Regulatory T Cells Interfere with Glutathione Metabolism in Dendritic Cells and T Cells

Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (Tregs) suppress proliferation of CD4+CD25− effector T cells (Teffs) by mechanisms that are not well understood. We have previously demonstrated a novel mechanism of Treg suppression, i.e. interference with extracellular redox remodeling that occurs during activation of T cells by dendritic cells. In this study, we demonstrate that Treg-mediated redox perturbation is antigen-dependent but not antigen-specific, is CTLA-4-dependent, and requires cell-cell contact. Furthermore, we show that Tregs use multiple strategies for extracellular redox remodeling, including diminished GSH synthesis in dendritic cells via decreased expression of γ-glutamylcysteine synthetase, the limiting enzyme for GSH synthesis. Tregs also consume extracellular cysteine and partition it more proficiently to the oxidation product (sulfate), whereas Teffs divert more of the cysteine pool toward protein and GSH synthesis. Tregs appear to block GSH redistribution from the nucleus to the cytoplasm in Teffs, which is abrogated by the addition of exogenous cysteine. Together, these data provide novel insights into modulation of sulfur-based redox metabolism by Tregs, leading to suppression of T cell activation and proliferation. Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (Tregs) suppress proliferation of CD4+CD25− effector T cells (Teffs) by mechanisms that are not well understood. We have previously demonstrated a novel mechanism of Treg suppression, i.e. interference with extracellular redox remodeling that occurs during activation of T cells by dendritic cells. In this study, we demonstrate that Treg-mediated redox perturbation is antigen-dependent but not antigen-specific, is CTLA-4-dependent, and requires cell-cell contact. Furthermore, we show that Tregs use multiple strategies for extracellular redox remodeling, including diminished GSH synthesis in dendritic cells via decreased expression of γ-glutamylcysteine synthetase, the limiting enzyme for GSH synthesis. Tregs also consume extracellular cysteine and partition it more proficiently to the oxidation product (sulfate), whereas Teffs divert more of the cysteine pool toward protein and GSH synthesis. Tregs appear to block GSH redistribution from the nucleus to the cytoplasm in Teffs, which is abrogated by the addition of exogenous cysteine. Together, these data provide novel insights into modulation of sulfur-based redox metabolism by Tregs, leading to suppression of T cell activation and proliferation. IntroductionA fundamental property of the immune system is to distinguish self from non-self. During maturation of T cells in thymus, autoreactive T cells are recognized and eliminated. However, a small fraction of autoimmune T cells escape to the periphery and cause damage to host tissues. By mechanisms that are not well understood, regulatory T cells (Tregs) 2The abbreviations used are: Tregregulatory T cellDCdendritic cellCysexextracellular cysteineTnnaïve TAPCantigen-presenting cellTCRT cell receptorTeffeffector T cellOVA323–329ovalbumin-(323–329)GSHinintracellular GSHCMFDA5-chloromethylfluorescein diacetate. are able to inhibit autoimmune T cells to maintain self-tolerance and immunosuppression (1.Wing K. Sakaguchi S. Nat. Immunol. 2010; 11: 7-13Crossref PubMed Scopus (850) Google Scholar, 2.Yan Z. Banerjee R. Biochemistry. 2010; 49: 1059-1066Crossref PubMed Scopus (68) Google Scholar, 3.Sakaguchi S. Annu. Rev. Immunol. 2004; 22: 531-562Crossref PubMed Scopus (2883) Google Scholar). Tregs also play important roles in antitumor responses as well as transplantation immunity. Dysregulation of Treg function has been shown to be involved in different kinds of immunological diseases ranging from the digestive to the central nervous system (4.Sakaguchi S. Yamaguchi T. Nomura T. Ono M. Cell. 2008; 133: 775-787Abstract Full Text Full Text PDF PubMed Scopus (3612) Google Scholar, 5.Randolph D.A. Fathman C.G. Annu. Rev. Med. 2006; 57: 381-402Crossref PubMed Scopus (109) Google Scholar).Tregs deploy various strategies to mediate their suppressive activity, including (i) secretion of immunosuppressive cytokines such as TGF-β and IL-10; (ii) cytolysis by granzyme secretion; (iii) metabolic disruption, e.g. by adenosine; (iv) suppression of dendritic cell (DC) function, e.g. via induction of indoleamine 2, 3-dioxygenase (6.Vignali D.A. Collison L.W. Workman C.J. Nat. Rev. Immunol. 