Allo-specific Treg-proliferative response and expansion. (A) Tregs were freshly purified and stimulated with γ-irradiated HLA-mismatched APCs plus rapamycin (100 ng/mL) in the presence or absence of IL-2 (10 U/mL) and IL-15 (10 ng/mL). Each bar represents mean ± SD of ³H-thymidine uptake (CPM) on day 6. Results are one representative of 5 individual experiments. (B) Expansion of allo-specific Tregs (2 × 10⁴/well in triplicate) stimulated with allogeneic DCs (10:1) plus rapamycin (100 ng/mL) in the presence or absence of IL-2 (10 U/mL) or IL-15 (10 ng/mL) for 12 days. Each bar represents mean ± SD of the absolute number of Tregs obtained by timed acquisition after 12 days of expansion. A representative of 5 experiments with similar results is shown. (C) Absolute number of Tregs 12 days after stimulation with allogeneic DCs in the presence or absence of rapamycin, IL-2 (10 U/mL), or IL-15 (10 ng/mL). (D) Bcl-2 expression by expanded Tregs in the presence of IL-2 and IL-15 (bold line) or IL-2 alone (thin line) gated on CD4⁺CD25⁺CD127⁻ Tregs. Data shown are representative of 3 experiments. *P < .05; **P < .01.