Figure 6. DTX1 increases Foxp3 stability through HIF-1α downregulation.

(a) HIF-1α promotes Foxp3 degradation. 293T cells were transfected with Foxp3, ubiquitin and HIF-1α, and cellular levels of Foxp3 determined. (b) DTX1 protects Foxp3 from HIF-1α-induced degradation. Foxp3-expressing DO11.10 cells were transfected with or without DTX1. Cells were incubated in normoxic (N) conditions for 16 h and kept at N conditions or switched to hypoxic (H) conditions for 4 h, and levels of DTX1 and Foxp3 were analysed. Right panel, quantification from three independent experiments. (c) Increased HIF-1α induction and decreased Foxp3 levels in hypoxia-treated Dtx1^−/− tTreg cells. Control and Dtx1^−/− tTregs were activated by CD3/CD28 plus IL-2 in N or H conditions for 72 h, and the levels of HIF-1α and Foxp3 were determined. (d) Expression of DTX1 and HIF-1α in CD4⁺ T cells from WT, Dtx1^−/−, Hif1a^−/− and Dtx1^−/−Hif1a^−/− mice. (e) Reduced Foxp3⁺ population in transferred Dtx1^−/− tTregs are restored by HIF-1α deficiency. CD45.2⁺ WT (+/+), Dtx1^−/−, Hif1a^−/− or Dtx1^−/−Hif1a^−/− tTreg cells were co-transferred with CD45.1⁺ CD4⁺CD25⁻ T cells into Rag1^−/− mice. Spleen and lymph nodes were isolated 1 week later, and the frequency of Foxp3⁺ cells in the CD4⁺CD45.2⁺ T-cell population was determined. Bottom panel, splenic Foxp3⁺ cells from three mice each group. The percentage of +/+ was set as 100%. (f) Hif1a knockout enables Dtx1^−/− tTregs to inhibit colitis. Colitis was scored in Rag1^−/− mice transferred with effector T cells, and WT (Foxp3^Cre), Foxp3^CreDtx1^f/f, Foxp3^CreHif1a^f/f or Foxp3^CreDtx1^f/fHif1a^f/f tTreg cells. (g) Decreased Foxp3⁺ population in transferred Dtx1^−/− iTregs are reversed by HIF-1α deficiency. CD45.2 WT (Foxp3^Cre), Foxp3^CreDtx1^f/f, Foxp3^CreHif1a^f/f or Foxp3^CreDtx1^f/fHif1a^f/f iTreg cells were administered to CD45.1 B6 mice, followed by ovalbumin immunization. The transferred CD45.2 iTreg cells were isolated from lymph nodes of mice killed at day 25, and the frequency of Foxp3⁺ cells was determined. Data are representative of three mice in each group. (h) HIF-1α deficiency restores the in vivo inhibitory activity of Dtx1^−/− iTreg cells. B6 mice were treated with WT, Dtx1^−/− (Cd4^CreDtx1^f/f), Hif1a^−/− (Cd4^CreHif1a^f/f), Dtx1^−/−Hif1a^−/− (Cd4^CreDtx1^f/fHif1a^f/f) iTreg cells or PBS as in Fig. 3. The contents of cytokines in the BAL were determined. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. BALF, bronchoalveolar lavage fluid. *P<0.05, **P<0.01, ***P<0.001 for paired t-test.