CD16⁺ monocytes alter Treg and Th proliferative responses in patients with ITP. PBMCs from healthy controls and ITP patients were depleted of CD16⁺ monocyte subsets by cell sorting or by magnetic bead selection. PBMCs before and after depletion were stained with CFSE and stimulated with anti-CD3. At day 7, intracellular expression of Foxp3, IL-17, and IFN-γ was detected by cytometry. The percentage of (A) Foxp3^hi as well as (B) total CD4⁺ proliferation as measured by frequency of divided CFSE^lo CD4⁺ cells and (C) IL-17 and (D) IFN-γ⁺ cells in divided CFSE^lo CD4⁺ T cells before and after depletion of CD16⁺ monocytes. The P values were calculated by paired t test and indicate that depletion of CD16⁺ cells improves Treg and CD4⁺IL-17⁺ Th development in both healthy controls and ITP patients. In contrast, the absence of CD16⁺ monocytes inhibits CD4⁺IFN-γ⁺ proliferative responses in ITP patients. (E-H) The addition of CD16⁺ monocytes to cocultures of T cells and CD14^hiCD16⁻ cells alters Treg and Th proliferative responses. Autologous CD3⁺ T cells, CD14^hiCD16⁻ and CD16⁺ monocyte subsets, were purified from PBMCs of healthy controls and ITP patients by cell sorting. After CFSE labeling, the T cells were cocultured with CD14^hiCD16⁻ cells with or without CD16⁺ monocytes (as indicated by + and −) in the presence of anti-CD3 for 7 days. The percentage of (E) Foxp3^hi (F) total CD4⁺ proliferation as the percent (G) IL-17 and (H) IFN-γ⁺ cells in divided CFSE^lo CD4⁺ T cells before and after addition of CD16⁺ monocytes. The indicated P values were calculated by paired t test. Whereas the addition of CD16⁺ monocytes inhibited the proliferative responses of Tregs and CD4⁺ IL-17⁺ cells, it stimulated the expansion of CD4⁺IFN-γ⁺ cells more significantly in ITP patients than in healthy controls.