CD28-mediated Lck and PI3K signals contribute to the suppression of iTreg generation. CD4⁺CD25⁻ cells were purified from a series of Tg mice on a CD28-deficient background that bears WT CD28 or unmutated CD28 in its cytosolic tail incapable of binding to Lck (CD28-Lck), PI3K (CD28-PI3K), or Itk (CD28-Itk). Purified CD4⁺CD25⁻ cells were then stimulated with plate-bound anti-CD3 and the indicated concentrations of anti-CD28 in the presence of TGFβ without (A) or with (B) additional IL-2 at a 2-ng/mL concentration. Four days after stimulation, cultured cells were harvested and measured for expression of surface CD4, CD25, and intercellular Foxp3, and CD4⁺CD25⁺Foxp3⁺ cells were considered to be iTregs. The data show the percentage relevance of iTreg generation with anti-CD28 to that without anti-CD28. The numbers are presented as the mean ± SD of percent relevance of pooled data from triplicate experiments.