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IMMUNOLOGY FINAL

CHAPTER 6: B CELL DEVELOPMENT

1. Where do the different steps of B-cell development occur?

IN THE BONE MARROW:
 Ig gene rearrangements
o heavy and light chains rearrange forming Immature B Cell – expresses IgM
 Ig specificity is determined
o Allelic Exclusion: ensures each B cell makes one specificity
 Both copies of chromosome rearrange at the same time  once there is a successful
rearrangement occurs the other chromosome allele is silenced
 Negative selection
o Immature B cells are produced by random gene rearrangements  broad range of specificities 
some of which will be autoreactive
o The pool of B cells that are NOT autoreactive = self tolerant
o Autoreactive cells get a second chance
 Undergo additional light chain rearrangements
 If fail recombo again, undergo apoptosis or become anergic (enters peripheral circulation
but doesn’t survive long)
 Formation of the immature B cell is promoted by SCF and IL-7 secreted by bone marrow stromal cells

IN THE SECONDARY LYMPHOID TISSUES (Spleen, etc)
 In primary follicles: Immature  Mature
 During infection: encounter cognate antigens
o THEN undergo class switching and somatic hypermutation
o differentiate into PLASMA cells or MEMORY cells

2. How, when and where are self-reactive B-cells removed (Negative Selection)?

How:
 if self reacts with bone marrow cell antigen  apoptosis
 if self reacts with soluble antigen  becomes anergic (doesn’t last long)
When/Where:
 After gene rearrangements in the bone marrow

3. What is the role of secondary lymphoid tissues in B-cell development?

 SLT = lymph nodes, spleen, peyer’s patches (small intestine)
 After leaving the bone marrow immature B cells travel to lymph nodes
 Attracted to the primary follicle by chemokine CCL21, CCL19, CXCL13
 IN PRIMARY FOLLICLE
o Interacts with follicular dendritic cells  becomes mature B cell
 MATURE B CELLS (naïve)
o Recirculate between lymph, blood, secondary lymphoid tissues

4. What happens in Germinal Centers?

 Mature/Naïve B cells travel through lymphoid tissues in search of
a match
 If find a match in the T CELL AREA will move into the B CELL
AREA OR the MEDULLARY CORDS
 GERMINAL CENTER = primary follicle becomes the area where B
cells proliferate/expand AFTER encountering antigen
o B cells undergo class switching and affinity
maturation here
o Also this is where B cells either become plasma cells or
memory cells
o Appear about one week after infection
o Causes lymph node swelling
 If enter the medullary cords become plasma cells
 DEFINITIONS:
o Isotype Switching: IgM  IgG, IgA, or IgE
o Affinity Maturation: process by which antibody affinity is increased
o Somatic Hypermutation: molecular process behind affinity maturation

5. How does Immunoglobulin Class Switching occur?

 CLASS SWITCH RECOMBINATION (CSR) of the C region of the Heavy Chain (variable region untouched)
 Induced by specific cytokines 0 IL-4, IFN gamma, TGF B
 Takes places in germinal centers

6. What is Somatic Hypermutation?

 Somatic hypermutation fine tunes antigen recognition sequences
 V regions within a single B cell clone acquire mutations
 Only mutations that increase antibody affinity are maintained
 Affinity improves with time (higher affinity binding 2 weeks after infection than immediately)
 PRIMARY VS MEMORY RESPONSE
o The secondary response is more efficient
 Ab affinity starts off high and somatic hypermutation continues throughout the
second response as well

7. What is Affinity Maturation?

 Process by which antibody affinity is increased (molecular basis = somatic hypermutation)

8. What are B-1 cells?

 Special Subset of “Innate” B cells
 Recognize repetitive structures (bacterial polysaccharides)
 In mouse found in peritoneal and pleural cavities
 Make low affinity antibodies that are polyreactive (bind many different antigens)
 Produce mainly IgM but CAN class switch
 Respond rapidly to antigen do NOT require T cell help
 Considered a cell of innate immunity

CHAPTER 7: T CELL DEVELOPMENT

1. Where do the different steps of T-cell development occur?

BONE MARROW
 Generated from stem cells
THYMUS
 Cortex: immature thymocytes
 Medulla: mature thymocytes
 Macrophages: remove failed thymocytes

 TCR gene rearrangements
o “race” between ab and yd
rearrangements
o >90% of time ab wins

 Comparison with BCR
TCR Beta chain and Ig Heavy TCR alpha chain and Ig light
Chain chain
- VDJ segments - VJ segments
- two attempts at recombination - several sequential
for each chromosome rearrangements
- TCR: successful rearrangement - TCR  rearrangement leads
of Beta chain = pre-T Cell to loss of delta chain
receptor
- pre- T cell receptor expresses
BOTH CD4 and CD8 (double
positive)
2. How, when and where are self-reactive T-cells removed (Negative Selection)?

