place
when the antigen concerned is freely circulating foreign material (not found intra cellular or
bound to cell surfaces). Such an antigen has been termed "exogenous" antigen and is taken up
by specific classes of APes such as B cells, macrophages or dendritic cens that are specialized
for processing exogenous antigen. The uptake of these antigens is either by receptor mediated
endocytosis (which may be purely fortuitous), or as a result of simple pinocytosis. The antigen
now lies in an endosome within the cytosol of the cell. In the endosome, under the influence of
an acid pH milieu, cathepsin - like proteases denature or fragment the original protein molecule.
The peptide fragments then bind within the cleft of the MHC Class II molecules which are also
found in the cytosol of the cell. The MHC Class II - antigen complex is then transported to the
cell surface in vesicles and displayed to the exterior. Such an MHC Class II-antigen complex is
recognized preferentially by the CD4+ T cell- and T cell help for antibody production is on its
way! (Discussed in the fonowing section). The remaining peptides of the original protein antigen
go into a lysosomal compartment for complete degradation.
Endoll"nQus
p",,,,,,nled In .noci.lion
""Ill MHC clu. I Ml11&en
..,Iij..,
p,'OOente<l "" surf"",e
In ... o<:i"l"", Wllh
MHC clUI II
-
-
·A·

/ Bind. witri
MHCclUl1I
In sol
Excl'l!l"IOUS
Endcae""'-" anlliM
Figure 12.1 Pathways of antigen procening.
For those proteins that are borne as a result of intracellular (such as viral) infection
or malignant change on a cell the pathway of processing is different (Pathway B, Figure
12.1). These antigens are known 8S "endogenous" antigens, being internal to the body's own
cells. Processing of '""endogenous" antigens occurs in the Golgi complex, an area specialized for
dealing with improperly folded proteins synthesized within the cell (Figure 12.1). Such internally
synthesized proteins need to be presented to the second major class of T cells, the cytotoxic
T cells. Endogenous antigen is denatured or fragmented in the Golgi compartment, where they
come into contact with MHC Class I molecules. The target peptide fragment binds into the
Immune Response Mechanisms II: Antigen Presentation ........... 99
ant igen binding gr oove on MHC Class I molecules and is t r anspor t ed t o t he ext er ior of t he cell
membr ane. Such a pept ide fr agment bound t o t he MHC Class I molecule is pr efer ent ially
r ecognized by t he cyt ot oxic T cell - and t he killer cyt ot oxic T cell has, t her efor e, been t ar get ed
ont o t he aber r ant cell.
However , some pr ot eins encoded by t he genes of an infect ing vir us ar e synt hesized in t he
cyt osol . How does t he cell get t hem int o t he Golgi compar t ment ? This is achieved by specialist
t r anspor t pr ot eins called TAP (t r anspor t er associat ed wit h ant igen pr ocessing) pr ot eins. Vir al
pr ot eins in t he cyt osol ar e degr aded by pr ot easomes int o vir al pept ides. The pept ides ar e picked
up by TAP pr ot eins embedded in t he membr ane of t he endoplasmic r et iculum. Using t he ener gy
of ATP, t he pept ides ar e pumped int o t he lumen of t he endoplasmic r et iculum wher e t hey
assemble wit h MHC class I molecule as descr ibed above. This complex t hen moves t hr ough t he
Golgi appar at us and is t r anspor t ed t o t he ext er ior of t he cell membr ane. Such a pept ide fr agment
bound t o t he MHC Class I molecule is also pr efer ent ially r ecognized by t he cyt ot oxic T cell.
Alain Townsend in 1985, showed conclusively t hat fr agment s of int er nal ant igen, t he vir al
nucleopr ot ein, wer e t he ant igens t hat wer e “seen” by cyt ot oxic T cells and t hese int er nal
ant igens init iat ed T cell cyt ot oxicit y against vir ally infect ed cells. He had exper iment al evidence
t o show t hat whole sur face ant igens, t her efor e, could not be r ecognized by cyt ot oxic T cells–
unpr ocessed as t hey wer e. Fr agment s of vir al genes (t he int er nal, nucleopr ot ein ant igens),
aft er being degr aded and pr ocessed wer e displayed in a suit able manner - fit t ed int o t he MHC
Class I-pept ide binding gr oove. The ant igen was t hen r ecognizable by killer T cells. These
pioneer ing exper iment s helped evolve t he cur r ent t hinking r egar ding killing of vir ally infect ed
or malignant cells by cyt ot oxic T cells. Cr edit also goes t o Bar uj Benacer r af and Hugh McDevit t
who, in 1960, showed t hat genes of t he MHC affect ed an animal’s abilit y t o mount an immune
r esponse t o cer t ain ant igens; and t o Don C.Wiley (1987), for elucidat ing t he ant igen binding
cleft on t he MHC molecule. The most st r iking feat ur e of his wor k was t he finding t hat t his
ant igen binding cleft was not , and has never been found empt y; in it Wiley and his colleagues
found for eign pept ide fr agment s - and pr esumed r ight ly t hat t his was pr ocessed ant igen.
Select ion of pept ides for sur face expr ession pr obably depends on what fit s most closely
int o t he ant igen binding gr oove of t he MHC Class I and Class II molecules. Because exogenous
ant igen evokes CD4+ T cell help, it pr efer ent ially binds t o MHC Class II ant igens ; wher eas,
endogenous ant igen needs t o at t r act t he at t ent ion of a cyt ot oxic T cell, it pr efer ent ially binds t o
t he MHC Class I molecule: class discr iminat ion in immunology!
Why does T cell recognition have to be so elaborate?
Why do T cells not r ecognize ant igen dir ect ly as B cells do? Why do ant igens need t o be
br oken down and pr esent ed wit h MHC molecules? Alain Townsend and his co-wor ker s, showed
t hat a cell cannot even assemble Class I MHC molecules pr oper ly, unless a pept ide is pr esent
dur ing t he final st ages of t he pr ot ein folding pr ocess. It seems t hat t hese omnipr esent pept ides
ar e fr agment s of t he body’s own pr ot eins and should t he need ar ise t hey ar e displaced by
for eign ant igen pept ides. Ther e ar e sever al r easons put for t h for t his complex chain of event s.
For one, t his scheme is consist ent wit h t he r ole of immune sur veillance t hat T cells play. Killer
T cells ar e known t o const ant ly monit or t he ot her cells of t he body for t he appear ance of
t umour or vir al ant igens and pr ompt ly eliminat e any cell expr essing t hem. By cont inuously
pr ocessing and pr esent ing t heir own ant igens, cells invit e inspect ion by t he immune syst em so
as t o det ect any ear ly aber r at ion. In t he case of vir ally infect ed cells, MHC r est r ict ing guides a
killer T cell t o t he culpr it cell - t he cell t hat is essent ially t he fact or y or manufact ur ing unit for
t housands of vir us par t icles. It ensur es t hat a for eign or ganism cannot elude it by being hidden
100 Immunology– Introductory Textbook
inside a cell and adopt ing a Tr ojan hor se st r at egy. Hidden ant igens can also be pr ocessed and
made visible! If, however , cells wer e t o r ecognize fr eely cir culat ing ant igens, t hey would all be
ut ilised by vir al par t icles r eleased by t he manufact ur ing unit s and not be available t o home in
on r enegade cells har bour ing vir us synt hesis machiner y. For t he equally complex scheme of
ant igen pr esent at ion for helper T cells, t he answer may be in t he t heor y of evolut ion. Cell -
mediat ed immunit y appear s t o be a defence mechanism much older t han man; even pr imit ive
sponges ar e endowed wit h t his abilit y. Hence T cells may have or iginat ed as killer cells and
may have acquir ed helper pr oper t ies lat er in evolut ion. Ther efor e, t he helper r ole seems t o
have super imposed it self int o cells alr eady pr ogr ammed t o r ecognize MHC molecules. Over
t he cour se of evolut ion, t hey have adapt ed t his funct ion t o guide t hem t o a sit e wher e t hey can
be most effect ive t o B cells which is, aft er all, t he pr imar y t ar get of T cell help.
Under st anding t he phenomenon of ant igen pr ocessing has helped invest igat or s manipulat e
t he immune syst em for clinical pur poses. Tr adit ionally, vaccines have consist ed of whole
pat hogenic or ganisms, live or killed or a pr ot ein ext r act such as a t oxin. For some diseases
such as malar ia t his is not feasible, besides whole cell vaccines may have r isky side effect s.
Vaccine scient ist s ar e now t r ying t o design synt het ic pept ides r epr esent ing a small fr act ion of
t he act ual ant igen, t o t r igger an immune r esponse. To do so t hese pept ides must st imulat e
bot h helper and cyt ot oxic T cells and bind t o MHC molecules. MHC based t echnology should
also make it possible t o develop compounds t hat bind ver y st r ongly t o t hose MHC molecules
t hat bind self ant igens and cause disease, as in aut oimmune r elat ed disease st at es. By blocking
t he binding of self ant igens on MHC molecules, such compounds might suppr ess t he aut oimmune
r esponse. Hence, some of t he immune syst em’s own pr ecision and power can be dir ect ed t o t he
fight against disease.
Mechanisms of Lymphocyte Activation
T cell activation
The fir st event t hat t r igger s T cell act ivat ion is T cell binding t o ant igen pr esent ed in
conjunct ion wit h MHC molecules. The ant igen binding cleft on MHC molecules shows var iabilit y,
t hus suppor t ing t he t heor y t hat t hey wer e made t o bind for eign pept ides. The T cell r ecept or
/CD3 complex, t her efor e int er act s wit h for eign pept ide. Simult aneously t he r egions of t he
MHC molecule t hat display t his ant igen, on t he sur face of t he APC int er act wit h t he appr opr iat e
CD4 or CD8 molecule on t he sur face of t he T cell (Figur e 12.2a/b).
Fig. 12.2a. The MHC class I–antigen-T8 cell
receptor interaction.
Fig. 12.2b. The MHC class II–antigen-T4
cell receptor interaction.
TCR TCR
CD8 CD4
MHC-I wi th
bound pepti de
MHC-II with
bound pepti de
Ant igen-presenti ng
cell
APC Nai ve
T8–lymphocyte
T4–lymphocyte
Immune Response Mechanisms II: Antigen Presentation ........... 101
This int er act ion t hen gener at es a ser ies of biochemical r eact ions which ar e aimed at
accomplishing t wo impor t ant event s : (i) t he secr et ion of int er leukin–2 (IL–2), by t he T cell and
(ii) t he expr ession of r ecept or s for IL–2 on t he
T cell sur face. The init ial int er act ion bet ween
T cel l r ecept or a n d a n t i gen s t i mu l a t es
membr ane bound phospholipase C t o hydr olyze
membr ane bound inosit ol (Figur e 12.3). This
r esult s in t he gener at ion of t wo biologically
act ive met abolit es: inosit ol t r iphosphat e (IP3)
and diacylglycer ol. The inosit ol t r iphosphat e
incr ea ses cyt opla smic fr ee ca lcium a nd t he
dia cylglycer ol a ct iva t es t he enzyme pr ot ein
kinase C. These t wo met abolit es, ar e r esponsible
for act ivat ing T cells. An act ivat ed T cell is t hen
able t o t r anscr ibe, t r anslat e and expr ess t wo
impor t ant gene pr oduct s: t he IL–2 molecule and
t he IL–2 r ecept or . Thus IL–2 is secr et ed and
t h e I L–2 r ecept or is expr essed on t h e cell
sur face. When IL–2 and it s r ecept or int er act on
t he cell membr ane, T cell pr olifer at ion occur s
(Figur e 12.4). Act ivat ed T cells secr et e sever al ot her lymphokines (discussed in Chapt er 13),
including int er fer on γ, which help r egulat e t he T cell cycle by t he int er leukin - int er fer on
cir cuit .
Figure 12.4. T cell proliferation.
Figure 12.3. Biochemical events in T cell activation
102 Immunology– Introductory Textbook
Co-stimulatory signals
In addit ion, cer t ain cell adhesion molecules have been ident ified t hat ar e necessar y for
efficient int er act ion bet ween T lymphocyt es and accessor y cells or t ar get cells. These co-
st imulat or y signals involve t he int er act ion of accessor y molecules on t he APC wit h t heir
cor r esponding ligands on t he T lymphocyt es (Figur e 12.5) These co-st imulat or y molecules ar e
only synt hesized when t oll-like r ecept or s on APCs bind t o pat hogen-associat ed molecular
pat t er ns of micr obes (Refer Chapt er 2: Innat e immunit y). Cell adhesion molecules and t heir
ligands ar e pr esent ed in Table 12.1.
Table 12.1: Cell adhesion molecules (on the APC) and their ligands on T cells
Dendritic cell or macrophage T lymphocytes
MHC I-peptide TCR/CD8
MHC II - peptide TCR/CD4
ICAM-1 LFA-1
B 7-1 and B7-2 CD28
LFA-3 CD2
The impor t ance of lymphocyt e funct ion associat ed (LFA – 1) molecule is evidenced by
exper iment s wit h monoclonal ant ibodies which block LFA - 1. This leads t o inhibit ion of cyt ot oxic
T cell act ivit y, nat ur al killer cell (NK) act ivit y and ant ibody dependant cellular cyt ot oxicit y
(ADCC). Pat ient s deficient in t his fact or suffer life t hr eat ening bact er ial and fungal infect ions.
The pr ot ein which is believed t o be t he ligand for LFA-1 is t he int er cellular adhesion molecule–
1 (ICAM–1). Ant ibodies t hat block ICAM–1 pr oduce t he same effect s as blockage of LFA–1.
Var ious cyt okines like IL–1 and IFN γ incr ease ICAM expr ession on t he B cell sur face. The CD2
ligand is t he LFA–3 molecule. Blockage of t hese
molecules also decr eases T cell killing act ivit y
by blocking T cell–t ar get cell adhesion. The CD4
a n d CD8 l i ga n d a s s oci a t i on s wi t h t h ei r
cor r esponding MHC molecules have alr eady
been descr ibed in pr evious sect ions.
Molecular int er act ions bet ween t he APC
and t he T8 - lymphocyt e pr oduce signals and
cyt okin es for a ct iva t ion of t h e n a ive T8–
l ymph ocyt e. On ce a ct i va t ed, s i gn a l s a n d
cyt ok i n es fr om effect or T4–l ymph ocyt es ,
pr imar ily T
h
1 cells, will t hen be able t o act ivat e
t he T8–lymphocyt e; t ur n on genes r esponsible
for t he pr olifer at ion and differ ent iat ion of t hat
T8-l ymph ocyt e i n t o a n effect or cel l : t h e
funct ioning cyt ot oxic T–lymphocyt e (CTL).
Figure 12.5 Co-stimulatory molecules that enhance
Tcell–APC interaction.
LFA-3
B7
MHC-II MHC-class I/ II
with bound pept ide
CD40
ICAM-1
APC*
* APCs can also be B cells bear ing ant igen
bound t o sur face lg (slg)
CD2
CD28
TCR
CD4/ CD8
CD40L
LFA-1
Act ivat ion of naive
T–lymphocyt e
Immune Response Mechanisms II: Antigen Presentation ........... 103
Interleukin - Interferon circuit
The int er leukin–int er fer on cir cuit is gr aphically depict ed in Figur e 12.6. Ant igens ar e
pr ocessed and pr esent ed t o T cells by ant igen pr esent ing cells such as macr ophages. This shift s
t he r est ing T lymphocyt es fr om t he G0 st at e t o t he ear ly G1 phase of t he cycle. G1 cells
synt hesize r ecept or inducing fact or s and ot her pr ot eins. Under t he influence of IL-1 fr om t he
accessor y cells, some of t he ear ly G1 cells pr oceed int o t he lat e G1 phase of t he cell cycle,
wher e IL–2 pr omot es expr ession of IL–2 r ecept or s on lat e G1 cells. Ot her lymphocyt es moving
in a similar cell cycle ar e st imulat ed by IL–1 t o secr et e IL–2. IL–2 in t ur n st imulat es IL–2
r ecept or bear ing cells t o pr olifer at e or act ively r eplicat e and ent er what is known as t he GS
(synt hesis) phase in T cell act ivat ion. Act ively r eplicat ing T lymphocyt es secr et e int er fer on γ
(IFN γ). IFN γ has a posit ive feed back effect on IL–1 pr oduct ion, ant igen pr ocessing and
expr ession of MHC Class II molecules, t hus pr omot ing accessor y cell funct ions r esult ing in a
cyclical incr ease of act ivat ed T cells pr edominant ly of t he T
h
1 subset .
Figure 12.6. The Inter leukin–Interferon circuit.
Activation of B Cells
It is now evident , t hat for many B cells t o make ant ibody t o a par t icular ant igen, T cell
help is essent ial. Such ant igens ar e also called T d e p e n d a n t ant igens. Ther e ar e ot her s,
not ably t he capsule of t he or ganism S treptococcus pneumoniae which ar e T-i n d e p e n d e n t
ant igens and can evoke ant ibody pr oduct ion fr om B cells wit hout T cell help.
Activation in response to T- dependant antigens
When T cell help is t o be r ender ed, it has been shown t hat T cells have fir st t o be act ivat ed
by a separ at e pr ocess which does not involve B cells, as we have just seen in t he pr evious
sect ion. Pioneer ing exper iment s by A. Mit chison in 1971, showed t hat , for helper T cells and B
104 Immunology– Introductory Textbook
cells t o collabor at e, t hey must , each in t ur n, r ecognize par t s of t he same ant igen molecule.
This, and ot her exper iment s have pr ovided compelling evidence t hat t he act ivat ed helper T cell
needs t o be physically close t o t he B cell.
The fir st signal for t he act ivat ion of a naive B-lymphocyt e occur s when B-cell r ecept or s,
t he sur face immunolobulin (slg), on t he sur face of t he B-lymphocyt e bind epit opes of T-dependant
ant igens having a cor r esponding shape. A second signal is also needed for t he act ivat ion of t he
naive B-lymphocyt e. This is pr ovided when a component of t he complement syst em , C3b binds
t o t he micr obial sur face. C3b is subsequent ly degr aded t o C3d which, in t ur n, binds t o a
complement r ecept or called CR2 on t he sur face of t he B-lymphocyt e (Figur e 12.7a). Act ivat ion
of t he naive B cell t r igger s a ser ies of met abolic event s in t he B cell, similar t o what has been
discussed for t he T cell; t he r esult is gene act ivat ion and incr eased pr oduct ion of MHC Class II
molecules, co-st imulat ory subst ances such as B7 and CD40 and recept ors for IL-2 and int erleukins
4 t o 6 ( IL-4, IL-5, IL-6); r equir ed for B cell gr owt h and differ ent iat ion.
Figure 12.7a. Activation of the naive B lymphocyte.
Figure 12.7b. T cell-B cell interaction.
B-lymphocyte
C3d
2
1
Receptor
for C3d
(CR2)
B-cell
receptor (sig)*
* slg = Surface Immunogl obuli n
Immune Response Mechanisms II: Antigen Presentation ........... 105
Ant igens bound t o sur face immunoglobulins on t he B cell ar e int er nalized and degr aded
t o oligopept ides which bind t o MHC Class II pr ot eins wit hin t he cell. The ant igen pept ide–
MHC Class II complex t hen goes t o t he cell sur face, wher e it is r ecognized by an ant igen
specific T cell r ecept or on t he act ivat ed helper T cell, pr imar ily t he T
h
2 cell (Figur e 12.7b). The
B cell, having displayed ant igen in conjunct ion wit h t he MHC Class II molecule, has now
moved fr om t he B0 st age t o t he B1 st age in it s cycle (Figur e 12.8). Her e co-st imulat or y molecules
such as CD28 and CD40L on t he T
h
2 cell bind t o B7 and CD40 molecules on t he act ivat ed B
lymphocyt e (Figur e 12.5). As a r esult of t his act ivat ed T helper cell - B cell int er act ion, t he T
h
2
cells pr oduce cyt okines such as IL-4, IL-5, IL-6. Collect ively t hese cyt okines enable act ivat ed B-
lymphocyt es t o proliferat e st imulat e act ivat ed B-lymphocyt es t o synt hesize and secret e ant ibodies
and pr omot e t he differ ent iat ion of B-lymphocyt es int o ant ibody-secr et ing plasma cells. T
h
2
cells also enable B-lymphocyt es t o under go affinit y mat ur at ion t hr ough somat ic hyper mut at ion
and t o swit ch ant ibody classes, t hat is, pr oduce eit her IgG, IgA, or IgE. This allows t he B-
lymphocyt es t o “fine-t une” t he shape of t he ant ibody for bet t er fit wit h t he or iginal epit ope and
t o pr oduce t he class of ant ibody of gr eat est value for t hat sit e.
B cells move int o t he B2 st age of act ivat ion and display r ecept or s for IL-4 and IL-5. When
t he r ecept or s and t he cor r esponding int er leukin molecules int er act , B cells gr ow and pr olifer at e
yielding sever al clones of B3 cells. B3 cells in t ur n display r ecept or s for IL-6 and ot her r elat ed
cyt okines. Int er act ion of t hese cyt okines wit h t heir r ecept or s on t he B cell pushes t he B cell
int o t he final differ ent iat ion st age– t he plasma cell, and ant ibody pr oduct ion ensues. A set of B3
cells however , r emain unst imulat ed by IL-6 and ot her r elat ed cyt okines, albeit wit h t he r ecept or s
displayed. This pool of B3 cells const it ut es t he me mor y ce lls. Subsequent exposur e of B cells
t o a nt igen will yield a much fa st er a nd gr ea t er a nt ibody r esponse since t he t er mina l
differ ent iat ion st ages befor e ant ibody pr oduct ion ar e all set . To go int o pr oduct ion, t hese B
cells only need r enewed fuelling wit h IL-6 and r elat ed cyt okines.
Ot her agent s which induce polyclonal B cell proliferat ion are Epst ein - Barr virus, Chlamydia
trachomatis and lipopolysacchar ides which r eact wit h non immunoglobulin r ecept or s on t he B0
cell.
Figure 12.8. Events involved in B-cell activation.
106 Immunology– Introductory Textbook
Activation in response to T- independent antigens
T-independent ant igens ar e usually lar ge car bohydr at e and lipid molecules wit h mult iple,
r epeat ing subunit s. B-lymphocyt es mount an ant ibody r esponse t o T-independent ant igens
wit hout t he r equir ement of int er act ion wit h T4-lymphocyt es. Bact er ial LPS, fr om t he gr am-
negat ive cell wall, and capsular polysacchar ides (as in S . pneumoniae) ar e examples of T-
independent ant igens. The r esult ing ant ibody molecules ar e gener ally of t he IgM isot ype and
do not give r ise t o a memor y r esponse. Ther e ar e t wo basic t ypes of T-independent ant igens:
TI-1 and TI-2.
TI -1 a n t i ge n s include lipopolysacchar ide (LPS) fr om t he out er membr ane of t he gr am-
negat ive cell wall and bact er ial nucleic acid. These ant igens act ivat e B-lymphocyt es by binding
t o t heir specific t oll-like r ecept or s (see chapt er 2) r at her t han t o B-cell sur face immunoglobulin
r ecept or s. Ant ibody molecules gener at ed against TI-1 ant igens ar e oft en called “nat ur al
ant ibodies” because t hey ar e always being made against bact er ia pr esent in t he body.
TI -2 a n t i ge n s , such as capsular polysacchar ides, ar e molecules wit h mult iple, r epeat ing
subunit s. These r epeat ing subunit s act ivat e B-lymphocyt es by simult aneously cr oss-linking a
number of B-cell r ecept or s.
™™™
CYTOKINES
CHAPTER – 13
T
he cour se of immunologic and inflammat or y pat hways is mediat ed by sever al hor mone
like soluble subst ances t hat ar e secr et ed by t he concer ned cell t ypes. These subst ances
have been given a gener al t er m: t he cyt ok i n e. Those secr et ed by lymphocyt es ar e called
lymp h ok i n e s , t hose pr oduced by monocyt es or macr ophages ar e called mon ok i n e s .
Cyt okines ar e low molecular weight , soluble pr ot eins t hat ar e pr oduced in r esponse t o an
ant igen and funct ion as chemical messenger s for r egulat ing t he innat e and adapt ive immune
syst ems. They ar e pr oduced by vir t ually all cells involved in innat e and adapt ive immunit y, but
especially by T helper lymphocyt es. The act ivat ion of cyt okine-pr oducing cells t r igger s t hem t o
synt hesize and secr et e t heir cyt okines. The cyt okines, in t ur n, ar e t hen able t o bind t o specific
cyt okine r ecept or s on ot her cells of t he immune syst em and influence t heir act ivit y: t his includes
bot h enhancing and suppr essing r esponses.
Cyt okines maybe char act er ized as p le i ot r op i c, r e d u n d a n t or mu lt i fu n ct i on a l.
• Pleiot r opic cyt okines can act on a number of differ ent t ypes of cells r at her t han a
single cell t ype.
• Redundant defines t he abilit y of a number of differ ent cyt okines t o car r y out t he
same funct ion.
• Mult ifunct ional cyt okines ar e able t o r egulat e a number of differ ent funct ions.
Cyt okines ar e oft en pr oduced in a cascade, as one cyt okine st imulat es it s t ar get cells t o
make addit ional cyt okines. Cyt okines can act syner gist ically or ant agonist ically.
Th e r e a r e t hr ee funct ional cat egor ies of cyt okines depending on whet her t hey
(a) Regulat e innat e immune r esponses
(b) Influence adapt ive immune r esponses
(c) St imulat e haemat opoiesis
The descr ipt ion of cyt okines below is not int ended t o be compr ehensive nor complet e; it
pr ovides a snapshot of some of t he mor e commonly known fact or s and t heir pr incipal act ivit ies.
Mor e new cyt okines and t heir r ecept or s ar e cont inuously being discover ed. A br ief descr ipt ion
of t he impor t ant int er leukins and int er fer ons is given in Table 13.1
A. Cytokines that Regulate Innate Immune Responses
Cyt okines t ha t r egula t e inna t e immunit y (see Cha pt er 2) a r e pr oduced ma inly by
macr ophages and dendr it ic cells alt hough t hey can also be pr oduced by T-lymphocyt es, NK
cells, and ot her cells. They ar e pr oduced pr imar ily in r esponse t o pat hogen-associat ed molecular
pat t er ns (PAMPS) such as lipopolysacchar ide (LPS), pept idoglycan monomer s, t eichoic acids,
and double-st r anded DNA. Most act on leukocyt es and t he endot helial cells t hat for m blood
vessels in or der t o pr omot e and cont r ol ear ly inflammat or y r esponses.
108 Immunology– Introductory Textbook
Tumor necrosis factor (TNF)
TNF-alpha is a key cyt okine in t he mediat ion of acut e inflammat ion. In excessive amount s
it is t he pr incipal cause of syst emic complicat ions such as t he shock cascade. Funct ions include
act ing on endot helial cells t o st imulat e inflammat ion and t he coagulat ion pat hway; st imulat ing
endot helial cells t o pr oduce select ins and ligands for leukocyt e int egr ins dur ing diapedesis;
st imulat ing endot helial cells and macr ophages t o pr oduce chemokines t hat cont r ibut e t o
diapedesis, chemot axis and t he r ecr uit ment of leukocyt es; st imulat ing macr ophages t o secr et e
int er leukin-1 (IL-1); act ivat ing neut r ophils and pr omot ing ext r acellular killing by neut r ophils;
st imulat ing t he liver t o pr oduce acut e phase pr ot eins; and act ing on muscles and fat t o st imulat e
cat abolism for ener gy conver sion. In addit ion, TNF is cyt ot oxic for some t umour cells; it int er act s
wit h t he hypot halamus t o induce fever and sleep; st imulat es t he synt hesis of collagen and
collagenase for scar t issue for mat ion; and act ivat es macr ophages. TNF is pr oduced mainly by
monocyt es, macr ophages, dendr it ic cells and T
h
1 cells.
Interleukin-1
The major cell sour ces of IL-1 ar e monocyt es and macr ophages, t hough sever al ot her cell
t ypes pr oduce int er leukin - like subst ances. Rest ing macr ophages and cir culat ing monocyt es
pr oduce lit t le IL-1. When macr ophages ar e st imulat ed, IL-1 act ivit y incr eases mar kedly.
St imulant s of IL-1 ar e par t icles like silica and adjuvant s such as LPS or mur amyl dipept ide
(MDP). All t he agent s t hat act ivat e T-lymphocyt es dir ect ly, such as ant igen in conjunct ion wit h
MHC molecules also st imulat e IL-1 r elease. T and B cell pr oduct s such as int er fer on γ, colony
st imulat ing fact or and ant igen - ant ibody complexes ar e pot ent st imulat or s of IL-1.
Int er leukin-1 r elease is blocked by dr ugs such as hydr ocor t isone and cyclospor in, pr obably
account ing for t he ant i inflammat ory and immunosuppressive propert ies of t hese drugs. Excessive
doses of t he macr ophage st imulat or s can have an inhibit or y effect on IL-1. Pr ost aglandin E2
has been shown t o have a negat ive effect on IL-1 pr oduct ion.
Biological properties of IL-1
(i) On T cell function
IL-1 augment s t he pr olifer at ion of medullar y t hymocyt es and incr eases t he pr oduct ion of
lymphokines in per ipher al T cells. It incr eases T lymphocyt e pr oduct ion of colony st imulat ing
fact or and ot her chemot act ic fact or s. Hence less differ ent iat ed T cells pr olifer at e and mor e
differ ent iat ed T cells display incr eased secr et or y act ivit y. IL-1 incr eases t he accessibilit y of t he
ant igen binding r ecept or and augment s ant igen st imulat ed CD4+ and CD8+ T cells t o pr oduce
IL-2. It t her efor e incr eases t he t umor icidal and bact er icidal funct ions of T cells.
IL-1 in conjunct ion wit h IL-2 or int er fer on, enhances non specific n a t u r a l k i lle r ce ll
act ivit y of lar ge gr anular lymphocyt es which in t ur n have t umor icidal act ivit y.
(ii) On B cell function
IL-1 augment s pr olifer at ion, differ ent iat ion and ant ibody pr oducing funct ions of B
lymphocyt es. It also induces expr ession of sur face bound immunoglobulin r ecept or s. IL-1
pr omot es B cell ant ibody pr oduct ion dir ect ly and by augment ing T cell helper funct ion.
(iii) On non-lymphocytic cells
IL-1 pr omot es t he gr owt h and funct ion of non lymphoid cells as well. IL-1 can act as an
endogenous pyr ogen, pr obably by st imulat ing pr ost aglandin pr oduct ion. This could explain t he
Cytokines 109
muscle wast ing and cachexia of chr onic febr ile illness. IL-1 st imulat es hepat ocyt es t o pr oduce
acut e phase pr ot eins such as fibr inogen,C- r eact ive pr ot ein, caer uloplasmin and α1 ant it r ypsin.
On inflammat or y cells, IL-1 causes neut r ophilia, is chemot act ic and induces r elease of
lysozyme and lact ofer r in. It st imulat es t he pr oduct ion of super oxide anion via t he hexose
monophosphat e (HMP) st unt . IL-1 incr eases bone r esor pt ion and collagen synt hesis and is
impor t ant in fibr osis and wound healing.
Cer t ain non macr ophage cells pr oduce IL-1 like subst ances. They ar e ker at inocyt es,
epit helial cells fr om t he or al mucosa and cor nea, ast r ocyt es fr om t he br ain and mesangial cells
fr om t he kidney.
Chemokines
Chemokines ar e a gr oup of cyt okines t hat enable t he migr at ion of leukocyt es fr om t he
blood t o t he t issues at t he sit e of inflammat ion. They incr ease t he affinit y of int egr ins on
leukocyt es for ligands on t he vascular wall dur ing diapedesis, r egulat e t he polymer izat ion and
depolymer iza t ion of a ct in in leukocyt es for movement a nd migr a t ion, a nd funct ion a s
chemoat t r act ant s for leukocyt es. In addit ion, t hey t r igger some whit e blood cells (WBCs) t o
r elease t heir killing enzymes for ext r acellular killing and induce some WBCs t o ingest t he
r emains of damaged t issue. Chemokines also r egulat e t he movement of B-lymphocyt es, T-
lymphocyt es, and dendr it ic cells t hr ough t he lymph nodes and t he spleen. Cer t ain chemokines
have also been shown t o suppr ess HIV, pr obably by binding t o chemokine r ecept or s (e.g. CCR5)
ser ving as t he second binding fact or for HIV on CD4
+
cells. When pr oduced in excess amount s,
chemokines can damage healt hy t issue as seen in r heumat oid ar t hr it is, pneumonia, ast hma,
adult r espir at or y dist r ess syndr ome (ARDS), and sept ic shock. Examples of chemokines include
IL-8, macr ophage act ivat ing fact or s: macr ophage inflammat or y pr ot ein (MIP-1a), MIP-1b;
monocyt e chemoat t r act ant pr ot ein (MCP-1), MCP-2, MCP-3, gr owt h r elat ed oncogene (GRO-a),
GRO-b, GRO-g, and t he molecule ‘r egulat ed on act ivat ion-nor mal T cell expr essed and secr et ed’
(RANTES). Chemokines ar e pr oduced by many cells including leukocyt es, endot helial cells,
epit helial cells and fibr oblast s.
Interleukin-12 (IL-12)
IL-12 is a pr imar y mediat or of ear ly innat e immune r esponses t o int r acellular micr obes.
It induces cell-mediat ed immunit y. It funct ions as a st imulant for t he synt hesis of int er fer on-
gamma by T-lymphocyt es and NK cells; incr eases t he killing act ivit y of CTLs and NK cells; and
st imulat es t he differ ent iat ion of naive T4-lymphocyt es int o int er fer on-gamma pr oducing T
h
1
cells. It is pr oduced mainly by macr ophages and dendr it ic cells.
Type I Interferons
Or iginally discover ed in 1957 by Isaacs and Lindenmann, int er fer ons wer e found t o consist
of a family of pr ot eins which shar e a unique ant ivir al pr oper t y. These same pr ot eins also exer t
non vir al funct ions such as t hose of an ant ipr olifer at ive and immunor egulat or y agent .
Types of Interferons
Int er fer ons can be classified accor ding t o t heir pr imar y cell or or igin
• int er fer on α fr om leukocyt es,
• int er fer on β fr om fibr oblast s,
• int er fer on γ fr om T lymphocyt es.
110 Immunology– Introductory Textbook
They can also be classified as t ype I and t ype II accor ding t o t he agent s t hat st imulat e
t heir r elease. Type I int er fer ons (IFN α and IFN β) ar e induced by vir al infect ion or ar t ificially
by double st r anded RNA (ds RNA). Lymphoblast oid cells pr oduce IFN α and β. Bot h α and β
int er fer ons ar e acid st able. Type II int er fer on (discussed lat er ), also known as IFN γ or immune
int er fer on, is st imulat ed by for eign ant igen or mit ogen. The genes coding for t he t hr ee int er fer ons
have been cloned and sequenced.
I n t e r fe r on α αα αα is pr oduced when leukocyt es and lymphocyt es ar e exposed t o vir uses. It
consist s of mult iple sub species t hat ar e ant igenically r elat ed. By r ecombinant DNA t echnology
it has been shown t hat mor e t han 20 dist inct int er fer on α genes and polypept ides exist .
In t er fer on β ββ ββ is ant igenically dist inct and is t he predominant species elaborat ed by fibroblast
cells. Epit helial cells and macr ophages also secr et e int er fer on β. Ther e ar e sever al t ypes of
IFN β species.
Mechanisms of action of the interferons
Int er fer ons act by binding t o specific r ecept or s on t he cell sur face. Subsequent t o binding,
t he int er fer on - r ecept or complex is int er nalized by r ecept or mediat ed endocyt osis. Type I
int er fer ons, pr oduced by vir t ually any vir us-infect ed cell, pr ovides an ear ly innat e immune
r esponse against vir uses. Int er fer ons induce uninfect ed cells t o pr oduce enzymes capable of
degr ading mRNA. These enzymes r emain inact ive unt il t he uninfect ed cell becomes infect ed
wit h a vir us. At t his point , t he enzymes ar e act ivat ed and begin t o degr ade bot h vir al and
cellular mRNA. This not only blocks vir al pr ot ein synt hesis, it also event ually kills t he infect ed
cell. They also pr omot e body defences by enhancing CTL, macr ophage, dendr it ic cell, NK cell
and ant ibody-pr oducing cell act ivit y.
Type I int er fer ons also induce MHC-I ant igen expr ession needed for r ecognit ion of ant igens
by CTLs; augment macr ophage, NK cell, CTL, and B-lymphocyt e act ivit y; and induce fever .
I nt er fer on-a lpha is pr oduced by T-lymphocyt es, B-lymphocyt es, NK cells, monocyt es/
macr ophages; int er fer on-bet a by vir us-infect ed cells, fibr oblast s, macr ophages, epit helial cells
and endot helial cells.
Immunoregulatory effects of Type I interferons
The immunor egulat or y effect s of int er fer ons in gener al ar e exer t ed on cells t hat car r y
out t he host s’ defence st r at egy - t he macr ophages, T and B cells and lar ge gr anular lymphocyt es
wit h nat ur al killer cell act ivit y, NK cells.
(a) On macrophages
Int er fer ons incr ease t he bact er icidal and t umor icidal capabilit ies of macr ophages and
augment t heir accessor y funct ion of ant igen pr esent at ion. The act ivat ion of macr ophages by
IFN α, β or γ is accompanied by incr eased expr ession of r ecept or s for t he Fc por t ion of t he
immunoglobulins (FcR). This incr eased expr ession of FcR pr omot es bot h incr eased phagocyt osis
of immune complexes and t he incr eased capacit y of macr ophages t o lyse ant ibody coat ed bact er ia,
vir uses, par asit es and t umour cells. This phenomenon is known as ant ibody dependant cellular
cyt ot oxicit y (ADCC).
All t hr ee int er fer ons have been r epor t ed t o eit her incr ease or decr ease synt hesis and
secr et ion of mult iple pr ot eolyt ic enzymes by macr ophages, depending on t he t ype of st imulus.
This endows t he host wit h t he capacit y t o dest r oy or det oxify invading agent s. By incr easing
t he int r acellular pr ot eolyt ic enzymes, ant igen pr ocessing also get s enhanced, as t his r equir es
pr ot eolyt ic cleavage of whole ant igens.
Cytokines 111
(b) On large granular lymphocytes and natural killer activity
Nat ur al killer (NK) act ivit y is a t er m used t o descr ibe cyt ot oxic act ivit y of lar ge gr anular
lymphocyt es wit hout pr ior sensit izat ion wit h specific ant igen. NK act ivit y is pr imar ily dir ect ed
against vir us infect ed cells, cer t ain t umour cell lines and nor mal effet e haemopoiet ic cells. All
t hr ee int er fer ons enhance NK cell act ivit y in lar ge gr anular lymphocyt es. Ther e ar e sever al
t heor ies r egar ding t he mechanisms by which t hese cells become mor e cyt ocidal under t he
influence of int er fer on. These include: incr eased expr ession of r ecognit ion st r uct ur es on t ar get
cells, incr eased binding bet ween t hese lymphocyt es and t ar get cells, incr eased met abolic act ivit y
and t he pr oduct ion of cyt olyt ic gr anules by lar ge gr anular lymphocyt es.
Animal st udies have demonst r at ed t hat int er fer on exer t s in vivo ant i t umour act ivit y.
Ant i t umour act ivit ies of int er fer on have been at t r ibut ed bot h t o ant i pr olifer at ive effect s as
well as t o augment at ion of host ant i t umour r esponses.
Interferon assays
Human int er fer on assays can be used for diagnost ic pur poses, as in t he diagnosis of vir al
infect ions and t uber culosis using t he ELISA or cyt okine assays such as t he ELISPOT (see
Chapt er 8). Ser um int er fer on levels r ise in a var iet y of vir al infect ions and aft er immunizat ion;
t hey may be incr eased dur ing high fever . Int er fer on has been det ect ed in CSF in cases of vir al
meningit is. Ser um of pat ient s wit h aut oimmune disease show r aised levels of IFN γ. St udies
have shown t hat neonat es have t he abilit y t o pr oduce α and β int er fer ons; pr oduct ion of γ
int erferon is usually deficient . Pat ient s receiving powerful immuno suppressives, as in t ransplant
pat ient s, have decr eased quant it ies of α and γ int er fer ons. Pat ient s wit h malignant lesions,
especially t he lymphocyt ic leukaemias and t hose on chemot her apeut ic dr ugs have a decr eased
abilit y t o pr oduce α and γ int er fer on. Diseases like ur aemia, syst emic lupus er yt hemat osus and
mult iple scler osis have an adver se effect on int er fer on pr oduct ion.
Therapeutic uses of interferon
(a) Interferon alpha
Int er fer on α was t he fir st of t he int er fer ons t o be used successfully in clinical t her apy.
Ther e ar e t wo t ypes of r ecombinant IFN-α available (2a and 2b); a newer for mulat ion is called
Pegylat ed Int er fer on. This dr ug was developed t o count er t he r apid br eakdown of t he or iginal
IFN α pr oduct s. It has five appr oved indicat ions in t he U.S. — hair y cell leukemia, AIDS-
r elat ed Kaposi’s sar coma, genit al war t s, chr onic hepat it is B and hepat it is C.
Alpha int er fer on’s biggest impact came in 1991 and 1992, when it was licensed for chr onic
hepat it is C and t hen for chr onic hepat it is B. Alpha int er fer on wor ks differ ent ly in each disease.
In hepat it is C t he vir us invades and dest r oys liver cells; int er fer on lower s t he vir us populat ion
t o a level wher e it no longer causes injur y. In hepat it is B, however , it is t he immune syst em’s
at t ack on vir us-infect ed cells t hat causes t he illness. Int er fer on helps by st imulat ing immune
cells t hat in t ur n r epel t he invasion. Not all pat ient s r espond t o int er fer on t her apy; some of
t hem r elapse when t he dr ug is st opped.
Alt hough a newer dr ug has now t aken it s place, alpha int er fer on was for a t ime t he main
t r eat ment for hair y cell leukemia, a r elat ively r ar e blood cancer t hat usually st r ikes older
men. It is also used t o shr ink t umor s and pr olong sur vival for some AIDS pat ient s disfigur ed by
t he pur ple lesions of Kaposi’s sar coma, a cancer t hat pr imar ily affect s t he skin.
112 Immunology– Introductory Textbook
Alpha int er fer on’s ant ivir al act ion has been impr essive against t he human papilloma vir us
in condylomat a acuminat a (genit al war t s), t he most common sexually t r ansmit t ed vir al disease.
In women, t he vir us is t hought t o be a pr ecur sor for cer vical cancer .
(b) Interferon beta
Ambulat or y pat ient s wit h r elapsing-r emit t ing mult iple scler osis who t ook high doses of
bet a int er fer on had about 30% fewer at t acks, and half as many sever e ones, as people who t ook
a placebo. As a r esult , t r eat ed pat ient s wer e hospit alized less oft en. Magnet ic r esonance imaging
showed a mar ked decr ease in t he number of new br ain lesions in t hese pat ient s. Side effect s of
t r eat ment wer e minimal, wit h about 75% of pat ient s on a high dose r epor t ing mild flu-like
sympt oms t hat diminished over t ime.
(c) Interferon gamma
Finally, gamma int er fer on is used t o t r eat chr onic gr anulomat ous disease, a r ar e, inher it ed
immune disor der found most ly in young men. It is also used t o t r eat malignant ost eopor osis.
Medical r esear cher s ar e act ively t est ing int er fer ons — pr imar ily alpha — in mor e t han 150
clinical t r ials involving pat ient s wit h var ious cancer s or HIV infect ion. Pr eliminar y indicat ions
ar e t hat alpha int er fer on may benefit pat ient s wit h ear ly st age melanoma, r enal cell car cinoma,
chr onic myeloid leukemia, and mult iple myeloma. The dr ug also appear s t o be a t eam player
when combined wit h ot her agent s; some skin cancer s, r espond especially well t o a combinat ion
of alpha int er fer on and r et inoids, which ar e used now t o t r eat acne.
S ide effects of interferon therapy
The most common side effect is t hat of flu-like sympt oms wit h t he fir st few inject ions. A
mor e ser ious side effect is depr ession; par t icular ly if t her e is a hist or y of depr ession. Typical
sympt oms of depr ession ar e unexplained sadness, spells of cr ying, insomnia, ear ly mor ning
awakening, loss of int er est in food, sex, hobbies, et c. Int er fer on may affect t he bone mar r ow
and can be sever e. Rar ely, t he t hyr oid gland may become over or under act ive. Ext r eme fat igue
or insomnia, ir r it abilit y, or excessive sweat ing may be t he war ning signs. Ot her t r oublesome
side effect s may include nausea, diar r hoea, t hinning of hair , ir r it at ion at inject ion sit e and
weight loss. For t he most par t t hese ar e r ever sible wit h t ime and occasional wit hholding of t he
medicat ion.
Interleukin-6 (IL-6)
Int er leukin-6 is a mult ifunct ional lymphokine t hat r egulat es immune r esponses, t he acut e
phase r eact ion and haemat opoiesis. It st imulat es t he liver t o pr oduce acut e phase pr ot eins; and
incr eases neut r ophil pr oduct ion. IL-6 aids B cell pr olifer at ion and induces t he t er minal
mat ur at ion of B cells int o immunoglobulin - secr et ing cells. In addit ion, IL-6 act ivat es T cells
and induces t heir pr olifer at ion. In associat ion wit h IL-2 and IFN γ, IL-6 induces differ ent iat ion
of cyt ot oxic T cells. IL-6 is pr oduced by many cells including T-lymphocyt es, macr ophages,
monocyt es, endot helial cells and fibr oblast s.
Interleukin-10 (IL-10)
IL-10 is an inhibit or of act ivat ed macr ophages and dendr it ic cells and as such, r egulat es
innat e immunit y and cell-mediat ed immunit y. IL-10 inhibit s t heir pr oduct ion of IL-12, co-
st imulat or molecules, and MHC-II molecules, all of which ar e needed for cell-mediat ed immunit y.
IL-10 is pr oduced mainly by macr ophages, and T
h
2 cells.The for mat ion of gr anuloma in infect ions
such as t uber culosis, lepr osy, hist oplasmosis, and coccidioidomycosis is a cyt okine-mediat ed
Cytokines 113
cellular r esponse. Because macr ophages have difficult y in r emoving t he micr obes t hat cause
t hese infect ions, t her e is a cont inuous secr et ion of cyt okines and chemokines t hat leads t o an
accumulat ion of densely packed macr ophages ar ound t he micr obes. The macr ophages r elease
fibr ogenic cyt okines such as TNF and IL-1 t hat lead t o t he for mat ion of gr anulat ion t issue and
scar t issue. The r esult ing mass is called a gr anuloma and is an at t empt by t he body t o “wall-off”
or localize t he infect ion.
B. Cytokines that Regulate Adaptive Immune Responses (Humoral and Cell-
Mediated)
Cyt okines t hat r egulat e adapt ive immunit y ar e pr oduced pr imar ily by T-lymphocyt es t hat
have r ecognized an ant igen specific for t hat cell. These cyt okines funct ion in t he pr olifer at ion
and differ ent iat ion of B-lymphocyt es and T-lymphocyt es aft er ant igen r ecognit ion.
Interleukin-2 (IL-2)
The cellular or igin of IL-2 is t he mat ur e T lymphocyt e. T helper and T cyt ot oxic subset s of
T cells can bot h pr oduce IL-2. Lar ge gr anular lymphocyt es also secr et e IL-2. Thr ee signals ar e
necessar y for IL-2 r elease : MHC r est r ict ion, int er act ion of ant igen and t he T cell r ecept or and
IL-1 (Figur e 12.6). The pat hway t o T cell act ivat ion and t he pivot al r ole played by IL-2 has been
discussed in t he pr evious chapt er .
Sever al subst ances t hat have be shown t o be suppr essive, seem t o exer t t heir effect s
pr imar ily by inhibit ing IL-2 gene expr ession. Included in t his cat egor y ar e glucocor t icoids,
cyclospor ine and pr ost aglandin E2. Impair ed pr oduct ion of IL-2 has been r epor t ed in sever al
pat ient s wit h deficiencies in cellular immunit y; as in leprosy, AIDS and in pat ient s wit h met ast at ic
cancer s.
Rest ing lymphocyt es do not bind t o IL-2. The development of a r esponse t o IL-2 r equir es
denovo acquisit ion of membr ane r ecept or s for IL-2. Upon appr opr iat e act ivat ion, T lymphocyt es
develop IL-2 r ecept or s in 6 hour s. For T cell pr olifer at ion t o occur , IL-2 needs t o couple wit h it s
r ecept or . The secr et ion of IL-2 by an act ivat ed T cell is st r ict ly r egulat ed by ext er nal st imuli
(ant igen pr ovocat ion).
Functions of IL-2 on T cell clones
The addit ion of IL-2 t o act ivat ed T cells pr omot es pr olifer at ion.The IL-2 dependant T
cells–pr edomina nt ly T helper cells -pr oduce ot her lymphokines such int er fer on, colony
st imulat ing fact or and int er leukin -3; t hey pr oduce fact or s for B cell gr owt h and differ ent iat ion.
IL-2 act ivat ed T cells also exhibit enhanced cyt ot oxicit y showing t hat besides clonal expansion,
IL-2 also pr omot es cellular funct ion such as delayed hyper sensit ivit y and T cell cyt ot oxicit y.
IL-2 action on non T lymphocytes
Alt hough t hey lack T cell mar ker s, lar ge gr anular lymphocyt es t hat exhibit nat ur al killer
cell act ivit y (see Chapt er :14), also r espond t o IL-2. These lymphocyt es begin t o gr ow, pr oduce
lymphokines and exhibit enhanced nat ur al killer act ivit y. Recept or s for IL-2 have also been
found on B cells. IL-2 can incr ease ant ibody pr oduct ion as well as pr olifer at ion in t hese cells.
Finally macr ophages can also be induced t o become cyt ocidal wit h lar ge doses of IL-2.
Interleukin-4 (IL-4)
Int er leukin-4 is a 20kd glycopr ot ein t hat affect s many cell t ypes including B cells. It pushes
B cells out of t he r est ing phase int o act ivat ion. It induces B cells t o expr ess MHC Class II
114 Immunology– Introductory Textbook
ant igens; CD23 which is a low affinit y r ecept or for IgE; and IL-4 r ecept or s. Ant ibody synt hesis
is also influenced by IL-4. IL-4 inhibit s t he swit ch fr om IgM t o IgG. IL-4 is a major st imulus for
pr oduct ion of IgE and t he development of T
h
2 cells for defence against helmint hs and ar t hr opods.
It also ant agoizes t he effect s of int er fer on-gamma and t hus inhibit s cell-mediat ed immunit y.
IL-4 is pr oduced mainly by T
h
2 cells and mast cells.
Interleukin-5 (IL-5)
IL-5 is a gr owt h and act ivat ing fact or for eosinophils as a defence against helmint hs and
ar t hr opods. It also st imulat es t he pr olifer at ion and differ ent iat ion of ant igen-act ivat ed B-
lymphocyt es and t he pr oduct ion of IgA. IL-5 is pr oduced mainly by T
h
2 cells.
Interferon gamma
Type II int er fer on, i.e. IFN γ, is pr oduced pr imar ily by T
h
1 cells, CD8
+
cells, and NK cells
as par t of t he immune r esponse and funct ions mainly t o pr omot e t he act ivit y of t he component s
of t he cell-mediat ed immune syst em. Int er fer on γ is an acid labile molecule. Ther e is only one
t ype of IFN γ.
IFN-gamma is t he pr incipal cyt okine for act ivat ing macr ophages. It also induces t he
pr oduct ion of MHC-I molecules, MHC-II molecules, and co-st imulat or y molecules by APCs in
or der t o pr omot e cell-mediat ed immunit y (act ivat ion of T lymphocyt es r equir es t hat t he T cells
r ecognize ant igen in conjunct ion wit h Class I and II MHC molecules); it also act ivat es and
incr eases t he ant imicr obial and t umor icidal act ivit y of monocyt es, macr ophages, neut r ophils,
and NK cells. IFN γ mor e t han t he ot her t wo int er fer ons, incr eases and induces IL-1 r elease
fr om monocyt es, t hus augment ing and amplifying immunologic r esponses. IFN-γ st imulat es
t he differ ent iat ion of T4-lymphocyt es int oT
h
1 cells and inhibit s t he pr olifer at ion of T
h
2 cells;
st imulat es t he pr oduct ion of IgG subclasses t hat act ivat e t he complement pat hway and pr omot e
opsonizat ion; augment s or inhibit s ot her cyt okine act ivit ies; and exer t s weak ant ivir al act ivit y.
For int er fer on assays and t her apeut ic uses of int er fer ons, including IFN – γ, r efer discussion
on Type I int er fer ons above.
Transforming growth factor-beta (TGF-beta)
TGF-β has a pr olifer at ive effect on many mesenchymal and epit helial cell t ypes. Under
cer t ain condit ions TGF-β will demonst r at e ant i-pr olifer at ive effect s on endot helial cells,
macr ophages, and T- and B-lymphocyt es. Such effect s include decr easing t he secr et ion of
immunoglobulin a nd suppr essing hema t opoiesis, myogenesis, a dipogenesis a nd a dr ena l
st er oidogenesis. Sever al member s of t he TGF-β family ar e pot ent inducer s of mesoder mal
differ ent iat ion in ear ly embr yos.
Interleukin-13 (IL-13)
IL-13 increases t he product ion of IgE by B-lymphocyt es, inhibit s macrophages, and increases
mucus pr oduct ion. IL-13 is made pr imar ily by T
h
2 cells.
C. Cytokines that Stimulate Haematopoiesis
Pr oduced by bone mar r ow st r omal cells, t hese cyt okines st imulat e t he gr owt h and
differ ent iat ion of immat ur e leukocyt es.
Cytokines 115
Colony-stimulating factors (CSFs)
Colony st imulat ing fact or s pr omot e t he pr oduct ion of colonies of t he differ ent leukocyt es
in t he bone mar r ow and enhance t heir act ivit y. Examples include gr anulocyt e macr ophage
colony st imulat ing fact or (GM-CSF), gr anulocyt e colony st imulat ing fact or (G-CSF), and
macr ophage colony st imulat ing fact or (M-CSF). In addit ion t o t heir r ole in pr omot ing pr oduct ion
of leukocyt e colonies, t he CSFs also appear t o pr omot e t heir funct ion. For example, when GM-
CSF binds t o r ecept or s on neut r ophils, eosinophils, and monocyt es, it act ivat es t hese cells and
inhibit s t heir apopt osis. GM-CSF incr eases adhesion of t hese cells t o capillar y walls dur ing
diapedesis, enhances t heir phagocyt osis and ext r acellular killing, and incr eases bot h super oxide
anion gener at ion and ant ibody-dependent cyt ot oxicit y. The var ious CSFs ar e pr oduced by T-
lymphocyt es, macr ophages, and ot her cells.
Bot h G-CSF and GM-CSF ar e available commer cially. They ar e used pr imar ily t o decr ease
neut r openic infect ions and t he r esult ing mor bidit y and mor t alit y as a r esult of chemot her apy
a nd or t ot a l body ir r a dia t ion in bone ma r r ow t r a nspla nt r ecipient s; in pa t ient s wit h
haemat ological and ot her malignancies.
Stem cell factor
St em cell fact or makes st em cells in t he bone mar r ow mor e r esponsive t o t he var ious
CSFs. It is made mainly by bone mar r ow st r omal cells.
Interleukin-3 (IL-3)
Int er leukin-3 is also called mult icolony st imulat ing fact or (mult i-CSF). It is pr oduced by T
cells and affect s gr owt h of macr ophages, gr anulocyt es and host cells.
Interleukin-7 (IL-7)
Int er leukin-7 affect s ear ly B cells. It induces pr olifer at ion but not mat ur at ion of cer t ain B
cell pr ogenit or s. It also st imulat es t he pr olifer at ion of ear ly T cells and augment s some in vit r o
T cell r esponses. Il-7 is pr oduced mainly my fibr oblast s and bone mar r ow st r omal cells.
Table 13.1 Interleukins and interferons: primary source and principal activity
Interleukins Principal Source Primary Activity
IL1 macrophages and other antigen Co-stimulation of APCs and T cells,
presenting cells (APCs) inflammation and fever, acute phase
response, haematopoiesis
IL-2 activated T
h
1 cells, NK cells proliferation of B cells and activated T
cells, NK functions
IL-3 activated T cells growth of haematopoietic progenitor cells
IL-4 T
h
2 and mast cells B cell proliferation, eosinophil and mast
cell growth and function, IgE and class II
MHC expression on B cells, inhibition of
monokine production
IL-5 T
h
2 and mast cells eosinophil growth and function
116 Immunology– Introductory Textbook
Interleukins Principal Source Primary Activity
IL-6 activated T
h
2 cells, APCs, other acute phase response, B cell proliferation,
somatic cells thrombopoiesis, synergistic with IL-1 and
TNF on T cells
IL-7 thymic and marrow stromal cells T and B lymphopoiesis
IL-8 macrophages, other somatic cells chemoattractant for neutrophils and T cells
IL-9 T cells haematopoietic and thymopoietic effects
IL-10 activated T
h
2 cells, CD8
+
T and inhibits cytokine production, promotes B
B cells, macrophages cell proliferation and antibody production,
suppresses cellular immunity, mast cell
growth
IL-11 stromal cells synergisitc haematopoietic and
thrombopoietic effects
IL-12 B cells, macrophages proliferation of NK cells, INF-γ
production, promotes cell-mediated
immune functions
IL-13 T
h
2 cells IL-4-like activities
Interferons Principal Source Primary Activity
IFN α and β macrophages, neutrophils and antiviral effects, induction of class I MHC
some somatic cells on all somatic cells, activation of NK cells
and macrophages
IFN γ activated T
h
1 and NK cells induces class I MHC on all somatic cells,
induces class II MHC on APCs and
somatic cells, activates macrophages,
neutrophils, NK cells, promotes cell-
mediated immunity, antiviral effects
™™™
CELL–MEDIATED IMMUNITY
CHAPTER – 14
W
hen t he pot ent defence mechanism available in ant ibody mediat ed immunit y was
fully elabor at ed, scholar s in immunology felt t hat t he complet e defence st r at egy of
t he host against invading micr o or ganisms had finally been wor ked out . It was soon r ealized
t hat t his for m of defence was woefully inadequat e when it came t o mount ing an at t ack on int r a
cellular bact er ia and vir uses, complex st r uct ur es such as par asit es and t he body’s own cells
when t hey became “effet e” or t ur ned malignant . In all of t hese inst ances, ant igen is not over t
but masked wit hin t he body’s own cell syst ems. Cell mediat ed immunit y has t her efor e been
t ar get ed t o:
• killing int r acellular or ganisms
• dest r uct ion of t umour cells
• r eject ion of gr aft t issue and
• a delayed t ype hyper sensit ivit y r eact ion aft er cont act wit h cer t ain ant igens such as
poison ivy and cer t ain t opically applied dr ugs.
Cell mediat ed immunit y involves t hr ee major defence st r at egies:
1. Act i va t i n g a n t i ge n -sp e ci fi c cyt ot oxi c T-lymp h ocyt e s (CTLs) t hat ar e able t o
lyse body cells displaying epit opes of for eign ant igen on t heir sur face. Examples ar e
vir us-infect ed cells, cells wit h int r acellular bact er ia and cancer cells displaying t umour
ant igens. This has been discussed in det ail in Chapt er : 12. A shor t summar y will be
pr ovided her e.
2. Act i va t i n g ma cr op h a ge , NK ce l l s a n d a n t i b od y d e p e n d a n t cyt ot oxi ci t y
enabling dest r uct ion of int r acellular pat hogens.
3. St i mu la t i n g cells t o secr et e a va r i et y of cyt ok i n es t hat influence t he funct ion of
ot her cells involved in adapt ive immune r esponses and innat e immune r esponses.
These have been det ailed in Chapt er 13 and will be summar ised her e.
1. Activating Antigen-specific Cytotoxic T-lymphocytes (CTLs)
The key st ages involved in cell mediat ed immunit y ar e:
Secretion of lymphokines by T
h
1 lymphocytes of the T4 lymphocyte subset
T
h
1-lymphocyt es r ecognize ant igens pr esent ed by macr ophages and funct ion pr imar ily t o
act ivat e and height en cell-mediat ed immunit y (Figur e: 14.1) by pr oducing cyt okines such as
int er leukin-2 (IL-2), int er fer on-gamma (IFN-gamma) and t umour necr osis fact or -bet a (TNF-
bet a). Collect ively t hese cyt okines enable T8-lymphocyt es t o pr olifer at e and differ ent iat e int o
cyt ot oxic T-lymphocyt es capable of dest r oying infect ed host cells and mut ant cells; act ivat e
cyt ot oxic T-lymphocyt es a nd NK cells; a ct iva t e ma cr opha ges ena bling t hem t o dest r oy
int r acellular pat hogens; st imulat e t he pr oduct ion of opsonizing and complement -act ivat ing
ant ibodies for enhanced at t achment dur ing phagocyt osis; act ivat e neut r ophils; st imulat e
118 Immunology– Introductory Textbook
incr eased pr oduct ion of monocyt es in t he bone mar r ow; and funct ion as chemoat t r act ant s for
phagocyt es.
Figure 14.1. The cell mediated immune response.
Cytotoxic T lymphocyte (CTL) activity
Ant igen r ecognit ion by cyt ot oxic T cells and t heir subsequent act ivat ion has been gr eat ly
discussed in pr evious chapt er s. Cyt ot oxic T cells kill vir ally infect ed t ar get cells by dir ect cell
lysis ot her wise known as a p op t osi s or pr ogr ammed cell deat h. For lysis t o occur t he t ar get
cells must car r y bot h t he same vir al ant igen and t he same MHC Class I ant igens as t he cells
t hat or iginally induced t he pr olifer at ion of T cyt ot oxic cells (Figur e 14.2). The T cyt ot oxic cells
r ecognize ant igen in conjunct ion wit h MHC Class I molecules, ar e st imulat ed by T
h
1 helper
cells and t hen t ur n cyt ot oxic. The MHC Class I ant igens appear t o be ideal r ecognit ion ant igens
t r igger ing cyt ot oxic T cells because t hey ar e pr esent on almost all cells. A wide var iet y of cells
can become vir ally infect ed or t ur n malignant , hence it is essent ial t hat cyt ot oxic T cells have
a wide r eper t oir e of t ar get cells against which t hey can mediat e t heir killing act ion. T cyt ot oxic
cells may t hus, be t he major effect or pat hway of cell mediat ed immunit y t o cer t ain vir uses; t his
happens just aft er a few hour s of vir uses infect ing cells—befor e t he vir uses can r eplicat e.
Figure 14.2. T cell - cytotoxicity - a schematic representation.
Cell–Mediated Immunity 119
Mechanism of cell lysis
CTLs and NK cells induce apopt osis by at least 2 differ ent pat hways:
Th e fi r st a n d most e ffe ct i ve p a t h wa y i n volve s i n t r a ce llu la r gr a n u le s . The CTLs
cont ain gr anules composed of pr ot eoglycans t o which chemokines ar e complexed. These gr anules
hold por e-for ming pr ot eins called per for ins and pr ot eolyt ic enzymes called gr anzymes in a
pr ot ect ed st at e. When t he TCR and CD8 of t he CTL binds t o t he MHC-I/epit ope on t he sur face
of t he vir us-infect ed cell, t his sends a signal t hr ough t he CD3 molecule which t r igger s t he
r elease of t he per for ins, gr anzymes, and chemokines.
For some year s it has been clear t hat killer cells do t heir job wit h gr eat efficiency, fir st
seeking a miscr eant t ar get cell, binding t o it and t hen exer cising some effect t hat finally causes
cell deat h, while spar ing innocent byst ander cells. It is now clear t hat having bound t o it s
vict im, t he killer cells shoot s it full of holes, mor e accur at ely, it fir es molecules of a let hal
pr ot ein at t he t ar get cell. It has been shown t hat a unique por e for ming pr ot ein is par t of t he
ar mament ar ium of T cyt ot oxic cells and NK cells.
It is now clear t hat ear ly in t he cell-killing pr ocess, gr anules in t he killer cell become
concent r at ed in t he par t of t he cell closest t o t he t ar get . The gr anule packaging or ganelle of t he
CTL - t he Golgi appar at us get s dir ect ed t owar ds t he cont act r egion and t he cell’s cyt oskelet on
is also r eor ient ed t owar ds t he t ar get cell. All t his happens only when T cell -t ar get cell cont act
is est ablished and is accompanied by an explosive incr ease in Ca++ (Figur e 14.3). This r esult s
in an exocyt osis of cont ent s fr om t he killer cell gr anules now sit uat ed at t he plasma membr ane
just apposing t he t ar get cell. The killer cell uses exocyt osis t o fir e a let hal agent cont ained in
it s gr anule int o t he t ar get cell membr ane. It has been shown t hat empt y gr anules ar e just shell
casings for t he pr oject ile. This pr oject ile molecule has now been ident ified as a por e for ming
pr ot ein and is oft en called “per for in”. Once cells ar e exposed t o t his 70 kd pr ot ein called per for in,
in t he pr esence of calcium ions, t hey lyse wit hin minut es. On t he ot her hand if calcium ions ar e
added t o per for in befor e it makes cont act wit h t he t ar get cell, t he killing act ivit y is complet ely
abolished.
The per for in molecules secr et ed by t he killer cell inser t int o t he t ar get cell membr ane
(Figur e 14.4). Ther e, wit hin t he cell membr ane, and in t he pr esence of Ca++ ions, t he individual
monomer ic molecules polymer ize int o a pr oduct t hat r esembles a cylinder or a r ing measur ing
5-20 nm in diamet er . For per for in t o damage t he t ar get cell, calcium mediat ed polymer izat ion
must t ake place ent ir ely wit hin t he t ar get cell membr ane. The r eason being, t hat only t he
per for in monomer can inser t int o t he t ar get cell. Any secr et ed per for in t hat spills int o t he
ext r a cellular space or blood st r eam, wher e calcium is abundant , immediat ely polymer izes,
vir t ually eliminat ing “accident al” injur y t o byst ander cells. On t he t ar get cell t he t ubular por es
cause leakage of wat er an ions and finally cell deat h by lysis. Per for in synt hesis has been
shown t o be st imulat ed in t he pr esence of int er leukin-2.
An int er est ing par allel has been dr awn bet ween per for in and t he t er minal pr ot eins of t he
complement cascade. Complement component s, C5, C6, C7, C8 and sever al C9 molecules
polymer ize t o for m por es much like t hose wr ought by per for in. Significant homology has been
found in t he sequence of amino acids bet ween C9 and per for in.
Ther e is now incr easing evidence t hat , t hough t he por e for ming pr ot ein or per for in is an
impor t ant met hod of cell lysis ( or it would not have evolved at all!), it is not t he cent r al
component in lymphocyt e mediat ed killing. A lymphot oxin like act ivit y has been det ect ed in T
cells; sever al ot her slow killing molecules (gr anzymes and caspases) have also been ident ified,
secr et ed by bot h T cyt ot oxic cells and NK cells. These molecules ar e ant igenically r elat ed t o
t he t umour necr osis fact or (TNF).
120 Immunology– Introductory Textbook
Figure 14.3. The perforin - mediated killing mechanism adopted by the killer lymphocyte. The killer
lymphocyte (1) recognizes the target cell and makes close contact with it (2). On contact the cell’s
granules and the Golgi complex that forms them are reoriented toward the target cell; perforin is
secreted and forms pores in the target-cell membrane. Having launched its lethal missiles, the killer
cell withdraws and goes on to kill again (3); the damaged target cell dies a programmed death within
minutes (4).
Figure 14.4. Target cell membrane damage by perforin. A rise in the lymphocyte’s calcium-ion level,
(1), brings about exocytosis, in which the granules fuse with the cell membrane (2) and disgorge
their perforin (3) into the small intercellular space abutting the target. Calcium there changes the
conformation of the individual perforin molecules, or monomers (4), which then bind to the target-cell
membrane (5) and insert into it (6). The monomers polymerize like staves of a barrel (7) to form pores
(8) that admit water and salts and kill the cell.
The per for in por es allow gr anzymes t o ent er . Cer t ain gr anzymes, in t ur n, can t hen act ivat e
t he caspase enzymes t hat lead t o apopt osis of t he infect ed cell. The caspases ar e pr ot eases t hat
dest r oy t he pr ot ein st r uct ur al scaffolding of t he cell (t he cyt oskelet on), degr ade t he cell’s
nucleopr ot ein and act ivat e enzymes t hat degr ade DNA.
Some act ivat ed T8-lymphocyt es also secr et e chemokines (see Chapt er 13) t hat suppr ess
r eplicat ion of HIV in T4-lymphocyt es. (a chemokine r ecept or is a cofact or for adsor pt ion of HIV
t o CD4+ cells. When t hat r ecept or is occupied by it s nat ur al chemokine, HIV cannot adsor b.)
Th e secon d p a t h wa y by which CTLs can t r igger apopt osis of t he infect ed cells is t hr ough
FasL/Fas int er act ions. CTLs have a r ecept or called FasL t hat can int er act wit h Fas molecules
Cell–Mediated Immunity 121
found on t he sur face of most cell t ypes. This FasL/Fas int er act ion t r igger s an int r acellular
t r ansduct ion t hat act ivat es t he caspase enzymes and leads t o dest r uct ion of t he cyt oskelet on
and chr omosome in t he infect ed cell. CTLs ar e not dest r oyed in t hese r eact ions, t hey can
funct ion over and over again t o dest r oy mor e vir us-infect ed cells.
Deat h by apopt osis does not r esult in r elease of cellular cont ent s such as inflammat or y
mediat or s or vir uses as does cell lysis. Inst ead, t he cell br eaks int o fr agment s t hat ar e
subsequent ly r emoved by phagocyt es. This r educes inflammat ion and also pr event s t he r elease
of vir uses and t heir spr ead int o uninfect ed cells. In addit ion, t he act ivat ed enzymes t hat degr ade
host DNA can also dest r oy micr obial DNA and t hus kill infect ious micr obes wit hin t he cell.
Mechanisms of Evading Cell-Mediated Immunity
A high r at e of mut at ion in some vir uses like HIV and t he hepat it is C vir us (HCV) changes
t he amino acid sequence and, t her efor e, t he shape of key epit opes. CTLs wit h TCRs made
against t he ear lier st r ains of t hese vir uses may no longer bind t o and lyse cells infect ed wit h
mut at ed st r ains.
Many vir uses such as t he cyt omegalovir us (CMV) and adenovir uses and some t umour
cells can block t he for mat ion of MHC-I molecules by t he infect ed cell. As a r esult , t he CTLs ar e
no longer able t o r ecognize t hat t he cell is infect ed and cannot kill it . Epst ein-Bar r vir us (EBV)
down r egulat es sever al host pr ot eins involved in at t aching vir al epit opes t o MHC-I molecules
and displaying t hem on t he host cell’s sur face.
Adenovir uses and EBV code for pr ot eins t hat block apopt osis of t he vir al infect ed cell.
2. Activating Macrophages, NK Cells and Antibody Dependant Cytotoxicity
Activated macrophages
Act ivat ed macr ophages ar e t he effect or cells in t he cell mediat ed immune r esponse t o
var ious micr o or ganisms. In t his effect or pat hway t he macr ophage ingest s t he micr o or ganism,
pr ocesses t he ant igen and t hen pr esent s t he ant igen on it s sur face in associat ion wit h MHC
Class II molecules, t o T
h
1 helper lymphocyt es (Figur e 14.5). Co-st imulat or y molecules such as
CD40L on t he T
h
1 cell t hen bind t o CD40 on t he macr ophage (Figur e 12.5). This t r igger s t he
T
h
1 cells t o secr et e sever al cyt okines including int er fer on-gamma (IFN-gamma). They bind t o
specific r ecept or s on t he macr ophage causing it s act ivat ion. On a subsequent pr esent at ion of
t he same ant igen by anot her macr ophage, t he sensit ized T
h
1cells ar e t r igger ed t o r elease
sever al chemokines which at t r act mor e macr ophages, gr anulocyt es and lymphocyt es t o t he
sit e of t he r eact ion. The abilit y of act ivat ed macr ophages t o kill micr o or ganisms is enhanced in
a non specific fashion. For example a macr ophage t hat has been act ivat ed t o kill int r a cellular
Listeria monocytogenes would also kill S almonella or ganisms mor e efficient ly.
Act ivat ion of macr ophages leads t o:
• incr eased pr oduct ion of t oxic oxygen r adicals, nit r ic oxide, and hydr olyt ic lysosomal
enzymes enabling t he killing of micr obes wit hin t heir phagolysosomes.
• causes t he macr ophages t o secr et e cyt okines such as TNF–alpha, IL–1, and IL–12.
TNF–alpha and IL–1 pr omot e inflammat ion t o r ecr uit phagocyt ic leukocyt es. IL–12
enables naive T4–lymphocyt es t o differ ent iat e int o T
h
1 cells.
• incr eases t he pr oduct ion of B7 co-st imulat or molecules (see Chapt er 12) and MHC–1
molecules by macr ophages for incr eased T-lymphocyt e act ivat ion.
122 Immunology– Introductory Textbook
• IFN–γ pr oduced by T
h
1 cells a lso incr ea ses t he pr oduct ion of opsonizing a nd
complement act ivat ing IgG t o promot e enhanced at t achment of microbes t o phagocyt es.
Figure 14.5. Role of activated macrophages in cell mediated immunity.
Natural killer cells
Nat ur al killer cells ar e lar ge gr anular lymphocyt es t hat car r y t he cell sur face mar ker s
CD56 and CD2. These cells appear t o have bot h ant i t umour and ant i micr obial act ivit y in vivo.
NK cells do not r equir e pr ior exposur e t o ant igen t o become cyt ot oxic and t heir act ivit y is not
MHC r est r ict ed. Cyt okines such as int er leukin–2 (IL–2) and int er fer on-gamma (IFN-gamma)
pr oduced by T
h
1 lymphocyt es act ivat e NK cells. NK cells also secr et e t he cyt okine IL–1 and in
r esponse t o IL–2, t hey become super killer s.
NK cells at t ach t o t heir t ar get cells by a r ecept or in a calcium independent manner (Figur e
14.6). Once t he NK cells ar e act ivat ed however , t hey r equir e calcium for lysis. They cause lysis
and apopt osis of t he t ar get cell by mechanisms
similar t o t hose descr ibed for CTLs above. The
main t ar get of NK cells ar e t umour cells and
cer t ain vir ally infect ed cells.
NK cells appear t o use a d u a l r e ce p t or
syst e m in det er mining whet her t o kill or not
kill human cells. The fir st r ecept or , called t he
killer -act ivat ing r ecept or can bind t o a number
of di ffer en t mol ecu l es u s u a l l y pr es en t on
nucleat ed human cells, and t his sends a posit ive
signal which enables t he NK cell t o kill t he cell
t o which it has bound unless t he second r ecept or
cancels t hat signal. This second r ecept or , called
t he killer -ihibit or y r ecept or r ecognizes MHC–I
molecules which ar e also usually pr esent on all
nucleat ed human cells. If MHC–I molecules ar e
expr essed on t he cell, t he killer -inhibit or y
r ecept or sends a negat ive signal t hat over r ides
t he kill signal and pr event s t he NK cell fr om
killing t hat cell.Vir uses oft en suppr ess class I
Figure 14.6. Role of NK cells in the cellular immune
response.
Cell–Mediated Immunity 123
MHC expr ession in cells t hey infect ; t he vir us-infect ed cell t her efor e becomes suscept ible t o
killing by NK cells. Tumour cells have r educed or no class I MHC expr ession, t hey t oo, become
suscept ible t o killing by NK cells. The cyt omegalovir us (CMV) evades t he immune syst em
because it can t r igger it s host cell t o pr oduce alt er ed MHC–I molecules t hat ar e unable t o bind
vir al epit opes, and t her efor e, ar e not r ecognized by CTLs. However , NK cells ar e also unable t o
kill t his infect ed cell because it is st ill displaying “MHC-I molecules” on it s sur face.
Antibody-dependant cell mediated cytotoxicity
NK cells ar e capable of ant ibody-dependant cellular cyt ot oxicit y (ADCC). When NK cells
ar e car r ying out ADCC, t hey ar e somet imes also r efer r ed t o as killer K cells (K cells). K cells
have r ecept or s on t heir sur face for t he Fc por t ion of IgG. The ant ibody, usually specific IgG
against ‘for eign’ cell ant igen, binds t o an Fc r ecept or on t he K cell and t o an ant ibody combining
sit e, via t he Fab por t ion, on t he t ar get cell (Figur e 14.7). The ant ibody act s like a br idge
bet ween t he specific ant igen bear ing t ar get cell and t he K cell. The K cell t hen r eleases por e-
for ming per for ins, pr ot eolyt ic enzymes called gr anzymes and chemokines t hat cause cell
apopt osis and cell lysis similar t o t he mechanism of killing descr ibed for CTLs.
Figure 14.7. Antibody dependant cell mediated cytotoxicity.
Monocyt es, neut r ophils and eosinophils can also par t icipat e in ADCC. Eosinophils, for
example, ar e effect ive killer s of ant ibody coat ed schist osomulae, t he lar val for ms of schist osomes.
The mechanism of killing involves binding of t he eosinophil t o t he lar va via an ant ibody molecule.
The eosinophil r eleases basic pr ot eins and ot her molecules fr om it s gr anules, leading t o for mat ion
of por es in t he t ar get cell membr ane. The or ganism dies because of t he leaky membr ane.
Eosinophils can be act ivat ed by bot h monokines and cer t ain lymphokines.
124 Immunology– Introductory Textbook
3. Secretion of cytokines
Cyt okines ar e soluble subst ances pr oduced by a var iet y of cell t ypes t hat amplify and
r egulat e a r ange of immune r esponses (Chapt er 13). Some cyt okines ar e cr ucial in cell mediat ed
immune react ions, whereas ot hers play a crit ical role in ant ibody responses. A variet y of cyt okines
affect cellular immunit y by
• mediat ing t he innat e immune r esponse
• r egulat ing haemat opoiesis
• dir ect ly influencing cell mediat ed immunit y
Cyt okines t hat affect t he inflammat ory response generally u p -r egu la t e in n a t e immu n it y.
These cyt okines also play a r ole in mediat ing r esist ance t o vir al infect ions and causing t he
car dinal signs of inflammat ion: pain, r edness, swelling, and heat . Included in t his gr oup ar e t he
Type I Int er fer ons (IFNa and IFNβ), t umor necr osis fact or (TNF), int er leukin-1, int er leukin-6,
and int er leukin-8.
Typ e I I F Ns include IFN α and IFN β. Type I IFN has sever al sour ces and sever al effect s.
For simplicit y, know t hat Type I IFN inhibit s vir al r eplicat ion in vir us-infect ed cells. IFN α has
been used t o t r ea t HI V, some for ms of ca ncer , a nd mult iple scler osis. I FN α is ma de
pr edominant ly by neut r ophils, and IFN β is made pr edominant ly by fibr oblast s.
Tu mor Ne cr osi s F a ct or (TNF) is made pr edominant ly by monocyt es and macr ophages
following st imulat ion wit h bact er ial LPS (TNF α) or by act ivat ed CD4+ T-cells (TNF β). TNF
ha s ma ny biologic funct ions including killing t umour s a nd inducing secr et ion of ot her
inflammat or y cyt okines. It is pr obably most impor t ant in inducing t he pr oduct ion of acut e
phase pr ot eins by t he liver . It also induces fever . This is one of t he fir st cyt okines t hat appear
dur ing an inflammat or y r esponse.
I n t er leu k i n -1 is also made by cells of t he monocyt e-macr ophage cell lineage. It has many
of t he same funct ions as TNF, but it t ypically appear s somewhat lat er in an inflammat or y
r esponse. Like TNF, IL-1 is an endogenous pyr ogen, so it induces fever . IL-1 is also a co-
st imulat or of CD4+ T-helper cells.
I n t er leu k i n -6, like IL-1, has 2 major funct ions: t o mediat e inflammat ion, and t o r egulat e
t he gr owt h and differ ent iat ion of lymphocyt es. IL-6 has a funct ion similar t o TNF in t hat it
induces t he synt hesis of acut e phase r eact ant s by t he liver . In addit ion, IL-6 also ser ves as a
gr owt h fact or for plasma cells.
I n t e r le u k i n -8, is pr oduced by monocyt es. IL-8 is a chemoat t r act ant for neut r ophils. It
also induces adher ence of neut r ophils t o vascular endot helial cells and aids t heir migr at ion
int o t issue spaces.
