Regulatory Lymphocytes of the Immune System. Dr. C. Piccirillo Canada Research Chair Department of Microbiology & Immunology McGill University MIMM-414A Lecture 2- Oct. 23, 2006

Induced regulatory T cells Peripherally-induced Thymic CD4+ T cell pool Peripherally-induced CD4+ Treg cells ( iTreg ) TCR + Peripheral differentiation signals APC _ Activated Effector T cell Autoimmunity Transplantation Tumor Immunity Infectious disease Piccirillo et al. Trends in Immunol. 2004.

A network of CD4+ regulatory T cells control immune reponses. A. INDUCED - immunization, experimental manipulation… NK-T cells CD8+ T cells  T cells DN T cells CD4+ Th1 cell-mediated cross-regulation of Th2 cells (IL-4 <-> IFN) Th3 - Induced by oral tolerance TGF-1 producing (IL-10) and TGF-1 dependent effect Tr1 - Induced by Ag + IL-10 and IL-10 dependent effect

B. NATURALLY-OCCURRING Peripherally-induced Endogenous in naïve repertoire: professional Thymic CD4+ T cell pool Thymically-derived naturally-occurring CD4+CD25+ Treg cells (nTreg ) Peripherally-induced CD4+ Treg cells ( iTreg ) Foxp3+ GITR+ CTLA-4+ CD25+ TCR TCR CD25 GITR CTLA-4 Foxp3 + Peripheral differentiation signals APC _ _ Activated Effector T cell Autoimmunity Transplantation Tumor Immunity Infectious disease Piccirillo et al. Trends in Immunol. 2004.

Lymph nodes from a 2 month old BALB/c Journal of Immunology 1995 155:1151 Lymph nodes from a 2 month old BALB/c

Naturally-occurring CD4+ regulatory T cells. Autoimmunity Dominant self-tolerance Unique lineage of CD25+ (IL-2R) T cells 1-10% of thymic or peripheral CD4+ T cells In resting, endogenous T cell repertoire Rodents, human and non-human primates Allergy Infectious disease CD4+CD25+ Treg cells Tissue regeneration Transplantation Mucosal Immunity Depletion Day3 thymectomy Genetic anomalies Irradiation Infections Drugs Gastritis Oophritis Orchitis Thyroiditis Pancreatitis Colitis Auto Abs Normal Immune responses Tumor Immunity

CD4+CD25+ nTreg cells Masterswitch of peripheral tolerance Immunity

Reliable biomarkers for CD4+CD25+ regulatory T cells? GITR CTLA-4 CD4+CD25+ Regulatory T cell NO marker after T cell activation. Only in the naïve T cell repertoire. Need better marker. Galectin-1 CD103 Ly6 4-1BB Neuropilin-1 Unidentified X,Y,Z ?? OX-40 Piccirillo et al. Trends in Immunology July 2004

T cell activation induces expression of functional T cell surface molecules Activated CD4+ T cell MHC/peptide CD40L TCR TCR CD25 Resting CD4+ T cell APC CD69 CD44 CD25 is not a marker of Treg after T cell activation. Only in the naïve T cell repertoire.

Phenotypic and functional characteristics Treg cells are partially-activated cells Treg cells are anergic. Hyporesponsive to TCR stimulation : restored by IL-2. Do not produce IL-2, IL-4, IFN-, IL-10 (), TGF1 ()

CD4+CD25+ regulatory T cells suppress T cell functions CD4+CD25+ regulatory T cells suppress T cell functions. Consensus elements 24h CD8+ Proliferation Cytokine Production CD8+/- CD8+/+ CD4+ or CD8+ T cells CPM 48h # CD4+CD25- (  ) or CD4+CD25+ ( ) T cells Requirement for TCR engagement Antigen non-specific & MHC unrestricted Induce cell cycle arrest (G1-S transition) (ng/ml) IFN- g IFN- CD8+ CD8+/- CD8+/+ CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production Angela M. Thornton and Ethan M. Shevach J. Exp. Med. 1998 188: 287-296.

