CD4⁺CD25^highFoxP3⁺ Treg-cell levels and proliferation of PBMCs after in vitro treatment with denileukin diftitox. PBMCs from a healthy donor were treated with up to 8 nM denileukin diftitox or media alone (0 nM) for 18 to 20 hours and then cultured in media containing 10 U/mL IL-2 for 3 days. (A) Cells were stained for CD4, CD25, and Foxp3 expression on days 1 (after 18 hours of denileukin diftitox) and 4. The percentage of CD4⁺CD25^highFoxp3⁺ cells are represented for each concentration. The percentage of CD4⁺CD25^highFoxp3⁺ cells from freshly isolated PBMCs before the addition of denileukin diftitox is represented by a dashed line (1.82%). Data are representative of 3 repeated experiments. (B) PBMCs similarly depleted of Treg cells with denileukin diftitox and then rested in IL-2 for 3 days were stimulated in a proliferation assay with 0.5 μg/mL soluble OKT3 or media alone (unstimulated) for 4 days. Proliferation was determined by [³H]thymidine incorporation during a final 18 hours of culture, and the data are represented as mean cpm plus or minus SD. *P < .01 compared with 0 nM. Data are representative of 3 repeated experiments.