HIF-1α Is a Metabolic Switch between Glycolytic-Driven Migration and Oxidative Phosphorylation-Driven Immunosuppression of Tregs in Glioblastoma

Abstract

The mechanisms by which regulatory T cells (Tregs) migrate to and function within the hypoxic tumor microenvironment are unclear. Our studies indicate that specific ablation of hypoxia-inducible factor 1α (HIF-1α) in Tregs results in enhanced CD8⁺ T cell suppression versus wild-type Tregs under hypoxia, due to increased pyruvate import into the mitochondria. Importantly, HIF-1α-deficient Tregs are minimally affected by the inhibition of lipid oxidation, a fuel that is critical for Treg metabolism in tumors. Under hypoxia, HIF-1α directs glucose away from mitochondria, leaving Tregs dependent on fatty acids for mitochondrial metabolism within the hypoxic tumor. Indeed, inhibition of lipid oxidation enhances the survival of mice with glioma. Interestingly, HIF-1α-deficient-Treg mice exhibit significantly enhanced animal survival in a murine model of glioma, due to their stymied migratory capacity, explaining their reduced abundance in tumor-bearing mice. Thus HIF-1α acts as a metabolic switch for Tregs between glycolytic-driven migration and oxidative phosphorylation-driven immunosuppression.

Keywords: fatty acid oxidation; glioblastoma; glycolysis; immunosuppression; migration; oxidative phosphorylation; regulatory T cell.

Figure 1.. Deficiency of HIF-1α Promotes the Mitochondrial Utilization of Glucose under Hypoxia

(A and B) FACS-sorted and expanded Tregs from C57BL/6 Foxp3-YFP-Cre mice (HIF-1α WT) or Foxp3-YFP-Cre HIF-1α^flox/flox mice (HIF-1α KO) were placed under normoxia or hypoxia overnight before being adhered to microplates, and oxygen consumption rate (OCR) was determined over time (A) and analyzed as bar graphs (B). A modified mitochondrial stress test was performed with an initial palmitate injection to determine palmitate-induced respiration and mitochondrial function of Tregs. Data were analyzed using Wave software from Agilent. n = 5 wells per condition were analyzed, representative of two independent experiments. Statistics were calculated as percent positive population ± SEM. One-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. See also Figures S1 and S2.

Figure 2.. HIF-1α KO Tregs Suppress CD8⁺ T Cell Proliferation Better than HIF-1α WT Tregs under Hypoxia due to Enhanced Glucose Oxidation

(A and B) Sorted and expanded Tregs were plated with proliferation-dye-labeled CD8⁺ T cells at decreasing ratios to determine their suppressive capability under (A) 21% O₂ and (B) 1% O₂. After 72 h, CD8⁺ T cell proliferation was analyzed. An n of 3 wells per ratio was analyzed, representative of three independent experiments. (C) Sorted and expanded Tregs were pre-treated with UK5099 (10 μm) or vehicle control for 24 h before a suppressor assay was run under 1% O₂. After 72 h, percent proliferation and expansion indexes were determined via flow cytometry. An n of 3 per condition was analyzed, from two independent experiments. (D) Sorted and expanded Tregs were pretreated with 1 mM DCA treatment overnight before suppressor assays were performed under 1% O₂. Statistics were calculated as percent positive population ± SEM. Unpaired t test analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Figure 3.. Inhibition of Either Lipid Uptake or Lipid Oxidation Prevents Immunosuppressive Capabilities of Regulatory T Cells

(A) Sorted and expanded Tregs were cultured for 72 h in the presence of the fatty acid oxidation inhibitor etomoxir (200 μM) or the fatty acid uptake inhibitor SSO (200 μM), and the expression of various Tregs markers was assessed via flow cytometry. (B and E) Sorted Tregs were cultured for 72 h in the presence of etomoxir (Eto; 200 μM; B) or SSO (200 μM; E), and Foxp3 retention was determined. (C and D) Percent CD8 T cell proliferation with co-culture of Eto pre-treated Tregs shown as a histogram (C) and analyzed as bar graphs with reducing Treg ratios (D) that were enumerated via flow cytometry. (F and G) Percent CD8 T cell proliferation with co-culture of SSO pre-treated Tregs shown as a histogram (F) and analyzed as bar graphs with reducing Treg ratios (G) that were enumerated via flow cytometry. (H and I) Sorted and expanded WT or HIF-1α KO Tregs were pretreated with (H) Eto (200 μM) or (I) SSO (200 μM) for 24 h before a suppressor assay was run under 1% O₂. After 72 h, expansion indexes were determined via flow cytometry. Flow cytometry statistics were calculated and shown as percent positive or MFI ± SEM; n = 5 per group in (A), and n = 3 per ratio in (B)–(I), representative of 2–3 independent experiments. A one-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. See also Figure S3.

