Expression of markers associated with CD4CD25⁺ Treg cells is similar irrespective of CD44^act levels after activation. (A) After 24-hour activation in vitro, cells were stained and analyzed by flow cytometry. Gating for histograms was done on the basis of Fl-HA binding, as indicated in the top panel. The HA-negative gate (left) is established based on the limits of KM81 blocking of the HA-positive population. Marker expression for total resting CD4CD25⁺ cells (gray line) is also shown for comparison. (B) Relative Foxp3 mRNA levels as measured by real-time quantitative PCR analysis in CD44^act+ and CD44^act– fractions of CD4CD25⁺ cells activated for 24 hours and separated as in Figure 3. Foxp3 levels in unactivated CD4CD25⁺ and CD4CD25^– T cells are shown for comparison. Data shown are the mean ± SD of 3 experiments. P = .003, comparing CD44^act+ Treg cells to unactivated CD4CD25^– cells; P = .007, comparing CD44^act– Treg cells to unactivated CD4CD25^– cells.