suppressive effect. Also, the addition of p286 peptide to the culture medium had no effects on either the proliferation of BDC2.5 T cells or the production of IL-10 and IFN2c, suggesting that the regulatory function of NR286 Treg was independent of their activation status. Our studies further showed that, in addition to soluble factors, suppression of BDC2.5 T cells was also dependent on cell contact between NR286 Treg and target cells; that is, the production of these soluble factors in cell cultures, in the absence of NR286 Treg, was insufficient to suppress BDC2.5 T cells. The GAD p286 peptide, similar to p206 and p221 peptides, has been demonstrated as a major immunogenic peptide in NOD mice [27]. Our previous studies showed that GAD peptide-specific CD4+ T cells are able to function as Treg [16], [17]. Consistent with our results, other studies also demonstrated that GAD-specific CD4+ T cells from NOD mice, or CD4+ T cells from transgenic mice (expressing TCRs derived from NOD mouse T cell hybridomas specific for GAD peptides) inhibited T1D [28],[29]. In addition, our current results further demonstrated that p286- specific T cells isolated from NOR mice, like GAD-specific T cells from NOD mice, functioned as Treg. However, the mechanisms underlying NR286 Treg regulatory function appeared to be different from GAD-specific T cells isolated from NOD mice, as they were dependent on IL-10 and independent of cell contact with target cells [16], [18], [28], [29]. Altogether, these results showed that, regardless of the mechanisms underlying their regulatory function, potent diabetes-inhibitory autoantigen-specif- ic Treg can be relatively easily induced in vivo using GAD peptides. Because the amino acid sequences of these GAD peptides are identical in both human and mouse, these results suggest the possibility their use to induce potent Treg as a therapy for treatment of T1D. This is further supported by recent phase II clinical trials showing that GAD-treated T1D patients showed expansion of Treg and enhanced preservation of islet cell mass [30]. NOR mice are a class II MHC I-Ag7-bearing recombinant congenic strain of NOD mice. However, unlike NOD mice, NOR mice do not develop diabetes, perhaps due to having a more robust Treg population [24], [25]. Our results suggest that the mechanisms underlying how GAD-specific Treg cells function may also differ between GAD-specific Treg isolated from these two different mouse strains. NOD mouse Treg, specific for GAD peptides, were IL-10-dependent and cell contact-independent, while NOR mouse GAD-specific Treg were IFN-c- and NO- dependent as well as cell-contact dependent [16], [17], [18], [23]. The reason for this difference is currently unclear. One possibility could be due to the culture conditions used to derive NR286 T cells. However, we consider this unlikely given that the same cell culture protocol was previously used by us to isolate T cells specific for different GAD peptides (p206 and p221) as well as for an unrelated peptide (a mimetic epitope, p79, that is highly active in stimulating BDC2.5 T cells). Isolated T cells either functioned as Treg or as non-pathogenic cells [16], [17], [18], [23], and did not lead to a biased production of NR286-like Treg. An alternative possibility is that the difference in non-class II MHC genes in NOR and NOD mice may contribute to the functional difference between NOD and NOR mouse Treg. It is possible that non-class II MHC background genes can influence the selection of Treg with varied underlying regulatory mechanisms in either NOD or NOR mice. Further studies are necessary to elucidate the potential mechanisms responsible for the observed differences in the regulatory mechanisms underlying different Treg in these mice. Although NR286 T cell regulatory function was dependent on cell-contact with target cells, their unique requirement of IFN2c and NO for their regulatory functions and the fact that they did Antigen-Specific Foxp32 Treg not express Foxp3 distinguishes them from Foxp3-expressing nTreg. In addition, production of NO, not IFN2c, was also dependent on cell contact between NR286 T cells and target cells. It is likely that a combined regulatory effect of IFN-c and NO may be directly involved in mediating suppression of target cells and inhibition of diabetes by NR286 Treg cells. The role of IFN2c in the immune response is complex [31]. Its pathogenic role as a Th1 cytokine in autoimmune disease development has been well documented [31–35]. However, IFN2c can also negatively regulate immune responses and contribute to immune regulation processes [31], [36], [37]. The reason why IFN-c plays these dual roles in the immune response is unclear. It is possible that IFN-c produced by pathogenic Th1 cells plays a pathogenic role in the absence of NR286 Treg [38], [39]. On the other hand, in the presence of Treg, such as NR286 T cells, IFN-c triggers NO production, which is dependent on cell-contact between Treg and target cells. The resulting NO may then serve as the effector molecule that suppresses pathogenic T cells and regulates autoimmune diseases. Alternatively, IFN-c may negatively affect the function of pro-inflammatory Th17 cells and, together with NO, suppress inflammatory responses [40]. Previous studies have demonstrated that NO, which can be produced by macrophages or dendritic cells, inhibits T cell proliferation [41], [42], [43]. It is possible that the recruitment of autoantigen-specific Treg, such as NR286 Treg, into an inflamed site, may facilitate the interaction between Treg and APCs at the site, and that the production of IFN2c and NO results in the suppression of autoimmune responses. In summary, the current studies have 1) identified a unique population of autoantigen-specific Treg (NR286 T cells) from a disease resistant strain of mice that can effectively suppress the proliferation of pathogenic T cells and inhibit T1D, 2) demon- strated that NR286 T cell function was dependent not only on the production of soluble factors, including IFN2c and NO, but also on cell contact with target cells, leading to the suppression of pathogenic T cell proliferation and diabetes development, and 3) provided evidence to support the hypothesis that NOR mice contain a novel population of GAD-specific Treg that could help prevent these mice from diabetes. Materials and Methods Mice NOR mice were purchased from the Jackson Laboratory (Bar Harbor, ME). BDC2.5 transgenic mice were a gift from Drs. D. Mathis and C. Benoist (Joslin Diabetes Center/Harvard Medical School, Boston, MA). All animals were housed in a specific pathogen-free environment and were used at 7–8 wk of age. Peptides and Immunization GAD65 p286 (p286–300) and BDC2.5 T cell-stimulating peptide p79 (1040–79)[23],[26] were synthesized at the Beckman Research Institute, City of Hope and purified using reverse-phase HPLC to a purity of .90%. Animals were immunized i.p. with 100 mg of peptide emulsified in an equal volume of incomplete Freund’s adjuvant (IFA)(Sigma, St. Louis, MO) on days 0 and 7. Spleens were removed from animals on day 14 for further experiments. Production of Class II MHC Tetramers Production and initial use of the I-Ag7 tetramers specific for either GAD peptides or p79 have been previously described [16], [22], [23]. PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e7863