Humoral immunity - B cell diversity Flashcards
What is the structure of an antibody?
- tetrameric protein - 2 identical heavy chains and light chains
- Light chains either kappa or lambda
- Each chain has a variable region - AA sequence differing between Ig molecules
- this region binds Ag (FAB)
- Hypervariable region has complementarity determining regions (CDRs) and framework regions
- Each chain also has a constant region - responsible for effector functions
How does clonal expansion start?
- Body generates over 100million different B cells making a different random Ig
- During an infection, a small number of B-cells will by chance be making an Ig that binds to the foreign Ag
- The cell that makes that Ig will be activated and multiply - Clonal expansion
- Most pathogens will have multiple epitopes, so there will be a number of activated B cells - gives polyclonal response
What is also required for activation?
- Direct involvement of CD4+ Th cells (Th1)
- Cytokines released by the Th cell
How do we generate diversity in the kappa light chain gene?
- Has 1 constant region segment, 35 V segments and 5 short J (joining) segments
- The J segments are close to C, but V segments are far away on the DNA
- There is a binding site for an endonuclease enzyme after each V segment and in front of each J segment - will randomly cut 1 V and 1 J
- The free ends are then ligated together (dsDNA joining) to give a functional protein
Briefly what is the process of getting the kappa chain protein from the germline DNA?
- Germline DNA -> DNA rearrangement -> active gene
- There is a promoter in front of each gene, where transcription will start - gives the precursor RNA (in nucleus)
- After splicing, we will have the mRNA in cytoplasm - will have V,D and J region
- mRNA is translated and then secreted
How many different variable regions can be generated from kappa light chain?
1C, 35V, 5J -> 175 possibilities
What are the events that occur during VDJ recombination?
- Recognition sequences on the DNA that binds recombinase enzyme
- One of the major proteins in the recombinase is RAG1/RAG2
- Binds to the recognition site on the V or J region
- Cut the DNA, discard the loop in the middle and then cap off the ends of the DNA using hairpin loops
- They then open up the ends and reattach them after a bit more processing
Why is there even more diversity generated from this junctional variation?
- before the free ends are ligated together, an exonuclease will process the ends
- Terminal deoxynucleotidyl transferase (TdT) randomly adds a few nucleotides
- This causes extra random variation at the site of N nucleotide action
- The V-J join and this further variation at the joins create the most variable region - CDR3
What is the importance of TdT?
- For generating Ig and TCR gene diversity
- As a leukaemia marker - In ALL cell is arrested in early development stage, where cells are still making TdT
- Useful enzyme in genetic engineering/ recombinant DNA work
Which bits of DNA (gene segments) encode which bit of kappa light chain polypeptide?
- V segment = variable region
- VJ join = CDRIII
- C = constant region
How do we make the lambda light chain gene?
- Slightly more complex
- Generates a similar number of possibilities
What diversity is generated by rearrangement of gene segments in the heavy chain?
- Same process as the light chain, however now there are also 20 Diversity (D) segments
- Exonuclease and TdT can add further variation at both the V-D and D-J junctions
- So more possibilities as 2 separate junctions
Which bits of DNA (gene segments) encode which bits of heavy chain polypeptide?
- V = variable
- VDJ = CDRIII
- C = constant
What is allelic exclusion?
- Any B-cell has two alleles of the Ig heavy chain, in theory it could make 2 different heavy chain proteins - never happens due to allelic exclusion
- Once one allele successfully rearranges and starts making heavy chain proteins, the gene rearrangement process for heavy chains turns off - one B cell only makes one heavy chain
- This is the same in light chains
- B-cell has 2 alleles of kappa and 2 alleles of lambda gene
- In theory could make 4 different proteins
- As soon as one allele makes the protein, the rearrangement process turns off
- One clone of B-cell only makes kapp or lambda
- Polyclonal B cells will be a mixture of cells making either kappa or lambda
What happens if a patient is only making one kind of chain?
Light chain restriction
