Thymocyte Development L14-16 Flashcards
1
Q
When is notch implicated in thymocyte development? (6)
A
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
2
Q
Describe α chain rearrangement on the TCR
A
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.
3
Q
What is Tdt?
A
Terminal deoxynucleotidyl transferase, it is expressed through arrangement and inserts N nucleotides leading to increase variation of rearrangement. Expressed in DN1 - DP
4
Q
What happens in non productive β chain rearrangement of the TCR
A
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.
5
Q
On productive rearrangement of the β chain on the TCR what happens?
A
The β chain is expressed together with the preTα and CD3 and is transported to the cell surface.
6
Q
What happens on pre Tα expression at the PM in DN3 of thymocyte development?
A
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.
7
Q
What does degradation of RAG2 lead to?
A
to allelic exclusion at the β locus. These signals also induced DN4 where rapid cell proliferation occurs and eventually CD4/8 are expressed.
8
Q
What does preTα signal constitutively via?
A
The cytoplasmic protein Lck but does not seem to require a ligand on thymocyte epithelium. Lck subsequently associated with the co-receptor proteins CD3.
9
Q
In mice what happens on lck-/-?
A
T cell development is arrested before CD4/8 DP and no α gene rearrangement can be made
10
Q
RAG1 and RAG2 are…?
And where are they expressed?
A
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)
11
Q
When is TCF1 expressed and what happens on KO?
A
In DN.
KO - T cells that make productive β chains do not proliferate = no efficient production of DP thymocytes.
12
Q
When does TCR α chain rearrangement begin?
A
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.
13
Q
Why does Tα chain rearrangement start after proliferation?
A
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.
14
Q
What happens a once α chain is rearranged and expressed?
A
Selection by self peptide:self MHC can begin
15
Q
During massive proliferation of thymocytes Rag1/2 are…?
A
REPRESSED
16
Q
ZAP70 is expressed when in thymocyte development?
A
From DN stage but promotes development of SP thymocytes from DP ones.
17
Q
When is fyn expressed in thymocyte development?
A
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
18
Q
What mutations prevent early thymocyte development passing through β selection checkpoint
A
Rag1 or 2 deficient TCRβ, preTα deficient CD3 deficient Lck, fyn deficient Zap70, syk deficient
19
Q
When is CD3 first expressed and what relies on its expression?
A
CD3 is expressed with the pTα:β heretofore complex but at very low levels. defects in CD3 means no β selection and proliferation
20
Q
Once early thymocytes progress to DN4 they can no longer become what type of T cells?
A
γ:δ (can still become this in DN3)
21
Q
Fyn -/- cells look like…?
A
Normal, Lck can compensate for fyn but not the other way round
22
Q
Lck/fyn -/- leads to?
A
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.
23
Q
What happens to thymic cellularity in αβ lineage blocked lck-fyn -/- mice?
A
There are at least 100fold fewer thymocytes as no DN4 and no proliferation occurs.
24
Q
How is preTα necessary for thymocyte development?
A
TCRβ and preTα form preTCR complex
PreTCR indicates a functional TCRβ chain made from rearrangements. Thymocytes lacking a preTCR signal are blocked at DN3.
25
The preTCR checkpoint of β selection controls?
DN to DP
allelic exclusion at the TCRβ locus.
Proliferation
Subsequent TCRα gene segment rearrangement
26
When β chain allelic exclusion occurs what else is switched off?
γ and δ gene rearrangement
27
If DP thymocytes are not activated for positive selection what happens?
Death, lots of cell death. Positive selection rescues from cell death
28
To allow self recognition VDJ joins need to be...?... When they are selected
Inframe joins to allow self MHC recognition
29
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
30
Thymocytes that express an αβ TCR that has very high affinity for self peptide/self MHC are...?
Negatively selected and die by apoptosis
31
DN1 subsets:
5 functionally distinct subsets DN1a-e based on CD24, CD117 and CD90 expression.
Most heterogenous group of DN
32
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
33
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
34
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
35
What does increased IL7 Lead to?
Reduced DP thymocytes. IL7 promotes survival of TCRβ DN but not DP
36
What does positive selection involve?
1. Rescue from apoptosis (up regulation of Bcl2 promotes survival
2. 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)
3. It is followed by commitment to the CD4 or CD8 lineage
37
Mouse MHC I genes
H-2K, H-2D, H-2L
38
Mouse MHC II genes
IA and IE
39
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
40
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.
41
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
42
What example demonstrated peptide:MHC II selects CD4 T cells?
2B4 (TCR) vs p.cytc(ag)/IEk(MHC II)
43
What peptide MHC example demonstrated selection of CD8 positive
HY (clonal TCR) vs HY/Db
44
Why is IE not expressed in MHCb haplotype?
