Figure 2.

Hypoxia and HIF-1α, but Not HIF-2α, Support CD8⁺ T Cell Acquisition of Effector Phenotype

(A) CD8⁺ T cells were isolated from spleens of HIF-1α^fl/fldlck^CRE (maroon) or HIF-2α^fl/fldlck^CRE (green) and littermate control (black) mice, and activated with αCD3/CD28 for 48 hr, then expanded for 5 days in IL-2 and subjected to 21% or 1% O₂ for 24 hr. Expression of CD44 and CD62L by flow cytometry is shown.

(B) Intracellular expression of IFNγ, TNFα, and granzyme B in HIF-1α^fl/fldlck^CRE (maroon) and HIF-1α^fl/fl control littermates (black) by flow cytometry after restimulation (n = 3, error bars represent SD).

(C) Intracellular expression of IFNγ, TNFα, and granzyme B in HIF-2α^fl/fldlck^CRE (green) and HIF-2α^fl/fl control littermates (gray) by flow cytometry after restimulation (n = 3, error bars represent SD).

(D) Surface expression of costimulatory molecules/checkpoint receptors CD137, OX40, GITR, PD-1, TIM-3, and LAG3 on CD8⁺ T cells isolated from HIF-1α^fl/fldlck^CRE (maroon) HIF-1α^fl/fl control littermates (black), activated and expanded as in (A) (n = 3, error bars represent SD).

(E) Same as in (D) on CD8⁺ T cells isolated from HIF-2α^fl/fldlck^CRE (green) or HIF-2α^fl/fl control littermate mice (gray) (n = 3, error bars represent SD).

(F) Relative mRNA levels of Vegfa (left, n = 3) and amount of VEGF-A in media (right, n = 4, error bars represent SD) on CD8⁺ T cells for the indicated time points after αCD3/CD28 activation.

(G) Amount of VEGF-A in media from CTLs expanded as in (A) and cultured under 21% O₂ or 1% O₂ for 24 hr (n = 4, error bars represent SD).

(H) Relative expression of Vegfa in total LLC tumor lysate and immune populations isolated from subcutaneous Lewis lung carcinoma (LLC) tumors 11 days after implantation (n = 10, error bars represent SD).

Grouped data were assessed by two-way ANOVA for multiple comparisons with Bonferroni correction. ^∗∗∗∗p < 0.00005, ^∗∗∗p < 0.0005, ^∗∗p < 0.005, ^∗p < 0.05. MFI, mean fluorescence intensity. See also Figure S2.