What are the pre-antigen exposure events (8)?

Where do they take place?

• Mechanisms of V_H, Vκ, and Vλ chain recombination
• RSS the 12/23 rule
• Junctional flexibility
• P and N nucleotide addition
• CDR1, CDR2 and CDR3: mechanisms of diversity
• Rearrangment & expression of H & L chains
• Allelic Exclusion
• Co-expression of IgM & IgD by naïve B cells

• Bone marrow

What are the post-antigen exposure events (6)?

Where do they take place?

• Affinity maturation
• Somatic Hyper Mutation
• Clonal selection
• Isotype (Class) switching
• Synthesis, assembly and/or secretion of Ig
• Expression of transmembrane & secreted Ig of the same specificity: alternative mRNA splicing

• Peripheral lymphoid organs

How many distinct segments encode the heavy chain? Name them?

How many distinct segments encode the light chain? Name them?

Heavy - 3 segments; V, D, J

Light - 2 segments; V, J

On what chromosomes are the heavy & light chain segments ?

Heavy-chain locus – chromosome 14 Kappa- light chain – chromosome 2 Lambda light chain – chromosome 22

What things give the immunoglobulin additional diversity?

• Junctional flexibility
• P & N nucleotide addition
• Somatic mutation

Decide if the following are DNA or RNA events?

1. Formation of the variable domain
2. Joining of the variable domain with the constant domains
3. IgM & IgD have the same specificity (within the variable region) but are of different classes
4. Isotype (or class) switching
5. Somatic hypermutation

1. DNA event
2. RNA event
3. RNA event (splicing)
4. DNA event
5. DNA event

(note: DNA events are irreversible, RNA events are reversible)

Somatic recombination can only occur between what two things?

What are the names of these segments?

• 12-spacer & 23-spacer

• Recombination Signal Sequences (RSSs)

What two nuclear phosphoproteins start the process of gene rearrangement?

What’s the process?

• RAG-1 & RAG-2

• Recognize the RSS
• Form the RSS synapse
• Remove hairpin loop structure

What’s the difference between P and N nucleotides?

• P nucleotides make a palindromic sequence in the final dbl stranded DNA, added by DNA polymerase, adding complement DNA according to the template

• N nucleotides added randomly to the end of single strands by TdT, not encoded by the germline

Name two things removal or addition of nucleotides at the end of a gene segment can do?

• Enhance antibody diversity
• Premature chain termination

What determines the antigen specificity of the Ig molecule?

Hypervariable regions

What is significant about CDR1, CDR2 & CDR3?

What is the difference between CDR3 and the other two?

• They are hypervariable regions 

• CDR3 is the result of random diversity (most hypervariable) while CDR1 & CDR2 are encoded for in the DNA & are variable

What is allelic exclusion?

Expression of a gene on one chromosomes prevents the expression of the allele on the other chromosome Only one H-C and one L-C are finally expressed after going through the process of immunoglobulin-gene rearrangement

What forms the functional B cell receptor?

The complex of immunoglobulin with Ig-α & Ig-β (the latter two have long cytoplasmic tails)

Antigen-induced B cell activation (clonal selection) leads to changes in the antibody the B cell produces, name the changes (4)?

• Ratio of secreted to membrane forms of Ig ↑
• Somatic hypermutation of variable regions occurs (in B cells only)
• Affinity maturation - generation of higher affinity antibodies
• Class switching occurs: retention of specificity, but diversification of function

Where do SHMs not occur?

Mutations do not extend into the portion of the gene encoding the constant region

Name the properties of isotype (or class) switching (4).

• It’s both T cell (cytokines) & microenvironment dependent (permanent switch)
• Involves translation of H-C constant region genes & loss of intervening DNA
• Maintains the selected variable region & thus specificity & affinity
• Increases the diversity of Ig function (Fc mediated)

What substance initiates class switching?

Activation Induced cytidine Deaminase (AID)

Where are cKit and Stem Cell Factor (SCF) located?

What about IL-7 and IL-7R?

• cKit is on the pro-B cell and SCF is on the bone marrow stromal cell. They join together as the B cell develops in the bone marrow

• IL-7 is from the bone marrow & IL-7R is on the B cell

With which signal transduction molecules do Igα and Igβ match up?

What 2 molecules make up the surrogate light chain?

• CD79a & CD79b

• VpreB (subs for a L-C variable region) & λ5 (subs for a L-C constant region)

Where does the cell commit to a B-cell lineage?

What are the two checkpoints for the making of a B cell?

Where is H-C gene diversity generated?

Where is L-C gene diversity generated?

Where do you have functional IgM?

