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MIMM 314 > Lecture 20 - Tregs (pt. 1) > Flashcards

Lecture 20 - Tregs (pt. 1) Flashcards

1
Q

3 positive consequences of Tregs

A
  1. maintain and control self-reactivity
  2. limit allergic response
  3. prevent tissue damage
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2
Q

2 negative consequences of Tregs

A
  1. suppress anti-tumour immunity
  2. limit clearance of chronic infection
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3
Q

what must Tregs be able to balance?

A

must be able to limit efficiacy of immune responses and preventing damage by immune system

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4
Q

4 different states of Treg activity

A
  1. Normally: stable and robust
  2. Chronic immune condition: fatigued and short-lived
  3. Functional plasticity: Tregs can induce inflammation too
  4. Inborn errors of immunity: genetics impact Tregs
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5
Q

recap: what is IL2Ra?

A

aka CD25 –> inducible on T cells as a result of TCR signaling
- Extracellular
- High affinity for IL2

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6
Q

describe the CD4 T cells that were found to express IL2Ra

A

IL2Ra was CONSTITUTIVELY expressed

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7
Q

what can we conclude from the fact that IL2Ra is constitutively expressed?

A

the cell is active and ready to go –> must have seen its antigen at some point and is now a memory cell

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8
Q

what percent of CD4s have constitutive IL2Ra?

A

5-10%

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9
Q

how can we selectively deplete IL2Ra?

A

COMPLEMENT-MEDIATED DEPLETION:
treat with MAb against CD25 antigen with complement protein, leaving only CD25 negative cells present

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9
Q

What type of cells can we use complement-mediated depletion on?

A

Spleen cells

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10
Q

Describe the experimental setup to see the role of constitutive IL2Ra and the conclusion

A

Transfer complement-mediated depleted spleen cells into immunodeficient mice:
- autoimmune disease in multiple organs

Normally, giving spleen cells to immunodeficient mice should replenish their immune cells but without IL2Ra they are not protected from self-reactivity –> induces autoimmune disease

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11
Q

What happens when spleen cells only receive the complement or Ab parts of the depletion mechanism?

A

nothing happens –> only depletes when combined

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12
Q

Overall, what is the role of IL2Ra?

A

maintain immune self-tolerance on activated T cells (if IL2Ra removed, lose self-tolerance)

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13
Q

what T cells do Tregs come from?

A

CD4+ T cells

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14
Q

how were Tregs originally described?

A

Tregs originally described as subset of CD4+ T cells that constitutively express IL2Ra

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15
Q

what is the master regulator of Treg development?

A

Foxp3 TF

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16
Q

what happens when there is a defect in Tregs?

A

severe, multi-organ autoimmunity

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17
Q

4 ways multi-organ autoimmunity can occur as a result of defective Tregs

A
  1. Day 3 thymectomy (time-dependent effect)
  2. IL2Ra/CD25 depletion
  3. Scurfy (foxp3 mutation)
  4. Foxp3 -/-
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18
Q

2 developmental/homeostatic signals required for Treg function

A
  1. TGFbeta
  2. co-stimulation
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19
Q

4 domains of foxp3

A
  1. Protein-binding domain
  2. Zn finger
  3. Leucine zipper
  4. Forkhead (DNA-binding domain)
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20
Q

significance of forkhead domain of Foxp3

A

allows TF to bind DNA so it can do its job

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21
Q

which domain is affected in Scurfy? how is it affected?

A

Forkhead domain is TRUNCATED

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22
Q

main function of Foxp3

A

generally acts as repressor

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23
Q

what types of genes does Foxp3 typically repress?

A

inflammatory and cytokine-expression genes

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24
Q

what is a consequence of Foxp3 turning off inflammatory and cytokine expression genes?

A

Tregs are ANERGIC

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25
Q

How many gene targets does foxp3 have?

A

targets 700 genes DIRECTLY and has indirect effect on the genes that interact with them

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26
Q

does foxp3 have effects by directly or indirectly affecting genes in the foxp3-dependent transcriptional programming?

