Phenotype of CD4⁺CD25^hi Tregs. (A) Purified CD4⁺ T cells from healthy donors were stained with anti-CD4 Cychrome and anti-CD25 PE, and the population was sorted into CD4⁺CD25^- and CD4⁺CD25^hi subsets as indicated in “Patients, materials, and methods.” The resultant purity of CD4⁺CD25^hi and CD4⁺CD25^- T cells is shown in panel C. (B) FoxP3 expression is restricted to the CD4⁺CD25^hi Treg population. Freshly sorted CD4⁺CD25^hi Tregs and CD4⁺CD25^int and CD4⁺CD25^- effector cells were isolated, and their expression of FoxP3 was characterized by intracellular staining with anti-FoxP3-APC. Data shown are representative of 3 different experiments. The isotype staining control is shown in the dotted line and the staining for FoxP3, in black. Numbers in each histogram indicate the percentage of positive cells and those in parentheses, the mean fluorescence intensity of staining. (C) Purified populations were stained with anti-TNFRI-FITC-, anti-TNFRII-APC-, anti-GITR-FITC-, anti-CD69L-FITC-, and anti-CCR4-APC-conjugated mAb. The gates were set using isotype control antibodies. Staining with the isotype-matched control mAb is indicated by the horizontal bracket. Data are representative of results from more than 10 different experiments. Numbers in each histogram indicate the percentage of positive cells and those in parentheses, the mean fluorescence intensity of staining. (D) TNF up-regulates expression of TNFRII on CD4⁺CD25^hi T cells. Freshly sorted CD4⁺CD25^hi T-regulatory cells and CD4⁺CD25^- effector cells were incubated overnight with TNF at 50 ng/mL^-1 or stimulated with immobilized anti-CD3, and their expression of CD25 and TNFRII was characterized. Numbers in each box indicate the percentage of positive cells and those in parentheses, the mean fluorescence intensity of staining.