Antibody Recognition and Generation of Diversity

Question 1

Paratope Structure

Answer 1

The region of the antibody that contacts the antigenic epitope.

• Composed of hypervariable regions (HV) aka complementarity-determining regions (CDRs)
  
  • Short ~10 AA regions with a highly variable AA sequence
  • Virtually every Ab contains a uniqure AA sequence in the HV region
  • Both the light and heavy chains contain 3 HV regions
• Framework regions provide structure support for the HVs

Question 2

Ig Structure & Gene Segments

Answer 2

Immunoglobulins consist of two major portions:

• Constant region: C_H, C_λ, C_𝛋
• Variable Region: formed by multiple gene segments coming together
  
  • Heavy chain: V_H, D_H, J_H (C_H)
  • Light chain:
    
    • V_λ, J_λ, C_λ (lambda locus)
    • V_𝛋, J_{𝛋 ,}C_𝛋 (kappa locus)

Variable region refers to the VDJ segment of the heavy chain & VJ segment of the light chain.

Variable gene segment refers to the V₁₄, V₂₂, etc.

Question 3

BCR Genes

Answer 3

• The heavy chain is encoded by 1 gene.
• The light chain is encoded by 1 of 2 genes:
  
  • kappa (𝛋) light chain
  • lambda (λ) light chain
• There are only 2 copies of each gene (H,𝛋, and λ)

Question 4

Ig Gene Rearrangement

Answer 4

Occurs in a precise order:

1. Heavy Chain
2. Light Chain
   
   • Kappa locus then
   • Lambda locus

Question 5

Heavy Chain Rearrangment

Answer 5

1. Randomly selected D-J segments joined
   
   • Intervening DNA deleted
2. Random V segment joined to the D-J complex
   
   • VDJ loop formed and intervening DNA excised
   • ⇒ rearranged heavy chain variable region formed
3. Transcription and RNA processing leads to splicing of the VDJ complex with the Cμ segment ⇒ gives rise to a functional mRNA for the μ (mu) heavy chain ⇒ pre-B-cell

Question 6

Light Chain Rearrangement

Answer 6

Both kappa genes are attempted first.

If unsuccessful, then lambda genes attempted.

1. V segment joined with J segment to form VJ complex.
2. Splicing of primary transcript joints the C gene to the VJ complex.
3. Forms mRNA transcript which is translated to produce a λ (lambda) or 𝛋 (kappa) light chain.
4. Light chain assembled with previously synthesized μ heavy chain in the ER to form complete IgM molecule.
5. IgM transported to the cell surface.

Question 7

Molecular Mechanism

Ig Gene Rearrangement

Answer 7

1. Recombination signal sequences recognized by V(D)J recombinase.
   
   • Recombinase enzyme composed of RAG-1 and RAG-2 (recombination-activating genes 1 and 2)
   • RAG genes specific for early stages of B (and T) cell development
2. V(D)J recombinase brings exons into apposition forming a hairpin loop
3. Intervening DNA randomly cleaved.
4. Exonucleases randomly remove nucleotides from the ends of the V, (D), J gene segments
5. Terminal deoxyribonucelotidyl transferase (TdT) takes nucleotides that are not part of the germ-line gene and adds them randomly to the sites of recombination
   
   • Added nucleotides called N-nucleotides
   • Process called junctional diversity

Question 8

Allelic Exclusion

Answer 8

Once a successful heavy chain (μ protein) is produced further rearrangement fo the heavy chain is inhibited on the other chromosome.

Genes on the other chromosome will only rearrange if a non-productive rearrangement took place on the first chromosome.

Similar events occur with light chain.

Allelic exclusion ensures:

1 receptor : 1 cell : 1 reactivity

Question 9

Factors Creating Ig Diversity

Answer 9

1. Multiple V, D, J segments
2. Combinatorial diversity
   
   • Different combinations of V, D, J
   • Different heavy and light chains combining
3. Diversity gene segments (D) code in all 3 frames
4. Junctional diversity
   
   • Exonucleases followed by TdT
   • Adds de novo N-nucleotides not germ-line encoded

Question 10

Somatic Hypermutation

Answer 10

Nucleotides in the hypervariable regions of both light and heavy chains undergo a very high rate of mutation.

Question 11

Antigen Selection

Answer 11

B-cells with higher affinity BCR (Ig) competes better for limited Ag binding which drives clonal proliferation.

Those with lower affinity BCR cannot compete for Ag and without stimulation die by apotosis.

Question 12

Affinity Maturation

Answer 12

Somatic hypermutation + atigenic selection allows for affinity maturation.

Allows the B-cell to undergo isotype switching.

Question 13

IgM & IgD

Expression

Answer 13

• Naive B-cells (prior to Ag exposure) express both membrane bound IgM and IgD.
• Activation of naive B cells results in the production of soluble pentameric IgM
• bound and free IgM and IgD produced through alternative RNA splicing and/or alternative polyadenylation
  
  • Do not require DNA rearrangement.
  • Allows for the generation of different polypeptides from a single gene

Question 14

Isotype Switching

Answer 14

• IgM producing B cells have not undergone isotype switching
• All heavy chain constant regions flanked by switch sites except C_δ​ (delta)
  
  • Promote looping out of DNA between VDKJ of variable region and subsequent heavy chain constant regions downstream
  • Intervening DNA deleted
  • Since DNA is lost, once isotype switching has occured the B-cell is unable to go back to the previous type of Ig
• Constant region closest to VDJ segment is made.
• Antigen specificity remains the same
• Light chain unchanged

Question 15

B-cell

DNA/RNA Alterations

Summary

Answer 15