Fig. 5: HIF-2α-KO cells have high expression of HIF-1α.

a CD45.2⁺ tTreg cells were transferred to CD45.1⁺ RAG-1-KO mice together with CD45.1⁺ Teff. CD45.2⁺ tTreg cells were re-isolated after 15 days, activated by TPA/A23187 (50/500 ng ml⁻¹), and the intracellular HIF-1α, Foxp3, and IL-17 determined. n = 3 (Hif2a^f/f), 4 (Cd4^CreHif2a^f/f). Data represent mean ± SEM. *P = 0.011 (IL-17A⁺), 0.0422 (HIF-1α⁺) by two-tailed unpaired t-test. b, c Hif1a in Hif2a^f/f and Cd4^CreHif2a^f/f naïve CD4⁺ T (b) and tTreg (c) cells was determined by quantitative PCR. n = 3. *P = 0.0116 b, ***P < 0.001 c by two-tailed paired t-test. d WT and HIF-2α-KO CD4⁺ naïve T cells were activated by CD3/CD28 (2/1 μg ml⁻¹) and HIF-1α levels were determined. e, f Hif2a^f/f and Cd4^CreHif2a^f/f tTreg cells were activated by CD3/CD28 (4/2 μg ml⁻¹) for 24 h under normoxic or hypoxic conditions (e), or iTreg cells were activated by CD3/CD28 (2/1 μg ml⁻¹) for 4 h (f), and HIF-1α levels were determined. Each sample f represents independent iTreg cells preparation. g Hif2a^f/f and Cd4^CreHif2a^f/f iTreg cells were activated as in f under hypoxia for 4 h. Cells were then treated with cycloheximide (100 μg ml⁻¹) and HIF-1α levels determined. h, i WT naïve CD4⁺ T cells (h) were activated by CD3/CD28 (2/1 μg ml⁻¹) in the presence of HIF-2α inhibitor PT2385 for 2 days, or activating WT iTreg cells (i) were treated with 10 μM PT2385, and HIF-1α levels determined. Results have been reproduced in three (d, h) or two e–i independent experiments. j WT tTreg cells were stimulated with or without 10 μM PT2385, and IL-17 production was quantitated. Data are technical triplicates, the experiment was independently reproduced twice. k Hif2a^f/f and Cd4^CreHif2a^f/f tTreg cells were analyzed for expression of Glut1 and Ccr4 by quantitative PCR. n = 4 (Glut1), n = 5 (Ccr4). *P = 0.0236 (Glut1) and 0.0488 (Ccr4), by two-tailed paired t-test. l The expression of Hif2a in Hif1a^f/f and Cd4^CreHif1a^f/f naïve CD4⁺ T and tTreg cells was determined. n = 3. *P = 0.0464, ***P < 0.001, by two-tailed paired t-test. Source data are provided as a Source data file.