Single-cell analysis of MAPK activation in ex vivo human CD4⁺CD25⁺ Treg cells and CD4⁺CD25⁻ T cells following TCR activation. (A) Ex vivo CD4⁺ T cells were left unstimulated or stimulated with crosslinked αCD3/CD28 mAbs for 10 minutes and stained for CD4, CD25, and phospho-ERK1/2. Histograms depicting levels of phospho-ERK were generated by gating on subsets of CD4⁺CD25^high or CD4⁺CD25⁻ T cells. (B) Geometric mean fluorescence intensities (MFIs) of cell populations stained with anti–phospho-ERK1/2 or (C) anti–phospho-p38 Abs were determined over a 60-minute time course following activation with αCD3/CD28 mAbs. The experiment was performed 3 times with similar results, and a representative analysis is shown. The inset is the fold change in MFI from resting to activation (at 10 minutes), with each point representing a separate experiment and horizontal bars representing the mean.