 Negative Selection by TCRs interacting with DENDRITIC CELLS, MACROPHAGES, OTHER APC’S in the
thymus
 Moderate binding  lives
 Tight binding  apoptosis and engulfed by macrophages

3. What is the role of “Positive Selection” of thymocytes?

 Positive Selection: T cells must be able to work effectively with
the individual’s own MHC molecules
 Each individual expresses up to 6 MHC CLASS I and 6 MHC
CLASS II molecules and each TCR must recognize one of those
molecules
 Positive selection occurs by TCRs interacting with MHC on
THYMIC CORTEX EPITHELIAL CELLS

 Binds MHC I  becomes CD8
 Binds MHC II  becomes CD4
 Double positive thymocytes become single positive thymocytes

4. What are “regulatory” T-cells?

 Suppress any surviving autoreactive T Cells
 “back up” defense against autoimmunity
 CD4 cells that express TF Foxp3 and CD25 on cell surface
 Have TCR specific to SELF antigen
 When encounter self antigen will suppress the proliferation of other naïve T
cells responding to antigen on the SAME APC
 Suppressive effects require contact between the two T cells and the
secretion of cytokines that inhibit differentiation of effector T
cells

CHAPTER 4: ANTIBODY STRUCTURE AND DIVERSITY

1. Understand OPSONIZATION and NEUTRALIZATION

 OPSONIZATION: coating of a pathogen with antibody  makes it easier for ingestion by phagocytes
 NEUTRALIZATION: antibodies directly inactivate a pathogen/toxin and prevent it from interacting with
human cells (toxin/ab complexes then ingested by macrophages)

2. Antibody structure (Light and Heavy chains, Constant and Variable regions)

 4 polypeptide chains (2 heavy, 2 light)
 Connected by covalent (S-S) and non-covalent bonds
 Divalent: 2 antigen-binding sites
 Repeating globular domains
 C-terminal domains are CONSTANT (C)
 N-terminal domains are VARIABLE (V)
o Variable domains of both heavy and light chains
contain three hypervariable loops
o Hypervariable loops determine antigen specificity
o Also called complementary-determining regions
o HV1, HV2, HV3 = CDR1, CDR2, CDR3

3. Five different Antibody Classes and their major functions

 Determined by heavy chains
ANTIBODY STRUCTURE FUNCTION
- First antibody made after infection
IgM Pentamer
- complement fixation
- becomes dominant antibody after acute infection
IgG
- neutralization, opsonization, complement fixation
- protects mucosal surfaces – GI tract, saliva, tears, urine, etc
- most abundant antibody (75% of all Ab’s in body)
IgA Dimer
- important defense against mucosal pathogens
- important for maintaining normal gut flora
- Mast cell and eosinophil degranulation
IgE
- allergies, parasites

IgD Coexpressed with IgM by naïve B cells

4. What is an Epitope?

 Epitope = minimal antigenic determinants (what antibodies
recognize)
o Linear epitopes: string of amino acids
o Discontinuous epitopes: depend on 3D protein structure
o Often on the surface of pathogens
 Antibodies vs TCR: TCR only recognize short processed fragments of
peptide, whereas Abs recognize 3D confirmation

5. How is antibody diversity generated?

 DNA RECOMBINATION
o Genetic diversity: germline organization of the heavy and light chain (one copy of each chromosome
inherited from each parent)
o Combinatorial diversity: random selection of one gene segment of each type (V, J, D)
 Heavy Chain = VJD
 Light Chain = VJ only
o Junctional diversity: random addition of nucleotides during joining
 FINAL PRODUCT:
o The VDJ region provides the specificity for antigen recognition
o All of the different C regions are still present
o This means the B cell can continue to differentiate and switch to different antibody isotypes during its
lifetime: from IgM to IgG, for example.
o However, different antibody isotypes made by the same B cell will have the same antigen specificity
since they have the same VDJ region. B cells are MONOSPECIFIC.