In t er leu k in -11 is produced by bone marrow st romal cells and shares t he funct ional act ivit y
of IL-6. It is also pr oduced by macr ophages and might be ant i-inflammat or y.
Ot h er ch emok in es such as macrophage inflammat ory prot ein (MIP-1a), MIP-1b, monocyt e
chemoat t r act ant pr ot ein (MCP-1), MCP-2, MCP-3 enable migr at ion of t hese cells t o t he sit e of
inflammat ion. They also induce cer t ain mor phologic, met abolic and funct ional changes in
macr ophages, t hat enhance t he cells’ abilit y t o kill micr o or ganisms and t umour cells.
Sever al cyt okines play a major r ole in haemat opoiesis in t he bone mar r ow. The best
cha r a ct er ized of t hese a r e gr a nulocyt e/monocyt e-colony st imula t ing fa ct or (GM-CSF),
gr anulocyt e-colony st imulat ing fact or (G-CSF), st em cell fact or (SCF), int er leukin-3, int er leukin-
Cell–Mediated Immunity 125
7 and int er leukin-9. Some of t hese cyt okines ar e being used t o t r eat pat ient s t hat have
deficiencies in haemat opoiesis, such as cancer pat ient s who ar e bone mar r ow suppr essed due
t o ant i-cancer t her apy.
GM-CSF a n d G-CSF ar e pr oduced by T-helper cells and pr omot e t he pr oduct ion of
gr anulocyt es (basophils, eosinophils, and neut r ophils)(G-CSF), as well as monocyt es (GM-CSF).
I n t er leu k i n -3 is also pr oduced by T-helper cells and incr eases t he pr oduct ion of basophils
and pr ogenit or cells.
I n t e r le u k i n -7 is pr oduced by bone mar r ow st r omal cells. It induces lymphoid st em cells
t o differ ent iat e int o pr ogenit or B cells. Under some condit ions IL-7 st imulat es t he pr olifer at ion
of t hymocyt es.
St e m Ce ll F a ct or is also pr oduced by bone mar r ow st r omal cells. It syner gizes wit h a
number of hemat opoiet ic gr owt h fact or s including IL-7, GM-CSF and G-CSF.
I n t e r le u k i n -9 is pr oduced by T-helper cells. In appear s t o incr ease er yt hr opoiesis and
mast cell division, and ser ve as an aut ocr ine gr owt h fact or for T-cells.
Specific cellular immune r esponses ar e mediat ed by T-lymphocyt es. Act ivat ion of T cells
r equir es t he pr esence of ant igen. Cyt okines ser ve as ‘second signals’ t o dr ive t he gr owt h and
differ ent iat ion of ant igen-act ivat ed lymphocyt es. Some cyt okines act pr edominant ly on T-cells,
ot her s on bot h T and B cells.
I n t e r le u k i n -2 is t he pr imar y gr owt h and differ ent iat ion fact or for T-cells. IL-2 causes
ant igen-pr imed T-helper cells t o pr olifer at e. In addit ion, it causes ant igen-pr imed cyt ot oxic T-
cells t o pr olifer at e and become aggr essively cyt ot oxic. IL-2 is pr oduced by CD4+ T-helper cells.
Typ e II In t er fer on or In t er fer on -γ (IFNγ) is produced by T-helper cells. It is an import ant
cyt okine for act ivat ing macr ophages. An act ivat ed macr ophage is mor e phagocyt ic, it pr ocesses
and pr esent s ant igen mor e efficient ly, it pr oduces mor e cyt okines, and becomes mor e bact er icidal
t han r est ing macr ophages. Act ivat ed macr ophages ar e an impor t ant mediat or of cellular
immunit y. IFNγ act s ant agonist ically against ot her cyt okines such as IL-4.
I n t e r le u k i n -12 has t he opposit e effect of IL-10. IL-12 is pr oduced by a number of cells
including B-cells, NK-cells, and macr ophages. IL-12 enhances IFN pr oduct ion. IL-12 may pr ove
t o be a key cyt okine in enhancing cell mediat ed immunit y.
For clinical t est s used t o assess cellular funct ion see Chapt er 22.
™™™
HYPERSENSITIVITY
CHAPTER – 15
A
t fir st glance it would appear as if all immunit y: humor al and cellular was gear ed
t owar ds defending t he host against unnat ur al ant igens. Not hing but good should
come out of a syst em so pr ecisely planned t o weed out aliens and mar auder s. However , it is
painfully evident t hat r eact ions t o put at ive for eign ant igens may be excessive and t hat if humor al
or cellular immunit y is swit ched on t o “high” for any lengt h of t ime, gr oss t issue damage can
occur . Such r eact ions have been apt ly t er med “h yp e r se n si t i vi t y” r eact ions. Ther e ar e t wo
ca t egor i es of a da pt i ve h yper s en s i t i vi t i es : i mmedi a t e h yper s en s i t i vi t y a n d del a yed
hyper sensit ivit y.
Coomb s a n d Ge ll classified hyper sensit ivit y int o four t ypes
Type I : Anaphylaxis
Type II : Ant ibody dependant cyt ot oxicit y
Type III : Immune - complex mediat ed disease
Type IV : Delayed t ype or cell mediat ed hyper sensit ivit y
Types I, II, III, and IV ar e r eact ions which involve t he humor al syst em of ant ibodies and
ar e somet imes t er med “I mme d i a t e h yp e r se n si t i vi t y”. Type IV hyper sensit ivit y involves
lymphocyt es and ot her cellular component s and r equir es a longer t ime cour se t o manifest .
Because of t his, t ype IV has been t er med d e la ye d t yp e or ce ll me d i a t e d h yp e r se n si t i vi t y.
Type I: Anaphylaxis
The most r apid hyper sensit ivit y r eact ion of t he immediat e t ype is known as anaphylaxis.
It is char act er ized by an explosive r esponse occur r ing wit hin minut es of applying a st imulus
a n d ca n be gen er a l i zed or l oca l i zed. Th e r ea ct i on s a r e medi a t ed by t h e r el ea s e of
phar macologically act ive subst ances fr om mediat or cells. The pr imar y act ion of t hese mediat or s
r esult s in cont r act ion of smoot h muscle, incr eased vascular per meabilit y and incr eased mucus
secr et ion. These agent s cause t he ea r ly p h a se of aller gic r eact ions t hat appear s wit hin minut es
aft er exposur e t o t he ant igen. La t e p h a se aller gic r eact ions may begin sever al hour s aft er
exposur e t o ant igen. It is t hought t hat basophils play a major r ole her e. Cell-bound IgE on t he
sur face of basophils of sensit ive individuals binds a subst ance called hist amine r eleasing fact or
(possibly pr oduced by macr ophages and B-lymphocyt es) causing fur t her hist amine r elease. That
t he ear ly and lat e phase r esponses ar e caused by dist inct mediat or s can be shown wit h inhibit or y
drugs. Arachadonic acid met abolism inhibit ors, such as indomet hacin, block only t he lat e response.
Sodium cr omoglycat e which blocks mast cell act ivat ion and degr anulat ion blocks bot h ear ly
and lat e r esponses.
Generalized anaphylaxis
When a small dose of ant igen such as egg whit e or het er ologous animal ser um is inject ed
int o a guinea pig for t he fir st t ime, t her e is no obvious effect . This, however , is called t he
Hypersensitivity 127
se n si t i zi n g d ose which, in effect , means t hat t he animal has pr oduced IgE ant ibodies t o t he
ant igen and t hese IgE ant ibodies ar e now at t ached by specific Fc r ecept or s t o mast cells and
basophils. A second inject ion of t he same ant igen given a week or t en days lat er , r esult s in a
dr amat ic onset of t he gener alized anaphylact ic r eact ion. The second dose of t he ant igen has
cr oss linked IgE ant ibodies sit uat ed on mast cells and basophils and caused degr anulat ion.
Hist amine, t oget her wit h ot her mediat or s r eleased fr om mast cell or basophil gr anules causes
mar ked vasodilat ion and leakage of int r a vascular fluids r esult ing in shock. The manifest at ions
of anaphylaxis var y in differ ent species. The guinea pig will scr at ch, sneeze, cough, may convulse,
go int o ext r eme br onchoconst r ict ion, asphyxiat e, collapse and die. On post mor t em, t he lungs
ar e char act er ist ically over inflat ed. In t he r abbit , t he shock or gan is t he hear t and r ight sided
hear t failur e is t he main cause of deat h. In humans, gener alized anaphylaxis pr esent s wit h
it ching er yt hema, vomit ing, abdominal cr amps, diar r hoea and r espir at or y dist r ess. In sever e
cases, lar yngeal oedema, vascular collapse and deat h can occur . Only a t imely int r avenous
inject ion of adr enaline t o count er smoot h muscle cont r act ion and capillar y dilat at ion can pr event
deat h.
In aller gic individuals, t he levels of IgE may be t housands of t imes higher t han in t hose
wit hout aller gies. Possibly t his is due t o a higher number of T
h
2 cells which pr oduce IL-4, a
cyt okine t hat can incr ease pr oduct ion of IgE, and a lower number of T
h
1 cells t hat pr oduce
gamma-int er fer on, a cyt okine t hat decr eases IgE pr oduct ion.
Local or Cutaneous Anaphylaxis
Upon inject ion of ant igen int o t he skin of a sensit ized animal, a local anaphylact ic r eact ion
will occur wit hin a few minut es. It consist s of a localized swelling and r edness - a wheal and
flar e r eact ion. Skin t est s in man, for aller gy t o a wide var iet y of ant igens is an example of t his
phenomenon. The local incr ease in vascular per meabilit y, t hat is char act er ist ic of t his r eact ion
may be demonst r at ed by t he use of t r acer dyes such as Evans blue which leak out of t he vessels
at t he r eact ion sit e and cause “blueing” of t he sur r ounding t issues. These r eact ions ar e mediat ed
by hist amine and ser ot onin and ar e quickly inact ivat ed by plasma hist aminases.
Passive Cutaneous Anaphylaxis
Localized anaphylaxis can be passively t r ansfer r ed and for ms t he basis of t he P r a u sn i t z-
Ku st n er (P.K.) r eact ion or t est . The P.K. t est is based on exper iment s done by t he t wo scient ist s
aft er whom t he t est is named. Kust ner was known t o be aller gic t o fish. Ser um fr om Kust ner
which pr esumably cont ained IgE ant ibodies t o fish was inject ed int o Pr ausnit z who was not
allergic t o fish. Aft er an obligat ory lat ent period it could be demonst rat ed t hat Prausnit z developed
a local r eact ion at t he sit e of inject ion, ever y t ime he at e fish!
In t he P.K. t est , ser um cont aining sensit izing ant ibody (IgE), is inject ed int r ader mally
int o a nor mal individual. The IgE fixes ont o t he local mast cells in t he skin. Aft er a lat ent
per iod (allowed for IgE t o fix t o mast cells), ant igen is administ er ed int r avenously wit h Evans
blue. Blueing of t he skin appear s wit hin minut es. This happens because ant igen cr oss links t he
IgE molecules on t he mast cell sur face, causes degr anulat ion and t he r esult ant vascular
per meabilit y leads t o blueing of t he sur r ounding t issues. The P-K t est demonst r at es definit ively,
t hat IgE ant ibodies ar e r esponsible for cut aneous anaphylaxis and t hat t his t ype of anaphylaxis
can be passively t r ansfer r ed. The t est is not done now for fear of t r ansmit t ing AIDS or hepat it is
B vir us.
128 Immunology– Introductory Textbook
I n a ct i ve cu t a n e ou s a n a p h yla xi s t he animal is induced t o pr oduce IgE by ant igen
administ r at ion. In passive cut aneous anaphylaxis pr e-made ant ibodies fr om anot her individual
ar e used t o elicit t he r eact ion.
In vitro models for anaphylaxis
In vit r o anaphylaxis was fir st demonst r at ed by Sir Henr y Dale, in what is now known as
t he Sch u lt z - Da le r e a ct i on , aft er it s aut hor s. Smoot h muscle st r ips fr om ileum or ut er us ar e
bat hed in physiologic buffer ed saline t o which ant ibody fr om a hyper sensit ive animal is added.
On addit ion of specific ant igen, smoot h muscle cont r act ions occur in t hese bit s of t issue in
vit r o. Bit s of lung or skin can be used t o demonst r at e t he same effect , which is due t o
degr anulat ion of t issue mast cells and r elease of vaso-act ive amines.
In man, t he t er m r eaginic or skin sensit izing ant ibodies has been used synonymously
wit h homocyt ot r opic ant ibodies. The ant ibodies t hat accomplish t his ar e most oft en IgE in all
species.
The mechanisms involved in anaphylaxis
Following t he fir st exposur e t o ant igen, t he animal r esponds by making ant ibody. Due t o
t he nat ur e of ant igen (aller gen) IgE is for med, which fixes ont o mast cells via r ecept or s t hat
exist on mast cells for t he Fc por t ion of t he IgE molecule. The animal displays no sympt oms but
is now consider ed sensit ized (Figur e 15.1 a). On second exposur e t o t he same ant igen, molecules
of ant igenic mat er ial seek out t he t issue fixed IgE ant ibodies and cr oss link adjacent ly placed
IgE molecules (Figur e 15.1 b). Br idging of adjacent Fab sit es on t he IgE molecules is r apidly
followed by t he br eakdown of phosphat idyl inosit ol t o inosit ol t r iphosphat e (IP3), t he gener at ion
of diacylglycer ol and incr ease in int r a cyt oplasmic fr ee calcium. These event s act ivat e pr ot ein
kinase C. This familiar biochemical cascade pr oduces membr ane - act ive “fusogens” such as
lysophosphat idic acid which facilit at es degr anulat ion and synt hesis of ar achidonic acid
met abolit es.
The p r e for me d or p r i ma r y me d i a t or s t hat ar e r eleased fr om t he gr anules ar e:
• hist amine.
• heparin
• eosinophil chemot act ic fact or
A for anaphylaxis (ECF-A)
• neut r ophil chemot act ic fact or (NCF-A)
• plat elet act ivat ing fact or
The n e wly syn t h e si ze d or secondar y met abolit es ar e :
• leukot r ienes B4, C4, D4 and E4
or slow r eact ing subst ance A (SRS-A)
• prost aglandins
• t hr omboxanes
Under nor mal cir cumst ances, t hese mediat or s help or chest r at e t he development of a
defensive acut e inflammat or y r eact ion. In a r un away r eact ion t heir br onchoconst r ict ive and
vasodilat or y r eact ions can be life t hr eat ening.
Hypersensitivity 129
Figure 15.1. Mechanisms involved in anaphylaxis, (a) first exposure to antigen,
(b) second exposure to antigen.
IgE receptors on mast cells
Mast cells and basophils have specific r ecept or s t hat for m non covalent bonds wit h t he Fc
por t ion of t he IgE molecule (Figur e 15.2). It is est imat ed t hat a basophil has 10,000 t o 40,000
such r ecept or s. The nor mal half life of IgE in ser um is t wo t o t hr ee days, however , IgE may
r emain fixed t o cells for weeks. Heat ing IgE t o 56
°
C for 30 minut es dest r oys it s abilit y t o bind
t o t he t ar get cell.
Ther e ar e t wo t ypes of IgE r ecept or s. The high affinit y IgE r ecept or on basophils and mast
cells has been t ermed Fc
E
RI; Fc
E
RII has low affinit y for IgE and is found on a variet y of leukocyt es,
including monocyt es, macr ophages, eosinophils, plat elet s and T and B cells. The high and low
affinit y r ecept or s for IgE ar e ant igenically dist inct ; t hey have differ ent st r uct ur es and ar e
130 Immunology– Introductory Textbook
encoded by separ at e genes. Fc
E
RI is composed of t hr ee polypept ide chains, wher eas Fc
E
RII may
play a major r ole in immunit y against par asit ic infect ions.
Figure 15.2. The Fc (IgE) receptor on the mast cell. The α chain binds the Fc portion of the
immunoglobulin IgE. The b chain traverses the membrane and the covalently linked g chains face the
cytoplasm.
Mediators of Immediate Hypersensitivity
Mast cells and basophils cont ain a number of pr efor med mediat or s wit h pot ent biologic
act ivit y. Mast cells cont ain a chymot r ypsin like enzyme, chymase; basophils cont ain a kallikr ein
like est er a se. Ot her media t or s a r e gener a t ed a ft er t he ma st cells a r e t r igger ed (See
Table 15.1).
Table 15.1: Chemical mediators of immediate hypersensitivity
Primary (pre-formed) Secondary (induced)
Mediator Functions Mediator Functions
Histamine Increased vascular permeability Leukotrienes: C4,D4,E4
Elevation of cAMP or SRS-A
Contraction of smooth muscle
Chemokinesis
ECF-A Chemotactic for eosinophils Platelet activating factor
NCF-A Chemotactic for neutrophils Lipid chemotactic, lipid
chemokinetic factor
Heprin Anticoagulant and Prostaglandin D2
anti-complement
Chymase Proteolysis Leukotriene B4
Thromboxanes
When released, t he mediat ors can cause increased vascular permeabilit y increased secret ion
by nasal and br onchial mucous glands and cont r act ion of smoot h muscle in br onchioles and
small blood vessels. Eosinophils and neut r ophils ar e summoned t o t he ar ea of injur y and
br adykinin is gener at ed by t he kallikr ein-like est er ase. The incr eased capillar y per meabilit y
leads t o an influx of plasma pr ot eins including ant ibody, complement and kinin gener at ing and
Contraction of human
bronchiole, increased
vascular permeability
Aggregation and increased
secretion
Neutrophil movement and
activation
Vasoactive smooth muscle
action
Increased vascular
permeability; eosinophil,
neutrophil chemotaxis,
neutrophil adhesion
Aggregate platelets
Hypersensitivity 131
coagulat ion pr ot eins int o t he t issues. All of t hese subst ances incr ease inflammat ion. The clinical
manifest at ions depend on t he sit es affect ed and include syst emic anaphylaxis, ur t icar ia, ast hma,
r hinit is and vasculit is.
A major gr oup of secondar y mediat or s, t he leukot r ienes, ar e pot ent br onchoconst r ict or s
and vasodilat or s (See Table:15.1). Leukot r ienes ar e for med aft er t he enzyme lipoxygenase act s
on ar achidonic acid. Also der ived fr om ar achidonic acid, by t he act ion of cyclooxygenase, ar e
pr ost aglandins which per for m r elat ed inflammat or y funct ions.
Regulatory Mechanisms of Immediate Hypersensitivity
Cr oss linking of IgE on mast cells r esult s in explosive degr anulat ion of t he cell. Act ivat ion
of enzymes for degr a nula t ion involves a complex ser ies of biochemica l st eps including
t ransmet hylat ion of membrane phospholipids, act ivat ion of prot ein kinases and adenylat e cyclase,
changes in int r acellular cAMP levels and t he opening of Ca++ channels. This pr ocess is r egulat ed
at sever al levels:
(i) The int ensit y of t he r eact ion depends on t he amount of IgE bound t o t he cells, t he
affinit y and concent r at ion of ant igen for IgE. If IgG has occupied a major it y of sit es on
t he mast cell, cr oss linking of IgE does not occur and degr anulat ion is pr event ed.
Such IgG molecules ar e called b lock i n g a n t i b od i e s .
(ii) Mediat or r elease is influenced by int r acellular cyclic nucleot ides. Agent s t hat induce
pr olonged elevat ions of int r a cellular cAMP lead t o decr ease in mediat or r elease;
agent s t hat incr ease cyclic GMP have t he opposit e effect . These agent s act via r ecept or s
on t he cell sur face (Figur e 15.3).
(iii) Hist amine can exer t negat ive feed back cont r ol by st imulat ing adenylat e cyclase
which incr eases cyclic AMP and t her eby pr event s fur t her r elease of hist amine.
(iv) Eosinophils at t r act ed t o t he sit e by chemot act ic fact or s ar e also t hought t o have a
cont r ol funct ion. They cont ain hist aminase which br eaks down hist amine and
phospholipase D, which act s on plat elet act ivat ing fact or . Enzymes fr om neut r ophils
also br eak down mediat or s.
Figure 15.3. Mediator release by mast cells is modulated by the cyclic nucleotides cAMP and cGMP.
Decreased cAMP or increased cGMP levels stimulate release, whereas increased cAMP inhibits
release. A number of physiologically active substances can trigger cell surface receptors, affecting
cyclic nucleotide production and, secondarily, mast cell release of mediators.
132 Immunology– Introductory Textbook
Atopic allergy
Near ly 10% of t he wor ld’s populat ion suffer fr om aller gies, which ar e localized anaphylact ic
r eact ions t o ext r insic ant igens such as pollens, foods, animal dander s and faeces fr om mit es in
house dust t o name just a few. Cont act of t he aller gen wit h cell bound IgE in t he br onchial t r ee,
t he nasal mucosa, skin and t he conjunct ival t issues r eleases mediat or s of anaphylaxis and
pr oduces t he sympt oms of hay fever , ast hma, ur t icar ia or aller gic r hinit is as t he case may be.
Cont act wit h food aller gens and cell bound IgE in t he gast r oint est inal t r act may cause diar r hoea
and vomit ing. Aller gen-IgE complex may diffuse out of an alr eady per meable gut and deposit in
joint s, skin or lung causing fur t her local anaphylact ic r eact ions. Thus eat ing pr awns or egg
may pr ecipit at e an ast hmat ic at t ack in a sensit ized individual.
Ther e is a st r ong familial disposit ion t o t he development of at opic aller gy, and t his may be
linked t o t he inher it ance of specific HLA haplot ypes, t hough culpr it HLA t ypes have not yet
been ident ified. It has been shown t hat t he higher t he level of ser um IgE, gr eat er is t he likelihood
of becoming at opic.
Clinical tests for allergy
Sensit ivit y is nor mally assessed by t he r esponse t o int r ader mal administ r at ion of aller gen.
The r elease of hist amine and ot her mediat or s pr oduces a wheal and flar e r eact ion in 30 minut es.
The immediat e wheal and flare react ion may be followed by a lat e phase react ion which somet imes
last s for 24 hour s. This r eact ion is t hought t o be mediat ed by NCF-A following cellular infilt r at ion
int o t he ar ea.
Ot her t est s include t he RI ST and t he RAST (descr ibed in Chapt er 8).
Th e r a p y for a t op i c d i se a se s t akes advant age of some of t he r egulat or y mechanisms of
immediat e hyper sensit ivit y ment ioned ear lier (Figur e 15.4). Examples include, diminishing
ant igen exposur e, inducing blocking IgG ant ibodies, use of mast cell st abilizer s such as sodium
cr omoglycat e; t her apy wit h cat echolamines such as epinephr ine and isopr ot er enol t o elevat e
cAMP and decr ease mediat or r elease and t he administ r at ion of ant ihist amines t o block t he
effect of hist amine on t ar get or gans. It may be not ewor t hy t hat bot h pr opr anolol and cimet idine
may induce aller gic manifest at ions in suscept ible individuals by lower ing cAMP and t her eby
pr omot ing mast cell mediat or r elease.
Figure 15.4. Possible modes of therapy for atopic diseases.
A new exper iment al appr oach t o t r eat ing and pr event ing Type-I hyper sensit ivit y involves
giving t he per son wit h aller gies inject ions of mon oclon a l a n t i b od i e s t hat have been made
a ga i n st t h e F c p or t i on of h u ma n I gE. This, in t ur n, blocks t he at t achment of t he IgE t o t he
Hypersensitivity 133
Fc r ecept or s on mast cells and basophils and t he subsequent r elease of hist amine by t hose cells
upon exposur e t o aller gen. In addit ion, t he ant i-IgE binds t o IgE-pr oducing B-lymphocyt es
causing apopt osis. The monoclonal ant ibody is a humanized hybr id molecule consist ing of a
mouse binding (Fab) por t ion at t ached t o a human const ant (Fc) por t ion and is known as r huMab
(r ecombinant human monoclonal ant ibody).
Type II-Antibody Dependant Cytotoxic Hypersensitivity
The combinat ion of IgM or IgG ant ibodies wit h ant igenic det er minant s on t he cell sur face
r ender s t he cell sur face coat ed or opsonized by ant ibody. Such an opsonized par t icle is r eadily
phagocyt osed by macr ophages which have Fc r ecept or s for IgG or IgM molecules on t heir
sur face (Figur e 15.5a). The ant ibody facilit at es t he associat ion bet ween t he phagocyt ic cell and
t he ant igen bear ing cell. If t his happens in excess and causes t issue damage it is one example
of t he ant ibody dependant cyt ot oxic hyper sensit ivit y.
Ther e ar e sever al ot her mechanisms which account for t ype II hyper sensit ivit y. These
include opsonizat ion via C3b r ecept or s which also exist on sur faces of macr ophages (see
Chapt er 7) and r esult ant phagocyt osis (Figur e 15.5b). Excessive cell lysis may occur when cell
sur face ant igen couples t o ant ibody, fixes complement and act ivat es t he ent ir e complement
cascade (Figur e 15.5c).
Anot her quit e dist inct mechanism in t ype II hyper sensit ivit y is t hat mediat ed by K cells,
in what is known as ant ibody dependant cellular cyt ot oxicit y-ADCC (see Chapt er 14), (Figur e
15.5d). It is quit e evident t hus far t hat t ype II hyper sensit ivit y may or may not involve t he
par t icipat ion of complement .
Figure 15.5. Mechanisms of antibody dependant cytotoxic hypersensitivity.
Type II reactions in blood transfusion and organ transplantation
The basis for ABO blood grouping
Of t he many var iant ant igens on t he human r ed cell membr ane, t he ABO ant igens for m
t he dominant syst em. Individuals wit h blood gr oup O display t he H subst ance on t he r ed cell
134 Immunology– Introductory Textbook
sur face (Figur e 15.6). The H subst ance is an oligosacchar ide anchor ed t o t he cell membr ane by
coupling t o a sphingomyelin called cer amide. This oligosacchar ide has a t er minal galact ose
r esidue coupled t o a fucose at posit ion 2. Individuals who ar e blood gr oup A pr oduce cer t ain
glucosyl t r ansfer ases encoded by an A gene. These enzymes act by adding N acet yl galact osamine
t o posit ion 3 of t he t er minal galact ose of t he H subst ance. Similar ly individuals who ar e blood
gr oup B posses enzymes encoded by t he B gene which couple anot her galact ose t o posit ion 3 of
t he galact ose r esidue in t he H subst ance. Individuals belonging t o t he AB blood gr oups possess
bot h genes and hence bot h ant igens on t he r ed cell sur face.
Figure 15.6. The basis of the ABO Blood Group System.
Nat ur ally occur r ing ant ibodies or i soh a e ma gglu t i n i n s in individuals ar e t ar get ed t o
t hose ant igens absent fr om t he r ed cell sur face. Thus a per son wit h blood gr oup A possesses
ant i B ant ibodies and vice ver sa. Per sons who ar e blood gr oup AB have neit her ant i A nor ant i
B ant ibodies and individuals wit h blood gr oup O, pr esent wit h bot h ant i A and ant i B ant ibodies.
These isohaemagglut inins ar e usually IgM. Since t hey ar e seemingly pr esent in t he cir culat ion,
wit hout ant igen pr ovocat ion, how wer e t hey induced? It has been t hought t hat bact er ial ant igens
of t he nor mal gut flor a cr oss r eact wit h blood gr oup ant igens. Hence, if an individual is blood
gr oup A, he will be t oler a nt t o a nt igens closely simila r t o A (see t heor ies of t oler a nce,
Chapt er 16) and will for m ant ibodies t o bact er ial ant igens t hat cr oss r eact wit h blood gr oup B.
Similar ly, an individual wit h blood gr oup O, is not t oler ant t o eit her ant igen and t her efor e will
pr oduce ant ibodies t o subst ance A and B. Per sons wit h blood gr oup AB ar e t oler ant t o bot h
ant igens and hence do not pr oduce t he appr opr iat e ant ibodies.
Mismatched Transfusion
On t r ansfusion of mismat ched r ed cells, t he donor r ed cells ar e r apidly coat ed wit h t he
host ’s isohaemagglut inins and sever e r eact ions ensue, which ut ilize complement . Since IgM is
involved, cr oss linking of just a few det er minant s is sufficient t o set off t he ent ir e complement
cascade (Figur e 15.7). Runaway complement act ivat ion wit h t he r esult ant t issue r eact ion is a
classic example of a t ype II hyper sensit ivit y r eact ion.
Hypersensitivity 135
Figure 15.7. Mismatched transfusion reactions DIC = Disseminated intravascular coagulation.
Rhesus incompatibility
The r hesus blood gr oups for m anot her major ant igenic syst em, t he r hesus ant igen is
designat ed RhD or just D. Ant ibodies t o RhD, unlike t he isohaemagglut inins, ar e not nat ur ally
occur r ing. An individual who does not display RhD on his r ed cell sur face (RhD ant igen negat ive),
needs t o be exposed t o t he D ant igen t o be able t o pr oduce ant i D ant ibodies. An individual who
has t he RhD ant igen on his r ed cell membr anes, is of cour se t oler ant t o t he ant igen and does
not pr oduce ant i D ant ibodies. A mot her who is RhD negat ive may give bir t h t o an RhD posit ive
child (t he RhD ant igen being inher it ed fr om t he fat her ). On deliver y of t he fir st child, dur ing
separ at ion of placent a, t her e is some r elease and admixing of foet al r ed cells in t he mat er nal
cir culat ion. The mot her being exposed t o t he RhD ant igen is sensit ized and begins t o pr oduce
ant i D ant ibodies. These ant ibodies ar e pr edominant ly IgG and ar e able t o cr oss t he placent a
in any subsequent pr egnancy. This IgG r eact s wit h foet al r ed cells, dur ing subsequent
pr egnancies, leading t o foet al r ed cell dest r uct ion in ut er o, t hr ough anot her t ype II mediat ed
hyper sensit ivit y r eact ion, r esult ing in haemolyt ic disease of t he newbor n. Foet al r ed cells
coat ed wit h mat er nal ant i D IgG and cir culat ing ant i D IgG in t he mat er nal blood can be
det ect ed in t he labor at or y by t he Coomb’s dir ect and indir ect t est s (see Chapt er :8).
To pr event haemolyt ic disease of t he newbor n, RhD negat ive mot her s ar e given passive
immunizat ion wit h small amount s of r eady made ant i D IgG ant ibodies at t he t ime of bir t h of
t he fir st child. These small amount s of ant i D IgG will complex wit h RhD on t hose foet al r ed
cells t hat escape int o t he mat er nal cir culat ion dur ing placent al separ at ion. This pr ecludes t he
pr esent at ion of foet al RhD ant igen t o t he mot her ’s immune syst em and sensit izat ion of t he
mot her t o RhD is pr event ed.
Organ transplants
A long st anding homogr aft may evoke ant ibodies in t he host which ar e dir ect ed against
sur face ant igens on t he t r ansplant ed t issue. These may be dir ect ly cyt ot oxic or cause adher ence
of phagocyt ic cells. They may also elicit non specific at t ack by K cells via ADCC. The ant ibodies
may lead t o plat elet adherence, when t hey are direct ed against ant igens on vascular endot helium.
All of t hese r eact ions ar e examples of Type II mediat ed hyper sensit ivit y r eact ions.
136 Immunology– Introductory Textbook
Autoimmune Type II Hypersensitivity
Many t ype II hyper sensit ivit y r eact ions ar e t he under lying mechanisms of aut oimmune
disease. Aut o ant ibodies t o t he pat ient ’s own r ed cells ar e pr oduced in aut oimmune haemolyt ic
anaemia. Red cells coat ed wit h bact er ial or vir al ant igens cause ant ibodies t o be dir ect ed against
t hem, wit h r esult ant r ed cell lysis.
Ser a of pat ient s wit h Hashimot os t hyr oidit is cont ain ant ibodies, which in t he pr esence of
complement , ar e dir ect ly cyt ot oxic for isolat ed human t hyr oid cells in cult ur e. In Goodpast ur es
syndr ome, aut o ant ibodies ar e dir ect ed t o glomer ular basement membr ane; t hey fix complement
and ser ious damage ensues. Ant ibodies t o acet ylcholine r ecept or s in myast henia gr avis is a
fur t her example of t ype II hyper sensit ivit y. In mult iple scler osis ant ibodies ar e made against
t he oligodendr oglial cells t hat make myelin in t he br ain and spinal cor d.
Type II Hypersensitivity due to drugs
Dr ugs coupled t o t he body’s own cells become ant igenic and t he humor al immune syst em
r ecognizes t he dr ug-cell complex as for eign, r esult ing in an ant ibody at t ack which dest r oys t he
cells as well. When t he dr ug is wit hdr awn, t he sensit ivit y is no longer evident . Wit h dr ugs such
as chlor pr omazine and phenacet in, r ed cells become coupling agent s and a t ype II haemolyt ic
anaemia is seen. Amidopyr ine and quinidine ar e known t o coat gr anulocyt es and a dr eaded
agr anulocyt osis could r esult . The classic example of dr ug induced t ype II hyper sensit ivit y, is
t hat of t hr ombocyt openic pur pur a, when sedor mid, a now banned sedat ive, known t o bind t o
plat elet s was used. This r eact ion, t oo, was shown t o be facilit at ed by complement .
Type III-Immune Complex Mediated Hypersensitivity
Pathogenesis of immune complex disease
The t er m immune complex disease r efer s t o a gr oup of diseases whose pat hogenesis
involves t issue damage fr om excessive ant igen-ant ibody r eact ions. The body may be exposed t o
excessive amount s of ant igen in a number of cir cumst ances, such as per sist ent infect ion wit h
micr obial agent s, aut oimmune r eact ions and r epeat ed cont act wit h envir onment al agent s. When
ant igen and ant ibody couple t o for m insoluble complexes at fixed sit es, t issue r eact ion and
t issue damage could well occur . If complement is involved, C3a and C5a, t wo pot ent vasoact ive
a mines, a r e r elea sed; t his ca uses incr ea sed va scula r per mea bilit y. I ncr ea sed va scula r
per meabilit y is a pr e r equisit e for t he deposit ion of immune complexes in t issues. If ant igen in
t he cir culat ing complex r eact s wit h IgE on cir culat ing basophils, it causes plat elet clumping
for ming micr ot hr ombi, degr anulat ion and t he subsequent r elease of hist amine, ser ot onin and
ot her chemot act ic fact or s. Chemot act ic fact or s lead t o influx of polymor phonuclear leukocyt es.
This in t ur n leads t o ext r acellular r elease of polymor ph gr anular cont ent s. These include
pr ot eolyt ic enzymes, kinin for ming enzymes and ot her pr ot eins which will damage t issues and
int ensify t he inflammat or y pr ocess. Lar ge insoluble complexes t aken up by macr ophages cannot
be r eadily digest ed and pr ovide a per sist ent act ivat ing st imulus (Figur e 15.8)
Clear ance of immune complexes in vivo nor mally occur s, ot her wise we would all be
suffer ing fr om immune complex disease! Clear ance depends on t he absolut e amount s of ant igen
and ant ibody which could det er mine r eact ion int ensit y. However , pr opor t ions of ant igen and
ant ibody also gover n t he nat ur e of complexes. Ext r eme ant ibody excess or ant igen excess does
not yield insoluble pr ecipit at ed complexes. An opt imum r at io of ant igen t o ant ibody pr ovides
for an insoluble complex (lat t ice), (Figur e 8.2). Complement fixat ion influences size and solubilit y
Hypersensitivity 137
of immune complexes (Figur e: 15.9). Binding of complement component , C1, t o immunoglobulin
pr event s Fc-Fc int er act ions needed t o for m lar ge insoluble aggr egat es. Smaller complexes
which have C3b at t ached t o t hem ar e clear ed away by macr ophages which have C3b r ecept or s
on t heir sur face. This clear ing away is aided by r ed cells bear ing t he CR1 r ecept or which
couples t o C3b in t he ant igen - ant ibody - C3b complex and t r anspor t s it away t o fixed
macr ophages in t he liver wher e t hey ar e inact ivat ed.
Figure 15.8. Schematic representation of Type III immune-complex mediated hypersensitivity.
(+) = stimulation of process.
Figure 15.9. An immune complex lattice can be disrupted by complement component C3b, which
weakens the forces between the Fc portion of the antibody molecules. This disruption solubilizes
the lattice and facilitates clearance of immune complexes from the circulation.
138 Immunology– Introductory Textbook
Where are immune complexes deposited?
When lar ge insoluble complexes for m, or when t he clear ing syst em is deficient , immune
complexes ar e deposit ed in t issues wit h r esult ant damage. Deposit ion can occur anywher e in
t he body, but some or gans such as t he kidney ar e affect ed mor e oft en t han ot her s. The
combinat ion of high blood flow, r apid filt r at ion and high blood pr essur e in r enal glomer uli
facilit at es deposit ion of immune complexes. Recept or s for complement component s ar e found
in t he glomer uli and in t he chor oid plexus - anot her favour ed sit e for deposit ion of immune
complexes. In addit ion, basement membr anes, whet her in t he glomer ulus or in t he der mal-
epider mal junct ion of t he skin, ar e negat ively char ged and will t her efor e r et ain posit ively
char ged immune complexes.
When t he abilit y of r ed blood cells t o act as car r ier s of immune complexes is impair ed,
complexes ar e mor e likely t o be deposit ed in blood vessels. Such an impair ment occur s in
pat ient s who have syst emic lupus er yt hemat osus, a disease in which r ed cells have decr eased
C3b r ecept or s. Likewise, if scavenger syst ems in t he liver or spleen ar e impair ed, immune
complexes cont inue t o cir culat e and deposit in ot her or gans.
Local reactions to Type III mediated Immune Complex Deposition
The Arthus Reaction
The Art hus react ion was described by Maurice Art hus who found t hat an acut e haemorrhagic
or necr ot ic pr ocess occur s locally, when ant igen is inject ed int o immunized individuals possessing
high t it r es of pr ecipit at ing ant ibody. This r eact ion is consider ed t he pr ot ot ype acut e, localized
immune complex mediat ed t issue injur y. The inject ed ant igen pr ecipit at es wit h ant ibody, oft en
wit hin a venule; subsequent ly, t he complex binds complement component C1q, t hereby act ivat ing
t he classical complement pat hway. The r eact ion gener at es pr ot eolyt ic cleavage pr oduct s such
as C3a, C3b and C5a. C3a and C5a cause mast cells t o r elease vasoact ive amines. Int r avascular
complexes cause plat elet aggr egat ion and fur t her r elease of vasoact ive amines fr om basophils.
The end r esult of all t his is sever e er yt hema and oedema. C5a is a power ful chemo at t r act ant
and polymor ph influx r esult s. Polymor phs r elease a wide var iet y of pr oduct s (see ear lier sect ion)
t hat degr ade basement membr ane and cause t issue damage. The Ar t hus r eact ion is t hus
pr imar ily mediat ed by complement act ivat ion and neut r ophil r eleased pr oduct s. The Ar t hus
r eact ion can be blocked by deplet ion of complement and neut r ophils.
Type III Reactions to inhaled antigens
Ar t hus t ype r eact ions in t he lung can occur t o a var iet y of inhaled ant igens. A classic
example is Far mer s lung, which occur s 6-8 hour s aft er exposur e t o mouldy hay. These pat ient s
have been found t o be sensit ized t o t her mophilic act inomycet es which gr ow in mouldy hay.
Inhalat ion of t he or ganism fr om t he hay, int r oduces ant igen int o t he lung and a immune
complex mediat ed hyper sensit ivit y r eact ion occur s. Similar r eact ions can occur fr om bir d and
r odent excr et a in bir d fancier s disease and in r at handler s disease r espect ively. Ther e ar e
many ot her quaint ly named condit ions of similar nat ur e due t o ext r insic aller gic alveolit is
r esult ing fr om inhalat ion of for eign ant igens.
Type III Reactions to tissue antigens
Type III r eact ions ar e associat ed wit h elephant iasis due t o Wuchereria bancrofti. The
dead par asit e, found in lymphat ic vessels, init iat es a t ype III r eact ion causing inflammat ion
and obst r uct ion t o lymph flow, sever e fibr osis and r esult ant elephant iasis.
Hypersensitivity 139
Vigor ous chemot her apy is known t o cause r eact ions due t o abr upt r elease of micr obial
ant igens in pat ient s wit h high ant ibody levels. Dr amat ic immune complex mediat ed r eact ions
manifest as er yt hema nodosum lepr osum in dapsone t r eat ed cases of lepr omat ous lepr osy and
in t he J ar isch - Her xheimer r eact ion of syphilis pat ient s t r eat ed wit h penicillin. In r heumat oid
ar t hr it is, ant ibodies t o self IgG get deposit ed in t he joint cont r ibut ing t o sever e synovit is.
Type III mediated S ystemic Immune Complex Disease
Ser um sickness was fir st descr ibed ear ly in t he cent ur y in pat ient s who r eceived hor se
ser um for t he t r eat ment of infect ious diseases. Typically, 8-12 days aft er t he administ r at ion of
lar ge amount s of ser um fr om a for eign species, t he pat ient may have a r ise in t emper at ur e,
t ender lymph nodes, painful and swollen joint s, an ur t icar ial r ash, gast r oint est inal dist r ess and
an enlar ged spleen. This is associat ed wit h lower ed ser um complement levels and leukocyt osis.
These changes r esult fr om deposit ion of immune complexes in t issues. To be pat hogenic, immune
complexes have t o be t he r ight size; not t oo big t o be scavenged by macr ophages and not t oo
small, so as not t o cause an inflammat or y r eact ion. The complexes lodge in blood vessels and
cause changes in vascular per meabilit y. This r esult s in separ at ion of capillar y endot helial cells
and exposur e of t he under lying basement membr ane t o which t he immune complexes at t ach.
Basement membr anes of skin, joint s, kidneys and t he hear t ar e par t icular ly affect ed.
In addit ion t o for eign ser um, sever al dr ugs can cause ser um sickness, including penicillin,
gold, penicillamine, sulfonamides, hydant oins, t hiazides, phenylbut azone, aminosalicylic acid
and st r ept omycin. These dr ugs act as hapt ens, complex wit h pr ot ein car r ier s and induce ant ibody
for mat ion. Repeat ed administ r at ion of for eign pr ot eins and dr ugs wit h r eposit or y or long act ing
char act er ist ics, such as penicillamine or gold, may induce chr onic ser um sickness. The major
complicat ions of ser um sickness ar e vasculit is, glomer ulonephr it is and neur it is.
Immune Complex Nephritis
Because r enal glomer uli ar e par t icular ly vulner able t o immune complex-mediat ed injur y,
glomer ulonephr it is is a common feat ur e of many immune complex diseases. Complexes for m
in t he cir culat ion and if t hey ar e lar ge enough, t hey ar e deposit ed in t he endot helial side of t he
glomer ular basement membr ane (Figur e 15.10). The smallest complexes manage t o filt er
t hr ough t o t he epit helial side. The deposit ions of immune complexes appear as lumpy gr anules
and cont ain ant igen, immunoglobulin and complement ; t hey ar e visible as lar ge amor phous
ma s s es by el ect r on mi cr os copy. Th e pa t h ogen es i s of i mmu n e compl ex medi a t ed
glomer ulonephr it is is illust r at ed in Figur e 15.10.
Gl omer u l on eph r i t i s h a s been a s s oci a t ed wi t h per s i s t en t b a c t e r i a l pr odu ct s .
Glomer ulonephr it is is well known a s a sequela t o infect ion wit h t ype 12 β ha emolyt ic,
“nephr it ogenic”, St r ept ococci. Glomer ulonephr it is can also occur aft er pneumococcal and
st aphylococcal infect ions.
Vi r a l infect ions wit h hepat it is B and Epst ein-Bar r vir us ar e associat ed wit h immune
complex disease. Ant igenic similar it y bet ween human and micr obial ant igens has been
hypot hesized as being t he cause of vigorous ant ibody format ion against host t issues and result ant
immune complex deposit ion.
Immune complex nephr it is has been associat ed wit h p a r a si t i c disease. Quar t an malar ia
in some Niger ian childr en leads t o an immune complex mediat ed nephr ot ic syndr ome. Chr onic
par asit oses such as leishmaniasis, t r ypanosomiasis, schist osomiasis and filar ia ar e par t icular ly
associat ed wit h chr onic glomer ulonephr it is. Chr onic st imulat ion of t he immune syst em and
polyclonal B cell st imulat ion has been incr iminat ed as possible aet iological fact or s in t hese
condit ions.
140 Immunology– Introductory Textbook
Figure 15.10. Pathogenesis of Immune-complex Nephritis.
Syst emic lupus er yt hemat osus (SLE), an a u t oi mmu n e disease is t he par adigm immune
complex disease. Immune complexes have been found in kidneys, blood vessels, skin and t he
cent r al ner vous syst em. Immune complexes isolat ed fr om lesions in SLE cont ain DNA and
ant ibody t o DNA. Vigor ous deposit ion of DNA-ant i DNA complexes is explained, in par t , by t he
finding t hat t her e ar e fewer CRI r ecept or s on r ed cells and monocyt es in t hese pat ient s and
hence immune complexes cannot be t r anspor t ed away t o t he r et iculo endot helial syst em.
Immune complex nephr opat hy can be found in ma li gn a n t disease such as lymphocyt ic
leukaemias and Hodgkins disease. Ga st r oi n t est i n a l d i sea se such Cr ohns disease, ulcer at ive
colit is, cir r hosis of t he liver and coeliac disease can be associat ed wit h immune complex t issue
injur y. Deposit ion of immune complexes can occur at ot her major filt r at ion sit es such as t he
chor oid plexus. Deposit ion also occur s at t he basement membr ane of t he der mal - epider mal
junct ion, and t he endot helial linings of blood vessels.
Detection of circulating immune complexes
Tissue bound complexes ar e usually visualized using immunofluor escent st aining. A
mult it ude of assays exist s for t he det ect ion of cir culat ing immune complexes.
One a ssa y is ba sed on t he t endency of complexes t o pr ecipit a t e in t he cold. The
cr yop r e ci p i t a t e s ar e t hen analysed for immuno globulin t ype, ant igen cont ent and pr esence
of complement .
A mor e sensit ive assay is based on t he abilit y of immune complexes t o bind C1q. C1q is
r adiolabelled and mixed wit h pat ient ’s ser um. The immune complexes bound t o C1q ar e
pr ecipit at ed out , t he amount of unbound C1q is quant ified as an indir ect indicat or of pr esence
of immune complexes.
Hypersensitivity 141
Ot her assays depend on t he abilit y of immune complexes t o bind t o cells t hat have r ecept or s
for t he Fc por t ion of IgG or for complement component s, such as t he Raji cell line or macr ophage
cell lines. The assays measur e t he amount of immune complexes bound t o cells. Close monit or ing
of cir culat ing immune complex levels helps t o evaluat e t her apy in a number of disease st at es.
Type IV-Cell Mediated or Delayed Type Hypersensitivity
Delayed hyper sensit ivit y is a cell mediat ed immune r eact ion in an individual pr eviously
sensit ized t o an ant igen. A skin react ion t ypically develops 12-48 hours aft er int radermal inject ion
of ant igen. The best known example of t his r eact ion is t he posit ive Mant oux r eact ion wher e
t uber culin is inject ed int o t he skin of an individual, in whom pr evious exposur e t o Mycobacterium
tuberculosis has induced a st at e of cell mediat ed immunit y (CMI). The r eact ion appear s aft er
sever al hour s and may t ake upt o 48 hour s t o r each a maximum, hence t he usage of t he t er m
“delayed” hyper sensit ivit y. Hist ologically, t he lesion consist s of a pr edominant ly mononuclear
cell infilt r at e - of t he monocyt e- macr ophage ser ies. This cont r ast s wit h t he essent ially neut r ophil
pr edominant char act er of t he Ar t hus r eact ion.
Similar lesions can be seen in or gans under going cell mediat ed immune r eact ions. For
example, t he gr anulomas t hat for m ar ound S chistosoma mansoni eggs in t he liver , ar e t he
r esult of cell mediat ed immune r eact ions. Cell mediat ed hyper sensit ivit y is r esponsible for t he
cavit at ion and caseat ion of t uber cular lesions and for t he gr anulomat ous skin lesions of t he
bor der line for m of lepr osy.
The skin r ashes of small pox and measles and t he lesions of her pes simplex have been
at t r ibut ed t o delayed t ype hyper sensit ivit y r eact ions wit h associat ed cyt ot oxic T cell damage t o
vir ally infect ed cells. Cell mediat ed hyper sensit ivit y is also evident in fungal diseases such as
candidiasis, der mat omycosis, coccidioidomycosis and hist oplasmosis and in par asit oses such as
leishmaniasis and schist osomiasis.
Cellular Reactions in type IV-Hypersensitivity : the chronic granuloma
The t ype IV hyper sensit ivit y r eact ion r esult s fr om an exagger at ed int er act ion bet ween
ant igen and a nor mal cell mediat ed immune r eact ion. Delayed hyper sensit ivit y t her efor e has
t he same mechanism as cell-mediat ed immunit y. T cells r ecognize ant igen t oget her wit h MHC
Class II molecules and ar e act ivat ed, r esult ing in pr olifer at ion and r elease of lymphokines. T8-
lymphocyt es once sensit ized t o an ant igen differ ent iat e int o cyt ot oxic T-lymphocyt es while T
h
1
t ype T4-lymphocyt es become sensit ized t o an ant igen and pr oduce cyt okines . When t his r eact ion
does not get r id of t he offending ant igen, per sist ing ant igen evokes a chr onic cell mediat ed
hyper sensit ivit y r eact ion. CTLs, cyt okines, and/or macr ophages t hen cause har m r at her t han
benefit . Cont inual r elease of lymphokines leads t o infilt r at ion of macr ophages wit h ar r ays of
epit helioid cells. Some cells fuse t o for m mult inucleat ed giant cells. Fur t her , t issue damage
occur s when K cells and NK cells join t he for ay against for eign ant igen and indiscr iminat e
cyt ot oxicit y r esult s. Hist ologically, t her e is evidence of lymphocyt es, macr ophages, epit helioid
cells and giant cells. Tissue necr osis is sur r ounded by t he above cellular r eact ion wit h ar eas of
fibr osis in t he per ipher y. This lesion r epr esent s t he chr onic gr anuloma and is an at t empt by
t he body t o wall off a sit e of per sist ent infect ion.
Unlike ot her for ms of hyper sensit ivit y, delayed hyper sensit ivit y does not involve ant ibody
and cannot be t r ansfer r ed fr om a sensit ized individual t o a non sensit ized individual wit h ser um
ant ibody.
142 Immunology– Introductory Textbook
Cutaneous Basophil Hypersensitivity
Cut aneous basophil hyper sensit ivit y is a t er m for a gr oup of delayed onset lymphocyt e
mediat ed r eact ions which have been st udied ext ensively in guinea pigs. They ar e also seen in
humans and wer e or iginally called J on e s - Mot e r eact ions. They differ fr om classic delayed
hyper sensit ivit y r eact ions in a number of ways. The skin lesions ar e int ensely infilt r at ed by
basophils as well as lymphocyt es, t hey ar e er yt hemat ous but lack t he indur at ion and fibr in
deposit s in classic delayed t ype react ions. This t ype of react ion is seen in human cont act dermat it is
and in skin and r enal allogr aft r eject ion. At pr esent it is not known what fact or s det er mine
whet her a r eact ion will t er minat e as a basophil hyper sensit ivit y or a classical delayed t ype
hyper sensit ivit y r eact ion.
Contact Hypersensitivity (contact dermatitis)
Cont act hyper sensit ivit y can occur in people who become sensit ized while wor king wit h
chemicals such as picr yl chlor ide and chr omat es, or who r epeat edly come int o cont act wit h
poison ivy. p-Phenylene diamine in cer t ain hair dyes, neomycin in t opically applied oint ment s
and nickel salt s as in nickel coat ed cost ume jeweller y can evoke a similar r eact ion. These
chemical subst ances bind t o body const it uent s t o for m ant igenic mat er ial capable of inducing a
delayed hyper sensit ivit y r eact ion. The r eact ion t o t hese neo-ant igens is char act er ized by a
mononuclear infilt r at e, accompanied by oedema of t he epider mis and micr o vesicle for mat ion.
A summar y of t he differ ent t ypes of hyper sensit ivit y is pr esent ed in Table: 15.2
Table 15.2: Comparison of different types of hypersensitivity
Type Descriptive Initiation Mechanism Examples
Name Time
I. IgE-mediated 2-30 mins Ag induces cross –
hypersensitivity linking of IgE bound to
mast cells with release
of vasoactive
mediators
II. Antibody-mediated 5-8hrs Ab directed against
cytotoxic hypersensitivity cell-surface antigens
mediates cell
destruction via ADCC
or complement
III. Immune-complex mediated 2-8hrs Ag-Ab
hypersensitivity complexes deposited
at various sites induces
mast cell degranulation
via Fcgamma, PMN
degranulation damages
tissue
IV. Cell-mediated 24-72hrs Memory T
h
1 cells
hypersensitivity release cytokines that
recruit and activate
macrophages
™™™
Systemic anaphylaxis,
local anaphylaxis, hay
fever, asthma, eczema
Blood transfusion
reactions, haemolytic
disease of the newborn,
autoimmune haemolytic
anaemia
Arthus reaction
(Localised); systemic
reactions disseminated
rash, arthritis,
glomerulonephritis
Contact dermatitis,
tubercular lesions
IMMUNOLOGIC TOLERANCE AND
AUTOIMMUNITY
CHAPTER – 16
O
ne of t he cent r al concept s of immunology has been t hat t he immune syst em should
be able t o dist inguish bet ween “self” and “non self”. Immune r eact ions t o “self” ant igens
const it ut es what is known as aut oimmunit y and is injur ious t o t he host . Ar ound 1900, Paul
Ehr lich post ulat ed t hat t he immune syst em acquir es a st at e of t oler ance t o self ant igens, in
addit ion he pr oposed t hat br eak down of t oler ance would lead t o self dest r uct ion, a condit ion
he descr ibed as “hor r or aut ot oxicus”. Over 40 year s ago, Owen demonst r at ed t oler ance in non
ident ical (dizygot ic) t win calves who shar ed t he same placent al cir culat ion. Even t hough each
calf had appr eciable number s of r ed cells fr om t he ot her t win (due t o t he common placent a),
t hey did not mount a r eact ion t o t he ot her ’s r ed cells. If t hey had not shar ed t he same placent al
cir culat ion, an infusion of t he ot her t win’s r ed cells in adult life, would have caused a sever e
immunological r eact ion.
About 50 year s aft er Paul Ehr lich, Macfar lane Bur net suggest ed t hat immune cells r eact ing
wit h ant igens dur ing development of t he foet us ar e dest r oyed by lymphoid or gans. This leads
t o t oler ance t o such ant igens since t he clones of cells r eact ing against such ant igens have been
delet ed dur ing foet al development . In t he last 15 t o 25 year s t her e have been major r evisions of
t hese classic concept s. It has become clear t hat at a finer level of det ail, t he law t hat a nor mally
funct ioning immune syst em does not r ecognize self is not absolut e. The r ecept or s of t he immune
syst em t hat per for m t he wor k of r ecognit ion can t hemselves be r ecognized by ot her r ecept or s.
Such “self r ecognit ion”, which was st r ict ly out lawed in t he ear ly year s, may for m t he basis of a
net wor k whose equilibr ium keeps t he body healt hy. Anot her impor t ant for m of self r ecognit ion
involves immuno compet ent cells which have been schooled t o r ecognize t he body’s own major
hist ocompat ibilit y ant igens. As discussed ear lier , t his for m of self r ecognit ion for ms t he key
r eact ion in many immunological pr ocesses beneficial t o t he host . Aut oimmune disease occur s
when t hese nor mal “aut oimmune” r eact ions ar e dist ur bed.
Forms of Normal Auto Recognition or Positive Autoimmunity
The idiotype - anti idiotype network
It is now clear t hat t he hyper var iable r egions of t he immunoglobulin molecules r eact wit h
ant igenic det er minant s also called epit opes. Impor t ant discover ies clear ly illust r at e t hat t hose
sit es on t he hyper var iable r egion of t he immunoglobulin molecule t hat bind t o ant igens ar e
t hemselves immunogenic: being complex pr ot ein molecules. Individual ant igenic sit es on t he
immunoglobulin hyper var iable r egion ar e called idiot opes. A set of idiot opes on a single
immunoglobulin molecule is called t he idiot ype. If an idiot ype is involved in ant igen binding, it
is called a par at ope. It st ands t o r eason t hat if an ant igen such as t he idiot ype exist s, t hen t he
human immune syst em must be equipped t o pr oduce ant ibodies t o idiot ypes. Hence it is now
144 Immunology– Introductory Textbook
clear t hat a net wor k exist s wher ein t he immune syst em pr oduces ant i idiot ypic ant ibodies t o
it s own idiot ypes. Cr edit for t hese discover ies goes t o Niels J er ne who in 1974, pr oposed t hat
nor mal aut oimmune r esponses t o self idiot ypes, might for m t he basis of immuno r egulat or y
net wor k syst ems. Homeost asis of t he immune syst em is t hus t hought t o be pr eser ved t hr ough
a funct ional assembly of idiot ype ant i idiot ype int er act ions.
Accor ding t o t his model, an ant igen induces pr oduct ion of an ant ibody (Ab1) char act er ized
by it s idiot ype (Id1). In t ur n, (Id1) st imulat es t he synt hesis of an ant i idiot ypic ant ibody (ant i-Id1
or Ab2), bear ing t he idiot ype Id2, t hat can, in t ur n, t r igger t he pr oduct ion of ant i Id2 or (Ab3)
(Figur e 16.1). Theor et ically, t hese idiot ype - ant i idiot ype r eact ions can cont inue indefinit ely.
However , t he int er act ions appear t o be limit ed. In addit ion, it has been post ulat ed t hat ant i
idiot ypes t hat ar e dir ect ed t owar ds par at opes (ant igen combining sit es), must st er eochemically
(by vir t ue of 3-D st r uct ur e), r esemble t hat par t of t he ant igen t hat locks wit h t he par at ope. This
is a r easonable post ulat e since bot h t he ant i idiot ype and t he ant igen combine wit h t he same
binding sit e on t he par at ope. Such ant i idiot ypes wer e t er med t he int er nal image set by J er ne;
Lindenmann called t hem homobodies.
Figure 16.1. Idiotype-Anti idiotype reactions. The idiotype network has been proposed as a
mechanism to regulate the immune response. The presence of a foreign antigen leads to production
of antibodies (Ab-1) that recognize the determinants of that antigen. Each of these antibodies
contains a collection of unique regions that are collectively known as the idiotype; the idiotype
includes the antigen binding site of the immunoglobulin molecule. The presence of Ab-1 leads to
production of a second type of antibody, anti-idiotype antibody (Ab-2), which recognizes the
idiotypes of Ab-1. Ab-2 also contains idiotypes and hence leads to production of a third type of
antibody, anti-anti-idiotype antibody (Ab-3). Although the network theoretically can continue
indefinitely, it appears to be limited.
It is gener ally believed t hat t he idiot ype ant i idiot ype net wor k cont r ibut es t o t he r egulat ion
of t he immune syst em. Besides dir ect ly int er act ing wit h B cell r ecept or s or wit h ant ibodies in
t he ser um, ant i idiot ype ant ibodies could modulat e t he immune r esponse by act ivat ing or
r epr essing differ ent T cell subset s. The concept emer ging, is t hat idiot ype - ant iidiot ype cir cuit s
may r egulat e immune r esponses at appr opr iat e levels: t he helper T cell st age, t he plasma cell
and t he level of t he for med ant ibody secr et ed int o t he cir culat ion.
Immunologic Tolerance and Autoimmunity 145
Recognition of S elf MHC by T cells
This has been discussed ext ensively in ear lier chapt er s. Suffice it t o say, t hat it has now
been demonst r at ed beyond any doubt t hat immunocompet ent cells of t he ver t ebr at e immune
syst em pr eser ve and expr ess t hr ough out life, a self r ecognit ion capacit y especially of MHC
molecules. This is now known t o be essent ial for t he nor mal funct ioning of t he immune syst em
in all it s diver sit y.
Tolerance
If t he nor mal immune syst em can r espond t o vir t ually any for eign subst ance, why does it
not r espond dest r uct ively t o self ant igens? The absence of har mful immune r eact ions t o ant igens
is t er med t oler ance. It has been shown t hat t oler ance can be induced in animals by var ying t he
dosage of t he immunogen. Rabbit s which ar e r epeat edly exposed t o low doses of a weak
immunogen, r emain t oler ant even t o a st r ongly immunogenic for m of t he same ant igen.
Similar ly, t oler ance can be induced even if high doses of t he immunogen ar e administ er ed t o
t he animal. Hence t her e seems t o be a low zon e and a h i gh zon e in t er ms of ant igen dosage
for t oler ance induct ion (Figur e 16.2). Exper iment s have shown t hat t he T cell is t he t ar get for
low zone ant igen t oler ance and bot h T cells and B cells ar e ir r esponsive at high ant igen dosage.
Figure 16.2. High zone and low zone tolerance.
How does an individual t oler at e self ant igens while gener at ing nor mal immune r eact ions
t o for eign ant igens? The answer t o t his quest ion is not known. St udies indicat e t hat t her e ar e
sever al mechanisms which may cont r ibut e t o t oler ance: clonal delet ion, clonal aner gy, r ecept or
blockade, non- immunogenic ant igens, ant i idiot ype ant ibodies, suppr essor T cell fact or s and
ot her r egulat or y fact or s.
Theories of Tolerance Induction
We can divide t he mechanisms t he immune syst em uses t o ensur e t he absence of self-
r eact ivit y (a u t oi mmu n i t y) int o t wo main t ypes:
• Ce n t r a l Tole r a n ce : t his occur s dur ing lymphocyt e development .
• P e r i p h e r a l Tole r a n ce : occur s aft er lymphocyt es leave t he pr imar y or gans.
146 Immunology– Introductory Textbook
Central Tolerance
Macfar lane Bur net pr oposed t he t heor y of clon a l select i on (Refer Chapt er 11). In a br ief
r ecapit ulat ion, t he t heor y st at es t hat clones of immune cells t hat ar e genet ically capable of
r eact ing wit h all pot ent ial immunogens ar e pr esent in t he immune syst em. Gener at ion of an
immune r esponse t hus r esult s fr om amplificat ion and pr olifer at ion of t hat clone of cells best
suit ed for a par t icular invading for eign ant igen. Bur net also pr oposed t hat t oler ance may occur
when foet al immunocyt es ar e exposed t o and r ecognize a specific ant igen - a pr ocess t hat leads
t o delet ion of t hose clones of immunocyt es. Suppor t for t he clonal delet ion t heor y came fr om
exper iment s conduct ed by Bi lli n gh a m a n d Me d a wa r . They infused lymphoid cells fr om one
inbr ed st r ain of mice (St r ain A) int o neonat al mice belonging t o anot her inbr ed st r ain (st r ain
B). Nor mally skin gr aft s fr om st r ain A mice would be r eject ed by st r ain B mice. However in
t his exper iment , neonat al st r ain B mice, having been exposed t o lymphoid cells fr om st r ain A
mice became t oler ant t o cell sur face ant igens of t he st r ain A mice. Ther eaft er st r ain B mice
could r eceive skin gr aft s fr om st r ain A mice and t oler at e t he gr aft .
Unfor t unat ely ear ly t est s t o pr ove t he clonal delet ion t heor y wer e unset t lingly negat ive.
Clinical immunologist s began finding ant ibodies against nor mal body pr ot eins such as insulin,
in t he blood of healt hy people. These ant ibodies ought not t o have been t her e if self r eact ive
clones of B cells ar e delet ed dur ing foet al development . Obviously some self r eact ive clones of
B cells wer e sur viving. Solid pr oof t hat clonal delet ions act ually occur r ed, appear ed in 1988 —
33 year s aft er Bur net fir st pr oposed t he idea. In independent st udies, P h i lli p a Ma r r a ck and
ot her wor ker s demonst r at ed t hat cer t ain self r eact ive clones of mat ur ing T cells wer e delet ed
fr om t he t hymus and never r eached t he r est of t he body. It is now appar ent t hat T cells ent er ing
t he t hymus ar e schooled by t wo select ion pr ocesses. In t he fir st pr ocess, also called p osi t i ve
se le ct i on , T cells ar e t aught t o r ecognize t he MHC pr ot eins; an essent ial lear ning st ep for
adequat e T cell funct ion (see Chapt er 3). The second st ep, also called n e ga t i ve se le ct i on , is
cr it ical for self t oler ance. Developing T cells ar e exposed t o a pot -pour r i of self ant igens. Young
T cells t hat t ake t he bait and bind t o self ant igens die. Danger ously self r eact ive T cells ar e
t her efor e weeded out in t he t hymus.
Ma t zi n ge r has pr oposed t hat dur ing neonat al life, whet her encount er wit h an ant igen
r esult s in t oler ance or an immune r esponse is det er mined by t he pr evailing host envir onment
pr oducing nonspecific cues ‘sensing’ danger . Neonat al T cells, she claims, ar e not int r insically
t oler isable but t he syst emic neonat al envir onment does pr edispose t o t oler ance. She has fur t her
suggest ed t hat t he cont r olled deat h pr ocess of apopt osis is cr it ical in pr event ing aut oimmunit y
when old or sur plus cells ar e disposed of.
Peripheral Tolerance
Clonal delet ion, however , is not t he ent ir e answer . Some invest igat or s believe, t hat it is
incr edible t hat ever y pot ent ial self ant igen in t he body must some how manifest it self in t he
t hymus so t hat T cells can be scr eened. Clon a l a n e r gy has t her efor e emer ged as a possible
alt er nat ive mechanism for self t oler ance. The t er m clon a l a n e r gy was coined by Gu st a v
Nossa l in t he mid - 1970s. He found t hat under cer t ain cir cumst ances, B cells in cult ur e could
not be st imulat ed by ant igen. The unr esponsive B cells did not die but per sist ed in a lazy or
aner gized st at e. Alt hough t hese exper iment s wer e elegant and infor mat ive, t hey do not r eveal
what ult imat ely happens t o aner gic, self r eact ive B cells especially in vivo. Almost 20 year s ago
P et er Br et sch er and Mel Coh n proposed t he t wo sign a l h yp ot h esis for lymphocyt e act ivat ion
Immunologic Tolerance and Autoimmunity 147
and t oler ance induct ion. They suggest ed t hat a B cell must r eceive t wo consecut ive signals in
or der t o be act ivat ed: signal 1 fr om t he ant igen and signal 2 fr om a second cell specific for t he
same ant igen, for example, a T helper cell. If a B cell was, however , r eact ing t o a self ant igen,
it would find it self alone and unable t o r eceive signal 2 fr om a T helper cell, except in t hose
exceedingly r ar e cir cumst ances when a T helper cell is also r eact ing specifically t o t he same
self ant igen. Thus self r eact ive B cells r eceiving only signal 1 fr om t he aut o ant igen could not
be act ivat ed t o r espond wit hout adequat e T cell help. Such a B cell was t her efor e aner gized
(Fig 16.3).
Figure 16.3. The two signal explanation for immunologic tolerance.
Alt hough t his model was or iginally pr oposed t o explain B cell t oler ance, Sch wa r t z a n d
J e n k i n s have now shown t hat a similar t wo signal t heor y can be used t o explain T cell
act ivat ion and t oler ance (Figur e 16.3). The essence of t he t wo signal model is t hat simply
binding of t he T cell r ecept or t o an ant igen molecule is not enough t o t r igger T cell act ivat ion.
The T cell must r eceive an addit ional signal of some kind fr om t he ant igen pr esent ing cell.
Wit hout t his second signal, a T cell becomes aner gized and st ays t hat way unless “r eset ” by t he
ant igen pr esent er s. It is t hought t hat only a few t ypes of specialized cells - macr ophages for
example, may be able t o send t his second signal needed t o give T cells a push int o it s act ive,
pr olifer at ive st at e. To dat e it is not known what pr ecise st eps ar e involved in t he wor king of t he
clonal aner gy t heor y.
Anot her mechanism of t oler ance induct ion is li ga n d -i n d u ce d a ct i va t i on or a n t i ge n
block a d e. In t his mechanism, ant igen, par t icular ly mult ivalent ant igen, int er act s wit h ant igen
r ecept or s on B cells, under cir cumst ances t hat int er fer e wit h t he pr ocesses of pat ching and
endocyt osis which ar e necessar y for clonal expansion. Cer t ain monovalent ant igens may block
148 Immunology– Introductory Textbook
B cell r ecept or s wit hout leading t o pat ch for mat ion and cer t ain mult ivalent ant igens and immune
complexes may immobilize membr ane r ecept or s and pr event pat ch for mat ion (Figur e 16.4).
Eit her of t hese int er act ions r ender s lymphocyt es unr eact ive t o subsequent ant igen exposur e.
In addit ion, occupancy of B cell r ecept or s by non immunogenic for ms of ant igen, could pr event
int er act ion wit h immunogenic for ms such as ant igen pr esent ed by macr ophages. Ant igen
blockade pr oduces r ever sible aner gy because it does not lead t o cell deat h.
Figure 16.4. Antigenic blockade by monovalent and multivalent antigens.
I mmune t oler a nce ma y a lso be induced by ot her mecha nisms. Some a nt igens a r e
sequest er ed fr om t he immune syst em in locat ions which ar e not fr eely exposed t o sur veillance.
These ar e t er med immunologically pr ivileged sit es. Examples of such sit es ar e t he eye, CNS
and t est is.
Though t he once popular suppr essor T cell t heor y for t oler ance seems t o have fallen int o
disfavour , some r esear cher s believe t hat t he “dat a and t he phenomena ar e st ill t her e”; however ,
t her e seems t o be insufficient exper iment al evidence t o pr ove t he suppr essor T cell phenomenon.
It is t he exist ence of a unique suppr essor cell t hat is most cont r over sial. The hist or y of t his fast
changing field only shows t hat one year ’s unfashionable concept can become t he next year ’s
dogma.
At t he molecular level, st udies now indicat e t hat binding of ant igen t o MHC gene pr oduct s
may play a r ole in immunologic t oler ance. MHC Class II gene pr oduct s on t he sur face of ant igen
pr esent ing cells, have been shown t o bind t o pr ocessed pr ot ein ant igens. It has been suggest ed
Immunologic Tolerance and Autoimmunity 149
t hat self ant igens may be differ ent iat ed fr om non self ant igens by t he way t hey bind t o MHC
Class II gene pr oduct s, aft er t hey have been pr ocessed wit hin t he ant igen pr esent ing cells.
Autoimmunity
Aut oimmune disease is defined as disease caused by immunologic r eact ion t o self ant igens.
Such diseases ar e classified eit her as or ga n sp eci fi c or syst emi c based on t he pr imar y locat ion
of t he injur y (Table 16.1).
Table 16.1: Classification of Autoimmune diseases
Disease Target of Antibody
Organ specific diseases
Myasthenia gravis Acetylcholine receptors
Graves’ disease Thyroid stimulating hormone receptor
Thyroiditis Thyroid
Insulin dependant diabetes with Insulin receptor
acanthosis nigricans
Insulin dependant diabetes with Insulin receptor
ataxia telangiestasia
Allergic rhinitis, asthma auto immune β 2-adrenergic receptor
abnormalities
Juvenile insulin dependant diabetes Pancreatic islet cells, insulin
Pernicious anaemia Gastric parietal cells, Vitamin B12 binding
site for intrinsic factor
Addison’s disease Adrenal cells
Idiopathic hypoparathyroidism Parathyroid cells
Spontaneous infertility Sperm
Premature ovarian failure Interstitial cells, corpus luteum cells
Pemphigus Intercellular substance of skin and mucosa
Bullous pemphigoid Basement membrane zone of skin and mucosa
Primary biliary cirrhosis Mitochondria
Autoimmune haemolytic anaemia Erythrocytes
Idiopathic thrombocytopenic purpura Platelets
Idiopathic neutopenia Neutrophils
Vitiligo Melanocytes
Osteosclerosis and Meniere’s disease Type II collagen
Chronic active hepatitis Nuclei of hapatocytes
Systemic diseases
Goodpasture’s syndrome Basement membranes
Rheumatoid arthritis Gamma globulin, Epstein-Barr virus-related
antigens, types II and III collagen
150 Immunology– Introductory Textbook
SjÖgren’s syndrome Gamma globulin
Systemic lupus erythematosus Nuclei, double-stranded DNA, single-stranded
DNA, ribonucleoprotein, lymphocytes,
erythrocytes, neurons, gamma globulin.
Scleroderma Nuclei, centromere
Polymyositis Nuclei, histadyl-tRNA synthetase, threonyl-
tRNA synthetase
Rheumatic fever Myocardium, heart valves, choroid plexus.
Mechanisms of Tissue Injury in Autoimmune Diseases
Thr ee mechanisms ar e pr incipally r esponsible for inflammat ion and t issue injur y in
aut oimmune disease
• Cell lysis and r elease of inflammat or y mediat or s t r igger ed by aut o ant ibodies
• Immune complex disease
• T-cell mediat ed damage
In t he fir st mechanism, cir culat ing ant ibodies r eact wit h modified or unmodified ant igens
on cell sur faces. The bound ant ibodies t hen st imulat e t he r elease of mediat or s of inflammat ion,
t r igger t he complement pat hway or act ivat e K cells, via ant ibody dependant cellular cyt ot oxicit y
(ADCC). The lat t er t wo pr ocesses r esult in cell lysis.
In t he second mechanism, complexes bet ween aut o ant ibodies and ant igens for m in t he
cir culat ion or in int er cellular fluids. These immune complexes deposit in var ious t issues,
including glomer uli, joint s and blood vessels; t hey subsequent ly fix complement and cause
inflammat ion and t issue injur y. The sit e of deposit ion is det er mined by t he physical pr oper t ies
of t he immune complex, such as it s size and char ge.
I n t he t hir d mecha nism, sensit ized T cells eit her injur e cells dir ect ly or r elea se
lymphokines t hat amplify t he inflammat or y r esponse. Alt hough t issue injur y caused by cell-
mediat ed mechanisms may be impor t ant in aut oimmune disease, it s r ole is cur r ent ly unclear .
Cell lysis by autoantibodies
Many aut oimmune diseases ar e pr imar ily init iat ed by t he int er act ion bet ween aut o
ant ibodies and cell sur face ant igens. One r esult of aut o ant ibody binding is cell dest r uct ion.
This occur s in Addison’s disease, due t o ant i adr enal cell ant ibodies; in Hashimot o’s t hyr oidit is,
due t o ant i t hyr oid ant ibodies; in sever al ot her endocr inopat hies and in some haemolyt ic
anaemias and leukopenias.
Alt er nat ively, aut o ant ibodies may bind t o cell sur face r ecept or s and int er fer e wit h t heir
funct ion. In myast henia gr avis, aut o ant ibodies bind t o acet ylcholine r ecept or s on t he mot or
end plat e. This leads t o dest r uct ion of t he r ecept or , defect ive neur o muscular t r ansmission and
r esult ant skelet al muscle weakness. Ant ibodies t o many hor mone r ecept or s may act as eit her
ant agonist s or agonist s. Ant ibodies t o t he insulin r ecept or , in pat ient s wit h diabet es mellit us,
may ant agonize insulin act ion by r educing t he availabilit y of t he r ecept or . In Gr ave’s disease,
t he long act ing t hyr oid st imulat or (LATS) is an aut o ant ibody t o t hyr ot r opin r ecept or ; it act s as
an agonist , causing excessive secr et ion of t hyr oid hor mones.
Immunologic Tolerance and Autoimmunity 151
Immune complex deposition
In a number of syst emic aut oimmune diseases, t issue injur y is mediat ed bot h by dir ect
aut o ant ibody binding and by immune complex deposit ion. In syst emic lupus er yt hemat osus
(SLE), t he prot ot ype syst emic aut oimmune disease, ant ibodies bind t o t he eryt hrocyt e, leukocyt e,
and plat elet ant igens, causing haemolyt ic anaemia, leukopenia and t hr ombocyt openia; ant i-
neuronal ant ibodies cont ribut e t o neurologic disease. Addit ionally, circulat ing immune complexes
consist ing of DNA- ant i DNA ar e deposit ed in t he glomer ulus of t he kidney and in ot her or gans
causing t issue injur y. Two significant fact s point t o t he r ole of immune complexes in SLE.
Fir st , pat ient s demonst r at e significant deplet ion of complement (C3) and neut r ophils as a r esult
of act ivat ion by complexes. Second, complement deficiencies which impair immune complex
clear ance (C1,C2 or C4,) ar e ver y st r ong pr edisposing fact or s for SLE.
In Rheumat oid ar t hr it is, t he aut o ant ibody called r heumat oid fact or , is usually an IgM,
which is dir ect ed t owar ds t he Fc por t ion of t he pat ient ’s own IgG. Complexes of r heumat oid
fact or IgM coupled wit h IgG, get deposit ed at var ious sit es leading t o t he char act er ist ic synovit is
and vasculit is of r heumat oid ar t hr it is. For a mor e complet e descr ipt ion of t he pat hogenesis,
diagnosis and t r eat ment of t he var ious aut oimmune diseases, t he r eader is dir ect ed t o r efer t o
t he many excellent t ext books of int er nal medicine available.
T-cell mediated damage
This t er m implies t hat t he r ecognit ion of aut oant igen by T cells leads t o t issue dest r uct ion
wit hout r equir ing t he pr oduct ion of aut oant ibody. Ther e ar e a number of ways t his can come
about :
• Dir ect T cell cyt ot oxicit y via CD8
+
CTL
• Self-dest r uct ion of t issue cells induced by cyt okines, e.g. TNF α
• Recr uit ment and act ivat ion of macr ophages leading t o byst ander t issue dest r uct ion
• Induct ion of t ar get t issue apopt osis by t he T cell membr ane pr ot ein F a sL
In most cases we do not know what t he r elat ive cont r ibut ion of t hese fact or s is.
The Aetiopathogenesis of Autoimmune Disease
How does a finely balanced, per fect ly t uned net wor k of immune r esponses t o for eign
ant igens br eak down t o cause an immunological r eact ion t hat is self dest r uct ive? Many t heor ies
and mechanisms have been pr oposed for t he gener at ion of aut o immune r esponses.
Exposure of sequestered antigens
If an ant igen is sequest er ed wit hin an or gan dur ing foet al development , t hen as per t he
t heor ies of clonal delet ion t he immune syst em does not acquir e t oler ance t o t his ant igen. No
aut oimmune r esponse develops as long as t hese ant igens r emain unexposed. Should t issue
damage or injur y occur t o expose t hese ant igens, aut o ant ibodies t o t hese sequest er ed ant igens
r a pidly a ppea r in t he cir cula t ion. Such a n a ut o a nt ibody r ea ct ion ha s been r epea t edly
