Rapamycin preserves a high CD27⁺T_REG/CD27^-T_REG ratio. (A) The expression of CD25 and CD27 on CD4⁺CD25⁺ T_REG expanded in the absence or presence of CsA or rapamycin was measured by flow cytometry. Freshly isolated CD4⁺CD25⁺ T cells (2.5 × 10⁴) were stimulated with 1 × 10⁵ γ-irradiated (30 Gy) allogeneic PBMCs and additional IL-2 and IL-15. CsA and rapamycin were added at the start of primary MLR at indicated concentrations. Cells were harvested at day 7, washed, rested, and analyzed for CD25 and CD27 expression at day 10 of culture. Dot plots show CD25 and CD27 expression of live-gated cells. One representative experiment of 3 independent experiments is shown. (B) CD27⁺T_REG/CD27^-T_REG subset ratios (y axis) within the CD25⁺ fraction were calculated for the ^ExpT_REG pool, the ^ExpT_REG CsA pool (cultured with 400 ng/mL CsA), and the ^ExpT_REG rapamycin pool (cultured with 100 nM rapamycin). The results of 6 independent experiments are shown. ^*P < .05 for difference between groups. (C) The intracellular expression profile of FOXP3 in the ^ExpT_REG pool, the ^ExpT_REG CsA pool (cultured with 400 ng/mL CsA), and the ^ExpT_REG rapamycin pool (cultured with 100 nM rapamycin) was determined by flow cytometry. Histograms show intracellular FOXP3 expression of live-gated cells. Dotted line indicates CD4⁺CD25^- cells; solid line, CD4⁺CD25⁺ cells.