Lecture 20 - Tregs (pt. 1) Flashcards
3 positive consequences of Tregs
- maintain and control self-reactivity
- limit allergic response
- prevent tissue damage
2 negative consequences of Tregs
- suppress anti-tumour immunity
- limit clearance of chronic infection
what must Tregs be able to balance?
must be able to limit efficiacy of immune responses and preventing damage by immune system
4 different states of Treg activity
- Normally: stable and robust
- Chronic immune condition: fatigued and short-lived
- Functional plasticity: Tregs can induce inflammation too
- Inborn errors of immunity: genetics impact Tregs
recap: what is IL2Ra?
aka CD25 –> inducible on T cells as a result of TCR signaling
- Extracellular
- High affinity for IL2
describe the CD4 T cells that were found to express IL2Ra
IL2Ra was CONSTITUTIVELY expressed
what can we conclude from the fact that IL2Ra is constitutively expressed?
the cell is active and ready to go –> must have seen its antigen at some point and is now a memory cell
what percent of CD4s have constitutive IL2Ra?
5-10%
how can we selectively deplete IL2Ra?
COMPLEMENT-MEDIATED DEPLETION:
treat with MAb against CD25 antigen with complement protein, leaving only CD25 negative cells present
What type of cells can we use complement-mediated depletion on?
Spleen cells
Describe the experimental setup to see the role of constitutive IL2Ra and the conclusion
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
What happens when spleen cells only receive the complement or Ab parts of the depletion mechanism?
nothing happens –> only depletes when combined
Overall, what is the role of IL2Ra?
maintain immune self-tolerance on activated T cells (if IL2Ra removed, lose self-tolerance)
what T cells do Tregs come from?
CD4+ T cells
how were Tregs originally described?
Tregs originally described as subset of CD4+ T cells that constitutively express IL2Ra
what is the master regulator of Treg development?
Foxp3 TF
what happens when there is a defect in Tregs?
severe, multi-organ autoimmunity
4 ways multi-organ autoimmunity can occur as a result of defective Tregs
- Day 3 thymectomy (time-dependent effect)
- IL2Ra/CD25 depletion
- Scurfy (foxp3 mutation)
- Foxp3 -/-
2 developmental/homeostatic signals required for Treg function
- TGFbeta
- co-stimulation
4 domains of foxp3
- Protein-binding domain
- Zn finger
- Leucine zipper
- Forkhead (DNA-binding domain)
significance of forkhead domain of Foxp3
allows TF to bind DNA so it can do its job
which domain is affected in Scurfy? how is it affected?
Forkhead domain is TRUNCATED
main function of Foxp3
generally acts as repressor
what types of genes does Foxp3 typically repress?
inflammatory and cytokine-expression genes
what is a consequence of Foxp3 turning off inflammatory and cytokine expression genes?
Tregs are ANERGIC
How many gene targets does foxp3 have?
targets 700 genes DIRECTLY and has indirect effect on the genes that interact with them
does foxp3 have effects by directly or indirectly affecting genes in the foxp3-dependent transcriptional programming?
mostly indirect, the directly targeted genes make up only a small part of foxp3 transcriptional program
why is autoimmunity induced by foxp3 mutation considered to be “catastrophic”?
can’t do much to fix, except give back Tregs
what does IPEX stand for?
I = immunodysregulation
P = polyendocrinopathy
E = enteropathy
X = x-linked syndrome
who does IPEX affect?
young boys
how does IPEX occur?
point mutations in foxp3, most commonly in DNA binding domain
what is the most common foxp3 mutation that causes IPEX? where is it located?
A384T –> in DNA binding domain
describe the variation of effects of foxp3 mutation
each mutation can have mild to severe effects depending on mutation location, HLA, environment, etc
2 genes that can be activated by foxp3?
- IL2Ra/CD25
- CTLA4
describe foxp3 activating IL2Ra/CD25 gene
foxp3 activates IL2Ra/CD25 to program Treg for optimal IL2 sensitivity
3 results of foxp3 activity
- Treg phenotype
- hyporesponsive/anergic
- suppressive
how does foxp3 make Tregs hyporesponsive/anergic?
turns off proliferation via turning of IL2 production (Tregs don’t make their own IL2!!)
