Preincubation with TNF suppresses the subsequent ability of CD4⁺CD25^hi Tregs to inhibit the proliferation of CD4⁺CD25^-T cells and decreases FOXP3 expression. (A) Freshly sorted CD4⁺CD25^- T cells and CD4⁺CD25^hi Tregs were incubated with TNF at 50 ng/mL in medium supplemented with IL-2 at 100 U/mL. After overnight incubation, cells were washed and examined for the capacity of CD4⁺CD25^hi Tregs to suppress proliferation of CD4⁺CD25^- T cells. Data are mean ± SEM from 6 different experiments. (B) FoxP3 expression is reduced by TNF incubation of CD4⁺CD25^hi Tregs. CD4⁺CD25^- and CD4⁺CD25^hi T cells were sorted as described and cultured overnight with IL-2 and with or without TNF (50 ng/mL). Real-time PCR was carried out in triplicate for FoxP3 mRNA and relative fold changes were normalized to GAPDH. TNF caused significant suppression of FoxP3 in CD4⁺CD25^hi Tregs after overnight incubation compared with that found in fresh cells (P < .01). (C) Intracellular FoxP3 staining was carried out with freshly isolated cells and the same cells after a 48-hour incubation with IL-2 alone or the combination of IL-2 and TNF (50 ng/mL). Similar results were noted after a 24-hour incubation. Data are representative of 3 different experiments. Numbers indicate the percentage of positive cells and those in parentheses, the mean fluorescence intensity of the stained cells.