Lecture 3 - Tolerance, T cells Flashcards
What are the mechanisms of central (thymic) tolerance?
- Deletion of self-reactive T cells
* Selection of Tregs
Compare the locations of central and periphery T cell tolerance
Central: thymus
Peripheral: outside the thymus
Compare the location of positive and negative selection in thymic tolerance
Positive: Cortex
Negative: medulla
Describe positive selection
After rearrangement of the TCR, the thymocyte interacts with cortical epithelial cells: TCR - MHC
If the interaction is weak (intermediate), the thymocyte receives a positive signal
This positive signal is required for the survival of the thymocyte
If the interaction doesn’t occur, the thymocyte receives no positive signal, and dies by neglect
Describe negative selection
Thymocyte interacts with medullary epithelial cell or DCs:
MHC:self-peptide - TCR
Outcomes:
• Deletion:
If the interaction is strong, bim is activated in the thymocyte, leading to apoptosis
• Treg development:
If the interaction between the mTEC and the thymocyte (MHC+Ag - TCR) is moderate, it is induced to become a Treg through the upregulation of FoxP3
• Survival:
If the TCR does not recognise the self-Ag, the T cell survives
How do thymocytes commit to the Treg pathway?
Upregulation of FOXP3
How are thymocytes deleted in negative selection?
Bim is upregulated in the thymocyte, leading to the formation of the apoptosome
Describe the affinity of the TCR for a self peptide-MHC complex that results in the induction of Tregs
Moderately high
Not high enough to be deleted, but higher than ‘normal’
At what stage does negative selection occur?
Can occur at any stage as long as the antigenic signal is adequately strong
e.g. Negative selection could occur if the thymocyte interacts with too great an affinity with a cTEC in the cortex
At what stage does positive selection of thymocytes occur?
DP
What are cTEC and mTEC?
cTEC: cortical thymic epithelial cells
mTEC: medullary thymic epithelial cells
What is the significance of AIRE?
Transcription factor that turns on the transcription of genes that are expressed all over the body, importantly tissue specific antigens (e.g. pancreas, kidney etc) that are not normally expressed in the thymus
AIRE is expressed in mTEC and the antigens are presented by DCs (as well as mTECs)
In effect, the entire body is represented in the thymus
Which disease results from a mutation in AIRE?
APECED
Autoimmune endocrinopathy-candidiasis-ectodermal dystrophy
Which cells express AIRE?
mTEC
Describe mTEC maturation, and the importance of this for T cell development
Over the course of development of mTEC, AIRE is differentially expressed
AIRE is expressed in the later stages of mTEC development
Soon after AIRE expression, the mTECs die
There is a ‘constant treadmill’ of mTECs developing
List the mechanisms of peripheral tolerance of T cells
- Tregs
- Anergy
- Deletion
- Ignorance
When do T cells encounter tissue-specific self-antigen?
They do not experience it as naïve T cells, as recirculation is limited to the lymphatics, LNs, and spleen
Cannot access organs e.g. the pancreas
Describe ‘Ignorance’
T cells are present in the periphery but are unaware of self antigen
Describe how ‘Ignorance’ can lead to autoimmunity
Ignorance involves the presence of mature T cells in the periphery specific for self-Ag, but they are not activated because they do not encounter the Ag
An infection with a microbe that contains an epitope shared by this self-Ag will result in the activation of these naïve ignorant T cells
The T cells, once activated, can access the organs where the self-Ag is expressed and cause auto-immunity
Describe the lymphocytic choriomeningitis virus model for ignorance
Demonstrates ignorance
Cross between two mice:
1. Anti-GP mouse; all T cells are CD8+ and recognise LCMV GP
- GP mouse; express GP under the insulin promoter, i.e. expressed in the pancreas
Result of cross:
T cells recirculate through secondary lymphoid tissues and ignore the Ag in the pancreas
– Ignorant T cells –
Mouse then injected with virus
The virus activates ignorant cells
The T cells can now access the kidneys, and destroy the islets
Mouse dies
These T cells were not anergic, because they could respond once they did encounter their Ag, but rather they are IGNORANT
Which inhibitory molecules are up-regulated, resulting in anergy of T cells in the periphery?Describe them
CTLA4
• Inhibitory receptor on T cell
PD-1
• Inhibitory molecule
• Prevent T cells from making cytokines
Characeterise the effect of DCs on naïve T cells in the resting environment and in an infectious environment
Resting:
• “Tolerogenic”
• No co-stimulation on DC (CD80/86)
Infectious
• “Immunogenic”
• Co-stimulation present on DC (through detection of Danger)
• T cells activated and bring about a response
Describe the signals required to activate a naïve T cell
Signal 1:
• Presentation of Ag in the context of MHC by the DC
• Recognition by TCR on T cell
Signal 2:
• Costimulation
• CD80, CD86 on DC
• CD28 on T cell
Signal 3:
• Cytokines
Compare the effect on T cells of ‘tolerogenic’ and ‘immunogenic’ DCs
Tolerogenic DC: ↓ TCR signalling down-regulation ↓ IL-7R down-regulation ↑ Bim ↓ Bcl2-down-regulated ↑ PD-1 (inhibitory molecule) ↓ Effector molecules IFN-γ & granzyme B
Immunogenic DC: ↑ TCR signalling enhanced ↑ BclX(L) upregulated ↓ Bim ↑ IL-7R ↑ Increased IL-2 production ↑ Effector molecules (IFN and granzyme B)
What sort of molecule is PD-1?
