Antibody and T cell receptor diversity

Question 1

Antibody structure: chains

Answer 1

2 identical heavy chains, 2 identical light chains

Each chain has Ig domains ~110 AA in length, held by disulfide bond

Question 2

IgG Ab structure

Answer 2

L chains: 2 Ig domains
H chains: 4 Ig domains

disulfide bonds between heavy & light, heavy & heavy

Question 3

Ig Variable regions

Answer 3

Sequence varies among antibodies; 1 Ig domain/chain

Question 4

Ig Constant regions

Answer 4

Sequence is the same among different antibodies; 3+ Ig domains/chain

CH: other effector functions
CL: no effector functions

Question 5

Light vs Heavy chain regions

Answer 5

Light: 1 V + 1 C per chain
Heavy: 1 V + 3 or more V per chain

Question 6

Antigen binding site

Answer 6

1 VL + 1 VH

Question 7

Hypervariable regions/CDRs

Answer 7

Greatest diversity in the H and L variable regions; ~10 AA long

Also called complemetarity-determining regions (CDRs) b/c bind to antigen

Question 8

CDR location on protein

Answer 8

extend away from protein to contact antigen

Question 9

Antigen binding pockets

Answer 9

Two per antigen, consist of VH and VL chain

Question 10

H chain isotypes

Answer 10

IgA, D, E, G, M

Question 11

L chain isotypes

Answer 11

𝜅 and 𝜆

Question 12

Secreted vs. Membrane-Associated Antibodies

Answer 12

Differ at C-terminal tail; Membrane = C𝜇4 followed by: cytoplasmic tail (3 aa, CY) + hydrophobic transmembrane region (26 aa, TM)
Secreted = C𝜇4 followed by: hydrophilic tail; uses poly(A) cleavage site before TM-CY exons, not included in primary transcript

Question 13

In the membrane-associated form, all isotypes are ____

Answer 13

Monomeric

Question 14

T Cell Receptor structure/function

Answer 14

Recognizes antigen in context of MHC

two chains: 𝛼𝛽 or 𝛾𝛿
each chain: V and C region

CDRs in variable region that bind antigens

Question 15

Mechanisms of Ab diversity

Answer 15

1. Multiple VH/L genes
2. Combinatorial association of different V, D, J gene segments
3. Junctional diversity by nucleotide addition
4. Combinatorial association of VH/L regions
5. Somatic hypermutation

Question 16

Ab gene rearrangement: Heavy chain

Answer 16

gene: V, D, J, C
CDR1/2 derived from VH segment
CDR3 derived from DH and JH segments

Question 17

Ab gene rearrangement: Light chain

Answer 17

gene: V, J, C
CDR1/2 derived from VL segment
CDR3 derived from JL segments

Question 18

Ab gene organization

Answer 18

Gene pieces assembled during lymphocyte development (3 loci, different chromosomes)
Heavy: (V, D, J), C
Light: (V, J), C

Question 19

Leader Sequence

Answer 19

Each V segment (V/D/J) has L (leader) sequence, codes for N-terminal signal peptide (targets protein to endoplasmic reticulum)

Question 20

VDJ recombination steps

Answer 20

1. chromatin opens, giving recomb enzymes access to genes
2. 2 gene segments brought together, double strand break
3. Nucs added/removed, ends ligated
4. RNA splicing of introns
5. each B cell has different combo of V gene segments/diversity due to addition of N/P nucs

Question 21

Recombination signals

Answer 21

RSSs - mediate recombination; 7 nt stretch (CACAGTG) separate by 12 or 23 spacer (1-2 turns, bring heptamers together), followed by concerved AT-rich stretch of 9 nt

12 bp spacer can only join to 23 bp segment

Question 22

VDJ recombination: deletion vs inversion

Answer 22

Deletion: heptamer synapses join together, gene is cut, creates a circle of intervening DNA

Inversion: when RSSs are 3’ of J𝜅 gene, intervening DNA is not removed, stays until RNA splicing

Question 23

Rearrangement and Transcription

Answer 23

Rearrangement brings enhancers close to promoter allowing transcription to occur

Question 24

Non-homologous end joining purpose

Answer 24

Repairs double strand breaks by joining the two broken ends together and ligating them without template

uses small ss region of homology to enhance repair

Question 25

Non-homologous end joining process

Answer 25

1. Ku70/80 binds to break, recruits DNA-dependent protein kinase (DNA-PK) 2. DNA-PK phosphorylates and activates Artemis endo-exonuclease 3. Artemis endonuclease trims unpaired DNA 4. Ligase complex ligates the DNA

