CD44^act is preferentially expressed on CD4CD25⁺ Treg cells after activation. (A) Absence of CD44^act expression on resting CD4CD25⁺ and CD4CD25^– T cells. (B) Kinetics of activation marker expression on CD25⁺ and CD25^– CD4 T cells. CD4 cells were separated into CD4CD25⁺ and CD4CD25^– fractions and activated at 1 × 10⁶ cells/well with increasing concentrations of plate-bound anti-CD3 with (open) or without (closed) 5 μg/mL anti-CD28. The percentage of CD69⁺ cells (top) and blast-positive cells (by scatter, middle) are nearly indistinguishable between populations, whereas the percentage of Fl-HA–binding cells differs significantly between CD4CD25⁺ and CD4CD25^– for both treatments (bottom; P ≤ .01, Wilcoxon test). (C) CD44^act expression on CD4CD25⁺ versus CD4CD25^– cells after 18-, 42-, and 72-hour activation with 5 μg/mL plate-bound anti-CD3/anti-CD28. Data shown are the mean ± SD from 3 independent experiments (*P ≤ .001; **P ≤ .01). FACS analyses from a representative experiment are shown below for the 18-hour time point with and without the HA-blocking anti-CD44 antibody, KM81.