Figure 4.

HIF-1α Is Necessary for Effector CD8⁺ T Cell Function and Migration

(A) CD8⁺ T cells were isolated from spleens of OT-1 HIF-1α^fl/fldlck^CRE (maroon) and littermate control (black) mice and activated with cognate peptide for 2 days, then expanded for 5 days in the presence of IL-2 and subjected to 21%, 5%, or 1% O₂ for 24 hr. Representative flow-cytometry histograms showing the expression of CD44 and CD62L (n = 3).

(B) Expression of intracellular granzyme B and the indicated costimulatory molecules/checkpoint receptors on CD8⁺ T cells prepared as in (A).

(C) Left: diagram outlining the in vivo migration experiment: representative flow-cytometry plots are shown for each pool before and after adoptive cotransfer of HIF-1α mutant (CD45.1^+/TdTomato⁺, maroon) and control (CD45.1⁺, black) OT-1 T cells into CD45.2⁺ B16-OVA tumor-bearing WT mice. Right: spleens, lymph nodes (LN), and tumors were collected 48 hr after the adoptive cell cotransfer and relative percentages of migrated cells of the indicated genotypes are shown (error bars represent SD, n = 6).

(D) 1 × 10⁶ OT-1 cells were transferred into recipient mice harboring B16-OVA tumors (n = 6): tumor volumes (left) and overall percent survival (right) are shown; statistical analysis by log-rank (Mantel-Cox) test; error bars represent SEM.

(E) Tumor growth curves of MC38 tumor cells subcutaneously injected into HIF-1α mutant and littermate controls. Mice were treated with either isotype control antibodies (left, n = 3) or a combination of αPD-1 and αCTLA4 blocking antibodies (right, n = 6), on days 5, 7, and 9 (dashed lines). Statistical analysis was performed by two-way ANOVA with Sidak correction for multiple comparisons. Tumor volumes are shown (mean values ± SEM).

^∗∗∗∗p < 0.00005, ^∗∗p < 0.005, ^∗p < 0.05; n.s., not significant. See also Figure S3.