Induction of Treg/Th17 T cells by MDSCs/monocytes. (A) CD4⁺ T cells were stimulated with anti-CD3/CD28/CD2 beads and cultured in the presence or absence of CD14⁺HLA-DR^−/low or CD14⁺HLA-DR⁺ cells. Intracellular staining (ICS) for IL-17 and Foxp3 was performed after 3 days gating on CD4⁺ T cells. Shown is a representative dot plot of 5 independent experiments. Numbers represent percentage of events within the respective quadrants. (B) qPCR analysis of Foxp3, RORc, and IL-17A expression. CD4⁺ T cells were stimulated with anti-CD3/CD28/CD2 beads and cultured in the presence of CD14⁺HLA-DR^−/low or CD14⁺HLA-DR⁺ monocytes for 3 days. CD4⁺ T cells were stained for IL-17 and Foxp3 expression. Three different CD4⁺ T helper subtypes (Foxp3⁺IL-17⁻ [+/−]/Foxp3⁻IL-17⁺ [−/+], and Foxp3⁻IL-17⁻ [−/−] cells) were isolated using FACS and analyzed for expression of Foxp3, RORc, IL17A, IL-10, and TGF-β mRNA. CD4⁺Foxp3⁺IL-17⁻ (+/−) cells were isolated from CD4⁺ T cells stimulated in the presence of CD14⁺HLA-DR^−/low cells, while Foxp3⁻IL-17⁺ (−/+) and Foxp3⁻IL-17⁻ (−/−) cells were derived from CD4⁺ T cells stimulated in the presence of CD14⁺HLA-DR⁺ monocytes. Expression was set relative to cyclophilin A mRNA. Shown are cumulative results of 2 independent experiments (*P < .05).