T-cell proliferation and differentiation in the spleen. (A) WT splenocytes stimulated with α-CD3ϵ + α-CD28 proliferated better than KI splenocytes as measured by tritiated thymidine incorporation (P < .001, **P < .03). This is representative of 3 experiments. (B) Stimulated WT (red) splenic T cells proliferated more rapidly than KI (blue) cells as evidenced by the lower carboxyfluorescein succinimidyl ester (CFSE) staining (72 hours). (C) FoxP3 expression in unstimulated CD4⁺, CD25⁺ cells was similar in WT (5.4%) and KI (4.0%) mice. (D) Increased percentages of FoxP3⁺ cells in purified KI splenic T cells stimulated with α-CD3ϵ/WT antigen-presenting cells for 3 days followed by culture in interleukin-2 for 3 days (WT 3.9% FoxP3⁺ vs KI 13.8% FoxP3⁺). Data are representative of 4 experiments.