Generation of Diversty

Question 1

Where does gene rearrangement take place for B cells?

Answer 1

In the bone marrow

Question 2

How many chromosomes contain genes that code for antibody heavy chains and kappa and lambda light chains?

Answer 2

3 separate chromosomes: 14, 2 and 22, respectively

Question 3

What is somatic recombination?

Answer 3

DNA rearrangement and deletion: VDJ for the heavy chain and VJ for the light chain
Occurs in somatic cells, not germline

Question 4

What is the mechanism of somatic recombination for antibodies and T cell development?

Answer 4

VDJ recombination

It results in the highly diverse repertoire of Abs and TCRs

Question 5

Mechanisms of B cell diversity

Answer 5

1. Somatic recombination: VDJ recombination for heavy chain, VJ recombination for light chain
2. Pairing various light chains with heavy chains
3. Junctional diversity (nucleotide addition via Tdt)
4. Somatic hypermutation (single point mutation in antibody idiotype)

Question 6

What are the steps of B cell development?

Answer 6

1. VDJ rearrangement on ‘H’ chain occurs in Pro-B cells to produce Heavy chain.
2. VJ rearrangement on ‘L’ chain occurs in Precursor B cells to produce Light chain.
3. After the re-arrangement, the B cells are now called Immature B cells.
4. Transcription of Immature B cell DNA to RNA followed by RNA splicing of introns occur. This brings the Combined V(D)J together with “C” segment that encodes for Constant region of heavy or light chains and forms mRNA.
5. This is followed by Translation to produce proteins – specific heavy chain and light chain.

Question 7

What do V, D and J stand for?

Answer 7

Variable
Diversity
Joining segments

Question 8

What is RSS?

Answer 8

Recombination signal sequence
Each gene segment (V,D and J) has an adjacent RSS
It produces a configuration that acts as a target for two proteins encoded by two Recombination Activating Genes: RAG1 and RAG2

Question 9

What do RAG1 and RAG2 do?

Answer 9

Recognize and bind to RSS and mediate VDJ recombination by nicking and opening chromosomes and filling harpin loops with nucleotides (ie. TdT enzyme leads to N-additions = junctional diversity)

Question 10

What is the total Ig potential diversity and the average number of B cells in an adult?

Answer 10

potential diversity: 5x10^13

avg 5x10^7

Question 11

What is the first isotype naive B cells express?

Answer 11

IgM
Often co-expression of IgM and IgD because the Cmu (constant region for IgM) is downstream from rearranged VDJ
IgM and IgD are generated by alternate splicing of RNA transcript and both have the same VDJ segments

Question 12

Explain the difference between membrane BCRs vs secreted Abs

Answer 12

A B cell membrane BCR is displayed with Ig-alpha and Ig-beta

Plasma cells generate soluble Ab proteins, which are the secreted forms of the receptor

Question 13

What is isotype switching?

Answer 13

DNA rearrangement between constant regions - exchange Cmu.

Question 14

What other changes do B cells undergo in lymphoid tissue?

Answer 14

1. Somatic hypermutation = single nucleotide point mutations. Targets rearranged gene segments encoding variable antibody region and may alter affinity of antibodies for the antigen
2. Isotype switching

Question 15

What is AID

Answer 15

= activation induced cytidine deaminase, involved in somatic hypermutation and isotype switching.
Expressed in activated mature B cells
Causes mutations that lead to diversity

Question 16

What is junctional diversity?

Answer 16

DNA sequence variations introduced by improper gene segment joining during VJ recombination

Question 17

What is allelic exclusion?

Answer 17

One allele of a gene is expressed while the other is silenced. Allows for mature B cells to express only one type of Ig