Optimal effects of CD16⁺ monocytes on Treg and Th proliferative require direct contact with T cells and release of soluble factors. Autologous CD3⁺ T cells, CD14^hiCD16⁻ and CD16⁺ monocyte subsets, from ITP patients were purified from PBMCs by cell sorting. Using a transwell plate, CFSE-labeled T cells were cocultured with CD14^hiCD16⁻ cells in the upper compartment, and CD16⁺ monocytes were added in the same compartment or in a separate lower compartment. After 7 days of stimulation in the presence of anti-CD3, the percent of (A) Foxp3^hi as well as (B) IL-17 and (C) IFN-γ⁺ cells in divided CFSE^lo CD4⁺ T cells before and after addition of CD16⁺ monocytes in the lower compartment (left panel) or to upper compartment is shown. CD16⁺ monocytes, when separated from cocultures of T cells plus CD14^hiCD16⁻ (left panel), are not as effective in altering Treg and Th responses (P > .05, paired t test) compared with that when added to the same compartment as T⁺CD14^hiCD16⁻ cells (P < .05). (D) Sorted ITP T cells and CD14^hiCD16⁻ cells were placed in the upper compartment and autologous CD16⁺ monocyte without (“Culture medium”) or with T cells were added to the lower compartment of the transwell system. After 7 days of stimulation in the presence of anti-CD3, only the cells in the upper compartment were harvested, and the levels of divided CD4⁺Foxp3 as well as (E) CD4⁺IL-17⁺ and (F) CD4⁺ IFN-γ⁺ cells were analyzed. As indicated by the P values (paired t test), CD16⁺ monocytes in direct contact with T cells can affect polarization of Th cells in a separate compartment.