Fig. 3: Iron promotes c-Maf expression in Treg cells by modulating HIF-2α expression.

a, b WT mice were administered iron dextran (37.5 mg/e.a) every five days for 3 weeks. The percentages of c-Maf⁺ Treg cells in the spleen were determined by flow cytometry. a Representative flow cytometric plots; (b). Summary of the percentages of c-Maf⁺ cells in splenic Treg cells from three independent experiments. c, d CD4 T cells were activated under iTreg differentiation conditions (anti-CD3+anti-CD28 + TGFβ1 + IL-2+anti-IFNγ+anti-IL4) with the indicated concentrations of ferrous sulfate or DFO. The expression of c-Maf and Foxp3 was determined by intracellular staining four days after T-cell activation. c Representative flow cytometry analysis; (d). Summary of the percentages of c-Maf⁺ Tregs (n = 7 biologically independent experiments for mock, and n = 5 for FeSO₄-treated mice). (e, f). As in (c), the transcriptomes of ferrous sulfate-treated iTreg cells were compared to those of iTreg cells without treatment by RNA-seq. e M-A plots showing the differentially expressed genes; Differentially expressed genes were determined by DESeq2 and are shown in red or blue (|LFC | ≥ 0.5, p < 0.05 in Wald test). f GO analysis of the differentially expressed genes using Metascape. p-values are calculated based on the cumulative hypergeometric distribution, and q-values are calculated using the Benjamini-Hochberg procedure. g The expression of HIF-1α and HIF-2α in YFP⁺Foxp3⁺CD4⁺ cells of Tfr1^fl/fl Foxp3-Cre^YFP/+ mice (Tfrc cKO) was determined by flow cytometry and compared with that in their counterparts (WT) of Tfrc^+/+Foxp3-Cre^YFP/+ mice. The data are representative of three independent experiments. h As in (c), one day after T-cell activation, iTregs were cultured with 50 μM FeSO₄ for 24 h, and the expression of HIF-2α and HIF-1α in Foxp3⁺ cells was determined by intracellular staining. The data are representative of three independent experiments. i, j As in (a), iTreg cells were treated with 50 μM ferrous sulfate with or without 50 μM PT2385 (an HIF-2α inhibitor). The expression of c-Maf was determined by intracellular staining. i Representative results. j Summary of c-Maf⁺% cells in iTreg cells based on four independent experiments. b, d, j Data shown are mean ± SD, ^*p < 0.05, ^**p < 0.01,^***p < 0.001 by two-tailed Student’s t test.