Regulatory Function of a Novel Population of Mouse 2 Autoantigen-Specific Foxp3 Regulatory T Cells Depends on IFN-c, NO, and Contact with Target Cells Cyndi Chen1, Chih-Pin Liu1,2* 1 Department of Immunology, Beckman Research Institute, City of Hope, Duarte, California, United States of America, 2 Department of Diabetes, Endocrinology, and Metabolism, Beckman Research Institute, City of Hope, Duarte, California, United States of America Abstract Background: Both naturally arising Foxp3+ and antigen-induced Foxp32 regulatory T cells (Treg) play a critical role in regulating immune responses, as well as in preventing autoimmune diseases and graft rejection. It is known that antigen- specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause autoimmunity and inflammation. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited their use in research to elucidate the mechanisms underlying their regulatory function and their potential role in therapy. Methodology/Principal Findings: Using a novel class II MHC tetramer, we have isolated a population of CD4+ Foxp32 T cells specific for the autoantigen glutamic acid decarboxylase p286–300 peptide (NR286 T cells) from diabetes-resistant non- obese resistant (NOR) mice. These Foxp32 NR286 T cells functioned as Treg that were able to suppress target T cell proliferation in vitro and inhibit type 1 diabetes in animals. Unexpected results from mechanistic studies in vitro showed that their regulatory function was dependent on not only IFN-gamma and nitric oxide, but also on cell contact with target cells. In addition, separating NR286 Treg from target T cells in transwell assays abolished both production of NO and suppression of target T cells, regardless of whether IFN-c was produced in cell cultures. Therefore, production of NO, not IFN-gamma, was cell contact dependent, suggesting that NO may function downstream of IFN-gamma in mediating regulatory function of NR286 Treg. Conclusions/Significance: These studies identified a unique population of autoantigen-specific Foxp32 Treg that can exert their regulatory function dependent on not only IFN-c and NO but also cell contact with target cells. Citation: Chen C, Liu C-P (2009) Regulatory Function of a Novel Population of Mouse Autoantigen-Specific Foxp32 Regulatory T Cells Depends on IFN-c, NO, and Contact with Target Cells. PLoS ONE 4(11): e7863. doi:10.1371/journal.pone.0007863 Editor: Derya Unutmaz, New York University School of Medicine, United States of America Received August 24, 2009; Accepted October 22, 2009; Published November 17, 2009 Copyright: 2009 Chen, Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by funding from Juvenile Diabetes Research Foundation International, American Heart Association, National Institute of Health, and the H.L. Snyder Medical Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cpliu@coh.org Introduction Inflammation plays a central role during the development of autoimmune diseases. It is known that CD4+ regulatory T cells (Treg) are able to induce effective immune tolerance that inhibits such inflammatory responses [1], [2], [3], [4]. Defective development or depletion of Treg in animals, such as the naturally arising Foxp3+CD4+CD25+ Treg (nTreg), results in the develop- ment of autoimmune diseases, including type 1 diabetes (T1D) [1], [2], [3], [4], [5], [6], [7]. Administration of Treg, expanded in vitro, to animals has been shown to inhibit autoimmune diseases, including T1D [8], [9]. Previous studies have demonstrated that different cell populations with varied phenotypes can function as Treg, regulating both immunity and autoimmunity [10], [11], [12], [13], [14]. Both Foxp3+ and Foxp32 Treg may play a pivotal role in the negative regulation of immune system function and inhibition of inflammation, as well as in the prevention of autoimmune diseases and graft rejection. Therefore, the use of Treg as a cellular therapy to induce immune tolerance in animals or humans may be a promising method to control inflammation and autoimmune diseases. In addition to the heterogeneous nTreg, whose antigen- specificity remains largely unknown, our studies and those of others have demonstrated that cytokine-dependent antigen- specific Treg can be expanded following treatment with antigens [12], [15], [16], [17], [18]. Unlike nTreg, whose function is cell- contact dependent [1], [2], [3], [11], [19], antigen-induced Foxp32 Treg may exert their function through the production of cytokines such as IL-10 [4], [13], [14], [16], [18]. It has been well-established that antigen-specific Treg are more potent than polyclonal Treg in suppressing pathogenic immune responses that cause inflammation [8], [9], [20], [21]. However, difficulty in identifying and isolating a sufficient number of antigen-specific Treg has limited the ability of researchers to elucidate the mechanisms underlying their regulatory function and their potential role in therapy. In studies to address this difficulty, we have generated autoantigen-specific class II MHC I-Ag7 tetramers to isolate several autoantigen-specific Treg from non-obese PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7863