Plate-bound anti-CD28 mAb reduces iTreg generation in vitro. CD4⁺CD25⁻ cells were purified from spleen and lymph node cells from normal B6 mice and stimulated with plate-bound anti-CD3 mAb plus anti-CD28 mAb in the absence of APCs. Exogenous TGFβ was also included in the culture with or without additional IL-2. Four days after stimulation, cultured cells were harvested and measured for CD4, CD25, and intracellular Foxp3. (A) Expression of CD25 and Foxp3 on gated CD4⁺ cells. (B) Data presented as the mean of percentage of Teffs (CD25⁺Foxp3⁻) and Tregs (CD25⁺Foxp3⁺) in total CD4⁺ cells in triplicate wells. (C) Data presented as the mean ± SD of absolute numbers of Teffs (CD4⁺CD25⁺Foxp3⁻) and Tregs (CD4⁺CD25⁺Foxp3⁺) in triplicate wells. The data represent 1 of 4 replicate experiments.