demonst r at ed: aut o ant ibody against sper m aft er vasect omy, against lens pr ot ein aft er eye
injur y, against hear t muscle ant igens aft er a myocar dial infar ct . These ant ibody r esponses ar e
t r ansient in most cases, appar ent ly br ief exposur e is insufficient for inducing aut oimmune
disease, which may r equir e per sist ent exposur e t o sequest er ed ant igen.
152 Immunology– Introductory Textbook
Altered self antigens and molecular mimicry
Alt er at ion of an ant igen t o which a host is t oler ant , may allow immune r eact ions t o t he
t oler ant ant igen t o develop. Exper iment ally it has been shown t hat r abbit s made t oler ant t o
bovine ser um albumin (BSA) can be st imulat ed t o pr oduce ant ibodies t o nat ive, unalt er ed BSA
when t hey ar e immunized wit h chemically alt er ed BSA. Alt er at ion of self ant igens occur s in
t he human syst em as a r esult of vi r a l or b a ct e r i a l i n fe ct i on s or t her apy wit h cer t ain d r u gs.
Ant ibodies t o t he I blood gr oup ar e for med following Mycoplasma pneumoniae infect ion.
Aut oimmune haemolyt ic anaemia is associat ed wit h administ r at ion of α met hyl dopa. The dr ug
is t hought t o modify t he r ed cell sur face in such a way t hat cer t ain B cell clones r eact against
t hese modified r ed cells, causing haemolysis. Similar ly, pr ocainamide and hydr alazine seem t o
pr ovoke t he pr oduct ion of ant inuclear ant ibodies.
A r elat ed mechanism t er med molecu la r mi mi cr y comes int o play when for eign ant igens
r esemble self ant igens. The encephalit is t hat somet imes r esult s fr om t he administ r at ion of t he
neur al r abies vaccines is t hought t o occur due t o an aut oimmune r eact ion t o human br ain
t issue, which cr oss r eact s wit h neur al t issue found in t he neur al vaccine. Similar ly, in r heumat ic
fever , st r ept ococcal ant igens induce ant ibodies which r eact wit h component s of t he myocar dium.
Pr esumably st r ept ococcal ant igens r esemble hear t ant igens enough, t o elicit an aut oimmune
r eact ion.
The idiotype anti-idiotype network
The concept of ant i idiot ypic ant ibodies being st er eochemically similar t o t he ant igenic
det er minant , since bot h bind t o t he one ant ibody, has been discussed ear lier in t his chapt er .
Ant i idiot ypic ant ibodies t hat st r uct ur ally r esemble a vir al ant igen, will be able t o bind t o t he
r ecept or for t he vir us on t he cell sur face. This can st imulat e a cyt ot oxic r eact ion against t he
r ecept or bear ing cells. Ther efor e ant i vir al ant ibodies may induce ant i idiot ype ant ibodies t hat
cr oss r eact wit h nor mal host t issues.Ther e ar e sever al examples of aut oimmune r eact ions t hat
might be account ed for by t he pr esence of cr oss r eact ive ant i idiot ype ant ibodies. Immunizat ion
of r abbit s wit h ant ibodies t o human t hyr ot r opin pr oduces ant i idiot ype ant ibodies t hat mimic
t hyr ot r opin r eact ion, by combining wit h t hyr ot r opin r ecept or s. This ant i idiot ype ant ibody
r esembles LATS found in pat ient s wit h Gr ave’s disease (Figur e 16.5).
Figure 16.5. Idiotypic mechanisms leading to autoimmunity.
Immunologic Tolerance and Autoimmunity 153
Abnormal expression of MHC antigens
Act ivat ed helper T cells ar e necessar y for pr oducing ant ibodies t o most ant igens, including
self ant igens. These cells become act ivat ed only when pr esent ed wit h ant igen by cells t hat
expr ess MHC Class II ant igens. Many self–r eact ive CD4+ T cells ar e eliminat ed in t he t hymus
dur ing mat ur at ion (see clonal delet ion). However , sever al ant igens such as neur al and endocr ine
ant igens ar e not pr esent in t he t hymus, t her efor e t hose T cells t hat ar e self r eact ive wit h
r egar d t o such ant igens cannot be scr eened and eliminat ed wit hin t he t hymus. Such T cells
escape int o t he per ipher al t issues and ar e able t o r ecognize t he above self ant igens when
pr esent ed in associat ion wit h MHC Class II ant igens.
This pot ent ial self r eact ivit y is usually pr event ed by t he limit ed dist r ibut ion of MHC Class
II ant igens, which ar e r est r ict ed t o just a few cell t ypes: t he macr ophages, T and B cells. These
ant igens can, however , be induced on ot her cells by vir al infect ion, t hr ough t he effect s of
int er fer ons (see Cha pt er 12). Dur ing a n infla mma t or y r esponse a n immunost imula t or y
envir onment is cr eat ed by t he r elease of cyt okines which r ecr uit and act ivat e pr ofessional
ant igen pr esent ing cells and pr ovide suppor t for T cell act ivat ion r at her t han aner gy. As a
r esult aut or eact ive T cells which wer e aner gic may become act ivat ed. This concept is cent r al t o
Mat zinger ’s Da n ge r Hyp ot h e si s. These t heor ies t her efor e pr ovide a possible mechanism for
t he induct ion of aut oimmune disease by infect ion or inflammat ion.
Genetic and other factors
Gen et ic fa ct or s are known t o play an import ant part in aut o immune disease. Aut oimmune
diseases t end t o occur in clust er s in cer t ain families. Hashimot o’s t hyr oidit is and SLE occur
more frequent ly among parent s, children and siblings. Furt hermore, t here are st rong associat ions
bet ween sever al aut oimmune diseases and par t icular HLA specificit ies for example, HLA DR3
in Addison’s disease and HLA DR4 in r heumat oid ar t hr it is. However , no single genet ic fact or
can account for any aut oimmune disease. Alt hough cer t ain HLA - DR ant igens ar e associat ed
wit h SLE, most individuals bear ing t hese ant igens ar e healt hy. Suscept ibilit y must t her efor e
be det er mined by mor e t han one genet ic fact or or by a combinat ion of fact or s.
Int erest ingly, cert ain inbred st rains of animals spont aneously develop aut oimmune disease.
Ther e is an obese line of chicken t hat spont aneously develop aut oimmune t hyr oidit is, and t he
New Zealand Black (NZB) mouse develops aut oimmune haemolyt ic anaemia. The hybr id of
NZB wit h NZW (whit e) develops LE cells, ant inuclear ant ibodies and fat al SLE. These animal
models pr ovide valuable insight int o t he st udy of aut oimmune diseases.
Cer t ain h or mon a l fa ct or s play a r ole in SLE and ot her aut oimmune diseases. Most
pat ient s wit h SLE ar e women in t heir child bear ing year s. In addit ion, pat ient s wit h SLE
excr et e excessive amount s of 16 α met abolit es of est r ones which have high oest r ogenic act ivit y.
In cer t ain st r ains of mice in which SLE develops, t he aut oimmune disease is aggr avat ed by
oest r ogens and r et ar ded by andr ogens.
Reviewing all t he exist ing evidence r egar ding aut oimmunit y, it is clear t hat spont aneous
aut oimmune disease is a mult ifact or ial phenomenon wit h immunologic, genet ic, vir ologic,
hor monal and ot her fact or s act ing singly or synchr onously t o pr oduce disease.
™™™
IMMUNOPOTENTIATION AND
IMMUNOSUPPRESSION
CHAPTER – 17
A
n under st anding of t he nor mal r egulat or y mechanisms of t he immune syst em has
yielded an insight int o t he many ways t he immune r eact ion may be enhanced or
inhibit ed, should t he need ar ise. Clinically, immunopot ent iat ion has been effect ive in combat ing
infect ious disease, in t he t r eat ment of immunodeficiency st at es and in t he sear ch for a cur e for
cancer .
Immunosuppr ession has found r elevance since t he bir t h of or gan t r ansplant at ion, and in
t he t r eat ment of aut oimmunit y and aller gy.
Immunopotentiation
Vaccination
Vaccinat ion has been used for over t wo cent ur ies as a means of exploit ing t he immune
syst em t o pr ot ect t he host against infect ious agent s. An under st anding of ant igen specificit y
and memor y has been used t o boost t he immune r esponse t o infect ious agent s by ar t ificially
exposing t he host t o small amount s of t he inact ivat ed infect ious agent . Immunizat ion has t hus
offer ed pr ot ect ion against a number of pot ent ially let hal infect ious diseases such as small pox,
poliomyelit is, per t ussis, dipht her ia, t et anus, mumps and r ubella t o name just a few.
Adjuvants
Responses t o many killed vaccines need t o be enhanced by subst ances t hat ar e collect ively
known as adjuvant s. An adjuvant , by definit ion, is a subst ance when incor por at ed int o or inject ed
simult aneously wit h ant igen pot ent iat es t he immune r esponse.
Mode of action of adjuvants
(i) On ant igen char act er ist ics: Some adjuvant s affect t he way in which ant igen is
pr esent ed. The immune r esponse is incr eased when pr ot ein ant igens ar e pr ecipit at ed
by alum. Ot her adjuvant s have a “depot ” effect , in t hat t hey pr event r apid disper sal of
ant igen fr om t he local t issues dr aining t he inject ion sit e. This r eser voir of ant igen is
t hen available eit her at an ext r a cellular locat ion or wit hin macr ophages. The most
common adjuvant of t his t ype is Fr eund’s incomplet e adjuvant , wher e ant igen, in t he
aqueous phase, is emulsified wit h par affin oil. Since par affin oil can pr oduce sever e
local r eact ions, it is not r ecommended for human use. Oils such as squalene and
peanut oil seem t o be bet t er t oler at ed when compar ed t o Fr eund’s incomplet e adjuvant .
Recent int er est has been focussed on t he use of liposomes, which ar e membr ane
bound lipid vesicles, as agent s for pr esent at ion of ant igen t o t he immune syst em.
Liposomes seem t o behave as st or age vacuoles for t he ant igen, t hey also ent er t he
macr ophage and ar e pr esent ed in a mor e immunogenic manner t o T cells.
Immunopotentiation and Immunosuppression 155
(ii) On host immune r esponse: Most adjuvant s, however , do not affect t he ant igenic
char act er ist ics but act on t he host immune r esponse. Vir t ually all adjuvant s st imulat e
macr ophages, a good example is F r e u n d ’s comp le t e a d ju va n t which is made fr om
incomplet e Fr eund’s adjuvant wit h t he addit ion of killed mycobact er ium or mor e
r ecent ly, wa t er soluble mur a myldipept ide. Such a djuva nt s a ct dir ect ly on t he
macr ophage or via t he T cell. Besides impr oving ant igen pr esent at ion, t hey enhance
t he accessor y signals r equir ed for lymphocyt e act ivat ion and pr olifer at ion.
Types of Adjuvants
(i) Or ganic adjuvant s: Or ganic adjuvant s include a var iet y of or ganic molecules obt ained
fr om bact er ia. Mur amyldipept ide (MDP) is a bact er ial pept idoglycan. MDP incr eases
bot h humor al and cellular immunit y. It st imulat es macr ophages and may, as a
consequence, r ecr uit T cell help in vivo.
(ii) Synt het ic adjuvant s: Synt het ic adjuvant s t hat incr ease host immunit y include
levamisole and isopr inosine. Levamisole was init ially int r oduced as an ant helmint hic
agent . It pot ent iat es humor al and cellular immunit y in a fashion t hat is T cell
dependant . It has been used wit h some success in t he t r eat ment of cancer and
r heumat oid ar t hr it is. Unfor t unat ely, t hese advant ages ar e not wit hout t he r isk of
side effect s of which agr anulocyt osis is t he most ser ious.
Isopr inosine is a complex cont aining inosine, a pur ine pr ecur sor . In vit r o isopr inosine
pr omot es T cell mit ogenesis. It s usefulness in t he t r eat ment of cancer has not been
est ablished.
(iii) Tuft sin : Tuft sin is a unique adjuvant t hat occur s nat ur ally and has been synt hesized
as well. It is a four amino acid pept ide-t hr eonine-lysine-pr oline-ar ginine, homologous
t o a sequence in t he const ant r egion of t he immunoglobulin heavy chain. Tuft sin
pr imar ily st imulat es macr ophages. Since it occur s nat ur ally it pr obably has a
physiologic r ole in host defence.
Lymphokines
The int er leukins and int er fer ons have been discussed ext ensively in Chapt er 13. By
r ecombinant DNA t echnology it is now possible t o har vest unlimit ed quant it ies of IL-1, IL-2
and IFN γ, IFN β, and IFN α.
IFN α was t he fir st t o be pr oduced in a lar ge scale manner . IFN α has pr oven adjuvant
pr oper t ies, as assessed by r educt ion in t umour size, especially in lymphomas. IFN α appear s t o
act bot h dir ect ly on t umour t issue and by act ivat ion of macr ophages. IFNs α, β and γ ar e all
used t her apeut ically in a number of clinical condit ions (see Chapt er 13).
Recombinant IL-2 was appr oved in 1992 for t he t r eat ment of met ast at ic and inoper able
renal cell carcinoma. It has been used experiment ally for malignant melanoma and HIV infect ion.
It appear s t o r est or e bot h humor al and cellular immunit y in nude (at hymic) mice. IL-2 also
appear s t o induce pr oduct ion of IFN α by T cells. Bot h t he int er leukins and t he int er fer ons ar e
not fr ee fr om side effect s such as fever , malaise, myalgias, ar t hr algias and fluid r et ent ion.
An alt er nat ive appr oach t o t he in vivo use of IL-2 has been t he induct ion of lymphokine
act ivat ed killer (LAK) cells in vit r o, by incubat ion of cells wit h IL-2. Under t hese condit ions
lymphocyt es acquir e cyt ot oxicit y against a br oad r ange of t umour cells. Cells act ivat ed in t his
manner have been used t o cause t umour r egr ession in mice. Clinical t r ials wit h t hese cells in
humans have shown pr omising r esult s.
156 Immunology– Introductory Textbook
Immunosuppression
Immunosuppr ession has been par t icular ly useful is pat ient s under going or gan t r ansplant s
and in t he t r eat ment of gr aft r eject ion, aut oimmunit y and aller gy. Cur r ent t r eat ment of gr aft
r eject ion, aut oimmunit y or aller gy is not ant igen specific. The following ar e some of t he agent s
used as immunosuppr essives.
Cytotoxic agents
Cyt ot oxic agent s such as cyclophosphamide, chlor ambucil, azat hiopr ine and met hot r exat e
block cell r eplicat ion and pr efer ent ially kill dividing cells. Cyclophosphamide and chlor ambucil
alkylat e DNA in bot h dividing and r est ing cells, leading t o cell deat h dur ing t he mit ot ic phase
of cell division. Azat hiopr ine and met hot r exat e block DNA synt hesis, pr efer ent ially killing
cells t hat ar e in t he S (DNA-synt hesis) phase of t he cell cycle. The major use of t hese dr ugs has
been t o kill malignant cells. Cyt ot oxic agent s, however , also suppr ess bot h humor al and cellular
immunit y. Because B cells or T cells t hat ar e st imulat ed by ant igen, go t hr ough act ive
pr olifer at ion-cyt ot oxic agent s exer t t heir killing act ion on t hese act ively dividing cells. This
may not be t he only way t hat t hese dr ugs cause immunosuppr ession, none t he less, t hey ar e an
impor t ant par t of t he immunosuppr essive t her apy given dur ing or gan t r ansplant at ion. They
have also been used wit h some success in t he t r eat ment of var ious aut oimmune diseases.
These dr ugs, however , have numer ous and ser ious side-effect s.
Glucocorticoids
Glucocor t icoids ar e pot ent immunosuppr essive and ant i inflammat or y agent s. They ar e
used r egular ly in t he t r eat ment of gr aft r eject ion, aller gies and ast hma. The immunosuppr essive
effect s of glucocor t icoids ar e poor ly under st ood, t hey appear t o r educe t he levels of cir culat ing
lymphocyt es and monocyt es and suppr ess t he pr oduct ion of IL-1 and IL-2. Glucocor t icoids have
a diver sit y of act ions and t he clinical benefit seen in aller gy and aut oimmunit y seems t o be due
t o t he var ious ot her act ions of t hese dr ugs t oget her wit h t heir immunosuppr essive pr oper t ies.
The chr onic use of glucocor t icoids is associat ed wit h ser ious side effect s t hat hamper t heir
cont inued use in some pat ient s.
Cyclosporine and Tacrolimus
Cyclospor ine is a cyclic polypept ide cont aining 11 amino acids, der ived fr om soil fungi.
J ust aft er implant at ion of a for eign gr aft , for eign ant igen sensit ive T cells begin t o get act ivat ed.
Cyclospor ine act s select ively on ant igen-sensit ive T cells in t he G0 t o G1 phase (see Chapt er
12), and blocks t he t r anscr ipt ion of lymphokine mRNA, t her eby suppr essing IL-2 pr oduct ion.
This effect ively blocks T cell act ivat ion and pr olifer at ion. Rest ing T cells which car r y memor y
for immunit y t o infect ious agent s ar e spar ed. It has also been shown t hat t he dr ug leads t o t he
development of act ive “suppr essor ” T cells, which could act ively maint ain t oler ance t o t he
gr aft ed t issue.
Cyclospor ine has been used successfully t o pr event gr aft r eject ion and in t he t r eat ment of
gr aft ver sus host disease. It has lit t le t oxicit y for dividing cells in t he gut and bone mar r ow.
Side effect s of cyclospor ine include nephr ot oxicit y and hepat ot oxicit y. It is also associat ed wit h
an incr ease in B cell lymphomas, t hough t he incidence r epor t ed is r elat ively low.
Tacr olimus is a macr olide ant ibiot ic t hat wor ks in a mechanism similar t o t hat of
cyclospor ine t o pr event allogr aft r eject ion.
Immunopotentiation and Immunosuppression 157
Antilymphocyte antibodies
Ant ilymphocyt e globulins ha ve been shown t o deplet e lymphocyt es a nd pr oduce
immunosuppr ession. They pr olong gr aft sur vival in r ecipient s, t hough long t er m effect s have
been var iable. Monoclonal ant ibodies t o t he T cell sur face ant igen, CD3 and t he IL-2 r ecept or
have been used for t r eat ing host r eject ion of allogr aft s. Bot h agent s have been associat ed wit h
sever al pr oblems including unpleasant side effect s.
Monoclonal ant ibodies t o ot her T cell sur face ant igens ar e being invest igat ed in animal
models, as pot ent ial immunosuppr essive agent s.
™™™
TRANSPLANTATION IMMUNOLOGY
CHAPTER – 18
T
he r eplacement of hopelessly diseased vit al or gans wit h healt hy donor or gans has
offer ed hope of a bet t er qualit y of life t o many pat ient s. The science of t r ansplant at ion
has moved fr om it s once falt er ing posit ion in t he 1960s t o t hat of a pr omising and life- enhancing
endeavour .
Classification of Grafts
The major classificat ion of gr aft s is based on t he r elat ionship bet ween t he donor and t he
r ecipient . Ther e ar e t hr ee main t ypes of gr aft s:
• a gr aft t aken fr om one locat ion in an individual and r et ur ned t o t he same individual
at a differ ent locat ion is called an a u t ogr a ft / a u t ologou s gr a ft .
• an a llogr a ft / a lloge n e i c gr a ft is t aken fr om one individual of a given species and
placed in anot her individual of t he same species.
• a gr aft bet ween t wo genet ically ident ical individuals, as in ident ical t wins is called an
i sogr a ft / i soge n e i c gr a ft .
• a xe n ogr a ft is t aken fr om an individual of one species and placed in an individual of
a differ ent species.
Gr aft ed cells, t issues or or gans may be placed in t heir nor mal locat ion as in an or t h ot op i c
gr aft . Gr aft s may be placed in an at ypical locat ion, when t hey called h et er ot op i c gr aft s. Almost
all clinical int erest is cent ered around allograft s and t he immunology of t ransplant at ion concerns
it self mainly wit h t he sur vival of t he allogr aft .
Immunology of Graft Rejection
First and second set reactions
When a gr aft is under going r eject ion for t he fir st t ime, t her e is ver y r apid invasion by
polymor phs and lymphoid cells, including plasma cells. Thr ombosis and acut e cell dest r uct ion
can be seen in t hr ee t o four days. This “fi r st t i me ” r eact ion t o a for eign gr aft is known as t he
fi r st se t r e a ct i on .
Se con d se t r e je ct i on occur s dur ing subsequent gr aft ing, t aken fr om t he or iginal donor
or a r elat ed subject . It does not occur if t he allogr aft has been donat ed by an ent ir ely unr elat ed
subject wit h a complet ely differ ent set of t issue ant igens. (Such gr aft s ar e r eject ed as fir st set of
r eact ions). The r ecipient of a second gr aft fr om a donor who has alr eady been r eject ed, will
mount an acceler at ed r eject ion of t he second gr aft . Since T cells ar e implicat ed in gr aft r eject ion,
it demonst r at ed t hat t hey ar e pr imed and r et ain memor y of t he fir st cont act wit h gr aft ant igens.
Transplantation Immunology 159
Dur ing r eject ion humor al ant ibodies, wit h specificit y for t he gr aft ant igens ar e pr oduced.
Such ant ibodies ar e pr oduced in a T cell dependant manner i.e., wit h T cell help.
Immune Mechanisms in Graft Rejection
Immune mechanisms in gr aft r eject ion ar e best illust r at ed using r eject ion of t he gr aft ed
kidney as a model.
A. Acute rejection
Ant igen pr esent ing cells (APC) in t he gr aft ed t issue pr ovide t he pr imar y st imulus. These
MHC Class II-posit ive cells ar e mainly dendr it ic cells and t o a lesser ext ent - monocyt es. These
cells ar e necessar y t o pr esent ant igen in a for m t hat r ecipient lymphocyt es can r ecognize.
These cells ar e somet imes called “passenger cells” since t hey come along wit h t he gr aft . If t hey
wer e not so r eadily available, for eign t issue ant igen would have t o be pr ocessed and pr esent ed
by r ecipient APCs. As shown in Figur e 18.1 a, t he ant igen pr esent ing cells bear ing MHC Class
II ant igens ar e r ecognized by CD4+ T cells or helper T cells. Toget her wit h IL-1 which is
secr et ed by t he “passenger APC”, t he CD4+ T cells get act ivat ed and pr olifer at e, const it ut ing
an in vit r o mixed lymphocyt e r eact ion (MLR). Once act ivat ed, t hese cells r elease IL-2, which is
an essent ial fact or in t he act ivat ion of CD8+ T cells (cyt ot oxic T cells) and B cells. Clonal
pr olifer at ion of cyt ot oxic T cells and B cells r esult s.
The pr ecise mechanism by which cyt ot oxic T cells kill gr aft cells is unclear . Dir ect
cyt ot oxicit y is a possibilit y as cyt ot oxic T cells r ecognize MHC Class I ant igens, which ar e
displayed on vir t ually all human cells. Anot her impor t ant consequence of T cell act ivat ion is
t he r elease of IFN. IFN induces incr eased expr ession of HLA-A,HLA-B and HLA-DR ant igens
on gr aft t issue, which makes t he gr aft mor e vulner able t o T cyt ot oxic mediat ed killing. It also
act ivat es monocyt es t o mediat e a dest r uct ive delayed hyper sensit ivit y r esponse against t he
gr aft (Figur e 18.1 b). In addit ion, act ivat ed CD4+ cells r elease IL-4, IL-5 and IL-6,which cause
B cells t o pr oduce ant ibody. Ant ibody mediat ed damage t akes place via complement act ivat ion
or by r ecr uit ment of ant ibody dependant cell mediat ed cyt ot oxic effect or cells (Figur e 18.1 b).
This t ype of acut e r eject ion occur s wit hin 10 days of t r ansplant at ion and is char act er ized
by dense cellular infilt r at ion. High dose cor t icost er oids ar e oft en used t o t r eat acut e r eject ion.
Cor t icost er oids funct ion t hr ough sever al mechanisms (see Chapt er 17). They ar e especially
useful in t r eat ing gr aft r eject ion because t hey r educe t he capacit y of APCs t o expr ess Class II
ant igens and t o r elease IL-1. They inhibit T cell act ivat ion in t he r ecipient and block r elease of
IL-2. They ar e also known t o pr oduce lymphocyt openia especially of CD4+ cells. If t he r esponse
t o cor t icost er oids is inadequat e, ant ilymphocyt e ant ibodies may be necessar y. Monoclonal
ant ibodies t o CD3 T cells and t hose against t he IL-2 r ecept or ar e used.
B. Hyperacute rejection
Hyper acut e r eject ion occur s wit hin minut es of t r ansplant at ion and is char act er ized by
sludging of r ed cells and micr ot hr ombi in t he glomer ulus. This t ype of r eject ion occur s in
individuals wit h pr e exist ing ant ibodies t o blood gr oup ant igens or in t hose t hat ar e pr e sensit ized
t o MHC Class I ant igens of t he donor , t hr ough blood t r ansfusions. Ant igen ant ibody complexes
formed during reject ion, fix complement and complement act ivat ion ensues, followed by act ivat ion
of t he clot t ing pat hway. This leads t o micr ot hr ombi wit hin glomer ular capillar ies, leading t o
sever e ischaemia and necr osis of t he gr aft . To dat e, t her e ar e no effect ive means t o t r eat t his
condit ion, except by ensur ing t hat pr e sensit izat ion has not t aken place.
160 Immunology– Introductory Textbook
Figure 18.1. (a) Cellular responses to grafted antigens
( b) Mechanisms of allograft rejection
APC = antigen presenting cell
DTH = delayed type hypersensitivity
MLR = mixed lymphocyte reaction
ADCC = antibody dependant cellular cytotoxicity
Transplantation Immunology 161
C. Chronic rejection
Chr onic r eject ion occur s mont hs t o year s aft er t r ansplant at ion. It is char act er ized by a
nar r owing of t he vascular ar t er ial lumen, owing t o gr owt h of endot helial cells t hat line t he
vascular bed. The act ual mechanism involved in t his over gr owt h of endot helial cells is unknown.
Monocyt e r elease of IL-1 and plat elet and endot helial cell r elease of plat elet der ived gr owt h
fact or s ar e t he t wo fact or s implicat ed. Init ially t he pr olifer at ing endot helial cell lesion is
r ever sible, but once it pr ogr esses t o fibr ot ic changes, it is unr esponsive t o t r eat ment and
pr ogr esses t o gr aft ischaemia, int er st it ial fibr osis and loss of r enal funct ion.
Precautions against graft rejection
The t yping and cr oss mat ching of t he ABO b lood gr ou p s is an essent ial t est per for med
on all r ecipient s and pot ent ial donor s. The ABO syst em is pr esent not only on r ed blood cells,
but also on t he vascular endot helium of t he gr aft . Hence r enal t r ansplant s ar e per for med only
bet ween ABO - compat ible pair s. Ver y r apid gr aft r eject ion occur s if t her e is ABO mismat ching
bet ween donor and r ecipient .
Ideally all r ecipient s and pot ent ial donor s need t o have a complet e t issue t yping pr ofile
done. The HLA-A, B, C and D/DR t issue ant igens ar e t yped (see Chapt er 10). The use of dr ugs
such as cyclospor ine A, have gr eat ly diminished t he effect s of HLA mismat ching. However ,
most t r ansplant specialist s will insist on a favour able degr ee of mat ching at t he DR locus.
St at ist ics show t hat mat ching at t he DR locus is of gr eat er benefit t han t he B loci which in t ur n
is mor e advant ageous t o gr aft sur vival t han t he A loci.
Anot her t est t hat is commonly done is t he mi xe d lymp h ocyt e cu lt u r e t est . This t est
evaluat es t he r ecipient ’s in vit r o pr olifer at ive r esponse t o mismat ched D/DR ant igens on t he
donor ’s cells, when donor and r ecipient cells (monocyt es fr om blood) ar e mixed. A mixed
lymphocyt e r eact ion (MLR) occur s if t her e is mismat ching at t he D/DR locus. However , if only
one haplot ype is mismat ched, a weak MLR r esult s and chances of gr aft sur vival ar e excellent
(90%). If a st r ong MLR occur s, even wit hin family member s, gr aft sur vival r at es fall t o 60%.
Some cent er s do not r ecommend t r ansplant at ion in such cases.
A cr os s ma t ch t e s t is used t o det er mine t he pr esence of any pr efor med ant ibodies
(pr esensit izat ion) t o donor HLA ant igens. The t est is done using t he pat ient ’s most r ecent
ser um and t he donor ’s lymphocyt es (fr om per ipher al blood). If t he donor ’s cells ar e killed by
t he pat ient ’s ser um, it indicat es a posit ive cr oss mat ch and t her efor e t he pr esence of pr efor med
ant ibodies t o donor cells. Posit ive cross mat ches are usually a cont ra indicat ion t o t ransplant at ion.
Tissue and Organ Transplantation
Corneal grafts
Cor neal gr aft s const it ut e some of t he most successful gr aft s done t o dat e. This is because
t hey ar e avascular and do not sensit ize t he pat ient . They sur vive wit hout immunosuppr essive
t her apy. Gr aft s of car t ilage ar e successful in t he same way and r emain pr ot ect ed by t he mat r ix.
Such t issues a r e r efer r ed t o a s i mmu n ol ogi c a l l y p r i vi l e ge d s i t e s ; due t o t heir poor
accessibilit y wit h r egar d t o t he immune syst em.
Kidney transplants
The fir st successful r enal t r ansplant was per for med at Pet er Bent Br igham Hospit al in
1954. Since t hen, t housands of successful kidney t r ansplant s have been car r ied out and sur vival
162 Immunology– Introductory Textbook
r at es ar e high. Advances in t issue t yping and impr oved under st anding of immunosuppr essive
t her apy, especially cyclospor ine, has cont r ibut ed t o t he success of r enal t r ansplant s. It has now
been fir mly est ablished t hat mult iple blood t r ansfusions pr ior t o gr aft ing, aids sur vival of t he
gr aft . The r eason for t his is not known, it may be due t o t he pr esence of blocking ant ibodies or
t o t he gener at ion of ant i idiot ypic suppr essor T cells, as a r esult of t he t r ansfusion.
Heart transplants
The fir st successful human hear t allogr aft was per for med in 1967. Car diac t r ansplant s ar e
indicat ed in pat ient s aged 40 or below wit h end st age cor onar y ar t er y disease, car diomyopat hy,
r heumat ic hear t disease or congenit al hear t disease. Absolut e cont r a indicat ions ar e sever e
pulmonar y hyper t ension, infect ion and cancer . The one year sur vival r at e for hear t t r ansplant s
st ands at ar ound 80%, mainly due t o t he int r oduct ion of cyclospor ine. Full HLA mat ching,
t hough ideal, is not possible. Single DR haplot ype compat ibilit y gives 90% sur vival aft er 3
year s, t his falls t o 65% when bot h haplot ypes ar e mismat ched.
Liver transplantation
The most common indicat ion for adult liver t r ansplant at ion has been hepat it is B ant igen
negat ive, post necr ot ic cir r hosis or chr onic act ive hepat it is. Infant s and childr en wit h congenit al
or development al anomalies of t he bile duct s, benefit fr om successful liver t r ansplant s. Ot her
indicat ions for liver r eplacement in childr en ar e inbor n er r or s of met abolism such as Wilson’s
disease and t yr osinaemia. The sur vival r at e for one year is 68% for adult s and 75% for childr en.
Common complicat ions aft er liver t r ansplant s ar e or gan ischaemia, infect ion and r eject ion.
Bone-marrow transplantation
Over t he past t wo decades, a llogen eic bon e ma r r ow t r a n sp la n t a t ion (BMT) has evolved
fr om an exper iment al pr ocedur e r eser ved for pat ient s wit h r efr act or y leukemia int o a r apidly
expanding ar ea of clinical invest igat ion t hat offer s pot ent ial cur e for pat ient s wit h aplast ic
anemia, acut e and chr onic leukemia, br east cancer , and select ed t ypes of lymphoma. The
object ive of BMT is t o pr ovide a healt hy st em cell populat ion t hat will differ ent iat e int o blood
cells t o r eplace deficient or pat hologic cells of t he host .
Pat ient s wit h acut e myeloid or lymphoblast ic leukemia may benefit fr om BMT. Pat ient s
wit h acut e myeloid leukemia t r ansplant ed in fir st r emission can now expect an appr oximat ely
50 t o 60% likelihood of long-t er m disease-fr ee sur vival. Similar pr obabilit ies ar e also achievable
aft er t r ansplant at ion of adult s wit h acut e lymphoblast ic leukemia in fir st r emissions. Pr obabilit y
of r elapse cor r elat es wit h r emission st at us at t he t ime of t he t r ansplant , r anging fr om 20% in
fir st r emission t o 60% wit h mor e advanced disease. Long-t er m sur vival for pat ient s wit h chr onic
myelocyt ic leukemia who r eceive BMT in t he phase of r emission is 60 t o 70%.
Pediat r ic BMT has expanded because of it s pot ent ial for cur ing childr en wit h genet ic
diseases (e.g., t halassemia, sickle cell anemia, immunodeficiencies, inbor n er r or s of met abolism).
Mar r ow can be pr ocur ed fr om unr elat ed living donor s. Relat ed donor s who ar e not HLA-
ident ical have been used wit h incr easing fr equency. Result s wit h eit her pr ocedur e suggest
long-t er m disease-fr ee sur vival pr obabilit ies of 30 t o 50% in pat ient s wit h acut e and chr onic
leukemia or aplast ic anemia; i.e., in most sit uat ions t he r esult s ar e somewhat infer ior t o t hose
wit h mar r ow fr om HLA-ident ical siblings.
Anot her opt ion for BMT is a u t ologou s t r a n sp la n t a t i on (r emoval of a pat ient ’s own
mar r ow when a complet e r emission has been induced, followed by ablat ive t r eat ment of t he
Transplantation Immunology 163
pat ient wit h t he hope of dest r uct ion of any r esidual t umor and r escue wit h t he pat ient ’s own
bone mar r ow). Since an aut ogr aft is used, no immunosuppr ession is necessar y ot her t han t he
shor t -t er m high-dose chemot her apy used for t umor er adicat ion and bone mar r ow ablat ion;
post t r ansplant pr oblems wit h GVHD ar e minimal. Indicat ions for aut ologous BMT ar e r elapsed,
chemot her apy-sensit ive lymphoma, in which a 30 t o 40% success r at e has been achieved, and
acut e leukemia in r emission, in which 20 t o 50% success r at es have been obser ved. Success
r at es ar e infer ior wit h mor e advanced disease and wit h r esponsive solid cancer s (e.g., br east or
ger m cell t umor s). Two major obst acles r emain for successful applicat ion of aut ologous BMT:
t he possibilit y of cont aminat ion of t he mar r ow inoculum wit h t umor cells, and t he absence of
gr aft -vs-t umor act ivit y (in cont r ast wit h t hat seen in allogeneic BMT), bot h of which cont r ibut e
t o t he obser ved higher r at es of t umor r ecur r ence.
Peripheral blood stem cell transplants
A r elat ively r ecent development in st em cell t r ansplant at ion is t he use of per ipher al blood
cells inst ead of st em cells fr om bone mar r ow. Per ipher al blood st em cells (PBSCs) ar e obt ained
fr om cir culat ing blood r at her t han fr om bone mar r ow, but t he amount of st em cells found in
t he per ipher al blood is much smaller t han t he amount of st em cells found in t he bone mar r ow.
Per ipher al blood st em cells can be used in eit her aut ologous or allogeneic t r ansplant s. The
major it y of PBSC t r ansplant s ar e aut ologous. However , r ecent clinical st udies indicat e t hat
PBSCs a r e being used mor e fr equent ly t ha n bone ma r r ow for a llogeneic bone ma r r ow
t r ansplant at ion.
The advant ages of PBSC t r ansplant s when compar ed t o bone mar r ow t r ansplant s ar e: in
allogeneic t r ansplant at ion, haemat opoiet ic and immune r ecover y ar e fast er wit h PBSCs which
r educes t he pot ent ial for disease r ecur r ence, pr imar ily gr aft -ver sus-host -disease. In aut ologous
t r ansplant at ion, t he use of PBSCs can r esult in fast er blood count r ecover ies. Also, some medical
condit ions exist in which t he r ecipient cannot accept bone mar r ow st em cell t r ansplant s, but
can accept PBSC t r ansplant s. Some possible disadvant ages t o PBSC t r ansplant ver sus bone
mar r ow t r ansplant at ion ar e: so much mor e fluid volume is necessar y t o collect enough PBSCs
t hat , at t he t ime of infusing t he new st em cells int o t he r ecipient , t he fluid can collect in t he
lungs or cause t empor ar y kidney pr oblems. Also, t he t ime commit ment for t he donor for a
PBSC t r ansplant is consider able. When t he PBSCs ar e being collect ed, sever al out pat ient
sessions ar e needed and each session last s appr oximat ely t wo-four hour s.
Other organs
Tr ansplant at ion of t he pancr eas and ot her endocr ine or gans and t he lung ar e being
int ensively r esear ched. The feasibilit y of such t r ansplant s will impr ove wit h advances in
t echniques, t her apy and suppor t ive car e.
Graft Versus Host Disease (GVHD)
When immunocompet ent donor cells ar e gr aft ed int o an immuno-compr omised host , gr aft
ver sus host disease can occur . The gr aft ed cells sur vive in t he immuno-compr omised host even
t hough t hey ar e hist o-incompat ible, as t he r ecipient is unable t o mount an immunological
r eact ion against t hem. In t ime, t hese immunologically compet ent cells r ecognize host ant igens
and r eact immunologically against t hem. Ther efor e, inst ead of a host r eact ing against t he
gr aft ; t he r ever se t akes place wher e t he gr aft mount s a r eact ion against an immuno-suppr essed
host , incapable of fight ing back.
164 Immunology– Introductory Textbook
A major pr oblem in allogeneic bone mar r ow t r ansplant at ion (BMT) is t he pr event ion and
cont r ol of GVHD. Sympt oms and signs of acut e GVHD ar e fever ; exfoliat ive der mat it is; hepat it is
wit h hyper bilir ubinemia; vomit ing; diar r hoea and abdominal pain, which may pr ogr ess t o an
ileus; and weight loss. Alt hough incr eased knowledge of t he major hist ocompat ibilit y complex
has aided under st anding of t he aet iology of GVHD, pat ient s who ar e mat ched at t he A, B, C,
and DR loci st ill have a 30 t o 60% incidence of GVHD. Alt hough t he int r oduct ion of cyclospor ine
in t he ear ly 1980s has gr eat ly r educed bot h t he incidence and sever it y of GVHD, it cont inues t o
be t he major cause of mor t alit y and sever e mor bidit y aft er allogeneic BMT.
About a t hir d t o half of BMT r ecipient s develop a mor e indolent , chr onic for m of GVHD.
Alt hough t he skin, liver, and gut remain t he organs primarily affect ed, ot her areas of involvement
(e.g., joint , lung) ar e also not ed. Int er est ingly, br onchiolit is oblit er ans similar t o t hat seen
aft er lung t r ansplant at ion can occur . Ult imat ely, 20 t o 40% of t he pat ient s die of complicat ions
associat ed wit h GVHD, t he incidence being higher when donor mar r ow is not fr om an HLA-
ident ical sibling. In pat ient s wit hout chr onic sequelae of GVHD, all immunosuppr ession can be
st opped 6 mont hs aft er BMT, making lat e complicat ions r ar e in t hese pat ient s, in cont r ast wit h
t he cont inued need for immunosuppressant s and result ing complicat ions in solid organ t ransplant
r ecipient s.
One ar ea of act ive clinical r esear ch aimed at r educing t he incidence of GVHD has been
t he r emoval of T cells fr om t he donor mar r ow wit h monoclonal ant ibodies or mechanical
separ at ion befor e r einfusion of t he mar r ow. T-cell deplet ion has been ver y effect ive in decr easing
bot h t he incidence and sever it y of GVHD; however , t he incidences of engr aft ment failur e and
r elapse ar e incr eased. A possible explanat ion is t hat t he cyt okines gener at ed in t he gr aft -vs-
host r eact ion pr omot e st em cell mult iplicat ion and mat ur at ion necessar y for engr aft ment .
Pat ient s who develop GVHD have significant ly lower r elapse r at es, suggest ing t hat T cells
r esponsible for GVHD ar e pr obably involved in a gr aft -vs-leukemia effect . Ot her agent s used t o
pr event or t r eat GVHD include met hot r exat e, cor t icost er oids, ant it hymocyt e globulin, and
monoclonal ant ibodies against ant igens expr essed on mat ur e T cells.
GVHD may also follow blood t r ansfusions in except ional cases, since even small number s
of donor T cells can induce t his r eact ion. Such sit uat ions include int r aut er ine foet al blood
t r ansfusions and t r ansfusions in immuno-suppr essed pat ient s (e.g., BMT r ecipient s, leukemia,
lymphoma, neur oblast oma, Hodgkin’s and non-Hodgkin’s lymphoma). Blood pr oduct s t o be
given t o pat ient s at r isk should be ir r adiat ed t o pr event development of GVHD.
The Enigma of the Foetal Graft
Pr egnancy in mammals act s much as a gr aft does, br inging int o dir ect cont act t wo
genet ically dist inct individuals in t he foet us; one half of all genes come fr om t he mot her and
t he ot her half fr om t he fat her . In t he case of t he foet us, however , t her e is no dr amat ic immune
r esponse by t he mot her against t he gr aft and pr egnancy is not compr omised. How t he foet us
escapes r eject ion and flout s t he laws of t issue t r ansplant at ion r emains unknown.
It has been hypot hesised t hat t he mat er nal immune syst em may be pr event ed fr om
r ecognizing t he foet al t issue as for eign and/or t he cells of t he mat er nal immune syst em may be
pr event ed fr om mount ing an immune r esponse in a number of ways.
Foet al t issue displays a unique t ype of MHC class I molecule encoded by t he gene HLA-G.
This molecule is consider ed t o be a non-classical MHC molecule because of it s low allelic var iat ion
(only t wo polymor phs ar e known) and r est r ict ed t issue dist r ibut ion (found only in t he placent a)
A st udy by Kovat s et al, localized t he expr ession of HLA-G t o t he t r ophoblast cells, t he embr yonic
Transplantation Immunology 165
cont r ibut ion t o t he placent a. It s funct ion in t he placent a may be inhibit or y as it may pr event
t he act ivat ion of decidual cyt ot oxic T cells and NK cells. (The decidua is t he mat er nal cont r ibut ion
t o t he placent a). St udies have shown t hat cells expr essing HLA-G ar e unable t o act ivat e T cells
and inhibit cyt ot oxic lysis mediat ed by NK cells.
Anot her lymphoid cell seems t o play an impor t ant r ole in a successful pr egnancy: t he
macr ophage. Classically act ivat ed macr ophages mediat e pr oinflammat or y r esponses, which
involve T
h
1 cell act ivat ion and cyt okine secr et ion. Cyt okines such as IL-1, IL-6 and IL-12 pr omot e
inflammat ion. In cont r ast , “alt er nat ively” act ivat ed macr ophages pr omot e ant i-inflammat or y
r esponses by secr et ing cyt okines, such as IL-10 and IL-1-r ecept or ant agonist , t hat down r egulat e
t he inflammat ory response and inhibit t he act ivat ion of T
h
1 lymphocyt es. Alt ernat ively, act ivat ed
macr ophages may cr eat e an immunosuppr essive envir onment in t he placent a.
Recent st udies have shown t hat a complement inhibit or , may be anot her way t hat t he
mat er nal immune syst em is r egulat ed t o pr omot e a successful pr egnancy.
Anot her mechanism for pr event ing foet al r eject ion t hat has r eceived some at t ent ion fr om
r esear cher s is t r ypt ophan cat abolism. Int er est ingly, it seems t hat t r ypt ophan cat abolism is
employed by t he immune syst em t o accomplish anot her goal: t o pr event r eject ion of t he foet us.
Tr ypt ophan depr ivat ion may r educe or inhibit t he immune r esponse in t wo ways: it may inhibit
lymphocyt e pr olifer at ion or it may halt t he manufact ur e of effect or pr ot eins.
Subsequent ly, under st anding t hese mechanisms may lead t o t r eat ment s t hat will help
women who exper ience r ecur r ent spont aneous abor t ions maint ain a pr egnancy and car r y a
baby t o t er m. Under st anding t hese pr ocesses may also help us in pr event ing r eject ion of donor
t issue gr aft s by t he r ecipient , since t he goals of a pr egnancy and t issue gr aft s ar e r elat ively t he
same: t o sust ain a for eign t issue wit hin an immunocompet ent host . However , all exper iment al
evidence so far appear s equivocal and despit e sever al decades of wor k t he enigma of t he foet al
gr aft st ill r emains.
™™™
TUMOUR IMMUNOLOGY
CHAPTER – 19
I
mmunology and cancer has had a long and somet imes fr ust r at ing associat ion. The idea
t hat immunologic pr ocesses could war d off malignant cells was r einfor ced in t he 1950s
when it was accept ed t hat one of t he impor t ant funct ions of cellular immunit y was t o execut e
sur veillance against malignant cells. The concept of immune sur veillance addr esses one
essent ial quest ion. Do t umour cells show differ ences fr om t heir nor mal cellular count er par t s
t hat t he immune syst em can r ecognize? If so, it would be int er est ing t o know whet her t umour
associat ed ant igens ar e specific for t he par t icular cancer involved or whet her t hey r epr esent
simple differ ent iat ion int o malignant cells. If t umour associat ed ant igens do exist , does t he
immune r esponse dir ect ed against t hese ant igens give r ise t o deat h of t he t umour cell?
Surface Antigens on Tumour Cells
Tumour ant igens fall int o 2 b r oa d ca t e gor i e s:
• t u mou r sp e ci fi c a n t i ge n s (TSA) ar e unique, found only on t umour cells and ar e
eminent ly posit ioned as t ar get s for immunologic at t ack.
• t u mou r -a ssoci a t e d a n t i ge n s (TAA), by cont r ast , ar e found an t umour cells and
some nor mal cells as well.
Unique Tumour Specific Antigens
Unique t umour specific ant igens ar e found only on t umour cells and not on ot her cells of
t he host . Ther e ar e sever al examples of unique t umour specific ant igens (TSAs)
(a) Virally Induced Antigens
The best example of unique TSAs ar e t hose induced by vir uses. In mammals, sever al RNA
vir suses (example: r et r ovir uses) and DNA vir uses (example: her pes vir uses) cause malignant
t r ansfor mat ion. The best example and t he gr eat est t r iumph so far in t umour immunology is
t hat of Ma r ek ’s d isea se in ch ick en s, caused by a her pes vir us. The vir us infect s t he lymphocyt e
and t r ansfor ms it ; a unique pr ot ein is found on t he membr ane of t he t r ansfor med lymphocyt e
when t he vir al DNA int egr at es wit h t he host cell genome. All t umour s due t o t his vir us car r y
t his sur face ant igen and ant ibodies t o t he pr ot ein ar e det ect able in host ser a. It is now possible
t o immunize animals pr ophylact ically against Mar ek’s disease, using eit her t he whole vir us or
t he TSA.
Tumour cells induced by RNA vi r u se s expr ess ant igens coded for by t he vir al pr ot ein in
addit ion t o t heir own cell sur face ant igens. These may be vir al envelope ant igens or int r a vir al
pr ot eins. Ant igens of vir ally induced t umour s show ext ensive cr oss r eact ivit y; immunizat ion of
animals wit h any of t hese vir uses pr ovides pr ot ect ion against a var iet y of similar vir uses.
Tumour Immunology 167
Common oncogenic RNA vir uses ar e t he human T cell leukaemia vir us (HTLV) in man and t he
feline leukaemia vir us (FeLV) in cat s. In many of t hese oncogenic vir uses, it has been shown
t hat vir al ant igens act ivat e cellular oncogenes leading t o cell t r ansfor mat ion. Some of t hese
oncogene pr oduct s will r ender malignant cells sufficient ly dispar at e fr om nor mal cells. This
could also explain t he ext ensive cr oss r eact ivit y bet ween t he TSAs of oncogenic vir uses.
(b) Chemically or Physically Induced Tumour Antigens
Tumour s induced by chemical car cinogens such as met hyl cholant hr ene (MCA) expr ess
unique and individually dist inct t umour ant igens. The ant igen is specific t o t he t umour pr oduced
in t hat MCA induced t umour s in one mouse will not shar e common ant igens wit h an MCA
induced t umour on anot her mouse, alt hough t hey ar e of t he same inbr ed st r ain. In fact mult iple
MCA induced t umour s on t he same mouse ar e all immunologically dist inct fr om each ot her .
The pr ecise mechanism account ing for t his phenomenon is not known. It may r esult fr om an
alt er ed expr ession of nor mal molecules on t he cell due t o gene mut at ions. Alt er at ions in bot h
major and minor hist ocompat ibilit y ant igens on t umour s have been descr ibed in chemical
car cinomas. The same mechanism is said t o oper at e in t umour s due t o ult r aviolet light or
r adiat ion. Gener at ion of an enor mous var iet y of dist inct t umour ant igens makes it unlikely
t hat immunizat ion against such cancer s will ever be possible.
(c) Antigens of Spontaneous Tumours
Spont aneous t umour s ar e t hose t hat have no known inducing agent . Though ant igens
differ ing fr om nor mal cellular ant igens have been ident ified, it has not been possible t o
char act er ize a unique TSA fr om such t umour s.
Tumour Associated Antigens
Though t umour associat ed ant igens ar e, t o a lar ge ext ent , specific t o t he t umour s t hat
display t hem, some nor mal cells may also expr ess such ant igens at par t icular st ages of
differ ent iat ion. Monoclonal ant ibodies have now per mit t ed t he char act er izat ion of t hese ant igens
and ar e now widely used t o diagnose some of t hese t umour s.
(a) Oncofoetal antigens
On coge n e s wer e discover ed in cancer -causing vir uses. Most oncogenes wer e act ually
pr esent in t he host cell, wher e t hey funct ioned in r egulat ed cell gr owt h. The host cell gene was
called a p r ot o-on coge n e. When t r ansduced by t he vir us and expr essed under t he cont r ol of a
vir al pr omot or , t he gene pr oduct cont r ibut es t o t he unr egulat ed gr owt h of t he t umour cell.
Since pr ot eins encoded by pr ot o-oncogenes ar e expr essed by nor mal cells, t heir over -expr ession
on t umour cells would qualify t hem as t umour -associat ed ant igens.
Tumour cells can somet imes “swit ch on” genes which ar e associat ed wit h gr owt h and
development of t he foet us. Consequent ly, a nt igens which a r e a ssocia t ed nor ma lly wit h
embr yogenesis and ar e not det ect able in t he adult begin t o appear dur ing t umour gr owt h. The
pr ot ot ype oncofoet al ant igen is ca r ci n o e mb r yon i c a n t i ge n (CEA), which is an impr oper ly
glycosylat ed glycopr ot ein found on foet al gut and human colon cancer cells, but not on nor mal
adult colonic cells. Such abnor mal glycosylat ion may be t he cause of t he obser vat ion t hat
cer t ain human gast r ic car cinoma cells display ABO blood gr oup ant igens differ ent fr om t he
host ABO blood gr oup.
168 Immunology– Introductory Textbook
Elevat ed ser um CEA also occur s in inflammat or y condit ions of t he colon and pancr eas
and also in cancer s of t he br east and pancr eas. Measur ement of ser um CEA is not useful for
diagnosing cancer , it is useful in monit or ing r esponse t o t r eat ment .
Alp h a fe t op r ot e i n is a secr et ed t umour ant igen and is t he foet al equivalent of albumin.
It is found in t he ser um of pat ient s wit h hepat omas and t er at omas and can be used as a mar ker
for t he pr esence of such cancer s. It is, however , not a suit able t ar get for t umour r eject ion.
(b) Differentiation antigens
Differ ent iat ion ant igens ar e unique t o t he hist ogenet ic t ype of t umour , r at her t han t o t he
pr ocess of cellula r t r a nsfor ma t ion. Thus, t her e a r e a nt igens common t o mela noma s or
neur oblast omas and t hey r eflect t he st age of differ ent iat ion at which par t icular t umour cells
ar e ar r est ed. Ther efor e, t issue specific t umour ant igens r epr esent differ ent iat ion ant igens of
t he st ages of cell lineage fr om which t umour s ar ise. Hence t hey will not be ent ir ely specific t o
t umour cells and populat ions of nor mal cells of t he same lineage will car r y similar ant igens. An
immune r esponse against such ant igens car r ies t he danger of at t ack against nor mal cells as
well.
Immune Responses to Tumour Antigens
Wher e t umour ant igens have been shown t o exist , bot h T cell cyt ot oxicit y and t umour
specific ant ibodies have been demonst r at ed.
Cellular mechanisms in tumour immunity
(a) T cells
T cell r esponses ar e t he most impor t ant means of cont r ol against ant igen bear ing t umour
cells. Bot h T cell populat ions : t he MHC Class II r est r ict ed T helper cells and t he MHC Class I
r est r ict ed T cyt ot oxic cells play a r ole in cont r ol of t umour gr owt h.
Since most t umour cells expr ess MHC Class I ant igens, t he T helper (CD4+T ) cells cannot
dir ect ly r ecognize t hese t umour cells. Ther efor e T helper cells ar e dependant upon ant igen
pr esent ing cells (APC), such as macr ophages, t o pr esent t he t umour ant igens in associat ion
wit h MHC Class II ant igens. This leads t o T helper cell act ivat ion and subsequent IL-2 secr et ion,
which in t ur n act ivat es t he cyt oxic T cell (CTL/CD8+T) cells, macr ophages, NK cells and B
cells. T helper cells also pr oduce lymphot oxin and t umour necr osis fact or , which may dir ect ly
lyse t umour cells.
The act ivat ed CTLs pr olifer at e in r esponse t o IL-2 pr ovided by t he T helper cell. They
t hen r ecognize t umour t ar get s in associat ion wit h t he ubiquit ous MHC Class I ant igen and
cause dir ect cell mediat ed cyt ot oxicit y.
(b) Macrophages
Macr ophages ar e impor t ant not only as ant igen pr esent er s; evidence suggest s t hat t hey
may also act as pot ent ial effect or cells mediat ing t umour lysis. Macr ophages act ivat ed by
macr ophage-act ivat ing fact or (MAF) become cyt olyt ic. MAF is a lymphokine secr et ed by T cells
following ant igen st imulat ion. Since macr ophage act ivat ion is not ant igen specific, t hey possibly
Tumour Immunology 169
play a r ole in lysing cells wit h var iant t umour ant igens, which have t her efor e lost t heir pot ent ial
t o act ivat e T helper cells.
(c) Natural killer (NK) cells
NK cells have t he abilit y t o kill a wide r ange of t umour t ar get s in vit r o. The pr ecise
mechanism of t his killing is unknown. Recognit ion and act ivat ion do not appear t o be ant igen
specific. Cyt olysis by NK cells is mediat ed by t he r elease of one or mor e cyt ot oxic fact or s. The
cyt ot oxic act ivit y of NK cells is augment ed by IL-2 and int er fer on, demonst r at ing t hat T cell
act ivat ion enhances NK act ivit y. NK cells r epr esent t he fir st line of host defence against t umour
gr owt h, at bot h pr imar y and met ast at ic sit es. It is also an effect or mechanism t hat can be
r ecr uit ed by T cells t o supplement t heir t umor icidal act ivit y.
Ther e ar e ot her cyt ot oxic cells t hat appear t o kill t umour cells apar t fr om t he NK cells.
These nat ur ally cyt ot oxic cells ar e r esist ant t o suppr ession by glucocor t icoids. Lymphokine
act ivat ed killer (LAK) cells can be induced by ver y high doses of IL-2. They ar e phenot ypically
differ ent fr om NK cells and kill a much br oader r ange of t umour t ar get s. The physiologic r ole
of t hese cell t ypes in cont r olling t umour gr owt h in vivo r emains t o be elucidat ed.
(d) B cells and antibody dependant cellular cytotoxicity
Ever since ant ibodies t o put at ive t umour ant igens have been det ect ed in ser um, a pot ent ial
r ole for host defence in cancer has been suggest ed. However , t he exact mechanism by which
t hey oper at e r emains unclear .
Ther e ar e t wo major mechanisms by which ant ibodies may mediat e ar r est of t umour
gr owt h. Complement fixing ant ibodies fix t o t umour ant igens, r ecr uit complement and cause
t umour cell lysis. Anot her mechanism is via ant ibody dependant cellular cyt ot oxicit y (ADCC),
wher e an immunoglobulin (usually IgG), for ms a br idge bet ween t he t ar get t umour cell and an
effect or or K cell (usually macr ophages or gr anulocyt es). This br ings t he killer (K) cell int o
pr oximit y wit h t he t umour cell and cell lysis ensues. Ther e is evidence t o suggest t hat ADCC is
t he mor e impor t ant mechanism of t he t wo descr ibed.
Mechanisms by which tumours escape immune surveillance
That t he immune mechanism plays a r ole in t he host -t umour r elat ionship is evidenced by
t he finding t hat immunosuppr essed per sons ar e mor e suscept ible t han nor mal individuals t o
cancer s. Leukaemias ar e a common occur r ence in r adiat ion vict ims. In immunosuppr essed
pat ient s and in t hose wit h immunodeficiency diseases, t her e is an incr eased incidence of cancer s.
Cancer s also seem t o hit older individuals when t he st r engt h of t he immune syst em begins t o
wane.
Sever al r easons have been suggest ed, fr om t ime t o t ime, t o explain t umour escape fr om
t he wat chful eye of immune sur veillance.
Poorly immunogenic variants
It is possible t hat var iant s wit h diminished expr ession of t umour ant igen ar e gener at ed as
a means of escape fr om t he immune mechanism. This phenomenon of “sneaking t hr ough” t he
immune sur veillance, select s out only t hose cells which sur vive, t o gr ow int o unchecked t umour
masses. Tumour s may also escape by changing t heir sur face ant igens, leading t o select ion of
t hose clones lacking t he or iginal sur face ant igen.
170 Immunology– Introductory Textbook
Rapid growth
If an immune r esponse is t o cause t umour r egr ession, it must dest r oy cells mor e r apidly
t han t he new cells t hat ar e gener at ed by t umour gr owt h. In some exper iment ally induced
t umour s, it has been shown t hat immune dest r uct ion is t oo slow t o do mor e t han just slow
down t umour gr owt h.
Blocking factors
It has been shown t hat incubat ion of t umour cells wit h host ser um pr event ed T cell
mediat ed killing of t umour cells by t he pr esence of ant ibody coat ing t he t umour cells. The
pr ecise nat ur e of t hese fact or s t hat block ant i t umour immune r esponses ar e not known.
Invest igat or s have found non cyt olyt ic ant ibody, immune complexes and fr ee t umour ant igens
t o act as pot ent ial blocking agent s. Ant ibodies and immune complexes mask t ar get ant igens
and pr event T cell r ecognit ion and fr ee t umour ant igens mop up cyt olyt ic ant ibody and T cell
r ecognit ion sit es, allowing in sit u t umour masses t o gr ow unchecked.
S uppressor mechanisms
The t umour it self may suppr ess t he immune r esponse. Deficient cellular immunit y can
be associat ed wit h r ecur r ence and disseminat ion of t umour s, alt hough cause and effect ar e
difficult t o dist inguish. This deficiency has been r epeat edly shown in var ious t umour s, most
dr amat ically in Hodgkin’s disease, which appear s t o involve a var iable defect in T-cell funct ion.
Decr eased IL-2 pr oduct ion, an incr ease in cir culat ing soluble IL-2 r ecept or s, and induced defect s
in ant igen-pr esent ing cell funct ion may also be involved. Defect ive funct ion of t he T cells
infilt r at ing t he t umour has been shown and can be over come by sufficient ant igen pr esent at ion
by ant igen-pr esent ing cells and appr opr iat e cyt okine suppor t . Deficient humor al immunit y is
commonly associat ed wit h neoplasms involving abnor mal B-cell der ivat ives (e.g., mult iple
myeloma, chr onic lymphocyt ic leukemia).
It is possible t hat t umour s pr oduce immunosuppr essive agent s t hat suppr ess t he immune
r esponse in vivo. Alpha fet opr ot ein is a t umour pr oduct known t o have immuno suppr essive
act ivit y. Pr ost aglandins r eleased by macr ophages of t umour bear ing host s have demonst r able
immunosuppr essive act ivit y.
Alteration in the expression of MHC coded antigens
The expr ession of MHC Class I gene pr oduct s on cell sur faces is essent ial for r ecognit ion
of for eign ant igen by CTLs. If MHC ant igens have an alt er ed expr ession, such cells will
successfully evade t he cyt ot oxic immune r esponse by CTLs.
Immunodiagnosis in Cancer
TAAs can be useful t umour mar ker s in t he diagnosis and management of var ious t umour s.
An ideal t umour mar ker is r eleased only fr om t umour t issue, is specific for a given t umour
t ype (t o dir ect diagnost ic assessment ), is det ect able at low levels of t umour cell bur den, has a
dir ect r elat ionship t o t he t umour cell bur den and t he mar ker concent r at ion in blood or ot her
body fluid, and is pr esent in all pat ient s wit h t he t umour . Most t umour s r elease ant igenic
macr omolecules int o t he cir culat ion t hat can be det ect ed by immunoassay. Alt hough useful in
monit or ing pat ient s for t umour r ecur r ence aft er t her apy, no t umour mar ker has undisput ed
specificit y or sensit ivit y for applicat ion in ear ly diagnosis or mass cancer scr eening pr ogr ams.
Ca r ci n oe mb r yon i c ant igen (CEA) is a pr ot ein-polysacchar ide complex found in colon
car cinomas and in nor mal foet al int est ine, pancr eas, and liver . A sensit ive immunoassay can
Tumour Immunology 171
det ect incr eased levels in t he blood of pat ient s wit h colon car cinoma, but t he specificit y is
r elat ively low because posit ive t est s also occur in heavy cigar et t e smoker s and in pat ient s wit h
cir r hosis, ulcer at ive colit is, and ot her cancer s (e.g., br east , pancr eas, bladder , ovar y, cer vix).
Monit or ing CEA levels may be useful for det ect ing cancer r ecur r ences aft er excision of a t umour
t hat had been associat ed wit h elevat ed CEA.
α αα αα-F e t op r ot e i n , a nor mal pr oduct of foet al liver cells, is also found in t he ser a of pat ient s
wit h pr imar y hepat oma, yolk sac neoplasms, and fr equent ly, ovar ian or t est icular embr yonal
car cinoma.
β ββ ββ-Su bu n i t of h u ma n ch or i on i c gon a d ot r op i n (β-HCG), measur ed by immunoassay, is
t he major clinical mar ker in women wit h gest at ional t r ophoblast ic neoplasia (GTN)-a disease
spect r um t hat includes hydat idifor m mole, nonmet ast at ic GTN, and met ast at ic GTN and in
about 2/3 of men wit h t est icular embr yonal or chor iocar cinoma. The βsubunit is measur ed
because it is specific for HCG.
P r ost a t e -sp e ci fi c a n t i ge n (PSA), a glycopr ot ein locat ed in duct al epit helial cells of t he
pr ost at e gland, can be det ect ed in low concent r at ions in t he ser a of healt hy men. Using an
appr opr iat e upper limit of nor mal, assays wit h monoclonal ant ibodies det ect elevat ed ser um
levels of PSA in about 90% of pat ient s wit h advanced pr ost at e cancer , even in t he absence of
defined met ast at ic disease. It is mor e sensit ive t han pr ost at ic acid phosphat ase. However ,
because PSA is elevat ed in benign pr ost at ic hyper t r ophy, it is less specific. PSA can be used t o
monit or r ecur r ence aft er pr ost at ic car cinoma has been diagnosed and t r eat ed.
CA 125 is clinically useful for diagnosing and monit or ing t her apy for ovar ian cancer ,
alt hough any per it oneal inflammat or y pr ocess can cause incr eased cir culat ing levels.
Ra d i ola b e le d mon oclon a l a n t i b od y B72.3, which r ecognizes a pancar cinoma ant igen
(one t hat r ecognizes car cinomas fr om all t issues) t er med TAG-72, is being used in t umour
localizat ion st udies t o find occult t umour deposit s. The clinical benefit of finding such occult
t umour s is under st udy.
The Prospects for Immunotherapy in Cancer
The use of va cci n e s especially for vir ally induced cancer s has become a r ealit y as in
Mar ek’s disease of chicks. The hepat it is B vaccine, alr eady widely in use, will be able t o eliminat e
hepat omas. Vaccines for t he Epst ein - Bar r vir us and human T cell leukaemia vir us ar e not , so
far available, but t he out look seems pr omising.
Passive cellular immunotherapy
Infusions of IL-2 may pr ove a useful adjunct t o cellular immunot her apy as t hey enhance
T cell and NK cell cyt ot oxicit y. Administ r at ion of lymphokine - act ivat ed killer (LAK) cells in
associat ion wit h IL-2 have been ext ensively st udied in vivo and in vit r o. Somet imes t he cells
ar e fir st exposed t o phyt ohemagglut inin, a lymphocyt e mit ogen, t o expand a br oad var iet y of
per ipher al lymphoid cells. The availabilit y of pur ified r ecombinant IL-2 in lar ge quant it ies has
made t he LAK cell plus IL-2 t echnique feasible, and some melanoma and r enal car cinoma
pat ient s have shown object ive r esponses.
Passive humoral immunotherapy
It may be possible t o use passive immunot her apy via mon oclon a l a n t i bod i es, especially
against t umour s showing unique t umour specific ant igens. Conjugat ion of cyt ot oxic dr ugs,
172 Immunology– Introductory Textbook
t oxins or r adio isot opes t o monoclonal ant ibodies was t hought t o be t he excit ing new way of
“homing in” ont o t umour cells, leaving nor mal cells unt ouched. Ant ilymphocyt e ser um has
been used in chr onic lymphocyt ic leukemia and in T-cell and B-cell lymphomas, r esult ing in
t empor ar y decr eases in lymphocyt e count s or lymph node size. Some st udies of mur ine
monoclonal ant ibodies against var ious ant igens associat ed wit h malignant melanoma and
lymphomas have shown significant r esponses; now “humanized ant ibodies” ar e used t o avoid
an immune r eact ion against mouse immunoglobulin. However , sever al t umour associat ed
ant igens ar e also displayed on nor mal cells and t he many agent s used as conjugat es ar e t oo
t oxic for human use. The new “magic bullet ” t her efor e, needs mor e r esear ch befor e it becomes
a r ealit y for t he t r eat ment of human cancer s.
Active specific immunotherapy
Appr oaches designed t o induce t her apeut ic cellular immunit y in t he t umour -bear ing host
ar e mor e pr omising t han passive immunot her apy t echniques. Inducing immunit y in a host
t hat failed t o develop an effect ive r esponse in t he fir st place r equir es special pr ocedur es t o
pr esent t he t umour ant igens t o t he host effect or s. Int act t umour cells, defined t umour ant igens,
or gener al immunost imulant s ar e used.
Au t och t h on ou s t u mor cells (cells t aken from t he host ) have been used—aft er irradiat ion,
neur aminidase t r eat ment , hapt en conjugat ion, or hybr idizat ion wit h long-t er m cell lines in
vit r o—in kidney car cinoma and malignant melanoma pat ient s, among ot her s.
Alloge n e i c t u mou r ce lls (cells t aken fr om ot her pat ient s) have been used in pat ient s
wit h acut e lymphoblast ic leukemia and acut e myeloblast ic leukemia.
De fi n e d t u mou r a n t i ge n -b a se d va cci n e s ar e among t he most pr omising appr oaches
in cancer immunot her apy. An incr easing number of t umour ant igens have been unequivocally
ident ified as t he t ar get of specific T cells gr own fr om cancer pat ient s.
Cellular immunit y (involving cyt ot oxic T cells) t o specific, ver y well defined ant igens can
be induced using shor t synt het ic pept ides in adjuvant or bound t o aut ologous ant igen-pr esent ing
cells in vit r o (ant igen pulsing). These ant igen-pulsed, ant igen-pr esent ing cells ar e r eint r oduced
int r avenously and st imulat e t he pat ient ’s T cells t o r espond t o t he pulsed pept ide ant igen.
Ear ly r esult s in clinical t r ials have shown significant r esponses.
Ant igen-specific immunit y can also be induced wit h r ecombinant vir uses (eg, adenovir us,
vaccinia vir us) expr essing such TAAs as CEA. These ant igen-deliver y vir uses ar e being t est ed
for ant i t umour effect iveness.
Nonspecific immunotherapy
Re comb i n a n t I n t e r fe r on s such as (IFN-α, IFN-γ and IFN-β) have ant i-t umour and
ant ivir al act ivit y. Depending on t he dosage, IFNs may eit her enhance or decr ease cellular and
humor al immune funct ions and may affect macr ophage and NK cell act ivit y. Human clinical
t r ials have indicat ed t hat IFNs have ant it umour act ivit y in hair y cell leukemia, chr onic
myelocyt ic leukemia, and AIDS-associat ed Kaposi’s sar coma. However , IFNs ar e quit e t oxic;
pat ient s may develop fever , malaise, leukopenia, alopecia, and myalgia.
Ba ct e r i a l a d ju va n t s (e.g, at t enuat ed t uber cle bacilli -BCG), or killed suspensions of
Corynebacterium parvum have been used in r andomized t r ials. They have been used wit h or
Tumour Immunology 173
wit hout added t umour ant igen t o t r eat a br oad var iet y of cancer s, usually along wit h int ensive
chemot her apy or r adiot her apy. Dir ect inject ion of BCG int o melanoma nodules almost always
leads t o r egr ession of t he inject ed nodules and, occasionally, of dist ant , noninject ed nodules.
Int r avesicular inst illat ion of BCG in pat ient s wit h super ficial bladder car cinoma has pr olonged
disease-fr ee int er vals, possibly as a r esult of immunologic mechanisms.
Ot her mi scella n eou s fa ct or s such as t umour necr osis fact or α (TNF α) and lymphot oxin
have been shown t o kill t umour cells in vit r o and in vivo. They also st imulat e t he funct ional
act ivit y of many t ypes of immune cells such as CTLs, NK cells and macr ophages, t her eby
augment ing t he immune r esist ance t o t umour s. Colony st imulat ing fact or s r egulat e pr olifer at ion
of gr anulocyt es and macr ophages, t hey also enhance t umor icidal act ivit y in macr ophages and
induce TNF pr oduct ion by monocyt es. These fact or s can also be used t o t r eat pancyt openia, a
major complicat ion of cancer chemot her apy, since t hey st imulat e nor mal haemat opoiet ic
pr ogenit or cells in vivo.
™™™
IMMUNITY AGAINST INFECTIOUS DISEASES
CHAPTER – 20
T
he development of act ive immunit y has it s or igins in t he long and legendar y fight
against micr obial infect ions and t oxins. The human body lives in const ant cont act
wit h bact er ia, vir uses, par asit es and fungi. Some of t hese associat ions ar e beneficial, ot her s
t ur n sour . Wit h t he under st anding of t he many var ied host defences against infect ion, was
bor n t he concept of act ive pr ot ect ion against infect ious disease. This chapt er deals wit h t he
mechanisms of host defence in t he face of const ant challenges by a shr ewdly manipulat ive sea
of micr o or ganisms.
Immune Responses to Bacterial Infections
The cynical among us may suggest t hat bact er ia have been, and will always r emain, one
jump ahead of man’s endeavour t o conquer infect ious disease. The varied and ingenious st rat egies
adopt ed by bact er ia ensur e t heir cont inued sur vival.
(a) Attachment and colonization
The mucous membr anes ar e const ant ly exposed t o micr o or ganisms fr om t he envir onment .
To colonize t he mucous membr anes bact er ia must have t he abilit y t o adher e closely t o membr ane
sur faces and mult iply t her e. It is now believed t hat S treptococcus mutans causes dent al car ies.
The or ganism has a const it ut ive enzyme, glucosyl t r ansfer ase, which is able t o conver t sucr ose
t o dext r an which is ut ilised by t he or ganism for adhesion t o t he t oot h sur face. At t achment is
most oft en mediat ed by sur face r ecept or s on bot h host and bact er ial cells. Gr oup A β haemolyt ic
S treptococci adher e by t he M ant igen, t he pr ot ein component of sur face fimbr iae. In Escherichia
coli, t he adhesive fact or is a pr ot ein filament on t he bact er ial sur face, designat ed t he K88
ant igen. Gonococci adher e t o epit helial cell sur faces by pili. These ar e just a few examples of
novel met hods of at t achment , some t o specific r ecept or s on human cells. At t achment ensur es
t hat t he or ganism is not washed off by body secr et ions such as saliva, mucous or ur ine and t he
or ganism need no longer compet e wit h nor mal flor a for a place t o pit ch it s t ent .
Since adherence t o epit helial cells of t he mucous membranes is so vit al t o est ablish infect ion,
t he host has devised sever al mechanisms t o over come t his ploy. The se cr e t or y a n t i b od y,
IgA, affords prot ect ion in secret ed body fluids such as t ears, saliva, nasal and int est inal secret ions.
IgA pr event s bact er ial adher ence t o mucosal sur faces. If an infect ious agent succeeds in dodging
t he IgA bar r ier , it is confr ont ed wit h t he immunoglobulin IgE, which is pr esent in mucosal
secr et ions. IgE mediat ed r elease of mast cell cont ent s helps enhance t he inflammat or y r esponse.
(b) Resistance to phagocytosis
The pr incipal host defence mechanism is ingest ion and dest r uct ion by phagocyt osis. This
is effect ively over come by t he capsular mat er ial t hat some bact er ia pr oduce. Capsulat ed st r ains
Immunity against Infectious Diseases 175
of t he pneumococcus and Bacillus anthracis ar e r esist ant t o phagocyt osis. The pr incipal fact or
r esponsible for t his r esist ance is t he polysacchar ide of t he capsule of pneumococcus and t he
polypept ide in t he capsule of B. anthracis.
The host defence mechanism oper at es t o cir cumvent t his pr oblem by ensur ing t hat
op s on i za t i on by a nt ibody or complement t a kes pla ce. Opsoniza t ion gr ea t ly fa cilit a t es
phagocyt osis and is t he basis of immunit y t o such infect ions. Opsonizat ion wit h C3b at t r act s
cells wit h t he CR I r ecept or , such as pr imat e r ed cells. Complexes wit h aggr egat es of r ed cells
ar e t hen t r anspor t ed t o t he liver for phagocyt osis. The lipopolysacchar ide of gr am negat ive
or ganisms act ivat es complement via t he alt er nat ive pat hway leading t o cell lysis. Biologically
act ive subst ances such as C3a and C5a aid chemot axis and enhance t he inflammat or y r esponse.
(c) Intracellular growth by bacteria
Some bact er ia ar e r esist ant t o int r acellular killing. Tuber cle and lepr osy bacilli and t he
Brucella species ar e able t o sur vive and mult iply wit hin phagocyt es and ar e t her eby easily
disseminat ed wit hin body t issues. These organisms defy t he killing mechanism wit hin phagocyt es
in a var iet y of ways. Mycobacterium tuberculosis inhibit s fusion of lysosome wit h phagosome,
Mycobacterium leprae has a r esist ant out er coat , some r icket t siae slip out of t he phagosome t o
sur vive undist ur bed in t he cyt osol.
Wher e ant ibody or complement have no access t o int r acellular or ganisms, t he human
immune mechanism r eact s using ce llu la r i mmu n i t y. Bot h cyt ot oxic T cells and act ivat ed
macr ophages play an impor t ant par t in cell mediat ed killing of int r acellular or ganisms. Chr onic
gr anulomat ous r eact ion r epr esent s an at t empt by t he body t o wall off per sist ent infect ion. The
act ivat ed macr ophage has an abundance of hydr olyt ic enzymes and densely packed macr ophages
ar e t he pr ominent feat ur e of chr onic gr anulomas.
(d) Microbial toxins and enzymes
A for midable ar r ay of t oxins and enzymes ar e pr oduced by micr o or ganisms t o pr omot e
t heir sur vival in t he human host . The pr oduct ion of coagulase by st aphylococci is closely r elat ed
t o vir ulence. Bact er ia coat ed wit h fibr in, as a r esult of coagulase act ion, r esist phagocyt osis.
Pseudomonas aeruginosa r eleases elast ases which inact ivat e C3a and C4a. Or ganisms such as
gonococci and meningococci pr oduce pr ot eases t hat split IgA dimer s. Sever al gr am negat ive
or ganisms pr oduce dr ug r esist ance enzymes which act against ant ibiot ics. The choler a t oxin
at t aches t o a specific r ecept or t he - GMI ganglioside, on t he ent er ocyt es, befor e t he t oxin
begins t o t ake effect .
Locally synt hesized IgA pr event s not only bact er ial colonizat ion, but also t oxin at t achment
t o it s r ecept or . Hence or al choler a vaccines which pr omot e local IgA synt hesis ar e consider ed
far mor e beneficial t han par ent er al IgG pr oducing choler a vaccines.
Immune Responses to Viral Infections
Vir uses escape much of t he ant ibody mediat ed immune r esponse since t hey ar e obligat e
int r acellular par asit es. Vir uses at t ach t o specific r ecept or s on t he cell sur face and ar e init iat ed
int o t he host syst em at sever al por t als of ent r y: skin and conjunct iva, dir ect ly int o t he blood or
via t he mucous membr anes lining t he upper r espir at or y, gast r oint est inal and genit o ur inar y
t r act s.
176 Immunology– Introductory Textbook
Vi r a l e sca p e me ch a n i sms have evolved in many ingenious ways. The adeno, ent er o,
influenza and par a influenza vir uses occur as many ant igenically dist inct ser ot ypes. Immunit y
against one ser ot ype offer s no pr ot ect ion against a subsequent unr elat ed ser ot ype. Fur t her ,
wit h infect ions such as influenza and t he common cold t he incubat ion per iod is shor t and t he
or gan of t r opism is pr act ically at t he por t al of ent r y. Ther e is lit t le t ime or exposur e of t he
vir us t o init iat e or int er act wit h t he immune syst em. Hu ma n i n t e r fe r on seems t he most
significant mechanism of defence against such infect ions.
Ant igenic shift and dr ift is a mechanism t he influenza vir us has evolved t o keep t he
defence syst em at bay. By t his ploy t he vir us is capable of const ant ly changing t he st r uct ur e of
it s sur face pr ot eins. This occur s by a n t i ge n i c d r i ft which r esult s fr om point mut at ions in t he
genome of t he vir us; or by a n t i ge n i c sh i ft wher e t her e ar e major changes in t he vir al genome
due t o r ecombinat ion bet ween human and animal vir uses. Ent ir ely new st r ains of vir uses t hus