Cellular origin of CD4+ regulatory T cells CD4+ Treg ? Endogenous Naturally-occurring Induced during Immune response

TReg cells are a normal product of thymic selection. Figure 1. TR cells may arise from relatively high-avidity interactions with self-peptide–MHC complexes, just below the threshold for negative selection (green area). This narrow avidity selection window ensures that Tr cells will constitute only a small fraction of the mature T cell pool and have a greater sensitivity to self-peptide–MHC than potentially pathogenic autoreactive T cells. Thymic development and TCR specificity: Unknown Diverse T cell repertoire Self-specific Cross-reactive to foreign? Altered negative selection?

Unique signals for CD4+CD25+ Treg cell homeostasis. MHC II engagement in periphery Foxp3 Requirement for co-stimulation: CD40/CD40L B7/CD28 IL-2 production IL-2-/- mice IL-2R signaling : IL-2Ra, STAT5, IL-2gc Functional correlate: survival, mechanism…? Other signals: TGF-b1

FoxP3 transcription factor FoxP3 spontaneous mutations induces autoimmunity: IPEX in humans: Immunodysregulation, polyendocrinopathy, enteropathy,X-linked syndrome Scurfy in mice. FoxP3-/- develop spontaneous autoimmunity- defective Treg cells FoxP3 is preferentially expressed in CD4+CD25+ T cells FoxP3 Tg have  cellular frequency of CD4+CD25+ Treg cells. FoxP3 Tg mice x CTLA-4-/- = resolved/delayed autoimmunity FoxP3 retroviral transduction in non-regulatory CD4+CD25- T cells induces regulatory potential. - Phenotypically and functionally similar to naturally occuring lineage. Genes induced by FoxP3 remain unknown.

More selective and faithful Fontenot et al. More selective and faithful marker than CD25

Hypothetical pathway to suppression. Self-reactive Teff cell √ Ag/TCR nTreg IL-2 transcription CD4+CD25+ IL-2 protein IL-2 driven expansion T cell differentiation & effector function

Antigen Presenting Cell CD4+CD25+ mediated suppression is contact-dependent. Requirement for APC? CD4+ CD25+ T cell Effector B Pathways to T cell suppression A X Antigen Presenting Cell Co-stimulation Adhesion CD4+CD25+ cells modulate co-stimulation? MHC class I/II, B7.1/2, CD40 expression is unaffected. Suppression is still operative with fixed LPS blasts. Not overcome with  numbers of APCs. Piccirillo et al. J.Immunology 167:1137-1140.

Antigen Presenting Cell Mechanism of CD4+CD25+ regulatory T cell function ? Cellular and molecular requirements of CD4+CD25+ nTreg cell suppressor function. Requires TCR engagement Antigen non-specific Cell-cell contact dependent Co-stimulation/APC independent T-T suppressor synapse Cytokine independent Suppress IL-2 mRNA in T cells. Suppression of effector functions proliferation inflammatory cytokines differentiation Effector molecules are unknown. nTreg Teff cell Antigen Presenting Cell CD4+CD25+ Suppressor Synapse APC CD4+ CD25+ CD4+ Teff

In vitro and in vivo role of cytokines in CD4+CD25+ regulatory T cell-mediated suppression? CD4+ CD25+ Effector T cell Cytokines ? IL-4, IL-10 Immunosuppressive effects on APC and T cells Suppression is cytokine independent Cytokine neutralization Absence of cytokines in suppressor supernatants Cytokine-deficient Treg cells Transwell chamber experiments Contribution of Transforming Growth Factor 1 (TGF-1) ? Piccirillo et al. J.Exp. Med. 196:237-250.

CD4+CD25+ Treg cells control bacterial-driven intestinal inflammation. CD4+CD45Rbhigh CD4+CD45Rblow (CD25+ subset) CD4+CD45Rbhigh Bacterially-driven, Th1 cell-mediated Inflammatory bowel disease (IBD) Colitis T cell infiltration of colon ->weight loss SCID Colitis No colitis Initial studies showed that anti-IL-10 or anti-TGF-B1 abrogated Treg-mediated suppression of disease. Suppressor T cell-derived TGF-1? Suppressor T cell-derived IL-10 ? Nakamura et al JEM 2001 Membrane-bound TGF Simon Read et al. JEM 2000.

