CD8+ T-cell activation, proliferation, and IFN-γ expression are suppressed when stimulated under hypoxia. (A-C) CD8+ T cells were cultured for 16 hours at 21%, 5%, or 1% O2, stimulated via CD3/CD28 as indicated at these oxygen tensions for 48 hours, then assessed for (A) viability, (B) CD25 expression, and (C) CD69 expression (representative histograms and summarized for n = 9 independent donors). (D) CD8+ T cells were cultured as in panel A, but stimulated for 6 days and assessed for proliferation by cell trace violet (CTV) dilution (representative histograms and summarized for n = 5 independent donors). (E-F) CD8+ T cells were cultured as in panel A, but stimulated for 5 hours in the presence of brefeldin A and monensin, and assessed for (E) CD107a trafficking and (F) intracellular granzyme B (GzmB) abundance (representative histograms and summarized for n = 4-5 independent donors). (G-H) Supernatants from CD8+ T cells cultured as in panel A were assessed for (G) IFN-γ and (H) TNF-α content (summarized for n = 7 independent donors, normalized to cytokine measured at 21% O2). (I) CD8+ T cells cultured as in panel A were restimulated at 48 hours via CD3/CD28 or with phorbol 12-myristate 13-acetate (PMA)/ionomycin as indicated, and assessed for intracellular IFN-γ abundance (summarized for n = 7 independent donors). (J-L) Sorted populations of naïve (CD45RA+CD62L+), CM (CD45RA−CD62L+), EM (CD45RA−CD62L−), and EMRA (CD45RA+CD62L−) cells were cultured as in panel A, and assessed for (J) CD25 expression, (K) IFN-γ secretion, and (L) intracellular IFN-γ abundance (summarized for n = 3-5 independent donors). P values were calculated by (B, C, D, J) 2-way analysis of variance (ANOVA) or (G) repeated measures ANOVA and Holm-Sidak’s post hoc test. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001.