• Early pro-B cell

• 1 -  pre BCR briefly present @ cell surface -see if fxnal H-C was made
• 2 - appearance of fxnal BCR @ cell surface (L-C made successfully) no more L-C gene rearrangement

• Pro-B cell population

• Pre-B cell population

•  Immature B cell

Binding of self results in what three things?

1. Deletion – physical removal from the repertoire
2. Anergy – paralysis of function
3. Receptor Editing – alteration of specificity (rearrangement of light chains)

Match for B Cell Tolerance

A. Receptor editing

B. Clonal deletion

C. Clonal anergy

1. 1. Immature B cell binds multivalent self antigen
2. Immature B cell binds soluble (monovalent) self antigen
3. New chance to make BCR with new specificity

A – 3

B – 1 (B cell expresses only IgM on surface)

C - 2 (IgM is retained inside the cell, still express IgD on surface)

A mature but naive B cell (IgM & IgD on surface) must be activated before it can differentiate into what?

What is the purpose of BCR activation?

• Ab producing cell or memory cell

• to initiate 2nd messenger systems, resulting in transcription of genes which up-regulate and/or encode adhesion mlcs, MHC mlcs & other proteins req to promote cell interaction w/ T cells.
• can also result in commitment to IgM production w/o T cell interaction

Why are lipid rafts impt to BCRs?

• Lipid rafts contain mlcs necessary for proper signal transduction
• Activation of BCR requires movement into lipid raft

What happens in BCR capping?

• Binding results in grouping of receptors w/i lipid rafts
• Initiates cytoskeleton rearrangement necessary for B/T interaction

Describe what happens after B cells become activated when their receptors are cross-linked by antigens.

• Receptor cross-linking activates Src family kinases (RTK)
• ITAMs are phosphorylated 
• PLC-γ is activated converts IP₂ → IP₃ + DAG
• DAG + Ca²⁺ activate PKC
• NF-KB, NFAT, AP-1 (TFs get activated, translocate to the nucleus (go from G₀ → G₁ outside into nucleus)
• New gene expression

Note: ITAMs (located in the cytoplasmic tails of Igα & Igβ) are activating motifs; ITIMs are inhibiting motifs; Tyr is impt in these motifs

What molecule removes inhibitory P on Src family kinases during signal transduction via the BCR?

• CD 45 phosphatase

What is the main purpose of the B cell co-receptor?

What are 3 markers that part of the co-receptor?

• ↓ the threshold for immunogen activation (positive)

• CR2/CD21 (complement receptor)
• CD81 - regulates motility & signal transduction
• CD19 - pan B cell marker, binds Src Kinase, Lyn when phosphorylated by CD21

What is the purpose of the negative co-receptor of the B cell?

• Ig/Ag binding  to BCR &  (IgG to F_cγR) turns off receptors i.e. it tells them there is no reason to activate, there is already secreted Ig against the Ag
• ITIM* is activated here

Note: this is not T₀, wouldn't have immune complexes already. At T₀ = no Ab, BCR bind Ag start making Abs

*Immunorecptor Tyr-based Inhibition Motif

T or F, B cells can undergo clonal expansion in the absence of T cell help?

T or F, low-level IgM secretion is possible?

T or F, you can still make memory cells, have class switching & fxnal diversity?

• True

• True

• False

Immunodeficient pts who lack both a thymus and T cells have a normal number of B cells but fail to make an Ab response to most Ags. The microbial Ags they are able to make an Ab response again are called ___?

thymus-independent antigens

In thymus-independent Ags what turns into B cells and then plasma cells?

Bacterial polysaccharides & lipopolysaccharides

Decide if it is TI-1 or TI-2

• Produces Ag specific Abs
• has mitogens, e.g. LPS (note: LPS is a PAMP, the PRR for it is a toll-like receptor #4)
• production of IgM, rarely IgG, IgA
• may be presented on non-classical MHC on γδ T cells
• Non-specific polyclonal activation
• Lack of memory response
• Most likely source of IgG anti-ABO activity in some pts.
• requires repeating epitopes

• TI-2 (Ag activates BCR → creation of specific Abs)
• TI-1
• TI-1
• TI-2
• TI-1 (via high dose of LPS)
• TI-1, 2
• TI-2
• TI-2

What marker is present in B-1 but not B-2 cells?

Which cells appear to be innate but are not?

Which cells are characterized by little or no cell-surface expression of IgD?

Which Ag(s) activate B-1 & B-2 cells?

Which Ag(s) activate only B-1 cells

Which cells produce Abs that tend to bind to bacterial polysaccharides, are polyspecific, low affinity & mainly IgM isotype?

