Soluble anti-CD28 mAb reduces iTreg generation in vitro. CD4⁺GFP⁻ cells from Foxp3^gfp reporter mice were purified by FACS and stimulated with anti-CD3 mAb plus irradiated TCD splenocytes as APCs in the absence or presence of TGFβ. Soluble anti-CD28 mAb was added at different concentrations into the culture. Four days after stimulation, cultured cells were harvested and measured for CD4, GFP, and intracellular Foxp3 expression. (A) Data showing the percentage of GFP⁺ (top panel) or Foxp3⁺ (bottom panel) cells on gated CD4⁺ cells. (B) Data presented as the mean of percentage of Teffs (GFP⁻) and Tregs (GFP⁺) cells in total CD4⁺ cells in triplicate wells. (C) Data presented as the mean ± SD of absolute numbers of Teffs (CD4⁺GFP⁻) and Tregs (CD4⁺GFP⁺) cells in triplicate wells. (D) In separate experiments, cultured cells were harvested and measured for CD4 and GFP expression and the culture supernatant was measured for IL-2 production. The data show the percentage of GFP⁺ on gated CD4⁺ cells (left y-axis), and IL-2 production (right y-axis). The data are presented as the mean ± SD in triplicate wells, and represent 1 of 3 replicate experiments.