Promoter deficiency in the IEb haplotype
45
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
46
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
47
If Cortical epithelial cells express MHC II what ℅ receptor is selected for?
Only CD4 T cells mature
48
If cortical epithelial cells express MHC I what SP cells mature?
Only CD8 T cells mature
49
Absence of β2n means
MHC I is very unstable.
50
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
51
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
52
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
53
No MHC II -
| No MHC I -
No MHC II - CD8 cells and few highly abnormal CD4
| No MHC I - no CD8 cells at all
54
Process of thymocyte development and coupling with migration in the lymph node
Progenitors from BM enter through HEV
Ligands that interact with notch1 are expressed to commit progenitors to T cell lineage
As cells differentiate through the DN stages they migrate through the corticomedullary junction and to the outer cortex. DN3 cells reside near the subcapsular region. As the progenitor matures further to the DP stage it migrates back through the cortex. The medulla contains only mature SP T cells that eventually leave the thymus
55
Lymph node organisation
Thymic lobes:
| Cortex- corticomedullary junction - cortex - subcapsular region
56
In the cortex of the thymus what cells express MHC for T cell selection?
Thymic cortical epithelial cells
57
What happens to thymocytes that have a TCR rearrangement that don't recognise MHC I or II?
They die by apoptosis
58
In the medulla of the thymus, what cells express MHC for TCR selection and what happens if this signal is very strong?
Dendritic cells and thymic macrophages. If the signal is too strong they undergo apoptosis.
59
What happens to mature naive positively selected CD4 and CD8 T cells?
They pass out of the thymus either returning to the blood, passing into the venues or via the lymphatic system
60
How was it shown that thymic cortical epithelial cells and thymic epithelium mediate positive selection
```
In MHC II negative mutants only CD8 cells mature
In MHC II mutant with a class II transgene expressed in thymic epithelium (so only thymic epithelium expresses MHC II) both CD8 and CD4 T cells mature
Mutant MHC II that cannot interact with cd4 only cd4 T cells mature
This showing that cortical epithelial cells are critical for positive selection
```
61
MHC II medullary epithelium does or does not allow CD4 positive selection
Does not
62
How is thymic cortical epithelium specialised for positive selection?
Unique proteases for MHC II (cathepsin L generates deferent peptides, thymic specific serine protease)
Unique proteasome for MHC I presentation (thymoproteasome includes β5t subunit - generates peptides that are optimal a for the positive selection of normal naive CD8 T cells)
Unusually high constitutive levels of macroautophagy - lots of cell destruction in this context.
63
Β2m -/- FTOC of mice shows
Class I chain is very unstable and short-lived. No CD8 T cells in thymic lobes of FTOC. Substitute in human β2m + peptides gives stable complex MHC I and peptides.
64
How was the avidity hypothesis determined
Using OT1 and SIINFEKL/Kb
Which is a very high avidity interaction. Ot1 thymocytes for SIINFEKL were deleted by negative selection. OT1 selected for the EIINFEKL/Kb weaker avidity.
This was dependent on β2m stability
65
Positive selected thymocytes have rapid up regulation of and migration where - shown by what technique?
CCR7 Which allows migration to the medulla shown by 2 photon imagine in intact thymic lobes (on positive selection with P14 a selective ligand that recognises LMCV a mouse virus peptide)
66
The antigen 5CC7 shows what on a non selecting background?
Random walk migration as the thymocytes scan for a matching peptide
67
Why are peptides that have too high avidity to self peptide/MHC negatively selected?
To prevent self tolerance and reaction to self.
68
Thymocyte positive selection results in...? (8)
1. Coordinated TCR and ℅ receptor expression
2. Generation of mature CD4 or CD8 T cells by down regulation of irrelevant ℅ receptor
3. Up regulation of TCR levels
4. Up regulation of Bcl2 to promote survival
5. RAG1 and RAG2 expression is turned off (so no subsequent TCR rearrangements)
6. TCRα gene segment rearrangement ceases
7. Expression of CD69, CD2, CD5 and CCR7 (allows recruitment from cortex to medulla)
8. Rapid migration to the medulla (CCL19 and 21)
69
What does the signal from preTα drive?
To get a mature αβ TCR
70
Rearrangement of the β chain occurs after interaction with which interleukin?
IL7
71
When strongly self reactive thymocytes are eliminated in the thymus what is it called?
Central tolerance
72
Where do Tregs get selected (in terms of avidity) and what CDs do they have?
Get selected on the interface of positive and negative selection and are CD25+ and CD4+
73
What other cells may be selected on the interface of positive and negative selection?
CD8αα intestinal intraepithelial cells and some NKT cells
74
How is apoptosis in negative selection detected?
TUNEL
Tdt- mediated dUTP biotin Nick end labelling
Monitors nicking between nucleosomes
75
Where are apoptotic thymocytes ingested and by what cells?
