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 doesnt 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 (doesnt 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, peyers 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 (nave)
o Recirculate between lymph, blood, secondary lymphoid tissues

4. What happens in Germinal Centers?

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

Negative Selection by TCRs interacting with DENDRITIC CELLS, MACROPHAGES, OTHER APCS 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 individuals 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 nave 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 Abs 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 nave 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 antibodys 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
Resembles membrane bound antibody
Immunoglobulin-like domains (2 per chain)
Variable Region (binds antigen) three
hypervariable loops = CDR1, 2, 3
Constant Region
Diversity results from gene rearrangement
DIFFERENT
- alpha and beta chains (not
heavy and light)
- variable region = Valpha and
Vbeta regions
- even more diverse than Ig
- 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 APCs, 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 APCs 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!