Lck activation through TCR and CD28 regulates iTreg generation. CD4⁺CD25⁻ cells isolated from CD28-WT and CD28-Lck Tg mice were stimulated with plate-bound anti-CD3 in the presence of TGFβ, IL-2, and the indicated concentrations of pp1. Cells were also stimulated without (A) or with (B) 40 μg/mL of plate-bound anti-CD28 mAb. Four days after stimulation, cultured cells were harvested and measured for the expression of surface CD4, CD25, and intracellular Foxp3. The data show mean ± SD of percent Tregs (CD25⁺Foxp3⁺ among gated CD4⁺ cells) in triplicate wells, and represent 1 of 3 replicate experiments.