CD16⁻ monocytes control TNF-α–mediated Helios⁻ Treg proliferation. Purified T cells were co-cultured with purified autologous CD14⁺CD16⁻ cells without or with CD16⁺ monocytes in the presence of neutralizing (A) anti–TNF-α or (B) anti-TNFRII, 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 the CFSE^lo CD4⁺ subset were analyzed. The P value was calculated by paired t-test comparing the effects of treatment with neutralization antibody relative to IgG control. In co-cultures without CD16⁺ cells, anti–TNF-α treatment decreased Helios⁺ proliferation but increased Helios⁻ Treg expansion, similar to data obtained when total monocytes were used. In the presence of CD16⁺ monocytes, Helios⁻ Tregs were expanded relative to IgG control. (B) The same assays were performed as in (A) except that for TNF-α blockade, anti-TNFRII was used instead of anti–TNF-α. In co-cultures without CD16⁺ cells, anti-TNFRII treatment only affected Helios⁻ Treg proliferation as indicated by P values, similar to data obtained when total monocytes (Figure 3A) were used.