Regulatory T cells

Control of potential T cell self-reactivity: tolerance

Random generation of an immense T-cell repertoire

(~1015 different TCRs):

Great opportunity to recognize every pathogenic antigen

BUT

Potential self-reactivity

TOLERANCE:

• Central

• Peripheral

95%5%

T-cell ESTRINSIC

Regulatory T cells

Tolerization mechanisms

T-cell INTRINSIC

Clonal deletion

(apoptosis) Anergy

Ignorance

Regulatory T cells

Regulatory T cells (Tregs) represent a population of T cells that are

specialized for the suppression of the immune response;

Treg cells are essential for:

Maintaining peripheral tolerance (preventing autoimmunity)

Limiting chronic inflammatory diseases (immune homeostasis)

However, they:

Limit beneficial immunity

Limit antitumor immunity

Regulatory T cells: an old story…

Early 1970s: T cell-mediated suppression

however:

- lack of markers to distinguish suppressor T cells

- difficulties in isolating suppressor T cells for further studies

- lack of robustness of some suppressive phenomena

1995: S. Sakaguchi - Regulatory T cells

Regulatory T cells: re-discovered recently…

Regulatory T cells subsets

CD4+CD25+Foxp3+ Treg

Tr1

Th3

Other minor subsets (CD8+, CD4-CD8-, --- T cells)

CD4+CD25+ Treg: origin

thymus-derived (Natural occurring Treg or nTreg)

induced in periphery ( CONVERSION)

Tcell

APC

Medium affinity

Highaffinity

Positive selection

Apoptosis/Tregs

% Foxp3

CD4+CD25+ Tregs

- constitute 10% of circulating CD4+ pool in normal mice

- they are anergic

- once activated, they do not produce IL-2

- they proliferate less than effector T cells in vitro after ag stimulation, but in vivo they are continuosly proliferating due to self-ag recognition

- they consume IL-2

- they suppress effector function of other T cells

- once activated in an ag-specific manner, their suppression is not limited to T cells with the same specificity

CD4+CD25+Foxp3+ Treg generation in the thymus

FoxP3 is induced in thymic precursor cells upon engagement with high-affinity TCR and other costimulatory factors resulting in FOXP3+ Treg cells. Different functions associated with Treg cell differentiation and function are shown in the boxes.

Generation of Treg in the thymus:

- strong TCR engagement

- signals to common --chain containing cytokyne receptors

- CD28 costimulatory signals

CD4+CD25+ Treg: markers

CD25 (IL-2 receptor --chain)

transiently expressed in activated T cells;

constitutively expressed in Tcells with regulatory abilitites

at the steady state, 10% of peripheral CD4+ cells express CD25

“…when CD4+ cell suspensions prepared from normal mice are depleted ofCD25+ cells and are then inoculated into athymic nude mice, all recipients spontaneously developed autoimmune disease…”(S. Sakaguchi et al., 1995)

BALB/C mouse

CD4+ cells CD4+CD25- cells

Athimic Nude mouse

CD25+ celldepletion

“…when CD4+ cell suspensions prepared from normal mice are depleted of CD25+ cells and are then inoculated into athymic nude mice, all recipients spontaneously developed autoimmune disease…”(S. Sakaguchi et al., 1995)

Depletion of CD25+ cells is sufficient to eliminate T cells with regulatory activity (Treg)

Autoimmunity

“CD4+CD25+ cells contribute to maintaining self-tolerance by down-regulating immune response”

Foxp3 (Forkhead box P3)

• is located on the X chromosome

• is exclusively expressed on Tregs (vs. CD25):all the T cells which express high levels of Foxp3 are Treg

BUT

Foxp3 is not expressed in every Treg

• forkhead/winged-helix transcription factor member

• its function is fundamental in Treg differentiation

Foxp3: IPEX and scurfy mice

IPEX (immune dysregulation, polyendocrinopathy,

enteropathy, X-linked)

• fatal autoimmune disease

• rare

• X-linked (as Foxp3 gene location!)

