Partially impaired CD4⁺CD25⁺ Treg function in p110γ^KOδ^D910A mice. (A-C) Cells from thymus, spleen, and lymph nodes of WT and p110γ^KOδ^D910A mice were stained with fluorescence-conjugated antibodies against surface marker CD4, et al. (A). Percentage of Foxp3⁺ cells gated from CD4⁺CD8⁻ cells in thymus or CD4⁺ cells in spleen and lymph nodes. (B,C) Mean fluorescent intensity of Foxp3 and GITR on CD4⁺CD8⁻CD25⁺ cells in thymus or CD4⁺CD25⁺ cells in spleen and lymph nodes. (D) Purified 5 × 10⁴ CD4⁺CD25⁺ and/or CD4⁺CD25⁻ cells from WT or p110γ^KOδ^D910A (−/−) mice were incubated with T-cell–depleted and mitomycin-treated splenocytes and 1 μg/mL α-CD3 in 96-well plate for 72 hours for measuring proliferation. (E,F) TNF-α and Il-4 produced by T cells shown in panel D. Data represent at least 2 experiments with triplicates for each sample (± SEM). ■ represents WT; □, p110γ^KO_δD910A.