Antigen-Specific Foxp32 Treg Figure 5. Blockade of IFN-c and NO production abolished NR286 T cell regulatory function. (A) CFSE-labeled CD4+ BDC2.5 T cells (26105/ well) were cultured under seven different culture conditions. In control cell cultures, BDC2.5 T cells were cultured in conditions of either (1) alone or (2) with p79 plus CD4+ cell-depleted irradiated APCs for activation. BDC2.5 T cells were also cultured with (3) an equal number of NR286 T cells. In some experiments, antibodies against (4) IFN-c or (5) IL-10 (24 mg/ml), and NO inhibitors, either (6) L-NMMA (active) or (7) D-NMMA (inactive) (0.2 mM), were added to the cultures to determine their effect on blocking the regulatory function of NR286 T cells. The number shown in each histogram represents the percentage of BDC2.5 T cells that underwent ,2 rounds of cell division. Data are from at least 2 experiments. (B) CFSE- labeled CD4+ BDC2.5 T cells (26105/well) were cultured under three conditions. Similar to those in (A), in control cell cultures, BDC2.5 T cells were cultured in conditions of either (1) alone or (2) with p79 plus CD4+ cell-depleted irradiated APCs for activation. BDC2.5 T cells were also cultured with (3) antibody against IFN-c (24 mg/ml), in the absence of NR286 T cells. (C) Cell culture supernatants from (A) were harvested for ELISA to detect the presence of IFN-c, IL-10, and NO. doi:10.1371/journal.pone.0007863.g005 possibility that anti-IFN2c blocked IFN2c production by BDC2.5 T cells, thereby enhancing its proliferation in the absence of NR286 Treg. Similarly, in the presence of the active NO inhibitor (L-NMMA), the percentage of non-dividing (or with reduced number of cell divisions) CD4+ BDC2.5 T cells also decreased to ,38% (Fig. 5A, condition 6). On the other hand, the addition of an anti-IL10 antibody or an inactive NO inhibitor analogue (D-NMMA) to the cell cultures did not restore BDC2.5 T cell proliferation (Fig. 5A, conditions 5 and 7, respectively). These results suggested that NR286 T cell regulatory function was dependent on IFN2c and NO production. Additional mechanistic studies were then performed to better understand the relative inhibitory effects on production of individual soluble factors (IFN2c vs. NO). Cell culture superna- tants were collected from seven different culture conditions for further analyses. These results showed that NR286 T cells significantly reduced the production of IFN2c in cell culture (Fig. 5C, conditions 2 vs. 3), while the production of NO was increased (Fig. 5C, conditions 2 vs. 3). In the presence of an anti- IFN-c antibody (Fig. 5C, condition 4), IFN-c was not detected in the cell culture supernatant. Unexpectedly, NO production (15 mM) in these cultures (Fig. 5C, condition 4) was also reduced compared to cultures without an anti-IFN2c antibody (condition 3), which contained more than 40 mM of NO despite a reduced amount of their IFN2c. These results suggested that blockade of IFN2c production also suppressed NO production. In addition, although more IFN2c was detected in cultures of p79-activated BDC2.5 cells without NR286 T cells (Fig. 5C, condition 2) than with NR286 T cells (condition 3), it did not lead to an increased NO production. These results suggested that IFN2c production alone would not induce NO production in the absence of NR286 T cells. In contrast to the IFN2c blockade effect on NO production, addition of the active NO inhibitor, L-NMMA, to cell cultures not only inhibited NO production in cell cultures but also restored IFN2c production (Fig. 5C, condition 6 vs. 3). As a negative control, the presence of the inactive NO inhibitor, D-NMMA, did not change the production of either IFN2c or NO (Fig. 5C, condition 7 vs. 3). In addition, although blockade of IL-10 restored IFN2c production, it also reduced NO production (Fig. 5C, condition 5 vs. 3). Altogether, these results implicated IFN-c and NO secretion as mediators of NR286 T cell regulatory function. In addition, these data suggested the production of IFN-c may precede that of NO PLoS ONE | www.plosone.org 5 November 2009 | Volume 4 | Issue 11 | e7863