Effect of denileukin diftitox on proliferation, apoptosis, and functionality of Treg cells. (A,B) A single intraperitoneal injection of 0.75 μg denileukin diftitox was administered to C57BL/6 mice. Flow cytometry analysis was performed on tissues on days 1, 3, 5, and 10 after injection. Animals were fed BrdU water for 5 days before being killed. At time of being killed, spleen (A) and bone marrow (B) cells from individual mice were stained for CD4⁺CD25⁺Foxp3⁺ Treg cells (individual values and means represented as percentage relative to naive mice, top), CD4⁺CD25⁺BrdU⁺ cells (mean values ± SD, middle), and CD4⁺CD25⁺AnnexinV⁺ cells (mean values ± SD, bottom). (C) Splenic CD4⁺CD25⁺ cells were isolated from naive mice, as well as from mice that were treated with 0.75 μg denileukin diftitox on day −7 or day −1 before being killed on day 0. Purified CD4⁺CD25⁻ effector T cells (5 × 10⁴) from naive mice were then cultured in the presence or absence of the various isolated Treg cells (CD4⁺CD25⁺) at a 1:1 ratio (with 10⁵ irradiated naive T-cell–depleted splenocytes as APCs) on an anti–CD3-coated 96-well plate. Mean proliferation (± SD) was measured by ³H-thymidine incorporation of triplicate wells.