Question 26

VDJ joining: Synapsis

Answer 26

1. Chromatin opens 2. Chromosome looping 3. Recombination enzymes (RAG1 and 2) form complex, recognize RSS sequences 4. RAG2 binds to hypermethylation in chromatin, recruits/activated RAG1

Question 27

VDJ joining: Cleavage

Answer 27

RAG1 nicks one strand, ss DNA covalently forms hairpin with other strand

Question 28

VDJ joining: Hair pin opening, end processing, joining

Answer 28

1. Ku70/80 binds to breaks, recruits DNA-PK (repair enzyme, activates Artemis) 2. Artemis endonuclease opens hairpins 3. DNA polymerase fills in nucleotides (P) 4. terminal transferase (TdT) adds nucs to broken DNA ends (N) 5. DNA ligase joins ends together

Question 29

Junctional diversity

Answer 29

P (hairpin cleavage + DNA polymerase) and N (TdT) nucleotide addition; CDR3 = greatest diversity

Question 30

Most important region for Ab binding specificity

Answer 30

CDR3

Question 31

Allelic Exclusion

Answer 31

Each B cell only makes one Ab; Only one chromosome undergoes rearragement Other is excluded (heterochromatized/methylated)

Question 32

Light chain isotype exclusion

Answer 32

If the 𝜅 locus undergoes a productive rearrangement, it inhibits 𝜆 rearrangement

Question 33

Affinity Maturation/Class Switching

Answer 33

During immune response, B cells produce Abs that progressively have a higher affinity for antigen Abs switch from IgM to G, A, E

Question 34

Switch Regions

Answer 34

1-10 kb long GC-rich regions between J and C gene segments Upstream of S region: I exon & I region promotor Cytokine stimulation triggers transcription (I, S, CH)

Question 35

Mechanism of Class Switching

Answer 35

1. CD40 reacts with B cells, induces AID 2. AID removes amino group from cytosines --> deaminated uracil, ultimately leading to double strand breaks at 𝜇 switch region and downstream S region 3. 𝜇 switch region joins downstream CH switch region, intervening DNA lost 4. RNA splicing yields mRNA with new CH region

Question 36

Activation-Induced Cytidine Deaminase Mechanism

Answer 36

1. AID removes amino group from cytosines --> deaminated uracil a) modify GC-rich tetranuc motifs, increase pol II stalling b) pol II stalling recruits AID c) RNA-DNA hybrid allows ss-DNA R loop, targeted by AID 2. Uracil N-glycosylase (UNG) removes U residues --> abasic 3. Ape I endonuclease nicks each abasic site 4. Exosome degrades RNA from D loop, AID + Ape convert + nick RESULT: ds break in two S regions

Question 37

Affinity Increase Cause

Answer 37

Somatic mutations in V region, enhance affinity for antigen (somatic hypermutation -- mutation rate 1000x higher that other genes) Require helper T cells and CD40:CD40L signaling Mutations cluster in CDRs, cause increased afffinity

Question 38

Somatic Mutation Mechanism

Answer 38

In V region, AID converts C -> U at AGCT hotspots, cause 3 changes: 1. DNA replication, U -> T 2. UNG excises bases and repairs with error prone system 3. Mismatch repair enzymes (MSH2/6, Exol) remove U nucs, replace surrounding DNA with error prone DNA pol

Question 39

T Cell Receptor Diversity

Answer 39

``` 𝛼𝛽 and 𝛾𝛿 pairs 𝛽, 𝛿 – VDJ; 𝛼, 𝛾 – VJ 𝛽, 𝛿 – VDJ; 𝛼, 𝛾 – VJ 𝛽: 50 V, 2 D, and 12 J segments 𝛼: 45 V and 50 J segments 𝛾, 𝛿: much smaller - 7 V genes total ``` V regions have CDR1-3 with CDR3 the most diverse 𝛽, 𝛾: 2 C regions 𝛼, 𝛿: single C region V(D)J joining and P/T diversity like antibody genes

Question 40

Severe Combined Immunodeficiency (SCID)

Answer 40

Severe mutations or deficiencies in recombination enzymes (RAG1, RAG2, Artemis, DNA-PK, DNA Ligase 4) result in a complete loss of B cells and T cells

Question 41

Omenn Syndrome

Answer 41

Hypomorphic mutations with reduced (not absent) function of RAG and Artemis genes less severe than SCID

Question 42

Hyper IgM Syndrome

Answer 42

Genetic defects in AID or UNG are defective in class switching and affinity maturation All IgM Can be caused by defects in CD40

Question 43

Lymphoid Tumors and Translocations

Answer 43

B and T cell tumors commonly have translocations of oncogenes into antibody loci AID ds breaks oncogenes near S sites, join in CSR break --> over expression of oncogene, uncontrolled growth Can have RAG-RAG or RAG-AID translocations