• CD5 (usually a marker for T cell lineage)

• B-1 cells

• B-1 cells

• TD & TI-1

• TI-2

• B-1 cells

What kinds of Ags are Thymus-dependent?

What cells do endocytosis, processing & presentation of Ag on MHC to T cells?

What role do the activated B cells have? (processing of Ag)

• soluble proteins, peptides & some (glyco)-lipids from whole cells, viruses, parasites

• APCs

• endocytosis via the BCR
• proteolysis to 13-18 aas
• presentation by MHC II to CD4+ T cell

Helper T cells interact w/ B cells

For B cells responding to TD Ags, multistep interactions btw T & B are req'd, what are they?

• Induction of affinity maturation
• Induction of class switching & memory (maintain variable region, switch F_c)

In B cell activation there are 2 signals. Signal 1 is ?

Signal 2 is?

• BCR binds with Ag (Ag specificity inherent in the 1st signal)
• CD40 on B cell binds CD40L on T cell (not expressed on mature, naive T cells; gets induced therefore only expressed on activated T cells)

Note: after signals 1 & 2 B cell is activated to divide & differentiate

T cell activation

1. Immature tissue resident DCs w/ intact & processed Ag on surface, migrate to ___ or ___?
2. Once there they lose phagocytic capacity & become ___, presenting Ag via MHC II mlcs to ____ T cells in T-cell rich paracortex.
3. Results in ___ T cells which migrate to ___ rich cortical region.

1. lymph node, spleen
2. APCs; mature, naive
3. activated; B cell

What sets up a polarity cytoskeleton rearrangement directed to site of binding w/ the B cell?

What cytoskeleton protein concentrates at the area of cell-cell contact?

Because there is a tight jxn where binding & localized release of cytokines to the B cell occurs, what is a consequence of this?

• MTOC

• talin

• don't have to make as much cytokine as you would if it was released randomly

The 1° focus for expansion of antigen-activated B cells is in the ____?

The 2° focus for expansion of antigen activated B cells is the ____?

• medullary cords

• germinal center

How do follicular dendritic cells (FDCs) differ from dendritic cells (in the germinal centers)?

• They will bind antigen but not process & present it.

What are the steps that occur in the two T-B interactions?

• B cells migrate to 1° follicles, which become germinal centers w/ B cell proliferation
• B cell centroblasts proliferate in the dark zone of the GC, undergoing SHM for diversification of Ig genes & thus BCR (helper T cells induce)
• Centrocytes that have undergone SHM migrate to light zone & compete for FDC-Ag. FDC can only present to B cells, not T cells, FDCs do NOT express MHC II
• Highest affinity Ig producing cells selected
• Express Bcl-2, prevents apoptosis, via interaction w/ MHC II helper T cells (CD40L on T_h & CD40 on Bcell) apical region of GC light zone
• Plasma cells and memory cells develop

Note: if CD40-L is mutated it won't respond to cytokine (tells us this is a key rxn)

What events occur during the 1st T-B interaction? 2nd T-B interaction?

• Ig production, germinal center formation

• Clonal selection; isotype switching, proliferation, memory

Note: Affinity maturation occurs

Where does Somatic Hypermutation occur for centrocytes (B cells) that are being activated?

What enzyme is responsible and what does it do?

The process in which high affinity BCRs are selected by antigen on FDCs is known as?

• In the dark zone of a germinal center

• Activation-Induced Cytidine Deaminase; deaminates DNA at cytidine residues (repair of induced DNA lesions → permanent base Δes

• Affinity maturation of the humoral response

What does it mean that T-B interaction requires cognate help?

• There is a piece of peptide presented on MHC to T cells that causes a recognition event. The T cell only focuses on the cell that presents the peptide to it. Without the peptide, there would be no T cell help

What is the role of T cell cytokines in T cell help?

Different T cells are making different cytokines which aid in isotype switching

Why is the 1° response short-lived (˜ 2 wks)?

What cells become memory cells (be specific)?

• Plasma cell differentiation is Ag-dependent

• Isotype switched B cells, they express membrane IgG, IgA, or IgE; resting cell stage (G₀)

What is the role of IL-10?

What is the role of Il-4?

• Helps centrocytes differ into plasma cells (current)

• Helps centrocytes differ into memory B cells (investment)

Why is apoptosis induced in most reactive T cell?

What happens upon antigen rechallenge?

• production of cytokines by T cells is potentially more harmful in maintaining an inappropriate response; activation induced death (AICD)/Fas pathway

• there is an increased speed & affinity of response

Note: both T & B memory cells express high levels of adhesion mlcs for recirculation & homing (latter is tissue specific)

What is the time course of events for plasma cells?