evolve; an immune r eact ion against t he or iginal st r ain is r ender ed ineffect ive against t he new
st r ain and major epidemics have r esult ed.
Var icella zost er and her pes vir uses r emain dor mant and pr ot ect ed in neur al ganglia.
Being in such pr ivileged sit es t hey ar e not exposed t o ser um ant ibody. Her pes vir uses have
evolved a met hod of spr ead, by cell t o cell cont act via int er cellular br idges, hence t her e is no
ext r acellular exposur e.
Ot her vir uses such as t he Epst ein - Bar r vir us have a life long associat ion wit h t he host ,
being at t ached t o r ecept or s on B cells.
The host ensur es adequat e defence in a var iet y of ways. The mucosa lining t he t r act s
which communicat e t o t he ext er ior is r eplet e wit h lymphocyt es, plasma cells and macr ophages.
Vir us specific cell mediat ed immunit y oper at es via T cells st at ioned for defence at mucosal
sit es. These T cells ar e also act ive against t hose vir uses t hat spr ead fr om cell t o cell and
r emain hidden fr om humor al fact or s. Cyt ot oxic T cells (CTLs) ar e dir ect ly cyt ot oxic t o cells
infect ed wit h vir uses. Vir us specific ant igens of infect ed cells ar e r ecognized by r ecept or s on T
cells in associat ion wit h MHC Class I ant igens. CTL pr olifer at ion, act ivat ion and t ar get cell
cyt ot oxicit y ensues. If cell t o cell spr ead of vir us occur s, T helper cells st imulat ed by ant igen,
secr et e IFN γ, which r ender s cont iguous cells immune t o vir al r eplicat ion. IFN γ also enhances
NK cell act ivit y and r esult ant non specific killing of t ar get cells. If vir al ant igen st imulat es
specific ant ibody, t his ant ibody is used t o mediat e ant ibody dependant cellular cyt ot oxicit y
(ADCC).
Secr et or y IgA in mucous secr et ions act s locally t o neut r alize vir us at t he por t al of ent r y,
pr event ing at t achment of vir us t o epit helial cells and subsequent invasion int o t issues. Ser um
ant ibodies IgG, IgM and IgA neut r alize vir uses dur ing t heir shor t per iod of vir aemia, as in t he
case of polio and r abies vir us infect ions. Blood bor ne vir uses like t he ar bovir uses, hepat it is B
virus and cyt omegalo virus come under ant ibody at t ack. Ant ibody mediat ed, complement induced
lysis of enveloped vir uses occur s. Ant i neur aminidase pr event s r elease of infect ious vir us and
count er s t he spr ead of vir al par t icles.
And finally t he neonat e is pr ot ect ed fr om let hal vir al infect ions by immunoglobulins in
br east milk and colost r um. For a compr ehensive view of host defence mechanisms against
vir al infect ions see Figur e 20.1.
Immunity against Infectious Diseases 177
Figure 20.1. Host defence mechanisms against viral infections. Macrophages (M) may either
enhance viral replication when susceptible or restrict viral growth when resistant. B cells after
antigen presentation by macrophages differentiate into memory cells and plasma cells. T cells have
a multifunctional role: they provide B cell help, cause cytotoxicity and secrete lymphokines.The
virally infected cell is hence subjected to complement mediated lysis, T cell cytotoxicity, NK cell
activity and ADCC.
Immune Responses to Parasitic Infections
Par asit es ar e biologically complex ent it ies, t hat ar e well adapt ed for sur vival in human
host s who ar e immunologically compet ent . Why ar e such host s unable t o mount an effect ive
defence st r at egy against even t he simplest pr ot ozoan par asit es? Because t her e is lit t le evidence
of immunologic pr ot ect ion against par asit ic infect ions, acquir ed specific immunit y has been
disput ed for a long t ime. Micr o or ganisms t o a cer t ain ext ent evade immune r esponses by r apid
mult iplicat ion. Par asit es, because of t heir complex life cycles r equir e t ime for mult iplicat ion
and t her efor e t hey have evolved var ious met hods of evasion - so successful t hat par asit es
sur vive in immune host s for year s.
178 Immunology– Introductory Textbook
The many ways in which par asit es block nor mal micr obicidal mechanisms is illust r at ed in
t he following figur e (Figur e 20.2).
Figure 20.2. Mechanisms of immune response evasion by parasites.
1. When par asit es such as t he Plasmodium species become int r cellular and ent er t he
liver , or when met acer car iae which do not mult iply in t he host get embedded in t he
eye or t he br ain t hey escape t he immune mechanism.
2. Similar ly when par asit es r eside and t hr ive solely in t he gut lumen t her e is lit t le
cont act wit h t he immune syst em unless t issue invasion occur s. Helmint hs wit hin t he
gut and Giardia lamblia ar e common examples of t his phenomenon.
3. Ot her or ganisms such as t he Schist osomes disguise t hemselves wit h host ant igens.
Schist osomes exhibit glycopr ot ein/glycolipid ant igens der ived fr om host r ed blood
cells as t he par asit es penet r at e t hr ough t he skin. Hence, host r esponses ar e not
dir ect ed t o t hese par asit es, only t o newly ent er ed schist osomulae. This phenomenon
has been t er med concomit ant immunit y.
4. Toxoplasma gondii and Trypanosoma cruzi live wit hin phagocyt ic cells. T.gondii
inhibit s phagosome-lysosome fusion. T.cruzi escapes fr om t he phagosome t o lie
dor mant in t he cell cyt osol.
Immunity against Infectious Diseases 179
5. Tr ypanosomes, leishmania and malar ia par asit es live wit hin lymphocyt es.
6. They inact ivat e host lymphocyt es and cause polyclonal B cell st imulat ion.
7. This r esult s in an abundance of ineffect ive and dir ect ionless ant ibodies.
8. Helmint hs such as t he Ascar is migr at e ar ound t he body st imulat ing var ious r esponses
and t hen move away fr om an est ablished r esponse, escaping t heir consequence by
ent er ing t he gut .
9. The malar ia par asit e exhibit s st age and species specific ant igens, shedding ant igens
at ever y st age. Ent amoeba hist olyt ica also r egular ly sheds it s sur face ant igens
confusing t he immune syst em even mor e.
10. Ant igenic var iat ion is t he best known example of t he evasion t act ic. Trypanosoma
brucei is par t icular ly successful at t his gambit . These or ganisms display sever al
glycopr ot ein sur face coat s each wit h a var iable ant igen t ype. When a r esponse is
mount ed against one t ype, anot her one is manifest r equir ing a whole new set of
ant ibodies.
11. Sever al par asit es inhibit cell or ant ibody binding, cause deplet ion of ant igen sensit ive
B cells and gener alized immunodepr ession. T. brucei, T. gondii, t he Plasmodia and
E.histolytica are a few examples of parasit es t hat cause generalized immunodepression.
The h ost ’s r e sp on se t o par asit ic infect ions is not t ot ally non exist ent . Innat e or nat ur al
immunit y plays an enor mous r ole as evidenced by t he fact t hat of all t he pr ot ozoa (animal and
human) t hat man comes int o cont act wit h, only few ar e pat hogenic t o humans. Gen et i c fa ct or s
also play a r ole in host suscept ibilit y t o par asit ic disease.
An t i b od y me d i a t e d i mmu n i t y is only par t ially effect ive and only in some cases.
P r e mu n i t i on is a t er m used t o descr ibe a cont r olled level of par asit emia, as in malar ia, which
r esult s fr om ant ibody act ion. The spor ozoit e and mer ozoit e st ages of plasmodium evoke ant ibody
r esponses t hat mediat e pr emunit ion. The immunoglobulin IgE, r epr esent s an impor t ant line
of defence. A ser ies of IgE molecules have been found t o coat wor ms and lead t o eosinophil
degr anulat ion. The major basic pr ot ein (MBP) r eleased, pr oduces wor m damage and ot her
vasoact ive amines enhance a local inflammat or y r esponse.
Cellular immunit y via T cyt ot oxic cells does not appear t o play a pr edominant r ole.
However,T cell relat ed lymphokines help act ivat e t he formidable macrophage, which is import ant
in t he int r acellular killing of par asit es such as T.gondii, Leishmania sp. and T.cruzi. Par asit es
have held sway over t he human host , in addit ion, by pr oducing damaging immunopat hologic
r eact ions such as liver gr anulomat a and aut oimmune car diac disease.
The over all impact of host -par asit e int er act ion seems t hus t o swing in favour of t he par asit e.
This is evidenced by t he finding t hat all at t empt s at successful vaccinat ion against par asit es
have so far failed. It r emains t o be seen if man’s ingenuit y when pit ched against t he par asit es’
impr essive ar r ay of weapons comes up t he vict or .
™™™
IMMUNIZATION
CHAPTER – 21
T
he gr eat est t r iumph of immunology has been t he successful use of immunizat ion
pr ocedur es in t he cont r ol of pot ent ially fat al infect ious diseases. The concept of
immunizat ion r ose fr om t he obser vat ion t hat individuals who r ecover fr om cer t ain diseases
ar e pr ot ect ed for life fr om r ecur r ences. The int r oduct ion of small quant it ies of fluid fr om act ive
small pox pust ules int o uninfect ed per sons (var iolat ion) was an effor t at mimicking nat ur al
infect ion. It is for t uit ous t hat t hese exper iment s wer e done in 1721 in an age when et hical
clear ance was not r equir ed! J enner int r oduced vaccinat ion in 1796 using cow pox t o pr ot ect
against small pox. This was t he fir st document ed use of live at t enuat ed vaccinat ion and t he
beginning of moder n immunizat ion. Hist or ical milest ones in immunizat ion ar e list ed in
Table 21.1.
Table 21.1: Milestones in Immunization
Variolation 1721
Vaccination 1796
Rabies vaccine 1885
Diphtheria toxoid 1925
Tetanus toxoid 1925
Pertussis vaccine 1925
Viral culture in chick embryo 1931
Yellow fever vaccine 1937
Influenza vaccine 1943
Viral tissue culture 1949
Polio vaccine (Salk) 1954
Polio vaccine (Sabin) 1956
Measles vaccine 1960
Tetanus immune globulin (human) 1962
Rubella vaccine 1966
Mumps vaccine 1967
Hepatitis B vaccine 1975
Licensure of first recombinant vaccine (Hepatitis B) 1986
Meningitis C vaccine 1999
Immunization 181
Primary and Secondary Immune Responses
The fir st exposur e t o a n a nt igen evokes a p r i ma r y r e s p on s e . Immedia t ely a ft er
int r oduct ion of immunogen lit t le or no ant ibody is det ect ed. This is called t he induct ive or
lat ent per iod (Figur e 21.1). Dur ing t his per iod, t he immunogen is r ecognised as for eign and
pr ocessed. The dur at ion of t his per iod is var iable and depends on t he t ype of ant igen used,
species of animal and r out e of immunizat ion.
Figure 21.1. Immune responses to primary and secondary stimulation.
Dur ing t he logar it hmic phase, ant ibody concent r at ion incr eases logar it hmically for 4-10
days, unt il it r eaches a peak. This peak ant ibody level usually t akes 4 t o 5 days for er yt hr ocyt es,
8-12 days for soluble pr ot eins and 2 t o 3 mont hs for t he t oxoid of Corynebacterium diphtheriae.
The log phase is followed by a st eady phase, wher e r at es of ant ibody synt hesis equal r at es
of ant ibody cat abolism. The decline phase follows wher e ant ibody synt hesis st eadily falls,
finally r eaching pr e immunizat ion levels. The ear ly pr imar y r esponse is char act er ized by a
pr edominance of IgM over IgG, IgM pr oduct ion is t r ansient and wit hin 2 weeks of init iat ion of
t he r esponse, IgG pr edominat es.
The secon d a r y i mmu n e r esp on se occur s upon second exposur e t o t he same immunogen,
weeks, mont hs or even year s lat er . The secondar y immune r esponse is accompanied by an
acceler at ed r esponse fr om alr eady commit t ed B lymphocyt es in t he memor y pool. Rapid
pr olifer at ion and differ ent iat ion int o plasma cells yields a higher ant ibody out put . The secondar y
r esponse is char act er ized by an init ial negat ive phase, which is due t o t he immediat e r eact ion
of pr e exist ing ant ibody wit h new immunogen. An enhanced r esponse follows due t o anamnest ic
r ecall of pr e commit t ed memor y cells. This enhanced r esponse under lies t he pr inciple of
administ er ing boost er doses aft er specific t ime int er vals dur ing immunizat ion pr ocedur es.
Immunologic memor y can last for year s, pr oviding long last ing immunit y t o cer t ain bact er ial
and vir al infect ions. The evolut ion of immunologic memor y is a funct ion of T helper cells. T
independent ant igens t her efor e cannot elicit memor y or for t hat mat t er a secondar y IgG
r esponse.
182 Immunology– Introductory Textbook
The Classification of Immunity
Immunit y can be classified as:
• Nat ur al/Innat e (Discussed in Chapt er 2)
• Acquir ed immunit y can be:
• Passively acquir ed immunit y
• Act ively acquir ed immunit y
Passively acquired immunity
Passive immunit y is acquir ed (i) by t he newbor n fr om t he mot her ; (ii) by administ er ing
pr efor med immunoglobulins t o an individual.
Maternal transfer of antibodies
The neonat e is endowed wit h a r elat ively immat ur e lymphoid syst em; t he pr emat ur e
baby wit h a gr ossly ineffect ive immune mechanism. In ear ly life, t he newbor n is t hus pr ot ect ed
by mat er nally der ived ant ibodies (IgG) t r ansfer r ed passively via t he placent a. Colost r um and
br east milk also affor d significant pr ot ect ion t o t he neonat e. The major immunoglobulin in
milk is t he secr et or y IgA, which r emains in t he gut of t he newbor n, pr ot ect ing t he int est inal
mucosal sur faces fr om ent er ic pat hogens. Int er est ingly, it has been found t hat t he secr et or y
IgA in t he br east milk is specific for bact er ial and vir al ant igens found in t he mot her ’s gut . It is
pr esumed t hat IgA pr oducing cells r esponding t o gut ant igens migr at e fr om t he gut mucosa t o
colonize br east t issue, now consider ed t o be a par t of t he mu cosa l a ssoci a t e d lymp h oi d
t i ssu e (MALT). Her e t he IgA pr oducing cells secr et e specific ant ibodies which appear in milk.
This cir cuit has also been t er med t he e n t e r o-ma mma r y a xi s .
Gamma globulins
Ant ibody, eit her as whole ser um or concent r at ed gamma globulins (immune globulin), is
obt ained fr om human volunt eer s who have r ecover ed fr om a specific infect ious disease or have
r eceived immunizat ion. The globulin consist s pr edominant ly of IgG. Administ r at ion of such
human immune globulin (HIG) offer s immediat e pr ot ect ion t o individuals who ar e at r isk,
par t icular ly wher e act ive immunizat ion may t ake 7-10 days for effect ive ant ibody pr oduct ion.
Passive immunizat ion is also useful t o t hose individuals who ar e unable t o pr oduce ant ibody for
t hemselves. Hazar ds associat ed wit h administ er ing human immune globulin (as pooled ser um)
include t r ansmission of blood bor ne vir uses - hepat it is B or C vir uses and t he human immuno
deficiency vir us (HIV). Pur ified IgG pr epar at ions ar e, however , fr ee of t hese vir us par t icles.
Ant ibodies may also be available fr om animal ser a. However , such immunoglobulins ar e
less desir able, as non human pr ot eins ar e clear ed away by t he host immune r esponse against
t hem. In addit ion, hazar dous immune r eact ions against animal pr ot eins may lead t o t he
development of anaphylaxis or ser um sickness. Neit her human nor animal immune globulin
should be administ er ed int r avenously for fear of anaphylact ic r eact ions.
Human immune globulin against var icella, t he var icella zost er immune globulin (VZIG) is
indicat ed in individuals wit h defect ive immune syst ems such as pr emat ur e infant s, childr en
wit h immunodeficiency diseases and pat ient s on st eroid t reat ment . The vaccinia immune globulin
is given t o pat ient s wit h disseminat ed vaccinia or ot her complicat ions of small pox vaccinat ion.
Rabies and hepat it is B immune globulin is administ er ed t o individuals who have been exposed
and ar e at r isk. Dipht her ia and bot ulism ant it oxin is used as t her apy in pat ient s who have
cont r act ed t he disease. The equine sour ce of t hese lat t er t wo ant ibodies is st ill widely used.
Immunization 183
Tet a nus a nt it oxin, now a huma n immune globulin, is given bot h pr ophyla ct ica lly a nd
t her apeut ically in appr opr iat e cases.
Ant ibodies (equine) for non infect ious condit ions is also available: ant ivenin against black
widow spider and snake venoms. Human immune globulin t o t he Rh blood gr oup is widely used
t o pr event haemolyt ic disease of t he newbor n.
Active Immunity
Besides suffer ing t he disease (and sur viving it !), vaccinat ion is t he only ot her way of
acquir ing act ive immunit y against an infect ious agent . The advant ages of act ive over passive
immunizat ion ar e due t o t he fact t hat t he individual’s immune syst em is st imulat ed t o pr oduce
an immune r esponse against a given ant igen. Host par t icipat ion ensur es t hat bot h t he humor al
and cellular component s of t he immune syst em ar e act ivat ed. Consequent ly, T cell help is
r ecr uit ed and immunologic memor y is available t o boost t he r esponse aft er subsequent exposur e
t o t he ant igen. Ant ibodies so for med ar e longer last ing as compar ed t o passively acquir ed
immunoglobulin.
Act ive immunizat ion may be per for med wit h eit her k i lled or li ve a t t e n u a t e d va cci n e s
and t oxoi d s .
The commonly used killed vaccines ar e:
• bact er ial vaccines for
t yphoid
choler a
per t ussis
plague
• vir al vaccines for
rabies
poliomyelit is (t he Salk vaccine)
hepat it is B
influenza
Live Attenuated Vaccines Include
• bact er ial
BCG – a live at t enuat ed Mycobacterium bovis for t uber culosis.
Ty21a – live or al at t enuat ed mut ant t yphoid bacillus
• vir al:
live vaccinia vir us for small pox
rubella
measles
mumps
polio (t he Sabin vaccine)
yellow fever vir us
Besides t hese, t her e ar e t oxoi d va cci n e s for :
dipht her ia
t et anus.
184 Immunology– Introductory Textbook
Polysaccharide vaccines for
• Haemophilus influenzae t ype B (Polyr ibosyl-r ibit ol-phosphat e, conjugat ed t o t et anus
pr ot ein)
• Neisseria meningitidis (a combinat ion vaccine against gr oups A,C,Y,W135 and t he
new Meningit is C vaccine)
• S treptococcus pneumoniae (a polyvalent 23 valent polysacchar ide vaccine and t he
new 7-valent vaccine)
Li ve a t t e n u a t e d va cci n e s have many a d va n t a ge s . At t enuat ion mimics t he nat ur al
behaviour of t he or ganism wit hout causing disease. The immunit y confer r ed wit h live at t enuat ed
vaccines is super ior because act ively mult iplying or ganisms pr ovide a sust ained ant igen supply.
The immune r esponse t akes place lar gely at t he sit e of nat ur al infect ion as in t he case of t he
live polio vaccine and t he or al t yphoid vaccine pr oducing an obviously advant ageous local
secr et or y IgA r esponse.
The h a za r d s of u si n g a t t e n u a t e d va cci n e s, t hough uncommon, must be document ed
as t he r isk of developing complicat ions is a ver y r eal one. A ver y small number of individuals
develop encephalit is following measles vaccine, however , t he danger of developing encephalit is
fr om nat ur al infect ion is far gr eat er . Ther e is t he possibilit y of t he at t enuat ed vir us r ever t ing
t o it s vir ulent for m; chances of t his decr ease if t he at t enuat ion incor por at es sever al gene
mut at ions inst ead of just one. Pr eser ving adequat e cold st or age facilit ies and maint aining t he
cold chain fr om t he labor at or y int o t he field, is a cont inuing pr oblem especially in t he t r opics.
Live at t enuat ed vaccines ar e not advised in pat ient s wit h an immunodeficiency disease, in
pat ient s on st er oid and ot her immunosuppr essive t r eat ment and for t hose under going
r a diot her a py. Ma ligna ncies such a s lymphoma s a nd leuka emia s a nd pr egna ncy a r e a ll
cont r aindicat ions t o t he administ r at ion of live at t enuat ed vaccines. The or al polio vaccine is
cont r aindicat ed for any member of a household wher e t her e is a pat ient wit h a lymphoma or
leukaemia, as t he live virus is shed in t he st ool of a vaccinat ed individual and poses a t ransmission
r isk. Table 21.2 list s t he cur r ent exper iment al and r est r ict ed use vaccines.
Table 21.2: Experimental and restricted use vaccines
Vaccine Status
Adeno virus Live attenuated, for military recruits
Anthrax For those with occupational exposure; military
Arboviruses: Experimental only 50-60% protection
Kyasanur Forest Disease Killed vaccines
Japanese encephalitis
AIDS Experimental
Cholera New oral vaccine restricted for travellers
Cytomegalovirus Experimental
Malaria Experimental Plasmodium falciparum malaria
vaccine based on the circumsporozoite protein
Gram negative bacteria Experimental and restricted
Leprosy Heat killed M. leprae + BCG. Clinical trials are
on
Rota virus Live oral tetravalent vaccine, withdrawn in 1998
due to rare complication with intussusception;
new vaccine trials now on.
Immunization 185
Newer approaches to vaccine preparation
Since t he pr ocess of at t enuat ion is cumber some and t ime consuming, sever al newer
appr oaches t o vaccine development ar e being r esear ched.
(a) S ub unit vaccines
Sub unit vaccines ar e being designed, which use only t he r elevant immunogenic por t ions
of t he or ganism. This has been possible using monoclonal ant ibodies and r adio labelling
t echniques. Sur face pr oject ions of t he influenza vir us, t he measles vir us and t he r abies vir us
elicit neut r alizing ant ibody and can be exploit ed for t his pur pose. If t he low immunogenicit y of
t hese sub unit s can be over come, such vaccines ar e st able, fr ee fr om ext r aneous pr ot eins and
nucleic acids and pr ecise in t heir composit ion.
(b) Biosynthesis of immunogenic proteins
Specific immunogenic sur face pr ot eins need t o be available in lar ge quant it ies for vaccine
pr epar at ion. The pr oblem of isolat ing and char act er izing ant igenic pr ot ein moiet ies has been
over come by cloning t he genes t hat code for t hese pr ot eins in bact er ial or eukar yot ic (yeast )
cells or in t he vaccinia vir us. In 1986, t he fir st r ecombinant (cloned) vir al vaccine was licensed
for use. The Hepat it is B sur face ant igen (HBsAg) is t he immunogen t hat st imulat es pr ot ect ive
immunit y. The gene t hat codes for HbsAg has been cloned most successfully in t he yeast cell.
The ant igen pr epar ed by gr owing t he yeast cells cont aining t he r ecombinant gene in mass
cult ur e is widely used as a safe and effect ive vaccine.
(c) S ynthetic peptide vaccines
A limit ed number of sit es on an or ganism ar e involved in evoking an immune r esponse. If
t hese sit es, consist ing mainly of pept ide fr agment s, can be synt hesized t hey pr ovide a possible
means of obt aining chemical polypept ides as vaccines. Unfor t unat ely, t his t ask has been made
har der by t he finding t hat immunogenic pept ides ar e not simple, linear sequences of amino
acids and t he final configur at ion of t he pr ot ein cannot always be synt hesized in t he for m t hat B
cells r ecognize. Besides, amino acid sequences of t hese pept ides ar e discont inuous and ar e
br ought t oget her by folding of t he molecule.
These a nd ot her st r a t egies such a s using a nt i idiot ypes a s va ccines a r e st ill ver y
exper iment al, t hough animal st udies have been encour aging.
™™™
IMMUNODEFICIENCY DISEASES
CHAPTER – 22
T
he t wo major ar ms of t he immune syst em-a n t i bod y (B-cell) mediat ed immunit y and
ce llu la r (T-ce ll) immunit y, help defend t he host against bact er ial, vir al, fungal and
pr ot ozoal infect ions. They also per for m t he impor t ant funct ions of immune sur veillance for
malignant cells. In t hese, t he many facet s of defence and sur veillance, t hey ar e ably aided by
t wo ot her a n ci lla r y syst e ms: comp le me n t and p h a gocyt osi s. Immunodeficiency disor der s
ar e consequent ly discussed under four main headings:
• Immunoglobulin (B-cell) immunodeficiency disor der s
• Cellular (T-cell) deficiency diseases
• Phagocyt ic dysfunct ion
• Complement deficiency st at es
Immunoglobulin (B-cell) Immunodeficiency Disorders are summarised in Table 22.1
(a) X-linked infantile hypogammaglobulinaemia
X linked hypo or agammaglobulinaemia was t he fir st immunodeficiency disor der t o be
descr ibed clinically. The condit ion is X linked and t he gene gover ning t he disor der has been
localized t o t he long ar m of t he X chr omosome. Because infant s ar e bor n wit h IgG t r ansfer r ed
fr om t heir mot her s, t he disease does not manifest unt il lat e in t he fir st year of life. The effect s
of t his condit ion usually appear in male infant s bet ween 9 mont hs and 2 year s of age.
The clinical cour se is mar ked by unusual suscept ibilit y t o pyogenic or ganisms namely,
Haemophilus influenzae, pneumococci, st r ept ococci, st aphylococci and meningococci. The
infect ions ar e mor e fr equent and sever e t han t hose of nor mal childr en and r ecur r ences ar e
common. The infect ion is slow t o r espond t o ant ibiot ics and br onchiect asis and pulmonar y
insufficiency ar e common sequelae. These childr en, however , have nor mal r esist ance t o common
vir a l infect ions, fungi, a nd most gr a m nega t ive or ga nisms; but t hey a r e suscept ible t o
poliomyelit is. Some childr en will manifest wit h sympt oms of r heumat oid ar t hr it is. Diar r hoea
and malabsor pt ion syndr ome ar e common, almost always caused by Giardia lamblia. Deat h is
due t o a fat al syndr ome, similar t o der mat omyosit is wit h neur ologic involvement . In sever al
pat ient s wit h t his syndr ome, echovir uses have been cult ur ed fr om blood, st ool and cer ebr ospinal
fluid.
Diagnosis is made by measur ement of t he ser um level of each class of immunoglobulin.
Ther e is usually less t han 100 mg/dl of IgG and levels of IgA, IgM, IgD and IgE ar e ext r emely
low or undet ect able. Examinat ion of whit e blood cells shows a t ot al deficiency of B cells. Test s
for cell mediat ed immune funct ion ar e nor mal. The lymphoid or gans ar e char act er ized by a
t ot al lack of ger minal follicles, B cells and plasma cells.
Immunodeficiency Diseases 187
Tr eat ment consist s of int r amuscular or int r avenous administ r at ion of gamma globulin
for life. Once t he diagnosis is made, all subsequent male offspr ing of t he mot her or mat er nal
aunt s should be scr eened by immunoelect r ophor esis ever y t wo mont hs dur ing t he fir st year of
life for t heir ser um immunoglobulin pr ofile. To t est for female car r ier s of t he gene, chr omosomal
analysis is done. The pr ognosis is good for pat ient s whose condit ion is diagnosed and t r eat ed
ear ly.
(b) Common variable immunodeficiency
Common var iable immunodeficiency (CVID) pr oduces hypogammaglobulinaemia t hat does
not appear t o be genet ically t r ansmit t ed. It affect s males and females equally. The condit ion
occur s at any age, usually aft er puber t y and is char act er ized by depr essed levels of IgG. IgG
levels ar e less t han 200 mg/dl and ot her immunoglobulins ar e also mar kedly decr eased. B cells
ar e usually pr esent but t hey do not funct ion nor mally. Defect s in cell mediat ed immunit y ar e
also obser ved.
Ther e appear t o be mult iple pat hogenet ic causes of combined var iable immunodeficiency.
Defect s include:
1. B cells do not r espond t o T cell help.
2. B cells synt hesize but cannot secr et e ant ibodies.
3. Helper T cells ar e absent .
4. Aut o ant ibodies t o B cells may be pr esent .
Pat ient s wit h CVID ar e subject t o t he same infect ions as t hose who have X linked
hypogammaglobulinaemia; t her e is chr onic involvement of sinuses and r espir at or y t r act . CVID
is also associat ed wit h sever al aut oimmune-like diseases, r esembling r heumat oid ar t hr it is,
idiopat hic t hr ombocyt openia, haemolyt ic anaemia and neut r openia. CVID is oft en associat ed
wit h sever e malabsor pt ion syndr ome which can be caused by G. lamblia infect ion or glut en
sensit ive ent er opat hy. Chr onic lung disease is a common feat ur e. These pat ient s cannot be
t r eat ed wit h st er oids for t heir aut oimmune-like disease due t o incr eased suscept ibilit y t o
infect ion. Ther e is gener alized lymphoid hyper plasia.
Pat ient s wit h CVID can have a nor mal life span. Women wit h t he disease can have nor mal
pr egnancy and nor mal babies who will lack mat er nal IgG.
Tr eat ment consist s of gamma globulin administ r at ion for life and vigor ous use of ant ibiot ics
dur ing infect ion.
(c) Selective IgA deficiency
Select ive IgA deficiency, one of t he most common immunodeficiencies occur s in one of
ever y 600 t o 800 Caucasian per sons. In t his condit ion, IgA in t he ser um is less t han 5 mg/dl, t he
levels of ot her immunoglobulins ar e nor mal. B cells bear ing sur face IgA ar e pr esent , indicat ing
t hat t he pr oblem is pr obably in t he secr et ion of t he IgA.
IgA deficiency is associat ed wit h many differ ent clinical syndr omes. The most fr equent
ar e t hose r elat ed t o sinus and pulmonar y infect ions due t o bact er ia and vir uses. Ther e appear s
t o be an incr ease in aut oimmune, gast r oint est inal, aller gic, connect ive t issue and malignant
diseases.
Most pat ient s wit h IgA deficiency have nor mal cellular immunit y. Despit e t he fact t hat
IgA deficiency pr edisposes t o a var iet y of diseases, most pat ient s ar e sur pr isingly healt hy.
188 Immunology– Introductory Textbook
Pat ient s wit h select ive IgA deficiency should not be given γ globulin since t hey may recognize
inject ed IgA as for eign. The classes of immunoglobulins pr esent , will r eact against t he inject ed
IgA, leading t o anaphylact oid r eact ions dur ing subsequent inject ions. Ther e is no specific
r eplacement t her apy for select ive IgA deficiency. Vigor ous ant ibiot ic t her apy is advocat ed for
infect ions.
(d) Immunoglobulin deficiency with elevated IgM
Immunoglobulin deficiency wit h an elevat ed IgM (150 t o 1000mg/dl) is char act er ized by
low levels of IgG and IgA; IgD may also be elevat ed. In some cases t he disease is X-linked, in
ot her s it appear s as an acquir ed disor der , affect ing bot h men and women. The clinical findings
ar e similar t o t hose seen in X-linked hypogammaglobulinaemia. In addit ion, t her e is a high
frequency of haemolyt ic anaemia, neut ropenia and t hrombocyt openia. The IgG and IgA deficiency
is t hought t o r esult fr om t he lack of T cells which cont r ol IgM t o IgG or IgA swit ching.
Tr eat ment includes ant ibiot ic t her apy for infect ions and globulin administ r at ion for specific
ant igens.
(e) Selective deficiencies of IgM or the subclasses of IgG
Select ive IgM deficiency r ar ely occur s in per sons wit h nor mal IgG or IgA levels. This
deficiency may pr ecede t he onset of CVID. Pat ient s wit h select ive deficiencies of t he IgG sub
classes have a decr ease in t ot al IgG, t he degr ee of which depends on t he sub class involved. The
decr ease is most pr ofound in t he case of IgG1 because 75% of all IgG is of t his subclass. Pat ient s
wit h IgG deficiency ar e especially pr one t o bact er ial infect ion wit h capsulat ed st r ains of
H.i n f l u en z ae a n d t h e pn eu mococcu s. Th e dia gn osis is ma de by t h e a bn or ma l ser u m
elect r ophor et ic pat t er n and confir med by quant it at ing t he IgG sub classes. Such pat ient s r espond
well t o γ globulins.
Table 22.1 Immunoglobulin (B-cell) immunodeficiency disorders
Designation
X-linked agamma-
globulinaemia
Common variable
immuno-
deficiency
(CVID)
Selective IgA
deficiency
Usual Phenotypic Expression
Functional Cellular
deficiencies Abnormalities
Antibody ↓ B cells
Antibody ±↓B cells
IgA antibody ↓IgA plasma
cells
±↓IgA B cells
Presumed
Level of Basic
Cellular Defect
Pre-B cells
B cells
Terminal
differentiation of
IgA cells
impaired
Known or
Presumed
Pathogenetic
Mechanism
Unknown.
Intrinsic B-cell
defect;
underproduction
of B cells
↓T helper cells;
auto-antibodies
to B cells
Unknown
Inheritance
X-linked
Unknown
Usually
unknown,
frequent in
families of
patients with
CVID
Immunodeficiency Diseases 189
Deficiencies of cell mediated (T cell) immunity are described in Table 22.2
Pat ient s wit h T cell immunodeficiencies ar e ext r emely suscept ible t o oppor t unist ic
infect ions. They manifest wit h impaired delayed hypersensit ivit y responses and may be inherit ed
or secondar y t o anot her disor der . Infect ions ar e much mor e likely in pat ient s wit h pur e T cell
deficiencies t han in t hose wit h pur e B cell deficiencies. Innocuous or ganisms such as Candida
albicans and Pneumocystis jir ovecii cause ser ious disease and such pat ient s ar e especially
suscept ible t o t he ent er ic bact er ia, vir uses and fungi. Vaccinat ion wit h cowpox or t he BCG may
lead t o a r apidly fat al out come.
(a) Congenital thymic hypoplasia (Di George Syndrome)
Congenit al t hymic hypoplasia r esult s fr om t he lack of nor mal development of t he t hir d
and four t h br anchial or phar yngeal pouches, which leads t o abnor malit y in t he gr eat vessels,
and t o t he absence of t he t hymus and t he par at hyr oid glands. It is not genet ically t r ansmit t ed
and r esult s fr om an int r aut er ine accident occur r ing befor e t he eight h week of pr egnancy. The
absence of t he t hymus leads t o deficiency in cell-mediat ed immunit y in affect ed childr en.
The T cell defect in pat ient s var ies fr om pr ofound t o mild. These childr en do not exhibit
delayed hyper sensit ivit y r eact ions. The lymph nodes lack par acor t ical lymphocyt es. Plasma
cells ar e pr esent and levels of immunoglobulin ar e nor mal. However , ant ibody r esponses t o
ant igens ar e not nor mal, since no T cell help is obt ained and secondar y r esponses ar e lacking.
As t he pat ient becomes older , T cell funct ion impr oves and usually by five year s of age, t her e is
no abnor malit y in cellular immunit y.
IgG deficiencies
with increased
IgM
Selective
deficiency of IgG
sub classes
κ chain deficiency
Transient
hypogamma-
globulinaemia
IgG heavy chain
deficiency
Antibody ↓IgG and IgA
plasma cells
↓IgM plasma
cells
±↓IgM and
IgG
B cells
One or more IgG ↓Plasma cells
subtypes ±↓Tcells
IgG (κ) ↓ κ+ Bcells
Antibody ↓Plasma cells
B cells normal
IgG1, IgG2, IgG4, None
and in some cases
IgE and IgA2
Failure of
immuno-
globulin class
switching
Unknown
Unknown
Impaired
terminal
differentiation
of B cells
Chromosome
deletion
X linked,
autosomal
recessive or
unknown
Unknown
Autosomal
recessive
Frequent in
heterozygous
individuals in
families with
various severe
combined
immune
deficiencies
Autosomal
recessive
Unknown
Unknown
Point mutation
↓T helper cells
Unknown
190 Immunology– Introductory Textbook
The condit ion is t r eat ed wit h t hymus t r ansplant at ion in t hose infant s who exper ience
fr equent infect ions.
(b) Severe combined immunodeficiency
Sever e combined immunodeficiency (SCID) disease is char act er ized by mar ked deplet ion
of t he cells t hat mediat e bot h B cell and T cell immunit y. SCID is invar iably fat al if left
unt r eat ed. Ther e ar e at least 5 var iant s of SCID (Table: 22.2). SCID is t r ansmit t ed eit her as an
aut osomal r ecessive t r ait or an X-linked r ecessive t r ait : (i) Many of t he cases inher it ed in an
aut osomal r ecessive manner ar e caused by a deficiency in t he enzyme adenosine deaminase
(ADA). (ii) Ot her pat ient s wit h an aut osomal r ecessive for m of SCID lack t he enzyme pur ine
nucleoside phosphor ylase (PNP). (iii) Anot her invar iant of SCID is r et icular dysgenesis, which
is a sever e combined immunodeficiency wit h gener alized gr anulocyt e deficiency. Newbor ns
wit h t his disease lack gr anulocyt es in t he blood and bone mar r ow and die of infect ion in t he
fir st few days of life (iv) In r ar e cases t he common t ype of SCID affect s t he long bones and
causes shor t limbed dwar fism and, (v) A for m of SCID in which immunogobulins ar e nor mal,was
for mer ly called Nezelof’s syndr ome, but is now t er med SCID wit h B cells.
Clinically, onset of infect ions occur s at 3 t o 6 mont hs of age: chr onic pulmonar y infect ions,
diar r hoea, moniliasis and failur e t o t hr ive ar e t he most common manifest at ions of SCID. No
t onsils ar e obser ved on physical examinat ion and t he lymph nodes ar e small t o absent despit e
chr onic infect ions. The t hymus is absent or vest igial. Ther e is a complet e absence of T cells and
ant ibody r esponses ar e low.
The pat hogenesis of t he common t ype of SCID is not known but is t hought t o be due t o a
deficiency in t he enzyme - ADA. Lymphocyt es lacking ADA have excessive dATP which blocks
t he enzyme r equir ed for making t he building blocks of DNA. SCID caused by ADA deficiency
can be diagnosed pr enat ally by amniocent esis, because fibr oblast s in t he amniot ic fluid also
show t he enzyme defect .
SCID due t o ADA deficiency can be successfully t r eat ed wit h bone mar r ow t r ansplant at ion.
Infusions of pur ified adenosine deaminase have also been successful.
(c) Wiskott - Aldrich syndrome
Wiskot t - Aldr ich syndr ome is a n X-linked r ecessive disea se a ffect ing boys a nd is
char act er ized by eczema, t hr ombocyt openia, incr eased suscept ibilit y t o infect ion and bloody
diar r hoea. These pat ient s display aner gy t o common skin t est s using bact er ial and fungal
ant igens. They lack isohaemagglut inins and cannot make ant ibody t o polysacchar ides. Ant ibody
t o pr ot ein ant igens is evident . Tot al IgG levels ar e nor mal, IgE and IgA levels ar e high, IgM is
low. These pat ient s cat abolize t heir immunoglobulins fast er t han nor mal individuals. The
par acor t ical ar eas of t he lymph nodes ar e deplet ed of lymphocyt es.
The disease is at t r ibut ed t o a mor phological abnor malit y of lymphocyt es. The condit ion
can be t r eat ed wit h bone mar r ow t r ansplant at ion.
(d) Immunologic deficiency with ataxia telangiectasia
At axia t elangiect asia is a pr ogr essive neur ologic disease t hat begins in ear ly childhood. It
is char act er ized by cer ebellar at axia, followed by incr easing t r emor and det er ior at ion of ment al
funct ion. The disea se is a ssocia t ed wit h defect s in cell media t ed immunit y a nd wit h
immunoglobulin deficiencies. It is inher it ed as an aut osomal r ecessive t r ait .
Immunodeficiency Diseases 191
Phagocytic Dysfunction Diseases
Pr imar y or int r insic phagocyt ic disor der s ar e r elat ed t o enzymat ic deficiencies wit hin t he
met abolic pat hway in t he phagocyt e, necessar y for killing bact er ia. Suscept ibilit y t o infect ion
in t hese disor der s may r ange fr om mild t o over whelming and fat al. They ar e suscept ible t o
bact er ial infect ion and fungal infect ion st r ikes t he mor e ser ious cases. They have no difficult y
wit h vir al or pr ot ozoal infect ions.
(a) Chronic granulomatous disease
Chr onic gr anulomat ous disease is an X-linked disor der which manifest s in t he fir st t wo
year s of life. Pat ient s ar e suscept ible t o infect ions wit h unusual or ganisms, nor mally of low
vir ulence, such as S taphylococcus aureus, S erratia marcescens and Aspergillus spp. Pat ient s
pr esent wit h dr aining lymphadenit is, hepat osplenomegaly, pneumonia, ost eomyelit is and
abscesses.
Due t o int r acellular enzyme deficiencies in t he gr anulocyt es, met abolism is impair ed
r esult ing in decr eased oxygen consumpt ion, diminished pr oduct ion of hydr ogen per oxide and
super oxide anion. The r esult is t hat int r acellular killing of bact er ia and fungi is impair ed.
Tr eat ment consist s only of t r eat ing t he var ious infect ions, whit e cell infusions have been
at t empt ed in some cases.
(b) Specific enzyme deficiencies
(i) Glu cose -6-p h osp h a t e d e h yd r oge n a se is complet ely lacking in t he leukocyt e, and
pr oduces a disease syndr ome similar t o chr onic gr anulomat ous disease. The disease
has a lat er onset , affect s bot h males and females and haemolyt ic anaemia is pr esent .
(ii) Deficiency of leukocyt e mye lop e r oxi d a se , needed for nor mal int r acellular killing
leads t o r ecur r ent candidial and st aphylococcal infect ions. The leukocyt e r espir at or y
bur st and super oxide anion for mat ion ar e, however , nor mal.
(iii) Modest r educt ion of leukocyt e bact er icidal act ivit y has been associat ed wit h t he
deficiency of leukocyt e a lk a li n e p h osp h a t a se.
(c) Chediak-Higashi Syndrome
Chediak-Higashi syndr ome is a mult i syst em aut osomal r ecessive disor der . The pat ient
pr esent s wit h r ecur r ent bact er ial infect ions, hepat osplenomegaly, par t ial albinism, cent r al
ner vous syst em abnor malit ies and a high incidence of lymphor et icular cancer . The basic defect
appear s t o be abnor mal int r acellular killing of or ganisms and lar ge gr anular inclusions in
whit e blood cells ar e evident . The killing defect consist s of delayed killing t ime even t hough
“r espir at or y bur st ” and oxygen consumpt ion ar e nor mal. Sever al leukocyt e enzymes and
micr ot ubule funct ion appear t o be deficient .
The pr ognosis is poor , most childr en do not sur vive t heir childhood.
(d) Lazy Leukocyte Syndrome
Pat ient s wit h defect ive neut r ophil chemot axis in associat ion wit h neut r openia have an
abnor mal in vivo inflammat or y r esponse. Such pat ient s ar e suscept ible t o sever e bact er ial
infect ions. The pr ognosis is unknown.
192 Immunology– Introductory Textbook
Table 22.2: Deficiencies of cell mediated (T cell) immunity
CMI: Cell mediated immunity LSC: Lymphocytic stem cell
HSC : Haematopoietic stem cell NK: Natural killer
Designation
Congenital
thymic
hypoplasia (Di
George
syndrome)
Severe
combined
immuno-
deficiency
(SCID)
(i) Adenosine
deaminase
(ADA)
deficiency
(ii) Purine
nucleoside
phosphorylase
(PNP)
deficiency
(iii) Reticular
dysgnesis
Wiskott
Aldrich
syndrome
Immuno-
deficiency with
ataxia
telangiectasia
MHC class II
Usual Phenotypic Expression
Functional Cel l ul ar
deficiencies abnormalities
CMI, impaired ↓ T cells
antibody
CMI, antibody ↓T cells
↓ B cells
CMI, antibody ↓ T cells
±B cells
CMI ± antibody ↓ T cells
CMI, antibody, ↓ T cells
phagocytes ↓ B cells
↓phagocytes
Antibody to ↓ T cells
certain antigens ↓ B cells
(mainly) (Progressive)
polysacchar-
ides), CMI
(progressive)
CMI, antibody ↓ T cells
(partial) ↓ plasma
cells
(mainly
those cells
producing
IgA, IgE
±IgG)
CMI ±antibody None
Presumed
Level of
Basic Cellular
Defect
Thymus
LSC
LSC or early
T cells
T cells
HSC
Unknown
Early T cells
and defective
terminal
differentiation
of B cells
T cells B cells
and antigen
presenting cells
Known or
Presumed
Pathogenetic
Mechanism
Embryopathy
of the 3
rd
and
4
th
pharyngeal
pouch areas
Unknown
Metabolic
effects of ADA
deficiency
Metabolic
effects of PNP
deficiency
Unknown
Defect in cell
membrane
glycoproteins
Unknown,
faulty thymic
epithelium,
DNA repair
defect
Defect of
promoter
binding protein
Inheritance
Usually not
familial
Autosomal
recessive or X
linked
Autosomal
recessive
Autosomal
recessive
Autosomal
recessive
X linked
Autosomal
recessive
Autosomal
recessive
Mai n
Associated
Features
Hypopara-
thyroidism,
abnormal facies,
cardio-vascular
abnormalities
Hypoplastic
anaemia
Neutropenia
Thrombocytopenia,
eczema,
lympho-reticular
cancers
Cerebellar
ataxia,
telangiectasia,
chromosomal
abnormalities,
raised serum
alpha-feto
protein levels
Intestinal
malabsorption
Immunodeficiency Diseases 193
Complement Deficiency States
A var iet y of complement deficiencies and abnor malit ies of complement funct ion have
been associat ed wit h incr eased suscept ibilit y t o infect ion. Complement fact or s ar e necessar y
for opsonizat ion, bact er ial killing and chemot axis. Many complement r elat ed disor der s ar e
associat ed wit h incr eased incidence of aut oimmune disease. The complement component
deficiencies and t he syndr omes t hey pr oduce ar e pr esent ed in Table 22.3.
Table 22.3: Complement related abnormalities and immunodeficiency
Complement deficiency Clinical syndromes
C1q Systemic lupus erythematosus (SLE)-like
syndrome. Increased susceptibility to bacterial
infections
C1r and C1s SLE like syndrome. Increased susceptibility to
bacterial infections
C2 SLE like disorders, anaphylactoid purpura,
dermatomyositis and increased susceptibility to
bacterial infections. Chronic renal disease.
Antibodies to DNA present.
C3 Increased susceptibility to pyogenic bacterial
infections and nephritis
C4 SLE like syndrome. Diminished chemotactic and
opsonic activity and impaired antibody responses.
C5 dysfunction and deficiency Defective chemotaxis leading to diarrhoea, recurrent
bacterial infections and failure to thrive
C6 Repeated episodes of meningococcal and gonococcal
infections
C7 Susceptibility to meningococcal and gonococcal
infections, autoimmune disease.
C8 Disseminated gonococcal and meningococcal
infections
C9 Haemolytic complement activity is reduced. No
clinical abnormality
C-1 inhibitor Results in hereditary angioedema; recurrent attacks
of non-pitting oedema of skin, gastrointestinal and
respiratory tracts. Laryngeal oedema can lead to
respiratory obstruction and death. Jejunal oedema
produces abdominal cramps and vomiting; colonic
involvement produces watery diarrhoea. Attacks
may be induced by tissue trauma such as dental
extraction.
Fr om Table 22.3, it can be seen t hat C3 is cr ucial for cont r olling bact er ial infect ions. C3 is
r equir ed for opsonizat ion and for t he ongoing pat hway t o C5 which is a vit al chemot act ic agent .
Individuals wit h inherit ed deficiencies of t he classical pat hway ie. C1, C4 and C2 exhibit increased
incidence of aut o immune disease r at her t han infect ious disease, demonst r at ing clear ly t hat
194 Immunology– Introductory Textbook
t he alt er nat ive pat hway is capable of t aking car e of host defence on it s own. The classical
pa t hwa y,which int er a ct s wit h a nt ibody, a ugment s clea r a nce of immune complexes. An
impair ment of t his funct ion due t o complement deficiency pr edisposes t o immune complex
deposit ion and consequent aut oimmune disor der s.
Acquired and Secondary Immunodeficiency
A var iet y of disor der s ar e associat ed wit h secondar y immunodeficiency (Table 22.4). The
pr ot ot ype secondar y immunodeficiency disor der is t he Acquir ed Immunodeficiency Syndr ome
(AIDS), caused by t he human immunodeficiency vir us (HIV). This will be discussed in det ail in
Chapt er 23.
Table 22.4: Disorders associated with secondary/acquired cellular deficiency
1. Chr omosomal disor der s:
Down’s syndr ome, Fanconi’s syndr ome
2. Infect ive disor der s: HIV, Lepr omat ous lepr osy,
Epst ein-Bar r vir us, chr onic mucocut aneous candidiasis,
secondar y syphilis, ot her vir al and par asit ic diseases
3. Neoplast ic disor der s: Thymoma, Hodgkins disease and ot her lymphomas, any
advanced malignant disease
4. Connect ive t issue disor der s: SLE, advanced r heumat oid ar t hr it is
5. Physical agent induced: Bur ns, X-ir r adiat ion
6. Ot her condit ions: sar coidosis, malnut r it ion, aging, inflammat or y bowel disease,
int est inal lymphangiect asia, r enal failur e, int r avenous dr ug use
7. Iat r ogenic causes: Chemot her apy, r adiot her apy, post -sur ger y.
Clinical Tests Used to Assess Immune Function
Tes t s for i mmu n e fu n ct i on a r e r equ i r ed t o di a gn os e pr i ma r y a n d s econ da r y
immunodeficiencies. Pr imar y immunodeficiency st at es ar e r ar e and oft en fat al. Secondar y
immunodeficiency st at es ar e associat ed wit h cer t ain disease st at es such as diabet es and ar e a
compl i ca t i on of i mmu n os u ppr es s i ve t h er a py, ch emot h er a py for ma l i gn a n cy, or ga n
t r a n s pl a n t a t i on a n d t r ea t men t for a u t oi mmu n e di s ea s es . Th e ba l a n ce bet ween
immunosuppr ession and fat al oppor t unist ic infect ions may be guided by such t est s.
Evaluation of B-lymphocyte function
The init ial scr eening t est for B-lymphocyt e funct ion is t he me a su r e me n t of se r u m
i mmu n ogl ob u l i n e s . Neit her ser um pr ot ein elect r ophor esis, immunoelect r ophor esis, nor
immunofixa t ion elect r ophor esis a r e sufficient ly sensit ive or qua nt it a t ive t o be useful.
Qu a n t i t a t i ve mea s u r emen t s of s er u m I gG, I gA a n d I gM wi l l i den t i fy pa t i en t s wi t h
panhypogammaglobulinemia as well as pat ient s who have a deficiency of an individual class of
immunoglobulin, such as select ive IgA deficiency. In a pat ient in whom t her e is a st r ong suspicion
of a humor al immunodeficiency based on clinical gr ounds, t he t ot al IgG may be nor mal. However ,
quant it at ive measur ement s of individual IgG subclasses may show deficiencies.
I n a ddit ion t o t he mea sur ement of ser um immunoglobulin concent r a t ions, some
a ssessmen t of a n t i bod y fu n ct i on is a necessar y par t of t he evaluat ion of humor al immunit y.
Ant ibody t it er s aft er immunizat ion wit h pr ot ein ant igens (e.g., t et anus or dipht her ia t oxoids)
Immunodeficiency Diseases 195
and polysacchar ide (e.g., pneumococcal capsular polysacchar ides) ar e most convenient . It should
be emphasized, however , t hat immunizat ion wit h live vir al vaccines should be avoided whenever
an immunodeficiency is suspect ed. If immunoglobulin levels and/or ant ibody t it er s ar e decr eased,
t he evaluat ion should pr oceed wit h mor e advanced t est s of B-lymphocyt e number s and funct ion
such as lymphocyt e phenot yping using flow cyt omet r y.
Evaluation of T-lymphocyte function
Test ing for defect s in T-lymphocyt e funct ion is r elat ively difficult because of t he lack of
inexpensive and r eliable scr eening t est s. De la ye d t yp e h yp e r se n si t i vi t y (DTH) sk i n t e st s
using a panel of ubiquit ous ant igens can be used as a scr eening t est in older childr en and
adult s. The pr esence of a posit ive DTH skin t est gener ally indicat es int act T-cell funct ion and
cell mediat ed immunit y.
Disease states that produce poorly reactive skin tests
• Immune deficiency st at es:
• congenit al
• acquir ed as in AIDS
• Malignancy
• Liver or kidney disease
• Over whelming infect ions – vir al
• bact er ial, such as t uber culosis
• fungal
• Ext r emes of age
• Malnut r it ion
However , t her e ar e some impor t ant limit at ions t o DTH skin t est ing. A posit ive DTH skin
t est t o some ant igens does not ensur e t hat t he pat ient will have nor mal cell mediat ed immunit y
t o all ant igens or microorganisms. For example, pat ient s wit h chronic mucocut aneous candidiasis
may have a limit ed defect in which cell mediat ed immunit y may be int act t o a wide var iet y of
micr oor ganisms except t o candida. Fur t her mor e, some nor mal individuals may have t r ansient ly
depr essed DTH r eact ions dur ing cer t ain vir al infect ions. And finally, a posit ive DTH skin t est
r equir es pr ior exposur e and sensit izat ion t o t he ant igen. Infant s and young childr en may not
have had sufficient pr ior exposur e t o have developed posit ive DTH skin t est s.
Thus, negat ive DTH skin t est s may not necessar ily r eflect abnor mal T-lymphocyt e funct ion.
Indirect informat ion about T-lymphocyt e funct ion may be obt ained by en u mer a t in g p er ip h er a l
blood T-lymp h ocyt es u si n g mon oclon a l a n t i bod i es. Wit h t he help of monoclonal ant ibodies
t o st age specific T cell ant igens (t ot al T-lymphocyt es -CD2 or CD3; T-helper lymphocyt es -CD4;
and T-cyt ot oxic lymphocyt es-CD8); var ious populat ions of T cells can be defined. Specific
monoclonal ant ibodies ar e used t o r eact wit h T cells at differ ent st ages of mat ur at ion and t o
ident ify t he differ ent fr act ional subset s. The t est is done by immuno fluor escence or immuno
per oxidase st aining. Defining and count ing populat ions wit h t he CD4+ and CD8+ mar ker yields
infor mat ion on T helper /T cyt ot oxic cell r at io which is nor mally, T helper : (65%) and T cyt ot oxic:
(35%). This r at io is alt er ed in cer t ain disease st at es such as AIDS.
Mor e specia lized t est s of T-cell fu n ct ion in clu de a n a ssessmen t of l y mp h o c y t e
p r oli fer a t i on in r esponse t o nonspecific mit ogens (e.g. phyt ohemagglut inin), specific ant igens
(e.g., candida) and/or mononuclear cells fr om an unr elat ed, hist o-incompat ible individual (mixed
leukocyt e r eact ion).
196 Immunology– Introductory Textbook
It is also possible, in specialized labor at or ies, t o measur e t he pr oduct ion of a number of
differ ent cyt ok i n e s t hat ar e involved in T- and B-lymphocyt e r egulat ion (e.g. Int er leukin 2,
int er fer on-gamma).
Evaluation of phagocytic function
The evaluat ion of phagocyt ic cells gener ally ent ails assessment of bot h t heir number and
t heir funct ion. For example, disor der s such as congenit al agr anulocyt osis or cyclic neut r openia
ar e char act er ized by r educt ions in phagocyt ic cell number in t he per ipher al blood and, t her efor e,
can be det ect ed by using a wh i t e b lood ce ll cou n t a n d d i ffe r e n t i a l .
Assessment of p h a gocyt i c ce ll fu n ct i on r equir es a number of differ ent assays. In vit r o
assays of dir ect ed cell movement (chemot axis), ingest ion (phagocyt osis), and int r acellular killing
(bact er icidal act ivit y) ar e available but usually r equir e specialized labor at or ies. Impor t ant ly,
t her e ar e simpler assays t hat indir ect ly assess phagocyt ic killing by measur ing t he met abolic
event s which accompany and/or ar e r esponsible for int r acellular killing. The most common of
t hese assesses t he abilit y of phagocyt ic cells t o r espond wit h an oxidat ive bur st by measur ing
t he r educt ion of nit r oblue t et r azolium (NBT t est ).
Evaluation of the complement system
Most of t he genet ically det er mined deficiencies of t he classical act ivat ing pat hway (C1, C4
and C2), of C3, and of t he t er minal component s (C5, 6, 7,8, and 9) can be det ect ed by using
ant ibody sensit ized sheep er yt hr ocyt es in a t ot al haemolyt ic complement assay since t his assay
r equir es t he funct ional int egr it y of C1 t hr ough C9. Deficiencies of alt er nat ive pat hway
component s Fact or s D, H and I and pr oper din can be det ect ed by a haemolyt ic assay t hat uses
unsensit ized r abbit er yt hr ocyt es which ar e pot ent act ivat or s of t he alt er nat ive pat hway. The
ident ificat ion of t he individual component which is deficient , r est s on specialized funct ional and
immunochemical t est s which ar e specific for each component .
™™™
IMMUNOLOGY OF HIV INFECTION
CHAPTER – 23
T
he HIV pandemic has emer ged as t he single most defining occur r ence in t he hist or y of
infect ious diseases of t he lat e 20t h and ear ly 21st cent ur ies. The immunopat hologic
effect s of HIV infect ion ar e dir ect ly r elat ed t o t he int er act ion of t he vir us wit h a r ecept or (CD4
sur face molecule) on CD4+ T cells or T helper cells. The CD4+ molecule is expr essed also on
t he sur face of monocyt es, macr ophages and cer t ain neur ons and glial cells fr om par t icular
ar eas of t he br ain, albeit wit h much less densit y. In addit ion t o t he CD4 r ecept or pr esent in
macr ophages, monocyt es, and T cells, macr ophage t r opic st r ains of HIV 1 also need t he pr esence
of a CCR5 r ecept or on t he cell sur face t o cause infect ion. Anot her r ecept or , CXCR4 a chemokine
r ecept or enhances binding and int er nalisat ion of lymphot r opic HIV.
The immunologic a bnor ma lit ies t her efor e r esult fr om int er fer ence in t he nor ma l
funct ioning of t hese CD4 bear ing cells. The car dinal manifest at ion of HIV infect ion is t he
deplet ion of t he CD4+ T cell populat ion. Vir t ually all t he immunologic abnor malit ies in AIDS
can be ascr ibed t o defect ive funct ioning by T helper cells.
Natural Course of Infection
HIV ent er s t he body and binds t o dendr it ic cells which car r y t he vir us t o T helper or CD4+
T cells in lymphoid t issue est ablishing t he infect ion. HIV specifically t ar get s and binds t o t he
CD4+ T-helper cells and macr ophages. Aft er infect ion, r eplicat ion of t he vir us occur s wit hin
t he T-helper cells. The cells ar e lysed and new vir uses ar e r eleased t o infect mor e T-helper
cells. Dur ing t he cour se of t he disease massive number s of vir us (>1 billion/day) ar e r eleased.
T- helper cells ar e infect ed, and r apidly dest r oyed bot h by vir us and by cyt ot oxic T cells. T-
helper cells ar e r eplaced wit h near ly a billion pr oduced per day. Over many year s (aver age may
be 10), t he T-helper cell populat ion is deplet ed and t he body loses it s abilit y t o mount an
immune r esponse against infect ions. Thus, we mount a ver y st r ong immune r esponse against
t he vir us for a long t ime, but t he vir us is pr oduced at a ver y high r at e and ult imat ely over comes
t he abilit y of t he immune syst em t o r espond.
The T helper funct ions t hat ar e ablat ed due t o HIV infect ion ar e:
• Act ivat ion of macr ophages
• induct ion of B cell funct ion
• induct ion of cyt ot oxic T cell funct ion
• induct ion of nat ur al killer (NK) cell funct ion
• secr et ion of int er leukins and ot her chemokines for ot her lymphoid cells
• secr et ion of haemat opoiet ic colony st imulat ing fact or s and
• secr et ion of fact or s t hat induce non lymphoid cell funct ion.
198 Immunology– Introductory Textbook
A summa r y of t he immunologic a bnor ma lit ies in pa t ient s wit h AI DS is given in
Table 23.1.
Table 23.1: Immunologic Abnormalities Associated with AIDS
Immunologic function Abnormality
Humoral functions • Elevated serum immunoglobulins pre-
dominantly IgG and IgA in adults,including IgM
in children.
• Increased spontaneous immunoglobulin secretion
by individual B cells.
• Decreased ability to mount a de novo antibody
response to a new antigen. Elevated serum levels
of immune complexes.
Cellular functions • Lymphopenia
• Selective T cell deficiency with reduction in the
CD4+ T cell subset (the helper-inducer sub set).
• Decreased or absent delayed cutaneous
hypersensitivity reactions.
• Decreased in vitro lymphocyte proliferative
responses to antigens and mitogens.
• Decreased T cell mediated cytotoxicity.
• Decreased NK cell activity.
Understanding the CD4+/ CD8+ test results
CD4+ and CD3+ counts
The nor mal CD4+ or helper T cell count is somewher e bet ween 500 and 1500 cells per
cubic millimet er of blood. In t he absence of ant i-HIV t r eat ment , t he CD4 cell count decr eases,
on aver age, about 50 t o 100 cells each year . Oppor t unist ic infect ions such as Pneumocystis
jirovecii pneumonia (PCP) can occur if t he CD4 count falls below 200. A lar ge number of ot her
infect ions can occur if it dr ops below 50 t o 100 cells. Please not e t hat t he CD3+ count r epr esent s
bot h t he CD4+ and CD8+ sub set s since bot h t hese lineages car r y t he CD3+ mar ker . The CD3+
% is t he per cent age of CD3+ cells wit hin t he t ot al lymphocyt e count . The CD4+/CD3+ or CD8+/
CD3+ per cent ages ar e t he per cent age of each subset wit hin t he pool of CD3+ T cells.
CD4+ %
In healt hy adult s, t he number of CD4+ cells make up bet ween 32% and 68% of t he t ot al
number of lymphocyt es – which would include CD4+ cells, CD8+ cells and B-cells. The CD4
per cent age is somet imes a mor e r eliable measur ement t han t he CD4+ count because it t ends
t o var y less bet ween measur ement s. For example, one per son’s CD4+ count may var y bet ween
200 and 300 over sever al mont hs while t heir CD4+ per cent age r emains const ant at , say, 21%.
Pr ovided t hat t he CD4+ per cent age st ays at 21% or higher , t he immune syst em st ill appear s t o
be funct ioning pr oper ly, r egar dless of what t he CD4+ count is. At t he same t ime, a CD4+
Immunology of HIV Infection 199
percentage at or below 13% - regardless of what the actual CD4+ count is - usually means that
the immune system is damaged.
CD8+ count and the CD4+/CD8+ ratio
CD8+ cells, also called cytotoxic T cells, playa major role in fighting infections such as
HIV. A healthy adult usually has between 150 and 1,000 CD8+ cells per cubic millimeter of
blood. Unlike CD4+ cells, people living with HIV tend to have higher-than-average CD8+ cell
counts. Laboratory reports also list the T-cell (CD4+/CD8+) ratio, which is the number of CD4+
cells divided by the number of CD8+ cell s. Since the CD4+ count is usually lower than normal
in people living with HIV, and the CD8+ count is usually higher, the ratio is usually low. A
normal ratio is usually between 0.9 and 6.0. Once anti-HIV therapy is started, an increase in
the 1'·cell ratio (i .e., a rising CD4+ count and a fall ing CD8+ count) is a sign that drug treatment
is working. See Figure 23.1 to see what a CD4+ test result looks like.
Figure 23.1 The CD4+ test result.
IMMUNITY AND MALNUTRITION
CHAPTER – 24
I
t is now a widely acknowledged obser vat ion t hat malnut r it ion is dir ect ly linked t o
immunodeficiency. The most suscept ible segment s of a given populat ion ar e infant s
and t he elder ly wher e infect ion, nut r it ional deficiency and impair ed immunit y ar e fr equent ly
encount er ed a nd ma y be ca usa lly r ela t ed. Migr a nt wor ker s a nd t heir fa milies in la r ge
met r opolit an cent r es, living in over -cr owded slums pose a major public healt h pr oblem wher e
t he int er play bet ween nut r it ion, immunit y and infect ion is most evident .
Infect ion is known t o be a fr equent complicat ion of malnut r it ion leading t o high mor bidit y
and mor t alit y especially in childr en. This associat ion bet ween malnut r it ion and infect ion has
led t o t he obvious infer ence t hat malnut r it ion leads t o immunodeficiency and incr eased
suscept ibilit y t o infect ion. Infect ion in it self causes an act ual loss of nut r ient s as a r esult of
vomit ing, diar r hoea, a t endency t o poor or inadequat e feeding and var ious diet ar y fads pr evalent
in many communit ies.
It has been obser ved t hat malnut r it ion in t he cr it ical ear ly mont hs of int r a ut er ine life
could have far r eaching effect s on a child’s immune syst em. Int r a ut er ine gr owt h r et ar dat ion
eit her due t o mat er nal malnut r it ion or t o a var iet y of ot her causes is associat ed wit h involut ion
of t he t hymus and impair ed neonat al immunit y. Recent obser vat ions have shown t hat t her e
ar e definit e changes in lymphoid or gans, number and funct ion of lymphoid cells and in t he
efficacy of humor al defence fact or s.
Immunological Changes in Malnutrition
(a) Morphological changes in the immunocompetent organs
The t hymus and ot her lymphoid or gans r eact mor e sever ely t o nut r it ional deficit s t han do
ot her t issues. Pr ot ein ener gy malnut r it ion r esult s in mar ked hist ological changes wit hin t he
t hymus: t her e is a r educt ion in t he size and weight of t he gland, deplet ion of lymphocyt es, loss
of cor t ico-medullar y differ ent iat ion and degener at ion of t he Hassall‘s cor puscles. Similar changes
ar e seen in t he t hymus dependant ar eas of t he lymph node and t he spleen. Concomit ant
infect ion such as measles pr oduces addit ional lymphoid at r ophy. It is possible t hat nut r it ional
depr iva t ion du r in g in t r a u t er in e gr owt h or in t h e n eon a t e ma y pr odu ce ir r ever sible
mor phological changes in t he t hymus and t hymus-dependant ar eas of ot her lymphoid or gans.
(b) Changes in T-lymphocyte function
Immune r esponses: bot h cellular and humor al depend heavily on efficient T cell funct ion.
Lymphoid at r ophy and impair ed mat ur at ion ar e evidenced in malnut r it ion. St udies have shown
t hat 15% of childr en wit h moder at e t o sever e pr ot ein-ener gy malnut r it ion show lymphopenia.
This is said t o be due t o impair ed differ ent iat ion in t he t hymus due t o decr eased t hymic hor mone
act ivit y.
Immunity and Malnutrition 201
Delayed cut aneous hyper sensit ivit y r eact ions following challenge wit h common ant igens
ar e decr eased in pr ot ein-ener gy malnut r it ion and impr ove wit h or al or int r avenous feeding
wit h pr ot ein and calor ie r ich mixt ur es. The mechanisms under lying t his deficit ar e unknown.
A combinat ion of fact or s such as ant igen r ecognit ion, pr ocessing, efficient funct ioning of T
lymphocyt es, r elease of lymphokines and mobilizat ion of polymor phs and macr ophages may
in flu en ce t h e ou t come of a skin t est in ma ln ou r ish ed in dividu a ls. Lymph ocyt e bla st
t r ansfor mat ion and lymphocyt e mediat ed cyt ot oxicit y ar e bot h abr ogat ed in sever e malnut r it ion
and int r aepit helial T cell populat ions ar e r educed.
Changes in T cell mediat ed immune r esponses in malnut r it ion ar e r apidly cor r ect ed aft er
nut r it ional supplement at ion, unless t he nut r it ional insult occur s dur ing int r a ut er ine life or in
t he neonat e.
(c) Changes in B lymphocyte function
The number of cir culat ing B lymphocyt es r emains unchanged dur ing malnut r it ion. Ser um
immunoglobulins ar e nor mal or modest ly elevat ed in malnut r it ion and is pr obably a r esponse
t o infect ion.
Ant ibody r esponse t o infect ious agent s is gener ally nor mal in pr ot ein ener gy malnut r it ion.
However , if an ant igen r equir es T cell help, and many infect ious agent s do, t hen ant ibody
r esponse t o r epeat ed infect ions is less t han sat isfact or y; since memor y is a T cell funct ion.
Impair ed ant ibody r esponses impr ove when nut r it ional supplement at ion is adequat e.
The finding t hat decr eased secr et or y IgA (sIgA) r esponse occur s over mucosal sur faces in
malnut r it ion is of immense fundament al and applied int er est . sIgA r esponses in nasophar yngeal
and ot her ext er nal secr et ions is low and specific IgA pr oduct ion dur ing vaccinat ion wit h measles
and polio vir uses is mar kedly r educed. These alt er at ions may be due t o decr eases in IgA bear ing
cells or due t o a lower ed t ur nover of t he IgA secr et or y component fr om an at r ophied mucosal
epit helium.
(d) Altered polymorphonuclear function
Alt hough t he t ot al number of polymor phs and leucocyt es is unchanged, t her e ar e significant
alt erat ions in polymorph funct ion during malnut rit ion. When nut rit ional deficiency is complicat ed
by infect ion, chemot act ic migr at ion of neut r ophils is mar kedly r educed; ingest ion is nor mal
but int r a cellular killing of bact er ia and fungi is deficient . Following phagocyt osis, in t he
malnour ished individual, t he neut r ophil does not exhibit t he char act er ist ic “r espir at or y bur st ”
and t he act ivit y of t he hexose monophosphat e shunt does not r ise. Similar changes ar e seen in
ir on deficiency anaemia. These abnor malit ies ar e r ever sed wit hin a few weeks of nut r it ional
supplement at ion.
(e) Complement function
Sever al st udies have demonst r at ed consist ent changes in t he complement syst em dur ing
malnut r it ion. Many of t he complement component s ar e pr oduced by t he liver which is oft en
affect ed as a r esult of pr ot ein depr ivat ion. Under nour ished childr en show r educed levels of C3,
C1, C2 and C5 and t he t ot al haemolyt ic act ivit y is r educed. Ther e is some evidence t o show
t hat t he alt er nat ive pat hway may also be affect ed dur ing malnut r it ion.
202 Immunology– Introductory Textbook
(f) Other factors
Ther e ar e mar ked changes in sever al non-specific host defence fact or s dur ing nut r it ional
depr ivat ion. Lower levels of lysozyme ar e found in plasma, t ear s, saliva and ot her secr et ions.
Met a pla sia of mucosa l epit helia , deficient mucus t r a pping a nd cilia l movement dur ing
malnut r it ion also influence suscept ibilit y t o infect ion. Int er fer on pr oduct ion is r educed dur ing
nut r it ional deficiency.
The int er act ions bet ween nut r it ion, immunit y and infect ion ar e impor t ant det er minant s
of mor bidit y in malnour ished individuals. Nut r it ional modulat ion of t he immune r esponse
may influence t he out come of an infect ious episode especially in t he elder ly, dur ing post oper at ive
r ecover y, in cancer pat ient s and ot her debilit at ing disease st at es and mor e impor t ant ly in
pr emat ur e and low bir t h weight infant s. In addit ion, cur r ent hypot hesis favour s t he use of
t est s for immunocompet ence as a funct ional index of nut r it ional st at us. As a pr ognost ic t est for
nut r it ional r ehabilit at ion and disease vulner abilit y t hese assays ar e bot h r eliable and sensit ive.
™™™
Index
A
Acquired immunodeflciency syndrome immu-
nologic abnormalities 8!lsociated with,
194, 198
acute phase proteins, 10
adjuvants, 154, 155
agglutination, 2
haemagglutination. 57
haemagglutination inhibition, 57
reverse passive haemagglutinalion. 57
techniques, 56
allergy/atopy
clinical tests fOf, 132 (see also
hypersensitivity)
therapy for, 132 (see also hypersensi-
tivity)
Allison, 4
alveolar macrophages, 12
anaphylaxis. 2
mechanisms and mediators of. 128· 130
(see also hypersensitivity)
regulatory mechanisms of, 131
type I hypersensitivity, 132
antibody/antibodies. 2·4 (see also
immunoglobulins)
diversity. 3
heterophile, 24
monoclonal. 3
structure. 3
antigen, 20 (see also immunogen)
Forssman, 24
antigenic determinant, 20
antigen presentation, 97
antigen processing, 97
antiserum
affinity. 22
avidity, 23
atopy (see also hypersensitivity)
autoimmunity
aetiopathogenesis of autoimmune
disease, 151
classification of autoimmune diseases,
149
mechanisms of tissue injury, 160
Bacterial flora , 7
B cells. 16
Behring, 1
B
toxins, antitoxins, 1
Bcnacerraf. 4
Bennet, 3
Billingham, 4
Bjorkman, 4
blood groups, 2
B-Iymphocytes
activation of, 103
biology of, 87
origin of, 87
bonding
electrostatic, 22
hydrophilic, 22
hydrophobic, 22
VanderWaals, 22
Bordet.2
Brent, 4
Buchner. 2
Burnet. 3
Bursa of Fabricius. 14
Caimette,2
cancer
c
immunology, 166 (see also tumour
immunology)
catalase, 9
CD4, 15 (see also T cells)
204
CDS, 15 (see also T cells)
cell adhesion Molecules
lCAM·I . 102
IFA· I. 102
cell mediated immunity
l'I clivatcd macrophages, 121
K cells and anti body dependant cell
mediated cytotoxicity, 121
natural killer cells. 122
T cytotoxic cells, 118
classification oflymphocytel:l. 19
clonal selection, :3
clonal selection theory, 88
Cohn, 3
complement, 2
activators of, 39
a lternative, 39
C2.11
C4, 41
cascade, 40
classical . 39
congenital deficiencies of, 49
control mechanisms, 46
me mhrane attack mechanis m, 44
receptor interactions. 47
the classical pathway, 41
the alternative complement pathway
factor A, 43
Cl
C3
factor B, 43
factor D, 43
factors I and H, 43
positive feedback mechanism, 41
properdin. 44
Clq, r , s, 11
cleavage products of, 12
complement cleavage products
biologic actions of, 48
cytolysis
molecular mechanism of, 45
receptor interactions
biological consequences of, 46
complement activation
biological consequences of, 46
complement fixati on test, 60
complement system
biologic significance of, 48
constant regions
functions of, 28
Coomb's test, 58
Counter immunoelectrophoresis, 54
crossed immunoelectrophoresis, 55
cross reactivity, 23
cytochrome. 10
Dausset,4
Davis. 4
Denis, 2
Doherty, 4
Douglas, 1
Dreyer, 3
Durham, 2
Edelman, a
Ehrlich,2
electrophoresis, 53
EUSA.63
D
E
applications of, 64
biotinJ avidin. 64
endogenous pyrogen, 11
eosinophils, 10
epitope.22
germinal centre. 16
Gorer,4
G
graft versus host disease, 163
Gruber, 2
Guerin, 2
H
hapten, 22
hexose monophosphate shunt, 10
histocompatibility
antigens. 4
complex. 4
H· 2.4
HLA,4
locus. 4
HLA typing. 77
mixed lymphocyte reaction, 78
uses of, 80
Hozumi,3
Index
lnde)(
humoral. I
hybridoma.3
hydrogen peroxide, 10
hypersensitivity. 2
Coombs and Gell classification, 126
hypersensitivity Type II
antibody dependant cytotoxic. 133
autoImmune type II. 136
due to drugH, 136
reactions in blood transfusion and
organ transplantation. 133
hypersensitivity type III
Arthus reaction. 138
circulating immune complexes, 140
detection of, 140
immune complex mediated, 136
hypersensitivity type N
delayed type hypersensitivity. 141
I
idiotype,3
idiotype-anti idiotype network. 113
lmmune responses
primary and secondary, 181
immunity, 1
active. 183
cellular, 2
definition, 7
humoral,2
innate. 5
non-specific, 5
passive, 182
dassification of. 182
specific, 5
immunization
milestones, 180
passive, 2
immunodeficiency diseases
B cell immunodeficiency, 186
Chediak-Higashi syndrome. 191
common variable immunodeficiency.
192
complement deficiency states, 193
Di George Syndrome, 189
secondaryfacquired cellular deficiency,
194
205
Be\'ere combined immunodeficiency,
190
Wiskott-Aldrich syndrome, 190
immunodiffusion. 51
immunoelectrophoresis, 53
immuno fluorescence, 67
immunoglobulins
characteristics of, 32
diversity, 33
Fab fragment, 25
Fc fragment, 25
Hand L chains. 25
heterogeneity of. 27
hinge region, 25
idiotypes, 27
IgA.30
I,D. 32
I,E.31
IgG .. 28
10M. 29
isotypes.26
papain cleavage, 26
pepsin cleavage. 26
sedmentation coefficient (8 value), 26
V and C regions, 25
diversity
somatic hypermutation. 36
somatic recombination, 34
immunologic testing
predictive theory, 69
immunosuppression
agents of, 154. 156
infectious diseases
immunity against. 174
inflammation, 1
factors, 5
interferons. II
interleukin-l,124
Jerne, 3
Kabat, 3
Kitasato,2
Koch,2
Kohler. 3
Kupffer cells, 12
J
K
206
lactic acid, 10
luctoferrin.9
lactoperoxidase. 7
Y.<IndlltA!iner,2
L
large granular lymphocytes, J 8
lattice formation, 51
Laurell's rocket electrophoresis, 54
linkagl! tli!lequilibrium. 76
lymph node, 17
lymph nodes, 16
lymphoid organs, 14
lymphokines.
types. functions of, 123
Iymphoreticular system, 12
Iysozymes. 6,7.8, 9. 10
macrophage. 7, 12
ml:tjor basic protein, 13
major histocompatibility complex, 14.77
(see also major histocompatibility com-
plex)
disease associations. 83
nomenclature and genetic organization
0(, 75 (see also human leucocyte
antigen)
antigens
Chl88 III MHC antigens. 77
class II MHC antigens, 77
class I MHC antigens, 77
structure and function of, 92
Mak.4
malnutrition
M
immunologic changes in, 200
Medawar,3
mesnngial cells, 13
Metchnikoff, 1
microglill, 13
Milstein, 3
monc1onal antibodies, 70
applications of. 73
principle of. 71
mononuclear phagocyte system, 12
mucosal associated lymphoid tissue (mucosal
associated lymphoid tissue), 18
mucus, G
myeloperoxidase,9
N
NADPH,IO
natural killer cells, 18
neutrophil granules, 12
Nissen, 1
null cells, J 8
Nutall, 1
opsonin, 1
osteoclast, 13
o
Oucht.erlony's double diffusion, 51
Owen, 4
paracortical area, 17
paratopc, 20
Pagteur,2
rabies
p
vaccines, 1
Paul·Bunnel ~ 9 t , 24
pfeiffer, 2
phagocytosis
theory, 1
phago. lysosome, 7
phagosome, 7
PMN,7
Porter, S
Portier, 2
Prausniu·Kustner reaction, 148
precipitation, 50
proteolytic enzymes, 1
prozone phenomenon, 50
R
radio immunlNlssay. 61
RAST, 62
RIST,62
respiratory burst, 10
Rh factor, 2
Richet.2
S
Schult%·Dale reaction, 128
sebaceous secretions, 6
secondary rt!sponse, 3
single radial immunodiffusion, 52
Snell. 4
Index
Index
somatic hypermutation, 3
somatic recombination, 3
Spermine. 7
s pleen, 17 ·
superoxidc anion, 9
superoxide dismutu.s6, 9
Suppressor T cells, 95
sweat. 6
T
T cell differentiation, 15
T cell receptor, 4, 15
Tr.ells. 17,18
T ceUs, subsets, 18
t-cytotoxiclsuppressor, 15
T. helper inducer, 15
thymocytes, 15
thymosin. 14
thymus, 13. 14
T·lymphocytes.
activation of, 100
CD2 molecule, 91
CD3 complex, 93
CD" and C08 molecules, 93
surface molecules of, 90
T cell receptor for antigen, 92
T ceJ1 receptor for antigen
genetic organization of, 92, 93
tolerance. 4
theories of. 145
Tonegawa,3
transfusion, 2
transplantation, 4
graft rejection, 158
tissues and organs, 161
Tuberculosis
BCG, 2
207
tumour immunology, 166 (see also cancer,
i mmunology)
v
vaccines
experimental and restricted use, 184
killed and live attenuated, 183
polysaccharide, 184
toxoids, 183
Weigert, 3
blots, 65
Widal, 2
Wiley. 4
1
WU,3
Zinkemagel, 4
w
z