2008; 8: 523-532Crossref PubMed Scopus (2142) Google Scholar, 7.Tang Q. Bluestone J.A. Nat. Immunol. 2008; 9: 239-244Crossref PubMed Scopus (814) Google Scholar, 8.Miyara M. Sakaguchi S. Trends Mol. Med. 2007; 13: 108-116Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar); and (v) perturbing DC-dependent extracellular redox remodeling, leading to restricted extracellular cysteine (Cysex) availability for naïve T (Tn) cells (9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar). A key role for CTLA-4 (cytotoxic T-lymphocyte antigen 4), a co-receptor expressed preferentially on Tregs, is implicated in the Treg suppression mechanism (1.Wing K. Sakaguchi S. Nat. Immunol. 2010; 11: 7-13Crossref PubMed Scopus (850) Google Scholar, 8.Miyara M. Sakaguchi S. Trends Mol. Med. 2007; 13: 108-116Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 10.Wing K. Onishi Y. Prieto-Martin P. Yamaguchi T. Miyara M. Fehervari Z. Nomura T. Sakaguchi S. Science. 2008; 322: 271-275Crossref PubMed Scopus (2107) Google Scholar). CTLA-4 interacts with CD80/CD86 (cluster of differentiation) on antigen-presenting cells (APCs) and transduces an intracellular inhibitory signal to APCs. Thus, one strategy for Treg-dependent immunosuppression is via down-regulation of APC function (1.Wing K. Sakaguchi S. Nat. Immunol. 2010; 11: 7-13Crossref PubMed Scopus (850) Google Scholar, 11.Mahnke K. Bedke T. Enk A.H. Cell. Immunol. 2007; 250: 1-13Crossref PubMed Scopus (80) Google Scholar).In addition to the T cell receptor (TCR)-antigen MHC class II interaction, co-stimulatory signals, and cytokines, T cell activation and proliferation also require a reducing microenvironment that is shaped mainly by APCs, especially DCs (9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar, 12.Angelini G. Gardella S. Ardy M. Ciriolo M.R. Filomeni G. Di Trapani G. Clarke F. Sitia R. Rubartelli A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1491-1496Crossref PubMed Scopus (298) Google Scholar, 13.Sido B. Braunstein J. Breitkreutz R. Herfarth C. Meuer S.C. J. Exp. Med. 2000; 192: 907-912Crossref PubMed Scopus (51) Google Scholar). Upon stimulation by T cells, DCs increase uptake of cystine via the xc− cystine transporter and, by a convoluted metabolic route involving the γ-glutamyl cycle, furnish Cysex, resulting in a relatively more reducing redox potential that is conducive to T cell proliferation (2.Yan Z. Banerjee R. Biochemistry. 2010; 49: 1059-1066Crossref PubMed Scopus (68) Google Scholar, 9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar). Furthermore, cysteine is needed by T cells for synthesis of GSH, which provides reducing power for DNA synthesis (14.Avval F.Z. Holmgren A. J. Biol. Chem. 2009; 284: 8233-8240Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and for cell cycle progression from the G1 to S phase (15.Suthanthiran M. Anderson M.E. Sharma V.K. Meister A. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 3343-3347Crossref PubMed Scopus (334) Google Scholar, 16.Messina J.P. Lawrence D.A. J. Immunol. 1989; 143: 1974-1981PubMed Google Scholar). Although extracellular cystine is relatively abundant, naïve T cells are inefficient at transporting cystine, the oxidized form of the amino acid cysteine, and depend on DC-derived cysteine to meet their metabolic needs (17.Garg, S. K., Yan, Z., Vitvitsky, V., Banerjee, R., (2010) Antioxid. Redox Signal., in pressGoogle Scholar). By controlling the Cysex level, DCs are able to affect intracellular GSH levels and subsequent redox signaling pathways in T cells (2.Yan Z. Banerjee R. Biochemistry. 2010; 49: 1059-1066Crossref PubMed Scopus (68) Google Scholar).The physiological relevance of redox remodeling is demonstrated by the dramatic increase in non-protein thiols in lymphoid tissues following immunization (18.Castellani P. Angelini G. Delfino L. Matucci A. Rubartelli A. Eur. J. Immunol. 