Fig. 5: Blocking dysregulated intracellular signaling pathways can reverse early metabolic and functional CD8+ T cell defects.

a PBMC from T1/early chronically-evolving patients were stimulated overnight with HCV-NS3 peptides in the presence or absence of the ROS scavenger resveratrol^38,39 (treated vs. untreated) and then stained with MitoSOX Red to assess mitochondrial superoxide content and with anti-phospho-ATM (Ser1981), phospho-p38 (Thr180), and phospho-p53 (Ser15) antibodies. Bars represent mean fold-change values + SEM derived from 6 patients. Representative overlay histograms are illustrated on the right. b PBMCs from T1/early chronically-evolving patients were stimulated for 40 h with HCV-NS3 peptides in the presence or absence of specific ATM (KU-55933), p53 (Pifithrin-α), AMPK (Dorsomorphin), and p38a (SB203580) inhibitors, followed by flow cytometry determination of GLUT-1 expression levels. Representative dot plots are illustrated on the right. Glucose uptake studied via incorporation of the glucose analog 2-NBDG (c) and PD-1 expression (d) have been measured as in b. e IFN-γ, TNF-α, and IL2 production by CD8+ T cells cultured as in b. Data are presented as the ratio between the percentage of cytokine positive CD8+ T cells detected in inhibitor-treated vs. untreated cultures (fold-change). f IFN-γ, TNF-α, IL2 single positive, as well as double-positive IFN-γ+/TNF-α+CD8+ T cells generated in short-term T cell lines upon 10-days stimulation with HCV-NS3 peptides in the presence or absence of the inhibitors specified in the legend to panel b. Data shown in all panels are presented as fold-change of treated vs. untreated CD8+ T cells. Horizontal lines in panels b to f represent median values; data were analyzed statistically with the Wilcoxon signed-rank test; NS = not significant.