Fig. 2: TfR1 promotes the differentiation of c-Maf⁺ Treg cells.

a–c CD4⁺ splenic cells from Tfrc^fl/fl Foxp3^YFP-Cre/+ and Tfrc^+/+Foxp3^YFP-Cre/+ (WT) female mice were gated and analyzed for YFP and Foxp3 expression. a Representative flow cytometric analysis with pregating on CD4⁺ cells; (b). Summary of the frequencies of YFP⁺ Foxp3⁺ cells in CD4⁺ T cells from the spleen (n = 10 mice) and lymph nodes (n = 10 for Tfrc^fl/fl Foxp3^YFP-Cre/+ mice and n = 9 for WT mice); (c). Histogram comparing Foxp3 levels in gated CD4⁺Foxp3⁺YFP⁺ cells from the spleen of either Tfrc^fl/fl Foxp3^YFP-Cre/+ or Tfrc^+/+Foxp3^YFP-Cre/+ female mice. The data are summary (b) or representative (a, c) of five independent experiments. d–g scRNA-seq analysis of CD4⁺CD25⁺ cells isolated from the spleens of Tfrc^fl/fl Foxp3^YFP-Cre/+ mice. TfR1-deficient cells (YFP⁺) and WT (YFP^-) cells were separated in silico based on detection of the YFP-IRES-CRE transcript. d Schematic of the experimental design. e t-SNE projection of all CD4⁺CD25⁺ cells isolated from Tfrc^fl/fl Foxp3^YFP-Cre/+ mice. YFP⁺ (Cluster A) and YFP^- (Cluster B) cells are indicated; f, t-SNE projection of all cells color-coded by normalized counts for Foxp3 (left), Cre-YFP (middle), or Maf (right); g, Violin plots comparing Cre-YFP (left) and Maf (right) expression between cluster A and cluster B. h, i As in (a), CD4⁺ FOXP3⁺YFP⁺ cells from the spleens of Tfrc^fl/fl Foxp3^YFP-Cre/+ mice were analyzed for the expression of CD62L and CD44 (upper panels) or c-Maf (lower panels). The frequencies of effector Treg cells (CD62L^low CD44^hi, upper panels) and c-Maf⁺ Tregs (lower panels) are indicated. (h Representative flow cytometric staining; (i). Summary of the c-Maf⁺ Treg cell and eTreg cell percentages (n = 8 mice for eTreg analysis; n = 9 mice for c-Maf⁺ Treg analysis based on 3 independent experiments). YFP⁺ cells from the Tfrc^+/+Foxp3^YFP-Cre/+ mice were included as controls. j As in (a), effector Treg cells in the spleen were gated as CD44^hiCD62L^low Foxp3⁺YFP⁺, and c-Maf expression was compared between TfR1-deficient and WT eTreg cells by flow cytometry. The data are representative of three independent experiments. b–i Data shown are mean ± SD, ^***p < 0.001, ^****p < 0.0001 by two-tailed Student’s t test.