Thymocyte Development L14-16 Flashcards
When is notch implicated in thymocyte development? (6)
Notch controls the development of many cell types and is involved in cell fate decisions
Critical for T vs B cell lineage commitment
Critical through early thymocyte development
Influences Tcrb gene rearrangement
Critical for β selection checkpoint
Implicated in αβ versus δγ lineage commitment
Describe α chain rearrangement on the TCR
The α chain undergoes successive rearrangements until positive selection or death intervenes. T cells have in frame rearrangements on both chromosomes so can produce two types of α chain. Possible because expression of TCR is not sufficient to shut off gene rearrangement continual rearrangement on both chromosomes can allow several difference α chains to be produced successively and simultaneously in each developing T cell and be tested for self peptide-self MHC reg omitting in partnership with the same β chain. The phase of gene rearrangement is 3-4 days in mouse and only stops on positive selection/cell death.
What is Tdt?
Terminal deoxynucleotidyl transferase, it is expressed through arrangement and inserts N nucleotides leading to increase variation of rearrangement. Expressed in DN1 - DP
What happens in non productive β chain rearrangement of the TCR
Non productive VDJ rearrangements (unlike in B cells) can be rescued by further rearrangement due to clusters of Dβ and Jβ upstream of the two Cβ genes.
On productive rearrangement of the β chain on the TCR what happens?
The β chain is expressed together with the preTα and CD3 and is transported to the cell surface.
What happens on pre Tα expression at the PM in DN3 of thymocyte development?
This expression (of preTα) at the DN3 stage of thymocyte development induces signals that causes phosphorylation and degradation of RAG2 therefore halting β chain rearrangement.
What does degradation of RAG2 lead to?
to allelic exclusion at the β locus. These signals also induced DN4 where rapid cell proliferation occurs and eventually CD4/8 are expressed.
What does preTα signal constitutively via?
The cytoplasmic protein Lck but does not seem to require a ligand on thymocyte epithelium. Lck subsequently associated with the co-receptor proteins CD3.
In mice what happens on lck-/-?
T cell development is arrested before CD4/8 DP and no α gene rearrangement can be made
RAG1 and RAG2 are…?
And where are they expressed?
Heterodimeric recombinases that initiate somatic recombination of VDJ joining. They are expressed in DN2 and into DN3 (and briefly into DP on α chain rearrangement)
When is TCF1 expressed and what happens on KO?
In DN.
KO - T cells that make productive β chains do not proliferate = no efficient production of DP thymocytes.
When does TCR α chain rearrangement begin?
No rearrangement of the α chain until after the proliferative phase ends. When RAG1 and RAG2 and transcribed again and a functional rag1:2 complex accumulates.
Why does Tα chain rearrangement start after proliferation?
To ensure that each cell has successfully rearranged it’s β chain to give lots of DP thymocytes. Once cells stop dividing each cell can independently rearrange it’s α chain genes so that one single functional β chain can be associated with many different α chains in progeny cell.
What happens a once α chain is rearranged and expressed?
Selection by self peptide:self MHC can begin
During massive proliferation of thymocytes Rag1/2 are…?
REPRESSED
ZAP70 is expressed when in thymocyte development?
From DN stage but promotes development of SP thymocytes from DP ones.
When is fyn expressed in thymocyte development?
Expressed from DP stage at increasing levels it is not required for αβ thymocyte development if lck is present but is crucial for iNKT development
What mutations prevent early thymocyte development passing through β selection checkpoint
Rag1 or 2 deficient TCRβ, preTα deficient CD3 deficient Lck, fyn deficient Zap70, syk deficient
When is CD3 first expressed and what relies on its expression?
CD3 is expressed with the pTα:β heretofore complex but at very low levels. defects in CD3 means no β selection and proliferation
Once early thymocytes progress to DN4 they can no longer become what type of T cells?
γ:δ (can still become this in DN3)
Fyn -/- cells look like…?
Normal, Lck can compensate for fyn but not the other way round
Lck/fyn -/- leads to?
Thymocytes blocked in DN no CD4 or CD8. No mature thymocytes migrate to the LNs or to peripheral LNs like the spleen (B cells are present).
On further investigation with flow cytometry on CD44 and CD25 shows thymocytes are blocked at DN3 specifically.
What happens to thymic cellularity in αβ lineage blocked lck-fyn -/- mice?
There are at least 100fold fewer thymocytes as no DN4 and no proliferation occurs.
How is preTα necessary for thymocyte development?
TCRβ and preTα form preTCR complex
PreTCR indicates a functional TCRβ chain made from rearrangements. Thymocytes lacking a preTCR signal are blocked at DN3.
The preTCR checkpoint of β selection controls?
DN to DP
allelic exclusion at the TCRβ locus.
Proliferation
Subsequent TCRα gene segment rearrangement
When β chain allelic exclusion occurs what else is switched off?
γ and δ gene rearrangement
If DP thymocytes are not activated for positive selection what happens?
