Rapamycin expands CD4⁺CD25⁺FoxP3⁺ Tr cells. (A) After 3 rounds of stimulation with APCs plus OVA (medium) or APCs plus OVA plus rapamycin (rapamycin), DO11.10 tg CD4⁺ T cells were left resting for 1 week with no further stimulation in the presence of IL-2 (50 U/mL). After 7 days cells were analyzed by FACS. Cells were gated on CD4⁺CD25⁺ cells and numbers represent percentages of the 3 different CD25⁺ subsets (ie, bright, dim, and low). Naive CD4⁺ T cells from a DO11.10 tg mouse were used as control. One representative experiment of 6 is presented. (B) Content of CD25^bright T cells in medium and rapamycin cultures in each of the 6 experiments is presented. ^*.001 < P ≤ .05. (C) A total of 1 × 10⁶ DO11.10 tg CD4⁺ T cells (containing 70 000 CD4⁺CD25^bright T cells) were cultured with APCs plus OVA (medium; □) or APCs plus OVA plus rapamycin (rapamycin; ▪). After 3 rounds of stimulation, the total number of CD4⁺CD25^bright T cells was determined by FACS. One representative experiment of 6 is presented.