(A) Splenocytes from DO11.10 TCR transgenic mice were stimulated with ova peptide (1 μg/ml). CD4⁺ T cells were purified by magnetic bead separation at the indicated time points and subsequently lysed. Samples were analyzed for PTEN expression by Western blot, and membranes were stripped and reprobed for β-actin as a loading control. Results are representative of 3 independent experiments. (B) Purified CD4⁺ T cells were retrovirally transduced, as described in Methods, with either MIGR1-NGFR empty vector or PTEN-containing virus. Cells were analyzed for expression of human NGFR 48 hours after infection. Data shown illustrate typical transduction efficiencies achieved using these vectors. The gate drawn shows NGFR-positive subsets used for comparison in subsequent experiments. (C) NGFR-positive cells were purified either by FACS sort or magnetic bead separation and cultured in the presence or absence of rIL-2 (20 U/ml) for 48 hours. Tritiated thymidine was added to cultures for the last 16 hours before harvesting. Data shown represent the mean ± SD of triplicate cultures and are representative of 4 independent experiments. (D) Cells as in C were analyzed for viability by 7-AAD incorporation. Data shown is representative of 4 independent experiments. (E) Purified CD4⁺ T cells were CFSE labeled before stimulation and retroviral transduction. After infection, cells were cultured in the presence of rIL-2 (10 U/ml) for 48 hours, and cells expressing identical levels of NGFR were analyzed for CFSE dilution by flow cytometry.