Lecture 6: B & T Cell Receptors

Question 1

How is B and T cell diversity achieved?

Answer 1

Through VDJ recombination: the random assembly of key genes that together make up the B or T cell receptor

Question 2

Why do B cells produce antibodies?

Answer 2

• neutralise toxins and pathogens
• promote phagocytosis
• activate compliment
• mediate immunity through Fc receptors

Question 3

Why is tight control of B and T cells important?

Answer 3

To prevent self-reactivity

Question 4

What is the B cell process leading to MHC II expression?

Answer 4

• BCR recognises specific peptide sequence in an antigen
• BCR/antigen complex induces a signalling cascade resulting in B cell activation
• B cell endocytoses the antigen and processes it into many peptide fragments to be presented on MHC II

Question 5

How are T cells recruited for B cell help?

Answer 5

Help for the B cell can come from any T cell that is specific for any of the presented peptides, where the T cell epitope is physically linked to the B cell epitope

Question 6

What sort of cells are B cells?

Answer 6

Professional APCs

Question 7

What are CDRs?

Answer 7

Complementary determining regions: 3 short segments in the V region with hyper variable amino acid sequences

Question 8

What are FRs?

Answer 8

Framework regions: 4 regions which alternate with CDRs to provide antibody with structure, and are less variable than CDRs

Question 9

What determines B cell receptor specificity?

Answer 9

VDJ genomic rearrangement, where large numbers of VDJ genes allows for massive diversity

Question 10

How many variable regions are generated per cell, and how does this impact Ig specificity?

Answer 10

Only 1 variable region is generated per B cell, so all Igs from a single b cell will have same specificity

Question 11

What are the large set of variable regions determined by?

Answer 11

Junctional diversity and somatic mutations

Question 12

What is the impact of affinity maturation?

Answer 12

Can increase the binding strength of antibodies via somatic mutations in the hyper variable regions, so improved affinity mean better antibodies

Question 13

Why is class switching used by B cells?

Answer 13

Allows them to alter the effector function of antibodies they produce

Question 14

In mice, what are the 2 light chains?

Answer 14

kappa and lambda

Question 15

In what order are the mice light chains used?

Answer 15

Kappa chains are used first as the gene is more complex, so can give rise to higher diversity

Question 16

Where is the joint between V and J located?

Answer 16

CDR3

Question 17

How does the V and J joint affect intron arrangement in the kappa chain?

Answer 17

J genes can be excised depending on where V joins to
eg. If V joins to J5, J1-4 are excised

Question 18

What segments do heavy chains have?

Answer 18

V, J, and extra D (diversity) segment

Question 19

What does the D segment do?

Answer 19

10-14 segments, which encodes amino acids 95-97 in the CDR3 to further expand the range of epitopes that could be recognised

Question 20

What do MH genes in the heavy chain do?

Answer 20

Determine the class of antibody produced

Question 21

What antibodies do naïve B cells produce?

Answer 21

IgM and IgM, which can class switch after activation to generate IgG, IgA and IgE

Question 22

In what order do the chains recombine?

Answer 22

First: heavy chain > D to J segment, then V to DJ segment
Second: kappa chain
Last: lambda (last resort)

Question 23

How many chances does each chain have to recombine successfully?

Answer 23

Heavy chain: once per chromosome, so 2 chances
Light chains: if unsuccessful, rescue attempt can be made if there is a 5’V and 3’J gene left

Question 24

What is an RSS?

Answer 24

Recombination signal sequence found at both 5’ and 3’ ends of V, D, and J genes

Question 25

What components do RSSs consist of?

Answer 25

- Heptamer: conserved region of 7 nucleotides - Nonamer: conserved region of 9 nucleotides - Spacer: less conserved region of 12/23 nucleotides

Question 26

What are RAG genes?

Answer 26

Recombination activating genes

Question 27

What is the function of RAG1 and RAG2 genes?

Answer 27

- RAG1 and RAG 2 function together to rearrange V(D)J genes - RAG1: binds both RSS and histone H3 - RAG2: guided by RAG1, is the enzyme with DNA cleavage activity

Question 28

How are RAG1 and RAG2 involved in enzymatic gene rearrangement?

Answer 28

- RAG1 and RAG2 recognise the conserved heptameter/nonamer sequences of the RSS - RAG1/2 complex at one site bind to the RAG1/2 complex at a different site, forming a loop of DNA - RAG2 proteins cleave the DNA and form hairpin loops

Question 29

What is the process of enzymatic gene rearrangement once RAG1/2 forms hairpin loops?

Answer 29

- DNA repair enzymes Ku70:Ku80 proteins bind the DNA at the ends and stabilise the strand break complexes - Signal joints: the signal join is ligated to form a loop, which dilutes as the cell divides OR - Coding joints: DNA protein kinase and Artemis binds to the closed loops of DNA covering the ends of the 2 sections to be recombined - combination of enzymes cleaves the hairpin loops to create raggedy ends - TdT adds random nucleotides to create some complementarity between both ends - DNA ligase and XRCC4 edit the ends (base excision and repair) to fully bind and ligate the DNA back together

Question 30

What is the purpose of the promoter at the end of every V gene?

Answer 30

It allows equal transcription of both genes

Question 31

In what way are enhancers needed?

Answer 31

Needed to initiate transcription of the gene, but are only in close proximity after V(D)J recombination

Question 32

What is allelic exclusion?

Answer 32

Rearrangement of the second chromosome is suppressed in cases where the first chromosome generates a functional antibody

Question 33

What is the purpose of allelic exclusion?

Answer 33

Ensures B and T cells have single specificity, as if the second allele was allowed to recombine then the cells would have dual specificity

Question 34

What processes are used for allelic exclusion

Answer 34

Asynchronous DNA replication, different feedback mechanisms, and histone modifications

Question 34

How does asynchronous DNA replication silence recombination?

Answer 34

One allele opens upon first, so the second allele has a extra mechanism to keep it closed. This process allows replication of only the gene that is being recombined first.

Question 35

How do feedback mechanisms silence recombination?

Answer 35

- When the first successful rearrangement is created, it will recombine with section of protein which creates a pre-B or T cell receptor. - Creates a surrogate light chains that join onto first recombination, which lets new receptor be expressed on the cell surface - If it is a functional receptor that has bound properly and has undergone selection, it will send signals into the cell that will halt rearrangement of second gene, so will inhibit the RAG complex so cannot bind or undergo recombination on the second allele

Question 36

How do histone modifications silence recombination?

Answer 36

Histone modifications keep the allele more closed and prevent transcription, so silence any further transcription of the second allele and therefore it cannot be opened up, RAG can't bind and can't be transcribed

Question 37

What chains do TCRs have?

Answer 37

- an alpha chain, containing V and J sections - a beta chain, containing V, D, and J sections

Question 38

Where is the delta locus located?

Answer 38

In the middle of the alpha locus

Question 39

Where is the TCR locus located?

Answer 39

Within the alpha locus, where the delta locus is within V regions