Theme 5b BCR/TCR gene rearrangement

Question 1

Which of the recombination gene segments is found only in antibody heavy chains?

Answer 1

diversity or D

Question 2

What is the 12/23 rule?

Answer 2

a 12bp RSS must pair with a 23bp RSS for VDJ recombination to occur.

Question 3

What is the RAG1/2 complex?

Answer 3

recombination activating gene 1 will bind to RSSs and RAG2 will stabilize the complex. The RAG1/2 complex is responsible for recognizing and cutting DNA at the RSSs.

Question 4

What are the five mechanisms of generating antibody diversity in naive B cells?

Answer 4

Combinatorial diversity:

1. Multiple gene segments (which V, D, and J segments are put together?)
2. Heavy chain/ light chain (which heavy chains will pair with which light chains, lambda vs kappa)

Imprecise cutting and joining:

3. P nucleotide addition (specific nucleotides are filled in between joints, causing asymmetric cleaving of DNA)
4. Exonuclease trimming (lose of nucleotides and change in reading frames)
5. N nucleotide addition (random nucleotides are filled between joints)

Question 5

Explain allelic exclusion

Answer 5

Ensures that only one copy of the heavy chain, and only one copy of the light chain are active (either maternal or paternal). leading to only a single specificity antibody being produced.

1. After heavy chain recombination and expression, recombination machinery of H-chain shuts down.
2. The heavy chain is paired with a surrogate light chain (SLC) to form a pre-BCR. If SLC binds then the recombination machinery starts up again.

2i. if the arrangement is non-productive apoptosis occurs.

3. Light chain recombination occurs and Igs are made.
4. Should the new BCR with the light chain be autoreactive, recombination machinery can be turned on again to salvage or inactivate the antibodies.

Question 6

Why is allelic exclusion less of an issue for TCRs than for BCRs?

Answer 6

If a TCR has specificity for two antigens, it will react the same for each one, releasing cytokines or initiating cytotoxic activity. But for BCRs, they will react by releasing some antibodies which would not be specific for the antigen in question, meaning that half of their resources are being wasted or even used to cause an autoimmune effect.

Question 7

What occurs after Ig rearrangement?

Answer 7

• Mature B cells undergo an mRNA splicing mechanism which allows both membrane-bound IgM and secreted IgD antibodies to be expressed at the surface.
• mRNA splicing removes the intervening spacer between the VDJ and C regions, allowing VDJC to be translated.

Question 8

What are Somatic Hypermutations?

Answer 8

Production of individual point mutations in Ig Heavy and light chains at hot spot sequence motifs.

It allows for tweaking of B cells to increase their detection/ affinity for an antigen. This in turn allows for them to access more cytokine survival signals. (clonal expansion)

Question 9

What is a difference between the binding site of BCRs and TCRs?

Answer 9

With antibodies, the CDR1,2 and 3 regions all form the antigen binding site.
With TCRs, the CDR1 and 2 bind only to MHC molecules and is coded by V segments, while only the CDR3 binds to the actual antigen and is coded for by the D & J segments.

Thus, the TCR V segment is less variable than the Ig V segments because the TCR must always bind to the MHC. To make up for this lack of diversity at the V segment, TCRs have more D and J segments than BCRs, leading to a roughly equal combinatorial potential.

Question 10

What chains in the TCR and BCR undergo exonuclease trimming in order to rescue a non-productive arrangement?

Answer 10

Alpha and Beta chains of TCR
Only the light chains of BCR