A

mostly indirect, the directly targeted genes make up only a small part of foxp3 transcriptional program

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27
Q

why is autoimmunity induced by foxp3 mutation considered to be “catastrophic”?

A

can’t do much to fix, except give back Tregs

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28
Q

what does IPEX stand for?

A

I = immunodysregulation
P = polyendocrinopathy
E = enteropathy
X = x-linked syndrome

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29
Q

who does IPEX affect?

A

young boys

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30
Q

how does IPEX occur?

A

point mutations in foxp3, most commonly in DNA binding domain

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31
Q

what is the most common foxp3 mutation that causes IPEX? where is it located?

A

A384T –> in DNA binding domain

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32
Q

describe the variation of effects of foxp3 mutation

A

each mutation can have mild to severe effects depending on mutation location, HLA, environment, etc

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33
Q

2 genes that can be activated by foxp3?

A
  1. IL2Ra/CD25
  2. CTLA4
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34
Q

describe foxp3 activating IL2Ra/CD25 gene

A

foxp3 activates IL2Ra/CD25 to program Treg for optimal IL2 sensitivity

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35
Q

3 results of foxp3 activity

A
  1. Treg phenotype
  2. hyporesponsive/anergic
  3. suppressive
36
Q

how does foxp3 make Tregs hyporesponsive/anergic?

A

turns off proliferation via turning of IL2 production (Tregs don’t make their own IL2!!)

37
Q

What does foxp3 do in addition to making Tregs anergic and suppressive?

A

conditions cells to sense and integrate environmental signals for rapid effector response

38
Q

2 types of Tregs

A
  1. natural/thymic Treg
  2. peripheral/post-thymic Treg
39
Q

describe the development of nTreg/tTreg

A

at cortico-medullary junction during selection, specific antigen and cytokine environment will allow Treg to acquire Foxp3

then foxp3+ Treg can go into periphery

40
Q

describe the development of iTreg/pTreg

A

in periphery, if conventional T cell sees antigen in context of IL10 or TGFbeta will allow Treg to acquire Foxp3

41
Q

what happens if conventional T cell does not see antigen in context of IL10 or TGFbeta?

A

will just be normal CD4+ T cell

42
Q

what % of CD4+ T cells become Tregs

A

1%

43
Q

how do we know Tregs exist in the thymus?

A

day 3 thymectomy = autoimmune disorder

44
Q

when in T cell development does foxp3 expression occur in thymus?

A

during DP to CD4+ SP transition

45
Q

describe the altered negative selection of Foxp3+ cells

A

TCR/pMHC affinity is high enough to delete self-reactive T cells but not high enough to delete Foxp3+ Treg cells

46
Q

why is it important that Tregs recognize self-antigens?

A

so they can mediate self-tolerance and induce Foxp3+

47
Q

what are Tregs biased towards?

A

self antigens

48
Q

what happens if TCR/pMHC affinity decreases?

A

Treg development is messed up

49
Q

6 things that are important to induce Treg production

A

1.TGFbeta
2. retinoic acid (carrots)
3. immature DC
4. aryl hydrocarbon receptor
5. stress
6. Ag

50
Q

why are immature DCs important for Treg production?

A

not strong enough to induce T cells but can still tolerize T cells

51
Q

What determines the specificity of Foxp3 expression?

A

epigenetic modifications of Foxp3 conserved non-coding regions

52
Q

what are the 3 conserved non-coding regions that can be epigenetically modified?

A
  1. FOXP3 promoter
  2. TGFbeta sensor (enhancer)
  3. TSDR
53
Q

What does TSDR stand for?

A

Treg-cell Specific Demethylated Region

54
Q

describe epigenetic modifications in Teff cells

A

lots of methylation, especially in TSDR

55
Q

describe epigenetic modifications in iTreg

A

TSDR is methylated but promoter and enhancer are more acetylated to allow TF binding

56
Q

describe Foxp3 expression in iTreg vs tTreg

A

iTreg –> short-lived and unstable
tTreg –> very stable

57
Q

describe epigenetic modifications in tTreg

A

no methylation and lots of acetylation in all 3 regions so TFs can access DNA2

58
Q

2 examples of TFs that bind tTreg foxp3

A
  1. STAT5
  2. SMAD3
59
Q

Where does STAT5 bind? (2)

A
  1. promoter
  2. TSDR
60
Q

describe STAT5

A

Downstream of IL2 signaling pathway –> since IL2Ra is constitutively expressed on Foxp3+ cells, it can constantly receive IL2 signal

61
Q

where does SMAD3 bind?

