Lecture 6: B & T Cell Receptors Flashcards
How is B and T cell diversity achieved?
Through VDJ recombination: the random assembly of key genes that together make up the B or T cell receptor
Why do B cells produce antibodies?
- neutralise toxins and pathogens
- promote phagocytosis
- activate compliment
- mediate immunity through Fc receptors
Why is tight control of B and T cells important?
To prevent self-reactivity
What is the B cell process leading to MHC II expression?
- 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
How are T cells recruited for B cell help?
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
What sort of cells are B cells?
Professional APCs
What are CDRs?
Complementary determining regions: 3 short segments in the V region with hyper variable amino acid sequences
What are FRs?
Framework regions: 4 regions which alternate with CDRs to provide antibody with structure, and are less variable than CDRs
What determines B cell receptor specificity?
VDJ genomic rearrangement, where large numbers of VDJ genes allows for massive diversity
How many variable regions are generated per cell, and how does this impact Ig specificity?
Only 1 variable region is generated per B cell, so all Igs from a single b cell will have same specificity
What are the large set of variable regions determined by?
Junctional diversity and somatic mutations
What is the impact of affinity maturation?
Can increase the binding strength of antibodies via somatic mutations in the hyper variable regions, so improved affinity mean better antibodies
Why is class switching used by B cells?
Allows them to alter the effector function of antibodies they produce
In mice, what are the 2 light chains?
kappa and lambda
In what order are the mice light chains used?
Kappa chains are used first as the gene is more complex, so can give rise to higher diversity
Where is the joint between V and J located?
CDR3
How does the V and J joint affect intron arrangement in the kappa chain?
J genes can be excised depending on where V joins to
eg. If V joins to J5, J1-4 are excised
What segments do heavy chains have?
V, J, and extra D (diversity) segment
What does the D segment do?
10-14 segments, which encodes amino acids 95-97 in the CDR3 to further expand the range of epitopes that could be recognised
What do MH genes in the heavy chain do?
Determine the class of antibody produced
What antibodies do naïve B cells produce?
IgM and IgM, which can class switch after activation to generate IgG, IgA and IgE
In what order do the chains recombine?
First: heavy chain > D to J segment, then V to DJ segment
Second: kappa chain
Last: lambda (last resort)
How many chances does each chain have to recombine successfully?
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
What is an RSS?
Recombination signal sequence found at both 5’ and 3’ ends of V, D, and J genes
What components do RSSs consist of?
- Heptamer: conserved region of 7 nucleotides
- Nonamer: conserved region of 9 nucleotides
- Spacer: less conserved region of 12/23 nucleotides
What are RAG genes?
Recombination activating genes
What is the function of RAG1 and RAG2 genes?
- 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
How are RAG1 and RAG2 involved in enzymatic gene rearrangement?
- 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
What is the process of enzymatic gene rearrangement once RAG1/2 forms hairpin loops?
- 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
What is the purpose of the promoter at the end of every V gene?
It allows equal transcription of both genes
In what way are enhancers needed?
Needed to initiate transcription of the gene, but are only in close proximity after V(D)J recombination
What is allelic exclusion?
Rearrangement of the second chromosome is suppressed in cases where the first chromosome generates a functional antibody
What is the purpose of allelic exclusion?
Ensures B and T cells have single specificity, as if the second allele was allowed to recombine then the cells would have dual specificity
What processes are used for allelic exclusion
Asynchronous DNA replication, different feedback mechanisms, and histone modifications
How does asynchronous DNA replication silence recombination?
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.
How do feedback mechanisms silence recombination?
- 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
How do histone modifications silence recombination?
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
What chains do TCRs have?
- an alpha chain, containing V and J sections
- a beta chain, containing V, D, and J sections
Where is the delta locus located?
In the middle of the alpha locus
Where is the TCR locus located?
Within the alpha locus, where the delta locus is within V regions
