FIGURE 3. Molecular signatures of Th17 cells and elevated IL-17 production are T cell specific.

A, One representative of eight samples stained for intracellular IL-17 in CD3⁺ T cells in PBMCs from ND (upper panels; n = 4) or T2D (lower panels; n = 4) donors. PBMCs were stimulated as indicated for 40 h. Brefeldin, PMA, and ionomycin were added 5 h before harvest. B, Summary of intracellular cytokine staining for IL-17 in CD3⁺ T cells from ND (white bars) and T2D (black bars) donors as shown in A (percent positive, top panels) or C (MFI). p values that were significant (p < 0.05) as measured by Mann–Whitney U test are shown on graph. n = 4 for each bar. Due to differences in isotype control Ab staining between media and PHA or CD3+28-stimulated samples, isotype staining in leftmost columns was used as a gating control for media-treated samples and PHA/CD3+28 isotype (middle columns) was used for PHA or CD3+28-stimulated samples (rightmost columns). In B, all comparisons between media (unstimulated) and stimulated values were statistically significant (p < 0.05) for the T2D cohort (media versus PHA, p = 0.0286; media versus CD3+28, p = 0.028). D, Intracellular IL-17 levels fail to correlate with age (top panels) or BMI (bottom panels) in all ND (○) and T2D (●) donors. p > 0.05 for all analyses as calculated by the Pearson test. Quantitation of active IL-17 (E) or RORC (F) promoters as measured by ChIP for DNA associated with hyperacetylated histone H3. P2T denotes PBMCs cultured in the presence or absence of indicated stimulus for 40 h, after which T cells were purified by negative selection (>98% pure) and analyzed. The non-T fraction had extremely low signals (fold GST control Ab <1: not shown). Data represent P2Ts from ND (white bars) and T2D (black bars) donors. Bars show average and SEM. n = 3–6. The p values above bars were calculated by Mann–Whitney U test.