IMMUNOLOGY

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for Dushyant & Meghna my greatest critics– my strongest supporters .

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very much like the many lectures I have given to students over the years. students often find themselves overwhelmed by the volume of reading required to understand basic concepts. As a teacher of immunology and clinical microbiology for undergraduate medical students. London Nandini Shetty . A new chapter dedicated to the immunology of HIV has been added in recognition of the devastating pandemic that has re-defined global health. nursing and for any one else in the life sciences who feels the need to explore this fascinating area of science. in keeping with scientific advances. Assimilating a fast changing. The second edition has been carefully updated. To this end I owe my own enjoyment of the subject to the many excellent articles in ‘Scientific American Medicine’. I have tried to present the basic tenets of the subject of immunology in a simple and understandable manner. I gratefully acknowledge these sources for the many ideas and figures that I have adapted for this book. ever expanding subject like immunology can be a daunting process. I have tried to evolve a story so that students may be inspired to read on and experience the wonder of scientific discovery. I hope the book will be of use to students of medicine. to the pictorial presentation of immunological concepts in Ivan Roitt’s excellent textbooks of Immunology and to ‘Basic and Clinical Immunology’ of the Lange Medical Publication series.PREFACE TO THE SECOND EDITION As textbooks and journals of immunology grow in size and number and the explosion of information floods the libraries. microbiology. with some chapters being rewritten incorporating several new illustrations.

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To this end lowe my own enjoyment of the subject to the many excellent articles in 'Scientific American Medicine'. very much like the many lectures I have given to students over the years. Assimilating a fast changing. I have tried to evolve a story so that students may be inspired to read on and experience the wonder of scientific discovery. I hope the book will be of use to students of medicine. London Nandini Shetty . As a teacher of immunology and clinical microbiology for undergraduatemedical students. to the pictorial presentation of immunological concepts in Ivan Roitt's excellent textbooks of Immunology and to 'Basic and Clinical Immunology' of the Lange Medica! Publication series. microbiology. ever expanding subject like immunology can be a daunting process. nursing and for anyone else in the life· sciences who feels the need to explore this fascinating area of science.PREFACE TO THE FIRST EDITION As textbooks and journals of immunology grow in size and number and the explosion of information floods the libraries. I gratefully acknowledge these sources for the many ideas and figures that I have adapted for this book. students often find themselves overwhelmed by the volume of reading required to understand basic concepts. I have tried to present the basic tenets of the subject of immunology in a simple and understandable manner.

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LIST OF ABBREVIATIONS Chapter 1 BCG C region Fab Fc HLA MHC T cell V region Chapter 2 CRP IL-1 IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 NADPH Chapter 3 CD GALT MALT MBP MPS NK PMN Chapter 4 DNP Dinitrophenyl Cluster of differentiation Gut associated lymphoid tissue Mucosal associated lymphoid tissue Major basic protein Mononuclear phagocyte system Natural killer Polymorphonuclear neutrophil C-reactive protein Bacille Calmette Guerin Constant region Fraction antigen binding Fraction crystallizable Human leukocyte antigen Major histocompatibility complex Thymus derived cell or thymocyte Variable region Interleukins-1 to 7 Nicotinamide adenosine dinucleotide phosphate hydrogen .

(xii) Chapter 5 CDR CH CL H chain L chanin VH VL Chapter 6 D region IVS J region Chapter 7 P SLE SRS-A Chapter 8 CEA CIE ELISA HBsAg HIV IEP IHA/PHA RAST RIA RIST RPHA SDS-Page Chapter 9 HAT HPRT PEG Chapter 10 GBM HTC Ir genes Is genes Glomerular basement membrane Homozygous typing cells Immune response genes Immune suppressor genes Hypoxanthine aminopterin thymidine Hypoxanthine phosphoribosyl transferase Polye thylene glycol Carcinoembryonic antigen Counter immunoelectrophoresis Enzyme linked immunosorbent assay Hepatitis B surface antigen Human immunodeficiency virus Immunoelectrophoresis Indirect/Passive haemagglutination Radio allergosorbent test Radio immunoassay Radio immunosorbent test Reverse passive haemagglutination Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis Properdin Systemic lupus erythematosus Slow reacting substance-A Diversity Intervening sequences Joining region Complementarity determining regions Constant heavy Constant light Heavy chain Light chain Variable heavy Variable light .

(xiii) MLR PLT Chapter 11 TCR Chapter 12 APC BCDF BCGF ICAM IFN IP3 LFA Tc Th Chapter 13 ADCC BAF LAF LPS MAF MDP MIF Multi-CSF Chapter 14 CMI EBV ECF ESP FACS GM-CSF HTLV 1 HTLV 2 LIF LT OAF PHA PPD PWM TNF Cell mediated immunity Epstein Barr virus Eosinophil chemotactic factor Eosinphil stimulatory promoter Fluorescence activated cell sorter Granulocyte-monocyte colony stimulating factor Human T cell leukemia virus 1 Human T cell leukemia virus 2 Lymphocyte inhibitory factor Lymphotoxin Osteoclast activating factor Phyto haemagglutinin Purified protein derivative Pokeweed mitogen Tumour necrosis factor Antibody dependent cellular cytotoxicity B cell activating factor Lymphokine activating factor Lipopolysaccharide Macrophage activating factor Muramyl dipeptide Migration inhibitory factor Multi-Colony stimulating factor Antigen presenting cell B cell differentiation factor B cell growth factor Intercellular adhesion molecule Interferon Inositol triphosphate Lymphocyte function associated antigen T cytotoxic T helper T cell receptor Mixed lymphocyte reaction Primed lymphocyte typing .

2. 2. 3 Long acting thyroid stimulator New Zealand black New Zealand white Eosinophil chemotactic factor-anaphylaxis Neutrophil chemotactic factor-anaphylaxis Prausnitz küstner . 3 Bovine serum albumin Idiotype 1. 3 LATS NZB NZW Chapter 17 LAK Chapter 18 GVH SCID Chapter 19 DNFB FeLV MCA Chapter 21 HIG VZIG Chapter 22 ADA AIDS CVID Adenosine deaminase Acquired immunodeficiency sundrome Combined variable immunodeficiency Human immunoglobulin Varicella zoster immune globulin Dinitroflurobenzene Feline leukemia virus Methyl cholanthrene Graft versus host Severe combined immunodeficiency Lymphokine activated killer Antibody 1. 3 BSA Id 1. 2. 2.(xiv) Chapter 15 ECF-A NCF-A PK Chapter 16 Ab 1.

........................... Cell-Mediated Immunity ... 180 22........................................... Milestones in Immunology ................................................ 143 17................................................................................ Immune Response Mechanismis II: Antigen Presentation and Processing......... Mechanisms of Lymphocyte Activation ............ 197 24.................................................................................... ix List of Abbreviations ......................... 1 Innate Immunity ....... Immunity Against Infectious Diseases ........................................................ 126 16............... 70 10....... 166 20............................................................................................................ 7.......................................................... 117 15.................. 9................................................................... Hypersensitivity ........ vii Preface to the First Edition ....................................... 5....................... 75 11............................. 8.......... Immunity and Malnutrition ......... Immunodeficiency Diseases ...............................................................................................CONTENTS Preface to the Second Edition ............................................................................................... 33 The Complement System ............................................. 2........................................................................................ 174 21..... Immunology of HIV Infection ..................... 12 Antigens and Immunogenicity .................................... Immunopotentiation and Immunosuppression ................................ Immunologic Tolerance and Autoimmunity .................................................. xi 1............................................................................ 6.................................................................................. Tumour Immunology ................................ Immunization .......... 200 Index ................................................................. 186 23..... 5 Immunobiology .................................................... 25 Immunoglobulins II: The Genetics of Antibody Diversity ................................................ 4.. 158 19.......................... 87 12.......... 144 18 Transplantation Immunology ................................... 20 Immunoglobulins I: Structure and Function ................................................................................................................................................................... 3... Immune Response Mechanismis I: B and T Lymphocytes ......................................... 39 Detection and Application of Antigen-Antibody Reactions ......................................................................................................................................................... Cytokines ........................................ The Major Histocompatibility Complex .. 203 ........................................... 97 13....................................... 107 14............................................................................................................. 50 Monoclonal Antibodies ............................

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von Behring was awarded the French legion of honour and the Nobel Prize in 1901.one of his great heroes and with Metchnikoff he established a continuing friendship that began in 1888. the complex chemicals associated with immunity have been the continuing subject of years of research. This friendship was unique because the two men were at opposite poles in explaining basic aspects of immunity -yet enjoying the stimulus of intellectual debate. the whole basis of immunity needed questioning. They thus forged a link between the two major theories of immunity. Metchnikoff hypothesized that the basis of inflammation was the cellular reaction and that vascular and nervous reactions were only of secondary importance. reported their observations. The phagocytic theory was the first to be developed in the 1880s a bold and imaginative concept — the fruit of Elie Metchnikoff’s deep knowledge of biology. Behring. As early as 1773.CHAPTER – 1 MILESTONES IN IMMUNOLOGY ith the evolution of the germ theory of disease. They introduced passive immunization into modern medicine and for this. could render bacteria susceptible to phagocytosis. theorists were already elucidating the mechanisms of host defence against infectious diseases and their possible prevention. Behring maintained cordial ties with Pasteur . Almroth Wright with his disciple Stewart Douglas observed that a humoral component which they designated opsonin. Finally in 1903. It was around this time that Louis Pasteur reported on the treatment of his first two patients with rabies using early vaccines. In 1908 he shared the Nobel Prize with Paul Ehrlich for his early contributions to the understanding of inflammation. He postulated further that these migrating cells which were able to move in order to meet an enemy were the major guardians of health against bacterial infections. In 1888 Metchnikoff left Russia to continue his work on phagocytosis in Pasteur’s Institute in Paris. Interest in immunology grew out of the everyday evidence that those individuals who survived: pocked and disfigured from the dread disease. small pox never did contract the infection again. Emil von Behring and Kitasato in 1890 inoculated animals with toxins of diphtheria and tetanus. After Metchnikoff died in 1916 other workers who had long cherished the theory that soluble substances in blood were also bactericidal. to produce neutralizing antitoxin serum. Nuttall and Nissen contributed substantially to the concept of soluble or humoral factors but found that these factors were not always bactericidal. And it could not be ignored that the blood of animals contained certain preformed bactericidal agents. the study of the mechanisms of immunity and the possible conquest of infectious diseases began almost simultaneously. much like snuff. Voltaire reported on an ancient Chinese custom where dried and powdered small pox scabs were inhaled. Behring found that certain diseases were the expression of the action of toxins which could be neutralized by antitoxins. in at attempt at preventing the disease. W . Although it would take several years before bacteria and viruses were fully characterized. Mere empirical methods of producing or increasing immunity were not sufficient. Since the time humoral and cellular components were shown to be interwoven in an intricate pattern.

At this time Robert Koch too had to taste failure in his attempts at providing a successful vaccine against tuberculosis. How antigens direct the various immunological processes was the chief point of contention among early theorists who had to choose between the instructive and the selective . Jean Baptiste Denis.it did not induce the formation of tubercles by intravenous. The same year Ferdinand Widal the Algerian born son of a French army surgeon described the agglutination reaction for typhoid fever—a test which is still widely used and bears his name. 5. From these early experiments the nature and function of complement mediated cytolysis were elucidated by Pfeiffer and later by Buchner and Bordet. Till the early 1900s the practice of transfusion of blood from man to man remained a risky. As serotherapy developed. Around this time Gruber and Durham described the diagnostic value of the agglutination reaction. Landsteiner received the Nobel Prize in 1930 for elucidating the blood groups and for his work on the Rh factor. It was in France that Albert Calmette and Camille Guerin developed an effective vaccine for tuberculosis by the methods of attenuation so dear to Pasteur and disdained by Koch. Carl Landsteiner discovered the blood groups. Excess receptors were liberated into the circulation as antibodies–the molecular basis of humoral immunity. it soon became evident that antigen. that reacted with toxins. performed what is considered to be the first transfusion of blood in man. To establish a live but attenuated strain of Mycobacterium tuberculosis they had to continually transfer or sub culture the organism till virulence was lost. The experiment was not well received by his fellow-physicians who were more accustomed to blood-letting than reversing the flow. but formed an effective prophylactic against tuberculosis. Elie Metchnikoff suggested that phagocytes were the prime detectors of foreign material— we trace the origins of cellular immunity to this hypothesis. In 1913 Charles Richet received the Nobel Prize in recognition of his work on anaphylaxis. when at the turn of the century. Pfeiffer in 1894-95. intraperitoneal or subcutaneous inoculation or even by ingestion. The Bacille Calmette Guerin (BCG) was thus born . as we know it today. Anaphylaxis. the foundations of modern immunology. They were rediscovered only in the 1940s and form a basis of the study of immunology today. on Jan. for his discovery of the whooping cough agent and for his enunciation of the diverse aspects of antigen – antibody reactions and the blood coagulation system. Thus. is remarkably close to this novel but original concept. Jules Bordet was awarded the Nobel Prize in 1920 for his pioneering work on complement.2 Immunology– Introductory Textbook Though eminently successful in passive immunization against diphtheria and tetanus. Paul Ehrlich proposed the pre–existence of receptors (which he called toxophores) on the living cell. physician to Louis the XIV. unpredictable business. In 1667. were laid by several European scientists over the last hundred or more years.Portier and Charles Richet in 1902. 1921 it was completely avirulent even at high doses for all animal species. discovered the phenomenon of in–vivo cytolysis of Vibrio cholerae when the organism and immune serum interacted intraperitoneally in the guinea pig. The twentieth century saw phenomenal progress in the understanding of immunological concepts.antibody reactions in vivo could have some harmful effects and even produce death. The prevention of tuberculosis was not to be found by the Germans. as we recognize it today. the most dramatic manifestation of hypersensitivity was first described by P. This odyssey took them thirteen years and 230 transfers later. Two of the most vital immunological hypotheses made in the 1800s went undetected for several decades. when agar cultures were mixed with their corresponding immune sera. von Behring failed abysmally when he tried to extend these principles to passive immunization against tuberculosis.

In 1970 T. Fc x 1) by enzymes that cleave polypeptides.Milestones in Immunology 3 theories. suggested that the germ line contains many variable (V) region genes and one constant (C) region gene. however. the most fundamental of which was his role in developing he concept of clonality and for his description of the idiotype network in the regulation of immune responses. Edelman who shared the Nobel Prize for their discovery in 1972. They also delineated the mechanism of shuffling of the many gene segments. yet it required that the cell have some means of rearranging genes in somatic cells. Tonegawa was awarded the Nobel Prize in 1987 for elucidating the mechanism by which the immune system generates an almost limitless variety of antibodies. Dreyer and Bennet in 1965. In 1984 Niels Jerne was also awarded the Nobel Prize for his theoretical contributions to immunology. We know today that during this second exposure the binding ability of antibody improves and we also know this to be the result of affinity maturation and somatic hypermutation. The theory was attractive. In the past 30 years the biological basis of the immune response was.T. They demonstrated that V and C genes were far apart in embryonic cells but much closer to each other in plasma cells. was demonstrated by Weigert and Cohn in 1970. In 1959 Edelman showed that the immunoglobulin molecule had 4 polypeptide chains: 2 of each kind and that each could be separated by chemical means. has enabled immunologists to prepare virtually unlimited quantities of antibodies that are chemically. remained for Burnet to draw together the new conceptualization and in 1957. This theory proposed. The secondary response is more powerful because antigenic memory leads to rapid clonal expansion during subsequent contact with the same antigen. or even much later in some instances. he asserted that “each cell and its clones can produce just one kind of receptor” to explain the specificity of the immune response and the exponential rise in antibody production following contact with antigen. Wu and E. As a cell matures. was found in 1970 by Hozumi and Tonegawa then at Basel. it selects one V gene out of many and combines it with the one C region gene. which combine to yield an immunoglobulin molecule. physically and immunologically completely homogenous. He called them light and heavy chains because of their size. At the same time Porter showed that the molecule could be cut into 3 different pieces ( Fab x 2. Macfarlane Burnet was awarded the Nobel Prize in 1960 which he shared with Peter Medawar. outer arms of the antibody molecule. Porter and Gerald M. Switzerland. The clonal selection theory also explained tolerance as the deletion or suppression of an entire clone of cells which could occur before or soon after birth. The production of monoclonal antibodies by somatic cell hybridization of antibody forming cells and continuously replicating cell lines was called the technique of hybridoma formation. All subsequent work has confirmed his conclusions with some elaborations. Somatic hypermutation. but in more complicated ways than Dreyer and Bennet suggested. by Paul Ehrlich almost a 100 years ago fell out of favour with the introduction of the template theory. in part. Evidence that immunoglobulin genes do undergo somatic recombination. The elucidation of antibody structure is credited to Rodney R. in essence. The first comprehensive attack against the template theory was launched in 1955 by Niels Kaj Jerne who recalled certain criticisms made by Macfarlane Burnet against the template theory. In 1970 Edelman showed that chemical differences responsible for the specificity of antigen binding were embodied in the amino acid sequences of their variable regions at the upper. clarified when the theory of clonal selection of antibody formation found acceptance. It. . For this invaluable contribution to immunology they were awarded the Nobel Prize in 1984. as another major source of the tremendous diversity of antibody specificity generated. This technique described by Georges Kohler and Cesar Milstein in 1975.A Kabat demonstrated the presence of hyper variable regions.

Peter Doherty with Rolf M. Benacerraf showed that genes of the HLA determining loci may control immune responses. With Snell in 1948 he showed that there were multiple alleles in the mouse H-2 locus and subsequently that this locus was genetically complex. Earlier Ray Owen had shown. Lechevalier HA and Solotorovsky M. Davis in 1984. the T cell receptor was studied and characterized by Tak W. 2. 1965. what has evolved as a precise and powerful tool created by nature to ensure the continued survival of the species could perhaps be manipulated to yield a better quality of life. The H-2 locus was shown to control the phenotype expression of certain cell surface markers in mice. Peter Medawar was awarded the Nobel Prize (with Burnet) in 1960. in his classic experiment with dizygotic twin calves. Parish. Existence of markers of biological individuality – what we now know as the histocompatibility antigens was first suggested by Gorer in 1937. New York. He suggested that specific individuality markers were associated with every cell of an organism and that an animal can be immunized against foreign grafts by first injecting it with cells derived from immunocompetent tissues of the donor. Three centuries of Microbiology Dover Publications Inc. found that T cells from mice infected with a meningitis virus destroyed virus-infected cells only from the same strain of mice. Ltd. In 1953. who demonstrated that loci determining blood group antigens and those controlling tumour rejection in mice were distinct. Bibliography 1. and Kappler & Reinherz. A. autoimmunity and responses to immunization could develop into strategies for protection against many crippling diseases. Medawar showed conclusively that such a mechanism was immunological. Zinkernagel. With this experiment he proved that resistance (or the lack of it) to foreign tissue is an immunological phenomenon. Immunology continues to fascinate and frustrate researchers the world over. . that dissimilar blood group antigens of one calf were tolerated by the other twin calf. Livingstone. A better understanding of tumour immunology.4 Immunology– Introductory Textbook Immunobiology of transplantation and tolerance was an active field of research in the 1940s and 50s. These workers cloned and sequenced a gene expressed and rearranged in T cells but not in B cells. Eventually. and they showed that T cells must recognize two signals on an infected cell—one from the virus and one from the cell’s own antigens—to destroy it. First glimpsed in experiments by Allison. using X-ray crystallography. Snell. Drawing from this experiment Medawar demonstrated that an inbred strain of mice – strain A could be made to tolerate skin grafts from another inbred strain – strain B by injecting the strain A animal soon after birth with strain B spleen cells. Edinburgh. For this new understanding of cellular immune mechanisms they shared the Nobel Prize in 1996.J. In 1950 Dausset identified the HLA ( Human Leukocyte Antigen) locus or MHC (major histocompatibility complex) as it is sometimes called. Medawar. revealed that the molecules of the major histocompatibility complex (MHC) bind to antigenic peptides and present these peptides to the T cell receptor. Their analysis showed that many of the T cell receptor sequences were homologous to those of immunoglobulin genes. History of Immunization. H. Brent and Billingham performed a series of dazzling experiments to explain the mechanisms that enable the host to recognize and destroy foreign cells. 1974. Elucidation of the structures of some HLA molecules by Bjorkman and Wiley in 1987.Mak and Mark M. Dausset and Benacerraf were awarded the Nobel Prize in 1980. and S. E.

• Gonorrhoea is a disease of man and chimpanzees and not of any other species. Escherichia coli meningitis. “The host is an island invaded by strangers with different needs. Within man. this may be because bactericidal IgM does not cross the placenta. there are certain well known racial differences in disease susceptibility: • dark skinned individuals have an increased susceptibility to coccidioidomycosis. (b) Age: The very young are more susceptible to many infections. The immune system faces the task of providing a defence mechanism to establish a state that is known as immunity to infection. recognition and destruction of mutant cells. in particular. different localities in which to raise their progeny”. • certain dark skinned people lack the red cell Duffy coat and are not susceptible to vivax malaria. removal of damaged or effete cells • Surveillance. • Bacillus anthracis is an infection of humans though not of chickens. . The contemporary definition of immunity is therefore: “All those physiological mechanisms that endow the animal with the capacity to recognize materials as foreign to itself and to neutralize. Among the non-specific defence mechanisms there are those that form a set of ill understood and perhaps grossly under-emphasized constitutional factors that make one species innately susceptible and another resistant to certain infections. eliminate or metabolize them with or without injury to its own tissues”. These responses have been widely classified as non–specific and specific. These constitutional factors can best be listed as– (a) Genetic: between species. T Immunological responses serve three broad functions: • Defence against micro organisms • Homeostasis. Genetic control of disease has been shown to be strongly associated with the major histocompatibility complex. There are a formidable range of infectious agents that can use the human body as a sanctuary to raise their offspring. Some species seem to be able to harbour organisms within their body tissues while the same organism may cause another species to succumb to such an infection. for example: • Mycobacterium leprae seems to infect humans and armadillos only. different food requirements. At the other end of the spectrum rickettsial infections and certain viral infections of children are more severe with age.CHAPTER – 2 INNATE IMMUNITY aliaferro said.

insulin and thyroxine do the opposite. Mucus secretions of the tracts that connect internal organs to external surfaces form an important form of defence. However.1). (d) There is a growing body of evidence that supports the hypothesis that immune processes can be influenced by neuroendocrine factors. oestrogens and progesterone depress immune responses. most bacteria fail to enter due to the low pH and direct inhibitory effects of lysozymes. Other mechanical factors which help protect these tracts are the washing . They entrap and immobilize bacteria and hence. stress and circadian rhythms modify the functioning of the immune system. A major form of defence in this context is the intact skin which is impermeable to most infectious agents. decrease in pH and a reduced influx of phagocytes . Sweat and sebaceous glands are potential points of entry for the infectious agent.infection can be a severe complication. Figure 2. overcrowding and undernutrition also increase susceptibility to infection. Immunologic cells have receptors for a whole range of hormones. (e) Environment: Poor living conditions. At a more physiologic level. It therefore seems reasonable to say that immune responses are finely tuned by neuro endocrine circuits. prevent adherence and colonization of epithelial surfaces. An exception is Staphylococcus aureus which commonly infects the hair follicle and glands. Hairs at the external nares. lactic acid and other fatty acids of sweat and sebaceous secretions. Natural barriers to infectious agents are simple yet effective means of innate defence (Figure 2. Immunologic organs are innervated by autonomic and primary sensorial neurons. the cough reflex and the ciliated mucus membrane of the respiratory tract help drive entrapped organisms upwards and outwards. androgens. hormone secretion is balanced by neural control. Natural barriers to infectious agents. In diseases such as diabetes mellitus where altered metabolism causes increase in blood glucose. whereas growth hormone. Corticosteroids.1.6 Immunology– Introductory Textbook (c) Metabolic: Hypoadrenal and hypothyroid states decrease resistance to infection. Steroid hormones are known to affect many modalities of the immune response.

Innate Immunity 7 action of tears. Many secretions contain bactericidal components such as acid in gastric juice. spermine and zinc in semen and lactoperoxidase in breast milk. and Clostridium difficile cause opportunistic infections. pathogens such as Candida spp. nasal secretions and saliva. acute phase proteins. Figure 2. ingestion and intracellular killing. requiring recognition. movement of PMNs out of blood vessels towards the irritant. Phagocytosis: a multiphasic act. mast cells. Normal flora can suppress the growth of many potentially pathogenic bacteria and fungi by competition for essential nutrients or by production of inhibitory substances such as colicins or acid. lysozyme in tears.2). attachment to microorganisms. saliva and urine. two main defensive operations come into play – phagocytosis and the bactericidal effect of soluble chemical factors (molecules such as complement proteins. and eosinophils) and natural killer cells (NK cells). When normal human flora is destroyed by broad spectrum antibiotics. and cytokines).2. Other cells may also be involved: cells that release inflammatory mediators (basophils. Phagocytosis The engulfment and digestion of infectious agents is assigned to two major cell populations : the polymorphonuclear neutrophil (PMN) and the macrophage (M). An important mechanism of defence is associated with normal bacterial flora of the body. . proteolytic enzymes in intestinal secretions. If microorganisms do penetrate the body. Pathogenic invasion of the vaginal flora is inhibited by lactic acid produced by the commensal vaginal flora. Phagocytosis is a multiphasic act (Figure 2.

. molecular components associated with microorganisms but not found as a part of eukaryotic cells. They are glycoprotein in nature and are also known as toll-Uke receptors and are found on the surface of various body defence cells_ They are so named because they recognize and bind to pathogen-associated molecular patterns.. Binding of the microbial molecule to the toll· like receptor sends a signal through the cytoplasm to the nucleus of the cell where it activates genes coding for the synthesis and secretion of cytokines. flagellin..noa.like and bind to externally diK played microbial sugars . P'those". . Several hydrolytic enzymes are now released into the phagolysosome which act optimally at a low pH. mennans.8 Immunology-Introductory Textbook Pattern Recognition and Ingestion Phagocytic cells are among the most important cells endowed with a variety of receptors capable of recognizing molecular patterns expressed on the surface of pathogens or pathogen ...ognized by a series of soluble pattern-recognition receptors in the blood that function as opsonins and initiate the complement pathways.the phagosome. sliOCiated molecular patterns (PAMPS). In all. lipopolysaccharide. These include bacterial molecules such as peptidoglycan. Most of these pattern recognition r eceptors (PRRa) are ledin. Most body defence cells bave patternrecognition receptors (PRRs) for common pathogen-associated molecular patterns (Figure 2. glycolipids. Lysosomal granules come into contact and finally fuse with the phagosome forming a pbagoly9080me.ted molecular paltern$ FigUN 2. and b. the innate immune system is thought to recognize approximately lQ3 molecular patterns.3) and so there is an immediate response against the invading microorganism . and zymosan . manDans. these are shared by a large group of infectious agents and are clearly differentiated from 'selr patterns.ptors on Defence Cells. pilin. There are also pattern-recognition molecules for viral double· stranded RNA (dsRNA) and fungal cell wall components such as lipoteichoic acids. Pathogenassociated molecular patterns can also be rec..3. There is evidence that an adherent particle may initiate ingestion by activating an actinmyosin contractile system which extends pseudopods around the particle_ The particle is eventually enclosed completely in a vacuole .ltcterial DNA. Palhogen-Associated Molecular Patterns Binding 10 Pattern-Recognition Rec. teichoic acids.

Innate Immunity 9 Intracellular Killing Intracellular killing utilizes two mechanisms: (i) Oxygen dependent mechanisms and (ii) Oxygen independent mechanisms (Table 2.. . • pcn\o~ c phuspbate + NAOP lI 02 burrst + generation of ::. cathespin C) Lysozyme Dflmage to microbial membranes Splits mucopeptide in bacterial cell wcll Deprives proliferating bacteria of iran nigest.ective mechanisms used by host + muny microbes supcroxidc 20 2 +2W di smutilse O~ + rhO~ ~ata]aFe 2H2.a .ion of k illed organisms Lacwferrin Prol. • • OH+Olr+ IO! Spontaneous Formation of further microbicidlli agents mye]operoxidllse H~O~ + CIH ~O .l Oxygen independent mechanisms Cationic proteins (incl. Table 2.+ H2O 0 OCI-+ Myeloperoxidase generation of microbicidal IIlQlecules Prot.eolytic enzymes variety of other hydrolytic em:rmes Oxygen-dependent mechanisms With the formation of the phagolYBoBome. ocr + H2 O 1 2 + CI.! +IO ~ 03+lhO~ . increased glycolysis and incr eased oxygen consumption with an exagger ated formation of hydrogen peroxide.1).uperoxide anion b_:ll~ NADPH+0 2 spontaneous di~mut:1otion NADpt +O~ 2~+2H' • H~O. there is a dramatic increase in activity of th e hexose monophosphate shunt.0:! 2 H:lO+ (}.lIlln l.flftf hexo slt mon ophosphille shunt cvtochmnHl Glucose + NADP' . lactic acid and a subsequent fall in pH .1: Oxygen Dependent and Independent Mechanisms Oxygen depenthllt 1tVr:.

10 Immunology-Introductory Textbook prerequisite for optimal functioning of hydrolytic enzymes. Human lHIefensins are proteins that play an important role in defending against microbial invaders along mucosal tracts. Extra cellular killing by eosinophils (using the complement pathway) has evolved as a way of defence against these parasites. It is thought to be particularly effective against Salmonella and Leishmania spp . Extra-cellular Killing Natural killer cells arc large granular Iympocytes. si nglet 02 (I0 2) and hydroxyl radicals (Om . Killing by Nitric Oxide Nitric oxide is known to be a physiologic mediator s imilar to factors that relax the endothelium . There are also a number of plasma proteins collectively called the "acute phase proteins" which show a dramatic increase during infection. These molecules are known as a. IL· l in turn induces the liver to release more CRP. Large parasites such as helminths cannot be physically phagocytosed. this they do by secreting a cytolysin called perforin that attacks the membrane of the infected cell. which is ultimately utilized to reduce molecular oxygen hound tocytoebrome. As a resul t OXYllen is converted to s uperoxide anion (OJ. lysozyme a muramidase which splits the peptidoglycan of the bacterial cell wall. These include C-reactive protein (CRP). Furthermore the combination of peroxide. cilusing a burst of oxygen consumption. During infection . Collectively the stimulation of all these pathways is called a "respiratory burst". Lysozyme and lactoferrin also constitute bactericidal or bacteriostatic factors which are oxygen independent and can function under anaerobic conditions.-defensins and act selectively on microbial lipid components. It is formed within most cells particularly neutrophils and macrophages and generates a powerful antimicrobial effect. The prime function of CRP is to bind to a number of micro organisms (in a calcium dependa nt fashion) which contain phosphorylcholine. a -I antitrypsin. Their main role is to kill virus infected cells. Soluble (Humoral) Bactericidal Factors Of the soluble bactericidal substances elabora ted by the body. Oxygen independent mechanisms As a r esu lt of t he oxygen dependent mechanisms the pH of the vacuole rises so as to allow several cationic proteins which are microbicidal to act optimally.all of which are powerful microbicidal agents .. This then enhances activation of . fibrinogen and caeruloplas min. pathogens known to live comfortably within the cell and yet escape phagocytic killing. The hexose monophosphate shunt generates NADPH. perhaps the most a bundant li nd widespread is the enzyme. microbial substances such as endotoxins stimulate the r elease of interle uk in -l (IL-l)-an endogenous pyrogen. serum amyloid A. hydrogen peroxide. Finally killed organisms are digested by hydrolytic enzymes and degraded products released to the exterior. Other substances such as the neutral proteinase (cathepsin G) are also powerful microbicidal agents. mannose-binding protein. Remember eosi nophilia can sometimes be an indirect clue that the patient has a parasitic infestation. myeloperoxidase and halide (en ions constitutes a potent halogenating system capable of kiUing both bacteria and viruses.

They are secreted into the extra-cellular fluid where they bind to receptors on uninfected cells.::ific immunity. many of these remarkable defence mechanisms are powerless in the face of overwhelming infection. all is not lost. However. CRP therefore acts as an opsonin. far more powerful and exquisitely precise are brought into play. other strategies of defence. coating organisms and triggering complement mediated lysis (see Chapter: 7). .a reflection of tbe waning innate or natural defence mechanism in the host.Innate Immunity 11 complement and thereby induces the acute inflammatory response. Should innate immunity fail for some reason. The bound interferon exerts an antiviral effect and prevents the uninfected cell from becoming infected. in the form of adaptive or acquired spe.t are infeded by viruses. Experience with chronically ill or debilitated patients indicates that many of these patients become "secondarily" infected . Interferons are anti·viral agents synthesized by cells tha.

The tertiary granules are the conventional lysozymes with acid hydrolases. They are present throughout the connective tissue and around the basement membrane of small blood vessels. lysozyme and a B12 binding protein. They enter the circulation as blood monocytes and finally settle in the tissues as mature macrophages. Metabolism by glycolysis enables the cell to function under anaerobic conditions. They are particularly abundant in the lung as alveolar macrophages and in the liver as Kupffer cells.hence they are often loosely called “pus cells”. The neutrophil granules are of three types. They are strategically placed in the lining of spleen sinusoids and lymph node medullary sinuses to filter foreign . The Mononuclear Phagocyte These cells are derived from bone marrow promonocytes. what is collectively termed.CHAPTER – 3 IMMUNOBIOLOGY T he cellular organelles of defence in the human body are found in. the lympho-reticular system. It is a non-dividing short lived cell with a multilobed nucleus and an array of granules. This is broadly classified into Internal Blood Tissues Thymus Lymph nodes Spleen External Respiratory tract Gastro-intestinal tract Genito-urinary tract The cellular constituents of the lympho-reticular system are: • Phagocytic cells Polymorphonuclear neutronphils Mononuclear phagocytes Eosinophils • Lymphocytes The Polymorphonuclear Neutrophil (PMN) This cell has a common haemopoietic stem cell precursor and is the dominant white cell in circulation. the primary azurophilic granule contains myeloperoxidase. some lysozyme and a family of cationic proteins. The secondary granules hold lactoferrin. They constitute the mononuclear phagocyte system (MPS). The polymorphs provide a major defence against pyogenic bacteria.

They have receptors for complement component C3b (see Chapter 7) and when activated produce a “respiratory burst” and concomitant generation of active oxygen metabolites. Lymphocytes derive from stem cells. Upon activation. Eosinophils also produce a “perforin” like protein which can produce membrane damage via transmembrane plugs. Initially stem cells arise from the yolk sac and the foetal liver. CD4 or CD8 markers. while an eosinophilic cationic protein together with a peroxidase have been identified in the granule matrix. Other enzymes found in the eosinophil include arylsulphatase B. such as LPS from Gram negative organisms and components of Mycobacterial and Gram positive cell walls. processing and presentation of antigen to the T-cell to elicit the specific immune response. This allows for adherence of eosinophils through their C3b receptors. Lymphoid Organs. Classification of lymphocytes on he basis of surface markers make use of two important classes of characteristics.1). lymph nodes and the aggregates of lymphoid tissue in the respiratory. namely the thymus and the bone marrow (Figure 3. There are more than 200 distinct CDs. The other function involves the initial recognition. the eosinophil then launches its extra cellular attack which includes release of MBP and cationic proteins both of which damage parasite membranes. microglia in the brain and osteosclasts in bone are also part of the MPS.Immunobiology 13 material. viruses and protozoa. but later in perinatal development. . some originate in the bone marrow. Mesangial cells in the renal glomerulus. gastro-intestinal and genito-urinary tracts. The macrophages feature predominantly in combating intracellular bacteria. Mononuclear phagocytes express a myeloid receptor (CD14) which serves as a recognition molecule for a wide variety of bacterial envelope molecules. Unlike polymorphs. The primary organs are the thymus and the bone marrow. Broadly. Thus mature T cells have CD3. classified commonly as Primary or central and Secondary or peripheral organs. the macrophage serves two major functions : to ingest and destroy particulate matter– a function greatly enhanced when the foreign matter is coated by complement or antibody. Most helminths activate the alternative complement pathway (see Chapter 7) and hence C3b is deposited all along the helminthic membrane. Ligation of this receptor leads to macrophage activation. The term opsonin is used to describe this coating with both antibody and complement to facilitate phagocytosis. and histaminase. and B cells have CDs 19-22. The immune system consists of a number of lymphoid organs. they have a long life span. Stem cells differentiate into lymphocytes in the primary lymphoid organs. Eosinophils Large parasites such as helminths cannot physically be phagocytosed and extracellular killing by eosinophils is largely the mode of defence involved. A major basic protein (MBP) is localized in the core of the granules. Lymphocytes and Lymphocyte Traffic Any discussion on lymphocytes takes its origins from a detailed study of the lymphoid organs. CD antigens represent families of surface antigens that can be recognized by specific antibodies produced against them. phospholipase D. These cells have distinctive granules which stain well with acid dyes. One is known as cluster designation (CD) and the other the antigen recognition receptors. the secondary organs are the spleen.

The Primary Lymphoid Organs The Thymus The thymus is responsible for the development of T-dependent lymphocytes. The thymus consists of two lobes surrounded by a thin capsule which extends into the substance of the gland to form septa–with the resultant formation of lobes and lobules. Furthermore. Peripheral portions of the lobule are heavily infiltrated with lymphocytes. the rate of mitotic activity is greater than in any other lymphoid tissue and yet. since B cells were known to originate in the Bursa of Fabricius in birds. The thymus is believed to perform two main functions.14 Immunology– Introductory Textbook Figure 3. Some interdigitating cells. This probably represents a system of homeostasis. foetal liver or . These humoral substances (one of which is called thymosin) may induce differentiation of lymphocytes directly within the thymus or may control differentiation in the periphery. A simplistic overview of the immune system. The thymus does not ontain any plasma cells. are strategically placed to meet foreign particles. Central portions have fewer lymphocytes and more epithelial cells. are also found in the thymus. which they display as surface markers. Thymic epithelial and interdigitating cells also influence T cell differentiation. derived from precursors in the bone marrow. Unlike other lymphoid organs it contains two tissue types: lymphoid and epithelial. Following infiltration of the thymus with pluripotential stem cells from yolk sac. the number of cells leaving it are comparatively less. All other lymphoid tissues. production of lymphocytes in the cortex and production of humoral substances in the medulla. the thymus is protected from antigen contact. These cells are rich in major histocompatibility antigens (MHC). This central lymphoid organ differs from other lymphoid tissues. * Bursal equivalent. It plays an important role in immunogenesis in the young and the T cells derived from it orchestrate the immune response throughout life. The assumption is that a large number of cells made in the thymus die within its substance.1.

. In addition. T. in addition. As the thymocytes pass to Stage II they exhibit CD5 CD4 and COB surface markers. During the process of maturation the T cells become immuno-competent : they learn to bind to specific antigens. T cell receptor genes become activated in both types of stage III cells.2).. Those that lose the COB surface marker become mature CD4+ T cells. T cells learn to differentiate between self and non self antigens. which forms part of an antigen binding receptor on the T cell. The T cells go through a selection process in the thymus based upon the T cell receptor that they posses. Stage 11 cells then lose the CD5 marker and differentiate further into one of two types of a Stage In cell. These two populations are called. Hence they arc schooled to recognize MHC antigens during thymic maturation.Immunobiology 15 spleen. 3. there are two separate populations of Stage III thymocytes released into the blood stream. allowing for the expression of the complete T cell antigen binding receptor complex (CD3-a ~) . In the blood stream they are .2. whereas those that lose the CD4 marker become COB T cells (Figure 3. these cells acquire new surface antigens as they undergo differentiation. when these antigens are presented in association with MHC molecules. Finally. T cells that recognize selfantigens are destined to die by apoptosis 01" programmed cell death . Stages of T cell differentilltion. the gene that encodes for the Il-chain of the T cell receptor becomes activated. During intra thymic maturation thymocytes lose or retain certain surface markers_ Stage I thymocytes express a specific antigen termed CD2 on their surface. They also ontain an activated gene known as the T gene.helper inducer and T-cytotoxic suppressor T cells. C02 Stage ~ o COAD4 1 Vb" ~ stage II Stage III /\ CD2U ' Flgu .

. A few B cells also come from the blood stream and many localize in the germinal centers of the eortex. There does not appear to be an organ in mammals that is equivalent to th+! Bu rsa of Fabricius.+fl-. The Bursal Equivalent and Bone Marrow Birds have another primary lymphoid organ in addition to the thymus. Stem cells enter the Bursa of Fabricius where they differentiate into Beells capable of producing antibody. Most of the blood lymphocytes arc T cells which migrate in and out of lymph nodes. H il~ Medullary cords Afferent lymphiltic1 "'1~'H--. situated near the cloaca." " '. A great majority of lymphocytes that traverse through the lymph nodes come from the blood stream.. .! (T cel!) Iru c:::'-. + f . These cells efficiently process antigen but cannot present it to T cellR. The germinal centers are hence peripheral bursal equivalents.16 Immunology-Introductory Textbook carried to the lymphoid system's peripheral organs where they reside in thymus dependent regions of the peripheral lymphoid system. germinal centers increase in size and thereafter contain many lymphoblasts and is the site of memory B cell proliferation.. . Cut section of lymph mode.. In the skin they are known as Langerhans cells . Langerhans cells pick up antigen in skin and carry it . Figure 3. Burscctomized birds are completely B cell deficient though T cell functional activity is unaffected . 3. Stem cells differentiate into B cells in the bone-marrow and in the peripheral lymphoid organs themselves.._ " " " .-_ Outar Cortex (8 cell arCiI) Primary tOllicl.Y. The bursa is derived from gut epithelium in the embryo. . ~. it is called the Bursa of Fabricius.3). . A sman percentage is generated in the lymph node from precursor cells. inusa. Secondary Lymphoid Organs Lymph nodes Cells and molecules are brought to the lymph nodes via afferent lymphatics (Figure 3.Gemtnill centre or secondary tolUd. When lymph nodes have been stimulated by antigen. Lymphocytes move through the sinuses and leave the lymph nodes through efferent lymphatics.Madu!IIrY . Cells of the dendritic cell lineage involved in T cell stimulation are bone marrow derived.Paril cortic.

C3 complex. The lymph nodes hence appear to be compartmentalized . They are called dendritic because of their morphology rather than any lineage relationship with dendritic cells. In addition. there is considerable uncertainty about their developmental origin. They present antigen to T cells which abound in the paracortical and sub cortical areas of the lymph mode. T cell function is closely associated and restricted by the presence of MHC antigens on antigen presenting cells.germinal centers with immobile B cells. may efficiently present antigen. Cut section of spleen.4. Their function appears to entrap antigen as an antigen. Figure 3. In fact. Follicular dendritic cells are found in germinal centres. The red pulp which surrounds the white is a thymic dependant area housing T-cells (Figure 3. now known as tissue dendritic cells or interdigitating cells. They cannot present antigen to T cells but are important in developing responses by B cells.4). para cortical areas with migrant T cells. as a stimulus to antibody production. All this appears to facilitate interactions between the different types of cells that are required for generating an immune response.Immunobiology 17 via afferent lymphatic vessels to lymph nodes. and present it to B cells. The para cortical areas contain these interdigitating cells bearing MHC class II molecules complexed to foreign antigen. They display C3 complement and IgG (Fc) receptors. In lymph nodes the cells. Spleen In immunological terms the white pulp of the spleen is the bursal equivalent and is the store house for B cells. Other Lymphoid Tissue The respiratory. alimentary and genito-urinary tracts are guarded immunologically by sub epithelial accumulations of lymphoid tissue which are not constrained by a connective . Dendritic cells in lymph are known as “veiled” cells. there are sinuses full of macrophages and a reticular network of dendritic cells that tends to hold antigen for a long time.antibody.

Binding of monoclonal antibodies is currently the most specific technique used to identify specific sub sets. It is believed that MALT forms a separate interconnected secretory system within which cells committed to IgA or IgE synthesis may circulate. plasma cells and phagocytes in the lung and in the lamina propria of the intestinal wall. their subsets and functions see Table 3. mucosal associated lymphoid tissue (MALT). Morphologically these cell types cannot be differentiated. Lymphocyte traffic There are three major types of lymphocyte circulation : (a) The seeding of stem cells from the foetal liver or bone marrow in the primary lymphoid organs and the subsequent differentiation and distribution of these cells to the peripheral lymphoid system. Immunocompetent cells involved in the immune response The primary cells involved in the immune system are the lymphocytes. can kill a number of tumour cell lines in a non specific manner. Phylogenetic evidence suggests that the development of immunologic competence coincides with the development of the lymphocyte. A majority of null cells are natural killer (NK) cells. lymphoid tissue found in the respiratory and urinary tracts: collectively they are termed. In addition there are a heterogenous group or groups of lymphocytes that are neither T or B cells. This includes the tonsils. Functionally. in vitro. are involved in cell mediated immune reactions and induce B cells to produce antibody. cytotoxic T cells (bearing the CD8 marker). the Payers patches and the appendix. these cells play pivotal roles in the immunological response to foreign antigen. or as clearly organized lymphoid follicles. Functionally however. These may occur as diffuse collections of lymphocytes. For a comprehensive classification of lymphocytes.18 Immunology– Introductory Textbook tissue capsule.1. . Large granular lymphocytes A portion of the lymphocytes circulating in blood lack the surface antigens of T or B cells. lymphocytes are divided into (a) T cells: These cell types regulate the immune response. Using conventional stains the lymphocyte is a morphologically featureless cell. (b) B cells: These cells differentiate into antibody producing plasma cells. (b) The recirculation of lymphocytes from blood to lymph to blood and (c) The distribution of effector cells to particular parts of the body. They are large granular lymphocytes that. These cells are sometimes called null cells. Sub sets of T cells Depending on the presence of surface markers (CD4 and CD8) T cells are broadly and functionally divided into helper T cells bearing the marker CD4.

T-cell receptor. CD20.Class I Large granular lymphocytes Null/ NK cells CD 56+ Not MHC restricted CD: Cluster differentiation designation .Class II Stimulate B cell to produce antibody. MHC. CD21. CD3.1: Classification of lymphocytes Lymphocytes and their sub-sets T cells T helper cell CD4+.Immunobiology 19 Table 3. CD3. macrophage activation Lyse antigen bearing target cell Antigen specific Differentiate into antibody producing and memory cells Kill tumour cells and show antimicrobial activity (mainly antiviral) Main cell surface markers Restrictions Functions T cytotoxic cell B cells Plasma cell and Memory B-cells CD8+. CD2+ MHC. CD23.T-cell receptor. Induce CD8+ Tcell cytokine secretion. CD2+ Ig+. CD19.

Hence there may be a cluster of amino acid sequences on the three dimensional structure constituting a series of epitopes. is referred to as an antigen. In contrast to this is the hapten. T Antigenic Determinants and Epitopes The part of the antibody molecule which contacts the antigen is termed the paratope. Figure 4.1 a and b. Each of these epitope clusters is what is meant by an antigenic determinant. Antigenic determinants on a single bacterial cell. As most antigens are protein in nature they exist in a folded. Consequently. which usually recognize intact antigen. . tertiary structure.CHAPTER – 4 ANTIGENS AND IMMUNOGENICITY he terms antigen and immunogen are often used synonymously. Antigens are recognised not only by antibodies but also by antigen specific T cell receptors. An illustration of the antigenic determinants on bacterial cells is shown in Figure 4. In contrast to immunoglobulins. Haptens are small well defined chemical groupings such as dinitrophenyl (DNP) which are not immunogenic on their own but will react with preformed antibodies. One which describes a molecule that provokes an immune response is called an immunogen and the other describes a molecule which reacts with the antibody produced or with the activated cellular constituents of cell mediated immunity. three dimensional. together with the major histocompatibility complex (MHC) Class I or Class II surface proteins (refer chapter 10). To make a hapten immunogenic it must be linked to a carrier molecule which is itself immunogenic. T cell surface receptors recognize processed antigen on the surface of antigen presenting cells. that part of the antigen molecule that makes contact with the paratope is called the epitope. However. imply two closely related entities. these terms antigen and immunogen.1.

An immunogenic molecule needs to possess a certain degree of chemical complexity.000 and more. Molecular size determines immunogenicity to some extent.000 are only weakly immunogenic or not immunogenic at all. for example. It is difficult to establish a definite threshold. The general rule is that particles with a molecular weight less than 10. Hence.2. adjuvants are used to enhance immunogenicity. so that the loop conformation was no longer present. Figure 4. Sometimes body constituents are recognized as foreign leading to autoimmune disease. Certain particles do not elicit an immune response – they merely undergo phagocytosis. Accessibility of antigenic determinants. Parts of the peptide chains which protrude significantly from the globular surface tend to be sites of high epitope density. Hence accessibility of these high epitope density areas to the recognition system determines the outcome of the immune response (Figure 4. do so when they are part of a larger particle. If the disulphide bond was disturbed. Only pure lipids are non immunogenic. however. but is highly immunogenic in the polymeric state. A solution of monomeric proteins may actually induce tolerance. Studies have also shown that the amino acid sequence is important to antigenicity. but crucial stretches of amino acids are present in an undisturbed fashion. carbon as coal dust. researchers have found that certain segments with high mobility (movement of 1 angstrom from the protein back bone position) were more antigenic than non mobile segments. Other workers have shown that the mobility of a segment of the antigen molecule influences its antigenicity. The most potent immunogens are macromolecular proteins with molecular weights of 10. can serve as antigens provided certain short. the lysozyme molecule is a good antigen in its native form which consists of several amino acids folded into a loop with the aid of a disulphide bond. Using the tobacco mosaic virus. Normally the body discriminates self from non self. Certain molecules that have no apparent complexity in structure. Several immunogens that do not induce an immune response in the pure form. For example. the antigenicity of the molecule would be dramatically reduced. .2). Experiments have shown that conformation of a molecule could be important to its antigenicity.Antigens and Immunogenicity 21 Requirements for immunogenicity The first and primary requirement for any molecule to qualify as an immunogen is that the substance be genetically foreign to the host. the general rule holds.

These intermolecular forces are: • electrostatic (attraction between oppositely charged ionic groups) • hydrogen bonding (reversible hydrogen bridges between hydrophilic groups) • hydrophobic bonding (similar to the manner in which oil droplets in water merge to form a single large drop. • Van der Waals forces : interaction between the electrons in the external orbits of the two different macro molecules. side chains of valine.22 Immunology– Introductory Textbook Antigens and antibodies interact by spatial complementarity and not by co-valent bonding. in essence. Hence those antibodies that fit closely to an antigenic determinant and are not easily dissociable are known as high affinity antibodies. The term affinity alludes to the strength of the bond between antigenic determinant and monovalent antibody. no different from the ‘non-specific’ interactions which occur between any two unrelated proteins. there are two commonly used terms that need definition: (i) affinity or strength of binding of antigen to antibody. the lock and key analogy. can be readily reversed. and like enzyme – substrate interactions. This may account for over 50% of the strength in an antigen antibody bond). leucine and phenylalanine tend to associate in an aqueous environment. The idea that antibody recognizes antigen through complementary shapes on paratope and epitope is simplistically illustrated using the “lock and key” analogy (Figure 4. Antigen and antibody interact by spatial complementarity.3. At this point of the discussion. These forces are. . The Forces that Bind Antigen to Antibody The forces that bind antigen to antibody become larger as intercellular distances decrease.3). Figure 4.

This is a function of multivalency i. An antiserum raised against a given antigen can cross react with a partially related antigen which bears an identical or similar determinant (Figure 4. multivalent antiserum has a higher avidity. monovalent and easily dissociated antiserum b. Cross reactivity of antibodies. Antiserum in Figure 4. in practice. Avidity of antiserum for antigen a. Antigen-antibody Specificity is not Absolute It is widely known that antigen-antibody reactions are exquisitely specific.4. binding is greater.4. .5. Each one of the many interactions is by itself a low affinity one.e. the term cross reactivity is widely used.4a. However.Antigens and Immunogenicity 23 (ii) avidity of antiserum for antigen refers to the overall strength of interaction between a large antigen with multiple epitopes and a polyvalent antibody molecule such as the IgM. An antibody raised to one determinant will not react with another determinant. an antigen with several determinants will elicit a variety of antibodies as illustrated in Figure 4. Figure 4.4b is multivalent. dissociation is not easy and it is of higher avidity than antiserum 4.5). Figure 4.

The Forssman antigen is another example which is found in the tissues of many species. Following an attack of infectious mononucleosis. . The Heterophile Antibody Response A number of similar antigen molecules are found in phylogenetically unrelated species. will react with Ag2 due to the identical determinant (hatched) shared by the two antigens. Antiserum to Ag1 will also react weakly to Ag3 because. Thus it is possible that each antibody will react not only with the antigen which stimulated its production. an infection by the Epstein-Barr virus.24 Immunology– Introductory Textbook Antiserum raised to Ag1. This is known as the heterophile antibody response and forms the basis of the Paul-Bunnel test for infectious mononucleosis. antibodies are produced which react not only to the virus but also to a completely unrelated antigen-the sheep red cell. This antigen is itself not found in man. but also with some quite unrelated molecule bearing similar looking determinants. the determinants though not identical. The cross reacting antigens of human heart tissue and the group A β-haemolytic Streptococcus is an example. however a great variety of other cells and tissues could sensitize man to this antigen. are similar in shape.

Besides.1). The amino terminal portion of the molecule is part of the variable or ‘V’ regions and the carboxy terminal portion the constant or ‘C’ regions. Digestion of the molecule by papain produces two Fab (antigen binding) fragments and one Fc (crystallizable) fragment. leaving the hinge region intact.CHAPTER – 5 IMMUNOGLOBULINS I: STRUCTURE AND FUNCTION he first real chemical information regarding the structure of antibodies was provided by Tiselius and Kabat in the early 1940s. designated H and L chains respectively. Hence the light chains have a variable (VL) and a constant (CL) region and so do the heavy chains have a variable region (VH) and a constant region (CH). hence papain acts at the hinge region to fragment the immunoglobulin molecule. hydrogen bonding and Van der Waal’s forces) . The hinge region is formed in the two heavy chains between the first and second C regions. Each polypeptide chain contains an amino terminal and a carboxy terminal. T Basic Structure of the Immunoglobulin Molecule Immunoglobulins are glycoproteins (Figure 5. The part of the antibody molecule that binds antigen is formed only by small numbers of amino acids in the V regions of the H and L chains. It has been shown that noncovalent forces (electrostatic. Amino acid sequence of the IgG molecule was determined by Edelman et al in 1969. In all these studies Bence Jones proteins which are monoclonal immunoglobulin light chains shed in plasma and urine from patients with plasmacytomas or multiple myelomas have been an invaluable source of large amounts of immunoglobulin molecules. CH2 and CH3 in order from the amino end to the carboxy end of the heavy chain. the disulphide bonds are not really responsible for holding the chains of an immunoglobulin molecule together because removal of these bonds retains the intactness of the molecule. The CH region is further divided into three areas and these are designated CH1. a few subtypes of immunoglobulin molecules do not have disulphide links between chains. These four polypeptide chains consist of two identical heavy chains and two identical light chains. It cleaves the Fc fragment from the remainder of the molecule. The enzyme pepsin strikes at a different point. The basic unit of the molecule is a four chain monomer. In the 1950s Porter used proteolytic enzymes such as papain to cleave the antibody molecule in an attempt at defining its structure and function. yet retain the basic four chain structure. The immunoglobulin molecule is linked together by several disulphide bonds from H to H chains. However. It is flexible and more exposed to enzymes and chemicals due to its high proline content. H to L chains and L to L chains. It leaves behind a large fragment designated F(ab)2 since it is still divalent with respect to antigen binding just like the parent antibody. Intrachain links also exist. These workers demonstrated that the fraction of serum proteins that migrated most slowly in electrophoresis contained most of the serum antibodies. which lacks the ability to bind antigen.

IgG3 and IgG4 have been described and similarly an IgA1 and IgA2 as well. IgA an α. IgE. larger is the S value.1. Isotypes Based upon the structure of their heavy chain constant regions. Likewise the light chain constant regions also exist in isotypic forms known as κ and λ. IgA and IgM have been further divided into subclasses by relatively minor differences in their CH regions. Structure of an immunoglobulin molecule. IgM as IgM κ or IgM λ and so on. Figure 5. IgG. Since these classes are all variants of the immunoglobulin molecule they are termed isotypic variants or isotypes. The 5 different isotypes are defined by differences in the amino acid sequences of their constant regions and therefore by antigenic differences in the CH regions of the molecule. Thus IgG exists as IgG κ or IgG λ . IgG2.26 Immunology– Introductory Textbook are far more important in maintaining the integrity of the molecule than the co-valent disulphide bonds. IgA. Therefore. immunoglobulins are classed into major groups termed classes. These classes are designated IgG. The CH region of the respective immunoglobulins are designated by greek alphabets. IgM a µ. on a single molecule. IgE or IgD. and IgD. All immunoglobulins are measured using a sedimentation coefficient (measured by Svedberg) or S value. IgA. Hence IgG has γ CH region. Immunoglobulins possess either κ or λ light chains. it is possible to raise an antiserum to any one isotype (say IgG) which can be absorbed to remove any cross reacting antibodies. IgM. IgE an ε and IgD a δ type heavy chain constant region. never mixed. an IgG1. . Higher the molecular weight of immunoglobulin. This antiserum will then be capable of reacting with IgG but not with IgM. Since the immunoglobulin molecule is a protein structure with antigenic differences in the various isotypes.

An antibody molecule is a Y-shaped protein made of four polypeptide chains. idiotypes are formed based on antigenic determinants in the variable region that distinguish one V domain from the other. (Chapter 16) Immunoglobulins in their natural state In their natural state immunoglobulins are folded into a three dimensional structure. Certain sequences in the variable region show quite remarkable diversity and are termed hypervariable regions. antigen binding sequences all appear as loops at one end of the variable domain. Such anti idiotypic sera are useful in detection and typing of the various idiotypes produced by the system. These hyper variable sequences have been localized to three segments each on the H and L chains. They are therefore in an ideal Figure 5. And just as an antisera can be raised to the different isotypes an anti idiotypic serum can be raised to the various idiotypes of immunoglobulin molecules.Immunoglobulins I: Structure and Function 27 The Variable Regions and Heterogeneity of Immunoglobulins The antibody population in any individual is incredibly heterogenous. this means that the normal immune system can generate an immunoglobulin for every possible immunogen that the system may encounter during its life time. Two heavy chains extend from the stem (blue). The consequences of this will be discussed later in the Jerne net work theory. It does this by constantly changing the amino acid sequences in the variable regions of the immunoglobulin molecule. Idiotypes Just as the C regions of the various isotypes have antigenic determinants different from each other. Significantly the hyper variable. clustered close to each other (Figure: 5. Characteristic folding pattern of the immunoglobulin molecule. The terminal 110 amino acid sequences of the L and H chains constitute the variable or heterogenous regions of the immunoglobulin molecule.2). A startling realization resulting from research in this area was that an individual could make antibodies to his own idiotypes (auto anti idiotypic sera). Not surprisingly.2. hot spots or complementarity determining regions (CDR). the immune system is equipped with the provision of rapidly changing and modifying the variable regions of immunoglobulins to suit a particular antigen. In effect. two light chains are confined to the arms (grey). Thus instead of carrying the enormous load of having to produce millions of billions of immunoglobulins to encounter every foreign agent. these areas of hypervariability are intimately involved in the formation of the antigen binding site. .

NK cells (natural killer) and placental syncytiotrophoblasts. Further. For example. the CH3 domain of IgG binds to Fc receptors on phagocytic cells. . Figure 5.3) Biological Role of the Constant Regions If the variable regions function as antigen binding sites. The apple is the antigen! Immunoglobulin Classes and Subclasses The following section describes the physical and biological characteristics of the 5 major immunoglobulin classes: IgG. also to Staphylococcal protein A.3. Hence it plays a vital role in the defence against infection. IgD and IgE exist in the basic four chain structural form. Being the immunoglobulin that can cross the placenta in humans it is responsible for the protection of the neonate in the first months of life. The three fingers of each hand constitute the six hypervarible regions which form the antigen binding site. since “polymorphs” also possess receptors for the Fc portion of immunoglobulin and for complement components. Besides sequence heterogeneity. IgG diffuses readily into extravascular spaces and hence provides a major defence against bacterial toxins and other blood borne infectious agents.28 Immunology– Introductory Textbook position to serve the function of antigen recognition and this has been confirmed by X-ray crystallographic analysis. During the secondary immune response it is the major immunoglobulin to be synthesized. Immunoglobulin G IgG constitutes 75% of the total serum immunoglobulins in humans. and the CH3 region of IgE binds to Fc receptors on homologous mast cells and basophils. IgM and IgA occur as complexes of this basic four chain unit. the CH2 regions of IgG and IgM fix complement most efficiently. complexes of antibody and bacteria also activate complement thereby chemotactically attracting polymorphonuclear phagocytic cells and promoting phagocytosis. This is reinforced by the presence of colostral IgG in breast fed infants. An analogy for the antigen binding site. (Figure 5. The complement binding site on the IgG molecule appears to be in the CH2 domain. Organisms coated with immunoglobulin G attract macrophages via their Fc receptors: thus enhancing phagocytosis. the hypervariable loops ensure tremendous diversity in combining specificity for antigen through variation in the shape and nature of the surface they create. what biological functions do the constant regions serve? The classes of antibody differ from each other in their ability to carry out certain secondary biological functions.

anti-B) and to the typhoid ‘O’ antigen. Such Fc receptors are also present on platelets and when complexed may lead to release of vasoactive amines. Figure 5. As will be discussed later the property of the Fc portion of IgG to bind to protein A on the surface of Staphylococcus aureus has been greatly exploited for use in diagnosis and research. IgM predominates in certain antibody responses such as the “natural” isohaemagglutinins (anti-A.4. since the IgM response is short lived its presence may be . are largely confined to the blood stream. IgM antibodies appear early in the response to infection and because of their size.5).the significance of which remains unclear. Electron microscopy studies reveal that it is shaped like a star. but has the ability to bind to guinea pig skin .4). The heavy chains of IgM are designated ‘µ’ chains. Structure of the IgM pentamer. furthermore. Since IgM does not cross the placenta its presence in cord blood indicates active foetal infection. but when it is attached to a bacterium.Immunoglobulins I: Structure and Function 29 In a similar way the extra cellular killing of target cells coated with IgG is mediated largely through recognition of the Fc portion of the IgG by surface receptors on NK cells. its antigen binding sites are bound to the bacterial surface. IgG is unable to bind firmly onto most cells. This changes the appearance of IgM to a crab like form and causes cross-linking of the different antigenic determinants or epitopes on the bacterial cell surface by the polyvalent IgM molecule (Figure 5. the IgM heavy chain therefore consists of regions CH1 to CH4. the J chain participates in the polymerization of IgM via a sulphydryl residue near the carboxy terminal. It exists as a pentamer of the basic four chain subunits held together by disulphide bonds. Immunoglobulin M IgM is the largest immunoglobulin in size (Figure 5. The size and valency of IgM makes it a very effective agglutinating and cytolytic agent. IgM antibodies tend to be of relatively low affinity but because of their valency they bind with high avidity to antigens with multiple epitopes. The µ chains bear an extra CH region. They are an important defence mechanism against bacteria. A relatively small molecule. This is so because it is the most efficient complement fixing immunoglobulin.

with the help of a cysteine rich polypeptide called the J chain which has a molecular weight of 15. nasal fluids. Figure 5. Figure 5. the secretory component.000.7).000 (Figure 5. Cleavage of the receptor releases the IgA. It is actively endocytosed and transported within the endocytic vacuole to the mucosal surface.30 Immunology– Introductory Textbook helpful in establishing an acute infection. IgA appears selectively in sero-mucus secretions such as saliva. The anchored IgM is the major antibody receptor used by B-lymphocytes to recognize antigen. mucosal associated lymphoid tissue (MALT). colostrum and in secretions of the lung. Structure of the IgA dimer. into sero-mucus secretions (Figure 5. It is present in these fluids as a dimer. stabilized against proteolysis by combination with another protein. The dimeric IgA is released from the plasma cell and binds strongly to a secretory component precursor present on the surface of the glandular epithelial cell.6). which is synthesized by local epithelial cells and has a single peptide chain of molecular weight 60. (a) IgM free and (b) bound to the bacterial surface. what is now known as. The IgA is synthesized locally by plasma cells and dimerized intra cellularly before secretion. tears. still attached to part of the receptor: termed the secretory piece. . genito-urinary and gastro-intestinal tracts. Monomeric IgM has a hydrophobic sequence stitched into the ‘C’ terminal of the heavy chain to anchor the molecule into the cell membrane of the B-lymphocyte.5. Immunoglobulin A IgA is actively secreted by.6.

It is possible that IgE acts in this way as a defence against helminthic infections. This would lead to an influx of plasma IgG. IgE is found mainly in the linings of the respiratory and gastro intestinal tracts. IgE also has the ability to attach to human skin where they are probably bound to mast cells. complement. but contributes to the protection of the newborn by being in abundance in colostrum. IgA is the prime functional unit of the mucosal associated lymphoid tissue (MALT). Since IgA is most abundant in body secretions it performs the role of defending the exposed external surfaces of the body against attack by micro organisms. . Infectious agents penetrating the IgA defences would combine with specific IgE on the mast cell surface to trigger the release of vasoactive agents and other factors chemotactic for granulocytes. IgA does not cross the placenta. Immunoglobulin E IgE is present in very low concentrations in serum. which are characterized by an exaggerated IgE response. for the symptoms of hay fever and extrinsic asthma.7. Formation of secretory IgA. IgA activates complement via the alternative pathway but is unable to do so via the classical pathway. This process is responsible for the wheal and flare skin reaction in allergy. IgE antibodies have a high affinity for mast cells and binding occurs via the Fc portion of the immunoglobulin molecule. Dimeric IgA binds to this receptor. The glandular cell on the mucosal surface synthesizes an immunoglobulin receptor inserted into the basal membrane. is endocytosed in a vacuole and transported to the mucosal surface. Cleavage of the receptor yields secretory IgA into the lumen with part of the receptor (as the secretory piece) still in place. IgA functions by inhibiting the adherence of coated micro organisms to the surface of mucosal cells thereby preventing entry into the body tissues. polymorphs and eosinophils causing an effective inflammatory response. On contact with specific antigens called allergens. the mast cells undergo degranulation with release of vasoactive amines.Immunoglobulins I: Structure and Function 31 Figure 5. The main physiological role of IgE would appear to be protection of external mucosal surfaces where release of vasoactive amines could perpetuate the acute inflammatory response. where they form constituents of MALT.

It has been suggested that they may operate as antigen receptors and in the control of lymphocytic activation and suppression. first line defence in bacteraemia IgA 11S 160.000 Most abundant in extravascular spaces. Table 5.000 Major Ig in sero-mucus secretions. defends external body surfaces IgE 8S 200. IgG3 and IgG4. The differences lie in the chains of the molecule. IgG2. IgD 7S 185. The main function of IgD has not yet been determined.32 Immunoglobulin D Immunology– Introductory Textbook This immunoglobulin has the basic four peptide structure.1. Major characteristics of the immunoglobulin classes have been summarised in Table 5. Certain functional and biological differences exist between the classes for eg: IgG3 does not combine with staphylococcal protein A. with IgM it is found abundantly on the surface of B-lymphocytes. IgG2 does not cross the placenta readily. IgA exists as IgA1 (constitutes 80-90% of total IgA). Raised in parasitic infections. Effective in agglutination.000 Protects external body surface. responsible for symptoms of allergy. combats microorganisms and their toxins IgM 19S 900. Because of an extended hinge region it is relatively liable to degradation by proteolytic enzymes. IgG4 does not bind to complement in the classical pathway nor does it bind to monocytes.1: Characteristics of the immunoglobulin classes Characteristics Sedimentation coefficient Molecular weight Functions IgG 7S 150. It is present in serum in trace amounts.000 Mostly present on surface of B lymphocytes Complement fixation classical alternative Cross placenta Fix to homologous mast cells / basophils Bind to macrophages and polymorphs ++ – + – +++ – – – – + – – – – – + – – – – + – + – – . IgA2 is unusual because it lacks H to L disulphide bonds.000 Early in immune response. Immunoglobulin Sub-classes IgG has four isotypic sub classes termed IgG1.

the germ line and somatic recombination theories. Since an antibody is an assembly of protein chains. they remember each infection so that a second exposure to the same organism is dealt with more efficiently. since each antibody is different depending on the antigen that provokes its formation. They also implied that these separate lists of information must somehow come together to form a contiguous. at a time when no means of rearrangement of genes in germ line cells was known of. Furthermore. they proposed that there were several hundred stretches of DNA that coded separately for different kinds of variable regions and one gene coded for the constant region in the germ line DNA (DNA in the sperm cell and ovum). How then does the immune system function on such a small defence budget demanding that only a small share of the genome and the body’s resources be ear marked for the production of antibodies? The paradox of a limited number of genes and an apparently limitless capacity to generate different antibodies has been a major puzzle for immunologists.CHAPTER – 6 IMMUNOGLOBULINS II: THE GENETICS OF ANTIBODY DIVERSITY he immune system is clearly essential for survival. It would appear that the genome must contain millions of antibody genes. Yet their idea proved to be essentially correct. the one gene. one polypeptide theory. These bits and pieces are shuffled in the DNA of the B-lymphocyte to yield the required antibody. without it death from infection would be inevitable. The molecules of the immune system maintain constant surveillance against invading micro organisms. Yet the total genetic complement of a mammal amounts to perhaps a million genes. This implies that millions of species of antibody molecules need to be synthesized. This proposal initially attracted considerable criticism since it opposed the central dogma of protein biosynthesis. coherent message. its structure is specified by a gene. they can distinguish and bind to every possible antigen that the host is likely to meet during its lifetime and some that it is not. Instead of assuming that the genetic information for an antibody light chain is specified by a continuous array of codons (a codon is a triplet of nucleotides which codes for an amino acid). T The Dreyer-Bennett Hypothesis William Dreyer and Claude Bennett in 1965 working at the Caltech laboratories made a radical proposal. Dreyer and Bennett claimed that two or more genes could control the production of one polypeptide chain. They recognize an almost limitless variety of foreign substances.and essentially two competing theories were put forward in explanation . The mechanisms responsible for shuffling of immunoglobulin DNA became clear when it was possible . The Generation of Antibody Diversity by Somatic Recombination The DNA in the germ cells (in the male sperm and the female egg) or in the early embryo contains several bits and pieces of the gene that dictates antibody formation.

Organization of the human light chain gene. Combining a light with a heavy chain can yield over 10 million distinct antigen binding sites. .1. The same principle holds for the lambda light chain. and four distinct J segments. The rest of the V region was coded for by a stretch of DNA located thousands of base . There are 5 joining (J) segments coding for amino acids 96-108.J and C segments. Thereafter it was discovered that there were a few hundred V segments in germ line DNA. each accompanied by a leader (L) sequence. In addition to V and J segments the genes for the heavy chain variable segment include a third fragment designated D (for diversity). DNA rearrangement joins 1 V region to 1 J region to the C region. Mouse germ line cells have about 20 D segments. There is only one constant (C) gene. illustrates the process of kappa light chain secretion. The intervening sequences (IVS) are removed by RNA splicing.pairs away. these segments can be brought together in over 10. This yields one kind of high chain. This interposed segment was called J for “joining”. As shown in Figures 6. These experiments by Tonegawa. Multiple variable (Vk) region bits exist in the germ line DNA. The C segment was also located about 1300 base pairs “upstream” and was found to code for the C region.1 and 6.2 the germ line DNA contains Figure 6. In principle. Many other permutations and combinations are possible.000 combinations. each differing slightly in amino acid sequence. The potential diversity of the heavy chains is even greater.34 Immunology– Introductory Textbook to sequence fragments of DNA after they were cloned in bacteria. Hozumi and others showed that i) V and C genes were farther apart in embryonic cells than in antibody producing cells and ii) a particular light chain had a small segment of DNA coding for a part of the V region: this fragment was called the V segment. Each light chain was assembled by combining the scattered V. The Assembly of a Functioning Immunoglobulin Gene The assembling of a functional immunoglobulin gene takes place in two stages of a process called Somatic Recombination.

In case of the heavy chain gene order the fusion order yields a V. it is then cleaved away as the antibody molecule passes across the membrane. J and C segments (See figure 6.Immunoglobulins II: The Genetics of Antibody Diversity 35 an array of segments from which permutations and combinations occur. followed by a spacer and a nonamer or nine nucleotide unit. This is then translated to form the protein molecule which is the antibody.D. These segments are fused by enzymes that delete all the intervening DNA. Certain signal sequences that guide the site of action of these splicing enzymes have been discovered. All introns have been excised.1. It employs a set of enzymes that can bring together distant V.2.cell DNA is unusual and may even be unique to the immune system. there are multiple variable (V H )regions with preceding leader (L) sequences. The second stage in this process relies on mechanisms of RNA splicing. RNA splicing removes the intervening sequences (IVS) to join the constant (Cµ) to the rest yielding a secreted IgM protein .3. just down stream from the V gene of a kappa chain there is a distinctive pattern composed of a heptamer or seven nucleotide unit. . Each of the V segments is preceded by a short coding sequence known as the “leader” sequence and this is thought to play a part in the transport of the antibody molecule through the cell membrane. After RNA splicing the mRNA contains a single V gene in combination with a single J which in turn tags on to a single C gene.D and J segments for a heavy chain. Fusion of gene segments in B. Organization of the human heavy chain gene. Just upstream of the J segment there is a complementary nonamer-spacer-heptamer pattern. coding for amino acids(1-95). An RNA transcript is obtained as shown in Figure 6. Similar to the k light chain V fragments.2). Single VH. Similar signal sequences are found in the heavy chain genes. with the selection of a V. a J coding for amino acids 96-108 and the C gene separated from the J gene by an intron of 1250 nucleotides. Figure 6.D and J segments deleting all the DNA that separates them.cell DNA contains randomly selected V and J segments for a light chain or V. These units could provide a template for the enzymes to cut and rejoin the double helix. sometimes termed intervening sequences (IVS) or introns. As shown in Figure 6.among the first immunoglobulins to be secreted by a stimulated plasma cell. a family of diversity (D H) segments increase the variety of possible combinations between segments. DH and JH regions recombine with each other at the DNA level. There are the 6 functional joining (JH) segments and in addition. Each B.

increase the number and variety of antigen binding sites and together with somatic recombination. Mutations are seen in the variable domain genes and in the immediately adjacent regions.D and J segments. IgA or . a rate several orders of magnitude greater than the average mutation rate for eukaryotic genes.joining inaccuracy and somatic mutation. Another major source of diversity is somatic hypermutation. The effects of DNA . There are at least two additional sources of variety that contribute to antibody diversity. V-D and D-J joining. a spacer of 12 bases (at the V end) and 23 bases (at the J end) and then a base paired nonamer. By altering individual nucleotide bases the mutations “fine-tune” the immune response creating immunoglobulin genes whose products better match the antigen. Sequences that facilitate V-J. additional base pairs are inserted in the process of combining segments. Figure 6. The characteristic sequences form a base paired heptamer. As a result. Estimates of the rate of mutations suggest that there should be one change in the V region for every three to 30 cell divisions. in some cases. develops into a cell that simultaneously produces IgM and IgD and is subsequently capable of switching to the production of IgG. Similar mechanisms exist for joining V-D and D-J segments. The mutational mechanism is called into action during proliferation of the selected B-cell clones. even if two antibodies are specified by the same set of gene segments. The process of somatic recombination creates a population of cells that vary widely in their antigen specificity.In contrast to kappa light chains. The site of the junction can vary by several base pairs. It seems likely that B cells or their predecessors carry enzymes that induce mutations in just these sets of genes. lambda light chains have multiple constant region genes arranged in tandem. Spontaneous genetic changes in the developing cells are known to occur. These characteristic sequences facilitate splicing by DNA repair enzymes bringing distant V-J regions into proximity. they may still have slightly different antigen binding sites. Both kinds of change can obviously alter the amino acid sequence of the polypeptide. Furthermore. but not in the constant domains. and the Heavy Chain Gene Order An immature B cell bearing only surface IgM. from which few cells are selected by any given antigen. One of these is a lack of precision in the DNA splicing machinery that fuses V. in addition.36 Immunology– Introductory Textbook Humans utilize lambda light chains in one third of their immunoglobulins. Immunoglobulin Class Switching in Individual B-cells. the total number is estimated at well over a billion different antigen binding types.3.

then mRNAs can be produced simultaneously for IgM and IgD by utilizing a transcript with the C µ region for IgM production and the C δ region for IgD production. Following the initial DNA re arrangement recombining VH. switch sites (S-sites) in front of the C µ and C α segments (Figure 6. Cγ1.4. DH and JH region a B cell can utilize alternative sites of RNA splicing to simultaneously produce IgM and IgD. the same B cell can further differentiate and switch to producing another heavy chain class. As can be seen from Figure 6. Similar switch sites (not shown in diagram) are found in front of each of the other C regions. This is followed by the ε C region and lastly the α C region. It is important to note that each of these heavy chain classes is associated with the same variable heavy chain region (VH) in a given cell and hence bears the same antigen binding specificity. As described earlier. Cγ2b and Cγ2a. This would necessitate a second DNA rearrangement at the highly homologous switch sites of S µ and S α in front of the C µ and C α regions. Alternatively such a B cell can further differentiate and switch to producing another class of immunoglobulins. J and C segments of the light chain gene. Alternatively. The µ constant (C µ) region is closely associated with the δ constant region (C δ).4. With transcription and RNA splicing all the intervening sequences are . The initial rearrangement of the V-D-J segments in the B cell DNA is similar to the light chain genes and has been described earlier. If RNA splicing occurs at the S µ site. Gene assembly for immunoglobulin class switching. this gene order was similar in principle to the V.Immunoglobulins II: The Genetics of Antibody Diversity 37 IgE. For example a DNA recombination can occur at the switch site (Sα) and bring about a V-D-J-C joining leading to IgA production. Such a B cell would be equipped to secrete both IgM and IgD. there are several VH segments followed by the DH and JH segments.4). The exact mechanism by which the different classes are produced became clearer when the heavy chain gene order was sequenced. Considerable space separates these two genes from the γ constant (C γ) cluster of genes which appear in the order of Cγ3. Studies on the various ‘C’ region segments showed the presence of highly homologous Figure 6. Similar homologous switch sites are found (not shown in this figure) in front of each of the ‘C’ regions.

