(A) Purified CD4⁺CD25⁺CD45RB^lo and CD4⁺CD25⁺CD45RB^hi cells from both wild-type and PTEN-ΔT mice were cultured at a constant density of 1 × 10⁵/well in the presence of rIL-2 (100 U/ml) for a 2-week period. At the indicated time points, total cell numbers were quantitated by trypan blue exclusion. Only PTEN-ΔT CD4⁺CD25⁺CD45RB^lo cells exhibited a significant increase in cell number with all other cell types showing overlapping live cell numbers over the examination period. Data are representative of 5 different experiments. (B) Purified CD4⁺CD25⁺CD45RB^lo cells from wild-type, PTEN-het (HET), and PTEN-ΔT mice were cultured in the presence of rIL-2 (100 U/ml) for 48 hours. Tritiated thymidine was added to cultures for the final 16 hours before harvesting. (C) PTEN-ΔT CD4⁺CD25⁺CD45RB^lo cells were CFSE labeled and cultured in the presence of rIL-2 (100 U/ml) for 10 days. CFSE dilution was analyzed at the indicated time points by FACS analysis. Results are representative of 4 separate experiments. FSC, forward scatter. (D) Purified wild-type and PTEN-ΔT CD4⁺CD25⁺CD45RB^lo cells and preactivated CD4⁺ T cell blasts were stimulated with titrated doses of rIL-2 as shown for 72 hours. Tritiated thymidine was added to cultures for the final 16 hours before harvesting. (E) CD4⁺ T cells were isolated from ER-Cre⁺/Pten^flox/flox mice and lysed immediately or after culture in the presence of rIL-2 (100 U/ml) and 4-OHT (1 nM). Samples were electrophoresed on an SDS-PAGE gel, transferred to nitrocellulose membranes, and probed as indicated. Purified CD4⁺ T cells from a littermate control mouse were used as a positive control (+ve ctrl) for PTEN expression. (F) Purified ER-Cre⁺/Pten^flox/flox CD4⁺CD25⁺ Tregs were CFSE labeled and cultured in the presence of rIL-2 (100 U/ml) and 4-OHT (1 nM) for 7 days. CFSE dilution was analyzed by FACS. Results are representative of 3 independent experiments. Data shown represent mean ± SD of triplicate samples.