Suppressive property of CD4⁺CD25^hi Tregs is abrogated by TNF. (A) CD4⁺CD25^- T cells (5 × 10⁴/well) or CD4⁺CD25^hi Tregs (5 × 10⁴/well) alone or mixed together at a ratio of 1:1 were stimulated with plate-bound anti-CD3 (1 μg/well). Recombinant TNF was added at the beginning of the culture at 50 ng/mL as indicated. After 72 hours, ³H-thymidine incorporation was determined. Data are the mean ± SEM of 6 independent experiments. (B) TNFRII cross-linking reverses the CD4⁺CD25^hi Treg-mediated suppression of the proliferation of CD4⁺CD25^- effectors. Previously activated T cells that had up-regulated surface TNFRII expression were used for in vitro regulatory assays as described in “Patients, materials, and methods.” Ninety-six-well microtiter plates were coated with anti-TNFRII mAb at 0.2 μg/well. CD4⁺CD25^- T cells (5 × 10⁴/well), or CD4⁺CD25^hi Tregs (5 × 10⁴/well) alone or mixed together at a ratio of 1:1 were stimulated with plate-bound anti-CD3 (1 μg/well), with or without anti-TNFRII. After 72 hours, ³H-thymidine incorporation was determined. (C) Culture supernatants were harvested and analyzed to determine the interferon γ content. Data are the mean ± SEM of 5 independent experiments. (D) Loss of suppressive function of CD4⁺CD25^hi Tregs by TNFRII cross-linking. CD4⁺CD25^- and CD4⁺CD25^hi Tregs were sorted as described. For assessing the suppressor function in the presence of TNFRII, 5 × 10⁵ of CD4⁺CD25^hi Tregs and 1 × 10⁶ CD4⁺CD25^- T cells were labeled with CFSE and cultured (5 × 10⁴/well) with plate-bound anti-CD3 mAb (1 μg/well) with or without anti-TNFRII mAb (0.2 μg/well). For assessment of suppression, labeled CD4⁺CD25^- T cells (5 × 10⁴) were cultured alone or at a 1:1 ratio with unlabeled CD4⁺CD25^hi Tregs. After 3 days, cell proliferation was assessed by CFSE dilution. Data are representative of 3 independent experiments.