Figure 2. MDSCs are highly efficient in promoting Th17 differentiation in the presence of IL-6 and TGF-β.

A. Naïve CD4⁺CD25⁻CD62L⁺ T cells (5×10⁵ per well in 24-well plates) were co-cultured with or without MDSCs in Th17–polarizing medium for 3 days. Cells were washed and examined for IL-17A or IFN-γ expression by intracellular staining using flow cytometry after stimulation with PMA plus ionomycin in the presence of BFA. CD4⁺Foxp3⁺ cells were also examined by FACS analysis. B. The percentage of CD4⁺IL-17A⁺ T cells (left) and total number of CD4⁺IL-17A⁺ T cells (right) after 3-day culture are shown. Cell proliferation in the presence or absence of MDSCs during Th17 cell polarization in the culture was analyzed using CFSE dilution assays (middle). C. 3 days after co-culture, supernatant was harvested and assessed for IL-17A levels by ELISA. D. Relative mRNA levels of il17a or rora were measured using real-time RT-PCR E. RORγt expression during Th17 differentiation was determined using intracellular staining and flow cytometry analysis (top, grey filled, isotype control; dashed line, without MDSCs; solid line, with MDSCs). The frequency changes of CD4⁺RORC⁺ T cells in the presence or absence of MDSCs are also shown (bottom). F. MDSCs were co-cultured with CD4⁺CD25⁻CD62L⁺ T cells at different ratios as indicated. The frequency of Th17 cells (upper) and IL-17A levels (lower) were analyzed. G. More efficient Th17 polarization by CD11b⁺Gr-1^low MDSCs. CD11b⁺Gr-1^low or CD11b⁺Gr-1^high cells were cultured with naïve CD4⁺ T cells. Th17 polarization was examined as described by analyzing the frequency of Th17 cells and IL-17A levels in the culture. The results shown are from three independent experiments.