Phenotype of CD4⁺CD25^hi Tregs from RA patients. (A) Purified CD4⁺ T cells 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. Purified populations were stained with anti-TNFRI-FITC-, anti-TNFRII-APC-, anti-GITR-FITC-, anti-CD69-FITC-, and anti-CCR4-APC-conjugated mAb as indicated. (B) Anti-TNF therapy down-regulates expression of TNFRII and GITR on CD4⁺CD25^hi T cells. Freshly sorted CD4⁺CD25^hi T-regulatory cells and CD4⁺CD25^- effector cells were isolated from 15 RA patients treated with infliximab and their phenotype was characterized. Numbers in each box indicate the percentages of positive cells and those in parentheses, the mean fluorescence intensity of the stained cells. These changes in phenotype were compared with the phenotype of healthy donors, shown in Figure 1.