Transforming Growth Factor 1 Requirement for Transforming Growth Factor 1 (TGF-1) ? RII Y TGF-1 TGF-R nTreg Y X DNRIITg Smad3-/- TGF-1-/- X Smad3 CD4+CD25- and CD4+CD25+ T cells produce TGF-b1? Piccirillo et al. J.Exp. Med. 196:237-250.

TGF- dependent in vivo? Is nTreg cell function TGF- dependent in vivo? CD4+CD25+ Treg cell-mediated control of mucosal inflammation. Mouse model of Inflammatory bowel disease (IBD) WT B6/Sv129 WT B6/Sv129 TGF-b1-/- 3-7 day old neonates Colitis T cell infiltration of colon Th1 response to gut bacteria Weight loss CD4+CD25- WT CD4+CD25+ CD4+CD25- TGF-b1-/-CD4+CD25+ CD4+CD25- B6 RAG-/- ? Colitis No colitis Kullberg M., and C.A. Piccirillo Euro. J. Immunol. 2005

TGF-1-/- CD4+CD25+ nTreg cells suppress IBD. A CD25– CD25+ cells cells — — WT — WT WT WT TGF-b1-/- — TGF-b1-/- Body weight (% of day 4 weight)     Days post cells CD25– CD25+ cells cells — — WT — WT WT WT TGF-b1-/- — TGF-b1-/- B Incidence of colitis (%)

TGF-1-/- CD4+CD25+ nTreg cells suppress colonic inflammation. B. A A. B. C. D. E. Grade of inflammation C. D. CD25– cells — WT WT WT — CD25+ cells — — WT TGF-b1-/- TGF-b1-/- B E. IFN-g / G3PDH mRNA ratio CD25– cells — WT WT WT — CD25+ cells — — WT TGF-b1-/- TGF-b1-/-

CD4+CD25+-mediated regulation of Smad3-deficient effector T cells in vivo. WT B6/Sv129 Smad3 -/- WT B6/Sv129 4-6 weeks old FACS sort WT CD4+CD25- CD4+CD25+ Smad 3-/- CD4+CD25- CD4+CD25+ CD4+CD25- B6 RAG-/- Colitis ? No colitis

Smad3-/- effector T cells are highly susceptible to suppression mediated by CD4+CD25+ T cells in vivo. A CD25– CD25+ — — WT Smad3-/- Smad3-/- WT Smad3-/- Smad3-/- WT WT Smad3-/- — WT —  Body weight (% of day 4 weight)      B Days post cells Powrie group observes abrogation of protection with TGFR-/- Effector T cells Why? Grade of inflammation

Tissue-specific CD4+CD25+ mediated disease protection in the absence of IL-10. IL-10-/- CD4+CD25+ CD4+CD25- Nude Gastritis IBD No Gastritis IBD develops ! No Gastritis IBD

Diversity of modes of action Context-dependent regulation of organ-specific autoimmunity. Tissue-specific differentiation of Treg? Any role for bacteria? IBD is a bacterially-driven disease, not gastritis. Lessons from germ-free mice. Genetic background Possible subsets of CD4+CD25+ Treg: Cytokine versus Contact Adaptable to inflammatory milieu. Induction of other Treg cells.

Re-evaluation of CTLA-4 autoimmune phenotype Could CTLA-4 deficiency result in defective Treg function and thus autoimmunity ? (rather than role of CTLA-4 on T cell autonomous regulation) CTLA-4 phenotype rescued by complementing CTLA-4-/- BM with wild type BM (mixed BM chimera) CTLA-4 deficient bone marrow can make CD25+CD4+ (function vs development) CTLA-4 is expressed constitutively on CD4+CD25+CD45RBlow cells. Read et al. J. Exp. Med. July 2000

CD4+CD25+ (CD45low) inhibit IBD Anti–CTLA-4 treatment abrogates the Treg function. Open: control Ig Closed: CTLA-4. Adoptive transfer of CD4 T cell into scid mice.

Possible Mechanisms of Action of CD4+ Regulatory T Cells Naturally-occurring Treg CD25 IL-10 TGF-1 Cellular contact A A+B B MHC: peptide Activated effector T cells TCR Naive CD4+ or CD8+ T cells APC Tolerance Autoimmunity Transplantation Tumor Immunity Infectious disease IL-10 TGF-1 Induced Treg CD25