Figure 4.. Regulatory T Cells Utilize Lipids for Their Mitochondrial Metabolism under Hypoxia and Prefer Lipids for Metabolism within Glioma

(A and B) Flow-cytometry-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were placed under 1% O₂ overnight before being adhered to microplates, and extracellular flux analysis was performed over time (A) and analyzed as bar graphs (B). Eto treatment (20 μm) was added immediately before assay to determine reliance on lipids for mitochondrial metabolism. (C–F) Wild-type C57/Bl6 mice were implanted with 4 × 10⁵ GL-261 astrocytoma cells, and after 2 weeks of tumor growth, T cell expression of surface fatty acid transporters was analyzed via flow cytometry. (C) Percent palmitic acid uptake and MFI of conventional CD4⁺, CD8⁺, and Treg subsets. (D) Percent 2-NDBG and MFI of 2-NBDG conventional CD4⁺, CD8⁺, and Treg subsets. (E) Tumor interstitial fluid was obtained from either the tumor hemisphere or the non-tumor hemisphere of mice, and FFA content was measured via colorimetric readout. (F) Data show the expression of fatty acid transporters CD36, SLC27A1, and SLC27A4 across different T cell subsets in the brains, DLN, and spleens of tumor-bearing mice (left); representative flow cytometry plots are shown on the right. Statistics were calculated as percent positive population ± SEM. Data in (A) and (B) were analyzed using Wave software from Agilent. Statistics were calculated as percent positive population ± SEM, n = 5 per group; results are representative of three experiments in (A) and (B) and two experiments in (C)–(F). One-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. See also Figure S4.

Figure 5.. In Vivo Treatment of Eto Causes a Survival Benefit in Immunocompetent Mice

(A) Survival of WT or immunodeficient Rag^0/0 mice implanted with 4 × 10⁵ GL-261 and treated with intracranial Eto administration beginning at day 7 after tumor implantation. (B) Quantification of Tregs and their ratios to other T cell subsets 48 hr after Eto treatment (day 9). (C) Survival of mice injected i.p. with 30 mg/kg Eto. (D) Flow-cytometric analysis of Treg infiltration and their ratios to other T cell subsets 48 h after second Eto treatment (9 days). Survival curves are from at least 7 mice per group from two independent experiments; statistical significance was calculated using log-rank analysis. Flow cytometry statistics were calculated and are indicated as percent positive population ± SEM; n = 5 per group, representative of two independent experiments in (B) and one experiment in (D). Unpaired t test analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. See also Figures S5 and S6A.

Figure 6.. Deficiency of HIF-1α Inhibits Glycolytic Metabolism of Tregs, Resulting in Reduced In Vitro Migration

(A and B) FACS-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were placed under 21% O₂ or 1% O₂ overnight before being adhered to microplates, and extracellular acidification rate (ECAR) was determined over time (A) and analyzed as bar graphs (B). (C) FACS-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were put under 21% O₂ or 1% O₂ overnight, and qRT-PCR was performed to determine the expression of PDK1 and LDHA. (D and E) Sorted and expanded HIF-1α WT Tregs were treated with 1 mM DCA overnight before being adhered to microplates, and metabolic flux was determined over time (D) and analyzed as bar graphs (E). Data were analyzed using Wave software from Agilent. In (A), (B), (D), and (E), an n of 5 wells per condition was analyzed, representative of two independent experiments. In (C), an n of 3 per condition was analyzed from three independent experiments. (F) Sorted and expanded Tregs from HIF-1α WT or HIF-1α KO mice were plated in Transwell inserts to measure their migration toward the chemokine CCL22 or GL-261 tumor cells under 1% O₂. (G) FACS-sorted and expanded HIF-1α WT or HIF-1α KO Tregs were injected with CCL22, and change in ECAR was measured. In (F), statistics were calculated as percent migration ± SEM. In (B) and (C), one-way ANOVA followed by Tukey’s post hoc analysis was used to calculate significance. In (D) and (E), unpaired t test analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. See also Figure S6B.

Figure 7.. Conditional Knockout of HIF-1α in Foxp3⁺ T Cells Inhibits Migration of Tregs to Brain Tumors In Vivo

(A) To test Treg migration in vivo, splenic-sorted and expanded Tregs were co-labeled with eFluor 450 (HIF-1α WT) and eFluor 670 (HIF-1α KO) cell proliferation dyes and injected i.v. at a 1:1 ratio into mice harboring GL-261. (B) After 48 h, the brain, spleen, and DLN of mice injected with Tregs were isolated, and the ratio of control to HIF-1α KO Tregs was determined. (C) HIF-1α WT or HIF-1α KO mice were implanted with 4 × 10⁵ GL-261 astrocytoma cells, and overall survival was determined. (D) After 2 weeks of tumor growth, Treg abundance was analyzed via flow cytometry. (E) Abundance of Foxp3⁺ cells from tumor-bearing mice was quantified in tissue sections. Kaplan-Meier curves are n = 7 per group from two independent experiments, and significance was calculated using log-rank analysis. Flow cytometry statistics shown as percent positive population ± SEM; n = 5 per group, representative of two experiments. In (B), the ratios of WT/HIF-1α KO Tregs were indicated as mean ± SEM. In (E), 3–5 fields per section were quantified for Foxp3⁺DAPI⁺ nuclei. n = 3 mice per group. Unpaired t test analysis was used to calculate significance. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. See also Figure S7.