Fig. 3: Mitochondrial metabolism is impaired in HCV-specific CD8+ T cells from chronically evolving acute patients.

a Percentage of mitochondrial depolarized virus-specific CD8+ T cells, detected with HLA-A2+ dextramers ex vivo after overnight anti-CD3/anti-CD28 stimulation, by staining with the mitochondrial membrane potential (MMP) sensitive dye JC-1 (see Methods section for details). Dextramer-positive virus-specific depolarized cells were quantified by subtracting the percentage of FL1high/FL2low cells (JC-1 staining) detected in the unstimulated samples from the percentage of the corresponding cellular subsets detected in the stimulated samples, as previously reported²³. b Mitochondrial superoxide levels determined ex vivo as in a with the MitoSOX Red dye. c Cytoplasmic reactive oxygen species (ROS) determined ex vivo, as in a, with the superoxide-specific dye DHE and the intracellular H₂O₂ specific dye H2DCFDA are shown on the left and on the right, respectively. a–c Data are presented as median fluorescence intensity (MFI) values; median values are indicated by horizontal lines. Different numbers of patients (represented by individual dots) were tested in each assay depending on dextramer-positive cell frequencies. a–c Differences between multiple groups were evaluated with the non-parametric Kruskal-Wallis test; p-values were corrected for pairwise multiple comparisons with the Dunn’s test. d Oxygen consumption rate (OCR) data determined on the same samples (n = 6 T1/early chronically evolving acute patients) utilized for ECAR analysis (see Fig. 2d) before (basal level) and after addition of the mitochondrial stressors oligomycin, FCCP and rotenone/antimycin A, which were used to calculate ATP production, maximal respiration capacity, spare respiratory capacity, coupling efficiency and proton leak, as indicated. e, OCR, ATP production, maximal respiration capacity and spare respiratory capacity were determined on the same samples (n = 4 T1/early self-limited acute patients) utilized for ECAR analysis in Fig. 2e, and were calculated as in d. In d and e, OCR values are given as the mean ± SD in the left-side and are presented as box-and-whisker plots (with median and 5–95 percentile) in the right-side. d–e Statistical analysis was performed with the Friedman test to compare different stimuli; p-values have been corrected for pair-wise multiple comparisons with the Conover’s test.