CD4⁺CD25⁻ T cells transduced with siRNA against FOXP3 proliferate to a greater extent than CD4⁺CD25⁻ T cells transduced with control siRNA against luciferase. CD4⁺CD25⁻ T cells transduced with siFOXP3 or siLuc were purified as ΔLNGFR⁺ cells, then labeled with CFSE and stimulated with different ratios of anti-CD3/anti-CD28–coated beads. Transduced T cells were stained with anti-CD4, anti-ΔLNGFR, and anti-FOXP3 Abs. (A) Representative FACS plots of FOXP3 expression in purified siFOXP3 and siLuc-transduced T cells 3 days after activation at a 1:32 bead to cell ratio. (B) FOXP3 expression in ΔLNGFR⁺ siFOXP3-transduced T cells and control siLuc-transduced T cells after anti-CD3/anti-CD28 bead stimulation (n = 4). (C) Average division index of siFOXP3-transduced T cells and control siLuc-transduced T cells (n = 4). (D) CD4⁺CD25⁻ T cells transduced with siFOXP3 or siLuc were stimulated with different ratios of anti-CD3/anti-CD28–coated beads at 5 × 10⁵ cells/mL. Supernatants were collected 20 hours later and analyzed for IFN-γ and IL-2 by ELISA (n = 4). Error bars represent SEM. *P < .05.