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Cellular and Molecular Immunology > 17. Peripheral Tolerance 2 > Flashcards

17. Peripheral Tolerance 2 Flashcards

1
Q

What is present in all of us?

A

Self-reactive T cells that have the potential to cause autoimmunity in the periphery.

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2
Q

What are the main mechanisms of peripheral tolerance?

A
  1. Ignorance
  2. Activation induced cell death
  3. Anergy
  4. Negative co-stimulation
  5. Tregs
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3
Q

What are the 4 main types of T regulatory cells?

A
  1. Thymically derived Foxp3+ Treg.
  2. Peripherally induced Foxp3+ Treg.
  3. Foxp3+ Treg induced in vitro.
  4. Foxp3- Treg.
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4
Q

What T cell subset are normally Tregs?

A

CD4+ T cells

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5
Q

Can other T cell subtypes be Tregs?

A
  1. Yes
  2. Mostly CD4+ T cells
  3. There are lots of different types like CD8+ Tregs
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6
Q

What is the function of Tregs?

A
  1. To regulate and suppress the immune response.
  2. Really important for maintaining peripheral tolerance.
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7
Q

How are thymically derived tTreg selected in the thymus?

A
  1. Using the affinity model
  2. Any T cells with high affinity for self antigens are negatively selected.
  3. Low to intermediate affinity T cells for self antigens are positively selected.
  4. Some T cells are released from the thymus despite having a high affinity for self antigens.
  5. Their affinity is not high enough to be negatively selected but it is over the normal threshold.
  6. These become tTreg cells.
  7. We are not sure of the mechanisms of this.
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8
Q

Apart from affinity, what else is required for tTreg devlopment?

A
  1. Interactions with the thymic epithelium in the cortex and the medulla.
  2. Co-stimulation from CD80/86.
  3. IL-2
  4. IL-7 and IL-15 when IL-2 is also lost.
  5. Common gamma chain in the cytokine receptor.
    (all these cytokines inc IL-2 use the gamma chain in their receptor)
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9
Q

What experimental evidence was used to prove tTregs contribute to peripheral tolerance?

A
  1. Take the thymus out of a neonatal mouse.
  2. These mice develop autoimmunity and T cell mediated tissue inflammation.
  3. So there is something in the thymus that maintains tolerance.
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10
Q

What happens to neonatal thymectomised mice when you add CD4+CD25+ T cells?

A
  1. The previous autoimmunity is prevented.
  2. This shows some cells in the CD4+CD25+ T cell population can regulate autoimmunity.
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11
Q

Why is CD25 not a marker for Treg cells?

A
  1. CD25 is always expressed on tTregs.
  2. But it is transiently expressed on other T cells.
  3. So it is not exclusive to Tregs
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12
Q

What is IPEX?

A
  1. Immune dysregulation, polyendocrinopathy enteropathy X-linked syndrome.
  2. A rare lymphoproliferative immune-mediated disorder.
  3. They have severe dysregulation of the immune system and T cell mediated inflammation due to failures in peripheral tolerance.
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13
Q

What does IPEX cause?

A
  1. Diabetes
  2. Thyroiditis
  3. Haemolytic anaemia
  4. Hyper IgE syndrome
  5. Dermatitis
  6. Splenomegaly
  7. Cytokine storm
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14
Q

What causes IPEX?

A

A mutation in Foxp3.

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15
Q

What are scurfy mice?

A
  1. mice with a Foxp3 mutation.
  2. Produce small sick mice that normally die within about 3-5 weeks.
  3. They have hyporesponsive T cells and multi-organ inflammation.
  4. There is a massive infiltration of inflammatory cells in the organs.
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16
Q

What do scurfy mice prove?

A

Foxp3 is essential for maintaining tolerance.

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17
Q

What happens if you experimentally knock Foxp3 out in a Treg?

A

It stops being a Treg.

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18
Q

What is Foxp3?

A
  1. The master regulator of Treg cells.
  2. It is a transcription factor required for tTreg development, maintenance and function.
  3. Regulates around 1400 different genes in CD4+ T cells. Both positive and negative regulation.
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19
Q

How does Foxp3 interact with TCR interactions?

A
  1. tTregs are selected through high affinity TCR interaction with self antigens.
  2. Foxp3 cooperates with and reinforces the gene expression patterns triggered in the thymus when they are selected to be tTregs.
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20
Q

What does Foxp3 do?

A
  1. It represses the production of pro-inflammation cytokines. This is vital as tTregs have Self-reactive TCR. They also don’t make IL-2.
  2. Foxp3 controls the expression of Treg associated genes like CTLA-4 and IL-10.
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21
Q

What happens in Tregs without Foxp3?

