Human CD4⁺CD25⁺ Treg cells have a reduced capacity to phosphorylate AKT following TCR stimulation. (A) Ex vivo CD4⁺ T cells were stimulated with crosslinked αCD3/CD28 mAbs for the indicated times and stained for CD4 and CD25, and geometric MFIs of cells stained with anti–phospho-AKT (Ser473) were determined in CD4⁺CD25^high or CD4⁺CD25⁻ T cells. (B) More than 90% pure CD4⁺CD25^high and CD4⁺CD25⁻ T cells were left unstimulated or stimulated with αCD3/CD28 mAbs for 10 minutes, lysed, and analyzed by Western blotting for amounts of AKT (Ser473) phosphorylation. Blots were reprobed with anti-p38 Abs to ensure equivalency of loading. (C) Ex vivo CD4⁺ T cells were stimulated and analyzed as in panel A, and MFIs following staining with anti–phospho-AKT (Thr308) were determined. (D-E) Ex vivo CD4⁺ T cells were stimulated as in panel A and then stained for CD4, FOXP3, and phospho-AKT (Ser473). (D) Histograms depicting levels of phospho-AKT were generated by gating on subsets of CD4⁺FOXP3⁺ or CD4⁺FOXP3⁻ T cells, and (E) MFIs of cells stained with anti–phospho-AKT (Ser473) were determined in CD4⁺FOXP3⁺ or CD4⁺FOXP3⁻ T cells. Panels A, C, and E represent a single experiment with the inset depicting the fold change in MFI from resting to activation (10 minutes) for all experiments, and with horizontal bars indicating means and asterisks indicating significance. For panels B and D, a single representative example of 3 experiments performed is depicted.