FIGURE 5. T cells require monocyte coculture to maintain Th17 phenotype.

A, Purified T cells (>97% pure) were incubated with the indicated stimuli for 40 h, then secreted IL-17 protein was measured by ELISA. n = 11 to 12 for each donor cohort. ChIP measuring association of acetylated histone H3 to the IL-17 (B) or RORC (C) promoter in purified T cells from ND (white bars) or T2D (black bars) donors. Cells were unstimulated or stimulated as indicated for 40 h. n = 4 for each donor cohort. D, Purified monocytes from ND (white bars) or T2D (black bars) donors were stimulated with Escherichia coli LPS for indicated time and analyzed by RT-PCR for expression of IL-1β (top panel) or TNF-α (bottom panel) mRNA. The p value indicating a significant difference between ND and T2D cohorts (p < 0.0001) was calculated by two-way ANOVA. n = 9 for ND donors; n = 8 for T2D patients. E, PBMCs, purified T cells, or CD14-depleted PBMCs from ND (white bars) or T2D (black bars) donors were incubated for 40 h in the presence or absence of anti-CD3 + anti-CD28 as indicated. Alternatively, CD14-depleted PBMCs + CD14⁺ monocytes (CD14⁻+M) or purified T cells and monocytes (M+T) from T2D donors were cocultured for 40 h with or without anti-CD3 plus anti-CD28. Supernatants were analyzed for IL-17. Bars show average and SEM. p values were calculated by Mann–Whitney U test and are shown above bars that are significantly different (p < 0.05). All other comparisons were insignificantly different (p > 0.05).