Rapamycin expands a sorted population of CD4⁺CD25⁺Tr cells. (A) CD4⁺CD25⁺ T cells isolated from spleens of Balb/c mice were sorted by FACS and the FACS profile of sorted CD4⁺CD25⁺ T cells is shown. Sorted cells were 93% CD4⁺CD25⁺ among which 46% were CD4⁺CD25^bright T cells. (B) Fold expansion of Balb/c CD4⁺CD25⁺ T cells 1, 2, and 3 weeks after culture in the presence of αCD3 plus αCD28 plus 1000 U/mL IL-2 (medium, □) or αCD3 plus αCD28 plus 1000 U/mL IL-2 plus rapamycin (rapamycin, ▪) was evaluated by direct cell counts. (C) After 3 rounds of stimulation with αCD3 plus αCD28 plus 1000 U/mL IL-2 (medium) or αCD3 plus αCD28 plus 1000 U/mL IL-2 plus rapamycin (rapamycin), Balb/c CD4⁺CD25⁺ T cells were left resting for 1 week with no further stimulation in the presence of low-dose 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). FITC indicates fluorescein isothiocyanate.