CD16⁺ monocyte subset in ITP patients is associated with peripheral Treg frequency and Th responses. Correlation between absolute CD16⁺ monocyte and (A) frequency of peripheral CD25^hiFopx3⁺ in CD4⁺ cells, (B) percent intracellular IL-17 expression in CD4⁺ cells after 5-hour PMA/ionomycin stimulation of whole blood, and (C) percent intracellular IFN-γ expression CD4⁺ cells after 5-hour PMA/ionomycin stimulation of sorted CD4⁺CD25⁻ cells. As indicated by P values, CD16⁺ monocytes are negatively correlated with CD25^hiFopx3⁺ as well as CD4⁺IL-17⁺ cell frequencies but positively with CD4⁺IFN-γ⁺ cells. Gating strategies for enumeration of CD4⁺CD25^hiFopx3⁺, CD4⁺IL-17⁺, and CD4⁺IFN-γ⁺ cells are shown in the supplemental Figure 3. (D) PBMCs labeled with CFSE were stimulated for 7 days, and the expression of CFSE in CD4⁺ cells within CD3 population is shown by the representative histogram. The frequency of CFSE^lo cells within CD4⁺ cells was used throughout the study to calculate the percentage proliferation of CD4⁺ cells. The gating strategy to analyze the frequency of Foxp3^hi (E) and IL-17⁺ (F) cells in divided (CFSE^lo) CD4⁺ subset in stimulated PBMC cultures. For analysis of “% Treg in CFSE^lo CD4⁺ cells,” only the high Foxp3 expressing cells were included (see supplemental Figure 4). (G) Correlation of percent CD16⁺ monocytes in PBMCs at the start of the cocultures and the frequency of Foxp3^hi (Tregs) in CD4⁺ divided CFSE^lo cells, and (H) percent IL-17⁺ cells in CFSE^loCD4⁺ cells at the end of the stimulated PBMC cultures of ITP patients, indicating an inverse relationship between CD16⁺ monocytes and Treg and CD4⁺IL-17⁺ frequencies.