. since whatever be the heavy chain class of the immunoglobulin produced. to state that one B cell produces one kind of antibody.4). Because similar homologous switch sites are found in front of the γ and ε regions. however. they will all react only to one antigenic determinant. Such a B cell would effectively switch to producing IgA with the same antigen specificity. since they all have similar variable regions and therefore similar antigen binding specificity. alternative production of immunoglobulins of all the above classes is possible.38 Immunology– Introductory Textbook removed yielding an mRNA consisting of V-V-D-J-C α (see Figure 6. It is essentially correct.

There are other non complement enzymes of serum or cellular origin which can activate the complement system midway for example : trypsin . bacteria and viruses to direct mediation of the inflammatory process. capable of interacting with each other. The system consists of at least 20 chemically and immunologically distinct plasma proteins. Once activated the complement components become enzymes and are designated with a bar such as C1 or Factor B . Each component is activated sequentially (similar to the triggered enzyme cascade phenomenon in coagulation). In addition. belonging to the plasma globulin fraction.like enzymes such as plasmin. C2. T Table 7. The two pathways converge at a point from which the final common pathway ensues. IgG Non-immunologic Trypsin-like enzymes DNA C-reactive protein Staphylococcal protein A.1). The individual proteins of the complement system are normally found in the serum as functionally inactive molecules. migration of leucocytes. phagocytosis and release of lysosomes from phagocytes. cobra venom. to terminate at the end point of complement activation which is cytolysis or cytotoxicity. resulting in the complement reaction. with antibody and with cell membranes. There are two parallel but entirely independent pathways leading to the terminal most active part of the complement system (Figure 7. Lipopolysaccharide.1: Activators of the complement system Classical Immunologic Antigen-antibody complexes Alternative IgA.CHAPTER – 7 THE COMPLEMENT SYSTEM he complement system is the primary humoral mediator of antigen antibody reactions. The two pathways are called the classical and the alternative pathways. these interactions lead to the generation of a wide range of biological activity from lysis of different kinds of cells. A complement component can sometimes be cleaved by an enzyme leading to the formation of cleavage products such as C4a and C4b. Following activation of this system. plant and bacterial polysaccharides. Components are designated by numerals C1. C3 etc and by names such as Factor B and Factor D. Trypsin-like enzymes. complement is able to recruit and enlist the participation of other humoral and cellular effector systems and induce histamine release from mast cells. . Each pathway is triggered by different substances.

The Complement Cascade. .40 Immunology– Introductory Textbook Figure 7.1.

2). yielding C4a and C4b (Fig 7. comprise the first functional unit of the complement pathway. each in the form of a triple helix (Figure 7. The polypeptide chains have been visualized as forming 6 sub units. C1r. Here. IgG3 is the most active. IgM is the most efficient immunoglobulin activator of the classical complement pathway. C1q is unique because its structure is very similar. a β globulin molecule. C1r and C1s held together by calcium bonds. to a site located in the CH2 region of the immunoglobulin molecule. by direct proteolytic attack of C1. C1s. The C1q molecule bears the sites which enable the C1 molecule to bind to the Fc region of IgG and IgM antibodies.1). non immunologic substances also activate complement. once activated.2). chemically.The Complement System 41 The Classical Complement Pathway The classical complement pathway is activated as described above (refer Table 7. Figure 7. It is therefore able to bind to 6 IgG molecules (Figure 7. Hence antigen antibody complexes or aggregated immunoglobulins are a must for activation of the classical complement system. Activation occurs by binding of the first complement component C1. following attachment or proteolytic attack. to that of collagen or basement membrane. Once C1s is activated the initial phase of the classical complement pathway is completed.Integrity of the C1 macromolecule and calcium ions are required for this process.3). C1q contains a total of 18 polypeptide chains of 3 distinct types. followed by IgG1 and IgG2. C1 The steps involved in the activation of C1. antibody and C1q or C1r are not necessary for progression of the complement reaction. C4 and C2 C1s cleaves C4. individual proteins of C1 are found only in disease states. activation occurs by direct binding of C1 or in the case of enzymes. Among the IgG sub classes. the earlier reactants including antigen. As listed. Once C1q binds to the immunoglobulin molecule. Activated C1s mediates the next phase of the complement cascade.2. acquires the ability to enzymatically activate C1s. C1 consists of 3 distinct protein molecules: C1q. C4b contains a labile binding site which . that are organized into a structure consisting of 6 peripheral globular portions connected by fibrillar strands to a central structure. (C1s) acquires proteolytic activity. Schematic representation of C1q. C1 is normally present as a complete entity. it also cleaves C2 and initiates the formation of the key enzyme C4b2a on the activator surface.

Schematic structure of C3 and its cleavage products. This enzyme acts to cleave C5.42 Immunology– Introductory Textbook binds to its activator in a transient manner. Figure 7. C4b2a is also termed C3 convertase as it cleaves and thereby activates C3. Magnesium ions are required for the formation of the C4b2a complex. Schematic model of C4: disulphide linkages between the three chains go into forming the C4 molecule. . earlier reactants are no longer necessary. Figure 7.3. C2a also has a labile binding site which allows it to bind to C4b . C3a is a biologically potent peptide and will be discussed later. Once it has been formed. The attachment of C3b to membranes in the vicinity of C4b2a molecules leads to the generation of the last enzyme of the classical pathway: C4b2a3b . The B chain together with the disulphide linked fragments remaining after Factor I cleavage is termed C3c. The enzymatic site resides in the C2 moiety of the complex.4). Activated C4b2a is a proteolytic enzyme that assumes the role of continuing the classical complement reaction. C3 Cleavage of C3 results in the formation of C3a and C3b (Figure 7.4.

which cleave it. H. Water can hydrolize C3 and form C3i. The reactions involved are highly specific and there is considerable turnover of the molecules involved i. however. It is clear. C4 and C5. for example. thus forming C3*. an enzyme C3bBb is generated. because of its major role in the resistance to infection. The Alternative Complement Pathway The alternative complement pathway constitutes the humoral component of natural defence against infections which can operate without antibody participation. This surface bound. several biologically active molecules are generated and it is evident that a relatively small stimulus to complement activation may lead to considerable generation of these molecules with the result that severe tissue reaction can ensue. viruses. I. This results in the accumulation of C3b and as shown in Figure 7. With the addition of factor D. This occurs following cleavage of a thio ester bond in C3. a molecule that functions in a manner similar to C3b. or plant (inulin) polysaccharides. bacterial (LPS). factors I and H. the enzyme zymosan : a yeast polysaccharide also causes C3 activation in the presence of magnesium ions. also known as the priming C3 convertase). yeast (zymosan). This steady level of C3b in the body is greatly modified by particulate activators of the alternative pathway. which acts on bound B to cleave it yielding Bb. by themselves perform the functions of initiation. It is possible that C3 in circulation is also cleaved by enzymes of the coagulation and fibrinolytic systems. there is a cyclical amplification of the critical C3b component. An initial requirement for activation is the presence of C3b. C3. C2. It is not yet known which structures these activators have in common as far as recognition by the pathway is concerned.1). and amplification of the pathway. which is continuously generated. Most of the newly generated C3b remains in the fluid phase. certain mammalian cells. and bypasses the need for C1. such as lipopolysaccharides and certain cells. C3. C4 and C2. and aggregates of immunoglobulins. recognition. which lead to the formation of several more complement enzymes. that recognition involves C3b. The C3b deposited on such activators is “protected” from destruction by factors I and H. Proteins of the alternative complement pathway • • • • P (properdin) – a γ2 globulin factor B – a β2 protein factor D – an α globulin factors I and H – β globulins The alternative complement pathway. D. are also not fully understood. and P. is considered to be the pathway that is more important to man. This surface bound enzyme is also called the amplifying C3 convertase and is able to cleave very large amounts of C3. the Fab portions of IgA or IgE (Table: 7. some binds to various cellular surfaces. which reacts with factors B and D to generate an enzyme able to cleave C3 into C3a and C3b (the C-3 cleaving enzyme. protected C3b interacts with factor B in the presence of magnesium ions to form C3bB. In either case this C3b is rapidly inactivated by control proteins. A variety of activators of this pathway have been described such as certain particulate polysaccharides. for example.The Complement System 43 The classical complement pathway thus consists of a series of enzyme substrate reactions in sequence.5.e. The mechanisms by which these heterogeneous groups of substances can initiate the activation of this pathway. bacteria. Six proteins. In addition. fungi. B. This is also called a positive . The alternative pathway was so named because activation proceeds in a manner different from that of the classical pathway.

which is present in glycoproteins. The activation reaction results . including synthesis of glycoproteins that contain sialic acid. glycolipids and polysaccharides increases the affinity of membrane associated C3b for H.1). Figure 7. Furthermore. Host cells are protected from the effects of complement activation by membrane proteins which prevent activation of classical and alternative pathways. Hence the cyclic amplifying system. The Membrane Attack Mechanism: C5 . The alternative pathway may be activated by an isolated protein obtained from cobra venom. in conjunction with the crucial “protected” surface represents the key events in activation of the alternative pathway. which are able to cleave C5 and initiate the final common membrane attack pathway (see Figure 7. Human parasites such as Leishmania major and Trypanosoma cruzi have evolved several strategies for evading recognition by the alternative pathway. Thus cells that have abundant sialic acid are non activators of the alternative pathway. bind to the surface of the activator particle in close proximity to each other.C9 The terminal portion of the complement sequence is termed the membrane attack system.1).5. This results in the formation of modified enzymes. if properdin (P) were to complex with C3bBb yielding C3bPBb . groups B and C Neisseria meningitidis and K1 Escherichia coli. It has been hypothesized that some bacterial species that carry capsular sialic acid are more pathogenic for neonates than organisms that lack this substance. This protein appears to represent cobra C3b. Cell surface sialic acid.44 Immunology– Introductory Textbook feed back mechanism which enhances C3b formation. The complement attack mechanism is initiated on cleavage of C5 by C4b2a3b . The positive feed back mechanism for C3b. either by C3bBb or C3bPBb . since C5b-9 must become membrane bound in order for membrane changes or damage to occur. rapid degradation of bound C3b and synthesis of a protein that competes with B for binding to C3b. C3b n Bb or C3b n PBb ( n > 1) . the enzyme becomes functionally more efficient. Many of the C3b molecules thus generated. C3 b n Bb or C3b n PBb or certain enzymes such as plasmin (see Figure 7. Bacteria with capsular sialic acid include type III group B Streptococcus.

this process is modulated by S protein which is a normal serum protein. The S protein acts by binding to the membrane at the same binding site as the C5 b67 complex. The Molecular Mechanism of Cytolysis Binding of antibody to a target cell triggers the complement cascade in which successive proteins of the complement system are activated as described in the previous section. The resulting C5b6789 complex. This complex has only transient ability to bind to membranes. represents the fully assembled cytolytic mechanism of the complement system. to the membrane bound C5 b678 complex.greatly enhanced by the attachment of the last complement component: C9. Membrane leakage begins at this stage.however. containing one molecule of C5b.C7.The Complement System 45 in generation of a biologically active peptide: C5a and a larger C5b. Figure 7. C5b has the ability to bind C6 and C7 forming a complex of C5 b67 . The cytolytic process is. . This is called the “one-hit” mechanism of complement mediated cell lysis. Each C5 b67 complex possesses a binding site for the molecule C8.C6. C8 and several C9 proteins then aggregate around the C5b67 complex binding together to form a cylindrical channel or a pore like structure. The mechanism of complement mediated membrane leakage. This permits rapid influx of ions because of a leaky membrane and ultimately leads to cell lysis (Figure 7. Just one such membrane . Eventually C5b binds to C6 and C7 and is inserted into the target cell membrane. However.6.6).C8 and several molecules of C9.attack complex ( or pore channel) of the complement system need be inserted into the target cell to cause cell lysis. thus naturally inhibiting it.

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Immunology– Introductory Textbook

Control Mechanisms of the Complement System
Uncontrolled activation of the complement system is prevented by the fact that active sites of the complement components are labile. In addition, several complexes such as
C3 bBb, C4 b2a and C4 b2a3 b dissociate in a time and temperature dependant fashion.

Other proteins limit runaway complement activation by binding to or enzymatically attacking certain specific complement components. C1 inhibitor is one such protein which inhibits enzymatic activity of C1 and its C1r and C1s subunits by rapidly forming firm, irreversible complexes. Another key control protein of the complement system is Factor I. Factor I attacks C3b free, in solution or on the surface of cells and cleaves the molecule. Another regulator that acts on C3b is Factor H, a serum protein that binds to C3b and accelerates the destruction of C3b by factor I. It also binds to C3b on intermediate complexes and inhibits the enzyme C3 bPBb . A C4 binding protein also exists which binds to C4b and facilitates its destruction by Factor I. A C8 binding protein inhibits binding of C9 to C5 b678 and prevents the membrane attack mechanism from going into operation. Human serum contains an enzyme the anaphylatoxin inactivator, an α globulin, which destroys the biological activities of C3a, C4a and C5a. The S protein binds to the C5 b67 complex and prevents membrane leakage.

Biological Consequences of Complement Activation
Besides the labile binding sites involved in the pathways, sites are generated or uncovered in the fragments of C3 and C4 as a consequence of proteolytic cleavage of the molecules during complement activation. Proteolytic cleavage also results in further breakdown products of the molecules C3 and C4. There are therefore several reactive sites generated in C3b, C4b and in their breakdown fragments. These reactive sites are recognised by various cells having specific receptors for these fragments. Table 7.2 lists the various complement fragments, their receptor specificity and the cells bearing these receptors. TABLE 7.2: SPECIFICITY AND CELLULAR DISTRIBUTION OF COMPLEMENT RECEPTORS
Receptor CR1 Complement fragment C3b Cell type Erythrocytes,PMNs; Blymphocytes; subsets of Tlymphocytes; monocytes; macrophages; dendritic cells B-lymphocytes PMNs, monocytes, macrophages PMNs, monocytes PMNs, monocytes, macrophages, mast cells

CR2 CR3 CR4 C3a and C5a

C3d C3bi C3d C3a, 4a, 5a

The Complement System

47

Results of Complement–Receptor Interactions
The major complement receptors are: (i) CR1 preferentially binds the initial C3 activation product C3b. These receptors are found on many cell types as listed in Table 7.3. The activator particle bearing the antibody-C3b complex is therefore attached to various cell types by these CR1 receptor molecules. The immune complex forms a bridge between the activator and the cell type. When the cell type involved is the erythrocyte (see Figure 7.7), there is an immune red cell adherence around the activator-antibody-C3b complex. This helps to regulate complement activation and aids in the clearance of immune complexes, as red cells “carry” these complexes to the liver for degradation. On certain lymphoid cells and phagocytic cells the receptors help to link the activator particle via the antibody-C3b link to phagocytic cells. This helps to augment phagocytic destruction of an activator particle that is coated (opsonized) by the antibody- C3b complex (Figure 7.7).

Figure 7.7. Biologic consequences of complement-receptor interactions

(ii) The receptor known as CR2, binds the terminal C3 cleavage products i.e. C3d.This receptor is confined to B lymphocytes. CR2 has also been shown to be the structure used by the Epstein-Barr virus to attach to and infect B cells. CR2 plays an important role in the presentation of antigen to specific B and T cells and in the control of B-cell proliferation. Evidence clearly suggests that CR2 is involved in the induction of a primary humoral response. (iii) The CR3 receptor binds the intermediate complement C3 cleavage product, C3bi. Interactions with C3bi and receptors on phagolytic cells enhance antibody dependant phagocytic responses, leading to destruction of the activator. Individuals genetically lacking CR3 are predisposed to recurrent life threatening bacterial infections. (iv) CR4 is a C3 receptor that binds C3bi and C3d. CR4 has an important role in host resistance to infection. C3bi-coated immune complexes have a high affinity for the

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CR4 receptors on phagocytic cells of the liver and spleen, to where they are transported and degraded. (v) Receptors for C3a and C5a bind to anaphylatoxins (see below).

Biologic Actions of Complement Cleavage Products
The low molecular weight fragments of C3, C4 and C5 - C3a, C4a and C5a respectively are known as anaphylatoxins. These hormone like peptides induce smooth muscle contraction, enhance vascular permeability, release vaso active amines from mast cells and basophils and induce lysosomal enzyme release from granulocytes. In addition, C5a is also chemotactic ie: it is able to induce the migration of leukocytes into an area of complement activation. The C5a molecule has a number of other properties, which include granulocyte aggregation, and activation of intracellular processes in certain cells, leading to various effects such as release of oxygen metabolites and SRS-A (slow reacting substance of anaphylaxis). Histamine induced effects of C3a, C4a and C5a are blocked by anti histamines. There are other biologic consequences of the complement activation system. These include the generation of a kinin, possibly a fragment of C2, which causes smooth muscle contraction and increased vascular permeability. This kinin does not function through release of histamine.

Biologic Significance of the Complement System
The sum total effect of the integrated complement system is that it is able to produce inflammation and facilitate the localization of an infective agent. (Figure 7.8 ). The C3b, C4b receptors on phagocytic cells help link phagocytic cells to activator particles that are coated with antibody and C3b or C3b alone. This coating and facilitation of phagocytosis has been called opsonization. The term opsonin is derived from the greek word “opsons” meaning to prepare food for: (phagocytes).

Figure 7.8. Biological effects of complement mediated reactions

The Complement System

49

A classic description of opsonin was given in 1906, by George Bernard Shaw, in his play “The Doctors Dilemma”. The lead character Sir Colenso Ridgeon was closely patterned after the British scientist Almroth Wright. “The phagocytes” Ridgeon says in the play, “won’t eat the microbes unless the microbes are nicely buttered for them. Well the patient manufactures the butter for himself alright, that butter, I call opsonin .” There are two principal classes of opsonin: specific serum antibody and complement component fragments. Other biologic effects such as smooth muscle contraction, vascular permeability and chemotaxis of leukocytes facilitate the acute inflammatory process. Evidence for the biologic importance of this system can be obtained from human and animal complement deficiency states. There is a marked increase in susceptibility to infection. Infections and other human disorders associated with congenital complement deficiencies are given in Table 7.3.

Table 7.3: Human disorders associated with congenital complement deficiencies
C1q C1r C1s & C4 C2 C3 C5 C6 C7 C1 inhibitor C3b inhibitor Systemic lupus erythematosus(SLE) or similar syndrome, hypogammaglobulinaemia, nephritis Renal disease, SLE or similar syndrome, recurrent infections, rheumatoid disease SLE Arthralgia, SLE or similar syndrome, nephritis, susceptibility to infection Recurrent infections with pyogenic bacteria SLE, recurrent infections, recurrent gonococccal infections Recurrent gonococcal and meningococcal infections and Raynaud’s phenomenon Recurrent gonococccal and meningococcal infections, SLE Hereditary angioedema Repeated infections, recurrent infections with pyogenic bacteria

maximal precipitation is formed in the zone of equivalence. For ease of description these antigen antibody reactions have been broadly classified into: • Precipitation • Agglutination • Complement fixation • Immuno assay using labelled reagents • Immuno histochemistry (Immunofluorescence) • Cytokine immunoassays (ELISPOTR ) • DNA innunoassays B Precipitation The classical precipitation reaction was first demonstrated by immunodiffusion in gel. is a term which is used to describe suboptimal precipitation or incomplete lattice formation which occurs in the region of antibody excess. It detects the reaction between soluble antigen and a potent antiserum mixed in the correct proportions. .CHAPTER – 8 DETECTION AND APPLICATION OF ANTIGEN–ANTIBODY REACTIONS asic knowledge regarding the molecular basis of antigen and antibody structure and function. A similar phenomenon occurs in the region of antigen excess and is sometimes called the post zone effect.2). The antigen-antibody precipitate that is so formed in any semi solid medium such as agar or agarose is also dependent on buffer electrolytes. This relationship is depicted schematically in Figure 8. pH and temperature. The prozone phenomenon (Figure 8. Figure 8. Much of this chapter will be devoted to the dynamics and clinical laboratory applications of antigen-antibody interactions. The zone of equivalence in antigen antibody reactions: where maximal precipitate is formed.1.1. will eventually influence diagnosis and therapeutics in clinical medicine. not in the zones of antigen or antibody excess. The formation of precipitation lines in any immunodiffusion system is highly dependent on relative concentrations of antigen and antibody.

Double diffusion can also be performed between one or more antigens and a single antibody for comparative purposes. Extreme antibody excess (left) or antigen excess (right). The three basic characteristic patterns of such reactions are shown in Figure 8. In single agar gel diffusion either antigen or antibody remains fixed and the other reactant is allowed to diffuse freely in the gel. however.3. It is still an important initial test for the detection of serologic reactions to various human. Double diffusion in agar can also be used for semi-quantification of antigen or antibody reactants as shown in Figure 8.2. The optimal ratio of antigen to antibody (middle) yields an insoluble precipitate in the classic precipitation test. Samples containing antigen and antibody are placed in opposing wells and allowed to diffuse toward one another in a moist chamber for 18. Gels can be dehydrated and stained by protein binding dyes like Coomassie brilliant blue and preserved indefinitely. The most important clinical application of immunodiffusion is the quantification of serum immunoglobulins. In double immuno diffusion both reactants are free to move towards each other and precipitate.4. Agar-gel diffusion. This method is still widely used today to detect and analyze antigen-antibody reactions. Small wells are punched out of the agar. Figure 8. . This important advance was soon followed by Ouchterlony’s classic description of double diffusion in agar layered on slides. which can be viewed by indirect light or with a magnifying lens (Figure 8. (i) Ouchterlony’s Double Diffusion This test is performed by pouring molten agar onto glass slides or into Petri dishes and allowing it to harden. Agar gel diffusion reactions may be classified as single or double.24 hours.Detection and Application of Antigen–Antibody Reactions 51 Figure 8. Two arcs from each well approximate with each other to form a line of precipitation. Antigen and antibody diffuse out of the wells in a radial fashion. a few millimetres apart. Movement within the gel may be linear or radial. does not lead to lattice formation. animal and plant antigens.3).5. Immunodiffusion or Agar Gel Diffusion Agar gel diffusion is the simplest and most direct means of demonstrating soluble antigenantibody reactions in the laboratory. In 1946 Oudin described a system of single diffusion of antigen and antibody in agar filled tubes.

Precipitin lines form with decreasing thickness until no longer visible at an antigen dilution of 1:32. antigen and antibody precipitate to form a ring. semiquantitative analysis of antigen. Radial diffusion is based on the principle that a quantitative relationship exists between the amount of antigen placed in a well (where the antibody is incorporated into the gel) and the resultant ring of precipitation (Figure 8. Precipitin lines completely cross owing to two separate interactions: R with αR and S with αS. Reaction patterns with double diffusion in agar R=antigen-R.4. An important application of this test is in the detection and semi-quantification of many antigens. The lines meet at a point.6). a characteristic spur is formed. αS = antibody to S Reaction of identity : Precisely similar precipitin lines have formed in the reaction between R and anti R. The area within the precipitin ring is proportional to the antigen concentration. In 1965.reacting antigens. Antigen x is serially diluted and placed in a ring of wells surrounding a central well containing antibody to x. Figure 8. the precipitin lines do not cross. showing that R and S are dissimilar and non cross. Mancini introduced a single diffusion technique for quantitative estimation of antigens. . Antibody is added to the agar which is then poured onto a slide or petri dish. showing that the two antigens are similar.52 Immunology– Introductory Textbook Figure 8.5. αR = antibody to R. Figure 8. when the point of equivalence is reached. The plates are left for 24 hours during which time antigen diffuses out of the wells and binds to antibody. (ii) Single radial immunodiffusion The double immunodiffusion is only semi quantitative. Reaction of partial identity: αR reacts with both R and R1. S=antigen-S. Single radial immuno diffusion. Wells are punched in the agar and dilutions of test antigen-S are put in the wells. R1=antigenR1.6. indicating that antigenic determinants are partially shared R and R1. Reaction of non-identity. Semi-quantitative analysis using double immunodiffusion. The whole process can be reversed to quantitate antibody.

paper. the monoclonal nature of Bence-Jones proteins in myeloma can be confirmed. allowing for separation of individual constituents. shows separation of normal human serum into 5 major electrophoretic bands: albumin. hypo or agammaglobulinemia and identification of L chains in the urine of patients with plasma cell dyscrasias or autoimmune disorders. (2) Antigens are separated by electrophoresis and the pH of the gel is chosen so that positively charged proteins move to the negative electrode and negatively charged proteins move to the positive electrode. Immunoelectrophoresis is also helpful in identifying increased amounts of proteins present in the cerebro spinal fluid in patients with various neurologic diseases. the trough is filled with antiserum which is left to diffuse.7. Human Figure 8. smaller molecular weight fragments traverse further.7. Large molecular weight substances move slowly and traverse a shorter distance in the given time period. Electrophoretic separation of normal human serum. Technique of immunoelectrophoresis : (1) Agar gel slab is prepared. agarose or cellulose acetate strips are used for this purpose. Immunoelectrophoresis permits the comparison of complicated mixtures of antigens found in human serum. interact and form precipitin arcs. Zone electrophoresis is extremely valuable in the diagnosis of human paraprotein disorders and disorders of the γ globulin fraction of serum such as hypo or agammaglobulinemia. Both identification and approximate quantification can thereby be accomplished for individual proteins present in serum. (4) The separated antigens and the antibodies in the trough diffuse. Thus with specific anti-κ or anti-λ antisera. The technique is illustrated in Figure 8. Technically. α1 globulin. Densitometer scanning converts the separated bands into peaks. Generally. .8. The medium in which this occurs is generally one that does not impede or enhance the flow of molecules in an electrical field. α 2 globulin. (3) After antigen separation. The applications of immunoelectrophoresis include diagnosis of paraproteinemias. Immunoelectrophoresis (IEP) Immunoelectrophoresis combines electrophoretic separation and immunoprecipitation of proteins. Figure 8. Figure 8. β globulin and γ globulin. the biologic fluid is spotted at the origin and subjected to an electrical current. urine or other biologic fluids. immunoglobulins fall into the γ globulin fraction of human serum. a trough and well are cut and the well is filled with antigen.Detection and Application of Antigen–Antibody Reactions 53 Electrophoresis Simple zone electrophoresis uses the principle of separation of individual proteins ( in a complex protein mixture such as human serum) in an electrical field.8. using human serum as antigen and antiserum raised against whole human serum as antibody.

(i) Counter immunoelectrophoresis The basic principle involves. Antigen and antibody are placed in wells and driven towards each other by electric current. Two variations of electroimmunodiffusion have received clinical applicability. (ii) Laurell’s rocket electrophoresis In this technique antiserum to the particular antigen or antigens is incorporated into an agarose medium and poured over a glass slide. However. the γ globulin fraction which contains all the immunoglobulins is driven in the opposite direction by electro endosmosis.54 Electroimmunodiffusion Immunology– Introductory Textbook In imm unodiffusion techniques described this far. as before. where antigen and antibody meet. a precipitin line forms in the zone of equivalence. This ensures that antibody does not migrate. Electrophoresis of the antigen into the antibody containing agarose is then performed. The slide is then placed in an electrophoresis tank and bathed in buffer solutions of optimal pH and connected to a power pack supplying the electrical current. As shown in Figure 8. This technique can produce visible precipitin lines within 30 minutes and is 10 times more sensitive than agar gel diffusion. This drives antibody towards the antigen and precipitin lines may be seen to be formed a few hours after beginning of electrophoresis. animal and human immunology laboratories for the detection of normal and abnormal proteins. The proteins in the given antiserum well also move towards the positive pole. They are (i) Counter immunoelectrophoresis (CIE) and (ii) Laurell’s rocket electrophoresis. γ globulins being relatively low molecular weight substances are hence carried along by electro endosmosis towards the antigen. However. pouring molten agar on a slide and punching wells into it a few millimetres apart. antigen and antibody are allowed to come into contact and to precipitate in agar purely by diffusion. it is only semi quantitative. Counter immuno electrophoresis. Figure 8.10). The specimen containing the unknown antigen is placed in a small well. Antigen and antibody are placed in the opposing wells.9. The system can be reversed to detect antibody in the clinical sample. In a clinical condition the biologic fluid is placed in one well and known antibody in the other. Electro endosmosis is a phenomenon in which molecules within the agar gel tend to move in the direction opposite to the flow of current when in an electrical field. facilitating the antigen. The resultant pattern of immuno-precipitation resembles a spike or rocket – hence the term rocket electrophoresis (Figure 8.antibody reaction. However. the chance of an antigen and antibody meeting and the speed of development of a precipitation line can be greatly enhanced by electrically driving the two together. CIE is widely used in many plant. the antigen solution being a protein moves towards the positive pole.9. This detects antigen in the clinical material. once the gel has solidified. .

Electrophoresis is performed and antigen is driven into the antibody containing gel.” Amount of antigen is directly proportional to height of the rocket. In this way each of several antigens in a mixture can be quantified (Figure 8. The principal application of this technique is to quantify antigens in biological fluids.11). as the antigen is driven into the agar containing the antibody. The technique is used in many immunology laboratories for the detection of antigens in human and animal biological fluids. The area under the peak is related to the concentration of antigen. (iii) Crossed immunoelectrophoresis One powerful variant of Laurell’s rocket system. Precipitin pattern forms in the shape of a “rocket. The rocket pattern occurs because precipitation occurs along the lateral margins of the moving boundary of antigen.Detection and Application of Antigen–Antibody Reactions 55 Figure 8. Gradually as the antigen is lost through precipitation its concentration at the leading edge diminishes and the lateral margins converge to form a sharp point. Antigen and antibody complex to form precipitin peaks. (a) Antigens are separated on the basis of electrophoretic mobility in agar gel (dotted vertical bands). laid over another gel.10. This system cannot be used to quantify immunoglobulins because their weak negative charge prevents their electrophoretic mobility in this system. which contains antibody and electrophoresis carried out in a direction at right angles to the first run to drive the antigen bands through the antibody containing gel. (b) A narrow longitudinal strip (dashed lines) containing the separated antigens is cut out as shown. The total distance of antigen migration (length of spike) for a given antiserum concentration is linearly proportionate to the antigen concentration. the crossed immunoelectrophoresis involves a preliminary electrophoretic separation of an antigen mixture in a direction perpendicular to that of the final “rocket stage”.11. Rocket electrophoresis (Laurells’ technique). Figure 8. . Varying concentrations of the antigen are placed in wells 1-4.

makes it a far more effective agglutinating agent. it is often coated onto a carrier particle. An example is the agglutination of group A red cells by anti-A sera in a simple slide agglutination test. Treponema pallidum haemagglutination (TPHA) for the diagnosis of syphilis is a widely used test that exploits this principle. serially diluted in test tubes. The diluents used are usually normal saline or any other suitable buffer solution. for most tests except for cold reacting antibodies or cold agglutinins which need refrigeration. Standardized amounts of known antigen are then added to all the tubes. thus rendering it particulate. Many antigens will spontaneously couple with red cells and form stable reagents for antibody detection. 1 in 20. (ii) Indirect/ passive agglutination When a soluble antigen needs to be used in an agglutination reaction. Tube agglutination reactions are used to detect specific antibody (in human or animal serum) in the presence of a constant amount of antigen.. agglutination is complete and tubes are examined for clumping. Coupling agents vary widely. Agglutination reactions may be classified as either direct or indirect (passive). may be used. to name just three. The agglutination of cells bearing only a small number of surface determinants may therefore be difficult to achieve. Important clinical applications of this technique are the Widal test for diagnosis of typhoid fever. laboratory medicine. alternatively dilutions such as 1 in 10. Besides these there are several modifications of the agglutination principle. 1 in 40 etc. the Brucella agglutination test for Brucellosis and the Weil Felix test for Rickettsiosis. the highest dilution of the test serum at which a positive agglutination reaction occurs. using two fold dilutions such as 1 in 2. a good example is tannic acid which increases the amount of most protein antigens coupled onto red cells. Similarly the higher avidity of multivalent IgM antibody relative to IgG..56 Agglutination Immunology– Introductory Textbook Whereas cross linking of multivalent protein antigens of a soluble nature leads to precipitation. Classically. cross-linking of cells or large particles by antibody directed against surface antigens leads to agglutination. what this means is that 1 part of serum is mixed with 1 part of a diluent in a 1 in 2 dilution. have a high degree of sensitivity and an enormous variety of substances are detectable through this method. a reasonable number of antibody links between two cells is required before the mutual repulsion is overcome. however. 1 in 4 or 1 in 8 etc.the serum to be tested is initially. After a few hours of incubation. a cell or insoluble particle is agglutinated directly by antibody.. A constant amount of coupled antigen is then added to each serum dilution and . Agglutination techniques (i) Direct agglutination In this simple direct technique. which have been used successfully in clinical. Essentially. In this way several species of bacteria (grown on culture) can be directly agglutinated and thus definitively diagnosed by specific antibody using a slide agglutination reaction. Since most cells are electrically charged. Agglutination tests are performed in tubes or microtiter plates which hold serially diluted serum samples. Soluble antigens can be passively adsorbed or chemically coupled to red blood cells of various species. in which case the test is called an indirect (passive) haemagglutination test (IHA or PHA). or one part of serum is mixed with 3 parts of diluent in a 1 in 4 dilution and so on. The tubes are incubated at suitable temperature (usually 37 C).e. The results are usually expressed as a titer i. Important advantages of agglutination are that reactions can be read visually.

This test is called the Rose-Waaler test. Tanned sheep red cells are coated (sensitized) with the amboceptor in sub agglutinating titers.Detection and Application of Antigen–Antibody Reactions 57 incubated. Thus viral haemagglutination can be used to detect presence of virus.03 µgs/ml. a negative reaction is evidenced by settling of red cells at the bottom of the tube or well as a button (Figure 8. a positive reaction is seen as clumping of red cells. Before interpreting the test reading it is important to ascertain that the given patient’s serum does not react with normal unsensitized sheep cells. Passive haemagglutination with protein sensitized cells can detect antibody at concentrations as low as 0. This occurs by virtue of the fact that rabbit IgG and human 7S IgG cross react (Figure 8. The latex agglutination test is widely used for detection of Rheumatoid factor. Among the commonest ones used are inert particles like latex in the latex agglutination (fixation) test or staphyloccal protien A (Figure 8. Since the reactants are reversed the test is sometimes called reversed passive haemagglutination (RPHA). These sensitized sheep cells are allowed to react with serial dilutions of human serum. when mixed with serum of patient with rheumatoid arthritis. A passive haemagglutination test is also used to detect rheumatoid factor. other than red blood cells.14). (iii) Haemagglutination inhibition Another category of agglutination involves spontaneous agglutination of red cells by certain viruses. Indirect (passive) haemagglutination (a) red cells are coated with antigen (b) specific antibody causes indirect (passive) haemagglutination. have been widely used in serology for the demonstration of agglutinating antibody.12.13). A modification of the above technique that is commonly used involves coupling of antibody onto red cells so as to detect antigen in the patient’s sample. the haemagglutination inhibition reaction can be used to detect the presence of . causing non specific clumping. alternatively. to cause clumping of red cells. This antiserum is also known as amboceptor. If latex particles are adsorbed with 7S IgG. Rheumatoid factor is a 19S IgM antibody (an auto antibody) directed against the patient’s own 7S IgG. Rheumatoid factor in human serum will react with rabbit anti sheep IgG coated onto the sheep red cells. After a few hours of incubation.12). the 19S IgM in these sera react with 7S IgG coated onto latex particles and cause visible clumping of the particles.Human rheumatoid factor (IgM) has been shown to react against the rabbit IgG raised against sheep red cells. Figure 8. Antibodies (IgG) against sheep red cells are raised in rabbits. This viral haemagglutination reaction can be specifically inhibited in the presence of antiviral antibody. Passive carriers of antigen.

the haemagglutinating sites on the viral particle thus lie complexed to antibody and are no longer available for reaction with red cells. an anti human immunoglobulin is added which is directed against the “incomplete” antibody.13.aureus binds avidly to the Fc portion of the IgG molecule. Principle of the Rose-Waaler test a) Sheep red cells sensitized with rabbit IgG raised against the sheep red cells (sub agglutinating doses) b) Human IgM (rheumatoid factor) reacts with rabbit IgG causing agglutination of red cells.as the two Fab arms are not long enough to bridge determinants on apposing red cells. (b) The Fab portion of the bound IgG complexes with antigen resulting in clumping of S. This happens because viral antigen reacts with antiviral antibody in patient’s serum.14. These antibodies are sometimes called “incomplete” or “blocking” antibodies.58 Immunology– Introductory Textbook specific antiviral antibody in patient’s serum. Figure 8.15 b). Co-agglutination (a) Protein A containing S. Coomb’s Test In some cases a short antibody molecule directed against a deeply located membrane antigenic determinant cannot agglutinate (Figure 8.15 a). A good example of such a determinant is the Rh antigenic determinant on red cells. To detect the presence of anti Rh antibodies. . Figure 8. already fixed to red cells.aureus cells. The addition of this second antibody leads to agglutination (Figure 8.

The infant is thus at risk of developing haemolytic disease of the newborn. is done by adding the sheep cell-amboceptor mixture (also known as sensitised sheep red cells). When added together. If antigen and antibody are specific for each other they will complex and utilise the complement as well. (ii) The indirect Coomb’s test is a 2 stage reaction for detection of circulating incomplete antibodies against the Rh determinant. The Coomb’s test is performed in two ways: (i) The direct Coomb’s test detects immunoglobulin already present on the red cell by addition of the antiglobulin (as described above).16). After a period of incubation the test is read looking specifically for the presence and degree of red cell lysis. Thus the consumption of complement in vitro. antigens or both. Complement Fixation Test The fixation of complement occurs during the interaction of antigen and antibody. such as the Rh positive infant born to an Rh negative mother. The patients serum is first incubated with red cells possessing the Rh determinant. can be used as a test to detect and measure antibodies. which are usually present in the mother’s serum after the birth of an Rh positive infant. The actual test is done in two stages: the first stage consists of adding the test reagents which are antigen and antibody (normally the biologic fluid such as human serum) and a standardized amount complement. The direct Coomb’s test is positive in an already sensitized individual. the antiglobulin is added and the appearance of agglutination indicates the presence of anti Rh antibodies in maternal serum. The second stage of the test.Detection and Application of Antigen–Antibody Reactions 59 Figure 8. amboceptor (antibody to sheep red cells) and complement. consisting of sheep red cells. The incomplete antibodies react with the Rh determinants and coat the red cells. The Coomb’s test is also used in the diagnosis of auto immune haemolytic anaemia. sheep red cell antigen complexes with antibody. Such an infant’s red cells are coated with anti Rh antibody produced by the Rh negative mother against her own baby’s Rh positive red cells. . The test depends on the use of a haemolytic indicator system. Once coating has taken place. The Coomb’s test (a) red cells are sensitized with “incomplete antibodies” (b) Antiantibody reacts with the “incomplete antibody” causing agglutination of red cells.15. utilizes complement and resultant red cell lysis occurs (Figure 8.

16. also known as binder-ligand assays. The Complement fixation test. Complement fixation tests have received widespread use in both clinical and research laboratories. Complement was hence freely available for utilisation by the haemolytic indicator system. therefore did not utilise complement. Immunoassay Using Labelled Reagents One of the most important analytic methods developed in recent years is the immunoassay of antigen and antibody using labelled reagents. Complement was hence not available to the haemolytic indicator system and sheep red cell lysis did not occur (Figure 8.16). Interpretation Occurrence of lysis indicates: test antigen and antibody in stage one did not complex. Results are shown as the highest serum dilution showing a positive reaction.60 Immunology– Introductory Textbook Figure 8. with resultant red cell lysis. The absence of lysis indicates: a positive test reaction since antigen was complexed to antibody (present in patient’s serum) and utilised most or all of complement. . though they have now been replaced by less cumbersome immunoassay tests. Lysis therefore indicates a negative test reaction since the patient’s serum did not contain the antibody in question.

Standard curve for hormone assays by RIA. This standardization procedure is described below: (i) For estimation of the hormone X (for e. Radio immunoassay Radio immunoassays has been used to quantify hormones such as insulin. In the past two decades ligand assays have revolutionized the quantification of hormones. (iv) A range of known concentrations of unlabelled pure X is added simultaneously with the titrated X* to the antibody and the mixture is incubated. (iii) The concentration of labelled hormone i.17). .18). thyroxine etc. This is done by using antibodies raised in rabbits or guinea pigs to the concerned hormones. Figure 8. the concentration of the hormone X in the patients sample can be ascertained. Using the graph (Figure 8. drugs. hence the sample containing X needs to be diluted so as to give a reading within this range (Figure 8. An initial step in the use of the RIA is to plot a standard graph for each hormone tested. X* is titrated so as to react with 70% of binding sites when added to anti X. The chief goal of an assay is to determine the concentration of some molecule of interest .17. The percentage of X bound to antibody is estimated in the presence of the clinical specimen.e. tumour markers. (vi) From the amount of bound X* measured out at various X concentrations a curve is constructed.(iv) with the patient’s sample. the experiment is repeated replacing X in the above step no.g. After the standardization is achieved.. The curve is linear over a relatively limited range.Detection and Application of Antigen–Antibody Reactions 61 The first ligand assay method to be developed was the radio immunoassay (RIA). which was introduced to detect human insulin using anti insulin antibodies.thyroxine) antiserum to X is raised in rabbit or guinea pig. in plasma. allergens and a variety of antigens and antibodies associated with infectious agents. The method was described by Berson & Yalow and in 1977.. Yalow was awarded the Nobel prize for her contributions to the field of binder-ligand assays. (v) Labelled X* + antibody is separated from free X by the addition of an anti-antibody. (ii) Pure hormone X is obtained and radio labelled to yield X*.the analyte. be it an antigen or an antibody.

is in the well coupled to anti IgE. The radio-immunosorbent test (RIST) detects total IgE in patients serum.18. This is treated with the patients serum.62 Immunology– Introductory Textbook Figure 8. . The bound IgE is then measured by addition of labelled anti human IgE raised in yet another animal. The specific allergen (eg: pollen extract) is coupled to a paper disc in a microtiter well. Solid phase radio immunoassay Solid phase radio immunoassays may be used to detect both antigen and antibody.carbonate plate with multiple wells. Anti human IgE raised in rabbits in used to coat the well (Figure 8. The radio allergo sorbent test (RAST) measures the amount of IgE to specific antigens (allergens) in the patient’s serum. The test is usually done in a microtiter poly . Figure 8.19. or is coupled via cyanogen bromide to micro-cellulose discs.18). washed and bound human IgE detected with labelled antihuman IgE similar to the procedure used for the RIST (Figure 8. A good example to illustrate this is the radio immunosorbent test (RIST) which detects total IgE in a patient’s serum. Measurements of radioactivity in the well are then recorded and quantified using a geiger counter. The solid phase is blocked with non-specific protein to decrease non-specific binding. Dilutions of the patient’s serum are added to the coated well. The radio allergosorbent (RAST) test to detect specific IgE. Excess serum is washed off ensuring that human IgE alone.19).

the problems of safe disposal and the prohibitive cost of reagents and equipment. The basic principle is very similar to the solid phase radi o immunoassay (Figure 8. Coat plate with antigen Wash Add test seru m (contains antibody) Wash Add enzyme labelled antihuman antibody Wash Add substratechromogen complex Measure degree of colour change Solid phase <J> Antigen <J>~Test ant ibody <J>: ~ <J> ~ ~ ~~ A. (a) The enzyme linked immunosorbent assay (ELISA) to detect antibody (b) The ELISA to detect antigen. Furthermore.p. a. Radiolabelled reagents have now been largely replaced by enzyme.20./-- ~ ~ 2n d antibody conjugat ed to an enzyme Colour change chromogen In Add substratechromogen complex Measure degree of colour change ~~~ Figure 8.labelled antibodies. the reaction can be quantified by measuring the density of colour using a simple colourimeter. Sensitize plate with capture antibody to a specific antigen Wash Add patients sample (contains antigen) Wash Add enzyme .e "libo'. ~ ~~ Antigen in que st ion Solid phase L< -----. .labelled 2nd antibody Wash ~c. ~~ COIO". Co lou r changes •• com pl ex is cleaved by enzym e b.".20 a and b).hom" 00 conjugat ed to an enzym e "libod.le"_ chromogen sub strate ~ ~:.'.Detection and Application of Antigen–Antibody Reactions 63 Enzyme labelled immunosorbent assays (ELISA) The use of radio labelled reagents carries with it the hazards of radioactivity. which when exposed to coloured substrate complexes release the colour.

Table 8.2). The ELISA and its many variations are widely used in clinical immunology (Table 8.2: Applications of the ELISA For antigen detection Hepatitis B surface antigen (HBsAg) HBeAg For antibody detection Anti Hbs Anti Hbc Antibodies to toxoplasma. This is called the signal antibody. The disposable device consists of chromatographic membrane with a liquid-phase antibody conjugated to a coloured substrate (eg: conjugated mouse monoclonal antibody) to the antigen to be detected.1. These assays have been shown to be several hundredfold more sensitive than the RIA. and can therefore be used to detect very small quantities of antigen or antibody. These tests use the principle of immunochromatography. Patient sample (stool extract for example) is introduced into the device and incubated at room . this forms a firm complex which is detected using a substrate-colour compound as in conventional ELISAs.64 Immunology– Introductory Textbook Commonly used enzyme labelled antibodies and their respective colour substrate complexes are given in Table 8. IgM.21). Table 8. The variation involves biotinylating the second antibody after which avidin conjugated to an enzyme is added. while the two solid-phase antibodies are a polyclonal antibody to antigen and a polyclonal antibody to mouse immunoglobulin in two stationary antibody zones (Figure:8. Antibodies rubella and other viruses Antibodies to HIV 1 and HIV 2 Immunochromatography (Immunocard tests) Immunoassays have been used in a variety of commercially available immunocard tests. IgA or IgE) can also be detected using the appropriate second antibody.1: Enzyme labelled antibodies with respective substrate complexes Antibodies conjugated to Horse radish peroxidase Alkaline phophatase β– galactosidase Substrates hydrogen peroxide + ortho phenylene diamine p–nitrophenyl phosphate o–nitro–phenyl–β–d– gal actopyranoside Biotin / avidin enhanced immuno assays A variation of the ELISA is a system which uses the strong affinity of compounds like biotin and avidin for each other. Class specific antibodies (IgG.

Detection and Application of Antigen–Antibody Reactions 65 temperature. The amplified gene product (by prior PCR) is added to the plate and the duplex DNA detected with an enzyme linked antibody against double stranded DNA. pylori). only one coloured line develops. sodium dodecyl sulphate (SDS) poly .21. the antigen mixture is subjected to electrophoretic separation in a gel using. for example. The specimen migrates via capillary action along the membrane until it reaches the first stationary specific monoclonal antibody to the antigen in question. Colour must always be seen at the control line. Helicobacter pylori faecal antigen tests. darker colour is strongly positive. Immuno blotting (Western blots) Western blots are used to detect antibody targeted to individual antigenic determinants in a crude whole cell antigen mixture. In the absence of antigen in the patient’s sample. producing a separate. two coloured lines on the test stick indicate the presence of antigen. as it migrates. Immunochromatography. Figure 8. Initially. If no colour is seen. Antigen binds to the stationary antibody. Reaction takes place and a coloured line appears. the test has failed and must be repeated. second coloured line. DNA immunoassays DNA enzyme immunoassays have been developed to detect a variety of micro organisms. Results are read by comparison of the intensity of colour at the test line with the control line – equivalent colour is positive. Thus. as a result of the reaction between the signal antibody and the antibody to mouse immunoglobulin. also dissolves the embedded signal antibody. These PCR-ELISAs are becoming very popular diagnostic tools for a variety of micro organisms where it is important to detect the specific pathogenic or antibiotic resistant gene in the organism. Commercial kits often use streptavidin coated microwell plates to which is added a biotinylated specific probe based on the gene to be detected (for eg you may want to detect the UreC gene of H. These tests are now widely available for diagnosis of Clostridium difficile toxins A and B. The excess signal antibody which does not bind to antigen migrates further until it reacts with the polyclonal antibody to mouse immunoglobulin. pregnancy tests and many other antigen detection tests. the signal antibody also binds to antigen. The specimen.

23a). when antibodies are allowed to bind to individual protein antigens. Colour changes indicate that antibodies have adhered to the proteins. Figure 8. much like the system used in an ELISA or RIA (Figure 8. In the direct immunofluorescent test the antibody to the tissue substrate is itself conjugated with the fluorescent dye and applied to cells or tissues fixed on a slide. where they bind nonspecifically. The separated protein bands are then transferred or blotted onto nitro-cellulose sheets by transverse electrophoresis. Such conjugates can combine with antigen present in a tissue section and the bound antibody can be visualized by means of a fluorescence microscope.22. In this way the distribution of antigen throughout a tissue and within cells can be demonstrated. Immunohistochemical techniques Immunofluorescence is essentially a histochemical or cytochemical technique for detection and localization of antigens. An immunoblot is considered positive if two or more proteins react.22). These nitro-cellulose sheets are then incubated with the patient’s serum. serum is incubated on nitrocellulose strips on which four recombinant viral proteins are blotted. Immunofluorescence tests can be either direct or indirect (Figure 8. Alternatively.66 Immunology– Introductory Textbook acrylamide gel electrophoresis (SDS -PAGE). antigens can be spotted onto a slide and detected by direct fluorescent labelled antibody (Figure 8.23 a and b). Fluorescent dyes such as fluorescein and rhodamine can be coupled to antibodies without destroying their specificity. The recombinant immunoblot assay (RIBA) used for the diagnosis of Hepatitis C uses the Western blot principle. Immunoblotting (Western blots). . Bound human antibody is then detected using enzyme labelled or radio labelled antihuman immunoglobulin.

Rapid identification of micro organisms in tissue or culture e.tissue substrate or antigen spotted onto a slide. Cytokine Immunoassays Detecting an immune response to the pathogen has been a successful method of diagnosis of infection for many years. When visualized under a fluorescent microscope. Detection of complement components in tissues. there are a number of limitations to using antibody detection for the diagnosis of acute infection: it is difficult to differentiate past infection from acute current infection. bound. This antibody is then treated with an anti human immunoglobulin labelled to a fluorescent dye.23. . Clinical applications of immunofluorescence are given in Table 8. The basis of immunofluorescence (a) direct immunofluorescence (b) indirect immunofluorescence.Detection and Application of Antigen–Antibody Reactions 67 Figure 8.3. Immunofluorescence techniques can be used to detect both antigen and antibody in clinical material. The unlabelled antibody is applied to the cell preparation. emitting a characteristic colour. However. Detection of specific tissue fixed antibody. Table 8. labelled anti globulin fluoresces.g. Chlamydia trachomatis Identification of tumour specific antigens. A classical example is tuberculosis(TB) where antibody detection has not played a role in diagnosis. Detection of immunoglobulins in tissues..22 b).3: Clinical applications of Immunofluorescence Identification of T and B cells in blood. and sometimes the antibody response is insufficient and cannot be used as a diagnostic method. Identification of transplantation antigens. The indirect immunofluorescence test is a double layered technique (Figure 8.

tuberculosis antigen). The T-spot or ELISPOT technology is a simple method that can be used in routine laboratories to detect pathogen specific T cells in blood. Until recently. T cells have been difficult to detect. So where there are antibodies there must also be activated T cells. The ELISPOT method for tuberculosis is sensitive and specific. wash and add conjugated second antibody IFN–γ Add substrate and count spots by eye or use a reader Each spot is an individual T cell that has released IFN–γ Figure 8.g. control the fight against intra-cellular pathogens such as M. the T lymphocytes produce γ interferon as a result of activation by antigen. tuberculosis. T cells. is able to detect active and latent TB in immunocompetent as well as immunosuppressed individuals and does not cross react with BCG.24). This γ interferon is captured by antibody to γ interferon which is pre-coated in wells of a microtitre plate. HOW THE T SPOTTM TECHNOLOGY WORKS Plasma White cells Gel barrier Crythnocytes and cosmophils Collect white cells using the commercially available tube or with Ficoll extraction Add white cells and TB antigens to wells. By a modification of the ELISA method the captured γ interferon is detected by a second anti-γ interferon antibody conjugated to a suitable substrate-indicator complex (Figure: 8. in particular. Cytokine immunoassays are being developed for a number of other viral and tumour antigens. M. Cytokine immunoassay– ELISPOT® techonology .68 Immunology– Introductory Textbook Our understanding of the immune response mechanisms tells us that antibodies are invariably formed with T cell help. When white cells from the patients blood are exposed to a specific antigen (for e.24. T cells release IFN–γ IFN–γ captured by antibody coated on well Incubate. viruses and tumour antigens.

The positive predictive value (PPV) of the new test is the proportion of individuals showing a positive result with the new test who actually have the disease.Detection and Application of Antigen–Antibody Reactions 69 Predictive Theory and Immunologic Testing When any test is used to make a decision there is some probability of drawing an erroneous conclusion. This divides results into four categories: • true positives (a+c) • true negatives (b+d) • false positives (b) • false negatives (c) Gold Standard test Positive New test Positive Negative a c Negative b d Diagnostic sensitivity of a new test is the proportion of true positives (as defined by a gold standard test) correctly identified by the new test. Specificity = d / b+d. NPV = d / c+d. Sensitivity = a / a+c. Diagnostic specificity is the proportion of true negatives (as defined by a gold standard test) correctly identified by the new test. The negative predictive (NPV) of the new test is the proportion of individuals showing a negative result with the new test who do not actually have the disease. PPV = a / a+b. .

The variability of sub specificities and cross reactivities in polyclonal antisera have plagued immunologists all along and forced them to remove unnecessary antibodies by repeated absorption. Kohler and Milstein shared the Nobel Prize in 1984. certain antisera are rare to come by and reference laboratories face the arduous task of keeping rare reference sera in constant supply.CHAPTER – 9 MONOCLONAL ANTIBODIES or many years antibodies have had a major role in the diagnosis of a wide variety of diseases. Several B cell clones are involved. England showed that cultured mouse myeloma cells could be fused to normal spleen cells of animals immunized with an antigen. in diagnostic laboratories to detect and quantify drugs. the solution grew out of a series of basic and completely unrelated experiments. Since these workers hybridized single cell mixtures. encompassing the whole antigen. thereby depleting the original antibody content and strength of the serum. each producing a slightly different antibody molecule to the myriad antigenic determinants. they concluded that subsequent antibody forming cell lines were clones of (progeny of ) this one cell (mouse myeloma) to one cell (normal spleen cell) hybridization. is termed a polyclonal antiserum. and 10 mgs/ml in serum or ascitic fluid of tumour bearing mice . with Niels Jerne who introduced the theoretical concept of clonality of the immune response. These findings have exceeded the wildest dreams of immunologists and have revolutionized serology. sheep red cells. secreted by many antibody producing B cell clones. a number of laboratories had been attempting to fuse mouse myeloma cells to each other or other cell lines. F The Genesis of Monoclonal Antibodies As it often happens in science. In other cases. mice. For this invaluable contribution to the progress of medicine. tumour antigens and circulating immunoglobulins. These cell lines grew continuously in culture because of the cancerous myeloma hybrid. To examine the genetic control of immunoglobulin production. Immunologic assays are used extensively. This phenomenon is referred to as heterogeneity of the antiserum and occurs even when apparently pure antigen and inbred strains of animals are used. In the course of such studies Georges Kohler and Cesar Milstein from Cambridge. . Such an antiserum. goat. hence the term monoclonal antibody. with or without an adjuvant into animals such as rabbits. as discussed in the previous chapter. bacterial and viral products. forming little tumour like masses in vitro . These clones were. These hybridomas continuously secreted antibody. producing antibody to a single determinant by a single hybridized B cell. These assays have been complicated because of antibodies of restricted reactivity and the heterogeneity of all antisera obtained by conventional methods. The yields are to the tune of upto a 100 ugs of antibody/ml of culture.giving rise to the term hybridoma. sheep etc. where they grew and produced an ascites which yielded large amounts of antibody. in fact. most commonly involve injecting whole antigen. The affinity and quantity of the antibody varies even from one bleed to another. recovered and injected into the peritoneal cavity of mice. in their case. they could be frozen away. These methods.

others to epitope “b”. Some antibodies generated may not fix complement and hence are not useful in complement fixation tests.1). Two to four days later the spleen is removed and teased apart to form a suspension of spleen cells. Non immunoglobulin producing myeloma cells are essential as the immunoglobulin desired is the one that the spleen cell is committed to produce . immunoelectrophoresis or agar gel diffusion. diploid cells do not have a very long life span in culture and will also die before long. in that. hybridomas become visible on gross examination and the supernatant of these clones are examined for specific antibody to various individual antigenic determinants usually by RIA or ELISA.Monoclonal Antibodies 71 The Principle of Monoclonal Antibody Production Mice are immunized with the whole antigen of interest (containing. The mixture of spleen and myeloma cells grows in wells of a microtiter dish. they cannot use exogenous hypoxanthine to synthesize purines. possess both the enzyme (HPRT+) and the ability to secrete immunoglobulin (Ig +). The hybrids are thus immortalized because the myeloma cell line contributes the property of growth in continuous culture and the spleen cell provides the hybrid with HPRT. This is done by growth in selective medium containing: H – hypoxanthine. Clones producing antibody are grown in mass culture and recloned. . two epitopes: a and b). However. is able to synthesize purines and pyramidines. Two to four weeks after fusion. Only one in every 2 x 105 spleen cells actually forms a viable hybridoma. This is important in the case of diagnostic reagents. The resultant ascitic fluid formed in the mice yields very high concentrations of antibody. A – aminopterin. The spleen cells on the other hand. providing a steady supply of reagents for diagnostic immunoassays. hence myeloma cells die after a period of time. it is therefore necessary to eliminate the unfused cells to allow recovery of the hybrid.and survives. The spleen cells are mixed with mouse myeloma cells that have been previously adapted to grow in continuous culture.myeloma immunoglobulin or hybrid immunoglobulins would pose many obvious problems. Polyethylene glycol (PEG) is added to promote the fusion between cell membranes and the cells are suspended in tissue culture medium. radial immunodiffusion. Because myeloma cells lack the enzyme HPRT. Drawbacks of Monoclonal Antibodies Since monoclonal antibodies produce antibodies to a single determinant. Besides. Spleen cells being normal. blood grouping and HLA typing. The mouse myeloma cells used are variants. the exact same antibody can be produced by different groups of workers indefinitely. since these are sensitive. they lack the enzyme hypoxanthine phosphoribosyl transferase (HPRT–) and are also non-secretors of immunoglobulin (Ig–). Some of these spleen cells will be committed to produce antibodies to epitope “a”. they do not form the lattice necessary for precipitation and so cannot be used in precipitation assays. let us say. These can either be frozen away or injected to induce tumours in mice. Aminopterin blocks endogenous synthesis of purines and pyramidines. large amounts of antibody can be produced in this way. quick and reliable assays. T – thymidine. a hybrid between a myeloma cell and a spleen cell which possesses the enzyme HPRT. This eliminates differences in results between laboratories. and given another injection to obtain a secondary response (Figure 9. Advantages of Monoclonal Antibodies Once a hybridoma has been established. being normal.

1. Figure 9. This kind of cross reactivity cannot be removed by absorption as in the case of conventional antisera. Interpretation of these results becomes confusing and difficult. They may recognize antigens of a low reactivity as in recipients of multiple transfusions or multiparous women.72 Immunology– Introductory Textbook Very high titer antibodies also pose certain difficulties. The principle of monoclonal antibody production. In this manner not only can . the immunoglobulin gene DNA can be introduced into myeloma cells. The Production of Hybrid Antibody Molecules With current technology it is possible to introduce DNA into cells and have it expressed as if it were part of the cells own genetic apparatus. By this process called DNA transfection.

and where it is not possible to either immunize humans or to generate an in vitro antibody response with human cells. Monoclonal antibodies are used to map areas of antigenic variation on the virus particle. recombinant antibodies can be produced with the desired antigen binding capacity and fused to a portion of a molecule with enzymatic function.Monoclonal Antibodies 73 myeloma cells produce monoclonal antibodies. Some Applications of Monoclonal Antibodies Besides a source of pure antibody for diagnosis of infectious diseases. with configurations corresponding to the protective antigen. With the help of monoclonal antibodies synthetic peptides have been produced as subunit vaccines. (iii) Analysis of putative protective antigens Monoclonal antibodies can define antigen structure and help identify certain antigenic determinants that evoke a protective immune response. Such a molecule could be used in immunoassays with no need for second antibody techniques. . This methodology may be useful in cases where it is necessary to have human immunoglobulin molecules. a tumour antigen) could be coupled to the gene for a toxin. monoclonal antibodies provide a means of: (i) Enumeration of human lymphocyte sub populations Anti-CD3 identifies all mature T cells. Using similar methodology. so that the final molecule could be used as a specific toxin to kill tumour cells. every antigenic drift or shift can be identified. The Anti Ia molecule inhibits T cell responses to macrophage processed antigen. the antigen combining portion is from the mouse gene and the C-region is from the human gene. Monoclonal antibodies have been used against the haemagglutinin glycoprotein that is exposed on the surface of the virus and these antibodies have also been found to neutralize the virus. For example. The gene for a specific antigen binding region (say. (ii) Analysis of viral antigens Monoclonal antibodies are used in the classification and diagnosis of viral diseases. the DNA can be custom . (v) Blood grouping Anti blood group monoclonals provide a more reliable standard reagent than conventional antisera. The best example of this is the dissection of the antigenic structure of the influenza virus. Non immunodominant determinants can also be identified and their reactivity analysed. Such functional mapping has also been done for polio virus and certain reoviruses. anti-CD4 identifies subsets containing T helper cells and anti-CD8 identifies cytotoxic T cells. In this way. In these molecules.altered to yield modified monoclonal antibodies. Anti CD8 inhibits killing by cytotoxic T cells. A cocktail of anti CD3 + complement kills T cells in human bone marrow and can be used to prevent graft versus host reaction. investigators have generated monoclonal antibodies that are hybrids of mouse and human immunoglobulin molecules. (iv) Analysis of immunologically competent cell surface molecules Monoclonal antibodies can be used to map and study the various regions of an immunoglobulin molecule and the basis for immunoglobulin variability.

Monoclonal antibodies to the TSH* and ACH* receptors are used to study Graves disease and myasthenia gravis. (vii) Diagnosis of Cancer Antibodies have been generated that distinguish between malignant and normal cells. (viii) In autoimmunity and immune deficiency Imbalances in the ratio of T helper and T suppressor subsets indicate immunodeficiency or autoimmunity.the new “magic bullet” therapy. Hybridoma technology has not only improved the quality and discriminating power of diagnostic and investigative serology.C and DR loci. it promises to provide new reagents that will be useful in the diagnosis and treatment of many disease processes.74 (vi) HLA typing Immunology– Introductory Textbook Monoclonal antibodies provide a reliable means of HLA antigen detection using individual specificities to the A. *TSH : Thyroid stimulating hormone ACH: Adrenocortico hormone HCG: Human chorionic gonadotrophin LH: Luteinising hormone LHRH: Luteinising hormone releasing hormone .B. It may be possible to deliver cytotoxic drugs conjugated to monoclonal antibodies right into tumour specific cell types . (ix) In the control of fertility Using monoclonal antibodies against hormones and reproductive tract antigens such as HCG*. attempts are being made to control fertility. LH* and LHRH*. (x) Monoclonal mutants Mutants lacking Fc structures are used for defining biologic roles of Fc domains and for in vivo neutralization of toxic drugs. These states are accurately monitored by monoclonals against specific T cell antigens. for example in cases of digoxin overdose. Malignant melanoma cells and acute lymphocytic leukemia cells are among the few that can be differentiated. Radioactive anti carcino-embryonic antigen is used to localize colonic tumours or secondaries on scanning.

More HLA related loci are continually being and identified. the terms MHC and HLA are often used interchangeably. In addition. . However. together with the HLA–D region. Consequently. in 1973 certain HLA antigens were found to be associated with specific diseases in high proportion. this region and the antigens encoded by this region are both known by the acronym HLA (human leukocyte antigen). HLA–B. J. HLA – E. Figure 10. it was realized that the major histocompatibility complex regulates several aspects of the human immune response. The vital role played by HLA antigens in tissue and organ transplants was soon appreciated. It was found that inbred mice of strain A would reject skin grafts from inbred mice of strain B and vice versa. In humans evidence for the existence of similar loci(a locus is a position on the chromosome where a given gene may be found) dates from the mid 1950s when leuko agglutinating antibodies (antibodies that agglutinate leukocytes). These membrane antigens are encoded by genes on chromosome 17 of the mouse. HLA – A. were found in recipients of multiple transfusions and in the sera of multiparous women. Though the terms HLA and MHC are sometimes used interchangeably. etc. The officially recognized genetic loci are HLA–A. H. HLA–G. in all species found to possess it. The chromosomal region analogous to the mouse H2 constitutes the “major histocompatibility complex” (MHC).CHAPTER – 10 THE MAJOR HISTOCOMPATIBILITY COMPLEX he discovery of the major histocompatibility complex in humans arose from studies of transplantation of tumours and grafts in mice. HLA-B.1 schematically depicts the current concept of the MHC. by producing antibodies against membrane antigens on the foreign grafted tissue. The complement region. An International Workshop meets every year to update the description and nomenclature of the MHC. HLA–C. in a region designated H2. showing the genetic regions containing the HLA loci in relation to each other. the acronym HLA is more often used to precede the individual antigens within the complex for example. which has been mapped between the HLA–B and HLA–DR regions contains genes determining the complement components C2 and C4 of the classical complement pathway and properdin and factor B F of the alternative pathway. T Nomenclature and Genetic Organization of the MHC The nomenclature of the MHC/HLA system is devised by the HLA Nomenclature Committee under the auspices of the World Health Organization. K. The entire histocompatibility complex occupies a segment on the short arm of chromosome 6. Such sera were found to agglutinate or lyse leukocytes from some persons but not others. L. In humans. HLA – F. Several additional genetic regions have been linked to the HLA complex. HLA – C. The HLA-D region consists of the following loci : HLA–DR (D-related) HLA –DQ and HLA–DP.

Genetic organization of the HLA system In the upper part of the figure the position of the HLA complex. is shown in relation to the other markers: PGM3= phosphoglucomutase 3. Currently. at a given HLA locus are expressed. B13 and so on. HLA–DR w1.76 Immunology– Introductory Textbook Figuere 10. HLA–B and HLA–C regions. HLA– A1. but . 10 being closely related to 25. This unit is referred to as the haplotype. HLA–A2 etc. GLO = glyoxylase. on the short arm of chromosome no: 6. The clinical significance of linkage disequilibrium is as yet unclear. the mechanism responsible for linkage disequilibrium remains the subject of many speculations. The precise nature of this advantage has not been defined. A cluster of closely linked complement genes: C4.. both alleles. for example. than would be possible by pure co incidental association. Pg5 = urinary pepsinogen. have been found to be a group of 2 or 3 closely related antigens.. Each allelic gene determines a different gene product or cell surface antigen which is also given the same number. and 2 complete sets of HLA antigens can be detected on cells. BF and C2 lies next to the HLA–B locus. HLA– A25(10). the most preferred explanation postulates that certain combinations of alleles at different HLA loci are advantageous to the survival of an individual.1. Officially recognized alleles at each locus are designated by the locus and a number. Linkage Disequilibrium This is a phenomenon that occurs when certain combinations of alleles are found with a frequency far exceeding that expected from pure random mating. Alleles that have been tentatively assigned to a given locus. However. one each from the maternal and paternal chromosomes. The combination of alleles at each locus on a single chromosome is usually inherited as a unit. The lower part of the figure shows the expanded version of the HLA complex. Because all HLA genes are co-dominant. The HLA system is extremely polymorphic. They are identified numerically and in brackets for eg. At each locus. but are not yet officially recognized are designated by a w (for workshop) placed before the number for example. The three class II loci are termed HLA–DP. Class I loci are the HLA–A. are the allelic (alternative forms) of the HLA–A locus that may exist. A11 and HLA–B27. As an example HLA–B8 and HLA –A1 are found together six to 21 times more often in the same individual in some population groups. HLA–DQ and HLA–DR from the 5' to the 3' end. having multiple different alleles at each known locus. Hence an individual can be typed: HLA–A1. In several instances HLA antigens initially thought to be a single antigen. one of several alternative forms (alleles) of a gene may be found. Since we inherit one chromosome from each parent we have two HLA haplotypes. for instance there are 30 or more different alleles at the HLA–A locus and at least 60 at the HLA– B locus.

. HLA – B. Typing for HLA Class I antigens: Microcytotoxicity assay.2). HLA typing Lymphocytotoxicity Availability of monoclonal antibodies to a variety of HLA antigens have made serological testing a popular method of HLA typing. HLA – C series.. Using the lymphocytotoxicity assay. where the antibody has bound) and in doing so. The Class II MHC antigens. Figure 10. The Class I MHC antigens. especially those that are prevalent in the environment of a given population. Complement is then added.2. HLA – DQ. HLA– DP antigens. If the serum contains an antibody specific to an HLA (Class I or Class II) antigen on the lymphocytes.The Major Histocompatibility Complex 77 it could represent an enhanced resistance to certain infections or diseases. the antibody will bind to this HLA antigen. Antigens of the Major Histocompatibility Complex (MHC) Based on their tissue distribution and structure.e. analogous to the Ia antigens in the mouse include the HLA – DR. The complement binds only to positive cells (i. the MHC antigens have been divided into two classes. The above reaction patterns allow the cell to be typed as HLA B27 +. HLA B8–. also termed the classic histocompatibility antigens include the HLA – A. lymphocytes are added to anti-sera which may or may not have antibodies directed to HLA antigens (shown schematically in Figure 10.

The damaged cells are not completely lysed but suffer sufficient membrane damage to allow uptake of vital stains such as eosin or fluorescent stains such as ethidium bromide. Currently. In an MLR.DR cells called the homozygous typing cells (HTC) is used. If these two lymphocyte populations possess differing (heterozygous) HLA . The HLA . using antisera of known specificity has long been used to type for HLA Class I and Class II antigens. an invitro transplant model is established which is an extremely useful indicator of possible rejection or Graft versus Host reaction. Thus the frequency of a particular sequence will determine the lengths of DNA produced by cutting with a particular enzyme.3).78 Immunology– Introductory Textbook causes membrane damage. . The most important use of this test is to detect specific donor-reactive antibodies present in a potential recipient prior to transplantation. Instead of using the patients mononuclear cells. as in 3H thymidine.DQ antigens are typed using a serologic procedure similar to the assay for Class I antigens. then it is concluded that the patient’s cells are not of the same type as the HTC. By testing the prospective donor and recipient. These stimulator cells are irradiated or treated with mitomycin C to prevent their proliferation in response to the unknown cells. a panel of known HLA . If the responder cells. many laboratories have changed to molecular genetic methods for HLA Class typing. the blast transformation can be measured by following the rate of uptake of 3H thymidine. the cells are stimulated to divide. after incubation with the stimulator cells. the problems of non-availability of certain antibodies has led to the introduction of DNA based methods. than the patients cells and the HTC are homozygous (similar). these assays are done on purified populations of B lymphocytes. The responder cells of the individual to be typed are cultured with the HTC (Figure 10. The cells used for the test are lymphocytes because of their excellent expression of HLA and ease of isolation compared to most other tissues. During blast transformation there is increased uptake of thymidine into the cells to facilitate increased DNA synthesis. If the thymidine can be labelled. This phenomenon is called blast transformation. If only base line DNA synthesis occurs. However.DR antigens. Historically. Molecular typing techniques RFLP Restriction Fragment Length Polymorphism (RFLP) methods rely on the ability of certain enzymes to recognise exact DNA nucleotide sequences and to cut the DNA at each of these points. Mixed Lymphocyte Reaction (MLR) The HLA .DR antigens also elicit a reaction called the mixed lymphocyte reaction (MLR).. Microscopic identification of the stained cells. indicates the presence of a specific HLA type. They become metabolically very active in the presence of a foreign HLA . This occurs when lymphocytes from one individual are cultured with those from another.DR antigen and begin to synthesize excessive amounts of DNA. this test. An important use of the MLC is in its use as a “cellular crossmatch” prior to transplantation especially for bone marrow transplants. display excessive DNA synthesis.DR and HLA . The typing sera are pretested to make certain that they do not detect the Class I antigens.

Typing for HLA Class II antigens: Mixed lymphocyte reaction.g.3. be denatured. The double stranded DNA is then denatured by heat into single stranded DNA. Sequencing DNA is now available using computerised and automated methodology. DR15. DR17. If the oligonucleotide is chosen to be close to a region of special interest like a hypervariable region of HLA-DR then the part of the DNA..The Major Histocompatibility Complex 79 Figure 10. Oligonucleotide primer sequences are then chosen to flank a region of interest. This cycle can then be repeated until there is sufficient of the selected portion of DNA to isolate on a gel and then sequence or type. So the lengths of DNA seen when DR15 is cut by a particular enzyme.g. reassociate with primers and produce four copies. will be amplified when DNA polymerase and deoxy-ribonucleotide triphosphates are added. Very small amounts of DNA can be used as a starting point. e. The first step in this technique is to obtain DNA from cells of an individual. The above reactions allow the cell to be typed as HLA Dw3 +. . are characteristic of DR15 and different to the sizes of the fragments seen when DR17 is cut by the same enzyme. Those two copies can then. The DNA for one HLA (Class II) antigen. will have these particular enzyme cutting sites (or “restriction sites”) at different positions to another antigen.. HLA-Dw2 –. The oligo. e. From one copy of DNA it is thus possible to make two. in turn. Polymerase Chain Reaction The Polymerase Chain Reaction (PCR) is a recently developed and revolutionary new system for amplifying the DNA nucleotide sequence of a particular region of interest in any individual.nucleotide primer is a short segment of complementary DNA which will associate with the single stranded DNA to act as a starting point for reconstruction of double stranded DNA at that site.

the oligonucleotide primers used to start the PCR have sequences complimentary to known sequences which are characteristic to certain HLA specificities. which are complementary for DNA sequences. Radioactive) oligonucleotide probes. because we possess 2 haplotypes each and because HLA expression is codominant it possible to type 2 antigens from each locus.The extra cellular portion of this Class I heavy chain is divided into three domains. the intra cellular portion is hydrophilic and has the carboxy terminal.4. Typing is done by using a set of different PCRs. the HLA DR gene region) is amplified in the PCR.4). Such a report could mean that the other HLA-B antigen was homozygous with the typed antigen or that typing was not possible by the methods available. α2 and α3 looped together by disulphide bonds (Figure 10. For example: In this test. will not be able to instigate the PCR for HLA-DR17. Uses of HLA Typing HLA typing is used primarily for determination of HLA compatibility prior to transplantation.000 daltons). the heavy chain domains α 1 and α 2 are most . the DNA for a whole region (e.g. Both the β2 microglobulin and the α3 domain of the heavy chain of the MHC Class I antigen are relatively non. designated α1. each with primers specific for different HLA antigens. Occasionally only one antigen can be typed when it is reported as HLA-B27.for example. the transmembrane portion is hydrophobic.500 daltons) known as β2– microglobulin which is encoded by a gene on chromosome 15. characteristic for certain HLA antigens. The other chain is a small protein (11. is heavy and glycosylated (44.4). The extracellular portion has an N terminal and is hydrophilic. Sequence Specific Oligonucleotide (SSO) Typing By this method. The primers which are specific to HLA-DR15. Structure and function of the MHC antigens MHC Class I Antigens The MHC Class I antigens consist of two polypeptide chains held together non-covalently (Figure 10. for anthropologic studies and to establish HLA-disease associations. One chain (the alpha chain). for paternity testing.polymorphic and demonstrate considerable amino acid homology with the CH3 portion of the constant region of the IgG molecule.g. for example.. The amplified DNA is then tested by adding labelled (e. X-ray diffraction studies of the HLA-A2 molecule have shown that Figure 10. These probes will then “type” for the presence of specific DNA sequences of HLA genes.80 Sequence Specific Priming (SSP) Immunology– Introductory Textbook There are a number of PCR based methods in use.. B. In general. Structure of the MHC Class I antigen.

However. Analysis of the protein stretches forming the groove show that there are hypervariable regions along the sides of the groove and to a lesser extent in the β pleated base. The two connected spheres represent a disulfide bond. In all studies concerning the MHC Class I antigen. Striated muscle cells and liver parenchymal cells are normally negative for Class I antigens or they may express only a low density of Class I molecules. The helices. the protein stretches of the base are found in a β pleated configuration(Figure 10. By a process called capping. only these antigens will form a cap. the HLA– B and HLA–C antigens will remain evenly distributed. it can be shown that the MHC Class I antigens are separate entities on the cell membrane. which form the sides. the structural configuration of the antigen is lost. The view is looking down on the top of the molecule. presumably representing processed antigen. the stretches of protein that form the walls of the groove lie in an alpha helix configuration.5). Figure 10. are shown as helical ribbons.5. If the β2 microglobulin is selectively removed. If a cell is exposed to antibody to HLA–A antigen. in inflammatory states these cells begin to express large numbers of Class I molecules. even distribution. and the N terminus is indicated. the MHC antigen on the cell surface will move from its usual. When exposed to specific antibody. showing the surface that faces away from the cell membrane. The base of the groove also contains parts of these α1 and α 2 domains. The β2 microglobulin helps to keep the configuration of the MHC Class I antigens. The groove has a base composed of eight β strands. These findings are consistent with the idea that MHC molecules bind and present processed antigens to responding T cells and that the T cell receptor co recognizes foreign antigen only if presented with the MHC Class I antigen. A separate exon codes for each of the three α domains and gene polymorphism exists within each of these exons. shown as thick arrows pointing in the amino to carboxyl direction. to a single small area or cap on the cell. particularly that of the groove between the α1 and α 2 domains. MHC Class I antigens are expressed on all cell types except erythrocytes and trophoblasts. If antibody to the β2 microglobulin is used. Polymorphism is found both along the edges of the helices and in the base. all antigens will cap as this molecule is common to all Class I antigens. . The sides of the groove are formed by the α1 and α2 domains.The Major Histocompatibility Complex 81 distal from the cell membrane and form a groove along the top surface of the molecule. The genes that determine a Class I antigen consist of several exons interspaced by introns. more so in the α 1 and α 2 regions. workers found that the groove always contained an unidentified peptide molecule. A schematic representation of the α2 and α3 domains of the MHC Class I antigen reveals a groove thought to be involved in antigen binding and presentation to T cells.