Macrophages ingest apoptotic thymocytes in the thymic cortex
76
How do macrophages recognise apoptotic thymocytes?
Changes in the cell surface
77
How was negative selection demonstrated in mice and what does this show about negative selection
Irradiation of MHCa mouse and reconstitute with bone marrow of MHCaxbF1 mouse. Skin graft of MHCb mouse onto MHCaxbF1 BM chimera. Toleration of the MHCb skin graft. Normally MHCa would reject MHCb donor derived cells so BM is responsible for negative selection of MHCb reactive cells because the BM is the only place MHCb is expressed in the chimeric mouse
78
What bone marrow derived cells mediate negative selection
DCs, pDCs, cDCs, macrophages and B cells that are BM derived
79
What enables DP thymocytes to pass through the cortex and the CMJ through lots of DCs to the medulla?
CCR7 to CCL19 and CCL21
80
How do you test the avidity hypothesis in vitro?
Surface plasmon resonance
MHC + peptide complex immobilised on gold plated surface
Soluble TCRs are allowed to flow over and bind to the complex.
receptor binding reaches equilibrium (when all sites are saturated)
Unbound receptors are washed away and cont washing removes TCRs and the dissoc from the MHCpeptide complex
81
What does surface plasmon resonance give? (Ru)
A graph of resonance units (Ru) over time
82
What does low and high Kd of peptide:MHC binding to TCR mean?
Low Kd - tight binding affinity
| High Kd - weak binding affinity
83
Agonist selection by high affinity ligands can lead
T cells with regulatory properties
84
Order of peptide avidity for OT1 TCR and whether peptide is an agonist or antagonist of mature T cell activation
```
SIINFEKL - agonist
EIINFEKL - agonist/antagonist
RGYNYEKL - antagonist
SIIRFEKL - antagonist
SIIKFEKL - none
RGYCYQGL - none
```
85
What role of medullary thymic epithelial cells play in negative selection?
Expression of certain tissue-specific or tissue-restricted auto antigens leading to elimination of potentially destructive auto reactive thymocytes centrally
86
What tissue specific auto antigens are expressed by mTECs?
Myelin components - proteolytic protein and myelin oligodendrocyte glycoproteins (auto antigens in MS)
Pancreatic β cell antigen insulin (TID auto antigen)
87
What regulates the autoantigens expressed by medullary thymic epithelium cells?
AIRE - autoimmune regulator
88
RNAseq of mTECs shows what?
They express most of the antigens in the body
89
What cells expressed AIRE?
A subset of epithelial cells in the medulla
90
What do patients with mutations in AIRE suffer from and what do they have autoantibodies to?
APECED - autoimmune polyendocrinopathy candidiasis ectodermal dystrophy
Involves autoantibodies against IL 17 which are known to be important in Mac recruitment in candidiasis
91
As well as IL17 autoantibodies patients with APECED have what...?
Destruction of endocrine tissues including insulin secreting cells
92
What do AIRE knock out mice exhibit?
Lack of deletion of T cells that are auto reactive to tissue specific organs (tend to be endocrine)
93
What are the (not well understood) mechanisms of AIRE in mTECs?
AIRE up regulates the expression of certain tissue- restricted antigens
AIRE doesn't act as a classical DNA bind TF
It targets non methylatedH3K4 (repressive Mark) switching on silent gene loci.
94
AIRE is thought to primarily act as a...
Docking platform for enzyme complexes that facilitate transcription by generating local double strand breaks promoting mRNA processing and relieving stalled RNA polymerase
95
If an immature DP or SP thymocyte is stimulated through the TCR what happens
Apoptosis
96
If a mature SP thymocyte is stimulated through the TCR what happens? And how long does this take?
Proliferation and final maturation takes less than 4 days
97
On final maturation of thymocytes what lipid molecule enables them to emigrate out of the thymus?
S1P in high conc in blood and lymph is recognised at high levels by S1P1 receptor on mature thymocytes.
98
What LN homing receptor do mature thymocytes express and what does it facilitate?
CD62L (L selectin)
It facilitates the localisation of mature naive T cells to the peripheral lymphoid organs after they have left the thymus.
99
What are superantigens?
Proteins produced by a variety of bacteria and viruses that trigger a very high (5-30%) frequency of mature T cells (normally 0.0001%)
100
Examples of superantigens are
Staphylococcal enterotoxin B
Staphylococcal toxic shock syndrome toxin 1
Mouse mammary tumour virus superantigen
Human endogenous retrovirus
101
Superantigens interact with which MHC molecule?
MHC II
102
In mice which MHC II binds viral SAgs better?
IE IS BETTER THAN IA
103
Where do SAgs bind on the TCR?
The Vβ domain of the TCR
104
Why is normal T cell triggering so low (0.0001%)?