• 4-7 days: production of plasma cells w/i 1° follicles
• 7-10 days: 1° response migration to GC for affinity mat, class switching, generation of Ig secreting plasma cells & memory cells
• 2-4 wks: plasma cells migrate to medulla & produce Ab; constant selection

Where are plasma cells found?

• Medullary cords of the lymph nodes
• Red pulp of the spleen
• Lamina propria of the GALT
• Bone marrow

What are the 4 phases of the Ab response after antigen challenge?

1. lag phase: no detectable Ab
2. log phase: Ab titer ↑es logarithmically
3. plateau phase: Ab titer stabilizes
4. decline phase: Ab is cleared or catabolized

Explain what happens during the second exposure to an Ag.

Even though you will have a 2° response to Ag A (IgG titer - keep Abs to things we've been exposed to, memory) will still have a 1° response b/c new B cells are seeing it for the 1st time (IgM)

Note: usually no disease symptoms the 2nd time

How does the cellular response help the humoral response?

Memory T cells can help memory B cells respond in a quicker fashion

Note: memory is faster w/ greater affinity

What is the difference btw affinity & avidity?

Affinity - is used to describe the strength of a single bond; eg. IgG₁

Avidity - is used to describe the combined (synergistic) strength of multiple bond interactions (fxnal affinity); eg. IgM

Note: you can have low affinity but high avidity

In a Precipitin rxn in which zone do you see precipitate & why?

• Precipitate forms in the zone of equivalence because a lattice forms btw Ag & Ab
• In the zone of Ab excess & zone of Ag excess, mlcs of Ag & Ab remain soluble → no precipitate

Ouchterlony: Double Immunodiffusion Assays

What are the 4 types of zone of equivalence lines you may see?

1. Identity: continuous line (looks like a boomerang); Ag A; Anti-A serum; A-anti-A ppt line
2. Nonidentity: two lines cross completely; Ag A & Ag B; Anti-A serum, Anti-B serum; A-anti A ppt line & B-anti-B ppt line
3. Mixture of antigens: 1 continuous line & 1 half line; Ag A & Ag A+B (no shared epitopes); Anti-A serum, Anti-B serum; A-anti-A ppt line (full) & B-anti-B ppt line (half - could be shifted based on Δ in conc.
4. Partial identity: continuous line w/ a spur @ 1 end; Ag A & Ag A/C (share some epitopes); Anti-A serum, Anti-C serum; A-anti-A and C-anti-C ppt line (spur) - Δ in conc. will not move the spur

Note: if you 2x the Ag conc. the zone of equivalence will shift to the left towards the Ab (Ag move from high to low conc.)

precipitin lines are only if you get a precipitated event

, or + means separate mlcs; / means same mlc

Antigenic determinants/epitopes can present in several ways on different species, what kinds of things can you see?

• epitopes can be conserved across species
• there can be identical & non-identical epitopes on related proteins
• cross-reactive recognition - slight recongnition; some binding w/ less affinity (can refer to the determinant or the protein

What's the difference btw precipitation & agglutination?

• precipitation - immune complex formation w/ mlc Ags (soluble Ag & soluble Ab)

• agglutination - immune complex formation w/ Ag particles (eg. RBCs, bacteria, latex particles) Beads or RBCs are coated w/Ag

Quantitative Hemagglutination Assay

What is a titer?

What is a prozone?

• titer - reciprocal of dilution

• prozone - Ab excess

Note: look at the dilution that was the last positive, the reciprocal is the titer

start with 1/2 in the 1st column if 2-fold deletion

results - look like a button if negative; fill in the well if positive

What is the Direct Coombs Test?

What is the Indirect Coombs Test?

• Test for the presence of antigen (biopsy, cells, tissue). Can use it to detect Abs that are already bound to a pt's RBCs
• specimen - you have Rh+ & baby RBCs
• reagent - anti human Ig
• + test - agglutination (pt had Abs)
• - test - no agglutination (no Abs)

• Test for the presence of antibody in the pt's serum (unbound).
• specimen - pt's serum
• reagent - Rh⁺, RBCs
• step 1 - pt's serum +RBCs & Rh⁺
• step 2 - bound from step 1 + anti-human Ig
• + test - agglutination (pt. has Abs in serum)

Explain what is happening in latex agglutination of rheumatoid factor screeing.

• Testing to see if pt. has Abs to RF in the serum.
• Use reagent - IgG bound to latex particles
• Add RF (tagged w/ IgM anti-IgG) & patient serum (want to know if IgM-RF present)
• + test - agglutination, pt. has Abs
• - test - no agglutination, pt didn't have Abs

What is the 1° purpose of generating Monoclonal Abs (mAbs)?

