Treg expansion cultures contain cytokine-producing FOXP3⁻ and FOXP3⁺ non-Tregs. (A) Fold expansion of magnetic bead–purified CD4⁺CD127^lowCD25⁺ T cells from 6 donors after stimulation with anti-CD3/CD28 and IL-2 in the absence (none) or presence of rapamycin. Error bars represent SEM. (B) Percentage of CD4⁺FOXP3⁺ T cells in the starting population and after expansion of CD4⁺CD127^lowCD25⁺ T cells as described in panel A. Horizontal lines represent the mean of each group. (C) Day 14 expansion cultures generated in the absence (none) or presence of rapamycin were restimulated for 5 hours with PMA/ionomycin. IFN-γ and IL-2 production was evaluated by intracellular staining. Data are representative of 6 independent experiments. The number in each quadrant represents the percentage of total population.