A
  1. tTregs have the potential to cause autoimmunity.
  2. Without Toxp3 self-reactive T cells precursors leave the thymus and can contribute to peripheral inflammation.
  3. In Foxp3 deficient mice activated T cells have same TCR as normally expressed by tTregs.
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22
Q

How does Foxp3 act as a marker for Tregs?

A
  1. Foxp3 enriches cells with suppressive capacity from the CD25+ population
  2. In Tregs it is stably expressed constantly.
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23
Q

What cells is Foxp3 transiently expressed in?

A
  1. Foxp3 is transiently expressed at low levels by activated CD4+ T cells.
  2. However these cells don’t have the Treg phenotype or function.
  3. Something is needed to stabilise the Treg phenotype.
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24
Q

What is needed to stabilise the Treg phenotype?

A

Epigenetic changes

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25
Q

How is Foxp3 expression induced and stabilised in tTregs?

A
  1. When the TCR is engaged by a self antigen the transcription factor REL enters the nucleus and binds CNS3 and the Foxp3 promoter.
  2. This activates Foxp3 expression.
  3. However Foxp3 expression is only stable if CNS2 is demethylated by IL-2 signalling.
  4. Foxp3 and RUNX1 binds to demethylated CNS2 which stabilises Foxp3 expression.
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26
Q

What is the make up of the Foxp3 locus?

A
  1. 3 conserved non-coding DNA sequences (CNS) that regulate expression.
  2. CNS1, CNS2 and CNS3
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27
Q

How is CNS2 demethylated?

A
  1. Through IL-2 signalling.
  2. This activates STAT5.
  3. Phosphorylated STAT5 and other transcription factors are then recruited to CNS2.
  4. This causes demethylation.
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28
Q

What epigenetic changes occur in the Foxp3 locus?

A
  1. Demethylation at CNS1 and CNS2 that allows transcription factors to bind.
  2. Histone modification at histone 3 lysine 4 trimethylation at CNS2.
  3. Causes heritable stable active Foxp3 locus and a stable Treg phenotype
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29
Q

What is tTreg differentiation dependant on?

A
  1. TCR signalling
  2. Foxp3 to maintain the phenotype
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30
Q

How was TCR engagement in tTreg cells in the periphery experimentally discovered?

A
  1. An inducible conditional knock-out mouse.
  2. This had a deletion in TCR alpha chain in Foxp3+ cells only.
  3. This deletion is inducible to allow for normal Treg differentiation in the thymus and then deletion of the TCR in the periphery.
  4. This resulted in the loss of activated tTreg cells and loss of capacity to suppress the immune response
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31
Q

How is TCR stimulation essential for Treg function?

A
  1. Around 136 genes in Tregs need continuous triggering of the TCR.
  2. These genes are essential for Treg function and phenotype.
  3. This includes negative co-stimulation molecules like CLTA-4 and LAG-3 and immunosuppressive cytokines like IL-10 and IL-35.
32
Q

What 2 things in tTreg development rely on TCR signalling?

A
  1. Differentiation of tTregs in the thymus
  2. Maintenance of the tTreg phenotype in the periphery.
33
Q

What mechanisms do Foxp3+ Treg cells use to suppress other cells?

A

Many different ones but there are 4 key mechanisms:
1. Inhibitory cytokines
2. Cytolysis
3. Metabolic disruption
4. Changing dendritic cell maturation or function.

34
Q

What needs to be remembered about Foxp3+ Treg mechanisms?

A

The precise mechanism of action depends on the context of the immune response.

35
Q

How do Foxp3+ Tregs use inhibitory cytokines to regulate immune cells?

A
  1. They make inhibitory cytokines that down regulates effector responses in other immune cells.
  2. Can be secreted or membrane-bound
  3. We don’t really know whether they are contact dependant or independent or both.
  4. Includes TGFß, IL-10, IL-35.
36
Q

How do Foxp3+ Tregs use cytolysis to regulate immune cells?

A
  1. They can secrete granzyme A or B.
  2. Produce perforin mediated pores.
  3. This can kill other immune effector cells.
37
Q

How do Foxp3+ Tregs use metabolic disruption to regulate immune cells?

A
  1. They can use many mechanisms.
  2. CD25 can enrich Treg populations as it is constitutively expressed.
  3. This means Tregs are cytokine sinks and prevent IL-2 binding to other T cells so they die from lack of IL-2 stimulation.
  4. Ectoenzymes like CD39 and CD73 that cleave extracellular substrates that make pericellular adenosine.
38
Q

What is CD25?

