Regulatory Lymphocytes of the Immune System. II Dr. C. Piccirillo Canada Research Chair Department of Microbiology & Immunology McGill University MIMM-414A Lecture 3- Oct. 25, 2006

_ _ NATURALLY-OCCURRING versus INDUCED Treg cells in the immune system. 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 _ _ IL-10, TGF-b1 iDC VitD Dexamethasone Activated Effector T cell Autoimmunity Transplantation Tumor Immunity Infectious disease Piccirillo et al. Trends in Immunol. 2004.

CD4+CD25+ nTreg cells Masterswitch of peripheral tolerance Immunity Non-self antigens Pathogens Tumors Allergens Grafts Self antigens

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

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 Suppress IL-2 mRNA in T cells. Suppression of effector functions proliferation inflammatory cytokines differentiation Effector molecules are unknown. Suppressive cytokines? nTreg Teff cell Antigen Presenting Cell CD4+CD25+ Suppressor Synapse APC CD4+ CD25+ CD4+ Teff

CD4+CD25+ Treg cell-mediated suppression? Role of cytokines in CD4+CD25+ Treg cell-mediated suppression? CD4+ CD25+ Effector T cell Cytokines ? IL-4, IL-10 Immunosuppressive effects on APC and T cells Suppression is cytokine independent in vitro Cytokine neutralization Absence of cytokines in suppressor supernatants Cytokine-deficient Treg cells Transwell chamber experiments Contribution of Transforming Growth Factor 1 (TGF-1) ? 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 IL-10 needed. Suppressor T cell-derived TGF-1? Powrie et al. JEM 1994 Simon Read et al. JEM 2000. Nakamura et al JEM 2001 Membrane-bound TGF ?

Potent immunosuppressive cytokine on various immune cell subsets Requirement for TGF-1 ? Potent immunosuppressive cytokine on various immune cell subsets Suppression of T, B and DC responses: proliferation, cytokine, MHC/Ag presentation and co-stimulation. Role is best exemplified in TGF-b1 knock-out mice which die of a fulminant, multi-organ, lymphoproliferative disease. 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. cells cells — — WT — WT WT WT TGF-b1-/- — TGF-b1-/-  Body weight (% of day 4 weight)    Days post cells

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

Regulation of immune responses via Foxp3 induction Any role for TGF-b1 in Treg responses? TGF-b1 iTreg nTreg Regulation of immune responses via Foxp3 induction + TGF-b1 - TGF-b1 CD45RBLow CD45RBHigh CD4+CD25- CD4+Foxp3+ IL-10+ CD4+Foxp3+ CD25-Rblow % Suppression Suppressor: Effector Cell

Tissue-specific CD4+CD25+ mediated disease protection in the absence of IL-10. Context-dependent regulation in vivo. Tissue-specific differentiation of Treg? Any role for bacteria? IBD is a bacterially-driven disease, not gastritis. Lessons from germ-free mice. Genetic background Subsets of CD4+CD25+ Treg? Cytokine versus Contact Adaptable to inflammatory milieu. Induction of other Treg cells. CD4+CD25- CD4+CD25+ CD4+CD25- IL-10-/- CD4+CD25+ CD4+CD25- Nude Gastritis IBD No Gastritis IBD develops ! No Gastritis IBD

Control of immune responses by CD4+CD25+ regulatory T cells. Infectious disease Immunity to intracellular pathogens ? ? CD4+CD25+ Regulatory T cells Belkaid/Piccirillo et al. Nature 420:502-7, 2002

Susceptibility and resistance to Leishmania major infection Non-healing Healing Th2 Th1 10 6 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 B6 Lesion size / parasite # Parasite number Acute 10 5 Silent Lesion size (mm) 10 4 Chronic 103 BALB/c 4 8 100 5 10 15 20 Weeks post-infection Weeks post-infection Chronic phase : Transmits back to vector Resistant to re-infection. Life-long immunity : concomitant immunity Site of immune pressure : IL-10IFN- IL-10-/- or anti-IL-10R -> Sterile cure. Role for nTreg cells?

