TNF-α inhibits Helios⁻ Treg proliferation through TNFRII. (A) Purified T cells were co-cultured with purified monocytes in the presence of neutralizing anti–TNF-α antibody or an isotype control (“IgG”) antibody, and after 7 days of stimulation with anti-CD3, the frequencies of Foxp3^hi (“Total Tregs”) as well as Foxp3^hiHelios⁺ (“Helios⁺ Tregs”) and Foxp3^hiHelios⁻ (“Helios⁻ Tregs”) in theCFSE^lo CD4⁺ subset were analyzed. The P value was calculated by paired t-test comparing the effects of treatment with anti–TNF-α relative to IgG control and indicates that Helios⁺ Treg proliferation is reduced, whereas Helios⁻ Treg proliferation is increased when TNF-α is neutralized through anti–TNF-α. (B) TNF-α was added at the start of the co-cultures of T cells and monocytes stimulated with anti-CD3 for 7 days. Treg (total, Helios⁺, and Helios⁻) proliferation was then compared in the absence (“Medium”) or presence (“TNF-α”) of cytokine by paired t-test. The calculated P values indicate that only Helios⁻ Treg proliferation is targeted by TNF-α, causing a decrease in proliferation of this subset. (C) Co-cultures of T cells and monocytes were treated with anti-TNFRI or (D) anti-TNFRII and stimulated with anti-CD3 for 7 days. Treg (total, Helios⁺, and Helios⁻) proliferation was then compared with IgG control antibody by paired t-test. The calculated P values indicate that only blocking with anti-TNFRII affects proliferation of Helios⁻ Tregs. (E) Percent increase in the numbers of Tregs (total, Helios⁺, and Helios⁻) in the presence of anti-TNFRI and anti-TNFRII relative to IgG control is shown for the studies presented in (C) and (D) and is consistent with a role for TNFRII, but not TNFRI, in Helios⁻ Treg development. (F) Representative dot plot of TNFRII and Helios expression in divided Foxp3^hi Tregs, showing surface expression levels of TNFRII on Helios^+/− Tregs. (G) Relative mean fluorescence intensity analysis of TNFRII expression on Helios^+/− Tregs is shown, indicating that Helios⁻ Tregs express higher levels of TNFRII compared with Helios⁺ Tregs.