Diminished AKT phosphorylation in CD4⁺CD25⁺ Treg cells results in decreased activation of downstream effectors. (A) More than 90% pure CD4⁺CD25^high and CD4⁺CD25⁻ T cells were left unstimulated or stimulated with αCD3/CD28 mAbs for the indicated times, lysed, and analyzed by Western blotting for amounts of phosphorylated FOXO1 and FOXO3a. Blots were reprobed with anti-p38 Abs to ensure equivalency of loading. Shown is a representative experiment of 3 performed. (B) Ex vivo CD4⁺ T cells were stimulated with αCD3/CD28 Abs for the indicated times, and MFIs following staining with anti–phospho-S6 (Ser235/236) were determined in CD4⁺CD25^high or CD4⁺CD25⁻ T cells. A single representative experiment is depicted, and the inset represents the fold change in MFI from resting to activation (10 minutes) for each separate experiment, with horizontal bars indicating means and the asterisk indicating significance.