A
  1. The alpha chain of the IL-2 receptor.
  2. This is a very high affinity receptor.
  3. CD25 is always expressed on Tregs.
39
Q

How is pericellular adenosine immune suppressive?

A
  1. It binds to the adenosine 2a receptor on other T cells which is immune suppressive.
  2. Adenosine promoters tolerogenic phenotype of dendritic cells.
  3. Upregualtes Cyclic AMP
  4. cAMP can transit through gap junctions. It is a potent inhibitory secondary messenger
40
Q

How do Foxp3+ Tregs change dendritic cell maturation or function to regulate immune cells?

A
  1. Changing DC function indirectly changes T cell responses.
  2. Tregs express CTLA-4 which competes with CD28 on T cells for CD80/86 preventing co-stimulation of T cells.
  3. CLTA-4 binding can trigger production of IDO by the APC.
  4. LAG-3 engagement with MHC2 on the DC can inhibit the maturation of the DC.
  5. LAG-3 can also down regulate CD80/86 expression to prevent co-stimulation.
41
Q

What does CTLA-4 deficiency result in?

A

Autoimmunity

42
Q

What is IDO?

A
  1. Indoleamine 2,3-dioxygenase
  2. Produces by APCs on Treg interaction.
  3. IDO catalyses tryptophan break down in the environment.
  4. Tryptophan is essential for T cell growth so this suppresses T cell growth.
43
Q

How do all these mechanisms in tTregs work together to maintain peripheral tolerance?

A
  1. The relative contribution of these mechanisms is likely to depend on context.
  2. The environment
  3. The cell types it is targeting
44
Q

Where else can Foxp3+ Treg be induced?

A

In the periphery

45
Q

What do peripherally induced Foxp3+ Tregs (pTreg) do?

A
  1. Maintain immune homeostasis and tolerance.
  2. Normally recognise non-self antigens.
46
Q

How are pTregs induced?

A
  1. Regular Foxp3- T cells are released from the thymus.
  2. most become normal T cells some will differentiate into Foxp3+ Tregs.
  3. These T cells encounter their high affinity antigens in the periphery and engage their TCR.
  4. They engage the TCR with high affinity and a lack of co-stimulation.
  5. This causes the induction of Foxp3+ pTregs.
47
Q

What antigens do pTregs normally recognise?

A
  1. Intestinal antigens
  2. Food antigens
  3. Commensal microorganism antigens.
  4. mostly not self antigens but not harmful.
48
Q

What conditions do pTregs arise in?

A
  1. When there is chronic antigen exposure.
  2. In homeostatic, non-inflammatory conditions.
49
Q

How is pTreg Foxp3 expression indued and stabilise?

A
  1. TCR stimulation causes REL to bind to CNS3.
  2. There is signalling from TGFß and a heterodimer of retinoic acid receptor and retinoid X receptor from TGFß and retinoic acid.
  3. TGFß causes SMAD 3 to bind to CNS1.
  4. The heterodimer of retinoic acid receptor and retinoid X receptor and retinoic acid binds to CNS1.
  5. CNS1 is essential for pTreg development and Foxp3 expression.
  6. IL-2 and STAT5 signalling causes CNS2 demethylation.
  7. Foxp3 and RUNX1 bind to demethylated CNS2 stabilising Foxp3 expression and pTreg phenotype.
50
Q

How can Foxp3+ Tregs be induced in vitro?

A
  1. Stimulate TCR of naive CD4+ T cells with TGFß and IL-2.
  2. This makes iTregs
  3. Then transfer these Foxp3+ iTregs made in vitro can rescue scurfy mice from lethal multi-organ inflammation.
  4. They helps autoimmunity but they have limitations
51
Q

Why are iTregs not currently a viable clinical therapy?

A
  1. TGFß induces the histone modifications at CNS1 which allows for Foxp3 transcription.
  2. However CNS2 remains methylated so Foxp3 expression is not stable.
  3. iTregs switch back to normal T cell phenotypes after some time in vivo and TGFß signalling is lost.
52
Q

What do IL-10 Foxp3- Tregs rely on?

A

IL-10 production

53
Q

What is IL-10?

A

A potent anti-inflammatory cytokine which can prevent inflammation and autoimmune pathology by limiting the immune response.

54
Q

What do IL-10 knock out mice show?

A
  1. Prolonged and exaggerated immune responses to antigen stimulation.
  2. Inflammation and tissue damage
  3. Especially to the gut flora causing colitis.
55
Q

What cells can express IL-10?

A

innate and adaptive immune cells, including CD4+ subsets.

56
Q

How can IL-10 secreting Tregs differentiate in vitro?

