L2 - Genetics of antigen recognition receptors (antibodies)

Question 1

What are the V & C regions of antibody & TCR polypeptide chains are encoded by?

Answer 1

Separate gene segments that rearrange during lymphocyte differentiation

Domains are encoded by very small mini genes

There are bits in-between V & C

Question 2

What are the bits in-between V & C?

Answer 2

D = diversity

J = joining

Question 3

What is the V region of H chains & TCR-beta encoded by?

Answer 3

V
D
J

Question 4

What is the V region of L chains & TCR-alpha encoded by?

Answer 4

V

J

Question 5

What do V, D & J encode?

Answer 5

V region of H chains & TCR-beta

Question 6

What do V & J encode?

Answer 6

V region of L chains & TCR-alpha

Question 7

Rearrangement of Ig genes in B cells

Answer 7

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

Question 8

How do B cells produce a functional immunoglobulin?

Answer 8

Involves recombination to bring gene segments together

Question 9

Light chain of an antibody

Answer 9

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

Question 10

What happens once V & J are next to each other for the light chain?

Answer 10

It gets transcribed into RNA like a normal gene making the light chain – can then fold up to form a unique antigen binding site

Question 11

Heavy chain of an antibody

Answer 11

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

Question 12

Order of breakage in the heavy chain

Answer 12

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

Question 13

What forms the binding site?

Answer 13

Variable regions of H & L chain

Question 14

Hierarchy of rearrangements for both heavy and light chains

Answer 14

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

Question 15

What chain has greater variability?

Answer 15

Greater variability in H chain as V, D & J

Question 16

What loci are the H, kappa & lambda chains encoded?

Answer 16

H chain – chromosome 14

Kappa chain – chromosome 2

Lambda chain – chromosome 22

Question 17

What isotope are most antibodies at the start?

Answer 17

IgM

Question 18

What does RSS stand for?

Answer 18

Recombination signal sequences

Question 19

What are RSS?

Answer 19

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)

Question 20

What are RAG genes?

Answer 20

Recombination activating gene

Question 21

What are the 2 types of RAG genes?

Answer 21

RAG-1 & RAG-2

Encode lymphoid-specific components of the recombinase

Question 22

Where are RAG genes expressed?

Answer 22

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

Question 23

What happens if theres mutations in the RAG genes?

Answer 23

Immunodeficiency

Question 24

How is the DNA broken and then reassembled to increase variability?

Answer 24

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

Question 25

What is allelic exclusion?

Answer 25

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

Question 26

How does allelic exclusion turn the gene off in 1 chromosome?

Answer 26

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

Question 27

Mechanisms for generation of antibody diversity

Answer 27

Multiple germ line genes Combinatorial diversity Junctional diversity Combinations of H & L chains Somatic hypermutation

Question 28

Multiple germ line genes

Answer 28

Multiple VH, V-kappa & V-lambda Also multiple D & J

Question 29

Combinatorial diversity

Answer 29

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

Question 30

Junctional diversity

Answer 30

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

Question 31

Combinations of H & L chains

Answer 31

Eg. 106 H and 104 L would give 1010 possible antibodies

Question 32

Somatic hypermutation

Answer 32

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

Question 33

What does somatic hypermutation involve?

Answer 33

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

Question 34

Membrane (BCR) vs secreted antibody

Answer 34

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

Question 35

What is the heavy chain in IgM?

Answer 35

Mu

Question 36

What is the heavy chain in IgD?

Answer 36

Delta

Question 37

What is the heavy chain in IgG?

Answer 37

Gamma

Question 38

What is the heavy chain in IgA?

Answer 38

Alpha

Question 39

What is the heavy chain in IgE?

Answer 39

Epsilon

Question 40

Why is IgM the first isotype expressed by each developing B cell?

Answer 40

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

Question 41

What does switching to other classes require?

Answer 41

Recombination Guided by switch regions & DNA breakage Also involves AID Cytokines & pathogens cause the switch