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Regulatory Lymphocytes of the Immune System. II

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.

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Regulatory Lymphocytes of the Immune System. II

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  1. 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

  2. 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 + Peripheral differentiation signals CD25 GITR CTLA-4 Foxp3 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.

  3. CD4+CD25+ nTreg cellsMasterswitch of peripheral tolerance Immunity Tolerance Non-self antigens Pathogens Tumors Allergens Grafts Self antigens

  4. 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.

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

  6. 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

  7. 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.

  8. CD4+CD25+ Treg cells control bacterial-driven intestinal inflammation. CD4+CD45Rbhigh CD4+CD45Rblow (CD25+ subset) Bacterially-driven, Th1 cell-mediated Inflammatory bowel disease (IBD) Colitis T cell infiltration of colon ->weight loss CD4+CD45Rbhigh 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? Nakamura et al JEM 2001 Membrane-bound TGF ? Powrie et al. JEM 1994 Simon Read et al. JEM 2000.

  9. RII 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. Y TGF-R TGF-1 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.

  10. 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

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

  12. TGF-1-/- CD4+CD25+ nTreg cells suppresscolonic inflammation. B. A. 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-/-

  13. 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

  14. 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

  15. Any role for TGF-b1 in Treg responses? TGF-b1 iTregnTreg 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

  16. 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 No IBD

  17. Control of immune responses by CD4+CD25+ regulatory T cells. CD4+ CD8+ T cells Infectious disease Immunity to intracellular pathogens ? ? CD4+CD25+ Regulatory T cells Belkaid/Piccirillo et al.Nature420:502-7, 2002

  18. 1.8 1.6 5 1.4 10 1.2 4 10 1 0.8 0.6 0.4 0.2 0 0 5 10 15 20 Susceptibility and resistance to Leishmania major infection Non-healing Healing Th2 Th1 6 10 B6 Lesion size / parasite # Parasite number Acute Silent Lesion size (mm) Chronic 103 BALB/c 100 0 4 8 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?

  19. CD4+ nTreg cells accumulate in sites of chronic infection. + + + + + + CD4+CD25- + + CD4+CD25+ 1 2 0 0 I-10 Chronic Dermal site 1 0 0 0 45-60% 8 0 0 CD25 8 months 6 0 0 4 0 0 CTLA-4 + Foxp3+ GITR+ CD45Rblow 2 0 0 pg/ml 0 CD4 4 1 . 5 1 0 IFN-g Cell Sorting 4 1 1 0 CD4+CD25+ CD4+CD25- 5 0 0 0 Cytokine Production 0 DCs Infected DCs L.major infected DC

  20. 200000 CD4+CD25- CD4+CD25+ 150000 CPM 100000 50000 0 -IL-2 +IL-2 CD4+CD25- 60000 50000 40000 CPM 30000 20000 CD4+CD25+ 10000 0 0 10000 20000 30000 40000 50000 CD4+CD25+ T cells from chronic sites are regulatory. L.major Infected macrophages IL10R # CD4+CD25± - -/+ -/+ Chronic site L. major Intradermally C57BL/6 RAG-/- CD4+CD25+ 1/10 CD4+CD25-

  21. 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

  22. 7 10 6 10 5 10 4 10 1000 100 10 CD25+ CD25- CD25-/ CD25+ 10/1 1 CD4+CD25+T cells from chronic sites prevent anti-parasite effector T cell function. IFN-g CD25+ Parasite number /ear CD25- CD25-/ CD25+ RAG-/- CD4

  23. IL-10 dependent and independent modes of disease control by nTreg cells. 2 1.5 1 0.5 -/+ IL-10-/- 0 0 2 4 6 8 10 12 14 16 Lesion size -/+ WT Parasite persistence is required for immunity to re-infection Implications : CD25- • 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 CD25- 102    10        

  24. 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

  25. 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

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

  27. 105 1.5 104 1 103 102 0.5 0 0 1 2 3 4 5 6 7 8 CCR5-/- CD4+CD25+ nTreg cells fail to promote parasite persistence. 7 weeks WT CD4+CD25+ Lesion size (mm) Parasite number CCR5-/- CD4+CD25+ 101 + + + WT CD4+CD25- + - - Weeks post infection WT CD4+CD25+ - + - CCR5-/- CD4+CD25+

  28. 70 60 50 40 30 20 10 0 CCR5 dependent homing of CD4+CD25+ Treg cells in sites of infection. 3 weeks WT CCR5-/- WT Skin 38% 4% CCR5-/- Lymph node WT CD4+CD25+ Ly5.1+ CD4+CD25+ Ly5.2+ CCR5-/- WT Spleen % CD4+CD25+ T cells CCR5-/- CD4+ effector T cells ND ND 3 10 1.5 5.5 Weeks post-infection

  29. 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)

  30. CD4+ nTreg cell function in health and diseaseDiversification 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.

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