6. What is Allelic Exclusion?

 “Race” to successful recombination
 First heavy chain (chromosome 14) rearranges on BOTH chromosome copies
o 2 chances to rearrange
o The first one to successfully rearrange wins  the other chromosome is silenced from the point on
 After heavy chain rearranges the light chain rearranges (chromosome 2 and 22)
o 4 chances to rearrange (K and gamma chains)
o Once one successful rearrangement others are silenced
o Immunoglobulins use  chains more frequently than  chains
 This process ensures each B cell only has one antigen specificity

7. What is Somatic Hypermutation?

 The process by which antibody’s affinity for antigen increases over time
 Random point mutations accumulate in V region (CDR1, CD2, CDR3) that strengthen antigen/antibody binding

8. CASE: Common Variable Immunodeficiency (CVID)

 PROBLEM = NO CLASS SWITCHING
 High susceptibility to bacterial infections
o Class switching from IgG to IgA is very important for fighting infections against bacteria with capsular
polysaccharides
o This type of class switching requires functional TNF receptors: BAFF-R, TACI, and BCMA
o At least 10% of cases caused by mutations in TACI
 Summary of Immunological Findings:
o Low levels of serum immunoglobulins (especially IgG, A, E but sometimes also IgM)
o Lack of antibody response to vaccines, e.g. Pneumovax
o Normal numbers of T and B lymphocytes
o Splenomegaly
o Predisposition to certain autoimmune disorders
o Predisposition to certain cancers (lymphoma, gastric carcinoma)
o Recurrent mucosal infections (otitis, sinusitis, tonsillitis, diarrhea, pneumonia, GI infections)
 Especially from encapsulated bacteria (both gram + and gram -)
 Etiology of Disease
o VARIABLE
 IgA alone, IgA and IgG, or IgG, A and M
 Age of onset is variable
 Either sporadic or inherited
o Known mutations are only a small minority of CVID cases
o Relatively common (between 1/20,000 and 1/50,000)
o Most common primary human immunodeficiency requiring treatment
o ~20% of cases are familial but most cases are sporadic
o Families with selective IgA deficiency are more likely to have members with CVID
 Treatment
o Intravenous immunoglobulin therapy at regular intervals
o Longterm treatment with broad spectrum antibiotics

CHAPTER 5: ANTIGEN RECOGNITION BY T CELLS

1. What is the TCR?

 T cell receptor – each T cell has only one TCR specificity
 Associates with CD3 in the ER  facilitates transport to the cell surface, when receptor encounters antigen
allows for signal transduction
 Gamma-delta T Cells
o Instead of alpha-beta antigen binding site = gamma delta
o More common in epithelium and GI tract

2. How are immunoglobulin molecules and T-cell receptors SIMILAR? How are they different?

SIMILAR DIFFERENT
Resembles membrane bound antibody - alpha and beta chains (not
 Immunoglobulin-like domains (2 per chain) heavy and light)
 Variable Region (binds antigen) – three - variable region = Valpha and
hypervariable loops = CDR1, 2, 3 Vbeta regions
 Constant Region - even more diverse than Ig
Diversity results from gene rearrangement - dual specificity (Self MHC
AND foreign peptide antigen)
3. Understand the functions of CD4+ T-cells (Th1 and Th2) and CD8+ T-cells

 CD8 = cytotoxic T cells  directly kills virus infected SELF cells (recognize MHC I) (intracellular pathogens)
 CD4 = Helper T Cells – extracellular pathogens
o TH1: activate macrophages  phagocytosis of extracellular pathogens
o TH2: promote differentiation of B cells to plasma cells  antibodies bind extracellular pathogens

4. Understand differences between how T cells and antibodies recognize antigen

 TCR: can only recognize processed peptides
 Antibodies: can recognize 3D structure on protein surface

5. What is the MHC? How is it “polymorphic”?

 MHC present foreign peptides to T cells

 Genes encoding HLA located on chromosome 6 (HLA = the cell surface protein encoded by MHC genes)

 Class I loci = HLA A, B, C
 Class II loci = HLA DP, DQ, DR

 Each locus has hundreds of potential allotypes – unique antigen binding groove depending upon allele
inherited
o Different allotypes recognize different antigens
 HLA typing – determines what alleles inherited for both class I and class II

 MHC Haplotype: the combination of MHC alleles found on a single chromosome 6 – inherited from each
parent
 MHC Haplotypes are co-dominantly expressed in offspring – all inherited alleles are found in all
expressing cells
 There are no two individuals with the exact same set of MHC genes

6. What is meant by “Heterozygote Advantage”?

 The more HLA alleles inherited, the greater the number of potential antigens recognized
 Heterozygous at ALL MHC loci  able to respond to the broadest range of foreign antigens