How are they made?

• To make a lot of Ig w/ one specificity & high affinity

1. B cells from mouse immunized w/Ag (wouldn't live long on own)
2. Fused w/ Myeloma cells (live long but don't make Ab of interest)
3. Grow the fused cells in drug-containing medium
4. Only hybrid cells live (myeloma cells have no resistance; B cells have resistance, only live 7 days; hybridomas resistance + duration
5. Select for Ag-specific hybridoma
6. Clone selected hybridoma cells

How can you detect mAbs?

What is the role of CD mlcs w/ mAbs?

• You can use Fluorescent Activated Cell Sorter

• Cells identified w/ mAbs specific for CD markers
• eg. if wanted T cells could bind Ab to CD3 & pick out T cells

Name the 4 types of therapeutic mAbs & how they work.

1. mouse: 1st use ok but person will make Abs to it so 2nd time wouldn't work
2. chimeric: F_v - mouse, F_c - human → recognized as foreign in F_v
3. humanized: keep just the hypervariable part for mouse
4. human: take Ig gene regions & put it into mouse (mouse w/ human Ig genes); immunize it; mouse makes human mAbs (thinks it's making its own product)

Note: #4 only works w/ human cells

What is the "filling" in a direct ELISA?

What is the "filling" in an indirect ELISA?

• antigen in pt's serum

• antibodies in pt's serum

Direct ELISA - fill in the blanks

1. ___ is adsorbed to well. What layer is this?
2. Pt sample added; complementary ___ binds to Ab. What layer is this?
3. Enzyme-linked ___ specific for test ___ is added & binds to ___. What layer is this?
4. Enzyme's ___ is added & rxn produces a product that causes a visible color Δ.

1. Ab; 1st layer "bread"
2. Ag; 2nd layer "filling"
3. Ab, Ag, Ag; 3rd "bread"
4. substrate

Indirect ELISA - fill in the blanks

1. ___ is adsorbed to well. What layer is this?
2. Pt antiserum added; complementary ___ binds to Ag. What layer is this?
3. Enzyme-linked anti-HISG is added & binds to bound ___. What layer is this?
4. Enzyme's ___ is added & rxn produces a product that causes a visible color Δ.

1. Ag; 1st layer "bread"
2. Ab; 2nd layer "filling"
3. Ab, 3rd "bread"
4. substrate

Classical Radioimmunoassay: Competition

Std: 60k cpm (counts/min)

have test Ab, pt Abs, Ag @ solid phase

1. Situation 1: measurement of radioactivity of sample shows 60k cpm
2. Situation 2: measurement of radioactivity of sample shows 30k cpm

1. No competition; there is no Ab in pt's serum 
2. Competition; Ab in pt serum is competing w/ radioactively labeled testAb

Western Blot testing for anti-HIVAb

• After HIV viral proteins are denatured, and Ags are separated by SDS-PAGE, they are transferred to a membrane incubated with ______?
• If the serum contains ___ which bind ___ they are made visible following incubation w/ an _____?

• a pt's serum
• Abs; viral proteins (Ag); enzyme-labeled anti-Ig Ab (and a chromogenic substrate that the enzyme will convert into a visible & insoluble product)
• Note: Integration alert!!! WB is used to test the positives from an ELISA (to eliminate FPs b/c ELISA is more sensitive than specific)

Complement Fixation Test

1. Ag mixed w/ test serum to be assayed for ___?
2. Std amnt of _____ is added?
3. Erythrocytes coated w/ Ab is added, what is the purpose of this step?
4. Amnt of RBC ___ is determined.
5. What indicates a positive test result (i.e. pt has Abs)?
6. Bonus: what has been split, what pathway activated?
7. What does the assay look like?

1. Ab
2. complement
3. indicator step; has nothing to do w/ disease - just used to see if lysis
4. lysis
5. No lysis (because the fixed complement has been consumed
6. C3b + C5b have been split; classical
7. dot in well is +; filled well (lysis) is -

• What is the composition of MHC class I? HLA markers?

• What is the composition of MHC class II? HLA markers?

• 0n what chromosome are all the MHC genes located?

• single polypep chain + non MHC protein β₂ microglobulin; HLA-A, HLA-B, HLA-C

• two polypep chains - α & β; HLA-DR, HLA-DP, HLA-DQ

• Chr 6

What is the 1° fxn of the MHC gene complex-encoded proteins?

Which cells recognize Ag presented by MHC I or II mlcs in the adaptive immune response?

How does this differ from the MHC role in the innate immune response?

• presentation of antigenic peptides

• T cells

• NK cells - KIR binds to MHC I - inhibitory signal

CD8 binds the ___ domain of MHC class I.