Because of requirement for exactly right V(D)J junctions on the β and α chains to recognised individual peptide:MHC combo.
105
Where do SAgs bind to trigger much T cell activation?
They bind away from the peptide binding groove
106
What does the mass triggering Of mature T cells by SAgs result in
Proliferation and secretion of cytokines such as TNFα leading to toxic shock in vivo
107
What happens when immature DP thymocytes express relevant RCR Vβ elements encounter SAg/MHC II complexes?
They are deleted
108
If a mouse expressed the superantigen Mtv-7 what happens to Vβ6+ mature thymocytes
If a mouse does not express mtv-7 what happens to Vβ6+ thymocytes?
In an mls 1a mouse (Mtv7 SAg) Vβ6+ mature thymocytes are absent from the medulla
In an mls 1b mouse (mtv7 -/-) Vβ6int thymocytes in the cortex and Vβ6hi thymocytes in the medulla
109
How is thymic negative selection been studied (3)
TCR transgenic mice
Mice expressing MMTV
inclusion of synthetic peptides in FTOCs of mutant thymuses lacking β2m or TAP1
110
How is positive/negative selection determined in the thymus?
Positive selection :
Low affinity ligand triggering of the TCR, partial LAT phosphorylation, recruitment of PLCγ1, generation of DAG and Ca2+. Results in sustained low level of ERK and NFAT activation
Negative selection :
Involves high affinity ligand triggering of the TCR
Total LAT phosphorylation, recruitment of Grb2 and Sos1. Results in transient high level ERK, p38 and Jnk activation
111
Where does pErk initially localise to in negative selection
The plasma membrane
112
Where does pErk initially localise to in positive selection?
The Golgi apparatus
113
What is one of the key differences between positive and negative selection?
How the MAP kinase signalling components are compartmentalised
114
What happens on weak TCR-pMHC interactions?
Death by neglect
115
What has kinetic proofreading predicted?
That the length of the TCR engagement determines positive and negative selecting signals
116
Surface plasmon resonance has shown the correlation of what?
| What has it not determined?
Ligand affinity, half-life and selection outcome
| But not determined the contribution of the ℅-receptor
117
How have peptides for OT1 shown the narrowness of the window between positive and negative selection?
The boarder peptide T4 is a negative selector at high concs and a positive selector at low concs and is a poor ag for peripheral OT1 T cells at all doses.
QR47 is 1.5x more potent than T4 and is a negative selector at all concs. Therefore showing the narrow window in which self reactive thymocytes can potentially escape negative selection.
118
Higher affinity ligands tend to have a longer interaction lifetime with the TCR what does this mean?
That there is sufficient time for lck to gain sufficient access to the TCR-CD3 complex to generate signals for negative selection. Shorter interactions - positive selection
119
What do negative selectors do to ZAP70?
Induce more efficient concentration of phosphorylated ZAP70 at the membrane. Suggesting enhanced phosphorylation of LAT may be as a result of increased ZAP70 kinase conc.
120
Activation of LAT in negative selection leads to?
Activation and translocation of RasGRP1 from the cytosol to the plasma membrane and recruitment of Grb2-Sos to phosLAT leading to activation of ras/raf/ERK cascade at the pm
121
In positive selection is there affects on Grb2-SOS localisation?
What happens?
No. Positive selection induce recruitment and activation of RasGRP1, ras, rad and ERK at the Golgi
122
Retention of ERK at the plasma membrane may lead to?
Phosphorylated JNK and other effector molecules to initiate negative selection successfully
123
How does TCR regulate the choice of CD4 Vs CD8?
Controlling expression of two transcription factors thPOK and runx3
124
In mice lacking ThPOK what happens?
MHC II restricted thymocytes are redirected to the CD8 lineage
125
When is ThPOK expressed?
Not in double positive thymocytes but strong TCR signalling at the CD4hiCD8low stage of development induces its expression
126
How does thPOK influence runx3
ThPOK repressed e press ion of Runx3. The expression of ThPOK and absence of runx3 leads to CD4 commitment and the ability to express cytokines genes typical of cd4 cells
127
If TCR signalling is insufficient dying positive selection what happens to ThPOK?
It is not induced and runx3 is expressed leading to silencing of CD4 expression and reexpression of Cd8 and the ability to express genes typical of CD8 T cells and encode proteins involved in target cell killing.
128
Commitment to treg lineage at CD4+CD8low stage of development is mediated by what?
FoxP3 and TCR SIGNALLING THAT IS STRONGER THAN INDUCING CONVENTIONAL CD4 commitment but not strong enough to induce deletion.
129
What surface proteins do treg cells express?
CD25 and CTLA4
130
What do the TCRs of future Treg cells recognise in the thymus?
Self antigens triggering expression of FoxP3