Death, lots of cell death. Positive selection rescues from cell death
To allow self recognition VDJ joins need to be…?… When they are selected
Inframe joins to allow self MHC recognition
What thymocytes get positively selected?
Those with an αβ TCR that recognises a self peptide/self MHC class I or class II complex in the thymus
Thymocytes that express an αβ TCR that has very high affinity for self peptide/self MHC are…?
Negatively selected and die by apoptosis
DN1 subsets:
5 functionally distinct subsets DN1a-e based on CD24, CD117 and CD90 expression.
Most heterogenous group of DN
DN2 subsets
Separable based on CD90 and CD117 expression levels DN2a and DN2b corresponding from when progenitors go from being T lineage specified to being T lineage committed
DN3 subsets
Last stage when both αβ and γδ can occur in FTOC DN3a-c subsets based on CD27 and CD28 cell size or metabolic state
What is the aim of thymic selection?
To allow only self-MHC restricted self tolerant T cells to leave the thymus.
Such mature naive T cells are useful for recognition of foreign antigens presented by self MHC I or II isoforms expressed but the individual as T cells circulate and pass through the the secondary lymphoid tissue
What does increased IL7 Lead to?
Reduced DP thymocytes. IL7 promotes survival of TCRβ DN but not DP
What does positive selection involve?
- Rescue from apoptosis (up regulation of Bcl2 promotes survival
- Initiation of T cell differentiation
- up reg of TCR levels
- expression of CD69, CD2 and CD5
- down regulation of heat stable antigen
- expression of ccr7 (promotes medulla recruitment) - It is followed by commitment to the CD4 or CD8 lineage
Mouse MHC I genes
H-2K, H-2D, H-2L
Mouse MHC II genes
IA and IE
Three states of MHC:peptide:TCR
Recognition all correct
No recognition: right peptide and TCR wrong MHC haplotype
No recognition: right MHC haplotype and TCR wrong peptide
Describe Bevan et al 1997 experiment showing thymocyte positive selection revealed by bone marrow chimeric mice:
MHCaxbF1 into irradiated MHCa and MHCb recipient.
T cells from F1 expressing both haplotypes. The original MHC phenotype prevails. New T cells respond only to original MHC haplotype expressed by APCs so shows that only MHCb recognising TCRs are selected in the MHCb mouse and vice versa - positive selection.
Generating T cell repertoire transgenic mice:
Clone rearranged TCR genes from a T cell clone of know specificity
Inject DNA into the male peony keys of a fertilised egg
Inject eggs into a pseudo pregnant female
With some luck some offspring will carry the transgene.
Breed male TCR transgenic mice with wt females to produce more mice in which most of the T cells will express the TCR transgene.
If you cross male TCR transgene with mice that lack rag1 or rag2 you get a monoclonal source of the original TCR genes being expressed
What example demonstrated peptide:MHC II selects CD4 T cells?
2B4 (TCR) vs p.cytc(ag)/IEk(MHC II)
What peptide MHC example demonstrated selection of CD8 positive
HY (clonal TCR) vs HY/Db
Why is IE not expressed in MHCb haplotype?
Promoter deficiency in the IEb haplotype
In Eb/b MHC II is never expressed so no lineage determination for what cells that recognise p.cytc
2B4 cells that recognise p.cytc
Dd/b MHC female will or will not present HY and select CD8 lineage?
Dd/d MHC female will or will not select present HY and CD8 lineage?
And why showed this?
Dd/b will select for cd8 because Db presents HY
Dd/d will not select for cd8 because Dd does not present HY
Von boehmer 1988
Suggests that there is a female peptide similar to the male HY antigen
If Cortical epithelial cells express MHC II what ℅ receptor is selected for?
Only CD4 T cells mature
If cortical epithelial cells express MHC I what SP cells mature?
Only CD8 T cells mature
Absence of β2n means
MHC I is very unstable.
How do you generate β2m deletion mutants by homologous recombination?
Three outcomes on integration into cell
DNA construct containing exons of β2m gene together with HSV thymidine kinase gene
Target gene interrupted by neomycin-resistance gene
1. DNA fails to integrate and cell is killed by G418 which is a neomycin analogue
2. DNA integrates at a random site in the genome expresses both neor and HSV-tk. Cell is killed by gangcyclovir (HSV-tk phosphorylates gangcyclovir causing it to become toxic and kill the cell
3. Homologous recombination replaced the WT β2m gene with interrupted copy so the cell is not killed by G418 or gangcyclovir
How do you generate β2m KO mice?
Transfer to β2m gene KO construct into ES cells.
Inject ES cells into mouse blastocyst
Reimposition blastocyst into pseudo pregnant female
Some offspring contain tissues (including germ cells) that derive from the injected cells
Breed chimeric mice to generate homozygous β2m deficient strain
What are bare lymphocyte syndromes?
Mutations that lead to absences of MHC molecules on lymphocytes and thymic epithelium cells
Mutations can be in TAP (lack of MHC I) no cd8
CIITA (transactivator for the class II locus) defects lead to lack of expression of MHC II molecules no cd4