CD4 binds the ___ domain of MHC class II.

• α₃

• β₂

On what tissues are MHC class I expressed?

On what tissues are MHC class II expressed?

• protein product expressed on the surface of all cells in the body (w/ exception of RBCs) i.e. expressed codominantly

• expressed 1° on hematopoeitic & APCs, other tissues under certain conditions; expressed codominantly

What is a haplotype?

Why are organ transplants so prone to rejection w/ regards to HLA matching?

• A combination of HLA alleles (co-dominant expression, 1 maternal & 1 paternal)

• The amount of polymorphism is great w/i the human species so most transplants are not completely matched (note: very difficult unless you're an identifcal twin)

Can new haplotypes arise and if so, how?

Yes, from recombination of maternal haplotypes. However it's usually at a very low frequency that new haplotypes arise (crossing over evens not common)

Which MHC mlcs are more diverse, class I or II?

Why have more than 1 MHC mlc?

• class II b/c 2 chains combining → additional polymorphism, i.e. "hybrid mlcs"
• note: this is not that common

• provides the body w/ the ability to "present" a larger # of Ags

How does the peptide binding cleft/groove differ btw MHC class I & II?

What happens as a result of this difference?

• In class I both ends are closed; in class II the cleft is open (note: the T cell recognizes aa residues that "stick up"

• class I mlcs bind shorter peptides (8-10 aas) vs. class II (13-18 aas)

Characteristics of binding of antigenic peptides: choose MHC class I or II

1. α₁ & β₁
2. closed @ both ends
3. endogenous
4. open @ both ends
5. α₁ & α₂
6. exogenous
7. lysosomal proteases
8. proteasome complex
9. 8-10 aas
10. 13-18 aas

1. II
2. I
3. I
4. II
5. I
6. II
7. II
8. I
9. I
10. II

From where does the variability in MHC II mostly come?

From which proteins do the peptides come?

To what does the peptide bind?

• the β-chain; the α-helix is conserved

• self or foreign protein

• 1 side binds MHC, the remaining binds the TCR

T or F the anchor residues in the MHC I mlc are available for binding the TCR, if F make it true.

F, they are already binding the MHC molecules, the ones in the "bulge" are available

What is CD1?

Which MHC mlc is it similar to and how?

What is its role?

What cells express CD1?

What does presentation of Ag require?

• an MHC-like protein

• MHC I; structurally it associates w/β₂m & Ag presenting cleft similar

• hydrophobic so allows for presentation of lipid Ags to T cells @ the cell surface

• APCs & they are specialized based on Ag (eg. LPS, Mycolicacid)

• endosome acidification (TAP independent (class I) & HLA-DM independent (class II)

Note: lipid Ags are also presented in fragments

APCs will have ___ mlcs binding antigenic peptide. The TCR will recognize BOTH the specific HLA eg. HLA-A & the ____. It will not recognize another ___ eg. HLA-B with the same peptide.

Bonus: What are the APCs?

• MHC (I & II)

• peptide

• HLA

• Dendritic cells
• Macrophages
• Activated B cells

Which cells in the body are capable of presenting Ag via MHC I?

What happens if the Ag is self?

What happens if the Ag is non-self or altered self? Result?

• virtually all cells except RBCs

• no immune response should be initiated

• immune response initiated; results in a target cell

Which cells are capable of processing & presenting foreign antigen via MHC II?

Co-stimulation via binding of ______ (B7-1/B7-2) on the APC to ____ on the T cell is one of the requirements for complete induction of an immune response.

• APCs (note: this is an impt step in the induction of the adaptive immune response)

• CD80/CD86; CD28

Fill in the Blanks

1. Most T cells recognize _____?
2. T cells recognize ____  & not conformational determinants
3. T cells recognize ____ & not soluble Ags (9-10 aas in binding groove)
4. CD4⁺ T cells recognize Ags sampled from _____ milieu presented by _____
5. CD8⁺ T cells recognize Ags sampled from ____ pools presented by MHC I

1. peptides
2. linear
3. cell-associated
4. extracellular; MHC II
5. cytosolic; MHC I

• Proteolytic degradation of proteins into peptides of a suitable size for binding to MHC mlcs (some lipids processed via CD1) is known as?

• the actual display of the processed peptide bound to MHC mlcs (or CD1 mlc) on the surface of an APC or target cell is known as?

• processed & presented Ags in the context of MHC mlcs are recognized by specific TCRs this is known as?

