L2 Genetics of antigen recognition receptors

what is the number of different possible antibodies in humans

more than 10⁹

how many genes are in the human genome

less than 30,000

what is the generation of an enormous antibody repertoire from

relatively few (~200) genes

what are V and C regions of antibody and TCR polypeptide chains encoded by

separate gene segments that rearrange during lymphocyte differentiation

what is the V region encoded by in the H chain and TCR beta

V, D and J (V is the biggest)

what is the V region encoded by in the L chain and TCR alpha

V and J gene segments

what are immunoglobulin genes found in the genome as

gene segments

how do B cells form functional Ig genes

during their development in the bone marrow

• the DNA containing the Ig gene segments is deliberately broken and the gene segments are rearranged (joined together)

what is the breaking and joining of Ig gene sequences in B cells called

Non-homologous end joining (NHEJ) recombination

what does each individual B cell perform the breakage and rearrangement of Ig gene segments

randomly

• different to that seen in any other B cell

Describe DNA recombination in the light chain of B cells

After DNA breaks, a single V and a single J gene segment are joined together to encode the V region of the light chain

what are the two loci containing the light chain

lambda and kappa

Describe DNA recombination in the heavy chain of B cells

a single random V, D and J gene segment are joined together in a single B cell to encode the V region of the heavy chain

Describe the process from the Germline DNA to the polypeptide chain (protein) through recombination

• Germline DNA → Somatic recombination → D-J joined rearranged DNA

• Somatic recombination → V-J or V-DJ joined rearranged DNA

• Transcription → primary transcript RNA

• Splicing → mRNA

• mRNA→ translation → polypeptide chain

what is the specific order that B cell development Ig rearrangements occur in

• Heavy chain gene segments rearrange

• Light chain gene segments rearrange

• if Kappa rearrangement is unsuccessful, then lambda gene segments rearrange

what is the process of H gene segments rearranging

D-J then V-DJ

• greater variability in H chains as V, D and J segments

what is the process of light chain gene segments rearranging

kappa segments (V-J) first

• if unsuccessful then lambda segments (V-J) rearrange

what is the number of functional gene segments in kappa light chain Variable

34-38

what is the number of functional gene segments in kappa light chain Joining

5

what is the number of functional gene segments in kappa light chain Constant

1

what is the number of functional gene segments in lambda light chain Variable

29-33

what is the number of functional gene segments in lambda light chain Joining

4-5

what is the number of functional gene segments in lambda light chain Constant

4-5

what is the number of functional gene segments in heavy chain Variable

38-46

what is the number of functional gene segments in heavy chain Diversity

23

what is the number of functional gene segments in heavy chain Joining

6

what is the number of functional gene segments in heavy chain Constant 

9

what chromosome is the H chain locus

14

what chromosome is the kappa chain locus

2

what chromosome is the lambda chain locus

22

what is V

variable

what is C

constant

what is D

diversity

what is J

joining

what are recombination signal sequences

Ig gene segment rearrangement is guided by special sequences flanking each of the V, D and J gene segments

what complex of enzymes does rearrangement involve

V(D)J recombinase

what activates the recombination process

recombination activating gene

what do RAG-1 and RAG-2 genes encode

lymphoid-specific components of the recombinase

what do mutations in RAG genes result in

immunodeficiency

what is allelic exclusion

• in each individual B cell, only one rearranged H chain gene from one chromosome (e.g allele) is expressed 

• only one rearranged light chain from one chromosome is expressed by each individual B cell

what is light chain isotype exclusion

each B cell expresses either a rearranged kappa or lambda light chain; never both

what does allelic exclusion ensure

each individual B cell produces just one randomly generated BCR/antibody that is different from the BCR/antibody made by every other B cell

what are the 2 mechanisms for generation of antibody diversity

• multiple gene segments for each chain multiple

• combinatorial diversity

• combinations of heavy and light chains

• junctional diversity

• somatic hypermutation

describe having multiple gene segments for each chain multiple as a mechanism for generation of antibody diversity

VH, Vkappa, Vlambda

• multiple D and J (no D for kappa and lambda)

describe combinatorial diversity as a mechanism for generation of antibody diversity

different V, D and J segments recombine to produce different sequences

• H chains are more diverse than L chains; with approximately 300 possible light chain combinations

give an example of combinatorial diversity

for H chain 50V X 27D X 6J gives 8100 combinations

describe an example of combinations of heavy and light chains as a mechanism for generation of antibody diversity

8100 possible heavy chains can pair with a possible 300L chains to give approx. 2.43 ×10⁶ possible BCR/antibodies

describe junctional diversity as a mechanism for generation of antibody diversity

• imprecise joining (small differences in sequences where V-D and D-J segments join)

• N regions (random addition of nucleotides at junctions of V-D and D-J by terminal transferase

describe somatic hypermutation (SHM) as a mechanism for generation of antibody diversity

• mutation frequency in antibody (H/L) V genes is orders of magnitude higher than that seen in all other areas of the genome

• approximately 10⁹ antibodies/BCRs possible

• Cytosine → Uracil performed by the enzyme, activation-induced deaminase (AID) - uracil is recognised by error-prone DNA repair pathways leading to mutations

where does SHM occur

in germinal centres (GC) as B cells recognise Ag and proliferate/become activated

how does AID act on DNA

to de-aminate cytosine to uracil

• uracil is recognised by error-prone DNA repair pathways leading to mutations (SHM)

following Ag recognition as each B cell differentiates, it will start to…

secrete its unique BCR as an antibody

• secreted form has an alternative constant region that lacks a transmembrane region

• as the original re-arranged VDJ regions arent altered, the secreted antibody has the same antigen specificity as the membrane BCR

• membrane and secreted forms produced by alternative RNA processing

what are membrane and secreted forms produced by

alternative RNA processing

what is the constant region of each heavy chain encoded by

a different C region gene segment (e.g Cmu, Cdelta, Cgamma etc)

• four gamma chain gene segments correspond to the four IgG subclasses; similarly two alpha chain gene segments

at the heavy chain locus, the Cmu segment is physically closest to the V, D and J gene segments and so what is the first class/isotype BCR, antibody expressed by each developing B cell

IgM

Cdelta is next to Cmu so, 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

further DNA recombination

• guided by switch regions

• enzyme AID (after B cell stimulated by Ag)

what happens after isotype switching

• looping out

• switch-region recombination

• further rearrangement may occur