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. 2019 Sep 12;10:2191. doi: 10.3389/fimmu.2019.02191

Figure 4.

Figure 4

TLR7 agonists improve glycolytic metabolism via mTOR signaling in CD8+ T cells. Purified CD8+ T cells were stimulated with an αCD3 antibody (5 μg/mL) with or without R848 (10 μg/mL) in the presence of rapamycin (2 μM) or Akti-1/2 (1 μM). (A) The expression of the glycolysis-related genes Glut-1, HK2, and LDHα was measured by real-time RT-PCR 24 h after stimulation. (B) The Glut-1 expression was detected by flow cytometry and uptake of glucose was measured by detecting MFI of the glucose analog 2-NBDG in CD8+ T cells 48 h after stimulation. (C) HK2 expression was detected by western blotting 24 h after stimulation. (D) Lactate production was measured by a specific enzyme assay after stimulation for 24 h. (E,F) Glycolysis in CD8+ T cells was measured by detecting MFI of Glut-1 and 2-NBDG and lactate production after treating the cells with rapamycin or Akti-1/2 for 48 h. The Glut-1 expression, 2-NBDG uptake, and lactate production in the αCD3+R848 stimulated cells were expressed as fold changes compared to the αCD3 stimulated cells after the corresponding treatment with rapamycin/Akti-1/2. Data are representative of three independent experiments. All data are presented as mean ± SD. The statistical relevance was determined by One-way ANOVA (A,B,D) or Two-way ANOVA (E,F): *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.