Antigen-Specific Foxp32 Treg Figure 2. Cytokine production and phenotype analyses of NR286 T cells. (A) Analysis of cytokine production by NR286 T cells (26105/well) in response to various concentrations of p286 plus APCs. Cell culture supernatant was harvested after 24 h cell culture for ELISA (lower detection limit, 8 pg/ml for IL-4 and 30 pg/ml for IL-10 and IFN-c). Data represent average 6 SEM, n = 3. (B) Phenotypic analysis of NR286 T cells. NR286 T cells were co-stained with anti-CD4 plus antibodies against indicated markers. Numbers in each quadrant represent the percentage of cells positively detected by antibodies. doi:10.1371/journal.pone.0007863.g002 Figure 3. NR286 T cells inhibit diabetes development. NOD/scid recipient mice were transferred with either NOD mouse splenocytes (Nspl)(16107 cells/mouse) alone, NR286 T cells alone (16107 cells/ mouse), or co-transferred with an equal numbers of both NR286 T cells and Nspl. The difference of diabetes onset and development between co-transferred mice and Nspl single transferred mice was significant (p,0.002).NOD mouse splenocytes were isolated from 7–8 wk old female NOD mice. At least 8 mice were studied in each group. doi:10.1371/journal.pone.0007863.g003 other T cells. The proliferation of CFSE-labeled CD4+ BDC2.5 T cells in response to stimulation by p79, a highly active synthetic peptide in stimulating the diabetogenic BDC2.5 T cells [26], was measured with and without the presence of NR286 T cells in the culture. The results showed that only ,11% of BDC2.5 T cells did not proliferate beyond more than two rounds of cell division in the presence of p79 (condition 1), compared to ,88% in the absence of p79 (condition 2) (Fig. 4A). Our results further showed that both non-activated (Fig. 4A, conditions 3–5) and p286-activated NR286 T cells (conditions 6–8) were able to suppress the proliferation of BDC2.5 T cells in response to p79. The percentage of non-dividing CFSE-labeled CD4+ BDC2.5 cells and cells with less than two rounds of cell division increased from ,11% to ,55% in the presence of an equal number of non-activated NR286 T cells (condition 3), and to ,59% in the presence of p286-activated NR286 T cells (condition 6). In addition, the suppression of BDC2.5 cell proliferation by NR286 T cells was concentration-dependent, that is, proportional to the number of NR286 T cells present in the cell culture containing BDC2.5 cells. The data showed that, compared to the 1:1 BDC2.5:NR286 T cell ratio, the percentage of cells with,2 rounds of cell division at 1:0.25 ratio decreased from ,55% to ,27% (condition 3 vs. 5; without p286 to activate NR286 T cells) and from ,59% to ,35% (condition 6 vs. 8; with p286 to activate NR286 T cells) (Fig. 4A). PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7863