Adaptive Development (B and T Cell Receptor Generation, Tolerance) Flashcards
How does endocytosis of an antigen and subsequent presentation allows potential help from T cell which recognise different epitopes?
a B cell receptor recognises a peptide sequence in an antigen (6aa long)
help for the B cell can come from any T cell that is specific for the presented peptides, not just the one the B cell initially recognises
important as T cell antigen may be in a deeper part of the protein (out of Ab reach)
Why are some regions of the antigen binding site of BCRs non variable?
variation is focussed on 3 short segments in the V region w hypervariable aa sequences
- known as complementarity determining regions (CDRs)
CDRS alternate w 4 framework regions (FRs) that provide Ig w structure
– if they were more variable the protein may not fold properly and become non functional
What is genomic rearrangement?
- genomic rearrangement
– V, D, and J selection determines B cell receptor specificity
– large number of genes = massive diversity
What is the significance of B cells possessing a large set of variable regions?
– determined by junctional diversity and somatic mutations
– only one generated per B cell
– all Ig from a single B cell have the same specificity
What is affinity maturation?
- can increase the binding strength of Abs via somatic mutations in the hyper variable regions
- improved affinity = better Abs
- somatic mutations can also create lower affinity Abs, but these will quickly be outcompeted
What is class switching?
allows B cells to irreversibly alter the effector function of Abs they produce
What is the genetic organisation of light chains in mice?
in mice there are two light chains (kappa (k) and lambda)
- each contains V, J, and C
k is used first as it can give rise to highest diversity as it is more complex
- any V gene can join to any J
- 95% of mouse Abs have a k light chain (70% in humans)
- ~350 V, 5 J,
lambda is used as a last resort and cannot generate as much diversity
- gene is split into sections. not any V can join any J, just ones in their section
- 3 V, 4 J
Outline lambda chain rearrangement.
the joint between the V and J region are always located within CDR3
- tru for k and heavy chains too
- helps further diversity found in Ig binding domains
lowest level of variation (accounts for 5% of Ig in mice)
Outline kappa chain rearrangement.
If V joins to J1 then J2-5 remain in the intron
If V joins to J5, then J1-4 are excised out from the chromosome and are lost
in this instance, the rearrangement is non-productive (eg the VJ gene is out of frame) then cell will rearrange on other chromosome
- if this also fails, cell will then try on lambda chain
What is the genetic organisation of the heavy chain?
larger locus and contains additional fragments
heavy chains have a diversity segment (D) which encodes aa within CDR3
- 10-14 segments in mice
- further expands range of epitopes
heavy chain also has multiple constant (C) genes which determine the class of Ab produced
- IgM and IgD in naive B cells (alternative splicing)
- class switching post activation
junctions (//) between regions sites for insertion of random aa
What is the order of recombination?
heavy chain recombines first
– J combines to D first then cut out remaining Ds to joining on of V
– can only happen once per chromosome as all D genes are cut out
cells will then rearrange kappa light chain
– if unproductive, a rescue attempt can happen if there is a 5’ V and a 3’ J left
lambda is used as last resort
- rescue attempt can be made if there is a 5’ V and a 3’ J left (human
- unlikely for mouse due to structure of genes (sections)
Taking into account other additions/ alterations which can affect diversity, there are possibly 10^10/10^11, explain why the body would not actually produce this many different BCRs.
a good portion of these BCRs would be unproductive, unreactive, or improperly folded
What are the mechanisms of recombination?
every V, D, and J genes has conserved flanking sequences
- recombination signal sequences (RSSs)
- found at 5’ and 3’ ends of the genes
highly conserved regions, eg
k structure:
V – (12 bp) – // – (23bp) – J
lambda structure:
V – (23 bp) – // – (12bp) – J
H chain structure:
V – (23 bp) – // – (12bp) – D – (12bp) – // – (23 bp) – J
sequences are palindromic
12 p 1 turn of DNA helix
restriction enzymes target these regions
recombination occurs between a 23 RSS and a 12 RSS
- means each gene is joined to correct gene
What is the function of RAG1/2?
RAG1 binds to both RSS and histone H3 (part of DNA structure
RAG2 guided by RAG1 and is enzyme w DNA cleavage activity
activity of RAG1/2 form a loop structure and bring the parts to be joined into close proximity
Outline the role of RAG1/2, and Ku70:80 in VDJ recombination step by step.