A
  1. IL-10
  2. IL-27
  3. IL-10 secreting DC
  4. Galectin 1 treated DC
  5. Vitamin D3 and dexamethasone
  6. CD46 ligation.
57
Q

What is another name for IL10 secreting Fosp3- Tregs?

A

T regulatory 1 cells

58
Q

What are the hallmarks of IL10 secreting Fosp3- Tregs?

A
  1. They secreted IL-10
  2. Anergic
  3. Immunosuppressive
  4. Secrete some IFNy
59
Q

Why are IL10 secreting Fosp3- Tregs harder to recognise then other Tregs?

A
  1. Several different IL-10 secreting phenotypes are described
  2. There is no master transcription factor or cell marker for them
60
Q

What stimulates IL10 secreting Fosp3- Treg differentiation in vivo?

A
  1. Chronic antigen exposure
61
Q

What are IL10 secreting Fosp3- Tregs good at?

A
  1. Good at limiting inflammatory responses to protect the body during microbial infection.
  2. Like M. tuberculosis
62
Q

How did studying bee keepers show how chronic antigen exposure induces IL-10 secreting Tregs?

A
  1. The bee keepers are chronically exposed to bee venom and their immune response to it was monitored.
  2. Quite quickly the proliferative response of the CD4+ T cells stops and anergy is induced.
  3. This anergy is maintained for most of the season.
  4. As the string reduce the cells start to proliferate again.
  5. There is a shift in cytokine production to increase IL-10 production and reduce IFN production.
63
Q

How could we use IL10 secreting Fosp3- Tregs in clinical therapies?

A
  1. To treat autoimmunity with a kind of reverse vaccine.
  2. You can use it to chronically stimulate the T cells with an antigen to turn them off.
  3. So when you encounter the antigen in real life there is limited inflammation and the immune regulatory mechanisms get triggered
64
Q

How was this kind of reverse vaccine idea proved experimentally in mice?

A
  1. Tg4 mice which 95% of the T cells are specific for myelin peptide MBP Ac1-9. This causes them to develop a condition like MS.
  2. IF the self antigen is repetitively administered the self reactive T cells can flip to being IL-10 producing Treg cells
  3. This actively prevents autoimmunity
  4. Overtime T cells lose the ability to proliferate and become anergic.
  5. They produce more IL-10 the more they are treated with self antigen this prevents autoimmunity.
65
Q

What was the results of the reverse vaccine experiment in Rag-deficient Tg4 mice?

A
  1. These mice have a RAG knockout so lack Foxp3 and spontaneously develop CNS autoimmunity.
  2. 100% of treated animals were protected by the vaccine.
  3. 100% of the control group developed autoimmunity.
  4. CD4+ T cell became anerguc
  5. IL-10 expression increases in CD4+ T cells
66
Q

How does IL-10 establish a negative feedback loop?

A
  1. IL-10 produced by Tregs in chronic antigen exposure.
  2. This acts on dendritic cells, making them more tolerogenic, so less inflammatory.
  3. This creates a negative feedback loop where the tolerogenic DC act to regulate immunity.
67
Q

What different Treg deficiencies lead to autoimmunity?

A
  1. Insufficient numbers of Tregs.
  2. Defective Treg function caused by mutation in a number of different genes.
  3. Effector cells are or becoming resistant to Tregs and continue causing autoimmunity.
68
Q

Are Treg involved in just immune tolerance?

A
  1. No
  2. They help tolerate and regulate many things.
69
Q

What scenarios are Tregs important in?

A
  1. Autoimmune disease.
  2. Allergy
  3. Tumour immunity
  4. Microbial infection
  5. Organ transplantation
  6. Fetal-maternal tolerance
  7. Immunometabolic diseases
  8. Tissue repair.
70
Q

Are Tregs always helpful?

A
  1. Treg can be good or bad depending on disease context.
  2. In autoimmunity they are good
  3. In tumour immunity, they are bad.
  4. There can be therapies that reduce or increase Treg function depending on disease context.
71
Q

Extra: What other T cells can IL10 regulate the responses of?

A
  1. Th1
  2. Th2
  3. Th17
72
Q

Extra: What is IL10 associated with in cancer?

A

Poor outcomes and poor survival

73
Q

Extra: Why are Tregs important in the intestines?

A
  1. Without them, unrestrained inflammatory responses can cause tissue damage.
  2. This happens in IBD.
  3. The immune system at mucosal surfaces needs to be tightly regulated. This is partially done by Tregs.
74
Q

Extra: Where are Foxp3+ Tregs found?

A

In every organ of the body

75
Q

Extra: What can a subset of intestinal Tregs aid?

A

Production of IgA at mucosal surfaces by Tfh cells.

Cellular and Molecular Immunology (17 decks)

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