Rapamycin fosters regulatory function of expanded CD4⁺CD25⁺ T cells. (A) Schematic representation of CD4⁺CD25⁺ T-cell expansion culture. (B) 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 and rapamycin. CsA and rapamycin were added at the start of the cultures at indicated concentrations. Cells were harvested at day 7, washed 3 times, and rested. At day 10, the cells were examined for suppressor function in coculture assays. ^ExpT_REGs derived from control (untreated), CsA-treated, and rapamycin-treated cultures were cocultured at the indicated suppressor-to-effector ratios (x axis) using 5 × 10⁴ responder CD4⁺CD25^- effector T cells and 5 × 10⁴ γ-irradiated (30 Gy) stimulator PBMCs. Proliferation at day 5 of culture as measured by ³H-thymidine incorporation is shown on the y axis. One representative experiment is shown (n = 6). ^*P < .05 for difference between groups. (C) CD4⁺CD25⁺ T cells were cultured as described for panel B. CsA and rapamycin were added at the start of primary MLR at indicated concentrations (x axis). Cells were harvested at day 7, washed, rested, and cocultured at a suppressor-to-effector ratio of 1:128 with 5 × 10⁴ responder CD4⁺CD25^- T cells and 5 × 10⁴ γ-irradiated (30 Gy) stimulator PBMCs (). Control cultures were performed without the addition of suppressor cells (▪). Proliferation at day 5 of culture, as measured by ³H-thymidine incorporation, is shown on the y axis. One representative experiment of 3 independent experiments is shown. Because of small sample sizes, the conditions in which treated cells were used were grouped together, resulting in 3 groups: untreated cells, CsA-treated cells, and rapamycin-treated cells. ^*P < .01 for difference between groups. NS indicates not significant.