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Regulatory Lymphocytes of the Immune System.

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. Thymic CD4 + T cell pool. Peripherally-induced CD4 + Treg cells ( iTreg ).

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Regulatory Lymphocytes of the Immune System.

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  1. 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

  2. Induced regulatory T cells 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.

  3. 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+ • Th1cell-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

  4. B. NATURALLY-OCCURRING 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 + Peripheral differentiation signals CD25 GITR CTLA-4 Foxp3 APC _ _ Activated Effector T cell Autoimmunity Transplantation Tumor Immunity Infectious disease Piccirillo et al. Trends in Immunol. 2004.

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

  6. 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 Mucosal Immunity Transplantation • Gastritis • Oophritis • Orchitis • Thyroiditis • Pancreatitis • Colitis • Auto Abs • Depletion • Day3 thymectomy • Genetic anomalies • Irradiation • Infections • Drugs Normal Immune responses Tumor Immunity

  7. CD4+CD25+ nTreg cellsMasterswitch of peripheral tolerance Immunity Tolerance

  8. Reliable biomarkers for CD4+CD25+ regulatory T cells? CD25 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

  9. 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.

  10. 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 ()

  11. CD4+CD25+ regulatory T cells suppress T cell functions.Consensus elements CD8+ 24h 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.

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

  13. 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?

  14. 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

  15. 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.

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

  17. 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

  18. 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.

  19. 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

  20. 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.

  21. 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 TGF-1? Suppressor T cell-derived IL-10 ? Nakamura et al JEM 2001 Membrane-bound TGF Simon Read et al. JEM 2000.

  22. RII • Requirement for • Transforming Growth Factor 1 (TGF-1) ? 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.

  23. 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

  24. TGF-1-/- CD4+CD25+ nTreg cells suppress IBD. A CD25–CD25+ cellscells — — 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 (%)

  25. 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-/-

  26. 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

  27. 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

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

  29. 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.

  30. 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

  31. 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.

  32. 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

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