• Ag processing

• Ag presentation

• MHC-restricted recognition

Ag processing & presentation via

MHC I - ENDOGENOUS

1. Intracellular Ag (CYTOSOLIC)
2. Proteasome (in cytosol, constitutive) recognizes Ag via Ubiquitin (poly-ubiquitination)
3. Ag processed to peptides in proteasome
4. IFN-γ in the proteasome helps to make better antigenic peptides  = immunoproteasome (preferred cleavage following hydrophobic or basic residues - near end positions - closed groove)
5. MHC class I heterodimer assembly in ER - chaperones for proper folding (Class I H-C stabilized by calnexin (disulfide bridge formation) until β-2 microgloblin binds; calnexin released, heterodimer of class I  & β₂m form peptide loading complex w/ calreticulin, tapasin (forms bridge btw TAP 1 & 2), TAP, ERp57, and PDI
6. peptide transport into ER via TAP 1 & 2, expression upregulated by IFN-γ, ATPase dependent (if not fxning won't get proteins on cell surface)
7. peptide binding to MHC class I = mature (if too long cleaved randomly by ERAP, dissociates from peptide-loading complex, exported from ER, travel through Golgi)
8. MHC class I present peptide @ cell surface to CD8⁺ T cell
9. End game: cell death

Note: restricted to peptides approx 8-10 aa in length

Note: viruses presented by DCs follow the MHC class I not II pathway

Ag processing & presentation via

MHC II - EXOGENOUS

1. extracellular Ag
2. forms an endocytic vesicle
3. peptide production in phagolysosome (acidified)
4. meanwhile MHC class II in vesicle, MIIC (from ER via Golgi); Invariant chain (Ii) binds MHC II preventing binding of peptides while in the ER (trafficking signal to endosome)
5. in vesicles Ii is cleaved, leaving CLIP
6. CLIP blocks binding of peptides to MHC class II in vesicles (until ready)
7. HLA-DM facilitates release of CLIP allowing peptides to bind (facilitated by HLA-DO)
8. peptide binding (exchange) by MHC class II
9. MHC class II presents peptide at cell surface to CD4⁺ T cell
10. End game: activation of B cell to secrete Ig to eliminate EC bacteria

Note: restricted to peptides approx 13-23 aa in length

Viruses can evade MHC I Ag presentation, give a couple of examples how? clinical significance?

• affect expression of TAP genes - no transport
• ER retention & degradation of MHC I
• Hold MHC I in Golgi
• Disrupt cleaving of peptides
• Translocation of MHC I to cytosol

• EBV -disrupts cleaving action of proteasome

Bacteria can evade MHC II Ag presentation, how?

• Escape endosomes
• neutralize endosome acidification
• block fusion w/ lysosome
• sequesters MHC II mlcs after vesicle fusion

ID either B or T cell

• have BCRs
• Recognize both linear & conformational antigenic determinants
• membrane bound only
• part of the adaptive immune response
• have TCRs
• Recognize only linear antigenic determinants
• part of the Humoral response
• part of the Cell Mediated response
• Membrane bound & secreted state
• Antigenic determinants processed & presented in context of MHC mlcs

• B cells
• B cells
• T cells
• Both
• T cells
• T cells
• B cells
• T cells
• B cells
• T cells

Structure of Heterodimeric TCR

• The 1° type is ___ (95%) & the 2° type is ___ (5%)
• What do they have that are not capable of signal transduction?
• What is CD3 analagous to on the B cell?
• Which domain contains CDR1, CDR2 & CDR3?
• How many Ag binding sites does a  TCR have?
• Which CDR is the most variable? what does it bind?
• Which CDR(s) bind MHC?
• What is the term for each T cell bearing a single specificity & single allele of TCR?
• TCR is encoded in gene segments that undergo _____ during T cell development to generate Ag-binding diversity

• αβ; γδ (note: all the stuff that makes the δ-chain is in the α-chain boxes; if you put together α-chain 1st can't make δ-chain, intervening DNA lost)
• short cytoplasmic tails
• CD79a/b
• the N terminus variable domain
• one 
• CDR3; antigenic peptide
• CDR1, CDR2
• allelic exclusion
• somatic recombination

TCR diversity

• Which CDR(s) is already for in our DNA?
• What occurs in CDR3?
• T or F, generation of Ag-binding diversity for TCR is similar to that used by the B cell for Ig diversity?
• What event does  not occur in TCR diversity that you see in BCR diversity?

• CDR1, CDR2
• joining regions for VJ (α-chain) & VDJ (β-chain) 
• T, uses RAG-1 & -2, P & N nucleotide addt'n, jxnal flexibility
• Somatic hypermutation

TCR chain rearrangement

• How many chances do you have to rearrange the β-chain?

• Which T cell type are intraepithelial lymphocytes, has limited diversity of the TCR, no CD4 or CD8 & has no Ag independent maturation in the thymus?

