Helios⁺ Treg proliferation is inhibited by CD16⁺ monocytes through IL-12. (A) Purified T cells were co-cultured with purified autologous CD14⁺CD16⁻ cells without or with CD16⁺ monocytes in the presence of neutralizing anti–IL-12 or isotype control (“IgG”) antibodies, 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 CFSE^loCD4⁺ subset were analyzed. The P value was calculated by paired t-test comparing the effects of treatment with anti–IL-12 relative to IgG control. In the presence of CD16⁺ cells, IL-12 neutralization increased total Treg and Helios⁺ Treg subset proliferation, but not the proliferation of Helios⁻ Treg, indicating that CD16⁺ monocytes inhibit Helios⁺ Treg development through IL-12. (B) IL-12 was added at the start of the co-cultures of T cells and monocytes, which were stimulated with anti-CD3 for 7 days. Treg (total, Helios⁺, and Helios⁻) proliferation was then compared in the absence (“Medium”) or presence (“IL-12”) of cytokine by paired t-test. The calculated P values indicate that total Treg and Helios⁺, but not Helios⁻, Treg proliferation, are inhibited by IL-12.