B-cell 2: Antibody diversity and isotope switching

Question 1

Receptor diversity

Answer 1

• single b-cells can make antibodies that only recognize a single epitope
• think about how many antigens there are - humans have a very large antibody repertoire
• how is initial diverse repertoire generated?

Question 2

Immunoglobulin light and heavy chain loci

Answer 2

• DNA: lambda light chain, kappa light chain, and heavy chain loci, found in different chromosomes
• peptide sequence that results in variable region is actually found in multiple gene segments that come together (for example, when making a lambda light chain variable region, a V (for variable) lambda segment must combine with one J lambda segment (and there are many many of each - many V, many J). This is called somatic recombination or VDJ rearrangement.
• Somatic recombination happens at DNA level before transcription

Lambda light chain: V and J
Kappa light chain: V and J
Heavy chain: V and D and J

Constant region (C segment) is found downstream in DNA (on light chain and heavy chains). The C region on the heavy chain determines the isotype of the antibody!!!

Question 3

Somatic recombination

Answer 3

• the number of different segment types!

V=variable, D=diversity, J=joining, C=constant
Light Chain
kappa
- 34-38 V
- 0 D
- 5 J
- 1 C
lambda
- 29-33 V
- 0 D
- 4-5 J
- 4-5 C

Heavy chain

• 39-46 V
• 23 D
• 6 J
• 9 C

Question 4

VDJ rearrangement

Answer 4

• Light chain forms (VJC)
• Heavy chain forms (VDJC)
• the two combine
• Recombination signal sequence (RSS) (non-coding sequences) on the DNA that are recognized by various proteins and make rearrangement possible

Question 5

Stages of VDJ rearrangement in B cells

Answer 5

- this occurs during the development of b-cells in the bone marrow
First H-chain
- first D-J rearranging 
- V- DJ rearranging
Then L-chain:
- VJ rearranging

Mature B-cell:
- assembled IgD and IgM that have the same antigen binding site

Question 6

Proteins needed for B-cell redevelopment

Answer 6

• many are needed
• many primary immune deficiencies where people don’t have functioning proteins –> profound immune defects: affects ability to make antibodies and T-cells (these same proteins are needed for T-cell development)

Question 7

Primary Diversification

- explain 4 ways antibodies are diversified

Answer 7

• multiple copies of VJD gene segments
• multiple combinations within heavy and light chains
• multiple combinations of light with heavy chains
• Junctional diversity (addition or subtraction of nucleotides during recombination)

Question 8

Secondary Diversification

• 2 types
• what are they mediated by?

Answer 8

• somatic hypermutation (affinity maturation)
• class switching (ABS stays the same)

–> both of these are mediated by an enzyme called activation-induced cytidine deaminase (AID)

Question 9

AID

Answer 9

Activation-induced cytidine deaminase

• initiates DNA lesions
• replaces with a completely different amino acid
• allows for selection of antibodies that bind with an even greater affinity to antigens (somatic hypermutation)
• deficiencies in enzyme leads to inability to develop functioning antibodies and inability to class switch (IgM to IgG)

Question 10

Affinity maturation

Answer 10

• Additional diversity is generated in activated B-cells
• Point mutations are introduced into rearranged V-region genes to select for more effective antibodies with improved affinity for antigen

Question 11

Isotype class switching

Answer 11

• once the Variable heavy chain region exons are determined for a given B-cell, there may be some minor modification (somatic hypermutation), but other wise the VDJ combination doesn’t change
• C-region isotypes can still change as B-cells mature and proliferate
• Isotype is determined by the heavy chain C-region

Question 12

Class switch recombination

Answer 12

• the first antigen receptors expressed (on cell surface) are IgM and IgD; the first secreted antibodies are IgM (out of plasma cells)
• Later in immune response the same V region can be expressed with different C-regions giving rise to different classes
• Switching from IgM to other classes occurs only once B cells have been stimulated by antigen

Question 13

Antibody Isotype and functional activity

Answer 13

IgG: opsonization, neutralization, antibody dependent cell-mediated cytotoxicity (ADCC), can also activate complement
**cross placenta

IgM: complement activation

IgE: sensitization of mast cells

IgA: across epithelium (secretions and mucosa)

Question 14

QUESTIONS:
- How is antibody diversity at the variable region generated?

• Point mutations can be introduced into rearranged V-region genes to select for more effective antibodies with improved affinity for antigen. What is this process called?
• What is the first antibody isotope produced by B-cells?
• What is the only antibody isotype that crosses the placenta?

Answer 14

Somatic recombination (VDJ rearragement)

Somatic hypermutation (affinity maturation)

IgM (also IgD but less clinically significant)

IgG