2008; 38: 2419-2425Crossref PubMed Scopus (60) Google Scholar). Additionally, Peyer's patches from the intestine show very low thiol staining because resident APCs from the lamina propria lack the xc− transporter for cystine. However, under inflammatory conditions as in inflammatory bowel diseases, infiltration of peripheral APCs with high xc− transporter expression allows Cysex accumulation, promoting activation and hyperreactivity of lamina propria T cells (19.Sido B. Lasitschka F. Giese T. Gassler N. Funke B. Schröder-Braunstein J. Brunnemer U. Meuer S.C. Autschbach F. Gastroenterology. 2008; 134: 179-191Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). We have demonstrated that Tregs suppress Cysex accumulation and that this is correlated with suppression of T cell activation and proliferation (9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar). However, the mechanism by which Tregs interfere with the redox signaling cross-talk between DCs and effector T cells (Teffs) is unknown.In this study, we demonstrate that Tregs decrease Cysex levels in an antigen-dependent but antigen-nonspecific and CTLA-4-dependent manner. We show that Tregs use multiple strategies for changing the extracellular redox potential, including modulation of DC and Teff GSH metabolism and competitive uptake of cysteine. This study provides the first mechanistic insights into how Tregs influence redox metabolism in DCs and, consequently, in Teffs.DISCUSSIONAlthough the requirement for a reducing microenvironment to support T cell activation and proliferation has been known for quite some time (12.Angelini G. Gardella S. Ardy M. Ciriolo M.R. Filomeni G. Di Trapani G. Clarke F. Sitia R. Rubartelli A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1491-1496Crossref PubMed Scopus (298) Google Scholar, 31.Ishii T. Hishinuma I. Bannai S. Sugita Y. J. Cell. Physiol. 1981; 107: 283-293Crossref PubMed Scopus (100) Google Scholar), redox modulation has emerged as an immunosuppressive strategy only recently (2.Yan Z. Banerjee R. Biochemistry. 2010; 49: 1059-1066Crossref PubMed Scopus (68) Google Scholar, 9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar). We have previously described this novel suppression mechanism whereby Tregs cause an oxidative shift in the extracellular redox environment during DC-dependent T cell activation (9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar). In this study, we have demonstrated that Tregs deploy multiple strategies for inhibiting DC-dependent extracellular redox remodeling (Fig. 6).Activation of Teffs requires interaction between the TCR and a specific antigen presented by MHC class II molecules. However, the antigen specificity for the suppressive action of Tregs is controversial. Although several studies suggest that Tregs suppress in an antigen-restricted manner in vitro and in vivo (32.Corthay A. Scand J. Immunol. 2009; 70: 326-336Crossref PubMed Scopus (408) Google Scholar, 33.Yu P. Gregg R.K. Bell J.J. Ellis J.S. Divekar R. Lee H.H. Jain R. Waldner H. Hardaway J.C. Collins M. Kuchroo V.K. Zaghouani H. J. Immunol. 2005; 174: 6772-6780Crossref PubMed Scopus (108) Google Scholar, 34.Tanchot C. Vasseur F. Pontoux C. Garcia C. Sarukhan A. J. Immunol. 2004; 172: 4285-4291Crossref PubMed Scopus (59) Google Scholar), others report that Tregs do not require specific TCR-antigen interaction for suppression of Teff proliferation (35.Szymczak-Workman A.L. Workman C.J. Vignali D.A. J. Immunol. 2009; 182: 5188-5192Crossref PubMed Scopus (42) Google Scholar, 36.Homann D. Holz A. Bot A. Coon B. Wolfe T. Petersen J. Dyrberg T.P. Grusby M.J. von Herrath M.G. Immunity. 1999; 11: 463-472Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). We have demonstrated that although remodeling of the extracellular environment by Tregs is antigen-dependent, it is not antigen-specific. These results are consistent with the observations of Vignali and co-workers (35.Szymczak-Workman A.L. Workman C.J. Vignali D.A. J. Immunol. 2009; 182: 5188-5192Crossref PubMed Scopus (42) Google Scholar), who have shown that Tregs can suppress Teffs derived from mouse strains with distinct antigen specificities. Hence, Tregs might harbor constitutive suppressive activity and mediate redox remodeling via a bystander suppression mechanism.Recent studies by Sakaguchi and co-workers (1.Wing K. Sakaguchi S. Nat. Immunol. 