Fresh peripheral blood nTregs were purified based on flow cytometric sorting of the CD4+, CD25-bright T cell subset (brightest 1/3 of CD25+ cells; Figure 6A/B) or by a bead based method based on a CD4+, CD25+, CD127- surface antigen expression profile (Figure C/D) then expanded for four days with CD3/CD28 beads and IL-2 with and without 10 ng/mL rapamycin. Both isolation methods gave similar results. CD4 and FOXP3 expression were then evaluated on day 4 and showed relative purity of the resulting Treg populations (90.8% and 90.5% in panel A and 85% and 83% as indicated in panel C). Tregs were then co-cultured at a 3:1 ratio of Tregs (Tr = Tregs expanded without rapamycin; TrRapa = Tregs expanded with rapamycin) to CFSE labeled L428 target cells. Samples were plated in triplicate in media without additional stimuli or inhibitors. After 6 or 48 hours as indicated, apoptosis was assessed in the CFSE labeled L428 target cell population by flow cytometric evaluation of annexin-V binding (early apoptotic cells) or propidium iodide (PI) staining (late apoptotic cells) in the 6 and 48-hour assays, respectively (panels B and D). Control samples consisted of CFSE-labeled L428 cells alone, L428 cells alone exposed to 10 μM staurosporine (6 hour cytotoxicity assays only) and nonactivated Treg-depleted T cells (Tc) plated at a 3:1 ratio with labeled L428 target cells. Representative dot plots for a 6-hour cytotoxicity assay, with annexin-V positive apoptotic target cells highlighted with percentages, are shown in panel B. Replicates from a 48-hour cytotoxicity assays are graphed in panel D. Statistical comparison of the indicated data sets was performed using a t-test (NS-not significant with P > .05, * denotes P < .02, ** denotes P = .003). The data shown in both of the timepoints are representative of three experiments using multiple donors.