A

enhancer

62
Q

general role of TGFbeta

A

immunosuppressive cytokine

63
Q

why is TGFbeta an immunosuppresive cytokine?

A

induces and maintains foxp3

64
Q

3 types of cells that CD4 Foxp3+ T regs act on?

A
  1. adaptive immune cells
  2. non-imune cells
  3. innate immune cells
65
Q

what type of immunity do Tregs suppress?

A

innate and adaptive

66
Q

where in the body do Tregs act?

A

in lymphoid AND non-lymphoid sites

67
Q

why is it important that Tregs act in non-lymphoid sites?

A

to suppress non-lymphoid sites of inflammation

68
Q

what is required for Tregs to be active?

A

must have seen antigen thru TCR

69
Q

are Tregs always active?

A

no –> must see antigen

70
Q

why is it important that Tregs are not activated against all responses all the time?

A

don’t want Tregs to be activated all the time and suppress all responses –> activation by antigen allows for control so only will suppress responses that trigger immunity

71
Q

is Treg response antigen-specific?

A

can be antigen-specific or non-antigen-specific

72
Q

how can Treg response be non-antigen-specific?

A

a specific Treg response may be made to be antigen-specific, but this same response may be able to act due to other antigens

73
Q

3 mechanisms that are suppressed by Tregs

A
  1. IL2 transcription in T cells
  2. proliferation
  3. differentiation/production of inflammatory cytokines
74
Q

in vitro, what is required for Treg activity?

A

cell-cell contacts

75
Q

6 mechanisms used by Tregs to suppress

A
  1. mess up adenosine metabolism of responding cells
  2. increase cAMP in responding cells via gap junctions
  3. IL2 consumption/sequestering
  4. secretion of anti-inflammatory cytokines: IL35, IL10, TGFbeta
  5. granzyme B and cytolysis
  6. CTLA4
76
Q

What gene is activated by foxp3 as a mechanism of suppression by Treg?

A

CTLA4

77
Q

How is CTLA4 used as a suppression molecule?

A

blocks B7.1/B7.2 to shut off signal 2 and block proliferation

78
Q

what happens CTLA4 deficient mice?

A

all cells deficient in CTLA4 –> lymphoproliferative disease

79
Q

what happens to mice deficient in CTLA4 only in Tregs?

A

only Tregs deficient in CTLA4 –> autoimmune disease

80
Q

what is the most critical cytokine for Foxp3+ Tregs?

A

IL2!!!

81
Q

Do Foxp3+ Tregs make their own IL2?

A

No, only get from nearby T cells (paracrine)

82
Q

if Treg is suppressing activated T cells, where is the IL2 coming from?

A

time plays a role –> Tregs use IL2 from T cells that have been activated to suppress other T cells

83
Q

what is the prediction and actual result if you block IL2 production?

A

predict: no T cell activation

actual: Treg pathways depend on IL2 even more than activating T cells so Tregs will be lost and self-reactive T cells can still be activated to induce autoimmunity

84
Q

5 ways to block IL2 production?

A

deficiencies in:
1. IL2
2. IL2Ra
3. CD40
4. CD28
5. B7.1, B7.2

85
Q

What happens if IL2 is neutralized in neonatal mice?

A

no more Tregs –> T1D autoimmunity

86
Q

why do we know that Tregs depend on IL2 more than activating T cells?

A

they constitutively express IL2Ra –> they are hungry for IL2!!!!

87
Q

what is the issue with tracking Tregs in humans?

A

Treg markers (CD25, Foxp3) are all activation markers –> can’t use these markers to differentiate btwn Tregs and other activated T cells

MIMM 314 (23 decks)

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