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. 2019 Feb 4;10:115. doi: 10.3389/fimmu.2019.00115

Figure 2.

Figure 2

Inhibition of glycolysis constrains Th17-lineage cells. PBMC were isolated from healthy donors. CD4+ T cells were isolated by magnetic separation and stimulated for 18 h with PMA and ionomycin in glucose-free RPMI supplemented with glucose (10 mM) (Gluc) or galactose (10 mM) (Gal). Representative plots of ECAR and OCR over time are shown (A). Memory CD45RO+CD4+ T cells were isolated by magnetic separation and cultured for 5 d in the presence of anti-CD3 and irrAPC in glucose-free medium supplemented with glucose or galactose. Cells were re-stimulated with PMA and ionomycin in the presence of brefeldin A; stained for CD161, IL-17, IFN-γ, CD3, and CD8 and analyzed by flow cytometry. Representative dot plots (gated on single, live, CD4 [CD3+CD8−] cells), accompanied by the frequencies of CD4+ T cells expressing CD161 and proinflammatory cytokines (n = 11) (B). Memory CD4+ T cells were cultured for 5 d with anti-CD3 and irrAPC in the presence or absence of rapamycin (Rapa). Cells were re-stimulated with PMA and ionomycin in the presence of brefeldin A, stained for CD161, IL-17, IFN-γ, CD3, and CD8 and analyzed by flow cytometry. The frequencies of CD4+ T cells (gated on live, single CD3+CD8− cells) expressing CD161, IL-17, or IFN-γ (n = 8–10) (C). *p < 0.05, **p < 0.01.