diabetic (NOD) as well as non-obese resistant (NOR) mice [16], [17], [22], [23]. NOR mice are a diabetes-resistant recombinant congenic strain of NOD mice [24], [25]. While both strains express the same disease-associated I-Ag7 molecules, NOR mice are resistant to T1D. Although the mechanism underlying disease resistance remains largely unclear, it is likely that NOR mice have a relatively more robust regulatory T cell function than their NOD counterparts [24]. In addition, the manner in which Treg exert their regulatory function may differ in NOR and NOD mice [17]. In order to better understand the function of autoantigen-specific Treg from NOR mice, as well as their role in T1D development, we have isolated NOR mouse CD4+ T cells specific for an immunodominant p286 (a.a. p286–300) peptide of a major autoantigen, glutamic acid decarboxylase (GAD), involved in T1D. We report our results from studies on these novel GAD p286-specific T cells. Results GAD p286-Specific CD4+ T Cells Isolated from NOR Mice Produce Both IFN-c and IL-10 GAD p286-specific CD4+ T cell line (NR286 T cells) were isolated from p286-immunized NOR mice, using a novel I-Ag7 tetramer (tetAg7/p286) specific for this peptide, to investigate their function and potential role in T1D development. Our results suggested the established NR286 T cell line was p286- specific, as cells were positively stained by tetAg7/p286, but not by a control tetramer (tetAg7/p206) specific for a different GAD peptide, p206; additionally, they produced IL-2 in response to p286, but not to p206 (Fig. 1A and 1B). In studies to examine their response to antigen stimulation, ELISA results showed that NR286 T cells produced IFN-c and IL-10, in a concentration-dependent manner, but not IL-4, in response to p286 stimulation (Fig. 2A). These results suggested that NR286 T cells may represent a novel population of T cells that are not only Th1-like, but also have acquired a Tr1-like cytokine secretion profile, producing both IFN-c and IL10 [4], [13], [14]. Antigen-Specific Foxp32 Treg Additional studies examined NR286 T cell phenotype, with results demonstrating that ,98% of NR286 T cells expressed CD25 (but not Foxp3), ,20% expressed CD40, and ,19% expressed CD45RB (Fig. 2B). NR286 T cells did not express CD44, CD62L, CD69, or CTLA-4 (data not shown). These results suggested that although NR286 T cells produced IL-10, they were different from the Foxp3-expressing nTreg. NR286 T Cells Inhibited Diabetes Caused by NOD Mouse Splenocytes Because NR286 T cells produced both IFN2c and IL-10, we examined whether they functioned as pro-inflammatory T cells or as Treg. Adoptive transfer experiments were performed to determine the in vivo role of NR286 T cells during diabetes development. NR286 T cells were adoptively transferred, either alone or with female NOD mouse splenocytes, into NOD/scid recipient mice. NOD mouse splenocytes were also transferred alone as controls. Recipient mice transferred with NOD mouse splenocytes alone developed diabetes as early as at 6 wk after the cell transfer, and all recipient mice became diabetic by 18 wk after cell transfer (Fig. 3). In comparison, transfer of NR286 T cells alone did not induce diabetes, suggesting they were not diabetogenic. Interestingly, NR286 T cells not only delayed diabetes onset by 7 wk, but also inhibited diabetes development when they were co-transferred with NOD mouse splenocytes (Fig. 3), as 50% of NOD/scid recipient mice that were co- transferred with NR286 T cells and NOD mouse splenocytes remained diabetes-free by the end of the experiment (22 wk after the cell transfer). In summary, these in vivo studies demonstrated that NR286 T cells do not act like pro-inflammatory T cells that induce diabetes; instead, they may function as Treg, with the potential to inhibit diabetes development. NR286 T Cells Suppressed In Vitro Proliferation of Pathogenic BDC2.5 T Cells In vitro experiments were then performed under eight different culture conditions to further determine whether NR286 T cells functioned as Treg to suppress antigen-specific proliferation of Figure 1. Isolation and characterization of CD4+ NR286 T cells. (A) FACS analyses of purified NR286 T cells stained with tetAg7/p286 tetramer and an anti-CD4 antibody or tetAg7/p206 (as a negative control). The numbers shown in each quadrant represent the percentage of cells stained by the tetramer. (B) IL-2 production by NR286 T cells (26105/well) in response to various concentrations of p286 or control p206 peptides plus NOR mouse APCs (46105/well). Peptides were 5-fold serially diluted from 100 mg/ml. HT-2 cells were used as the indicator in a MTT assay. Data represent average 6 SEM, n = 3. doi:10.1371/journal.pone.0007863.g001 PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e7863