CD4+ nTreg cells accumulate in sites of chronic infection. 1 2 Chronic Dermal site I-10 CD4+CD25+ + 1 45-60% 8 CD25 8 months 6 CTLA-4 + Foxp3+ GITR+ CD45Rblow 4 2 pg/ml CD4+CD25- + CD4 4 1 . 5 1 IFN-g Cell Sorting 4 1 1 CD4+CD25+ CD4+CD25- 5 Cytokine Production DCs Infected DCs L.major infected DC

CD4+CD25+ T cells from chronic sites are regulatory. 10000 20000 30000 40000 50000 60000 CPM 100000 150000 200000 -IL-2 +IL-2 CD4+CD25+ CD4+CD25- L.major Infected macrophages IL10R # CD4+CD25± - -/+ -/+ Chronic site CD4+CD25+ L. major Intradermally C57BL/6 RAG-/- 1/10 CD4+CD25-

IL-10+ Treg / IFNg+ Teff cells CD4+CD25+T cells control immunity to pathogens. Model of cutaneous L.major infection. 3 Wks Silent 5 Wks Acute 9 Wks Chronic 1. Rapid nTreg accumulation 2. Prevent effector T cell functions. 3. Promote susceptibility to infection 4. Remain in chronic site 5. Favor persistence of pathogen IL-10+ Treg / IFNg+ Teff cells 12 % 65 % 50% Ly5.1 CD25

CD4+CD25+T cells from chronic sites prevent anti-parasite effector T cell function. CD25+ IFN-g CD25+ 1 10 100 1000 4 5 6 7 CD25-/ CD25+ 10/1 Parasite number /ear CD25- CD25- CD25-/ CD25+ RAG-/- CD4

IL-10 dependent and independent modes of disease control by nTreg cells. 0.5 1 1.5 2 -/+ IL-10-/- Lesion size -/+ WT Parasite persistence is required for immunity to re-infection Implications : CD25- 2 4 6 8 10 12 14 16 Parasite Long-term maintenance of infectious reservoirs. Host Role of parasite persistence in immunity ? Weeks post infection 109 3 10 3 10 3 10 108 107 RAG-/- 106 CD25- CD25+ WT CD25- CD25+ IL-10-/- Parasite number 105 104       103 102 CD25-    10        

Homing of Treg cells to the infected sites Preferential tropism for Treg cells to infected sites? Chemokine-mediated selective recruitment of CD4+CD25+T cells ? Journal of Experimental Medicine Oct. 2006

CCR5 CCR5 is required for CD4+CD25+ nTreg cell chemo-attraction but not suppressive activity in vitro. CCR5 gene expression A. C. D. B. Resting Activated Gated on Foxp3+ cells CCR5

CCR5-/- mice are resistant to L.major infection.

CCR5-/- CD4+CD25+ nTreg cells fail to promote parasite persistence. 0.5 1 1.5 102 103 104 105 7 weeks WT CD4+CD25+ Lesion size (mm) Parasite number CCR5-/- CD4+CD25+ 101 + + WT CD4+CD25- + 1 2 3 4 5 6 7 8 Weeks post infection WT CD4+CD25+ + - - CCR5-/- CD4+CD25+ - + -

CCR5 dependent homing of CD4+CD25+ Treg cells in sites of infection. 3 weeks WT CCR5-/- 10 20 30 40 50 60 70 WT Skin CCR5-/- 38% 4% WT Lymph node CD4+CD25+ Ly5.1+ CD4+CD25+ Ly5.2+ CCR5-/- WT Spleen % CD4+CD25+ T cells CCR5-/- CD4+ effector T cells ND ND 1.5 3 5.5 10 Weeks post-infection

Summary nTreg cells home preferentially to sites of inflammation: nTreg cells express CCR5 and responds to its ligands. CCR5-mediated signals may drive the early recruitment of nTreg cells in sites of infection. CCR5 mediated homing into sites of pathogen infection regulates pathogen persistence. Pathogen persistence may itself provide a major benefit to the host by maintaining life long immunity to re-infection. Blockade of CCR5 chemotaxis may hinder nTreg/Teff balance and provoke anti-pathogen immune responses. Mechanism of immune evasion ? Other receptors: CCR4 and CCR6 ( tumors and CNS homing)

CD4+ nTreg cell function in health and disease Diversification versus adaptability model Subset Diversification CD4+CD25+ nTreg Adaptability - Foreign Pathogens Tumors Grafts Self Autoimmunity Teff Loss of tolerance Genetic determinants Innate signals Adaptive signals Increased immunity Current Drug Targets 2006.