Inhibitory molecule
What sort of molecules are IFN-γ and granzyme-B?
What happens to them in the ‘tolerogenic’ response?
These are effector molecules of T cells
They are poorly up-regulated in the tolerogenic response
What is the role of IL-7R?
PRO-SURVIVAL
Found on the surface of T cells
Ligation with IL-7 results in Bcl-2 (pro-survival) intracellularly
IL-7R present in naïve T cells and activated T cells
IL-7R is down-regulated in T cells in response to tolerogenic DCs
Differentiate between ‘anergy’ and ‘ignorance’
Anergy:
• T cells are present and ‘in contact’ with Ag, but are non-responsive to it
Ignorance:
• T cells are present, but do not come into contact with the Ag for which they are specific
• If they came into contact with the Ag, they would be able to respond
List the mechanisms of anergy of T cells in peripheral tolerance
- Down-regulation of TCR
- Down-regulation of TCR signalling
- Up-regulation of inhibitory molecules (CTLA4, PD-1)
Describe what happens to T cells in deletion
T cells are simply eliminated
What are RIP-mOVA mice?
Transgenic mice that express membrane bound ovalbumin under the insulin promoter (i.e. in the beta cells of the pancreatic islets)
mOVA: Membrane bound Ovalbumin
Describe what was learnt about peripheral tolerance from the RIP-mOVA mouse
- mOVA is expressed in the pancreatic islets of the mouse
- DCs take up and process the antigen (OVA)
- DCs migrate to LNs
- DC presents OVA to CD4+ and CD8+ T cells in the context of MHC class I & II (cross-presentation)
5. Response in T cells • TCR signalling down-regulation • IL-7R down-regulation • Bim up-regulation • Bcl-2 down-regulation
- Deletion of T cells
Describe the importance of Antigen dose in peripheral tolerance
Antigen dose determines whether Ignorance or deletion occurs
Experiment: Two Tg mice: 1. RIP-OVA(lo) • OVA is present in very small amounts • T cells ignorant of Ag
- RIP-OVA(hi)
• OVA present in great amounts
• T cells deleted
How long does T cell development in the thymus last?
Several weeks
Which cells are responsible for positive and negative selection?
Positive selection: cTEC
Negative selection: mTEC, DCs
How are Tregs induced in T cell development?
Thymocyte interacts with mTEC / DC with moderately high affinity
i.e. higher than ‘intermediate’, but not so high as to be deleted through negative selection
FoxP3 expression up-regulated in T cell
T cell differentiates down the Treg lineage
What are the various ways that Ag can access the thymus for presentation to developing thymocytes?
- Enters through blood (then presented by DCs)
* AIRE expression in mTECs
Which cells express AIRE?
mTEC
What is the effect of AIRE on mTECs?
In AIRE KO, there are more mTECs (lower death rates, as AIRE also regulates apoptosis)
There is still other tissue specific Ag being expressed
This indicates that there are other genes that regulate the expression of tissue spec. Ag
What is the importance of DCs in the medulla of the thymus?
DCs capture the dying mTECs, process the tissue specific Ag, and present it to thymocytes
Why do people with AIRE mutation get candida infection?
No thymic tolerance to many things, including cytokines
Inhibition of certain cytokines, such as IL-17 and IL-22 in this case, reduces capacity to respond to fungal infections
What is required of the DC for up-regulation of co-stimulatory molecules, such that it can activate T cells?
Detection of danger
Through pattern recognition receptors e.g. TLRs, NLRs etc
Characterise the following: • Bcl-2 • Bim • Bax • Bak • BclX
Bcl-2: pro-survival Bim: pro-apoptotic Bax: pro-apoptotic Bak: pro-apoptotic BclX: pro-survival
In MHC KO mice, what is the effect on naïve T cells?
Naïve T cells need weak signalling through TCR by MHC to survive in the periphery
What happens when T cells have down-regulated TCRs?
Struggle to survive / die, as it needs weak signalling through the TCR by MHC to survive
What is the role of IL-2 in T cells?
Once T cells are activated, it starts to make IL-2 which signals in an autocrine manner
Autocrine signalling of IL-2 induces Bcl-X (pro-survival)
Which T cells are liable to cause auto-immunity?
Ignorant auto-reactive T cells
When do thymocytes become DP?
After receiving success signals that the B chain of the TCR was successfully rearranged, the cells undergo proliferation, and then upregulate both CD4 and CD8
Then, the alpha chain is rearranged
Will any tissue specific Ag be presented in the medulla of the thymus in AIRE KO mice?
Yes, there are other TFs (yet to be identified) that seem to regulate the expression of tissue specific Ags in the thymus
Also, Ags can circulate in through the blood which are taken up and presented by DCs
How do DCs in the medulla of the thymus get the TSAs from the mTECs?
Expression of AIRE causes the mTECs to die
The DCs phagocytose the dead cells and process all of the Ags for presentation
Describe how anergy and deletion occur in peripheral T cell tolerance
- DCs migrate to LNs w/ Ag form periphery
- Ag presented to T cells in the absence of signal 2 (co-stimulation)
3. Tolerisation of T cells: ↓ TCR ↓ TCR signalling ↓ IL-7R ↓ Perforin and IFN-gamma ↑ PD-1 ↑ CTLA-4 ↑ bim ↓ Bcl-2