MHC class I molecules bind peptide fragments derived from proteolytically degraded proteins endogenously synthesized by a cell. hypervariable regions are located primarily along the groove which suggests that foreign antigen rests within this groove and that Fig. T cells recognize foreign antigen in conjunction with the MHC antigen. The α chain intracellular region can be phosphorylated. When T lymphocytes are exposed to a viral antigen. Class I antigens are the principal antigens recognized by the host during tissue graft rejection. which is divided into at least three sub regions : HLA–DP. activated T cells.6.6). they will recognize it only if associated with a Class I antigen. MHC class I molecules specifically bind CD8 molecules expressed on cytotoxic T lymphocytes. As with Class I antigens. In summary MHC class I expression is widespread on virtually every cell of the body. nor will they kill cells bearing the correct Class I antigen and a different viral antigen. the sides of the α1 and β1 domains form a groove in which the sides are in an α helix configuration and the base contains β pleats. These T lymphocytes will not kill target cells bearing the same viral antigen and a different Class I antigen.e. consists of an α chain (34. The detailed structure of an HLA– DR molecule has been elucidated and serves as a prototype for the Class II molecule.82 Functions of the MHC Class I antigens Immunology– Introductory Textbook In cell mediated cytolysis the Class I antigens are the target antigens recognized by the killer/ cytotoxic T lymphocytes. these viral antigens are presumably the peptides found within the groove formed between the α1 and α2 domains. As shown in figure 10.HLA–DQ and HLA–DR. Structure of the MHC Class II antigen. β2. . including monocytes/macrophages. The MHC Class II antigen.000 daltons) and a β chain (29. a transmembrane hydrophobic region and an intracellular hydrophilic region.7 both chains contain extracellular domains termed α1.000 daltons) in non covalent association (Figure 10. Positions of disulphide bonds and carbohydrate linkages are also illustrated in the figure. This is consistent with the protective function of cytotoxic T lymphocytes which continuously survey cell surfaces and kill cells harbouring metabolically active microorganisms. In humans the Class II antigens are encoded by the HLA–D gene region. The true physiologic role of the MHC Class I antigens lies in the cell mediated lysis of virus infected cells.. Both chains are anchored in the cell membrane and display an extracellular hydrophilic region. α2 and β1. 10. dendritic cells and most notably on B cells. Again. inflammatory states cause many other tissues to express Class II antigens. Small peptides are transported into the endoplasmic reticulum where they associate with nascent MHC class I molecules before being routed through the Golgi apparatus and displayed on the surface for recognition by cytotoxic T lymphocytes. i. MHC Class II antigens Class II antigens are found chiefly on surfaces of immunocompetent cells. like the Class I antigen. Like in the MHC Class I molecule. The cytotoxic T lymphocytes elicited by such an exposure are restricted in their killing to those target cells which bear both the same viral antigen and the same Class I antigen as were present on the cell that first stimulated their proliferation.

” This is consistent with the functions of helper TH lymphocytes which are locally activated wherever these cells encounter macrophages. dendritic cells. In summary.7.” including macrophages. The resulting peptide fragments are compartmentalized in the endosome where they associate with MHC class II molecules before being routed to the cell surface for recognition by helper T lymphocytes. or B cells that have internalized and processed antigens produced by pathogenic organisms. dendritic cells. The same happens for the DP and DQ molecules. As shown in Figure 10. and B cells. .7.The Major Histocompatibility Complex 83 The genetic organization of the MHC Class II antigens is a little more complex (Figure 10.disease associations.7). In the physiological situation foreign antigen is recognised in conjunction with Class II molecules by certain groups of T cells: the CD4+ T cells or T–helper cells. Disease and the Major Histocompatibility Complex Diseases associated with HLA antigens have several characteristics : (a) they are of unknown cause and unknown pathophysiologic mechanism.1 lists the diseases showing positive HLA . Figure 10. MHC class II molecules bind peptide fragments derived from proteolytically degraded proteins exogenously internalized by “antigen presenting cells. Whether T cells recognize antigen in conjunction with Class I or Class II molecules. Functions of the MHC Class II antigens The MHC Class II antigens are principally responsible for an in vivo correlate of the mixed lymphocyte reaction (MLR) –the graft versus host reaction. DQ and DR regions and their constituent α and ß chain genes. although one of the latter may be a pseudogene (ie: it does not determine a product). with a hereditary pattern of distribution. MHC class II expression is restricted to “antigen presenting cells. The DR α chain can combine with any of the ß chains to produce a DR molecule. it follows that T cells must be equipped with receptors both for the MHC antigens as well as foreign antigens. is known as the MHC Restriction Phenomenon. Hence MHC Class II molecules play an important part in presenting processed antigen to T cells and aid collaboration between T and B cells. the HLA –DR gene region has one locus for the α chain and three loci for the β chain. Table 10. (b) they are associated with immunologic abnormalities and (c) they have little or no effect on reproduction. Genetic organization of the MHC Class II antigen showing the DP. This phenomenon. α genes encode the α chains and β genes code for β chains. when T cell activity is restricted to those cells which bear antigen in conjunction with either the MHC Class I or Class II antigens.

8 3.7 10.1 2.6 11.6 6.7 2.84 Immunology– Introductory Textbook Table 10.8 6. Cw6 B5 DR4 DR3 DR2 B8 DR3 Dw3 B35 DR5 Bw47 Relative Risk 69.5 13.1 37.7 3.5 3.8 5.2 18.0 6.2 15.8 3.7 9.2 2.0 10. Bw57.9 3. B14 DR5 DR3 B13.3 7.5 3.4 17. campylobacter) Psoriatic arthritis (cenral) Psoariatic arthritis (peripheral) Juvenile rheumatoid arthritis Juvenile rheumatoid arthritis pauciarticular Rheumatoid arthritis Sjogren’s sundrome Systemic lupus erythematosus Gastro-intestinal Gluten sensitive enteropathy Chronic active hepatitis Ulcerative colitis Haematologic Idiopathic haemochromatosis HLA Antigen B27 B27 B27 B27 B27 B28 B 27 B38 B27 DR5 DR4 DR3 DR2 / DR3 DR3 DR3 B5 A3 B14 A3.8 0.3 3.0 5.4 Pernicious anaemia Skin Dermatitis herpetiformis Psoriasis vulgaris Behcet’s disease Endocrine Insulin dependent diabetes mellitus Graves disease Addison’s disease Subacute thyroiditis (deQuervain) Hashimoto’s throiditis Congenital adrenal hyperplasia .6 4.1: Diseases with positive HLA associations Disease Rheumatic Ankylosing spondylitis Reiter’s syndrome Acute anterior uveitis Reactive arthritis (yersinia. B17.7 26.0 8. salmonella.5 3.7 90.

1 2.8 4. Several hypotheses have been advanced to explain HLA disease associations. . there may be linkage disequilibrium between alleles at a particular disease associated locus and the HLA antigen associated with that disease: this is so for HLA–A3 and idiopathic haemochromatosis.7 85 The prototype HLA –disease association is that of ankylosing spondylitis with HLA–B27. the immune system fails to recognise the pathogen as foreign and fails to mount an immune response against it. Since then numerous reports have been published describing the role of the human MHC in the control of immune responsiveness and disease susceptibility. Ninety per cent of American Caucasian patients with ankylosing spondylitis possess HLA–B27.2 3.0 2.3 12. Another possible explanation is that the HLA antigen itself plays a role in disease. In the 1960’s.3 5.7 2.The Major Histocompatibility Complex Disease Neurologic Myasthenia gravis (without thymoma) Multiple sclerosis Bipolar affective disorder Narcolepsy Schizophrenia Renal Idiopathic membranous glomerulonephritis Goodpasture’s syndrome Minimal change nephrotic syndrome IgA nephropathy Polycystic kidney disease Infectious Tuberculoid leprosy (Indians) Paralytic polio Low vs high response to vaccinia virus HLA Antigen B8 DR2 B16 DR2 A28 DR3 DR2 DR7 DR4 B5 B8 B16 Cw3 Relative Risk 3.7 15.6 6. In some cases a disease may be associated with antigens determined by 2 different HLA loci (refer Table 10. There are two general explanations for HLA and disease associations.3 130.3 2.1). it was discovered that the mouse MHC (called H2) played a part in the genetic susceptibility of mice to certain leukaemias and in their immune response to certain antigens.9 4. as explained by one of the following models: (a) by being a poor presenter of a certain viral or bacterial antigens (b) by providing a binding site on the surface of the cell for a disease provoking virus or bacterium (c) by providing a possible means of transport for the virus to enter the cell (d) by having close molecular similarity to the pathogen. Firstly.

In gluten enteropathy. and present it as an imunogen to the body. Whatever the explanation for HLA and disease association. but to varying extents in different diseases. a disease associated with HLA DR2 and DR3. it is clear that the HLA system and other non-linked genes operate concurrently to produce disease. In multiple sclerosis and ankylosing spondylitis. a specific gene product is thought to act as an abnormal receptor for gliadin.86 Immunology– Introductory Textbook It is most likely more than one mechanism is involved. which shows a high association with HLA DR3. the wheat protein. not only in the patients but also in their parents and siblings. cell mediated immunity is often depressed. . Complement (C2) levels are known to be low in systemic lupus erythematosus.

The foetal liver continues to be a major site for production of the erythroid/ myeloid series including B cells.Barr virus. a hindgut lymphoid organ was the site of early development of antibody producing cells. The amino or antigen binding end is extracellular and strategically placed to receive foreign antigen. Studies in birds showed that the bursa of Fabricius. IgD molecules appear on their surface membranes along with IgM and the IgD becomes the predominant membrane bound isotype found. This process begins during the 8th week of human gestation. Immature B cells initially display membrane bound IgM monomers. Stem cells then populate the bone marrow. B cells develop from a pluripotential stem cell that can give rise to all of the different types of haemopoietic cell types. Resting B cells display higher levels of surface IgD. Immature B cells rapidly acquire receptors for C3d. Monoclonal antibodies are now available which can identify cell surface markers and thereby accurately stage the B cell in its development. thereafter B cells are continuously produced in the bone marrow throughout life. Histocompatibility Class I antigens are present on immature B lymphocytes. Receptors for Fc(gamma) and C3b also show an increase. In mammals. The trans membrane region is hydrophobic in nature.CHAPTER – 11 IMMUNE RESPONSE MECHANISMS I: B AND T LYMPHOCYTES The Origin of B Lymphocytes he B lymphocyte and its secreted end product. Immature B cells respond negatively to cross linkage of their IgM molecules by multivalent antigen. The latter two then decrease during the terminal stages of B cell differentiation. C3b and Fc portions of IgG molecules. until well into the second trimester. when haemopoietic stem cells migrate to the liver from the yolk sac. . T The Biology of B Lymphocytes B lymphocytes display immunoglobulins as integral proteins of their cell membranes. They are anchored in the cell membrane with the carboxy terminal embedded into the membrane. Activated B cells also display receptors for interleukin-2 (IL-2) and other factors needed for full differentiation of B cells. IgD molecules are not found on newly formed B cells. the antibody molecule has been recognized as a powerful immunologic tool for over a century. As B cell development proceeds. Figure 11. Epstein . Thus early exposure to antigen eliminates B cell responsiveness leading to tolerance towards that antigen.1 shows the full complement of receptor molecules on the B cell surface. These membrane bound immunoglobulins are the B cell receptors for antigen and they differ from secreted immunoglobulins in several ways. cells of B lineage are initially generated in the foetal liver. Activated B cells express increased amounts of HLA-D region encoded molecules. This lineage of cells was therefore termed B cells. as activation of B cells commences IgD is lost and other receptors that aid B cell activation appear on the cell surface.

It was only in the 1960s that immunologists resumed their search and brought this theory into its own. An antigen–antibody complex interacts with white cells and more antibody to the same antigen is produced.They release thousands of antibody molecules every second. After antigen or mitogen stimulation B cells can proceed along one of two pathways. Yet. They can differentiate into plasma cells and secrete large amounts of immunoglobulin or they can divide and return to a resting stage.2). he failed to win validation in scientific circles. The plasma cell seldom divides and has an average life span of less than 4 days. viruses.1. The Clonal Selection Theory How do cells make antibodies in such enormous variety? One antibody can neutralize just one type of antigen . David Talmage . The conceptual father of the clonal selection theory was Paul Ehrlich. He suggested that any animal possesses in its armamentarium. It was Niels Jerne who introduced the concept of clonality and formulated a notion similar to Ehrlich’s side chain theory. Plasma cells are the terminally differentiated state of B lymphocytes. Over a hundred years ago he postulated that a white blood cell’s surface bore receptors with side chains to which foreign substances became chemically linked. Receptor molecules on the B Cell surface. sizes and chemical compositions.the antibody -into the circulation (Figure 11. Over 30 years of intensive research has led to the doctrine that is most widely accepted today and is called the Clonal Selection Theory of antibody formation. Over 40% of the total proteins produced by plasma cells are immunoglobulins. These latter cells are called memory B cells and can rapidly differentiate into plasma cells following second exposure to the same antigen.and antigens come in an incredible diversity of shapes.88 Immunology– Introductory Textbook Figure 11. For decades after Paul Ehrlich proposed his theory. pollen grains. The antibody forming machinery of these cells are turned up full force. small numbers of preformed antibodies against all antigens. incompatible blood cells and even some novel man made molecules. the human system is capable of manufacturing a practically unlimited range of antibodies against bacteria and their toxins. This binding prompted the cell to produce and secrete copies of this bound receptor .

or offspring. He proposed that binding of an antigen with an antibody -cum-receptor on the cell surface.Immune Response Mechanisms I: B and T lymphocytes 89 in 1957 went a bit further. Figure 11. Figure 11.ure 11.3.2. In retrospect it is obvious that Jerne and Talmage laid the foundations of the clonal selection theory . The Clonal Selection Theory. to say that white cells are selected for proliferation when the antibody they synthesize matches the invading antigen. He asserted that each cell and its clones. can produce just one kind of antibody and he coined the term clonal selection to describe his theory. Ehrlich’s side chain theory (a) Combination of antigen with preformed receptors (b) Cell is triggered to produce and secrete more of these receptors.3 illustrates the clonal selection concept. Central to Burnet’s thinking was the tenet that one cell produced just one kind of antibody. it remained for Macfarlane Burnet to crystallize the concept of clonal selection. . triggers the cell to multiply and manufacture more of the same receptor. Fig.

certain surface molecules begin to be expressed. Table 11. each B-lymphocyte becomes genetically programmed. The clonal selection theory together with an understanding of the genetics of antibody diversity has provided many answers regarding the mechanisms which control the production of the vast repertoire of antibodies that the immune system is endowed with. In 1960. CD2+ Restrictions Functions MHC–Class II Stimulate B cell to produce antibody.90 Immunology– Introductory Textbook The human system is endowed with antibody producing cells bearing an entire library of antigen binding receptors (the immunoglobulin molecule). if an antigen overwhelmed the metabolic capabilities of the cells. Niels Jerne also received the Nobel Prize for his theoretical contributions in conceptualizing the idea of clonality in immunology. macrophage activation Lyse antigen bearing target cell T cytotoxic cell or T8 cell CD8+. The most important of these surface molecules is listed in Table 11. This induces the cell to proliferate. Induce CD8+ T–cell cytokine secretion. In 1984. prolonged exposure to antigen is not necessary to maintain antibody production. This occurs during development. An understanding of T cell function has been possible because of the identification and characterization of these surface molecules. CD3–T–cell receptor. If an antigen enters the system it matches with one of the receptors and binds to it. or T cells mediate 2 general types of immunologic functions: effector and regulatory. These functions also require the synthesis of lymphokines (cytokines).a journey that takes three days. Each cell will then generate more clones secreting the same antibody. T-Lymphocytes The thymus derived lymphocytes. to produce several clones of its kind. CD2+ MHC–Class I . The regulatory functions are represented by their ability to amplify cell mediated cytotoxicity by other T cells and by the “help” they render in the production of immunoglobulins by B cells. Molecules of that B-cell receptor are placed on its surface where it can react with epitopes of an antigen. Finally. to make a unique B-cell receptor. The effector functions are mediated by (i) secretion of soluble substances called lymphokines or cytokines and (ii) by their ability to kill other cells (cytotoxicity). each capable of secreting more of just that one particular type of receptor (antibody) matched for the invading antigen. Once clones of antibody producing cells have been generated.1: Molecules on the T cell surface T cells subsets T helper cell or T4 cell Main cell surface markers CD4+. clonal selection explained immunologic tolerance as the complete deletion of an entire clone of cells. As T cells gain maturity. through a process called gene translocation. Stem cells migrate to the thymus and move from cortex to medulla and out into the periphery . Macfarlane Burnet shared the Nobel Prize with Peter Medawar for his accomplishments in understanding immunologic tolerance.1. which would occur before or after birth. the second response to the same antigen is more intense since there are many more cells awaiting to bind antigen. CD3–T–cell receptor. Once an entire cadre of cells has been generated.

Collectively these cytokines enable activated B-lymphocytes to proliferate. the T helper or T4 cells. intracellular bacteria. CD3 and CD8 molecules. is not water tight and it has been shown that T4 cells take part in cytotoxicity targeted at cells bearing MHC Class II molecules. The two primary types are Th1 cells and Th2 cells. These cytotoxic T cells are effector cells derived from T8-lymphocytes . Cytotoxic T-Lymphocytes) Cytotoxic T cells consititute one of the body’s major defences against viruses. This demarcation. Th2 lymphocytes Th2-lymphocytes recognize antigens presented by B-lymphocytes. stimulate activated B–lymphocytes to synthesize and secrete antibodies. In the thymic medulla. CD3 and CD4 molecules on their surface and 35% display CD2. CD4 and CD8. activate cytotoxic T– lymphocytes and NK cells. Th1 lymphocytes Th1–lymphocytes recognize antigens presented by macrophages and function primarily to activate and heighten cell-mediated immunity by producing cytokines such as interleukin-2 (IL-2). 4. render B cell help. interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta). 65% of the cells possess CD2. Another major function of the cytokines produced by Th2 cells is to enable B– lymphocytes to activate eosinophils and produce increased amounts of IgE against helminths. T4-Lymphocytes (T4-Helper Cells.Immune Response Mechanisms I: B and T lymphocytes 91 Each of the molecules listed is detectable by monoclonal antibodies and plays an important role in T cell differentiation and function. In peripheral lymphoid tissue. followed by CD3. the CD2 molecule is the first to appear. activate macrophages enabling them to destroy intracellular pathogens. there are different types of T4-lymphocytes based on the cytokines they produce. they secrete cytokines to propagate this function and also to amplify cell mediated immunologic reactions. two distinct lineages become evident: one bearing CD2. Broadly. and function as chemoattractants for phagocytes. stimulate the production of opsonizing and complement-activating antibodies for enhanced attachment during phagocytosis. 10. and enable antibody producing cells to switch the class of antibodies being produced. stimulate increased production of monocytes in the bone marrow. however. Collectively these cytokines enable T8-lymphocytes to proliferate and differentiate into cytotoxic T– lymphocytes capable of destroying infected host cells and mutant cells. CD3 and CD4 molecules and the other bearing CD2. promote the differentiation of B-lymphocytes into antibody– secreting plasma cells. CD4+ Cells) Functionally. and 13 that promote antibody production. As shown in Figure 3.2. T8-Lymphocytes (T8-Cells. CD8+Cells. The T cytotoxic (T8) cells act as specific killer cells. In the thymus all T cells learn to recognize self MHC gene products and this helps them to react with antigen in conjunction with MHC gene products in the periphery. IgE acts as an opsonizing antibody enabling eosinophils to attach to helminths for extracellular killing. activate neutrophils. 5. CD4+ T cells or T4 cells constitute the helper T cells and CD8+ T cells or T8 cells form the cytotoxic/suppressor T cells. They produce cytokines such as interleukins 2. stem cells do not display any surface molecules. CD3 and CD8 antigens. (Both T4-lymphocytes and T8– lymphocytes can exhibit Th1 or Th2 cytokine profiles). and tumour cells. As T cells move into the thymic cortex.

Figure 11. The T cell receptor is a heterodimer composed of 2 chains. and the spleen.The two chains are termed α and β chains and are integral membrane proteins extending 4-5 amino acids into the cytoplasm. native antigen. much like the constant region of the immunoglobulin molecule. that the variable regions are strategically placed and constructed to bind with foreign antigen. each of molecular weight 4050. . the lymph nodules. the B cell receptor is capable of recognizing and reacting against free.4. The T Cell Receptor for Antigen Few problems in immunology have been as difficult to solve and as controversial as that of characterizing the T cell receptor for antigen. The B cell receptor for antigen has long been identified as the IgM monomer bound on its surface. Studies in the 1960s and early 1970s clearly demonstrated that T cells too. The other domain exhibits far more variability than the immunoglobulin molecules and is analogous to the immunoglobulin variable region.92 Immunology– Introductory Textbook during cell-mediated immunity. The T cell receptor for antibgen. Each chain is folded into two domains. Therefore it followed that T cells must have a receptor capable of recognizing antigen.4). therefore. in order to become cytotoxic. Further. This interaction between APCs and naive T8-lymphocytes occurs primarily in the lymph nodes. were antigen specific. It stands to reason. naive T8-lymphocytes must become activated by cytokines produced by antigen-presenting cells (APCs). The molecules comprising the T cell receptor and the genes that code for them have been characterized. linked by a disulphide bond on the T cell surface (Figure 11. one having a relatively constant structure.000 daltons. However.

Surface CD4 and CD8 Molecules The CD4 and CD8 molecules play an important role in T cell activation. while the genes coding for the β chain are present on the 7th chromosome. analogous to that described for the immunoglobulin molecule (refer Chapter:6). The CD3 molecules transduce the antigen recognition signal received by the α – β heterodimer to the inside of the cell. joining region regions (J) and constant region regions (C). These genetic loci can be further divided into regions similar to the gene arrangement for the immunoglobulin molecule (refer Chapter 6). are capable of recognizing an enormous range of antigens. There is no evidence of polymorphism or gene rearrangement in the case of these molecules and they appear identical in cells having different antigen specificities. Both the CD4 and CD8 molecules and the genes that encode them have been identified. Antigen diversity is accounted for not only by rearrangement of gene segments as with the immunoglobulin molecule. diversity segment genes (D). Therefore. Therefore they do not seem to participate in antigen recognition. for example.4). but also by interactions between the α and β chains of the T cell receptor molecule.2. This provides the diversity to account for the existence of over a million distinct T cell clones per individual. The polymorphic genetic organization of the T cell receptor is shown in Table 11. The genetic locus containing the α chain genes is present on chromosome no:14. Each of the CD3 molecules is an integral part of the T cell membrane and extends into the cytoplasm farther than either the α or β chains of the receptor. The three peptide molecules of the CD3 complex have been called CD3/γ CD3/δ and CD3/ε (Figure: 11.2: Genetic organization of the T cell receptor Gene regions V region D region J region C region α chain 60 – 40 1 β chain 21 2 12 2 Rearrangements occur between gene segments. Other regions also. . Table 11. each having a different antigen specificity. The CD3 Complex In all immunocompetent T cells the T cell antigen receptor is non covalently but intimately linked with closely related molecules called the CD3 complex. Each gene region is comprised of gene clusters.Immune Response Mechanisms I: B and T lymphocytes 93 Genetic Organization of the T Cell Receptor A study of the genetic organization of the T cell receptor (TCR) led to the understanding that T cells too. are highly polymorphic and have several gene clusters. The regions have been named: the variable region genes (V). there are 60 V region genes for the α chain and 21 for the β chain. different antigen specificities are created when a single α chain interacts with different β chains. Separate sets of genes encode the α and β chains.

and (ii) for the cytolytic effector function of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). they play a part in recognizing the constant regions of the MHC Class I and II molecules. the α and β 2 microglobulin chain of the MHC Class I antigen and the α and β chains of the MHC Class II antigen (Figure 11. what is now known as. refer Chapter 12). Both CD2 and CD58 are members of the Immunoglobulin gene superfamily ( see below). These are the heavy and light chains of the immunoglobulin molecule. It has been found that certain sequences (about 110 amino acids long) are common to all three structures. common to these structures. is the primary role of these molecules.94 Immunology– Introductory Textbook The CD4 and CD8 molecules have been shown to function as recognition markers for the MHC gene products. An additional feature. another cell surface glycoprotein widely expressed on various cell types including hematopoietic and epithelial cells. are that many are 2 chain molecules with strong interdomain non covalent interactions.5). The Immunoglobulin Gene Superfamily All the molecules involved in antigen recognition described so far are members of. The presence of CD4 on T cells indicates that the T cell is programmed to recognize and interact with cells bearing MHC class II molecules. the α and β chains of the T cell receptor. it has been suggested that recognition of cell surface molecules (immune or otherwise). This is consequent to the polymorphic gene structure and characteristic gene rearrangements seen in all of these molecules.2. They seem to behave like extra “glue”. The success of these molecules indicate that the forces of evolution will make sure that they are widely exploited in nature. The importance of CD2 function in the normal human immune response has been well documented: (i) recognition involving helper T cells and antigen presenting cells. thus guiding the T cell receptor onto the antigen (Figure 12. It has been hypothesized that the CD4 and CD8 molecules are not involved in activation of T cells. . but can increase interaction between T cells and target cells. It is also thought that since they are structurally constant. It is believed that all three molecules evolved from a single primordial ancestral gene. so that activation can occur. This characteristic β pleated folding of short protein stretches is a shared feature between all three molecules. For cells seeing antigen for the first time this “glue” may play an important role. The CD2 Molecule The CD2 molecule on T lymphocytes is a transmembrane surface glycoprotein which facilitates cell-cell contact. It is obvious that all of these molecules have certain common characteristics . they all recognize and bind foreign antigen and to do this they are equipped with variable regions as part of their structures. Since molecules unrelated to the immune system also fulfil criteria for inclusion into the “super gene family”. Besides these. a “gene superfamily”. This is borne out by the finding that the CD4 and CD8 molecules are closely associated with the T cell receptor and actually bind to the constant region of the MHC Class I and II molecules. while T cells bearing CD8 molecules recognize cells displaying MHC Class I molecules as part of their antigen specificity. The amino terminal domain of CD2 (domain 1) mediates its adhesion function by binding to LFA-3 (CD58). homologous sequences have been conserved around disulphide bonds and in the characteristic antiparallel β pleated strands.

and Th2 help for antibody production. Tr1 Cells Tr1 cells resemble Tr cells in several ways. and. the most promising recent candidates have been given other names. Tr1 cells are abundant in the intestine.5. which is the α chain of the receptor for interleukin-2 (IL-2). most immunologists believe that some form of suppression exists. They require IL-10 for their formation and once mature. that is. although they do not express large amounts of CD25 on their surface. Examples of these are: • Tr cells • Tr1 cells • Th3 cells Tr Cells Most CD4+ T cells belong to either the Th1 or Th2 subsets. The Immunoglobulin gene superfamily Regulatory (Suppressor) T cells The existence of specific suppressor T cells and the phenomenon of immunologic suppression has fallen into disfavour in the present decade. they secrete large amounts of β interleukin 10 (IL-10) and often some transforming growth factor-beta (TGF-β) as well. possibly. If activated. Of the original suppressor T cells (Ts cells).Immune Response Mechanisms I: B and T lymphocytes 95 Figure 11. They express a transmembrane protein called CD25. they also express the α β (alpha-beta) T-cell receptor for antigen (TCR) and can only be activated if it binds to the peptide-class II MHC molecule. These have been termed T-regulatory (Tr) cells. secrete large amounts of it. Both these lymphokines are powerful immunosuppressants inhibiting both Th1 help for cellmediated immunity (including graft-versus-host disease) and inflammation. Like other T cells. The antigenic peptides recognized by their TCRs tend to be self-peptides and perhaps the major function of Tr cells is to inhibit other T cells from mounting an immune attack against self components. Like other T cells their cell surface molecules have been characterised. However. the action of CD8+ cytolytic T lymphocytes (CTL). to protect the body against autoimmunity. However some 5–10% of them do not. and their chief function may be to make . The reason for the suppressor cell controversy is that no one has been able to clone a unique suppressor T cell.

. but unlike Tr1 cells.96 Immunology– Introductory Textbook the host tolerant to the many antigens that are part of its diet. This work is relevant for patients who risk rejection of organ transplants. and for the management of patients with autoimmune disorders like lupus erythematosus. they suppress immune responses to ingested antigens. Further research is needed to elaborate the relationships between these and other T cells that suppress immune responses. Also like Tr1 cells. Th3 Cells Th3 cells are also prevalent in the intestine. their main lymphokine is TGF-β. It is believed that patients who develop Crohn’s disease lack the down regulation characterised by IL-10 via the Tr1 cells making them intolerant to a number of dietary and other antigens. insulin-dependent diabetes mellitus (IDDM-type I diabetes) and Crohn’s disease.

It is therefore obvious that T cells and B cells need to communicate and interact with each other and with other antigen presenting cells. soluble factors and various cell adhesion molecules. W Antigen Presentation Very early studies have shown that T cells and B cells recognize different parts of an antigen. antigen must first be processed by an antigen presenting cell and then presented in association with MHC Class I or Class II molecules. B cells can recognize native. The Pathways of Antigen Processing Macrophages were the first accessory or antigen presenting cells to be identified. More often than not. T cells do not recognize native antigen. T cells are blind to native antigen and will “see” antigen only when processed and presented together with an MHC molecule. or both may occur. In other words. unmodified antigen and this antigen need not be presented in conjunction with cellular MHC antigens. If a hapten is coupled with a carrier particle to yield an immunogen.CHAPTER – 12 IMMUNE RESPONSE MECHANISMS II: ANTIGEN PRESENTATION AND PROCESSING. they break . they also kill virus infected and malignant cells directly. Several antigen presenting cells (APCs) do more than simply capture antigen and display it on their surface. These T cells help B cells to proliferate and secrete antibodies and are popularly known as T helper (Th) cells. Cellular immunity is mediated by T cells. though this is not a rigid rule as we shall see in the following sections. which after stimulation proliferate and differentiate into antibody producing plasma cells. which become activated to secrete a number of substances important in the immune response. To reiterate a general rule of thumb that has been discussed in previous chapters: the CD8+ T cell recognizes antigen in association with an MHC Class I molecule and a CD4+ T cell recognizes antigen in conjunction with an MHC Class II molecule. Humoral immunity is mediated by B cells. Though most B cells do require T cell help for an adequate antibody response and for memory cells to be generated. In contrast. fulfilling the role of T cytotoxic lymphocyte(Tc/CTL) or killer T cells. CD4+ T cells act as helper cells and CD8+ T cells act as cytotoxic cells. MECHANISMS OF LYMPHOCYTE ACTIVATION hen cells of the immune system encounter an antigen. are another set of T cells. Enhancing and supplementing the activity of B cells. Later dendritic cells (specialized cells found in the lymph nodes and spleen) and B cells themselves were added to the principal list of potential antigen presenting cells. This they do through receptors. a humoral immune response or cellular immune response. B cells react with the hapten to produce hapten specific antibodies whereas T cells respond predominantly to parts of the carrier molecule. Presentation of antigen to T cells differs from presentation of antigen to B cells.

obscuring its shape and leaving only its distinctive amino acid sequence as the target. In the endosome. cathepsin . p.lion ""Ill MHC clu..e ~Jl-------':':ii MHC clUI / Bind.98 Immunology-Introductory Textbook down antigen into peptide fragments before presenting it. Such an antigen has been termed "exogenous" antigen and is taken up by specific classes of APes such as B cells. This explains why investigators found that T cells have no interest in native antigen shape. the cytotoxic T cells. under the influence of an acid pH milieu. Endogenous antigen is denatured or fragmented in the Golgi compartment. For those proteins that are borne as a result of intracellular (such as viral) infection or malignant change on a cell the pathway of processing is different (Pathway B....o<:i"l"". an area specialized for dealing with improperly folded proteins synthesized within the cell (Figure 12..1 Pathways of antigen procening. A scheme that describes the antigen processing pathway takes into account all of the above data and is illustrated in Figure 12. The uptake of these antigens is either by receptor mediated endocytosis (which may be purely fortuitous).onh~n p". This distinctive target peptide fragment processed within the APe. The MHC Class II . is then transported to the l'iurface of the cell and displayed in the antigen binding cleft or groove thatis an integral part of both MHC Class I and Clagg II mulecules (Refer chapter 10). witri MHCclUl1I In . Wllh II .noci.Iij. Such an MHC Class II-antigen complex is recognized preferentially by the CD4+ T cell. describes the events that take place when the antigen concerned is freely circulating foreign material (not found intra cellular or bound to cell surfaces). I Ml11&en El<"iI"""'-'~ .and T cell help for antibody production is on its way! (Discussed in the fonowing section). Such internally synthesized proteins need to be presented to the second major class of T cells. The antigen now lies in an endosome within the cytosol of the cell. These antigens are known 8S "endogenous" antigens. Endoll"nQus . The peptide fragments then bind within the cleft of the MHC Class II molecules which are also found in the cytosol of the cell.v 'B· Endcae""'-" anlliM Figure 12.1).1). being internal to the body's own cells.antigen complex is then transported to the cell surface in vesicles and displayed to the exterior. Pathway A.nled In . or as a result of simple pinocytosis. macrophages or dendritic cens that are specialized for processing exogenous antigen.'OOente<l "" surf"".1. where they come into contact with MHC Class I molecules.like proteases denature or fragment the original protein molecule...nl~n In sol - ~thw. a process that requires both time and energy. Processing of '""endogenous" antigens occurs in the Golgi complex. Figure 12. The target peptide fragment binds into the ..y ·A· Excl'l!l"IOUS . The remaining peptides of the original protein antigen go into a lysosomal compartment for complete degradation..nt~n ~lhw'...

Fragments of viral genes (the internal.fitted into the MHC Class I-peptide binding groove. Such a peptide fragment bound to the MHC Class I molecule is also preferentially recognized by the cytotoxic T cell. In the case of virally infected cells. The most striking feature of his work was the finding that this antigen binding cleft was not. therefore. 99 antigen binding groove on MHC Class I molecules and is transported to the exterior of the cell membrane. Such a peptide fragment bound to the MHC Class I molecule is preferentially recognized by the cytotoxic T cell . It seems that these omnipresent peptides are fragments of the body’s own proteins and should the need arise they are displaced by foreign antigen peptides. Killer T cells are known to constantly monitor the other cells of the body for the appearance of tumour or viral antigens and promptly eliminate any cell expressing them. It ensures that a foreign organism cannot elude it by being hidden . and has never been found empty.... The peptides are picked up by TAP proteins embedded in the membrane of the endoplasmic reticulum. endogenous antigen needs to attract the attention of a cytotoxic T cell. There are several reasons put forth for this complex chain of events.. How does the cell get them into the Golgi compartment? This is achieved by specialist transport proteins called TAP (transporter associated with antigen processing) proteins. been targeted onto the aberrant cell. it preferentially binds to MHC Class II antigens . For one. Credit also goes to Baruj Benacerraf and Hugh McDevitt who. By continuously processing and presenting their own antigens. nucleoprotein antigens).Wiley (1987). the peptides are pumped into the lumen of the endoplasmic reticulum where they assemble with MHC class I molecule as described above. and to Don C.. for elucidating the antigen binding cleft on the MHC molecule. in 1960. some proteins encoded by the genes of an infecting virus are synthesized in the cytosol. the viral nucleoprotein. could not be recognized by cytotoxic T cells– unprocessed as they were.. Because exogenous antigen evokes CD4+ T cell help.. in it Wiley and his colleagues found foreign peptide fragments .. Viral proteins in the cytosol are degraded by proteasomes into viral peptides. Selection of peptides for surface expression probably depends on what fits most closely into the antigen binding groove of the MHC Class I and Class II molecules. These pioneering experiments helped evolve the current thinking regarding killing of virally infected or malignant cells by cytotoxic T cells.and the killer cytotoxic T cell has. whereas.. it preferentially binds to the MHC Class I molecule: class discrimination in immunology! Why does T cell recognition have to be so elaborate? Why do T cells not recognize antigen directly as B cells do? Why do antigens need to be broken down and presented with MHC molecules? Alain Townsend and his co-workers. this scheme is consistent with the role of immune surveillance that T cells play. after being degraded and processed were displayed in a suitable manner .Immune Response Mechanisms II: Antigen Presentation .the cell that is essentially the factory or manufacturing unit for thousands of virus particles.. showed that a cell cannot even assemble Class I MHC molecules properly. Using the energy of ATP. showed conclusively that fragments of internal antigen. This complex then moves through the Golgi apparatus and is transported to the exterior of the cell membrane. The antigen was then recognizable by killer T cells. However. cells invite inspection by the immune system so as to detect any early aberration. were the antigens that were “seen” by cytotoxic T cells and these internal antigens initiated T cell cytotoxicity against virally infected cells. therefore. showed that genes of the MHC affected an animal’s ability to mount an immune response to certain antigens. unless a peptide is present during the final stages of the protein folding process.and presumed rightly that this was processed antigen. MHC restricting guides a killer T cell to the culprit cell . Alain Townsend in 1985. He had experimental evidence to show that whole surface antigens.

on the surface of the APC interact with the appropriate CD4 or CD8 molecule on the surface of the T cell (Figure 12.2b.100 Immunology– Introductory Textbook inside a cell and adopting a Trojan horse strategy. thus supporting the theory that they were made to bind foreign peptides. For some diseases such as malaria this is not feasible. cells were to recognize freely circulating antigens. The T cell receptor /CD3 complex. Hidden antigens can also be processed and made visible! If. after all. Therefore. For the equally complex scheme of antigen presentation for helper T cells. the helper role seems to have superimposed itself into cells already programmed to recognize MHC molecules. Understanding the phenomenon of antigen processing has helped investigators manipulate the immune system for clinical purposes. Over the course of evolution. however. To do so these peptides must stimulate both helper and cytotoxic T cells and bind to MHC molecules. such compounds might suppress the autoimmune response. besides whole cell vaccines may have risky side effects. MHC based technology should also make it possible to develop compounds that bind very strongly to those MHC molecules that bind self antigens and cause disease. 12. live or killed or a protein extract such as a toxin. The MHC class II–antigen-T4 cell receptor interaction. vaccines have consisted of whole pathogenic organisms.2a/b). Cell mediated immunity appears to be a defence mechanism much older than man. they have adapted this function to guide them to a site where they can be most effective to B cells which is.2a. therefore interacts with foreign peptide. Mechanisms of Lymphocyte Activation T cell activation The first event that triggers T cell activation is T cell binding to antigen presented in conjunction with MHC molecules. the primary target of T cell help. Hence T cells may have originated as killer cells and may have acquired helper properties later in evolution. Hence. MHC-I with bound peptide MHC-II with bound peptide Antigen-presenting cell Naive T8–lymphocyte APC T4–lymphocyte TCR TCR CD8 CD4 Fig. the answer may be in the theory of evolution. Vaccine scientists are now trying to design synthetic peptides representing a small fraction of the actual antigen. Traditionally. Fig. . even primitive sponges are endowed with this ability. 12. The MHC class I–antigen-T8 cell receptor interaction. The antigen binding cleft on MHC molecules shows variability. they would all be utilised by viral particles released by the manufacturing units and not be available to home in on renegade cells harbouring virus synthesis machinery. to trigger an immune response. By blocking the binding of self antigens on MHC molecules. some of the immune system’s own precision and power can be directed to the fight against disease. as in autoimmune related disease states. Simultaneously the regions of the MHC molecule that display this antigen.

which help regulate the T cell cycle by the interleukin . are responsible for activating T cells..3.4... This results in the generation of two biologically active metabolites: inositol triphosphate (IP3) and diacylglycerol. T cell proliferation occurs (Figure 12. by the T cell and (ii) the expression of receptors for IL–2 on the T cell surface.. The initial interaction between T cell receptor and antigen stimulates membrane bound phospholipase C to hydrolyze membrane bound inositol (Figure 12. Activated T cells secrete several other lymphokines (discussed in Chapter 13)...4). These two metabolites. An activated T cell is then able to transcribe.. 101 This interaction then generates a series of biochemical reactions which are aimed at accomplishing two important events : (i) the secretion of interleukin–2 (IL–2). The inositol triphosphate increases cytoplasmic free calcium and the diacylglycerol activates the enzyme protein kinase C... . T cell proliferation. Biochemical events in T cell activation the cell membrane.interferon circuit. When IL–2 and its receptor interact on Figure 12. Thus IL–2 is secreted and the IL–2 receptor is expressed on the cell surface.3). translate and express two important gene products: the IL–2 molecule and the IL–2 receptor.. including interferon γ. Figure 12.Immune Response Mechanisms II: Antigen Presentation .

Patients deficient in this factor suffer life threatening bacterial and fungal infections. The CD4 and CD8 ligand associations with their corresponding MHC molecules have already LFA-3 CD2 been described in previous sections.peptide ICAM-1 B 7-1 and B7-2 LFA-3 TCR/CD8 TCR/CD4 LFA-1 CD28 CD2 The importance of lymphocyte function associated (LFA – 1) molecule is evidenced by experiments with monoclonal antibodies which block LFA . Antibodies that block ICAM–1 produce the same effects as blockage of LFA–1. primarily Th1 cells. Blockage of these molecules also decreases T cell killing activity by blocking T cell–target cell adhesion. . Table 12. signals and cytokines from effector T4–lymphocytes. certain cell adhesion molecules have been identified that are necessary for efficient interaction between T lymphocytes and accessory cells or target cells.1.1. Cell adhesion molecules and their ligands are presented in Table 12.102 Co-stimulatory signals Immunology– Introductory Textbook In addition.5) These co-stimulatory molecules are only synthesized when toll-like receptors on APCs bind to pathogen-associated molecular patterns of microbes (Refer Chapter2: Innate immunity). Once activated.1: Cell adhesion molecules (on the APC) and their ligands on T cells Dendritic cell or macrophage T lymphocytes MHC I-peptide MHC II .lymphocyte produce signals and cytokines for activation of the naive T8– lymphocyte. Various cytokines like IL–1 and IFN γ increase ICAM expression on the B cell surface. This leads to inhibition of cytotoxic T cell activity. turn on genes responsible for the proliferation and differentiation of that T8-lymphocyte into an effector cell: the functioning cytotoxic T–lymphocyte (CTL). will then be able to activate the T8–lymphocyte. These costimulatory signals involve the interaction of accessory molecules on the APC with their corresponding ligands on the T lymphocytes (Figure 12. B7 MHC-II MHC-class I/II with bound peptide CD40 ICAM-1 CD28 TCR CD4/CD8 CD40L LFA-1 Activation of naive T–lymphocyte APC* * APCs can also be B cells bearing antigen bound to surface lg (slg) Figure 12. The protein which is believed to be the ligand for LFA-1 is the inter cellular adhesion molecule– 1 (ICAM–1).5 Co-stimulatory molecules that enhance Tcell–APC interaction. natural killer cell (NK) activity and antibody dependant cellular cytotoxicity (ADCC). The CD2 ligand is the LFA–3 molecule. Molecular interactions between the APC and the T8 .

.6. There are others... Mitchison in 1971. 103 Interleukin . Activation of B Cells It is now evident.Interferon circuit The interleukin–interferon circuit is graphically depicted in Figure 12.. Actively replicating T lymphocytes secrete interferon γ (IFN γ). antigen processing and expression of MHC Class II molecules.6.. Under the influence of IL-1 from the accessory cells. thus promoting accessory cell functions resulting in a cyclical increase of activated T cells predominantly of the Th1 subset.dependant antigens When T cell help is to be rendered. some of the early G1 cells proceed into the late G1 phase of the cell cycle.. as we have just seen in the previous section. Pioneering experiments by A. Such antigens are also called T dependant antigens.. Figure 12. G1 cells synthesize receptor inducing factors and other proteins.. it has been shown that T cells have first to be activated by a separate process which does not involve B cells. for helper T cells and B .. IFN γ has a positive feed back effect on IL–1 production.. T cell help is essential. The Inter leukin–Interferon circuit. where IL–2 promotes expression of IL–2 receptors on late G1 cells.Immune Response Mechanisms II: Antigen Presentation . Antigens are processed and presented to T cells by antigen presenting cells such as macrophages. that for many B cells to make antibody to a particular antigen. showed that. Activation in response to T. notably the capsule of the organism Streptococcus pneumoniae which are T-independent antigens and can evoke antibody production from B cells without T cell help. This shifts the resting T lymphocytes from the G0 state to the early G1 phase of the cycle. IL–2 in turn stimulates IL–2 receptor bearing cells to proliferate or actively replicate and enter what is known as the GS (synthesis) phase in T cell activation. Other lymphocytes moving in a similar cell cycle are stimulated by IL–1 to secrete IL–2.

C3d Receptor for C3d (CR2) 2 1 B-lymphocyte B-cell receptor (sig)* * slg = Surface Immunoglobulin Figure 12. and other experiments have provided compelling evidence that the activated helper T cell needs to be physically close to the B cell. binds to a complement receptor called CR2 on the surface of the B-lymphocyte (Figure 12. . the result is gene activation and increased production of MHC Class II molecules. The first signal for the activation of a naive B-lymphocyte occurs when B-cell receptors. C3b is subsequently degraded to C3d which. IL-6). This. C3b binds to the microbial surface. they must. in turn.104 Immunology– Introductory Textbook cells to collaborate. on the surface of the B-lymphocyte bind epitopes of T-dependant antigens having a corresponding shape. A second signal is also needed for the activation of the naive B-lymphocyte.7a). Activation of the naive B cell triggers a series of metabolic events in the B cell. IL-5. required for B cell growth and differentiation.7a. co-stimulatory substances such as B7 and CD40 and receptors for IL-2 and interleukins 4 to 6 ( IL-4. similar to what has been discussed for the T cell. recognize parts of the same antigen molecule. the surface immunolobulin (slg). each in turn.7b. Figure 12. T cell-B cell interaction. Activation of the naive B lymphocyte. This is provided when a component of the complement system .

As a result of this activated T helper cell . remain unstimulated by IL-6 and other related cytokines. IL-5. Other agents which induce polyclonal B cell proliferation are Epstein . has now moved from the B0 stage to the B1 stage in its cycle (Figure 12. The B cell.Barr virus. and antibody production ensues... Events involved in B-cell activation. This pool of B3 cells constitutes the memory cells. that is. where it is recognized by an antigen specific T cell receptor on the activated helper T cell. the Th2 cells produce cytokines such as IL-4..7b).5). B3 cells in turn display receptors for IL-6 and other related cytokines. albeit with the receptors displayed... Figure 12. B cells move into the B2 stage of activation and display receptors for IL-4 and IL-5. When the receptors and the corresponding interleukin molecules interact. produce either IgG. To go into production.. primarily the Th2 cell (Figure 12....Immune Response Mechanisms II: Antigen Presentation .B cell interaction. having displayed antigen in conjunction with the MHC Class II molecule. Here co-stimulatory molecules such as CD28 and CD40L on the Th2 cell bind to B7 and CD40 molecules on the activated B lymphocyte (Figure 12. Chlamydia trachomatis and lipopolysaccharides which react with non immunoglobulin receptors on the B0 cell. these B cells only need renewed fuelling with IL-6 and related cytokines.8). This allows the Blymphocytes to “fine-tune” the shape of the antibody for better fit with the original epitope and to produce the class of antibody of greatest value for that site. 105 Antigens bound to surface immunoglobulins on the B cell are internalized and degraded to oligopeptides which bind to MHC Class II proteins within the cell. . IgA. The antigen peptide– MHC Class II complex then goes to the cell surface.. Subsequent exposure of B cells to antigen will yield a much faster and greater antibody response since the terminal differentiation stages before antibody production are all set. Th2 cells also enable B-lymphocytes to undergo affinity maturation through somatic hypermutation and to switch antibody classes. Collectively these cytokines enable activated Blymphocytes to proliferate stimulate activated B-lymphocytes to synthesize and secrete antibodies and promote the differentiation of B-lymphocytes into antibody-secreting plasma cells. A set of B3 cells however. or IgE. Interaction of these cytokines with their receptors on the B cell pushes the B cell into the final differentiation stage– the plasma cell. B cells grow and proliferate yielding several clones of B3 cells.8. IL-6.

These antigens activate B-lymphocytes by binding to their specific toll-like receptors (see chapter 2) rather than to B-cell surface immunoglobulin receptors. The resulting antibody molecules are generally of the IgM isotype and do not give rise to a memory response. repeating subunits. Antibody molecules generated against TI-1 antigens are often called “natural antibodies” because they are always being made against bacteria present in the body. B-lymphocytes mount an antibody response to T-independent antigens without the requirement of interaction with T4-lymphocytes. from the gramnegative cell wall. Bacterial LPS. TI-2 antigens. There are two basic types of T-independent antigens: TI-1 and TI-2.independent antigens Immunology– Introductory Textbook T-independent antigens are usually large carbohydrate and lipid molecules with multiple. . TI-1 antigens include lipopolysaccharide (LPS) from the outer membrane of the gramnegative cell wall and bacterial nucleic acid. pneumoniae) are examples of Tindependent antigens. and capsular polysaccharides (as in S. repeating subunits. are molecules with multiple. such as capsular polysaccharides.106 Activation in response to T. These repeating subunits activate B-lymphocytes by simultaneously cross-linking a number of B-cell receptors.

soluble proteins that are produced in response to an antigen and function as chemical messengers for regulating the innate and adaptive immune systems. A brief description of the important interleukins and interferons is given in Table 13. They are produced primarily in response to pathogen-associated molecular patterns (PAMPS) such as lipopolysaccharide (LPS). redundant or multifunctional. Cytokines are low molecular weight. as one cytokine stimulates its target cells to make additional cytokines. Cytokines can act synergistically or antagonistically. and other cells. NK cells. those produced by monocytes or macrophages are called monokines. and double-stranded DNA.1 T A. • Redundant defines the ability of a number of different cytokines to carry out the same function. it provides a snapshot of some of the more commonly known factors and their principal activities. peptidoglycan monomers. • Multifunctional cytokines are able to regulate a number of different functions. These substances have been given a general term: the cytokine. are then able to bind to specific cytokine receptors on other cells of the immune system and influence their activity: this includes both enhancing and suppressing responses. Most act on leukocytes and the endothelial cells that form blood vessels in order to promote and control early inflammatory responses. More new cytokines and their receptors are continuously being discovered. but especially by T helper lymphocytes. Those secreted by lymphocytes are called lymphokines. The cytokines. • Pleiotropic cytokines can act on a number of different types of cells rather than a single cell type.CHAPTER – 13 CYTOKINES he course of immunologic and inflammatory pathways is mediated by several hormone like soluble substances that are secreted by the concerned cell types. There are three functional categories of cytokines depending on whether they (a) Regulate innate immune responses (b) Influence adaptive immune responses (c) Stimulate haematopoiesis The description of cytokines below is not intended to be comprehensive nor complete. . Cytokines maybe characterized as pleiotropic. They are produced by virtually all cells involved in innate and adaptive immunity. teichoic acids. The activation of cytokine-producing cells triggers them to synthesize and secrete their cytokines. in turn. Cytokines are often produced in a cascade. Cytokines that Regulate Innate Immune Responses Cytokines that regulate innate immunity (see Chapter 2) are produced mainly by macrophages and dendritic cells although they can also be produced by T-lymphocytes.

Functions include acting on endothelial cells to stimulate inflammation and the coagulation pathway. T and B cell products such as interferon γ. It increases T lymphocyte production of colony stimulating factor and other chemotactic factors. enhances non specific natural killer cell activity of large granular lymphocytes which in turn have tumoricidal activity. Interleukin-1 The major cell sources of IL-1 are monocytes and macrophages. differentiation and antibody producing functions of B lymphocytes. probably accounting for the anti inflammatory and immunosuppressive properties of these drugs. This could explain the .108 Tumor necrosis factor (TNF) Immunology– Introductory Textbook TNF-alpha is a key cytokine in the mediation of acute inflammation. IL-1 promotes B cell antibody production directly and by augmenting T cell helper function. TNF is produced mainly by monocytes. IL-1 increases the accessibility of the antigen binding receptor and augments antigen stimulated CD4+ and CD8+ T cells to produce IL-2. Prostaglandin E2 has been shown to have a negative effect on IL-1 production.antibody complexes are potent stimulators of IL-1. Stimulants of IL-1 are particles like silica and adjuvants such as LPS or muramyl dipeptide (MDP). probably by stimulating prostaglandin production. dendritic cells and Th1 cells. IL-1 in conjunction with IL-2 or interferon. IL-1 activity increases markedly. macrophages.like substances. Excessive doses of the macrophage stimulators can have an inhibitory effect on IL-1. stimulating the liver to produce acute phase proteins. In excessive amounts it is the principal cause of systemic complications such as the shock cascade. Resting macrophages and circulating monocytes produce little IL-1. It also induces expression of surface bound immunoglobulin receptors. stimulating endothelial cells to produce selectins and ligands for leukocyte integrins during diapedesis. (ii) On B cell function IL-1 augments proliferation. Interleukin-1 release is blocked by drugs such as hydrocortisone and cyclosporin. stimulating endothelial cells and macrophages to produce chemokines that contribute to diapedesis. (iii) On non-lymphocytic cells IL-1 promotes the growth and function of non lymphoid cells as well. IL-1 can act as an endogenous pyrogen. All the agents that activate T-lymphocytes directly. TNF is cytotoxic for some tumour cells. Biological properties of IL-1 (i) On T cell function IL-1 augments the proliferation of medullary thymocytes and increases the production of lymphokines in peripheral T cells. colony stimulating factor and antigen . such as antigen in conjunction with MHC molecules also stimulate IL-1 release. stimulates the synthesis of collagen and collagenase for scar tissue formation. and activates macrophages. activating neutrophils and promoting extracellular killing by neutrophils. stimulating macrophages to secrete interleukin-1 (IL-1). It therefore increases the tumoricidal and bactericidal functions of T cells. though several other cell types produce interleukin . and acting on muscles and fat to stimulate catabolism for energy conversion. chemotaxis and the recruitment of leukocytes. In addition. Hence less differentiated T cells proliferate and more differentiated T cells display increased secretory activity. it interacts with the hypothalamus to induce fever and sleep. When macrophages are stimulated.

They increase the affinity of integrins on leukocytes for ligands on the vascular wall during diapedesis. astrocytes from the brain and mesangial cells from the kidney. It stimulates the production of superoxide anion via the hexose monophosphate (HMP) stunt. It is produced mainly by macrophages and dendritic cells. MIP-1b. IL-1 causes neutrophilia. MCP-3. They are keratinocytes. asthma. adult respiratory distress syndrome (ARDS). MCP-2.reactive protein. . endothelial cells. regulate the polymerization and depolymerization of actin in leukocytes for movement and migration. and stimulates the differentiation of naive T4-lymphocytes into interferon-gamma producing Th1 cells. monocyte chemoattractant protein (MCP-1). When produced in excess amounts. In addition. chemokines can damage healthy tissue as seen in rheumatoid arthritis. and septic shock. and dendritic cells through the lymph nodes and the spleen. IL-1 stimulates hepatocytes to produce acute phase proteins such as fibrinogen. macrophage activating factors: macrophage inflammatory protein (MIP-1a). epithelial cells and fibroblasts. interferons were found to consist of a family of proteins which share a unique antiviral property. Chemokines are produced by many cells including leukocytes. These same proteins also exert non viral functions such as those of an antiproliferative and immunoregulatory agent. CCR5) serving as the second binding factor for HIV on CD4+ cells. Examples of chemokines include IL-8. • interferon γ from T lymphocytes. increases the killing activity of CTLs and NK cells. caeruloplasmin and α1 antitrypsin. pneumonia. Certain chemokines have also been shown to suppress HIV. is chemotactic and induces release of lysozyme and lactoferrin. GRO-g.C. and function as chemoattractants for leukocytes. growth related oncogene (GRO-a).g. On inflammatory cells. and the molecule ‘regulated on activation-normal T cell expressed and secreted’ (RANTES). It functions as a stimulant for the synthesis of interferongamma by T-lymphocytes and NK cells. Chemokines also regulate the movement of B-lymphocytes. Certain non macrophage cells produce IL-1 like substances. Types of Interferons Interferons can be classified according to their primary cell or origin • interferon α from leukocytes. • interferon β from fibroblasts.Cytokines 109 muscle wasting and cachexia of chronic febrile illness. epithelial cells from the oral mucosa and cornea. IL-1 increases bone resorption and collagen synthesis and is important in fibrosis and wound healing. It induces cell-mediated immunity. they trigger some white blood cells (WBCs) to release their killing enzymes for extracellular killing and induce some WBCs to ingest the remains of damaged tissue. Tlymphocytes. GRO-b. Chemokines Chemokines are a group of cytokines that enable the migration of leukocytes from the blood to the tissues at the site of inflammation. Interleukin-12 (IL-12) IL-12 is a primary mediator of early innate immune responses to intracellular microbes. Type I Interferons Originally discovered in 1957 by Isaacs and Lindenmann. probably by binding to chemokine receptors (e.

The genes coding for the three interferons have been cloned and sequenced. and induce fever. β or γ is accompanied by increased expression of receptors for the Fc portion of the immunoglobulins (FcR). parasites and tumour cells. Interferon-alpha is produced by T-lymphocytes. Lymphoblastoid cells produce IFN α and β. Epithelial cells and macrophages also secrete interferon β. macrophages. the interferon . dendritic cell. Subsequent to binding. epithelial cells and endothelial cells. They also promote body defences by enhancing CTL. NK cells. These enzymes remain inactive until the uninfected cell becomes infected with a virus. Type I interferons also induce MHC-I antigen expression needed for recognition of antigens by CTLs. Interferon α is produced when leukocytes and lymphocytes are exposed to viruses. By increasing the intracellular proteolytic enzymes. CTL.receptor complex is internalized by receptor mediated endocytosis. macrophage. Type I interferons (IFN α and IFN β) are induced by viral infection or artificially by double stranded RNA (ds RNA). Interferons induce uninfected cells to produce enzymes capable of degrading mRNA. Type I interferons. viruses. antigen processing also gets enhanced. This endows the host with the capacity to destroy or detoxify invading agents. (a) On macrophages Interferons increase the bactericidal and tumoricidal capabilities of macrophages and augment their accessory function of antigen presentation. produced by virtually any virus-infected cell. T and B cells and large granular lymphocytes with natural killer cell activity. This not only blocks viral protein synthesis. All three interferons have been reported to either increase or decrease synthesis and secretion of multiple proteolytic enzymes by macrophages. NK cell. is stimulated by foreign antigen or mitogen. Type II interferon (discussed later). as this requires proteolytic cleavage of whole antigens. and B-lymphocyte activity. Immunoregulatory effects of Type I interferons The immunoregulatory effects of interferons in general are exerted on cells that carry out the hosts’ defence strategy . It consists of multiple sub species that are antigenically related. the enzymes are activated and begin to degrade both viral and cellular mRNA. This phenomenon is known as antibody dependant cellular cytotoxicity (ADCC). By recombinant DNA technology it has been shown that more than 20 distinct interferon α genes and polypeptides exist. Interferon β is antigenically distinct and is the predominant species elaborated by fibroblast cells. monocytes/ macrophages.the macrophages. it also eventually kills the infected cell. augment macrophage. There are several types of IFN β species. B-lymphocytes. This increased expression of FcR promotes both increased phagocytosis of immune complexes and the increased capacity of macrophages to lyse antibody coated bacteria. At this point. The activation of macrophages by IFN α. depending on the type of stimulus. Both α and β interferons are acid stable. Mechanisms of action of the interferons Interferons act by binding to specific receptors on the cell surface. NK cells. fibroblasts. also known as IFN γ or immune interferon. interferon-beta by virus-infected cells. provides an early innate immune response against viruses. NK cell and antibody-producing cell activity. .110 Immunology– Introductory Textbook They can also be classified as type I and type II according to the agents that stimulate their release.

genital warts. These include: increased expression of recognition structures on target cells. they may be increased during high fever.Cytokines 111 (b) On large granular lymphocytes and natural killer activity Natural killer (NK) activity is a term used to describe cytotoxic activity of large granular lymphocytes without prior sensitization with specific antigen. systemic lupus erythematosus and multiple sclerosis have an adverse effect on interferon production. however. it is the immune system’s attack on virus-infected cells that causes the illness. especially the lymphocytic leukaemias and those on chemotherapeutic drugs have a decreased ability to produce α and γ interferon. Alpha interferon works differently in each disease. a newer formulation is called Pegylated Interferon. as in the diagnosis of viral infections and tuberculosis using the ELISA or cytokine assays such as the ELISPOT (see Chapter 8). have decreased quantities of α and γ interferons. Serum interferon levels rise in a variety of viral infections and after immunization. a relatively rare blood cancer that usually strikes older men. Alpha interferon’s biggest impact came in 1991 and 1992. Although a newer drug has now taken its place. Anti tumour activities of interferon have been attributed both to anti proliferative effects as well as to augmentation of host anti tumour responses. There are two types of recombinant IFN-α available (2a and 2b). It is also used to shrink tumors and prolong survival for some AIDS patients disfigured by the purple lesions of Kaposi’s sarcoma. It has five approved indications in the U. — hairy cell leukemia. Interferon has been detected in CSF in cases of viral meningitis. when it was licensed for chronic hepatitis C and then for chronic hepatitis B. In hepatitis B. interferon lowers the virus population to a level where it no longer causes injury. Not all patients respond to interferon therapy. a cancer that primarily affects the skin. This drug was developed to counter the rapid breakdown of the original IFN α products. Diseases like uraemia. Therapeutic uses of interferon (a) Interferon alpha Interferon α was the first of the interferons to be used successfully in clinical therapy. All three interferons enhance NK cell activity in large granular lymphocytes. increased metabolic activity and the production of cytolytic granules by large granular lymphocytes. as in transplant patients. Patients with malignant lesions. Studies have shown that neonates have the ability to produce α and β interferons. Patients receiving powerful immuno suppressives. In hepatitis C the virus invades and destroys liver cells. some of them relapse when the drug is stopped. . AIDSrelated Kaposi’s sarcoma. Serum of patients with autoimmune disease show raised levels of IFN γ. certain tumour cell lines and normal effete haemopoietic cells. Interferon helps by stimulating immune cells that in turn repel the invasion. NK activity is primarily directed against virus infected cells. production of γ interferon is usually deficient. increased binding between these lymphocytes and target cells. Interferon assays Human interferon assays can be used for diagnostic purposes. There are several theories regarding the mechanisms by which these cells become more cytocidal under the influence of interferon. alpha interferon was for a time the main treatment for hairy cell leukemia.S. Animal studies have demonstrated that interferon exerts in vivo anti tumour activity. chronic hepatitis B and hepatitis C.

diarrhoea. hobbies. IL-6 induces differentiation of cytotoxic T cells. Magnetic resonance imaging showed a marked decrease in the number of new brain lesions in these patients. macrophages. regulates innate immunity and cell-mediated immunity. a rare. In addition. Preliminary indications are that alpha interferon may benefit patients with early stage melanoma. insomnia. respond especially well to a combination of alpha interferon and retinoids. IL-10 is produced mainly by macrophages. (c) Interferon gamma Finally. which are used now to treat acne. Side effects of interferon therapy The most common side effect is that of flu-like symptoms with the first few injections. the most common sexually transmitted viral disease. leprosy. It stimulates the liver to produce acute phase proteins. IL-6 activates T cells and induces their proliferation. some skin cancers. endothelial cells and fibroblasts. It is also used to treat malignant osteoporosis. and MHC-II molecules. and increases neutrophil production. A more serious side effect is depression. and half as many severe ones. irritability. particularly if there is a history of depression. sex. as people who took a placebo. or excessive sweating may be the warning signs. IL-6 aids B cell proliferation and induces the terminal maturation of B cells into immunoglobulin . Side effects of treatment were minimal. and Th2 cells. all of which are needed for cell-mediated immunity. monocytes. The drug also appears to be a team player when combined with other agents. renal cell carcinoma. the virus is thought to be a precursor for cervical cancer. (b) Interferon beta Ambulatory patients with relapsing-remitting multiple sclerosis who took high doses of beta interferon had about 30% fewer attacks. histoplasmosis.secreting cells.The formation of granuloma in infections such as tuberculosis.112 Immunology– Introductory Textbook Alpha interferon’s antiviral action has been impressive against the human papilloma virus in condylomata acuminata (genital warts). the thyroid gland may become over or underactive. and coccidioidomycosis is a cytokine-mediated . gamma interferon is used to treat chronic granulomatous disease. IL-6 is produced by many cells including T-lymphocytes. Rarely. In women. loss of interest in food. Extreme fatigue or insomnia. and multiple myeloma. irritation at injection site and weight loss. etc. Interleukin-6 (IL-6) Interleukin-6 is a multifunctional lymphokine that regulates immune responses. treated patients were hospitalized less often. spells of crying. Typical symptoms of depression are unexplained sadness. Interferon may affect the bone marrow and can be severe. As a result. inherited immune disorder found mostly in young men. Other troublesome side effects may include nausea. the acute phase reaction and haematopoiesis. chronic myeloid leukemia. IL-10 inhibits their production of IL-12. costimulator molecules. early morning awakening. For the most part these are reversible with time and occasional withholding of the medication. Interleukin-10 (IL-10) IL-10 is an inhibitor of activated macrophages and dendritic cells and as such. thinning of hair. Medical researchers are actively testing interferons — primarily alpha — in more than 150 clinical trials involving patients with various cancers or HIV infection. In association with IL-2 and IFN γ. with about 75% of patients on a high dose reporting mild flu-like symptoms that diminished over time.

Receptors for IL-2 have also been found on B cells. IL-2 needs to couple with its receptor. IL-2 action on non T lymphocytes Although they lack T cell markers. seem to exert their effects primarily by inhibiting IL-2 gene expression. T helper and T cytotoxic subsets of T cells can both produce IL-2. Included in this category are glucocorticoids. IL-2 activated T cells also exhibit enhanced cytotoxicity showing that besides clonal expansion. they produce factors for B cell growth and differentiation. The resulting mass is called a granuloma and is an attempt by the body to “wall-off” or localize the infection. Interleukin-4 (IL-4) Interleukin-4 is a 20kd glycoprotein that affects many cell types including B cells. colony stimulating factor and interleukin -3. It pushes B cells out of the resting phase into activation. there is a continuous secretion of cytokines and chemokines that leads to an accumulation of densely packed macrophages around the microbes. Resting lymphocytes do not bind to IL-2. cyclosporine and prostaglandin E2. Three signals are necessary for IL-2 release : MHC restriction. also respond to IL-2. B. Because macrophages have difficulty in removing the microbes that cause these infections. Large granular lymphocytes also secrete IL-2. Impaired production of IL-2 has been reported in several patients with deficiencies in cellular immunity. These cytokines function in the proliferation and differentiation of B-lymphocytes and T-lymphocytes after antigen recognition. Interleukin-2 (IL-2) The cellular origin of IL-2 is the mature T lymphocyte. large granular lymphocytes that exhibit natural killer cell activity (see Chapter:14). produce lymphokines and exhibit enhanced natural killer activity.Cytokines 113 cellular response. The macrophages release fibrogenic cytokines such as TNF and IL-1 that lead to the formation of granulation tissue and scar tissue. as in leprosy. The secretion of IL-2 by an activated T cell is strictly regulated by external stimuli (antigen provocation). It induces B cells to express MHC Class II .6). IL-2 also promotes cellular function such as delayed hypersensitivity and T cell cytotoxicity. AIDS and in patients with metastatic cancers. Finally macrophages can also be induced to become cytocidal with large doses of IL-2. Cytokines that Regulate Adaptive Immune Responses (Humoral and CellMediated) Cytokines that regulate adaptive immunity are produced primarily by T-lymphocytes that have recognized an antigen specific for that cell. The pathway to T cell activation and the pivotal role played by IL-2 has been discussed in the previous chapter. Several substances that have be shown to be suppressive. Functions of IL-2 on T cell clones The addition of IL-2 to activated T cells promotes proliferation. Upon appropriate activation. interaction of antigen and the T cell receptor and IL-1 (Figure 12. The development of a response to IL-2 requires denovo acquisition of membrane receptors for IL-2. For T cell proliferation to occur. These lymphocytes begin to grow.The IL-2 dependant T cells–predominantly T helper cells -produce other lymphokines such interferon. IL-2 can increase antibody production as well as proliferation in these cells. T lymphocytes develop IL-2 receptors in 6 hours.

neutrophils. Under certain conditions TGF-β will demonstrate anti-proliferative effects on endothelial cells. increases and induces IL-1 release from monocytes.114 Immunology– Introductory Textbook antigens. CD23 which is a low affinity receptor for IgE. adipogenesis and adrenal steroidogenesis. Transforming growth factor-beta (TGF-beta) TGF-β has a proliferative effect on many mesenchymal and epithelial cell types.and B-lymphocytes. It also induces the production of MHC-I molecules. including IFN – γ. CD8+ cells. stimulates the production of IgG subclasses that activate the complement pathway and promote opsonization. and IL-4 receptors. and exerts weak antiviral activity. Several members of the TGF-β family are potent inducers of mesodermal differentiation in early embryos. IL-4 inhibits the switch from IgM to IgG. inhibits macrophages. IFN-γ stimulates the differentiation of T4-lymphocytes intoTh1 cells and inhibits the proliferation of Th2 cells. IFN-gamma is the principal cytokine for activating macrophages. IL-5 is produced mainly by Th2 cells. IFN γ more than the other two interferons. Interleukin-5 (IL-5) IL-5 is a growth and activating factor for eosinophils as a defence against helminths and arthropods. IL-4 is a major stimulus for production of IgE and the development of Th2 cells for defence against helminths and arthropods. and NK cells as part of the immune response and functions mainly to promote the activity of the components of the cell-mediated immune system. Antibody synthesis is also influenced by IL-4. C. Interleukin-13 (IL-13) IL-13 increases the production of IgE by B-lymphocytes. and T. Cytokines that Stimulate Haematopoiesis Produced by bone marrow stromal cells. . Interferon γ is an acid labile molecule. IL-13 is made primarily by Th2 cells. and co-stimulatory molecules by APCs in order to promote cell-mediated immunity (activation of T lymphocytes requires that the T cells recognize antigen in conjunction with Class I and II MHC molecules). Interferon gamma Type II interferon. There is only one type of IFN γ. and increases mucus production. macrophages. MHC-II molecules. Such effects include decreasing the secretion of immunoglobulin and suppressing hematopoiesis. augments or inhibits other cytokine activities. IL-4 is produced mainly by Th2 cells and mast cells. thus augmenting and amplifying immunologic responses. i. IFN γ. For interferon assays and therapeutic uses of interferons. It also stimulates the proliferation and differentiation of antigen-activated Blymphocytes and the production of IgA.e. these cytokines stimulate the growth and differentiation of immature leukocytes. and NK cells. is produced primarily by Th1 cells. It also antagoizes the effects of interferon-gamma and thus inhibits cell-mediated immunity. it also activates and increases the antimicrobial and tumoricidal activity of monocytes. myogenesis. refer discussion on Type I interferons above. macrophages.

It is produced by T cells and affects growth of macrophages. enhances their phagocytosis and extracellular killing. Both G-CSF and GM-CSF are available commercially. Il-7 is produced mainly my fibroblasts and bone marrow stromal cells. They are used primarily to decrease neutropenic infections and the resulting morbidity and mortality as a result of chemotherapy and or total body irradiation in bone marrow transplant recipients. and increases both superoxide anion generation and antibody-dependent cytotoxicity. For example. GM-CSF increases adhesion of these cells to capillary walls during diapedesis. granulocyte colony stimulating factor (G-CSF). IgE and class II MHC expression on B cells. inflammation and fever. it activates these cells and inhibits their apoptosis. It induces proliferation but not maturation of certain B cell progenitors. Interleukin-7 (IL-7) Interleukin-7 affects early B cells. eosinophil and mast cell growth and function. In addition to their role in promoting production of leukocyte colonies. eosinophils. NK cells activated T cells Th2 and mast cells Primary Activity Co-stimulation of APCs and T cells.1 Interleukins and interferons: primary source and principal activity Interleukins IL1 Principal Source macrophages and other antigen presenting cells (APCs) activated Th1 cells. granulocytes and host cells. inhibition of monokine production eosinophil growth and function IL-2 IL-3 IL-4 IL-5 Th2 and mast cells . and monocytes. The various CSFs are produced by Tlymphocytes. haematopoiesis proliferation of B cells and activated T cells. when GMCSF binds to receptors on neutrophils.Cytokines 115 Colony-stimulating factors (CSFs) Colony stimulating factors promote the production of colonies of the different leukocytes in the bone marrow and enhance their activity. acute phase response. Stem cell factor Stem cell factor makes stem cells in the bone marrow more responsive to the various CSFs. NK functions growth of haematopoietic progenitor cells B cell proliferation. It also stimulates the proliferation of early T cells and augments some in vitro T cell responses. in patients with haematological and other malignancies. It is made mainly by bone marrow stromal cells. Examples include granulocyte macrophage colony stimulating factor (GM-CSF). and macrophage colony stimulating factor (M-CSF). Interleukin-3 (IL-3) Interleukin-3 is also called multicolony stimulating factor (multi-CSF). and other cells. Table 13. macrophages. the CSFs also appear to promote their function.

INF-γ production. B cells. promotes cellmediated immunity. neutrophils and some somatic cells activated Th1 and NK cells Interferons IFN α and β Primary Activity antiviral effects. activation of NK cells and macrophages induces class I MHC on all somatic cells. mast cell growth synergisitc haematopoietic thrombopoietic effects and IL-7 IL-8 IL-9 IL-10 IL-11 IL-12 stromal cells B cells. other somatic cells thymic and marrow stromal cells macrophages. promotes cell-mediated immune functions IL-4-like activities IL-13 Th2 cells Principal Source macrophages. APCs. B cell proliferation. induction of class I MHC on all somatic cells. macrophages proliferation of NK cells. activates macrophages. NK cells. antiviral effects IFN γ . thrombopoiesis. suppresses cellular immunity. macrophages CD8+ T and Immunology– Introductory Textbook Primary Activity acute phase response.116 Interleukins IL-6 Principal Source activated Th2 cells. other somatic cells T cells activated Th2 cells. induces class II MHC on APCs and somatic cells. promotes B cell proliferation and antibody production. synergistic with IL-1 and TNF on T cells T and B lymphopoiesis chemoattractant for neutrophils and T cells haematopoietic and thymopoietic effects inhibits cytokine production. neutrophils.

activate neutrophils. complex structures such as parasites and the body’s own cells when they became “effete” or turned malignant. stimulate . In all of these instances. A short summary will be provided here. cells with intracellular bacteria and cancer cells displaying tumour antigens. Activating macrophage. W 1. It was soon realized that this form of defence was woefully inadequate when it came to mounting an attack on intra cellular bacteria and viruses. Collectively these cytokines enable T8-lymphocytes to proliferate and differentiate into cytotoxic T-lymphocytes capable of destroying infected host cells and mutant cells. interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNFbeta). activate macrophages enabling them to destroy intracellular pathogens.1) by producing cytokines such as interleukin-2 (IL-2). 2. Cell mediated immunity involves three major defence strategies: 1. scholars in immunology felt that the complete defence strategy of the host against invading micro organisms had finally been worked out. Activating antigen-specific cytotoxic T-lymphocytes (CTLs) that are able to lyse body cells displaying epitopes of foreign antigen on their surface. This has been discussed in detail in Chapter: 12.CHAPTER – 14 CELL–MEDIATED IMMUNITY hen the potent defence mechanism available in antibody mediated immunity was fully elaborated. Cell mediated immunity has therefore been targeted to: • killing intracellular organisms • destruction of tumour cells • rejection of graft tissue and • a delayed type hypersensitivity reaction after contact with certain antigens such as poison ivy and certain topically applied drugs. 3. These have been detailed in Chapter13 and will be summarised here. Stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses. NK cells and antibody dependant cytotoxicity enabling destruction of intracellular pathogens. antigen is not overt but masked within the body’s own cell systems. Examples are virus-infected cells. Activating Antigen-specific Cytotoxic T-lymphocytes (CTLs) The key stages involved in cell mediated immunity are: Secretion of lymphokines by Th1 lymphocytes of the T4 lymphocyte subset Th1-lymphocytes recognize antigens presented by macrophages and function primarily to activate and heighten cell-mediated immunity (Figure: 14. activate cytotoxic T-lymphocytes and NK cells. stimulate the production of opsonizing and complement-activating antibodies for enhanced attachment during phagocytosis.

a schematic representation. For lysis to occur the target cells must carry both the same viral antigen and the same MHC Class I antigens as the cells that originally induced the proliferation of T cytotoxic cells (Figure 14. The cell mediated immune response. hence it is essential that cytotoxic T cells have a wide repertoire of target cells against which they can mediate their killing action. Cytotoxic T cells kill virally infected target cells by direct cell lysis otherwise known as apoptosis or programmed cell death. T cell . The MHC Class I antigens appear to be ideal recognition antigens triggering cytotoxic T cells because they are present on almost all cells. T cytotoxic cells may thus.1. The T cytotoxic cells recognize antigen in conjunction with MHC Class I molecules.2. A wide variety of cells can become virally infected or turn malignant.cytotoxicity .2). Figure 14. and function as chemoattractants for phagocytes. are stimulated by Th1 helper cells and then turn cytotoxic. this happens just after a few hours of viruses infecting cells—before the viruses can replicate.118 Immunology– Introductory Textbook increased production of monocytes in the bone marrow. . Figure 14. be the major effector pathway of cell mediated immunity to certain viruses. Cytotoxic T lymphocyte (CTL) activity Antigen recognition by cytotoxic T cells and their subsequent activation has been greatly discussed in previous chapters.

All this happens only when T cell -target cell contact is established and is accompanied by an explosive increase in Ca++ (Figure 14. These granules hold pore-forming proteins called perforins and proteolytic enzymes called granzymes in a protected state. The reason being. and chemokines. binding to it and then exercising some effect that finally causes cell death. more accurately. immediately polymerizes. . that only the perforin monomer can insert into the target cell. they lyse within minutes. C7. The perforin molecules secreted by the killer cell insert into the target cell membrane (Figure 14. Complement components. There.4). There is now increasing evidence that. secreted by both T cytotoxic cells and NK cells. When the TCR and CD8 of the CTL binds to the MHC-I/epitope on the surface of the virus-infected cell. The killer cell uses exocytosis to fire a lethal agent contained in its granule into the target cell membrane. Significant homology has been found in the sequence of amino acids between C9 and perforin. It has been shown that a unique pore forming protein is part of the armamentarium of T cytotoxic cells and NK cells. the individual monomeric molecules polymerize into a product that resembles a cylinder or a ring measuring 5-20 nm in diameter. and in the presence of Ca++ ions. the killing activity is completely abolished. C8 and several C9 molecules polymerize to form pores much like those wrought by perforin. For some years it has been clear that killer cells do their job with great efficiency. This results in an exocytosis of contents from the killer cell granules now situated at the plasma membrane just apposing the target cell. It is now clear that having bound to its victim. It is now clear that early in the cell-killing process. On the target cell the tubular pores cause leakage of water an ions and finally cell death by lysis. where calcium is abundant. though the pore forming protein or perforin is an important method of cell lysis ( or it would not have evolved at all!). The granule packaging organelle of the CTL . within the cell membrane. calcium mediated polymerization must take place entirely within the target cell membrane. virtually eliminating “accidental” injury to bystander cells. while sparing innocent bystander cells. C6. These molecules are antigenically related to the tumour necrosis factor (TNF). The CTLs contain granules composed of proteoglycans to which chemokines are complexed. several other slow killing molecules (granzymes and caspases) have also been identified. A lymphotoxin like activity has been detected in T cells. C5. Once cells are exposed to this 70 kd protein called perforin. This projectile molecule has now been identified as a pore forming protein and is often called “perforin”. the killer cells shoots it full of holes. it fires molecules of a lethal protein at the target cell.Cell–Mediated Immunity 119 Mechanism of cell lysis CTLs and NK cells induce apoptosis by at least 2 different pathways: The first and most effective pathway involves intracellular granules. Any secreted perforin that spills into the extra cellular space or blood stream. in the presence of calcium ions. granules in the killer cell become concentrated in the part of the cell closest to the target. first seeking a miscreant target cell. it is not the central component in lymphocyte mediated killing. It has been shown that empty granules are just shell casings for the projectile. An interesting parallel has been drawn between perforin and the terminal proteins of the complement cascade. granzymes. Perforin synthesis has been shown to be stimulated in the presence of interleukin-2. this sends a signal through the CD3 molecule which triggers the release of the perforins.3). On the other hand if calcium ions are added to perforin before it makes contact with the target cell. For perforin to damage the target cell.the Golgi apparatus gets directed towards the contact region and the cell’s cytoskeleton is also reoriented towards the target cell.

in which the granules fuse with the cell membrane (2) and disgorge their perforin (3) into the small intercellular space abutting the target. can then activate the caspase enzymes that lead to apoptosis of the infected cell. Calcium there changes the conformation of the individual perforin molecules. degrade the cell’s nucleoprotein and activate enzymes that degrade DNA.) The second pathway by which CTLs can trigger apoptosis of the infected cells is through FasL/Fas interactions.120 Immunology– Introductory Textbook Figure 14. On contact the cell’s granules and the Golgi complex that forms them are reoriented toward the target cell. brings about exocytosis.3. HIV cannot adsorb. The perforin . Certain granzymes. in turn. perforin is secreted and forms pores in the target-cell membrane. which then bind to the target-cell membrane (5) and insert into it (6). Having launched its lethal missiles. the damaged target cell dies a programmed death within minutes (4). The perforin pores allow granzymes to enter.4. The monomers polymerize like staves of a barrel (7) to form pores (8) that admit water and salts and kill the cell. A rise in the lymphocyte’s calcium-ion level. the killer cell withdraws and goes on to kill again (3). The killer lymphocyte (1) recognizes the target cell and makes close contact with it (2). or monomers (4). Figure 14. Some activated T8-lymphocytes also secrete chemokines (see Chapter 13) that suppress replication of HIV in T4-lymphocytes. When that receptor is occupied by its natural chemokine. CTLs have a receptor called FasL that can interact with Fas molecules . (1).mediated killing mechanism adopted by the killer lymphocyte. (a chemokine receptor is a cofactor for adsorption of HIV to CD4+ cells. The caspases are proteases that destroy the protein structural scaffolding of the cell (the cytoskeleton). Target cell membrane damage by perforin.