• characterized by Foxp3 mutation and hyperresponsive CD4+ cells

Scurfy mice

The scurfy mutation can be rescued by a transgene encoding Foxp3 allele

Foxp3 in Treg development and function

Foxp3 KO mice have lower numbers of CD4+CD25+ cells

Expression of a Tg encoding Foxp3 convert naive T cells to Treg-like:- confers suppressor ability- CD25/CTLA4/GITR expression- repress IFN---IL-4/IL-2 production

Conditional deletion of Foxp3 in mature peripheral Tregs results in - loss of suppressor function- production of IL-2 and other pro-inflammatory cytokines

Control of Treg function by Foxp3Interaction with TFs

Control of Treg function by Foxp3Target genes

Foxp3 controls directly or indirectly nearly 700 genes

Foxp3 binds directly to nearly 10% of them

Among target genes:

• signal transduction genes • transcription factors (!!!!!!!) • cytokines (e.g.Il2) • cell surace molecules • enzymes for cell metabolism • miRNA

Foxp3 functions as an activator as well as a repressor of the transcription depending on the target

CTLA-4 (Cytotoxic T lymphocyte antigen)

• CD28-family receptor

• It binds the same ligands as CD28 (CD80 and

CD86)

• higher affinity than CD28

• T cell inibitory receptor ----CTLA4 KO die

prematurely for multiorgan infalmmation

• Constitutively expressed (vs. transient expression in other T)

• CTLA4 expression is controlled by Foxp3

In nTregs:

• required for their in vivo and in vitro suppression, (down-regulation of CD80 and CD86 on APC)

GITR (glucocorticoid-induced tumor necrosis factor receptor)

• Expressed in activated T cells or in Tregs

• role of GITR in attenuating the suppressive activity of CD4+ CD25+ T cells

IL-2 and Tregs

Mechanisms of suppression

Mechanisms of suppression: inhibitory cytokines

TGF-- and IL-10:

- fundamental for iTreg mediated suppression

- Their contribution to nTreg mediated suppression is debated

IL-35:

- Recently discovered in mice- Not produced by human Tregs

Mechanisms of suppression: cytotoxicity

Cell-contact dependent mechanism

Mechanisms of suppression: metabolic disruption

Competition for cytokines (es IL-2 deprivation)

Delivery of a negative signal to Teff: - upregulation of cAMP- -T cell proliferation and IL-2 production- generation of pericellular adenosine- -T cell function

Cell-contact dependent mechanism (or close proximity)

Mechanisms of suppression: functional modification of APC

Repression of APC function/ maturation:

- - CD80/CD86 expression via CTLA4

- indoleamine 2,3-dioxygenase (IDO) production

- LAG-3 dependent block of maturation

Relative contribution of different mechanisms of suppression

Hyp 1: operate synergistically and sequentially

Hyp 2: different mechanism for different scenario (contextual model)

Hyp 3: one/few critial and many accessory mechanism (hierarchical model)

Induced or adaptive regulatory T cells (iTregs)

• Foxp3+ iTregs

• Tr1

• Th3

Role of Treg in infectious disease

• limit the magnitude of effector response (advantage/disadvantage for the host)

• limit collateral tissue damage (advantage)

Treg function

Effector response

Outcome of an infection

Infection activates both Tregs and effector function

Nature Immunology 6, 353 - 360 (2005)

Ovarian tumors are infiltrated by both effector (CD4+CD25-) T cells and by an excess of TREG cells (CD4+CD25+). These TREG cells impede the function of the effectors in combating the tumor. The chemokine CCL22, produced by the tumor cells, binds to its receptor (CCR4) on the TREG cells and mediates their recruitment into the tumor.

Role of Tregs in cancer

Treg suppress host immune responsepreventing autoimmunity

Limit anti-tumor immunity