CD4⁺CD25⁺ T cells cultured in the presence of rapamycin, but not CsA, are able to inhibit ongoing T-cell responses. Freshly isolated CD4⁺CD25⁺ T cells (2.5 × 10⁴) were stimulated with 1 × 10⁵ γ-irradiated (30 Gy) HLA-mismatched stimulator PBMCs and additional IL-2 and IL-15 in the absence or presence of CsA (400 ng/mL) and rapamycin (100 nM). Cells were harvested at day 7, washed 3 times, and rested. At day 10, the cells were examined for suppressor function in suppression coculture assays. ^ExpT_REGs derived from control (untreated), CsA-treated, and rapamycin-treated cultures (x axis) were cocultured from day 0 (T = 0) () or day 2 (T = 2) (▪) of MLR at a suppressor-to-effector ratio of 1:16 using 5 × 10⁴ responder CD4⁺CD25^- effector T cells and 5 × 10⁴ γ-irradiated (30 Gy) stimulator PBMCs. Proliferation at day 5 of culture was measured by ³H-thymidine incorporation and is depicted as a percentage of inhibition on the y axis. A representative experiment is shown. ^*P < .05 for difference between groups.