1, RAG 1/2 recognise conserved heptamer/nonamer sequences of RSSs
- RAG complex at one site (eg D gene) bind to the RAG complex from a different site (eg J gene)
– this forms a loop of DNA - RAG2 cleaves DNA and forms hairpin loops
- DNA repair enzymes Ku70:Ku80 bind DNA at ends and stabilises strand break complexes
5a. signal joint is ligated together to form a loop, which dilutes out as cell divides
Outline the role of Artemis and TdT in VDJ recombination.
5b. DNA protein kinase and Artemins binds to closed loops of DNA, covering ends of the two loops being recombined
- this cleaves the hairpin loops, introducing P bases, creating raggedy ends
- TdT adds random, non-templated nucleotides, including non-complementary bases
- complementary bases pair before mismatches are repaired
- DNA ligase and XRCC4 edit ends through base excision and repair to fully bind and ligate DNA back together
new section of DNA will always be different even if genes are the same
Artemis and TdT do not act on signal joints, only coding joints
What happens if TdT adds too many nucleotides?
region will be too long, creating a frameshift in CDR3
– meaning it will be non-functional
generation of greater diversity at a cost
Where are promoters and enhancers of Ig genes located?
every V genes has a promoter of 25bp at 5’ end that is shared between H and L chains
- results in equal transcription of both
an enhancer is needed to initiate transcription of the gene
- they are only close enough after VDJ recombination events have taken place
If recombination is successful on the first chromosome, what happens to the second one?
allelic exclusion
- rearrangement is suppressed
ensures B and T cells have a single specificity
- dual is a problem, as cell could have one receptor that recognises non-self and the other recognises self
- if it were to be activated, would cause major autoimmune reaction
How is allelic exclusion carried out?
- initiation
- allele asynchrony- one allele opens up first, the other has mechanisms to keep it closed
- feedback
- after first rearrangement, will recombine w section of protein which creates pre B or pre T receptor = surrogate light/alpha chains that join onto first recombination which allows it to be expressed on surface
- if it is functional (binds to heavy chain, stable) it will send signals to halt rearrangement of second allele - maintenance
- second allele is silenced
- cant be opened up so RAG cant enter
What is the structure of a TCR?
- no alternative constant regions
- never secreted
- heterodimeric joined by a disulphide bond
- short cytoplasmic tail
- antigen binding site made up of both subunits
What does VDJ recombination look like in T cells?
the mechanisms that control the recombination events are the same between B and T cells
a chain contains V and J
b chain contains V, D, and J
V has 23
J has 12
D has both
this means that D region can potentially be skipped
- higher diversity
What is different between rescue events between T and B cells?
rescue can happen on all T cell alleles, but in B cells this will only occur on the light chain
T cell a chain has more options than light chains for rescue of rearrangement events, due to D being able to be skipped on beta
and T cells have two sets of clusters
Where is the delta locus located?
located within the alpha locus, with the V regions interspersed
some of the V segments are shared between a and d
- rearrangement of the a chain results in deletion of the d locus from the chromosome
At the DN2/DN3 stages, TCRγ, TCRδ, and TCRβ gene loci start undergoing somatic recombination
Rearrangement of the TCRγ and TCRδ loci occurs simultaneously.
Rearrangement of the TCRβ locus is independent of the γδ loci.
If both TCRγ and TCRδ loci successfully rearrange and produce a functional γδ TCR before the TCRβ locus, the thymocyte expresses the γδ TCR on its surface.
These cells bypass the pre-TCR checkpoint and commit to the γδ lineage.
If a functional TCRβ chain rearranges first, it pairs with the pre-Tα chain to form the pre-TCR complex
What are the different outcome of alternative recombination in the delta locus?
V-J
V-D-J
V-D-D-J
joining of V-J directly is often limited due to other control mechanisms
What is somatic hypermutation?
occurs mainly in the CDR regions after clonal expansion, and can happen again after reactivation (secondary/tertiary immune response
mutation rate in variable regions is 10_3 bp/generation (10_8 in other genes)
selection of higher affinity clones for selective expansion
– lower affinity clones are outcompeted and die
enhanced diversity by 1-2 orders of magnitude
What is the mechanism of hypermutation?
involved are RNA pol II and activation-induced cytosine deaminase (AID)
AID is responsible for deamination of bases in DNA
- C residue is replaced w U
– doesnt belong in DNA
– must be repaired
What is the cost of hypermutation?
many cells will mutate to non-functional Igs, or will lower affinity for receptor