What does foxp3 do in addition to making Tregs anergic and suppressive?
conditions cells to sense and integrate environmental signals for rapid effector response
2 types of Tregs
- natural/thymic Treg
- peripheral/post-thymic Treg
describe the development of nTreg/tTreg
at cortico-medullary junction during selection, specific antigen and cytokine environment will allow Treg to acquire Foxp3
then foxp3+ Treg can go into periphery
describe the development of iTreg/pTreg
in periphery, if conventional T cell sees antigen in context of IL10 or TGFbeta will allow Treg to acquire Foxp3
what happens if conventional T cell does not see antigen in context of IL10 or TGFbeta?
will just be normal CD4+ T cell
what % of CD4+ T cells become Tregs
1%
how do we know Tregs exist in the thymus?
day 3 thymectomy = autoimmune disorder
when in T cell development does foxp3 expression occur in thymus?
during DP to CD4+ SP transition
describe the altered negative selection of Foxp3+ cells
TCR/pMHC affinity is high enough to delete self-reactive T cells but not high enough to delete Foxp3+ Treg cells
why is it important that Tregs recognize self-antigens?
so they can mediate self-tolerance and induce Foxp3+
what are Tregs biased towards?
self antigens
what happens if TCR/pMHC affinity decreases?
Treg development is messed up
6 things that are important to induce Treg production
1.TGFbeta
2. retinoic acid (carrots)
3. immature DC
4. aryl hydrocarbon receptor
5. stress
6. Ag
why are immature DCs important for Treg production?
not strong enough to induce T cells but can still tolerize T cells
What determines the specificity of Foxp3 expression?
epigenetic modifications of Foxp3 conserved non-coding regions
what are the 3 conserved non-coding regions that can be epigenetically modified?
- FOXP3 promoter
- TGFbeta sensor (enhancer)
- TSDR
What does TSDR stand for?
Treg-cell Specific Demethylated Region
describe epigenetic modifications in Teff cells
lots of methylation, especially in TSDR
describe epigenetic modifications in iTreg
TSDR is methylated but promoter and enhancer are more acetylated to allow TF binding
describe Foxp3 expression in iTreg vs tTreg
iTreg –> short-lived and unstable
tTreg –> very stable
describe epigenetic modifications in tTreg
no methylation and lots of acetylation in all 3 regions so TFs can access DNA2
2 examples of TFs that bind tTreg foxp3
- STAT5
- SMAD3
Where does STAT5 bind? (2)
- promoter
- TSDR
describe STAT5
Downstream of IL2 signaling pathway –> since IL2Ra is constitutively expressed on Foxp3+ cells, it can constantly receive IL2 signal
where does SMAD3 bind?
enhancer
general role of TGFbeta
immunosuppressive cytokine
why is TGFbeta an immunosuppresive cytokine?
induces and maintains foxp3
3 types of cells that CD4 Foxp3+ T regs act on?
- adaptive immune cells
- non-imune cells
- innate immune cells
what type of immunity do Tregs suppress?
innate and adaptive
where in the body do Tregs act?
in lymphoid AND non-lymphoid sites
why is it important that Tregs act in non-lymphoid sites?
to suppress non-lymphoid sites of inflammation
what is required for Tregs to be active?
must have seen antigen thru TCR
are Tregs always active?
no –> must see antigen
why is it important that Tregs are not activated against all responses all the time?
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
is Treg response antigen-specific?
can be antigen-specific or non-antigen-specific
how can Treg response be non-antigen-specific?
a specific Treg response may be made to be antigen-specific, but this same response may be able to act due to other antigens
3 mechanisms that are suppressed by Tregs
- IL2 transcription in T cells
- proliferation
- differentiation/production of inflammatory cytokines
in vitro, what is required for Treg activity?
cell-cell contacts
6 mechanisms used by Tregs to suppress
- mess up adenosine metabolism of responding cells
- increase cAMP in responding cells via gap junctions
- IL2 consumption/sequestering
- secretion of anti-inflammatory cytokines: IL35, IL10, TGFbeta
- granzyme B and cytolysis
- CTLA4
What gene is activated by foxp3 as a mechanism of suppression by Treg?
CTLA4
How is CTLA4 used as a suppression molecule?
blocks B7.1/B7.2 to shut off signal 2 and block proliferation
what happens CTLA4 deficient mice?
all cells deficient in CTLA4 –> lymphoproliferative disease
what happens to mice deficient in CTLA4 only in Tregs?
only Tregs deficient in CTLA4 –> autoimmune disease
what is the most critical cytokine for Foxp3+ Tregs?
IL2!!!
Do Foxp3+ Tregs make their own IL2?
No, only get from nearby T cells (paracrine)
if Treg is suppressing activated T cells, where is the IL2 coming from?
time plays a role –> Tregs use IL2 from T cells that have been activated to suppress other T cells
what is the prediction and actual result if you block IL2 production?
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
5 ways to block IL2 production?
deficiencies in:
1. IL2
2. IL2Ra
3. CD40
4. CD28
5. B7.1, B7.2
What happens if IL2 is neutralized in neonatal mice?
no more Tregs –> T1D autoimmunity
why do we know that Tregs depend on IL2 more than activating T cells?
they constitutively express IL2Ra –> they are hungry for IL2!!!!
what is the issue with tracking Tregs in humans?
Treg markers (CD25, Foxp3) are all activation markers –> can’t use these markers to differentiate btwn Tregs and other activated T cells