2010; 11: 7-13Crossref PubMed Scopus (850) Google Scholar, 10.Wing K. Onishi Y. Prieto-Martin P. Yamaguchi T. Miyara M. Fehervari Z. Nomura T. Sakaguchi S. Science. 2008; 322: 271-275Crossref PubMed Scopus (2107) Google Scholar) demonstrate an essential role for CTLA-4 in immunosuppression by Tregs. Foxp3, a transcription factor needed for Treg function, regulates CTLA-4 expression. The addition of anti-CTLA-4 antibody or disruption of the CTLA-4 gene in Tregs blocks Treg suppression and causes a variety of autoimmune diseases (10.Wing K. Onishi Y. Prieto-Martin P. Yamaguchi T. Miyara M. Fehervari Z. Nomura T. Sakaguchi S. Science. 2008; 322: 271-275Crossref PubMed Scopus (2107) Google Scholar, 37.Sakaguchi S. Wing K. Onishi Y. Prieto-Martin P. Yamaguchi T. Int. Immunol. 2009; 21: 1105-1111Crossref PubMed Scopus (650) Google Scholar). In this study, we have shown that Treg-mediated redox remodeling is both contact- and CTLA-4-dependent. Administration of anti-CTLA-4 antibody blocked the Treg effect on Cysex, GSHex, GSHin, and γ-glutamyl synthetase expression, suggesting that CTLA-4 plays an important role in several aspects of redox remodeling by Tregs. By interacting with CD80/CD86 on DCs, Treg-derived CTLA-4 triggers several signaling pathways in DCs, including activation of indoleamine 2, 3-dioxygenase expression and induction of the Foxo3 transcription factor (27.Grohmann U. Orabona C. Fallarino F. Vacca C. Calcinaro F. Falorni A. Candeloro P. Belladonna M.L. Bianchi R. Fioretti M.C. Puccetti P. Nat. Immunol. 2002; 3: 1097-1101Crossref PubMed Scopus (992) Google Scholar, 28.Fallarino F. Grohmann U. Hwang K.W. Orabona C. Vacca C. Bianchi R. Belladonna M.L. Fioretti M.C. Alegre M.L. Puccetti P. Nat. Immunol. 2003; 4: 1206-1212Crossref PubMed Scopus (1044) Google Scholar, 29.Dejean A.S. Beisner D.R. Ch'en I.L. Kerdiles Y.M. Babour A. Arden K.C. Castrillon D.H. DePinho R.A. Hedrick Nat. Immunol. 2009; PubMed Scopus Google Scholar). Tregs are known to the maturation and antigen-presenting of DCs by expression of CD80/CD86 co-stimulatory on DCs L. H. F. Eur. J. Immunol. 2000; PubMed Scopus Google Scholar, N. J. S. J. Immunol. 2004; 172: PubMed Scopus Google that GSH synthesis in DCs is by Tregs, which is consistent with GSH by DCs and, consequently, Cysex via the γ-glutamyl The regulatory cross-talk between Tregs and APCs has been Foxo3 is via CTLA-4 signaling and Foxo3 is a and of redox signaling A.S. Beisner D.R. Ch'en I.L. Kerdiles Y.M. Babour A. Arden K.C. Castrillon D.H. DePinho R.A. Hedrick Nat. Immunol. 2009; PubMed Scopus Google Scholar, I. K.W. 2002; PubMed Scopus Google Scholar), a potential between this transcription factor and the γ-glutamyl cycle of levels requires the availability and of cysteine, the amino acid that GSH synthesis. cysteine is oxidized to cystine in the extracellular the of cysteine in the is of cystine H. M. D. Biol. Chem. 1989; PubMed Scopus Google Scholar). However, the cystine transporter activity in naïve T cells is very low with the cysteine transporter activity T. Sugita Y. Bannai S. J. Cell. Physiol. 133: PubMed Scopus Google Scholar), on APCs to meet their cysteine needs (2.Yan Z. Banerjee R. Biochemistry. 2010; 49: 1059-1066Crossref PubMed Scopus (68) Google Scholar, 9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar, 12.Angelini G. Gardella S. Ardy M. Ciriolo M.R. Filomeni G. Di Trapani G. Clarke F. Sitia R. Rubartelli A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1491-1496Crossref PubMed Scopus (298) Google Scholar). DCs the xc− cystine transporter and cystine Cysex accumulation during T cell activation (9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar, 12.Angelini G. Gardella S. Ardy M. Ciriolo M.R. Filomeni G. Di Trapani G. Clarke F. Sitia R. Rubartelli A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1491-1496Crossref PubMed Scopus (298) Google Scholar). This is by Tregs at multiple including of cysteine and oxidation to However, the in vivo of this competitive mechanism for Cysex is because Teffs Tregs, which only of T cells S. Annu. Rev. Immunol. 2004; 22: 531-562Crossref PubMed Scopus (2883) Google plays essential roles in T cell including DNA synthesis (15.Suthanthiran M. Anderson M.E. Sharma V.K. Meister A. