Antigen-Specific Foxp32 Treg Figure 4. NR286 T cells suppress proliferation of BDC2.5 T cells. (A) Inhibition of CFSE-labeled BDC2.5 T cell proliferation co-cultured with NR286 T cells. CD4+ BDC2.5 T cells were isolated from BDC2.5 TCR transgenic mice and labeled with CFSE (26105/well), then cultured under eight different conditions (indicated as the numbers in parenthesis shown in each histogram). In control cultures, BDC2.5 T cells were cultured alone either (1) without p79 or (2) with p79 (0.1 mg/ml) plus CD4+ cell-depleted irradiated APCs from NOD mice. Cells also were co-cultured in the presence of either NR286 T cells (conditions 3–5) or with NR286 T cells plus p286 (conditions 6–8) for 4 days. NR286 T cells were added to cell cultures with a 2- fold serial dilution starting at a 1:1 ratio (BDC2.5 T cells: NR286 T cells). The numbers shown in each histogram represent the percentage of cells that underwent ,2 rounds of cell division. Data are representative from at least n = 3. (B) Cell culture supernatant was harvested from 4-day cell cultures under the same eight different conditions as shown in (A). Levels of IL-10 detected in conditions 3, 4, and 6–8 were significantly reduced compared to condition 2 (p,0.01). NO production in conditions 3–8 was significantly increased compared to conditions 1 and 2. doi:10.1371/journal.pone.0007863.g004 In addition to measuring the suppressive effect of NR286 T cells on BDC2.5 T cell proliferation, cytokine production under the different culture conditions was also analyzed. ELISA was used to analyze supernatants collected from cell cultures under the various conditions with or without p286-activation of NR286 T cells. As predicted, the supernatant from all conditions, except BDC2.5 T cells alone, contained a comparative amount of IFN-c (Fig. 4B). None of the cultures produced detectable levels of IL-4. In addition, compared to cultures containing BDC2.5 T cells activated by p79 plus antigen presenting cells (APCs)(Fig. 4B, condition 2), the production of IL-10 was significantly reduced in the presence of either p286-activated or non-activated NR286 T cells, especially when NR286 T cells were present at a ratio of 1:1 or 1:0.5 (Fig. 4B, conditions 3, 4, 6 and 7). While the reason for this reduction has not yet been identified, it could simply have been that the IL-10 produced by BDC2.5 T cells was suppressed by NR286 T cells. We were unexpectedly also able to detect a significant amount of nitric oxide (NO) present in the supernatants in all cell cultures containing NR286 T cells (Fig. 4B, conditions 3–8). In comparison, the supernatant from cell cultures containing BDC2.5 T cells alone, either with or without p79, did not produce significant amounts of NO. These results demonstrated that NR286 T cells were able to function as Treg in vitro by suppressing the target T cell proliferation; soluble factors other than IL-4 may be involved in this suppressive process. The Regulatory Function of NR286 T Cells is Dependent on Production of Both IFN2c and NO In order to further understand how NR286 T cells may function as Treg, we investigated the mechanisms underlying their suppression of BDC2.5 T cell proliferation. In particular, cytokine or NO blockade experiments were performed under seven different culture conditions to examine whether NR286 T cell regulatory function was dependent upon secretion of soluble factors detected in cell cultures (Fig. 5A). Neutralizing antibodies against either IFN2c or IL-10, or inhibitors against NO were added to the cell cultures to block the potential effect of these soluble factors on BDC2.5 T cell proliferation in the presence of NR286 T cells. The results showed that blockade of IFN-c and NO, but not IL-10, resulted in a significant increase in BDC2.5 T cell proliferation. In the presence of an anti-IFN2c antibody, the percentage of CFSE-labeled CD4+ BDC2.5 T cells with less ,2 rounds of cell division decreased from ,57% to 30% (Fig. 5A, condition 3). In control experiments, our results showed that anti- IFN2c blockade alone did not alter BDC2.5 T cell proliferation (Fig. 5B, condition 2 vs. 3,). Therefore, these results excluded the PLoS ONE | www.plosone.org 4 November 2009 | Volume 4 | Issue 11 | e7863