and help trigger NO production, resulting in suppression of the target BDC2.5 T cells. In Vitro Regulatory Function of NR286 T Cells Is Cell- Contact Dependent In order to examine whether NR286 T cell regulatory function depended solely on cytokine production, transwell assays were performed to determine whether NR286 T cells were still able to suppress cell proliferation without being in direct contact with the target BDC2.5 T cells. In these assays, NR286 T cells were cultured in the upper well and were separated from BDC2.5 T cells (cultured in the lower well) of transwell plates. CD4+ BDC2.5 T cells were labeled with CFSE and cultured either alone or stimulated with p79 in the presence of CD4+ cell-depleted APCs. Unexpectedly, neither p286-activated nor non-activated NR286 T cells suppressed BDC2.5 T cell proliferation (Fig. 6A, conditions 3 and 4 vs. condition 2). The addition of an anti-IFN-c antibody or the NO inhibitor L-NMMA had no effect on either restoring or enhancing proliferation of BDC2.5 T cells (Fig. 6A, conditions 5 and 6). IFN-c and NO production were also assessed under the same culture conditions. IFN-c was detected in the supernatant of cell cultures containing BDC2.5 T cells stimulated with p79 either in the absence or presence of NR286 T cells (Fig. 6B, conditions 2, 3, and 4). As expected, addition of an anti-IFN-c antibody blocked the production of IFN-c in cell cultures, and the NO active inhibitor L-NMMA had no effect on IFN2c production (Fig. 6B, condition 5 and 6). In comparison, no significant levels of NO were detected in any of the conditions tested (Fig. 6B). These results suggested that cell contact between NR286 T cells and target cells was necessary for NO but not IFN-c production and for suppression of BDC2.5 T cell proliferation. Antigen-Specific Foxp32 Treg Our data demonstrated that NR286 T cell regulatory function was not only dependent on IFN-c and NO, but also on cell contact with target cells; similarly, NO production was also dependent on cell contact. These results provide evidence to support the conclusion that NO may be an effector molecule downstream of IFN2c in mediating NR286 T cell regulatory function, which is also dependent on cell contact between NR286 T and target cells. Discussion We report here a novel population of Foxp3-negative GAD p286-specific NR286 Treg isolated from NOR mice. While Th1- like NR286 T cells secreted significant amounts of IFN-c, they functioned as Treg. Their regulatory function was dependent not only on IFN-c, but also on NO and cell-contact with target cells. Similar to Foxp3-expressing CD4+CD25+ nTreg, NR286 Treg expressed CD25 and were cell-contact dependent; however, they did not express Foxp3. In addition, separating NR286 Treg cells from target T cells abolished both NO production and suppression of target T cells, regardless of whether IFN2c was produced in cell cultures. These results showed that production of IFN2c alone by antigen-activated pathogenic T cells, such as BDC2.5 T, was insufficient to induce NO production unless Treg, such as NR286 T cells, were also present. Our studies did not distinguish the producers of IL-10, IFN2c or NO in cell culture studies showing the suppression of BDC2.5 T cell proliferation by NR286 Treg. However, our results demon- strated that suppression of BDC2.5 T cells occurred only when NR286 Treg were present in cell cultures. In addition, blockade of IFN2c alone, in the presence of NR286 Treg, did not alter BDC2.5 T cell proliferation. Even if these soluble factors produced by activated BDC2.5 T cells contributed to the suppression of BDC2.5 T cells, the presence of NR286 Treg was required for the Figure 6. Inhibition of BDC2.5 T cell proliferation is cell contact dependent. (A) Transwell analyses of BDC2.5 T cell proliferation. CD4+ BDC2.5 T cells were isolated from BDC2.5 TCR transgenic mice and labeled with CFSE (56105/well). BDC2.5 T cells were cultured either (1) alone or (2) activated with p79 in the transwell lower well (L.W.). In the upper wells (U.W.), an equal number of (3) NR286 T cells alone, (4) NR286 T cells plus p286, or NR286 T cells in the presence of either (5) anti-IFN-c or (6) the active NO inhibitor L-NMMA were cultured for 4 days. The CFSE-labeled BDC2.5 T cells (L.W.) were harvested and cell proliferation analyzed by FACS. Data are from at least 2 experiments. (B) Cell culture supernatants obtained from the six cell culture conditions were harvested for analysis of IFN-c and NO production. doi:10.1371/journal.pone.0007863.g006 PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e7863