7. Know how CD4+ and CD8+ T-cells recognize antigen (presentation by MHC I vs MHC II)

 MHC I  CD8
 MHC II  CD4

8. Be able to explain the MHC class I AND MHC class II antigen processing pathways

 Peptides bind MHC noncovalently

CLASS I CLASS II
HLA GENES A, B, C DR, DP, DQ
TISSUE EXPRESSION All cells APC’s, activated T Cells
T CELL SUBSET REACTIVITY CD8 CD4
SOURCE OF PEPTIDES Endogenous pathway Exogenous Pathway

Endogenous Pathway (MHC I)
o Intracellular pathogens
o Degraded by proteasome  transported across ER by TAP  binds MHC I while in ER  Loaded MHC
I transported to cell surface

Exogenous Pathway (MHC II)
o Extracellular pathogens taken up by endocytosis
o Degraded by phagolysosomes  bind MHC II within export vesicles

CHAPTER 8: T CELL IMMUNITY

 How T Cells enter Lymph nodes
o ROLLING  ACTIVATION/TETHERING  ARREST  EXTRAVASION
CIRCULATING T CELL ENDOTHELIAL CELL
ROLLING L Selectin GlyCam1 and CD34
(Addressins)
ACTIVATION/ARREST LFA-1 ICAM1

 T Cell / APC Interaction
o “SMAC”
o Co-Stimulatory Signals
 Signal One: TCR binds MHC + peptide
 Signal Two: B7 on APC interacts with CD28 on T cell

1. What are the 3 types of antigen-presenting cells and how do they differ?
 Dendritic Cells
o Direct infection or phagocytosis of
virally infected cells/allergens
o move to a draining lymphatic
vessel and undergo maturation
(increase MHC expression)
o enter lymph node via AFFERENT
lymphatic
o Settle in the T CELL area of the
draining lymph node and present
antigen on MHC II
o When mature have lots of B7 (high
co-stimulator delivery)
 Macrophage
o Phagocytose bacteria and present
on MHC II
 B Cells
o Presents microbial toxins and
soluble antigens
o Looking for a specific epitope
(other APC’s are nonspecific)

2. What is the role of IL-2 in T-cell growth?

 IL-2 induces a positive feedback loop for T Cell proliferation
 Cyclosporin and Rapamycin interfere with IL-2 production/signaling

3. How do CTL recognize and kill infected cells?

 DIRECTLY kills
 TCR binds MHC I + peptide
 Releases perforin and granzymes
 Induces apoptosis
 Kills several infected cells in a short time

4. What are the functions of Th1 cells?

 Promotes cell mediated immunity by activating
macrophages
 Good for VIRAL, BACTERIAL (intracellular)
INFECTIONS
 TH1 cells travel to INFECTED TISSUE
 Macrophage activation
o IFN y cytokine
o CD40 Ligand binds CD40 on macrophage

5. What are the functions of Th2 cells?

 Promotes humoral immunity by activating B cells
 Good for PARASITIC (extracellular) INFECTIONS and
many viral/bacterial infections
 TH2 cells travel to LYMPHOID TISSUE
 B Cell activation
o Cooperative interaction: B and T cells recognize
different epitopes of the same pathogen
o TH2 Cytokines: IL-4, IL-5, IL-6
o CD40 ligand binds CD40 on B cell

6. Case Study: Leprosy – understand both forms
(Lepromatous and Tuberculoid Leprosy)

 INTRACELLULAR BACTERIUM (infects macrophages)  TH1 is best!

LEPROMATOUS LEPROSY TUBERCULOID LEPROSY
TH1 Response  cell mediated immunity
TH2 Response  B cell activation &
• MORE APPROPRIATE FOR THIS
antibody production
CD4 T Cell • Very high serum immunoglobulins PATHOGEN!!!
• Normal serum immunoglobulin levels
• Low T-cell responses to M. leprae
• Strong T-cell responses to M. leprae
• Severe disease course • LESS severe disease course
• Highly infectious skin lesions • Few organisms present (“paucibacillary”)
• Many organisms present • Organisms are walled off in a granuloma
Clinical
(“multibacillary”) in macrophages (partially successful immune response)
Picture
• Disseminated infection with • Local inflammation, peripheral nerve
widespread nerve damage damage
• Cutaneous nodules
Cytokine
• IL4, IL5, IL-10 • IL2, IFNy, LT
Patterns
Therapy? • Stimulate a TH1 response!