Antigen Receptor Development

Question 1

Describe the germline configuration of immunoglobulin gene families.

Answer 1

• there are 3 gene families: kappa light chain, lambda light chain, and heavy chain
• located on different chromosomes
• each has a variable region and constant region

Question 2

What are the segments of the variable region for light chains?

Answer 2

V-segments are located at the 5’ end

J-joining segments are located near the 3’end

Question 3

What are the segments of the variable region for heavy chains?

Answer 3

V-segments are located at the 5’end
D-diversity segments are located downstream
J-joining segments are located further downstream near the 3’ end

Question 4

What do the J and D segments code for?

Answer 4

the 3rd CDR on the 3’ end of the antigen binding site

Question 5

What is the order of gene rearrangement for heavy chains?

Answer 5

1. D-J rearrangement
2. V-DJ rearrangement
3. VDJ gene will now be the only V region expressed in that cell
4. alternative splicing => VDJ-C

Question 6

What is the order of gene rearrangement for light chains?

Answer 6

1. V-J rearrangement
2. alternative splicing => VJ-C
3. kappa or lambda light chain joins heavy chain => IgM

Question 7

Describe B cell specificity.

Answer 7

A single B cell (and its progeny) will only express ONE antigen-binding specificity (1 Vh and 1 VL)

Question 8

What is allelic exclusion?

Answer 8

In Ig-producing cells, only one allele is expressed to ensure antigen specificity

Question 9

Describe plasma cell specificity.

Answer 9

Plasma cells can only make 1 kind of antibody:
1 heavy chain type
1 light chain type
=> B cells may violate this rule (express both IgM and IgD)

Question 10

List the stages of B cell development.

Answer 10

• HSC
• pro-B cell (early, late)
• pre-B cell (large, small)
• immature B cell
• mature B cell

Question 11

Describe gene rearrangement during the HSC stage of B Cell development.

Answer 11

all genes are in germline configuration

- no rearrangement occurs

Question 12

Describe gene rearrangement during the pro-B cell stage of B Cell development.

Answer 12

• D-J rearrangement of the heavy chain (early)

- V-DJ rearrangement of the heavy chain (late)

Question 13

Describe gene rearrangement during the pre-B cell stage of B Cell development.

Answer 13

• large: now that VDJ gene is produced, mu heavy chain is transiently expressed on the surface, associated with a surrogate light chain (pre-B receptor)
• small: V-J rearrangement of the light chain

Question 14

What components make up the pre-B cell receptor?

Answer 14

mu heavy chain + surrogate light chain + Ig-alpha + Ig-beta

Question 15

Which B cell developmental stage is the first time there is Ig expression?

Answer 15

pre-B cell large

Question 16

Describe gene rearrangement during the immature B cell stage of B Cell development.

Answer 16

Now that the VJ gene is expressed, the newly produced kappa/lambda light chain will join the mu heavy chain on the surface => IgM on the surface

• no response to antigens
• critical control point for gene rearrangement (can no longer make another V region)

Question 17

Describe gene rearrangement during the mature B cell stage of B cell development.

Answer 17

immature B cell enters the peripheral lymphoid tissue
begins to express IgD (with the same antigen specificity as IgM)
- responsive to antigens

Question 18

What is the mechanism for Ig gene rearrangement?

Answer 18

1. Recombination Signal Sequence (RSS) is located downstream of V, on both sides of D, and upstream of J.
2. RAG1/RAG2 recognize the RSS.
3. RAG loops out the intervening DNA between V/J or J/D
4. RAG excises the intervening DNA => circularizes => BREC/TREC => deleted
5. DNA Ligase ligates the ends

Question 19

What is the RSS?

Answer 19

RSS is made of 7-9 bps separated by a 12 or 23 bp spacer

- highly conserved

Question 20

What are BRECs/TRECs?

Answer 20

B-Cell Recombination Excision Circle

• excised intervening DNA gets circularized
• as cell proliferates, the BREC/TREC will get diluted and eventually deleted

Question 21

What is the purpose of quantifying BRECs/TRECs?

Answer 21

to see if B cells and T cells are functioning and producing Ig and proliferating

Question 22

How do you get transmembrane IgM vs secreted IgM?

Answer 22

alternative splicing of the primary RNA transcript leads to either a transmembrane exon added after the C region or a secretion exon added after the C region

Question 23

How can one cell co-express IgM and IgD?

Answer 23

• primary RNA transcript contains all C-region genes
• alternative splicing leads to choosing either a Cmu or a Cdelta
• the VDJ region remains the same, thus conserving antigen specificity

Question 24

What are the 3 mechanisms by which the body increases antibody diversity?

Answer 24

1. Combinatorial Joining of VDJ segments
2. Junctional Diversity by adding N sequences
3. Combinations of H and L chain proteins

Question 25

What is combinatorial joining of VDJ segments?

Answer 25

every B cell clone expresses a unique VDJ/VJ combination | facilitates increased diversity and antigen specificity

Question 26

What is N-region addition (junctional diversity)?

Answer 26

- After RAG excises the intervening DNA, enzyme Tdt randomly adds nucleotides on the excised ends - only occurs in the heavy chain - on either end of the DJ segment

Question 27

What is a consequence of N addition?

Answer 27

- nonfunctional antibody due to frameshift or stop/nonsense codon

Question 28

What happens if a B cell receptor is non-functional?

Answer 28

apoptosis

Question 29

What are the possible effects of an anti-self B cell receptor?

Answer 29

apoptosis, anergy, or receptor editing

Question 30

What is anergy?

Answer 30

state of nonfunctionality of B cells that have anti-self receptors that have low autoreactivity - decreases IgM expressed on the surface - can bind to self proteins, but does not lead to a T cell activity

Question 31

What is receptor editing?

Answer 31

If a B cell produces an anti-self receptor, when the Ig binds to a self-antigen a signal is sent to the cell to change the receptor - RAG is activated to create a new genetic recombination (since there are still upstream Vs and downstream Js available) - this only occurs on the light chain - cell continues to mature - if still anti-self, apoptosis

Question 32

Describe the structure of T Cell Receptors.

Answer 32

2 different chains (alpha and beta) | 2 domains each (variable and constant)

Question 33

Which terminal corresponds to which domain of the TCR?

Answer 33

- N domain = variable | - C domain = constant

Question 34

Alpha chains and Beta chains correspond to light or heavy?

Answer 34

``` alpha = light (VJ) beta = heavy (VDJ) ```

Question 35

What is an isotype switch?

Answer 35

A B cell can be induced to change the heavy chain it expresses if there are still downstream C regions available - occurs after B cell is activated and presents to T cells

Question 36

What is the mechanism for an isotype switch?

Answer 36

1. enzyme AID (activation-induce cytidine deaminase) recognizes switch regions upstream of C regions 2. loop out, excise, ligate 3. if there are downstream C regions available, you can create a new Ig isotype (with the same antigen specificity)