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 3343-3347Crossref PubMed Scopus (334) Google Scholar, J. J. Mol. Med. 2009; PubMed Scopus Google Scholar). of levels and the redox T cell DNA and T cell activity J. 2006; PubMed Google Scholar). levels also influence T cell signal pathways involving and Eur. J. PubMed Scopus Google Scholar). of cell results in GSH levels by GSH to the nucleus to S. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). GSH is in the nucleus during the of cell proliferation and is more cells GSH regulates protein via which also protein thiols from oxidation J. C. A. J. J. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). Although of GSH is by the at the data suggest that GSH is in the nucleus in naïve T cells but is more in T cells activation and proliferation. the cell cycle is correlated with redox 2007; PubMed Scopus Google Scholar), and of GSH by Tregs might one mechanism of of Teff proliferation. Although the of GSH from the to the for Teff proliferation is not it is that the inhibitory effect of Tregs on is by exogenous cysteine at a under cell study that Tregs deploy multiple strategies for perturbing the extracellular redox environment during T cell both DCs and cells. of the signaling pathways that Treg and the redox metabolic responses in this study might of novel IntroductionA fundamental property of the immune system is to distinguish self from non-self. During maturation of T cells in thymus, autoreactive T cells are recognized and eliminated. However, a small fraction of autoimmune T cells escape to the periphery and cause damage to host tissues. By mechanisms that are not well understood, regulatory T cells (Tregs) 2The abbreviations used are: Tregregulatory T cellDCdendritic cellCysexextracellular cysteineTnnaïve TAPCantigen-presenting cellTCRT cell receptorTeffeffector T cellOVA323–329ovalbumin-(323–329)GSHinintracellular GSHCMFDA5-chloromethylfluorescein diacetate. are able to inhibit autoimmune T cells to maintain self-tolerance and immunosuppression (1.Wing K. Sakaguchi S. Nat. Immunol. 2010; 11: 7-13Crossref PubMed Scopus (850) Google Scholar, 2.Yan Z. Banerjee R. Biochemistry. 2010; 49: 1059-1066Crossref PubMed Scopus (68) Google Scholar, 3.Sakaguchi S. Annu. Rev. Immunol. 2004; 22: 531-562Crossref PubMed Scopus (2883) Google Scholar). Tregs also play important roles in antitumor responses as well as transplantation immunity. Dysregulation of Treg function has been shown to be involved in different kinds of immunological diseases ranging from the digestive to the central nervous system (4.Sakaguchi S. Yamaguchi T. Nomura T. Ono M. Cell. 2008; 133: 775-787Abstract Full Text Full Text PDF PubMed Scopus (3612) Google Scholar, 5.Randolph D.A. Fathman C.G. Annu. Rev. Med. 2006; 57: 381-402Crossref PubMed Scopus (109) Google Scholar).Tregs deploy various strategies to mediate their suppressive activity, including (i) secretion of immunosuppressive cytokines such as TGF-β and IL-10; (ii) cytolysis by granzyme secretion; (iii) metabolic disruption, e.g. by adenosine; (iv) suppression of dendritic cell (DC) function, e.g. via induction of indoleamine 2, 3-dioxygenase (6.Vignali D.A. Collison L.W. Workman C.J. Nat. Rev. Immunol. 2008; 8: 523-532Crossref PubMed Scopus (2142) Google Scholar, 7.Tang Q. Bluestone J.A. Nat. Immunol. 2008; 9: 239-244Crossref PubMed Scopus (814) Google Scholar, 8.Miyara M. Sakaguchi S. Trends Mol. Med. 2007; 13: 108-116Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar); and (v) perturbing DC-dependent extracellular redox remodeling, leading to restricted extracellular cysteine (Cysex) availability for naïve T (Tn) cells (9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar). A key role for CTLA-4 (cytotoxic T-lymphocyte antigen 4), a co-receptor expressed preferentially on Tregs, is implicated in the Treg suppression mechanism (1.Wing K. Sakaguchi S. Nat. Immunol. 2010; 11: 7-13Crossref PubMed Scopus (850) Google Scholar, 8.Miyara M. Sakaguchi S. Trends Mol. Med. 2007; 13: 108-116Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 10.Wing K. Onishi Y. Prieto-Martin P. Yamaguchi T. Miyara M. Fehervari Z. Nomura T. Sakaguchi S. Science. 