Helios^+/− Treg proliferation is differentially controlled by monocyte subsets. Autologous CD3⁺ T cells, CD14⁺CD16^- and CD16⁺ monocyte subsets were purified from PBMCs of healthy volunteer controls. T cells were CFSE-labeled and co-cultured with CD14⁺CD16⁻ cells with or without CD16⁺ monocytes (as indicated by + and −) in the presence of anti-CD3 for 7 days. (A) The gating strategy to analyze the frequency of Foxp3^hi in divided (CFSE^lo) CD4⁺ subset is shown. (B) Representative dot plot of Helios and Foxp3 expression gated on divided CFSE^lo CD4⁺ cells. Gating strategy for cells expressing Foxp3^hiHelios⁺ and Foxp3^hiHelios⁻ is indicated. (C) The percentage of Foxp3^hi in CFSE^lo CD4⁺ subset (“Total Tregs”) as well as Foxp3^hiHelios⁺ (“Helios⁺ Tregs”) and Foxp3^hiHelios⁻ (“Helios⁻ Tregs”) before and after addition of CD16⁺ monocytes is shown. The P value was calculated by paired t-test and indicates that addition of CD16⁺ cells decreases Treg development in healthy normal volunteers (P = .021), as per our previously published data,²⁶  as well as Helios⁺ Treg development (P = .015), but has a less obvious (not statistically significantly) effect on Helios⁻ Treg proliferation.