Effect of denileukin diftitox on murine and human Treg cells in vitro. (A) Whole murine splenocytes from C57BL/6 mice were treated with the indicated doses of denileukin diftitox for 2 hours, then washed, and incubated at 37°C for 72 hours. Splenocytes were stained for CD4⁺CD25⁺Foxp3⁺ Treg cells. The plots shown were gated on CD4⁺ T cells. Percentage of CD25⁺Foxp3⁺ cells is indicated in the upper right quadrant of each plot. (B-D) PBMCs from healthy human donors were treated with 290 ng/mL (5 nM) denileukin diftitox for 72 hours. Cells were stained for CD4 and CD25. CD4⁺CD25⁺ cells were gated into CD25^high and CD25^low subpopulations based on CD25 mean fluorescent intensity. The low threshold for the CD25^high gate was set above the population of CD4⁻CD25⁺ cells; the low threshold for the CD25^low gate was set above the isotype control. (B) Percentage CD25^high, CD25^low, and CD25⁺ of total CD4⁺ T cells is shown in the left, center, and right graphs, respectively, for untreated and denileukin diftitox-treated samples from 6 donors. (C) Representative plots for untreated and denileukin diftitox-treated samples from one donor are shown. Percentage CD25^high of total CD4⁺ T cells is indicated on each plot. (D) CD4⁺CD25⁺ cells were isolated from untreated and denileukin diftitox-treated PBMC samples by FACS. Purified CD4⁺CD25⁻ effector T cells (2.5 × 10⁴) from untreated PBMCs were then cultured in the presence or absence of the isolated CD4⁺CD25⁺ cells at a 1:1 ratio (with irradiated untreated PBMCs as antigen-presenting cells [APCs; 1.25 × 10⁵]) on an anti–CD3-coated 96-well plate. Mean proliferation (± SD) was measured by ³H-thymidine incorporation of triplicate wells.