2008; 322: 271-275Crossref PubMed Scopus (2107) Google Scholar). CTLA-4 interacts with CD80/CD86 (cluster of differentiation) on antigen-presenting cells (APCs) and transduces an intracellular inhibitory signal to APCs. Thus, one strategy for Treg-dependent immunosuppression is via down-regulation of APC function (1.Wing K. Sakaguchi S. Nat. Immunol. 2010; 11: 7-13Crossref PubMed Scopus (850) Google Scholar, 11.Mahnke K. Bedke T. Enk A.H. Cell. Immunol. 2007; 250: 1-13Crossref PubMed Scopus (80) Google Scholar).In addition to the T cell receptor (TCR)-antigen MHC class II interaction, co-stimulatory signals, and cytokines, T cell activation and proliferation also require a reducing microenvironment that is shaped mainly by APCs, especially DCs (9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar, 12.Angelini G. Gardella S. Ardy M. Ciriolo M.R. Filomeni G. Di Trapani G. Clarke F. Sitia R. Rubartelli A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1491-1496Crossref PubMed Scopus (298) Google Scholar, 13.Sido B. Braunstein J. Breitkreutz R. Herfarth C. Meuer S.C. J. Exp. Med. 2000; 192: 907-912Crossref PubMed Scopus (51) Google Scholar). Upon stimulation by T cells, DCs increase uptake of cystine via the xc− cystine transporter and, by a convoluted metabolic route involving the γ-glutamyl cycle, furnish Cysex, resulting in a relatively more reducing redox potential that is conducive to T cell proliferation (2.Yan Z. Banerjee R. Biochemistry. 2010; 49: 1059-1066Crossref PubMed Scopus (68) Google Scholar, 9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar). Furthermore, cysteine is needed by T cells for synthesis of GSH, which provides reducing power for DNA synthesis (14.Avval F.Z. Holmgren A. J. Biol. Chem. 2009; 284: 8233-8240Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and for cell cycle progression from the G1 to S phase (15.Suthanthiran M. Anderson M.E. Sharma V.K. Meister A. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 3343-3347Crossref PubMed Scopus (334) Google Scholar, 16.Messina J.P. Lawrence D.A. J. Immunol. 1989; 143: 1974-1981PubMed Google Scholar). Although extracellular cystine is relatively abundant, naïve T cells are inefficient at transporting cystine, the oxidized form of the amino acid cysteine, and depend on DC-derived cysteine to meet their metabolic needs (17.Garg, S. K., Yan, Z., Vitvitsky, V., Banerjee, R., (2010) Antioxid. Redox Signal., in pressGoogle Scholar). By controlling the Cysex level, DCs are able to affect intracellular GSH levels and subsequent redox signaling pathways in T cells (2.Yan Z. Banerjee R. Biochemistry. 2010; 49: 1059-1066Crossref PubMed Scopus (68) Google Scholar).The physiological relevance of redox remodeling is demonstrated by the dramatic increase in non-protein thiols in lymphoid tissues following immunization (18.Castellani P. Angelini G. Delfino L. Matucci A. Rubartelli A. Eur. J. Immunol. 2008; 38: 2419-2425Crossref PubMed Scopus (60) Google Scholar). Additionally, Peyer's patches from the intestine show very low thiol staining because resident APCs from the lamina propria lack the xc− transporter for cystine. However, under inflammatory conditions as in inflammatory bowel diseases, infiltration of peripheral APCs with high xc− transporter expression allows Cysex accumulation, promoting activation and hyperreactivity of lamina propria T cells (19.Sido B. Lasitschka F. Giese T. Gassler N. Funke B. Schröder-Braunstein J. Brunnemer U. Meuer S.C. Autschbach F. Gastroenterology. 2008; 134: 179-191Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). We have demonstrated that Tregs suppress Cysex accumulation and that this is correlated with suppression of T cell activation and proliferation (9.Yan Z. Garg S.K. Kipnis J. Banerjee R. Nat. Chem. Biol. 2009; 5: 721-723Crossref PubMed Scopus (113) Google Scholar). However, the mechanism by which Tregs interfere with the redox signaling cross-talk between DCs and effector T cells (Teffs) is unknown.In this study, we demonstrate that Tregs decrease Cysex levels in an antigen-dependent but antigen-nonspecific and CTLA-4-dependent manner. We show that Tregs use multiple strategies for changing the extracellular redox potential, including modulation of DC and Teff GSH metabolism and competitive uptake of cysteine. This study provides the first mechanistic insights into how Tregs influence redox metabolism in DCs and, consequently, in