Figure 1.

Hypoxia Promotes CD8⁺ T Cell Glycolytic Metabolism in an HIF-1α- but Not HIF-2α-Dependent Fashion

(A) qRT-PCR of mRNA levels of Hif1a and Hif2a on magnetically isolated splenic CD8⁺ T cells before and after activation with αCD3/CD28 for the indicated time points (n = 3). US, unstimulated. Error bars represent SD.

(B) Immunoblots showing HIF-1α and HIF-2α expression in T cells collected at the indicated time points after activation. Densitometric analyses are shown at the bottom.

(C) Deletion efficiency of Hif1a and Hif2a in genomic DNA from CD8⁺ lymphocytes isolated from HIF-1α^fl/fldlck^CRE or HIF-2α^fl/fldlck^CRE mice (n = 4, error bars represent SD).

(D) CFSE (carboxyfluorescein succinimidyl ester) dilution assay 72 hr after in vitro activation (n = 3, error bars represent SD).

(E) Proliferation index and percent survival of isolated CD8⁺ T lymphocytes 4 days after activation (n = 4, error bars represent SD).

(F) CD8⁺ T cells were isolated from spleens and activated with αCD3/CD28 for 48 hr, and then expanded for 3 days in IL-2 and subjected to 21% or 1% O₂ for 24 hr. Western blotting was performed on nuclear fractions; densitometric analyses are shown.

(G) CD8⁺ T cells from HIF-1α^fl/fldlck^CRE (maroon), HIF-2α^fl/fldlck^CRE (green), and littermate controls (black for HIF-1α^fl/fl, gray for HIF-2α^fl/fl) were isolated, activated, expanded for 5 days in the presence of IL-2, and cultured for 24 hr under 21% versus 1% O_2. qRT-PCR was performed for Hk2, Pdk1, Mct4, and Pgk (n = 3, error bars represent SD).

(H) Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD8⁺ T cells prepared as in (G) were measured by flux analysis, under basal conditions and after injection (dashed line) of oligomycin and FCCP (stressed) (n = 4 per genotype, error bars represent SEM).

(I and J) Supernatants of CD8⁺ T cells cultured as in (G) were measured for glucose (I) and lactate (J) levels (n = 6, error bars represent SD).

(K) Representative flow-cytometry histograms of viability staining and percent survival of CD8⁺ T cells cultured as in (G) (n = 4). Grouped data were assessed by two-way ANOVA for multiple comparisons with Bonferroni correction.

Horizontal lines are the mean values. ^∗∗∗∗p < 0.00005, ^∗∗∗p < 0.0005, ^∗∗p < 0.005, ^∗p < 0.05. See also Table S1 and Figure S1.