Stimulation-induced increases in CD8+ T-cell metabolism, ROS production, and nuclear NFAT translocation are impaired under hypoxia. (A) CD8+ T cells were cultured for 16 hours at 21% or 1% O2, stimulated via CD3/CD28 at these oxygen tensions as indicated for 24 hours in the presence of fully labeled 13C-glucose or 13C-glutamine, and assessed for fractional isotopic labeling of indicated metabolites by gas chromatography-mass spectrometry (GC-MS, summarized for n = 4 independent donors). (B) CD8+ T cells were cultured for 16 hours at 21% or 1% O2, stimulated via CD3/CD28 at these oxygen tensions as indicated for 48 hours, then assessed for excreted lactate by measurement of supernatant concentration (summarized for n = 5 independent donors). (C-G) CD8+ T cells cultured as in panel B were assessed after 24 hours for (C) mitochondrial membrane potential (Δψm, summarized for n = 6 independent donors), (D) mROS production (summarized for n = 8 independent donors), (E) total cellular ROS abundance (summarized for n = 8 independent donors), (F) nuclear (summarized for n = 5 independent donors), and (G) total NFAT abundance (summarized for n = 4 independent donors) within isolated nuclei and whole cells, respectively, all by flow cytometry. (F-G) Data are shown as change (Δ) calculated as MFI of stimulated sample/MFI of matched unstimulated sample. P values were calculated by (A-E) 2-way ANOVA and Holm-Sidak’s post hoc test or (F) paired t test. ∗P < .05, ∗∗P < .01.
