Regulatory T cells

Control of potential T cell self-reactivity: tolerance Random generation of an immense T-cell repertoire (~ 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 High affinity 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 of CD25 + 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 + cell depletion

“…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 Foxp3 Interaction with TFs

Control of Treg function by Foxp3 Target 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, (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 response preventing autoimmunity Limit anti-tumor immunity