• increases the production of B7 co-stimulator molecules (see Chapter 12) and MHC–1 molecules by macrophages for increased T-lymphocyte activation. IL–12 enables naive T4–lymphocytes to differentiate into Th1 cells. and IL–12. the cell breaks into fragments that are subsequently removed by phagocytes. and hydrolytic lysosomal enzymes enabling the killing of microbes within their phagolysosomes. They bind to specific receptors on the macrophage causing its activation. the sensitized Th1cells are triggered to release several chemokines which attract more macrophages. the activated enzymes that degrade host DNA can also destroy microbial DNA and thus kill infectious microbes within the cell. NK Cells and Antibody Dependant Cytotoxicity Activated macrophages Activated macrophages are the effector cells in the cell mediated immune response to various micro organisms. This triggers the Th1 cells to secrete several cytokines including interferon-gamma (IFN-gamma). nitric oxide. Mechanisms of Evading Cell-Mediated Immunity A high rate of mutation in some viruses like HIV and the hepatitis C virus (HCV) changes the amino acid sequence and. Many viruses such as the cytomegalovirus (CMV) and adenoviruses and some tumour cells can block the formation of MHC-I molecules by the infected cell. CTLs are not destroyed in these reactions. For example a macrophage that has been activated to kill intra cellular Listeria monocytogenes would also kill Salmonella organisms more efficiently. they can function over and over again to destroy more virus-infected cells. Co-stimulatory molecules such as CD40L on the Th1 cell then bind to CD40 on the macrophage (Figure 12. granulocytes and lymphocytes to the site of the reaction. Epstein-Barr virus (EBV) down regulates several host proteins involved in attaching viral epitopes to MHC-I molecules and displaying them on the host cell’s surface. In addition. Death by apoptosis does not result in release of cellular contents such as inflammatory mediators or viruses as does cell lysis. • causes the macrophages to secrete cytokines such as TNF–alpha. As a result.5). This FasL/Fas interaction triggers an intracellular transduction that activates the caspase enzymes and leads to destruction of the cytoskeleton and chromosome in the infected cell. This reduces inflammation and also prevents the release of viruses and their spread into uninfected cells. 2. the CTLs are no longer able to recognize that the cell is infected and cannot kill it. the shape of key epitopes. Activating Macrophages. . Activation of macrophages leads to: • increased production of toxic oxygen radicals. CTLs with TCRs made against the earlier strains of these viruses may no longer bind to and lyse cells infected with mutated strains. The ability of activated macrophages to kill micro organisms is enhanced in a non specific fashion. TNF–alpha and IL–1 promote inflammation to recruit phagocytic leukocytes. Adenoviruses and EBV code for proteins that block apoptosis of the viral infected cell. Instead.5). processes the antigen and then presents the antigen on its surface in association with MHC Class II molecules. On a subsequent presentation of the same antigen by another macrophage.Cell–Mediated Immunity 121 found on the surface of most cell types. In this effector pathway the macrophage ingests the micro organism. therefore. to Th1 helper lymphocytes (Figure 14. IL–1.

Cytokines such as interleukin–2 (IL–2) and interferon-gamma (IFN-gamma) produced by Th1 lymphocytes activate NK cells. the killer-inhibitory receptor sends a negative signal that overrides the kill signal and prevents the NK cell from killing that cell. and this sends a positive signal which enables the NK cell to kill the cell to which it has bound unless the second receptor cancels that signal. The main target of NK cells are tumour cells and certain virally infected cells. They cause lysis and apoptosis of the target cell by mechanisms similar to those described for CTLs above. . This second receptor. they become super killers. NK cells appear to use a dual receptor system in determining whether to kill or not kill human cells. Figure 14. These cells appear to have both anti tumour and anti microbial activity in vivo. NK cells also secrete the cytokine IL–1 and in response to IL–2. If MHC–I molecules are expressed on the cell. Role of activated macrophages in cell mediated immunity. called the killer-ihibitory receptor recognizes MHC–I molecules which are also usually present on all nucleated human cells.122 Immunology– Introductory Textbook • IFN–γ produced by Th1 cells also increases the production of opsonizing and complement activating IgG to promote enhanced attachment of microbes to phagocytes. called the killer-activating receptor can bind to a number of different molecules usually present on nucleated human cells.5. NK cells do not require prior exposure to antigen to become cytotoxic and their activity is not MHC restricted. Role of NK cells in the cellular immune response. The first receptor. Natural killer cells Natural killer cells are large granular lymphocytes that carry the cell surface markers CD56 and CD2.Viruses often suppress class I Figure 14.6). Once the NK cells are activated however. NK cells attach to their target cells by a receptor in a calcium independent manner (Figure 14.6. they require calcium for lysis.

leading to formation of pores in the target cell membrane.Cell–Mediated Immunity 123 MHC expression in cells they infect. the virus-infected cell therefore becomes susceptible to killing by NK cells.7. for example. and therefore. are effective killers of antibody coated schistosomulae. the larval forms of schistosomes. on the target cell (Figure 14. via the Fab portion. The organism dies because of the leaky membrane. K cells have receptors on their surface for the Fc portion of IgG. The antibody. Eosinophils can be activated by both monokines and certain lymphokines. However. Eosinophils. The antibody acts like a bridge between the specific antigen bearing target cell and the K cell. . The eosinophil releases basic proteins and other molecules from its granules. Figure 14. NK cells are also unable to kill this infected cell because it is still displaying “MHC-I molecules” on its surface. Monocytes. Antibody dependant cell mediated cytotoxicity. The K cell then releases poreforming perforins. Tumour cells have reduced or no class I MHC expression. Antibody-dependant cell mediated cytotoxicity NK cells are capable of antibody-dependant cellular cytotoxicity (ADCC). become susceptible to killing by NK cells.7). neutrophils and eosinophils can also participate in ADCC. The cytomegalovirus (CMV) evades the immune system because it can trigger its host cell to produce altered MHC–I molecules that are unable to bind viral epitopes. are not recognized by CTLs. proteolytic enzymes called granzymes and chemokines that cause cell apoptosis and cell lysis similar to the mechanism of killing described for CTLs. The mechanism of killing involves binding of the eosinophil to the larva via an antibody molecule. When NK cells are carrying out ADCC. they are sometimes also referred to as killer K cells (K cells). binds to an Fc receptor on the K cell and to an antibody combining site. they too. usually specific IgG against ‘foreign’ cell antigen.

some forms of cancer. Interleukin-6. Tumor Necrosis Factor (TNF) is made predominantly by monocytes and macrophages following stimulation with bacterial LPS (TNF α) or by activated CD4+ T-cells (TNF β). has 2 major functions: to mediate inflammation. Included in this group are the Type I Interferons (IFNa and IFNβ). is produced by monocytes. tumor necrosis factor (TNF).124 Immunology– Introductory Textbook 3. that enhance the cells’ ability to kill micro organisms and tumour cells. and interleukin-8. and heat. TNF has many biologic functions including killing tumours and inducing secretion of other inflammatory cytokines. interleukin-3. IFN α has been used to treat HIV. A variety of cytokines affect cellular immunity by • mediating the innate immune response • regulating haematopoiesis • directly influencing cell mediated immunity Cytokines that affect the inflammatory response generally up-regulate innate immunity. whereas others play a critical role in antibody responses. These cytokines also play a role in mediating resistance to viral infections and causing the cardinal signs of inflammation: pain. It is probably most important in inducing the production of acute phase proteins by the liver. Interleukin-11 is produced by bone marrow stromal cells and shares the functional activity of IL-6. Interleukin-1 is also made by cells of the monocyte-macrophage cell lineage. interleukin-6. so it induces fever. swelling. The best characterized of these are granulocyte/monocyte-colony stimulating factor (GM-CSF). MIP-1b. IL-1 is also a costimulator of CD4+ T-helper cells. MCP-2. but it typically appears somewhat later in an inflammatory response. In addition. like IL-1. and IFN β is made predominantly by fibroblasts. It is also produced by macrophages and might be anti-inflammatory. MCP-3 enable migration of these cells to the site of inflammation. It has many of the same functions as TNF. Interleukin-8. and multiple sclerosis. Other chemokines such as macrophage inflammatory protein (MIP-1a). IL-6 has a function similar to TNF in that it induces the synthesis of acute phase reactants by the liver. It also induces adherence of neutrophils to vascular endothelial cells and aids their migration into tissue spaces. Secretion of cytokines Cytokines are soluble substances produced by a variety of cell types that amplify and regulate a range of immune responses (Chapter 13). IL-8 is a chemoattractant for neutrophils. IL-6 also serves as a growth factor for plasma cells. Type I IFN has several sources and several effects. monocyte chemoattractant protein (MCP-1). Some cytokines are crucial in cell mediated immune reactions. IFN α is made predominantly by neutrophils. and to regulate the growth and differentiation of lymphocytes. For simplicity. They also induce certain morphologic. IL-1 is an endogenous pyrogen. Type I IFNs include IFN α and IFN β. granulocyte-colony stimulating factor (G-CSF). metabolic and functional changes in macrophages. stem cell factor (SCF). It also induces fever. know that Type I IFN inhibits viral replication in virus-infected cells. interleukin-1. This is one of the first cytokines that appear during an inflammatory response. redness. Like TNF. Several cytokines play a major role in haematopoiesis in the bone marrow. interleukin- .

and serve as an autocrine growth factor for T-cells. In addition. In appears to increase erythropoiesis and mast cell division. Stem Cell Factor is also produced by bone marrow stromal cells. Activated macrophages are an important mediator of cellular immunity. GM-CSF and G-CSF. Some of these cytokines are being used to treat patients that have deficiencies in haematopoiesis. Interleukin-7 is produced by bone marrow stromal cells. it produces more cytokines. Interleukin-2 is the primary growth and differentiation factor for T-cells. and becomes more bactericidal than resting macrophages. It induces lymphoid stem cells to differentiate into progenitor B cells. others on both T and B cells. Activation of T cells requires the presence of antigen. An activated macrophage is more phagocytic. IL-12 enhances IFN production. Cytokines serve as ‘second signals’ to drive the growth and differentiation of antigen-activated lymphocytes. For clinical tests used to assess cellular function see Chapter 22. IL-12 may prove to be a key cytokine in enhancing cell mediated immunity. such as cancer patients who are bone marrow suppressed due to anti-cancer therapy. Type II Interferon or Interferon-γ (IFNγ) is produced by T-helper cells. NK-cells. IL-12 is produced by a number of cells including B-cells. Under some conditions IL-7 stimulates the proliferation of thymocytes. and macrophages. Interleukin-12 has the opposite effect of IL-10. it processes and presents antigen more efficiently. and neutrophils)(G-CSF). Interleukin-3 is also produced by T-helper cells and increases the production of basophils and progenitor cells. IL-2 causes antigen-primed T-helper cells to proliferate. GM-CSF and G-CSF are produced by T-helper cells and promote the production of granulocytes (basophils. Specific cellular immune responses are mediated by T-lymphocytes. it causes antigen-primed cytotoxic Tcells to proliferate and become aggressively cytotoxic.Cell–Mediated Immunity 125 7 and interleukin-9. IL-2 is produced by CD4+ T-helper cells. Some cytokines act predominantly on T-cells. It is an important cytokine for activating macrophages. IFNγ acts antagonistically against other cytokines such as IL-4. It synergizes with a number of hematopoietic growth factors including IL-7. as well as monocytes (GM-CSF). . Interleukin-9 is produced by T-helper cells. eosinophils.

It is characterized by an explosive response occurring within minutes of applying a stimulus and can be generalized or localized. Such reactions have been aptly termed “hypersensitivity” reactions. It is thought that basophils play a major role here. there is no obvious effect. Nothing but good should come out of a system so precisely planned to weed out aliens and marauders. These agents cause the early phase of allergic reactions that appears within minutes after exposure to the antigen. such as indomethacin. III. However. gross tissue damage can occur. Late phase allergic reactions may begin several hours after exposure to antigen. increased vascular permeability and increased mucus secretion. The reactions are mediated by the release of pharmacologically active substances from mediator cells. This. A Type I: Anaphylaxis The most rapid hypersensitivity reaction of the immediate type is known as anaphylaxis. Type IV hypersensitivity involves lymphocytes and other cellular components and requires a longer time course to manifest. block only the late response. That the early and late phase responses are caused by distinct mediators can be shown with inhibitory drugs. type IV has been termed delayed type or cell mediated hypersensitivity.complex mediated disease Type IV : Delayed type or cell mediated hypersensitivity Types I. however. Coombs and Gell classified hypersensitivity into four types Type I : Anaphylaxis Type II : Antibody dependant cytotoxicity Type III : Immune . it is painfully evident that reactions to putative foreign antigens may be excessive and that if humoral or cellular immunity is switched on to “high” for any length of time. Because of this. The primary action of these mediators results in contraction of smooth muscle. is called the .CHAPTER – 15 HYPERSENSITIVITY t first glance it would appear as if all immunity: humoral and cellular was geared towards defending the host against unnatural antigens. II. Sodium cromoglycate which blocks mast cell activation and degranulation blocks both early and late responses. Arachadonic acid metabolism inhibitors. Cell-bound IgE on the surface of basophils of sensitive individuals binds a substance called histamine releasing factor (possibly produced by macrophages and B-lymphocytes) causing further histamine release. Generalized anaphylaxis When a small dose of antigen such as egg white or heterologous animal serum is injected into a guinea pig for the first time. There are two categories of adaptive hypersensitivities: immediate hypersensitivity and delayed hypersensitivity. and IV are reactions which involve the humoral system of antibodies and are sometimes termed “Immediate hypersensitivity”.

In severe cases.) reaction or test. The second dose of the antigen has cross linked IgE antibodies situated on mast cells and basophils and caused degranulation. and a lower number of Th1 cells that produce gamma-interferon.Hypersensitivity 127 sensitizing dose which. the shock organ is the heart and right sided heart failure is the main cause of death. diarrhoea and respiratory distress. Possibly this is due to a higher number of Th2 cells which produce IL-4. that is characteristic of this reaction may be demonstrated by the use of tracer dyes such as Evans blue which leak out of the vessels at the reaction site and cause “blueing” of the surrounding tissues. After a latent period (allowed for IgE to fix to mast cells). Histamine. It consists of a localized swelling and redness . The IgE fixes onto the local mast cells in the skin. vomiting. The manifestations of anaphylaxis vary in different species. These reactions are mediated by histamine and serotonin and are quickly inactivated by plasma histaminases.K. serum containing sensitizing antibody (IgE). the lungs are characteristically over inflated. test is based on experiments done by the two scientists after whom the test is named. a cytokine that can increase production of IgE. Blueing of the skin appears within minutes. Kustner was known to be allergic to fish. together with other mediators released from mast cell or basophil granules causes marked vasodilation and leakage of intra vascular fluids resulting in shock.K. abdominal cramps. . The P-K test demonstrates definitively. collapse and die. On post mortem. asphyxiate. In humans. may convulse. laryngeal oedema. Passive Cutaneous Anaphylaxis Localized anaphylaxis can be passively transferred and forms the basis of the PrausnitzKustner (P. The guinea pig will scratch. for allergy to a wide variety of antigens is an example of this phenomenon. cough. causes degranulation and the resultant vascular permeability leads to blueing of the surrounding tissues. means that the animal has produced IgE antibodies to the antigen and these IgE antibodies are now attached by specific Fc receptors to mast cells and basophils.K. the levels of IgE may be thousands of times higher than in those without allergies. The local increase in vascular permeability. Only a timely intravenous injection of adrenaline to counter smooth muscle contraction and capillary dilatation can prevent death. The P. results in a dramatic onset of the generalized anaphylactic reaction. In the rabbit. that IgE antibodies are responsible for cutaneous anaphylaxis and that this type of anaphylaxis can be passively transferred.a wheal and flare reaction. vascular collapse and death can occur. generalized anaphylaxis presents with itching erythema. a local anaphylactic reaction will occur within a few minutes. Serum from Kustner which presumably contained IgE antibodies to fish was injected into Prausnitz who was not allergic to fish. antigen is administered intravenously with Evans blue. A second injection of the same antigen given a week or ten days later. a cytokine that decreases IgE production. every time he ate fish! In the P. in effect. go into extreme bronchoconstriction. Local or Cutaneous Anaphylaxis Upon injection of antigen into the skin of a sensitized animal. is injected intradermally into a normal individual. sneeze. Skin tests in man. In allergic individuals. This happens because antigen cross links the IgE molecules on the mast cell surface. test. The test is not done now for fear of transmitting AIDS or hepatitis B virus. After an obligatory latent period it could be demonstrated that Prausnitz developed a local reaction at the site of injection.

On second exposure to the same antigen. These events activate protein kinase C. In passive cutaneous anaphylaxis pre-made antibodies from another individual are used to elicit the reaction. . Bridging of adjacent Fab sites on the IgE molecules is rapidly followed by the breakdown of phosphatidyl inositol to inositol triphosphate (IP3). The animal displays no symptoms but is now considered sensitized (Figure 15. Bits of lung or skin can be used to demonstrate the same effect.1 b). which is due to degranulation of tissue mast cells and release of vaso-active amines. the animal responds by making antibody. D4 and E4 or slow reacting substance A (SRS-A) • prostaglandins • thromboxanes Under normal circumstances. The preformed or primary mediators that are released from the granules are: • histamine. On addition of specific antigen.active “fusogens” such as lysophosphatidic acid which facilitates degranulation and synthesis of arachidonic acid metabolites.128 Immunology– Introductory Textbook In active cutaneous anaphylaxis the animal is induced to produce IgE by antigen administration.1 a). The mechanisms involved in anaphylaxis Following the first exposure to antigen. Smooth muscle strips from ileum or uterus are bathed in physiologic buffered saline to which antibody from a hypersensitive animal is added. In man. after its authors. the term reaginic or skin sensitizing antibodies has been used synonymously with homocytotropic antibodies. This familiar biochemical cascade produces membrane . molecules of antigenic material seek out the tissue fixed IgE antibodies and cross link adjacently placed IgE molecules (Figure 15. which fixes onto mast cells via receptors that exist on mast cells for the Fc portion of the IgE molecule. these mediators help orchestrate the development of a defensive acute inflammatory reaction. The antibodies that accomplish this are most often IgE in all species. smooth muscle contractions occur in these bits of tissue in vitro. C4. Due to the nature of antigen (allergen) IgE is formed. • heparin • eosinophil chemotactic factor A for anaphylaxis (ECF-A) • neutrophil chemotactic factor (NCF-A) • platelet activating factor The newly synthesized or secondary metabolites are : • leukotrienes B4. In a run away reaction their bronchoconstrictive and vasodilatory reactions can be life threatening. In vitro models for anaphylaxis In vitro anaphylaxis was first demonstrated by Sir Henry Dale.Dale reaction. in what is now known as the Schultz . the generation of diacylglycerol and increase in intra cytoplasmic free calcium.

however. eosinophils. including monocytes.Hypersensitivity 129 Figure 15. The high affinity IgE receptor on basophils and mast cells has been termed FcERI. they have different structures and are . FcERII has low affinity for IgE and is found on a variety of leukocytes. There are two types of IgE receptors. (a) first exposure to antigen. Mechanisms involved in anaphylaxis. It is estimated that a basophil has 10. IgE receptors on mast cells Mast cells and basophils have specific receptors that form non covalent bonds with the Fc portion of the IgE molecule (Figure 15.000 to 40. (b) second exposure to antigen. The high and low affinity receptors for IgE are antigenically distinct.2). Heating IgE to 56° C for 30 minutes destroys its ability to bind to the target cell.1.000 such receptors. The normal half life of IgE in serum is two to three days. platelets and T and B cells. macrophages. IgE may remain fixed to cells for weeks.

lipid chemokinetic factor Prostaglandin D2 Leukotriene B4 Thromboxanes When released.1: Chemical mediators of immediate hypersensitivity Primary (pre-formed) Mediator Histamine Functions Increased vascular permeability Elevation of cAMP Contraction of smooth muscle Chemokinesis Chemotactic for eosinophils Chemotactic for neutrophils Anticoagulant and anti-complement Proteolysis Mediator Leukotrienes: C4. neutrophil adhesion Aggregate platelets ECF-A NCF-A Heprin Chymase Platelet activating factor Lipid chemotactic. basophils contain a kallikrein like esterase. Other mediators are generated after the mast cells are triggered (See Table 15.1). Figure 15. eosinophil. FcERI is composed of three polypeptide chains.E4 or SRS-A Secondary (induced) Functions Contraction of human bronchiole. Mediators of Immediate Hypersensitivity Mast cells and basophils contain a number of preformed mediators with potent biologic activity. The increased capillary permeability leads to an influx of plasma proteins including antibody. neutrophil chemotaxis. chymase.130 Immunology– Introductory Textbook encoded by separate genes. The b chain traverses the membrane and the covalently linked g chains face the cytoplasm. Table 15. The Fc (IgE) receptor on the mast cell. whereas FcERII may play a major role in immunity against parasitic infections.2. The α chain binds the Fc portion of the immunoglobulin IgE. complement and kinin generating and .D4. Mast cells contain a chymotrypsin like enzyme. Eosinophils and neutrophils are summoned to the area of injury and bradykinin is generated by the kallikrein-like esterase. increased vascular permeability Aggregation and increased secretion Neutrophil movement and activation Vasoactive smooth muscle action Increased vascular permeability. the mediators can cause increased vascular permeability increased secretion by nasal and bronchial mucous glands and contraction of smooth muscle in bronchioles and small blood vessels.

secondarily. Also derived from arachidonic acid. are potent bronchoconstrictors and vasodilators (See Table:15. They contain histaminase which breaks down histamine and phospholipase D. Activation of enzymes for degranulation involves a complex series of biochemical steps including transmethylation of membrane phospholipids. changes in intracellular cAMP levels and the opening of Ca++ channels. affecting cyclic nucleotide production and. Enzymes from neutrophils also break down mediators. the affinity and concentration of antigen for IgE. urticaria.1). cross linking of IgE does not occur and degranulation is prevented. Decreased cAMP or increased cGMP levels stimulate release. (iv) Eosinophils attracted to the site by chemotactic factors are also thought to have a control function.3. by the action of cyclooxygenase. Regulatory Mechanisms of Immediate Hypersensitivity Cross linking of IgE on mast cells results in explosive degranulation of the cell. the leukotrienes. are prostaglandins which perform related inflammatory functions. agents that increase cyclic GMP have the opposite effect. All of these substances increase inflammation. (ii) Mediator release is influenced by intracellular cyclic nucleotides. The clinical manifestations depend on the sites affected and include systemic anaphylaxis. Such IgG molecules are called blocking antibodies. Leukotrienes are formed after the enzyme lipoxygenase acts on arachidonic acid. A number of physiologically active substances can trigger cell surface receptors.Hypersensitivity 131 coagulation proteins into the tissues. whereas increased cAMP inhibits release. activation of protein kinases and adenylate cyclase. A major group of secondary mediators. (iii) Histamine can exert negative feed back control by stimulating adenylate cyclase which increases cyclic AMP and thereby prevents further release of histamine. which acts on platelet activating factor. This process is regulated at several levels: (i) The intensity of the reaction depends on the amount of IgE bound to the cells. If IgG has occupied a majority of sites on the mast cell. Mediator release by mast cells is modulated by the cyclic nucleotides cAMP and cGMP. Figure 15. rhinitis and vasculitis. mast cell release of mediators. Agents that induce prolonged elevations of intra cellular cAMP lead to decrease in mediator release. . asthma. These agents act via receptors on the cell surface (Figure 15.3).

greater is the likelihood of becoming atopic. which are localized anaphylactic reactions to extrinsic antigens such as pollens. skin and the conjunctival tissues releases mediators of anaphylaxis and produces the symptoms of hay fever. Clinical tests for allergy Sensitivity is normally assessed by the response to intradermal administration of allergen. foods. and this may be linked to the inheritance of specific HLA haplotypes. It may be noteworthy that both propranolol and cimetidine may induce allergic manifestations in susceptible individuals by lowering cAMP and thereby promoting mast cell mediator release.4). There is a strong familial disposition to the development of atopic allergy. asthma. diminishing antigen exposure. This reaction is thought to be mediated by NCF-A following cellular infiltration into the area. animal danders and faeces from mites in house dust to name just a few. The release of histamine and other mediators produces a wheal and flare reaction in 30 minutes. Thus eating prawns or egg may precipitate an asthmatic attack in a sensitized individual. in turn. Therapy for atopic diseases takes advantage of some of the regulatory mechanisms of immediate hypersensitivity mentioned earlier (Figure 15. therapy with catecholamines such as epinephrine and isoproterenol to elevate cAMP and decrease mediator release and the administration of antihistamines to block the effect of histamine on target organs. Figure 15. The immediate wheal and flare reaction may be followed by a late phase reaction which sometimes lasts for 24 hours. skin or lung causing further local anaphylactic reactions. blocks the attachment of the IgE to the . the nasal mucosa. Possible modes of therapy for atopic diseases. Examples include. use of mast cell stabilizers such as sodium cromoglycate. Other tests include the RIST and the RAST (described in Chapter 8). Contact with food allergens and cell bound IgE in the gastrointestinal tract may cause diarrhoea and vomiting. inducing blocking IgG antibodies. Allergen-IgE complex may diffuse out of an already permeable gut and deposit in joints. Contact of the allergen with cell bound IgE in the bronchial tree. This. though culprit HLA types have not yet been identified. A new experimental approach to treating and preventing Type-I hypersensitivity involves giving the person with allergies injections of monoclonal antibodies that have been made against the Fc portion of human IgE. urticaria or allergic rhinitis as the case may be.132 Atopic allergy Immunology– Introductory Textbook Nearly 10% of the world’s population suffer from allergies. It has been shown that the higher the level of serum IgE.4.

Another quite distinct mechanism in type II hypersensitivity is that mediated by K cells. It is quite evident thus far that type II hypersensitivity may or may not involve the participation of complement. The antibody facilitates the association between the phagocytic cell and the antigen bearing cell. These include opsonization via C3b receptors which also exist on surfaces of macrophages (see Chapter 7) and resultant phagocytosis (Figure 15. (Figure 15. If this happens in excess and causes tissue damage it is one example of the antibody dependant cytotoxic hypersensitivity. in what is known as antibody dependant cellular cytotoxicity-ADCC (see Chapter 14).5. There are several other mechanisms which account for type II hypersensitivity. Such an opsonized particle is readily phagocytosed by macrophages which have Fc receptors for IgG or IgM molecules on their surface (Figure 15.5b). Excessive cell lysis may occur when cell surface antigen couples to antibody. Type II reactions in blood transfusion and organ transplantation The basis for ABO blood grouping Of the many variant antigens on the human red cell membrane. The monoclonal antibody is a humanized hybrid molecule consisting of a mouse binding (Fab) portion attached to a human constant (Fc) portion and is known as rhuMab (recombinant human monoclonal antibody).5c).5a). Mechanisms of antibody dependant cytotoxic hypersensitivity. Figure 15.Hypersensitivity 133 Fc receptors on mast cells and basophils and the subsequent release of histamine by those cells upon exposure to allergen. Type II-Antibody Dependant Cytotoxic Hypersensitivity The combination of IgM or IgG antibodies with antigenic determinants on the cell surface renders the cell surface coated or opsonized by antibody. the anti-IgE binds to IgE-producing B-lymphocytes causing apoptosis. In addition. Individuals with blood group O display the H substance on the red cell . fixes complement and activates the entire complement cascade (Figure 15. the ABO antigens form the dominant system.5d).

how were they induced? It has been thought that bacterial antigens of the normal gut flora cross react with blood group antigens. the donor red cells are rapidly coated with the host’s isohaemagglutinins and severe reactions ensue. Individuals who are blood group A produce certain glucosyl transferases encoded by an A gene.7). he will be tolerant to antigens closely similar to A (see theories of tolerance. Figure 15.6. Chapter 16) and will form antibodies to bacterial antigens that cross react with blood group B. Persons who are blood group AB have neither anti A nor anti B antibodies and individuals with blood group O. an individual with blood group O. Naturally occurring antibodies or isohaemagglutinins in individuals are targeted to those antigens absent from the red cell surface. Since IgM is involved. Runaway complement activation with the resultant tissue reaction is a classic example of a type II hypersensitivity reaction. These enzymes act by adding N acetyl galactosamine to position 3 of the terminal galactose of the H substance. Mismatched Transfusion On transfusion of mismatched red cells. Individuals belonging to the AB blood groups possess both genes and hence both antigens on the red cell surface. without antigen provocation. is not tolerant to either antigen and therefore will produce antibodies to substance A and B.6). present with both anti A and anti B antibodies. The basis of the ABO Blood Group System. Similarly individuals who are blood group B posses enzymes encoded by the B gene which couple another galactose to position 3 of the galactose residue in the H substance. Hence. if an individual is blood group A. Thus a person with blood group A possesses anti B antibodies and vice versa. Since they are seemingly present in the circulation. Persons with blood group AB are tolerant to both antigens and hence do not produce the appropriate antibodies. cross linking of just a few determinants is sufficient to set off the entire complement cascade (Figure 15. which utilize complement. . The H substance is an oligosaccharide anchored to the cell membrane by coupling to a sphingomyelin called ceramide. These isohaemagglutinins are usually IgM.134 Immunology– Introductory Textbook surface (Figure 15. This oligosaccharide has a terminal galactose residue coupled to a fucose at position 2. Similarly.

during separation of placenta. An individual who does not display RhD on his red cell surface (RhD antigen negative). An individual who has the RhD antigen on his red cell membranes.Hypersensitivity 135 Figure 15. Rhesus incompatibility The rhesus blood groups form another major antigenic system. RhD negative mothers are given passive immunization with small amounts of ready made anti D IgG antibodies at the time of birth of the first child. To prevent haemolytic disease of the newborn. These may be directly cytotoxic or cause adherence of phagocytic cells. These small amounts of anti D IgG will complex with RhD on those foetal red cells that escape into the maternal circulation during placental separation. All of these reactions are examples of Type II mediated hypersensitivity reactions. The antibodies may lead to platelet adherence. Mismatched transfusion reactions DIC = Disseminated intravascular coagulation. are not naturally occurring.7. They may also elicit non specific attack by K cells via ADCC. Foetal red cells coated with maternal anti D IgG and circulating anti D IgG in the maternal blood can be detected in the laboratory by the Coomb’s direct and indirect tests (see Chapter:8). is of course tolerant to the antigen and does not produce anti D antibodies. This precludes the presentation of foetal RhD antigen to the mother’s immune system and sensitization of the mother to RhD is prevented. during subsequent pregnancies. . there is some release and admixing of foetal red cells in the maternal circulation. needs to be exposed to the D antigen to be able to produce anti D antibodies. A mother who is RhD negative may give birth to an RhD positive child (the RhD antigen being inherited from the father). These antibodies are predominantly IgG and are able to cross the placenta in any subsequent pregnancy. Antibodies to RhD. The mother being exposed to the RhD antigen is sensitized and begins to produce anti D antibodies. leading to foetal red cell destruction in utero. through another type II mediated hypersensitivity reaction. Organ transplants A long standing homograft may evoke antibodies in the host which are directed against surface antigens on the transplanted tissue. unlike the isohaemagglutinins. resulting in haemolytic disease of the newborn. This IgG reacts with foetal red cells. the rhesus antigen is designated RhD or just D. On delivery of the first child. when they are directed against antigens on vascular endothelium.

This reaction. too. Red cells coated with bacterial or viral antigens cause antibodies to be directed against them. When the drug is withdrawn. Sera of patients with Hashimotos thyroiditis contain antibodies. the sensitivity is no longer evident. Type III-Immune Complex Mediated Hypersensitivity Pathogenesis of immune complex disease The term immune complex disease refers to a group of diseases whose pathogenesis involves tissue damage from excessive antigen-antibody reactions. C3a and C5a. In multiple sclerosis antibodies are made against the oligodendroglial cells that make myelin in the brain and spinal cord. An optimum ratio of antigen to antibody provides for an insoluble complex (lattice). with resultant red cell lysis. When antigen and antibody couple to form insoluble complexes at fixed sites. In Goodpastures syndrome. two potent vasoactive amines. proportions of antigen and antibody also govern the nature of complexes. (Figure 8. Chemotactic factors lead to influx of polymorphonuclear leukocytes. autoimmune reactions and repeated contact with environmental agents. resulting in an antibody attack which destroys the cells as well. are directly cytotoxic for isolated human thyroid cells in culture. However. If antigen in the circulating complex reacts with IgE on circulating basophils. otherwise we would all be suffering from immune complex disease! Clearance depends on the absolute amounts of antigen and antibody which could determine reaction intensity. Amidopyrine and quinidine are known to coat granulocytes and a dreaded agranulocytosis could result. This in turn leads to extracellular release of polymorph granular contents. The classic example of drug induced type II hypersensitivity. tissue reaction and tissue damage could well occur. are released. Large insoluble complexes taken up by macrophages cannot be readily digested and provide a persistent activating stimulus (Figure 15. degranulation and the subsequent release of histamine. red cells become coupling agents and a type II haemolytic anaemia is seen. auto antibodies are directed to glomerular basement membrane.136 Autoimmune Type II Hypersensitivity Immunology– Introductory Textbook Many type II hypersensitivity reactions are the underlying mechanisms of autoimmune disease. The body may be exposed to excessive amounts of antigen in a number of circumstances. Antibodies to acetylcholine receptors in myasthenia gravis is a further example of type II hypersensitivity. These include proteolytic enzymes. kinin forming enzymes and other proteins which will damage tissues and intensify the inflammatory process. they fix complement and serious damage ensues. was shown to be facilitated by complement.8) Clearance of immune complexes in vivo normally occurs. is that of thrombocytopenic purpura. Complement fixation influences size and solubility . If complement is involved.2). With drugs such as chlorpromazine and phenacetin. this causes increased vascular permeability. Auto antibodies to the patient’s own red cells are produced in autoimmune haemolytic anaemia. which in the presence of complement. Type II Hypersensitivity due to drugs Drugs coupled to the body’s own cells become antigenic and the humoral immune system recognizes the drug-cell complex as foreign. such as persistent infection with microbial agents. a now banned sedative. it causes platelet clumping forming microthrombi. known to bind to platelets was used. Extreme antibody excess or antigen excess does not yield insoluble precipitated complexes. serotonin and other chemotactic factors. when sedormid. Increased vascular permeability is a pre requisite for the deposition of immune complexes in tissues.

Hypersensitivity 137 of immune complexes (Figure: 15. Schematic representation of Type III immune-complex mediated hypersensitivity.9. Figure 15. This clearing away is aided by red cells bearing the CR1 receptor which couples to C3b in the antigen .8. C1. . Binding of complement component.C3b complex and transports it away to fixed macrophages in the liver where they are inactivated.9). to immunoglobulin prevents Fc-Fc interactions needed to form large insoluble aggregates. (+) = stimulation of process. An immune complex lattice can be disrupted by complement component C3b. This disruption solubilizes the lattice and facilitates clearance of immune complexes from the circulation. which weakens the forces between the Fc portion of the antibody molecules. Smaller complexes which have C3b attached to them are cleared away by macrophages which have C3b receptors on their surface.antibody . Figure 15.

but some organs such as the kidney are affected more often than others. The dead parasite. Receptors for complement components are found in the glomeruli and in the choroid plexus . rapid filtration and high blood pressure in renal glomeruli facilitates deposition of immune complexes. which occurs 6-8 hours after exposure to mouldy hay. initiates a type III reaction causing inflammation and obstruction to lymph flow. severe fibrosis and resultant elephantiasis. often within a venule. immune complexes are deposited in tissues with resultant damage. These patients have been found to be sensitized to thermophilic actinomycetes which grow in mouldy hay. subsequently. The combination of high blood flow. thereby activating the classical complement pathway. when antigen is injected into immunized individuals possessing high titres of precipitating antibody. This reaction is considered the prototype acute. The Arthus reaction can be blocked by depletion of complement and neutrophils. the complex binds complement component C1q. immune complexes continue to circulate and deposit in other organs. Type III Reactions to inhaled antigens Arthus type reactions in the lung can occur to a variety of inhaled antigens. C5a is a powerful chemo attractant and polymorph influx results. When the ability of red blood cells to act as carriers of immune complexes is impaired. introduces antigen into the lung and a immune complex mediated hypersensitivity reaction occurs. C3a and C5a cause mast cells to release vasoactive amines. are negatively charged and will therefore retain positively charged immune complexes.another favoured site for deposition of immune complexes. Polymorphs release a wide variety of products (see earlier section) that degrade basement membrane and cause tissue damage. The injected antigen precipitates with antibody.138 Where are immune complexes deposited? Immunology– Introductory Textbook When large insoluble complexes form. Similar reactions can occur from bird and rodent excreta in bird fanciers disease and in rat handlers disease respectively. In addition. C3b and C5a. A classic example is Farmers lung. Intravascular complexes cause platelet aggregation and further release of vasoactive amines from basophils. The Arthus reaction is thus primarily mediated by complement activation and neutrophil released products. Inhalation of the organism from the hay. . The reaction generates proteolytic cleavage products such as C3a. localized immune complex mediated tissue injury. Such an impairment occurs in patients who have systemic lupus erythematosus. Deposition can occur anywhere in the body. basement membranes. or when the clearing system is deficient. There are many other quaintly named conditions of similar nature due to extrinsic allergic alveolitis resulting from inhalation of foreign antigens. Type III Reactions to tissue antigens Type III reactions are associated with elephantiasis due to Wuchereria bancrofti. a disease in which red cells have decreased C3b receptors. found in lymphatic vessels. whether in the glomerulus or in the dermalepidermal junction of the skin. if scavenger systems in the liver or spleen are impaired. Local reactions to Type III mediated Immune Complex Deposition The Arthus Reaction The Arthus reaction was described by Maurice Arthus who found that an acute haemorrhagic or necrotic process occurs locally. The end result of all this is severe erythema and oedema. Likewise. complexes are more likely to be deposited in blood vessels.

In rheumatoid arthritis. Viral infections with hepatitis B and Epstein-Barr virus are associated with immune complex disease. Glomerulonephritis is well known as a sequela to infection with type 12 β haemolytic. The major complications of serum sickness are vasculitis. joints. may induce chronic serum sickness. . gastrointestinal distress and an enlarged spleen. Streptococci.10). phenylbutazone. In addition to foreign serum. several drugs can cause serum sickness. penicillamine. trypanosomiasis. Complexes form in the circulation and if they are large enough. schistosomiasis and filaria are particularly associated with chronic glomerulonephritis. gold. Basement membranes of skin. This results in separation of capillary endothelial cells and exposure of the underlying basement membrane to which the immune complexes attach. thiazides. To be pathogenic. These drugs act as haptens. glomerulonephritis and neuritis. Chronic stimulation of the immune system and polyclonal B cell stimulation has been incriminated as possible aetiological factors in these conditions. Antigenic similarity between human and microbial antigens has been hypothesized as being the cause of vigorous antibody formation against host tissues and resultant immune complex deposition. This is associated with lowered serum complement levels and leukocytosis. antibodies to self IgG get deposited in the joint contributing to severe synovitis. Glomerulonephritis has been associated with persistent bacterial products. The pathogenesis of immune complex mediated glomerulonephritis is illustrated in Figure 15. immunoglobulin and complement. Immune complex nephritis has been associated with parasitic disease. including penicillin. The depositions of immune complexes appear as lumpy granules and contain antigen. an urticarial rash.10. they are deposited in the endothelial side of the glomerular basement membrane (Figure 15. Dramatic immune complex mediated reactions manifest as erythema nodosum leprosum in dapsone treated cases of lepromatous leprosy and in the Jarisch . immune complexes have to be the right size. such as penicillamine or gold. Typically. “nephritogenic”. Chronic parasitoses such as leishmaniasis. These changes result from deposition of immune complexes in tissues. Type III mediated Systemic Immune Complex Disease Serum sickness was first described early in the century in patients who received horse serum for the treatment of infectious diseases. aminosalicylic acid and streptomycin. 8-12 days after the administration of large amounts of serum from a foreign species. The complexes lodge in blood vessels and cause changes in vascular permeability. painful and swollen joints. hydantoins.Hypersensitivity 139 Vigorous chemotherapy is known to cause reactions due to abrupt release of microbial antigens in patients with high antibody levels. complex with protein carriers and induce antibody formation. Glomerulonephritis can also occur after pneumococcal and staphylococcal infections. Repeated administration of foreign proteins and drugs with repository or long acting characteristics. kidneys and the heart are particularly affected. The smallest complexes manage to filter through to the epithelial side. Quartan malaria in some Nigerian children leads to an immune complex mediated nephrotic syndrome. sulfonamides. not too big to be scavenged by macrophages and not too small. so as not to cause an inflammatory reaction. the patient may have a rise in temperature. they are visible as large amorphous masses by electron microscopy. tender lymph nodes. glomerulonephritis is a common feature of many immune complex diseases. Immune Complex Nephritis Because renal glomeruli are particularly vulnerable to immune complex-mediated injury.Herxheimer reaction of syphilis patients treated with penicillin.

. antigen content and presence of complement. blood vessels. Systemic lupus erythematosus (SLE). the amount of unbound C1q is quantified as an indirect indicator of presence of immune complexes. and the endothelial linings of blood vessels. Gastrointestinal disease such Crohns disease. in part. ulcerative colitis. Immune complexes isolated from lesions in SLE contain DNA and antibody to DNA. Immune complex nephropathy can be found in malignant disease such as lymphocytic leukaemias and Hodgkins disease. Deposition of immune complexes can occur at other major filtration sites such as the choroid plexus. A multitude of assays exists for the detection of circulating immune complexes.140 Immunology– Introductory Textbook Figure 15. by the finding that there are fewer CRI receptors on red cells and monocytes in these patients and hence immune complexes cannot be transported away to the reticulo endothelial system. The cryoprecipitates are then analysed for immuno globulin type. The immune complexes bound to C1q are precipitated out. skin and the central nervous system.epidermal junction. C1q is radiolabelled and mixed with patient’s serum. One assay is based on the tendency of complexes to precipitate in the cold. Pathogenesis of Immune-complex Nephritis. Deposition also occurs at the basement membrane of the dermal .10. Immune complexes have been found in kidneys. Vigorous deposition of DNA-anti DNA complexes is explained. an autoimmune disease is the paradigm immune complex disease. cirrhosis of the liver and coeliac disease can be associated with immune complex tissue injury. Detection of circulating immune complexes Tissue bound complexes are usually visualized using immunofluorescent staining. A more sensitive assay is based on the ability of immune complexes to bind C1q.

tissue damage occurs when K cells and NK cells join the foray against foreign antigen and indiscriminate cytotoxicity results. Cell mediated hypersensitivity is also evident in fungal diseases such as candidiasis. Similar lesions can be seen in organs undergoing cell mediated immune reactions. CTLs. macrophages. Some cells fuse to form multinucleated giant cells.macrophage series. This lesion represents the chronic granuloma and is an attempt by the body to wall off a site of persistent infection. the granulomas that form around Schistosoma mansoni eggs in the liver. Close monitoring of circulating immune complex levels helps to evaluate therapy in a number of disease states. The skin rashes of small pox and measles and the lesions of herpes simplex have been attributed to delayed type hypersensitivity reactions with associated cytotoxic T cell damage to virally infected cells. coccidioidomycosis and histoplasmosis and in parasitoses such as leishmaniasis and schistosomiasis. When this reaction does not get rid of the offending antigen. Continual release of lymphokines leads to infiltration of macrophages with arrays of epithelioid cells. For example. in whom previous exposure to Mycobacterium tuberculosis has induced a state of cell mediated immunity (CMI). dermatomycosis. T cells recognize antigen together with MHC Class II molecules and are activated. This contrasts with the essentially neutrophil predominant character of the Arthus reaction. Tissue necrosis is surrounded by the above cellular reaction with areas of fibrosis in the periphery. epithelioid cells and giant cells. Histologically. Cell mediated hypersensitivity is responsible for the cavitation and caseation of tubercular lesions and for the granulomatous skin lesions of the borderline form of leprosy. Type IV-Cell Mediated or Delayed Type Hypersensitivity Delayed hypersensitivity is a cell mediated immune reaction in an individual previously sensitized to an antigen. Further. Unlike other forms of hypersensitivity. and/or macrophages then cause harm rather than benefit. Delayed hypersensitivity therefore has the same mechanism as cell-mediated immunity. The reaction appears after several hours and may take upto 48 hours to reach a maximum. such as the Raji cell line or macrophage cell lines. resulting in proliferation and release of lymphokines. The assays measure the amount of immune complexes bound to cells. the lesion consists of a predominantly mononuclear cell infiltrate . delayed hypersensitivity does not involve antibody and cannot be transferred from a sensitized individual to a non sensitized individual with serum antibody.of the monocyte. Histologically. cytokines. . there is evidence of lymphocytes. are the result of cell mediated immune reactions. A skin reaction typically develops 12-48 hours after intradermal injection of antigen.Hypersensitivity 141 Other assays depend on the ability of immune complexes to bind to cells that have receptors for the Fc portion of IgG or for complement components. The best known example of this reaction is the positive Mantoux reaction where tuberculin is injected into the skin of an individual. T8lymphocytes once sensitized to an antigen differentiate into cytotoxic T-lymphocytes while Th1 type T4-lymphocytes become sensitized to an antigen and produce cytokines . Cellular Reactions in type IV-Hypersensitivity : the chronic granuloma The type IV hypersensitivity reaction results from an exaggerated interaction between antigen and a normal cell mediated immune reaction. persisting antigen evokes a chronic cell mediated hypersensitivity reaction. hence the usage of the term “delayed” hypersensitivity.

asthma.2: Comparison of different types of hypersensitivity Type Descriptive Name IgE-mediated hypersensitivity Initiation Time 2-30 mins Mechanism Examples I. They are also seen in humans and were originally called Jones . Ag induces cross – linking of IgE bound to mast cells with release of vasoactive mediators Ab directed against cell-surface antigens mediates cell destruction via ADCC or complement Ag-Ab complexes deposited at various sites induces mast cell degranulation via Fcgamma. glomerulonephritis III. These chemical substances bind to body constituents to form antigenic material capable of inducing a delayed hypersensitivity reaction. eczema II. The reaction to these neo-antigens is characterized by a mononuclear infiltrate. A summary of the different types of hypersensitivity is presented in Table: 15. they are erythematous but lack the induration and fibrin deposits in classic delayed type reactions. The skin lesions are intensely infiltrated by basophils as well as lymphocytes. haemolytic disease of the newborn. accompanied by oedema of the epidermis and micro vesicle formation. They differ from classic delayed hypersensitivity reactions in a number of ways. p-Phenylene diamine in certain hair dyes. Antibody-mediated cytotoxic hypersensitivity 5-8hrs Blood transfusion reactions. systemic reactions disseminated rash.2 Table 15. autoimmune haemolytic anaemia Arthus reaction (Localised). neomycin in topically applied ointments and nickel salts as in nickel coated costume jewellery can evoke a similar reaction. tubercular lesions . or who repeatedly come into contact with poison ivy. This type of reaction is seen in human contact dermatitis and in skin and renal allograft rejection. At present it is not known what factors determine whether a reaction will terminate as a basophil hypersensitivity or a classical delayed type hypersensitivity reaction. Contact Hypersensitivity (contact dermatitis) Contact hypersensitivity can occur in people who become sensitized while working with chemicals such as picryl chloride and chromates.Mote reactions. local anaphylaxis.142 Cutaneous Basophil Hypersensitivity Immunology– Introductory Textbook Cutaneous basophil hypersensitivity is a term for a group of delayed onset lymphocyte mediated reactions which have been studied extensively in guinea pigs. hay fever. Cell-mediated hypersensitivity 24-72hrs Contact dermatitis. PMN degranulation damages tissue Memory Th1 cells release cytokines that recruit and activate macrophages Systemic anaphylaxis. arthritis. Immune-complex mediated hypersensitivity 2-8hrs IV.

Autoimmune disease occurs when these normal “autoimmune” reactions are disturbed. a condition he described as “horror autotoxicus”. then the human immune system must be equipped to produce antibodies to idiotypes. Another important form of self recognition involves immuno competent cells which have been schooled to recognize the body’s own major histocompatibility antigens. It stands to reason that if an antigen such as the idiotype exists. Owen demonstrated tolerance in non identical (dizygotic) twin calves who shared the same placental circulation. Around 1900. Such “self recognition”. Individual antigenic sites on the immunoglobulin hypervariable region are called idiotopes. Important discoveries clearly illustrate that those sites on the hypervariable region of the immunoglobulin molecule that bind to antigens are themselves immunogenic: being complex protein molecules.anti idiotype network It is now clear that the hypervariable regions of the immunoglobulin molecules react with antigenic determinants also called epitopes. It has become clear that at a finer level of detail. Immune reactions to “self” antigens constitutes what is known as autoimmunity and is injurious to the host. Paul Ehrlich postulated that the immune system acquires a state of tolerance to self antigens. A set of idiotopes on a single immunoglobulin molecule is called the idiotype. If an idiotype is involved in antigen binding. Hence it is now . it is called a paratope. This leads to tolerance to such antigens since the clones of cells reacting against such antigens have been deleted during foetal development. the law that a normally functioning immune system does not recognize self is not absolute.CHAPTER – 16 IMMUNOLOGIC TOLERANCE AND AUTOIMMUNITY ne of the central concepts of immunology has been that the immune system should be able to distinguish between “self” and “non self”. Over 40 years ago. may form the basis of a network whose equilibrium keeps the body healthy. The receptors of the immune system that perform the work of recognition can themselves be recognized by other receptors. they did not mount a reaction to the other’s red cells. Even though each calf had appreciable numbers of red cells from the other twin (due to the common placenta). About 50 years after Paul Ehrlich. In the last 15 to 25 years there have been major revisions of these classic concepts. would have caused a severe immunological reaction. which was strictly outlawed in the early years. O Forms of Normal Auto Recognition or Positive Autoimmunity The idiotype . Macfarlane Burnet suggested that immune cells reacting with antigens during development of the foetus are destroyed by lymphoid organs. As discussed earlier. in addition he proposed that break down of tolerance would lead to self destruction. an infusion of the other twin’s red cells in adult life. this form of self recognition forms the key reaction in many immunological processes beneficial to the host. If they had not shared the same placental circulation.

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clear that a network exists wherein the immune system produces anti idiotypic antibodies to its own idiotypes. Credit for these discoveries goes to Niels Jerne who in 1974, proposed that normal autoimmune responses to self idiotypes, might form the basis of immuno regulatory network systems. Homeostasis of the immune system is thus thought to be preserved through a functional assembly of idiotype anti idiotype interactions. According to this model, an antigen induces production of an antibody (Ab1) characterized by its idiotype (Id1). In turn, (Id1) stimulates the synthesis of an anti idiotypic antibody (anti-Id1 or Ab2), bearing the idiotype Id2, that can, in turn, trigger the production of anti Id2 or (Ab3) (Figure 16.1). Theoretically, these idiotype - anti idiotype reactions can continue indefinitely. However, the interactions appear to be limited. In addition, it has been postulated that anti idiotypes that are directed towards paratopes (antigen combining sites), must stereochemically (by virtue of 3-D structure), resemble that part of the antigen that locks with the paratope. This is a reasonable postulate since both the anti idiotype and the antigen combine with the same binding site on the paratope. Such anti idiotypes were termed the internal image set by Jerne; Lindenmann called them homobodies.

Figure 16.1. Idiotype-Anti idiotype reactions. The idiotype network has been proposed as a mechanism to regulate the immune response. The presence of a foreign antigen leads to production of antibodies (Ab-1) that recognize the determinants of that antigen. Each of these antibodies contains a collection of unique regions that are collectively known as the idiotype; the idiotype includes the antigen binding site of the immunoglobulin molecule. The presence of Ab-1 leads to production of a second type of antibody, anti-idiotype antibody (Ab-2), which recognizes the idiotypes of Ab-1. Ab-2 also contains idiotypes and hence leads to production of a third type of antibody, anti-anti-idiotype antibody (Ab-3). Although the network theoretically can continue indefinitely, it appears to be limited.

It is generally believed that the idiotype anti idiotype network contributes to the regulation of the immune system. Besides directly interacting with B cell receptors or with antibodies in the serum, anti idiotype antibodies could modulate the immune response by activating or repressing different T cell subsets. The concept emerging, is that idiotype - antiidiotype circuits may regulate immune responses at appropriate levels: the helper T cell stage, the plasma cell and the level of the formed antibody secreted into the circulation.

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Recognition of Self MHC by T cells This has been discussed extensively in earlier chapters. Suffice it to say, that it has now been demonstrated beyond any doubt that immunocompetent cells of the vertebrate immune system preserve and express through out life, a self recognition capacity especially of MHC molecules. This is now known to be essential for the normal functioning of the immune system in all its diversity.

Tolerance
If the normal immune system can respond to virtually any foreign substance, why does it not respond destructively to self antigens? The absence of harmful immune reactions to antigens is termed tolerance. It has been shown that tolerance can be induced in animals by varying the dosage of the immunogen. Rabbits which are repeatedly exposed to low doses of a weak immunogen, remain tolerant even to a strongly immunogenic form of the same antigen. Similarly, tolerance can be induced even if high doses of the immunogen are administered to the animal. Hence there seems to be a low zone and a high zone in terms of antigen dosage for tolerance induction (Figure 16.2). Experiments have shown that the T cell is the target for low zone antigen tolerance and both T cells and B cells are irresponsive at high antigen dosage.

Figure 16.2. High zone and low zone tolerance.

How does an individual tolerate self antigens while generating normal immune reactions to foreign antigens? The answer to this question is not known. Studies indicate that there are several mechanisms which may contribute to tolerance: clonal deletion, clonal anergy, receptor blockade, non- immunogenic antigens, anti idiotype antibodies, suppressor T cell factors and other regulatory factors. Theories of Tolerance Induction We can divide the mechanisms the immune system uses to ensure the absence of selfreactivity (autoimmunity) into two main types: • Central Tolerance: this occurs during lymphocyte development. • Peripheral Tolerance: occurs after lymphocytes leave the primary organs.

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Central Tolerance
Macfarlane Burnet proposed the theory of clonal selection (Refer Chapter 11). In a brief recapitulation, the theory states that clones of immune cells that are genetically capable of reacting with all potential immunogens are present in the immune system. Generation of an immune response thus results from amplification and proliferation of that clone of cells best suited for a particular invading foreign antigen. Burnet also proposed that tolerance may occur when foetal immunocytes are exposed to and recognize a specific antigen - a process that leads to deletion of those clones of immunocytes. Support for the clonal deletion theory came from experiments conducted by Billingham and Medawar. They infused lymphoid cells from one inbred strain of mice (Strain A) into neonatal mice belonging to another inbred strain (strain B). Normally skin grafts from strain A mice would be rejected by strain B mice. However in this experiment, neonatal strain B mice, having been exposed to lymphoid cells from strain A mice became tolerant to cell surface antigens of the strain A mice. Thereafter strain B mice could receive skin grafts from strain A mice and tolerate the graft. Unfortunately early tests to prove the clonal deletion theory were unsettlingly negative. Clinical immunologists began finding antibodies against normal body proteins such as insulin, in the blood of healthy people. These antibodies ought not to have been there if self reactive clones of B cells are deleted during foetal development. Obviously some self reactive clones of B cells were surviving. Solid proof that clonal deletions actually occurred, appeared in 1988 — 33 years after Burnet first proposed the idea. In independent studies, Phillipa Marrack and other workers demonstrated that certain self reactive clones of maturing T cells were deleted from the thymus and never reached the rest of the body. It is now apparent that T cells entering the thymus are schooled by two selection processes. In the first process, also called positive selection, T cells are taught to recognize the MHC proteins; an essential learning step for adequate T cell function (see Chapter 3). The second step, also called negative selection, is critical for self tolerance. Developing T cells are exposed to a pot-pourri of self antigens. Young T cells that take the bait and bind to self antigens die. Dangerously self reactive T cells are therefore weeded out in the thymus. Matzinger has proposed that during neonatal life, whether encounter with an antigen results in tolerance or an immune response is determined by the prevailing host environment producing nonspecific cues ‘sensing’ danger. Neonatal T cells, she claims, are not intrinsically tolerisable but the systemic neonatal environment does predispose to tolerance. She has further suggested that the controlled death process of apoptosis is critical in preventing autoimmunity when old or surplus cells are disposed of.

Peripheral Tolerance
Clonal deletion, however, is not the entire answer. Some investigators believe, that it is incredible that every potential self antigen in the body must some how manifest itself in the thymus so that T cells can be screened. Clonal anergy has therefore emerged as a possible alternative mechanism for self tolerance. The term clonal anergy was coined by Gustav Nossal in the mid - 1970s. He found that under certain circumstances, B cells in culture could not be stimulated by antigen. The unresponsive B cells did not die but persisted in a lazy or anergized state. Although these experiments were elegant and informative, they do not reveal what ultimately happens to anergic, self reactive B cells especially in vivo. Almost 20 years ago Peter Bretscher and Mel Cohn proposed the two signal hypothesis for lymphocyte activation

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and tolerance induction. They suggested that a B cell must receive two consecutive signals in order to be activated: signal 1 from the antigen and signal 2 from a second cell specific for the same antigen, for example, a T helper cell. If a B cell was, however, reacting to a self antigen, it would find itself alone and unable to receive signal 2 from a T helper cell, except in those exceedingly rare circumstances when a T helper cell is also reacting specifically to the same self antigen. Thus self reactive B cells receiving only signal 1 from the auto antigen could not be activated to respond without adequate T cell help. Such a B cell was therefore anergized (Fig 16.3).

Figure 16.3. The two signal explanation for immunologic tolerance.

Although this model was originally proposed to explain B cell tolerance, Schwartz and Jenkins have now shown that a similar two signal theory can be used to explain T cell activation and tolerance (Figure 16.3). The essence of the two signal model is that simply binding of the T cell receptor to an antigen molecule is not enough to trigger T cell activation. The T cell must receive an additional signal of some kind from the antigen presenting cell. Without this second signal, a T cell becomes anergized and stays that way unless “reset” by the antigen presenters. It is thought that only a few types of specialized cells - macrophages for example, may be able to send this second signal needed to give T cells a push into its active, proliferative state. To date it is not known what precise steps are involved in the working of the clonal anergy theory. Another mechanism of tolerance induction is ligand-induced activation or antigen blockade. In this mechanism, antigen, particularly multivalent antigen, interacts with antigen receptors on B cells, under circumstances that interfere with the processes of patching and endocytosis which are necessary for clonal expansion. Certain monovalent antigens may block

MHC Class II gene products on the surface of antigen presenting cells. Antigenic blockade by monovalent and multivalent antigens.148 Immunology– Introductory Textbook B cell receptors without leading to patch formation and certain multivalent antigens and immune complexes may immobilize membrane receptors and prevent patch formation (Figure 16. Examples of such sites are the eye. Either of these interactions renders lymphocytes unreactive to subsequent antigen exposure. At the molecular level.4). Some antigens are sequestered from the immune system in locations which are not freely exposed to surveillance. It has been suggested . occupancy of B cell receptors by non immunogenic forms of antigen. The history of this fast changing field only shows that one year’s unfashionable concept can become the next year’s dogma. studies now indicate that binding of antigen to MHC gene products may play a role in immunologic tolerance. however.4. These are termed immunologically privileged sites. Antigen blockade produces reversible anergy because it does not lead to cell death. It is the existence of a unique suppressor cell that is most controversial. have been shown to bind to processed protein antigens. In addition. Figure 16. could prevent interaction with immunogenic forms such as antigen presented by macrophages. there seems to be insufficient experimental evidence to prove the suppressor T cell phenomenon. CNS and testis. some researchers believe that the “data and the phenomena are still there”. Though the once popular suppressor T cell theory for tolerance seems to have fallen into disfavour. Immune tolerance may also be induced by other mechanisms.

Such diseases are classified either as organ specific or systemic based on the primary location of the injury (Table 16.1). Table 16. asthma auto immune abnormalities Juvenile insulin dependant diabetes Pernicious anaemia Addison’s disease Idiopathic hypoparathyroidism Spontaneous infertility Premature ovarian failure Pemphigus Bullous pemphigoid Primary biliary cirrhosis Autoimmune haemolytic anaemia Idiopathic thrombocytopenic purpura Idiopathic neutopenia Vitiligo Osteosclerosis and Meniere’s disease Chronic active hepatitis Systemic diseases Goodpasture’s syndrome Rheumatoid arthritis Basement membranes Gamma globulin. insulin Gastric parietal cells. corpus luteum cells Intercellular substance of skin and mucosa Basement membrane zone of skin and mucosa Mitochondria Erythrocytes Platelets Neutrophils Melanocytes Type II collagen Nuclei of hapatocytes Target of Antibody . types II and III collagen Acetylcholine receptors Thyroid stimulating hormone receptor Thyroid Insulin receptor Insulin receptor β 2-adrenergic receptor Pancreatic islet cells. Autoimmunity Autoimmune disease is defined as disease caused by immunologic reaction to self antigens.Immunologic Tolerance and Autoimmunity 149 that self antigens may be differentiated from non self antigens by the way they bind to MHC Class II gene products. Epstein-Barr virus-related antigens. after they have been processed within the antigen presenting cells. Vitamin B12 binding site for intrinsic factor Adrenal cells Parathyroid cells Sperm Interstitial cells.1: Classification of Autoimmune diseases Disease Organ specific diseases Myasthenia gravis Graves’ disease Thyroiditis Insulin dependant diabetes with acanthosis nigricans Insulin dependant diabetes with ataxia telangiestasia Allergic rhinitis.

the long acting thyroid stimulator (LATS) is an auto antibody to thyrotropin receptor. ribonucleoprotein. lymphocytes. sensitized T cells either injure cells directly or release lymphokines that amplify the inflammatory response. Antibodies to the insulin receptor.150 SjÖgren’s syndrome Systemic lupus erythematosus Immunology– Introductory Textbook Gamma globulin Nuclei. In myasthenia gravis. via antibody dependant cellular cytotoxicity (ADCC). such as its size and charge. auto antibodies bind to acetylcholine receptors on the motor end plate. threonyltRNA synthetase Myocardium. due to anti adrenal cell antibodies. joints and blood vessels. In the second mechanism. choroid plexus. Antibodies to many hormone receptors may act as either antagonists or agonists. circulating antibodies react with modified or unmodified antigens on cell surfaces. These immune complexes deposit in various tissues. single-stranded DNA. causing excessive secretion of thyroid hormones. Although tissue injury caused by cellmediated mechanisms may be important in autoimmune disease. in patients with diabetes mellitus. The bound antibodies then stimulate the release of mediators of inflammation. This leads to destruction of the receptor. complexes between auto antibodies and antigens form in the circulation or in inter cellular fluids. The site of deposition is determined by the physical properties of the immune complex. gamma globulin. centromere Nuclei. double-stranded DNA. may antagonize insulin action by reducing the availability of the receptor. heart valves. in several other endocrinopathies and in some haemolytic anaemias and leukopenias. in Hashimoto’s thyroiditis. Alternatively. its role is currently unclear. erythrocytes. due to anti thyroid antibodies. it acts as an agonist. including glomeruli. In Grave’s disease. defective neuro muscular transmission and resultant skeletal muscle weakness. Scleroderma Polymyositis Rheumatic fever Mechanisms of Tissue Injury in Autoimmune Diseases Three mechanisms are principally responsible for inflammation and tissue injury in autoimmune disease • Cell lysis and release of inflammatory mediators triggered by auto antibodies • Immune complex disease • T-cell mediated damage In the first mechanism. In the third mechanism. trigger the complement pathway or activate K cells. Nuclei. The latter two processes result in cell lysis. One result of auto antibody binding is cell destruction. Cell lysis by autoantibodies Many autoimmune diseases are primarily initiated by the interaction between auto antibodies and cell surface antigens. auto antibodies may bind to cell surface receptors and interfere with their function. . they subsequently fix complement and cause inflammation and tissue injury. This occurs in Addison’s disease. neurons. histadyl-tRNA synthetase.

antibodies bind to the erythrocyte. antineuronal antibodies contribute to neurologic disease. e. the auto antibody called rheumatoid factor. No autoimmune response develops as long as these antigens remain unexposed. is usually an IgM. Exposure of sequestered antigens If an antigen is sequestered within an organ during foetal development. TNF α • Recruitment and activation of macrophages leading to bystander tissue destruction • Induction of target tissue apoptosis by the T cell membrane protein FasL In most cases we do not know what the relative contribution of these factors is. apparently brief exposure is insufficient for inducing autoimmune disease. auto antibodies to these sequestered antigens rapidly appear in the circulation. Additionally. These antibody responses are transient in most cases. diagnosis and treatment of the various autoimmune diseases. Should tissue damage or injury occur to expose these antigens. perfectly tuned network of immune responses to foreign antigens break down to cause an immunological reaction that is self destructive? Many theories and mechanisms have been proposed for the generation of auto immune responses.Immunologic Tolerance and Autoimmunity 151 Immune complex deposition In a number of systemic autoimmune diseases. The Aetiopathogenesis of Autoimmune Disease How does a finely balanced. patients demonstrate significant depletion of complement (C3) and neutrophils as a result of activation by complexes. For a more complete description of the pathogenesis. and platelet antigens. the prototype systemic autoimmune disease. get deposited at various sites leading to the characteristic synovitis and vasculitis of rheumatoid arthritis. leukopenia and thrombocytopenia. Complexes of rheumatoid factor IgM coupled with IgG. . the reader is directed to refer to the many excellent textbooks of internal medicine available. which may require persistent exposure to sequestered antigen. complement deficiencies which impair immune complex clearance (C1. leukocyte. There are a number of ways this can come about: • Direct T cell cytotoxicity via CD8+ CTL • Self-destruction of tissue cells induced by cytokines. In Rheumatoid arthritis. tissue injury is mediated both by direct auto antibody binding and by immune complex deposition. In systemic lupus erythematosus (SLE). against lens protein after eye injury. against heart muscle antigens after a myocardial infarct.C2 or C4. which is directed towards the Fc portion of the patient’s own IgG. Second. Two significant facts point to the role of immune complexes in SLE. causing haemolytic anaemia.) are very strong predisposing factors for SLE. circulating immune complexes consisting of DNA. T-cell mediated damage This term implies that the recognition of autoantigen by T cells leads to tissue destruction without requiring the production of autoantibody. then as per the theories of clonal deletion the immune system does not acquire tolerance to this antigen. Such an auto antibody reaction has been repeatedly demonstrated: auto antibody against sperm after vasectomy.anti DNA are deposited in the glomerulus of the kidney and in other organs causing tissue injury.g. First.

has been discussed earlier in this chapter. Therefore anti viral antibodies may induce anti idiotype antibodies that cross react with normal host tissues. unaltered BSA when they are immunized with chemically altered BSA. may allow immune reactions to the tolerant antigen to develop. streptococcal antigens induce antibodies which react with components of the myocardium. procainamide and hydralazine seem to provoke the production of antinuclear antibodies. Autoimmune haemolytic anaemia is associated with administration of α methyl dopa. Presumably streptococcal antigens resemble heart antigens enough. Immunization of rabbits with antibodies to human thyrotropin produces anti idiotype antibodies that mimic thyrotropin reaction.152 Altered self antigens and molecular mimicry Immunology– Introductory Textbook Alteration of an antigen to which a host is tolerant. This anti idiotype antibody resembles LATS found in patients with Grave’s disease (Figure 16. causing haemolysis. Alteration of self antigens occurs in the human system as a result of viral or bacterial infections or therapy with certain drugs. which cross reacts with neural tissue found in the neural vaccine. A related mechanism termed molecular mimicry comes into play when foreign antigens resemble self antigens. Idiotypic mechanisms leading to autoimmunity. Similarly. to elicit an autoimmune reaction.5). The idiotype anti-idiotype network The concept of anti idiotypic antibodies being stereochemically similar to the antigenic determinant. in rheumatic fever. Antibodies to the I blood group are formed following Mycoplasma pneumoniae infection. . The drug is thought to modify the red cell surface in such a way that certain B cell clones react against these modified red cells. Figure 16.5. The encephalitis that sometimes results from the administration of the neural rabies vaccines is thought to occur due to an autoimmune reaction to human brain tissue.There are several examples of autoimmune reactions that might be accounted for by the presence of cross reactive anti idiotype antibodies. This can stimulate a cytotoxic reaction against the receptor bearing cells. Anti idiotypic antibodies that structurally resemble a viral antigen. since both bind to the one antibody. Similarly. Experimentally it has been shown that rabbits made tolerant to bovine serum albumin (BSA) can be stimulated to produce antibodies to native. by combining with thyrotropin receptors. will be able to bind to the receptor for the virus on the cell surface.

be induced on other cells by viral infection. Interestingly. most individuals bearing these antigens are healthy. In certain strains of mice in which SLE develops. there are strong associations between several autoimmune diseases and particular HLA specificities for example. several antigens such as neural and endocrine antigens are not present in the thymus. This concept is central to Matzinger’s Danger Hypothesis. This potential self reactivity is usually prevented by the limited distribution of MHC Class II antigens. Autoimmune diseases tend to occur in clusters in certain families. hormonal and other factors acting singly or synchronously to produce disease. certain inbred strains of animals spontaneously develop autoimmune disease. through the effects of interferons (see Chapter 12). antinuclear antibodies and fatal SLE. However. These theories therefore provide a possible mechanism for the induction of autoimmune disease by infection or inflammation. patients with SLE excrete excessive amounts of 16 α metabolites of estrones which have high oestrogenic activity. the autoimmune disease is aggravated by oestrogens and retarded by androgens. children and siblings. These animal models provide valuable insight into the study of autoimmune diseases.DR antigens are associated with SLE. Most patients with SLE are women in their child bearing years. Furthermore. In addition. However. HLA DR3 in Addison’s disease and HLA DR4 in rheumatoid arthritis. which are restricted to just a few cell types: the macrophages. and the New Zealand Black (NZB) mouse develops autoimmune haemolytic anaemia. These cells become activated only when presented with antigen by cells that express MHC Class II antigens. Hashimoto’s thyroiditis and SLE occur more frequently among parents. Genetic and other factors Genetic factors are known to play an important part in auto immune disease. Although certain HLA . . however. Reviewing all the existing evidence regarding autoimmunity. Certain hormonal factors play a role in SLE and other autoimmune diseases. As a result autoreactive T cells which were anergic may become activated. There is an obese line of chicken that spontaneously develop autoimmune thyroiditis. it is clear that spontaneous autoimmune disease is a multifactorial phenomenon with immunologic. virologic. During an inflammatory response an immunostimulatory environment is created by the release of cytokines which recruit and activate professional antigen presenting cells and provide support for T cell activation rather than anergy.Immunologic Tolerance and Autoimmunity 153 Abnormal expression of MHC antigens Activated helper T cells are necessary for producing antibodies to most antigens. The hybrid of NZB with NZW (white) develops LE cells. including self antigens. T and B cells. Such T cells escape into the peripheral tissues and are able to recognize the above self antigens when presented in association with MHC Class II antigens. no single genetic factor can account for any autoimmune disease. therefore those T cells that are self reactive with regard to such antigens cannot be screened and eliminated within the thymus. Many self–reactive CD4+ T cells are eliminated in the thymus during maturation (see clonal deletion). Susceptibility must therefore be determined by more than one genetic factor or by a combination of factors. These antigens can. genetic.

by definition. which are membrane bound lipid vesicles. Oils such as squalene and peanut oil seem to be better tolerated when compared to Freund’s incomplete adjuvant. where antigen. Liposomes seem to behave as storage vacuoles for the antigen. Adjuvants Responses to many killed vaccines need to be enhanced by substances that are collectively known as adjuvants. tetanus. diphtheria. and in the treatment of autoimmunity and allergy. Other adjuvants have a “depot” effect. it is not recommended for human use. . Recent interest has been focussed on the use of liposomes. A Immunopotentiation Vaccination Vaccination has been used for over two centuries as a means of exploiting the immune system to protect the host against infectious agents. is emulsified with paraffin oil. they also enter the macrophage and are presented in a more immunogenic manner to T cells. The immune response is increased when protein antigens are precipitated by alum. The most common adjuvant of this type is Freund’s incomplete adjuvant. mumps and rubella to name just a few. pertussis. Immunosuppression has found relevance since the birth of organ transplantation. This reservoir of antigen is then available either at an extra cellular location or within macrophages. should the need arise. in the aqueous phase. Clinically. Since paraffin oil can produce severe local reactions. immunopotentiation has been effective in combating infectious disease. poliomyelitis. Immunization has thus offered protection against a number of potentially lethal infectious diseases such as small pox. as agents for presentation of antigen to the immune system.CHAPTER – 17 IMMUNOPOTENTIATION AND IMMUNOSUPPRESSION n understanding of the normal regulatory mechanisms of the immune system has yielded an insight into the many ways the immune reaction may be enhanced or inhibited. An adjuvant. is a substance when incorporated into or injected simultaneously with antigen potentiates the immune response. An understanding of antigen specificity and memory has been used to boost the immune response to infectious agents by artificially exposing the host to small amounts of the inactivated infectious agent. in that they prevent rapid dispersal of antigen from the local tissues draining the injection site. in the treatment of immunodeficiency states and in the search for a cure for cancer. Mode of action of adjuvants (i) On antigen characteristics: Some adjuvants affect the way in which antigen is presented.

malaise. recruit T cell help in vivo.Immunopotentiation and Immunosuppression 155 (ii) On host immune response: Most adjuvants. Clinical trials with these cells in humans have shown promising results. It appears to restore both humoral and cellular immunity in nude (athymic) mice. IL-2 and IFN γ. Unfortunately. especially in lymphomas. In vitro isoprinosine promotes T cell mitogenesis. Types of Adjuvants (i) Organic adjuvants: Organic adjuvants include a variety of organic molecules obtained from bacteria. IL-2 also appears to induce production of IFN α by T cells. It stimulates macrophages and may. It is a four amino acid peptide-threonine-lysine-proline-arginine. they enhance the accessory signals required for lymphocyte activation and proliferation. arthralgias and fluid retention. by incubation of cells with IL-2. myalgias. a purine precursor. By recombinant DNA technology it is now possible to harvest unlimited quantities of IL-1. It has been used with some success in the treatment of cancer and rheumatoid arthritis. (iii) Tuftsin : Tuftsin is a unique adjuvant that occurs naturally and has been synthesized as well. Lymphokines The interleukins and interferons have been discussed extensively in Chapter 13. MDP increases both humoral and cellular immunity. An alternative approach to the in vivo use of IL-2 has been the induction of lymphokine activated killer (LAK) cells in vitro. It potentiates humoral and cellular immunity in a fashion that is T cell dependant. and IFN α. however. a good example is Freund’s complete adjuvant which is made from incomplete Freund’s adjuvant with the addition of killed mycobacterium or more recently. homologous to a sequence in the constant region of the immunoglobulin heavy chain. Isoprinosine is a complex containing inosine. It has been used experimentally for malignant melanoma and HIV infection. Muramyldipeptide (MDP) is a bacterial peptidoglycan. Its usefulness in the treatment of cancer has not been established. β and γ are all used therapeutically in a number of clinical conditions (see Chapter 13). IFNs α. . Recombinant IL-2 was approved in 1992 for the treatment of metastatic and inoperable renal cell carcinoma. these advantages are not without the risk of side effects of which agranulocytosis is the most serious. Under these conditions lymphocytes acquire cytotoxicity against a broad range of tumour cells. Such adjuvants act directly on the macrophage or via the T cell. as assessed by reduction in tumour size. water soluble muramyldipeptide. IFN α appears to act both directly on tumour tissue and by activation of macrophages. IFN α has proven adjuvant properties. (ii) Synthetic adjuvants: Synthetic adjuvants that increase host immunity include levamisole and isoprinosine. do not affect the antigenic characteristics but act on the host immune response. Besides improving antigen presentation. Levamisole was initially introduced as an anthelminthic agent. Tuftsin primarily stimulates macrophages. Both the interleukins and the interferons are not free from side effects such as fever. as a consequence. IFN α was the first to be produced in a large scale manner. Virtually all adjuvants stimulate macrophages. Cells activated in this manner have been used to cause tumour regression in mice. Since it occurs naturally it probably has a physiologic role in host defence. IFN β.

Cyclosporine acts selectively on antigen-sensitive T cells in the G0 to G1 phase (see Chapter 12). thereby suppressing IL-2 production. however. Glucocorticoids Glucocorticoids are potent immunosuppressive and anti inflammatory agents. foreign antigen sensitive T cells begin to get activated. They are used regularly in the treatment of graft rejection. azathioprine and methotrexate block cell replication and preferentially kill dividing cells. Cyclosporine has been used successfully to prevent graft rejection and in the treatment of graft versus host disease. chlorambucil. Glucocorticoids have a diversity of actions and the clinical benefit seen in allergy and autoimmunity seems to be due to the various other actions of these drugs together with their immunosuppressive properties. leading to cell death during the mitotic phase of cell division. Just after implantation of a foreign graft. Current treatment of graft rejection. This may not be the only way that these drugs cause immunosuppression. have numerous and serious side-effects. Cytotoxic agents Cytotoxic agents such as cyclophosphamide. The following are some of the agents used as immunosuppressives. This effectively blocks T cell activation and proliferation. It has little toxicity for dividing cells