L2 - Genetics of antigen recognition receptors (antibodies) Flashcards
What are the V & C regions of antibody & TCR polypeptide chains are encoded by?
Separate gene segments that rearrange during lymphocyte differentiation
Domains are encoded by very small mini genes
There are bits in-between V & C
What are the bits in-between V & C?
D = diversity
J = joining
What is the V region of H chains & TCR-beta encoded by?
V
D
J
What is the V region of L chains & TCR-alpha encoded by?
V
J
What do V, D & J encode?
V region of H chains & TCR-beta
What do V & J encode?
V region of L chains & TCR-alpha
Rearrangement of Ig genes in B cells
Genes rearrange during B cell development to form a functional gene
The genes in B cells are closer than in every other type of cell in the body
DNA is different in B cells than every other cell
B cells break their DNA in a way no other cells do (apart from T cells) & move genes so they are closer
How do B cells produce a functional immunoglobulin?
Involves recombination to bring gene segments together
Light chain of an antibody
After DNA breaks, a single V & a single J gene segment are joined together to encode the V region of the light chain
The 2 exons come together
DNA is broken randomly to bring 2 of each together randomly
Leads to different BCRs in every cell
B cell has to decide to break its DNA at one locus – either lambda or kappa
What happens once V & J are next to each other for the light chain?
It gets transcribed into RNA like a normal gene making the light chain – can then fold up to form a unique antigen binding site
Heavy chain of an antibody
Similar mechanism but extra sequence section with the D region
2 breaks needed
A single V, D & J gene fragment are joined together to encode the V region of the heavy chain
Order of breakage in the heavy chain
Each B cell first breaks between D & J to bring them together
Then is sequentially broken again & the DJ joins the V
Lots of combinations – every V can go with every D & J
What forms the binding site?
Variable regions of H & L chain
Hierarchy of rearrangements for both heavy and light chains
First H chain genes – D-J then V-D
Then light chain genes – kappa first (V-J)
If kappa rearrangement unsuccessful then lambda genes rearrange
What chain has greater variability?
Greater variability in H chain as V, D & J
What loci are the H, kappa & lambda chains encoded?
H chain – chromosome 14
Kappa chain – chromosome 2
Lambda chain – chromosome 22
What isotope are most antibodies at the start?
IgM
What does RSS stand for?
Recombination signal sequences
What are RSS?
DNA rearrangement is guided by special sequences flanking the V, D & J regions = RSS
Involves a complex of enzymes = V(D)J recombinase (recombination activating gene – RAG)
What are RAG genes?
Recombination activating gene
What are the 2 types of RAG genes?
RAG-1 & RAG-2
Encode lymphoid-specific components of the recombinase
Where are RAG genes expressed?
Only in B & T cells
Recognise the RSS to do the breaking
Can put RAG genes into cells that don’t express them to show they break DNA
What happens if theres mutations in the RAG genes?
Immunodeficiency
How is the DNA broken and then reassembled to increase variability?
RSS are recognised by RAG
DNA is then bent & broken & reassembled
When its reassembled the enzyme TDT randomly puts a few base pairs on before it reassembles it – increases variability even more
All get reassembled slightly differently – encodes a slightly different variable region
What is allelic exclusion?
When a B cell makes a heavy chain, it tells the other chromosome not to do the same process again
Also turns off further light chain rearrangement once a light gene rearranges successfully
Each B cell only has 1 BCR – isn’t made on both chromosomes
How does allelic exclusion turn the gene off in 1 chromosome?
Turns of the RAG genes in that chromosome
Ensures every B cell only has 1 L chain & 1 H chain
All time dependent – heavy chain goes first & then the light chain
Mechanisms for generation of antibody diversity
Multiple germ line genes
Combinatorial diversity
Junctional diversity
Combinations of H & L chains
Somatic hypermutation
Multiple germ line genes
Multiple VH, V-kappa & V-lambda
Also multiple D & J
Combinatorial diversity
Different V, D & J segments recombine to produce different sequences
Eg. 40V x 25D x 6J = 6,000 combinations
H chains potentially more diverse than L chains
Junctional diversity
Includes:
– Imprecise joining – small differences in position of V-D & D-J join
– N regions – random addition of nucleotides at junctions of V-D & D-J by terminal transferase
Combinations of H & L chains
Eg. 106 H and 104 L would give 1010 possible antibodies
Somatic hypermutation
Mutation frequency in antibody VH genes orders of magnitude higher than normal spontaneous mutation rate
Occurs in germinal centres as B cells recognise Ag & proliferate/become activated
What does somatic hypermutation involve?
Involves the enzyme AID – activation-induced deaminase
AID acts on DNA to de-aminate cytosine to uracil
Uracil is then recognised by error-prone DNA repair pathways leading to mutations
Leads to even more variation in the receptors
Membrane (BCR) vs secreted antibody
As individual B cells differentiate, they start to secrete their unique BCR as antibody (soluble form)
Secreted form has an alternative hydrophilic C-terminus but same specificity as membrane Ig (BCR)
Membrane & secreted forms produced by alternative RNA processing
If the BCR binds to the antigen we want it to on the surface, then we can release the BCR as an antibody
What is the heavy chain in IgM?
Mu
What is the heavy chain in IgD?
Delta
What is the heavy chain in IgG?
Gamma
What is the heavy chain in IgA?
Alpha
What is the heavy chain in IgE?
Epsilon
Why is IgM the first isotype expressed by each developing B cell?
C-mu is physically the closest to the V,D and J genes
C-delta is then next to C-mu: hence IgD can be co-expressed with IgM by differential processing of the RNA from the two C region genes
What does switching to other classes require?
Recombination
Guided by switch regions & DNA breakage
Also involves AID
Cytokines & pathogens cause the switch