• Which T cell type is the 1° T cell pop. responding to APCs & target cells, , express co-receptorsfor CD4 or CD8^*, express both constitutive & inducible co-stimulatory & adhesion mlcs, require CD3 for TCR expression & signal transduction & undergo Ag indep. maturation in the thymus?

^*CTLA4 has expression of both, thymic education

• 2

• γδ

• αβ

• Which T cell secretes cytokines to strengthen a response?
• Which ones recognize glycolipids & peptides presented by CD1?
• Which ones are cytotoxic?
• Which ones are cell-mediated?
• Which ones are humoral?

• CD4⁺
• Natural Killer
• CD8⁺
• Th1 (part of CD4)
• Th2 (part of CD4)

Relationship btw TCR & CD3

• What is expressed with TCR?

• What happens if one part of the complex is missing?

• T or F, TCR gets secreted?

• Signal transduction complex, CD3

• αβ won't come to the surface

• F, TCR never gets secreted

Which statement is correct?

• A. CD4 is a co-receptor mlc for TCR but CD8 is not
• B. CD8 is a co-receptor mlc for TCR but CD4 is not
• C. CD4 & CD8 expression is linked w/ TCR expression but is not necessary for TCR signal transduction events
• D. CD4 & CD8 expression is not linked w/ TCR expression but is necessary for TCR signal transduction events
• E. Weakens Ag/MHC-TCR avidity

The correct answer is choice D

Co-stimulatory & accessory mlcs

• CD28 on T cell binds __ & __ on APCs.

• LFA-1 on APC binds ___ on T cell

• CTLA-4 induced on T cell binds __ & ___ (downregulatory - post-activation)

• ___ induced on T cells binds CD40 on B cells, marcrophages

• CD86; CD80

• ICAM-1

• CD86; CD80

• CD40L

T cell development - thymic education

• Trafficking of CD34⁺ from the thymus to the bone marrow requires that what 2 things be attached?

• The Pro-T receives 2 signals, what are they and what do they do?

• c-kit & CD44

• IL-7/IL-7R & SCF/c-Kit; directs TCR gene rearrangement (DNA)

Progressive expression of surface markers

• Lineage committment & MHC recognition is what type of selection?
• Where does this occur?
• What other unique event occurs there?
• T or F, you want self-recognition during this selection, ie. tight binding?
• T or F, a substantial majority of T cells survive during this selection?

• What type of selection involves self reactive T cells being eliminated?
• Where does this occur?
• What CD markers are expressed here?
• T or F, during this selection you want tight binding?

• positive selection
• cortex of the thymus
• induction of the expression of both CD4 & CD8 on all cells
• True b/c has to recognize MHC
• False, 95% of T cells die

• Negative selection
• Medulla of the thymus
• either CD4 or CD8
• False, tight binding = death

• Will you have a double positive (DP), double negative (DN) or single positive (SP) thymocyte as a pre-TCR?

• What event causes a thymocyte to become SP?

• DP

• after positive selection - MHC recognition

• What is AIRE?

• Is this important for peripheral or central tolerance?

• a transcription factor expressed in the medulla of the thymus, impt for prevention of autoimmune disease, inititates transcription of self-antigens such as insulin that are usually only expressed in peripheral tissues so that self-reactive T cells are eliminated before they can leave the thymus

• central tolerance

• What is central tolerance?

• What is peripheral tolerance?
• What is the purpose of immune privileged sites?

• Newly developing T & B cells are rendered non-reactive to self; occurs while cells are still present in the 1° lymphoid organs; result of negative selection

• Generated after the cells reach the periphery (note: when a T cell sees Signal 1 w/o Signal 2, it results in anergy
• periph tol: certain sites do not have activated T cells (eye, brain, ovary, testes) immune response to Ag is not destructive
• cells in these sites express CD95L (Fas-L) which binds to CD95 (Fas) (cytotoxic T cells), causes cell apoptosis of T cell (CD95)
• IDO - cause catabolism of Trp, deleterious for WBC life or proliferation & another recogntion to B cells that get activated. complement deposited → MAC → lysis
• (note: periph tol specific for fetal tissue)

• What is the role of regulatory T cells (T_reg)?
• One form expresses CD4⁺, CD25⁺  and ___?
• How does this process work?

• This is important for peripheral or central tolerance?

• They suppress the immune responses of other cells

• Foxp3 (note: these are naturally occurring)

• expression of tranx factor foxp3 (located inside of cell) reqd for T_reg development
• Majority of these T_regs are found within CD4⁺ cells & express CD25⁺(IL-2)

• peripheral