Ferritin heavy chain supports stability and function of the regulatory T cell lineage

Abstract

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Keywords: FOXP3; Ferritin Heavy Chain; Iron Metabolism; Regulatory T Cells; Ten–eleven Translocation Enzymes.

Figure 1. T_reg cell ferritin expression is a stable property of human and mouse T_reg cells.

(A) FTH and β-Actin protein expression, detected by western blot in whole-cell extracts from human T conventional (CD4⁺CD45RA⁺CD127⁺CD25^–; T_conv) and T_reg (CD4⁺CD127^–CD45RA⁺CD25^hi) cells after two weeks of expansion with anti-CD3, anti-CD28 mAb and IL-2. (B) Relative quantification of FTH protein expression, normalized to β-Actin, detected by western blot as in (A). n = 3 independent experiments. (C) FTH and histone H3 protein expression detected by western blot in whole-cell extracts from sorted mouse naive T cells (CD4⁺Foxp3^-CD44^lowCD62L^high; T_N), (CD4⁺Foxp3⁺GFP⁺) T_reg cells and memory T cells (CD4⁺Foxp3⁻CD44^highCD62L^low; T_M), by western blot. (D) Relative quantification of FTH, normalized to histone H3, protein expression, detected by western blot as in (C). Data were normalized to FTH expression in T_N cells, pooled from four independent experiments. (E) Schematic representation of the protocol used for the generation of iT_reg and representative flow cytometry dot plots of mouse iT_reg generated from sorted naive T cells (T_N), stimulated with anti-CD3 and anti-CD28 mAb plus IL-2 and TGFβ for 5 days. Control T_conv cells were subjected to the same experimental conditions, without TGFβ. (F) FTH and histone H3 protein expression, detected by western blot in whole-cell extracts from iT_reg and T_conv generated as depicted in (E). Data pooled from three independent experiments with similar trend. (G) The relative level of Fth mRNA expression, quantified by qRT-PCR, using Arbp0 as housekeeping gene. Data pooled from three independent experiments, with similar trend. (H–J) Schematic representation of the protocol used (top panels), representative flow cytometry dot plots (middle panels) and corresponding quantification of percentage (%) in CD4⁺ cells and cell number (Nbr.) (bottom panels) of live (TCRβ⁺CD4⁺ Foxp3⁺) GFP⁺ T_reg cells in (H) thymus, (I) spleen and (J) mesenteric LN (MLN). (H) Data from N = 8 mice per genotype, per organ, from two independent experiments, with similar trend. (I, J) Data from N = 12 mice per genotype, per organ, from three independent experiments, with similar trend. (K) Schematic representation of the experimental approach (top panel) used to monitor the expression of the GFP-hCre transgene in the thymus, spleen, and MLN of (CD4⁺GFP⁺) T_reg cells. Representative flow cytometry histograms of GFP-hCre (bottom left panel). Relative quantification of GFP-hCre expression (bottom right panel), represented as mean fluorescence intensity (MFI). Data from N = 8 mice per genotype, pooled from two independent experiments with similar trend. (L) Schematic representation of the experimental approach (left panel) used, representative flow cytometry dot plots (top panel) and corresponding quantification of percentage (%) (bottom left panel) and cell number (Nbr.) (bottom right panel) of live (TCRβ⁺CD4⁺ GFP⁺) Nrp1⁺ and Nrp1^-T_reg cells in MLN. Data from N = 8 mice per genotype, pooled from two independent experiments with similar trend. Data information: Data in (B, F, G) are presented as mean ± SD. Data in (D) are presented as mean ± SEM. Circles in (H–L) correspond to individual mice. P values in (B, F–J) determined using unpaired t test with Welch’s correction, in (D, H–J) using ordinary one-way ANOVA, and in (K, L) using Two-way ANOVA with Sidak’s multiple comparisons test. NS not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure.

Figure 2. FTH expression in T_reg cells prevents transdifferentiation into inflammatory ex-T_reg cells.

(A) Schematic representation of experimental approach (left panel) and representative flow cytometry dot plots of (CD4⁺CD25⁺GFP⁺) T_reg cells from the lymph nodes before and after sorting. (B) Volcano plot representation of RNA sequencing data of genes overexpressed (red) or under-expressed (blue) in (CD4⁺CD25⁺GFP⁺) T_reg cells sorted from Foxp3^GFP-FthΔ/Δ and control Foxp3^GFP (N = 5 per genotype) mice. P values determined by Benjamini and Hochberg adjusted probabilities. (C) Heatmap representation of T_reg transcriptional signature genes differentially expressed in T_reg cells sorted from Foxp3^GFP-FthΔ/Δ vs. Foxp3^GFP mice, as illustrated in (A, B). (D, E) Pathway enrichment analysis of genetic programs overexpressed (D) or under-expressed (E) in T_reg cells sorted from Foxp3^GFP-FthΔ/Δ vs. Foxp3^GFP mice, as illustrated in (A, B). Data were analyzed using g:SCS multiple testing correction method with a significance threshold of 0.05. (F) Heatmap representation of individual genes associated with oxidative stress-responsive programs, differentially expressed in T_reg cells sorted from Foxp3^GFP-FthΔ/Δ vs. Foxp3^GFP mice, as illustrated in (A, B). Source data are available online for this figure.

Figure 3. FTH enforces T_reg cell lineage stability.

(A) Schematic representation of Foxp3^{GFP-Fth∆/∆-tdT} mice used to monitor the transition of (CD4⁺GFP⁺tdT⁺) T_reg cells into (CD4⁺GFP^-tdT⁺) ex-T_reg cells that repressed GFP expression while retaining the expression of a tdT transgene. (B–D) Percentage of circulating: (B) T_reg and (C) ex-T_reg cells in Foxp3^{GFP-Fth∆/∆-tdT} and control Foxp3^GFP-tdT mice, and (D) Percentage of ex-T_reg cells among CD4⁺tdT⁺ cells, calculated as the ratio of CD4⁺GFP^-tdT⁺/CD4⁺tdT⁺ cells in the same mice as (B, C). Data from N = 4–6 mice per genotype was pooled from three independent experiments with a similar trend. (E–G) Percentage and number of splenic (E) T_reg cells, (F) ex-T_reg cells and (G) relative percentage of ex-T_REG cells over total CD4⁺tdT⁺ cells. Data from N = 4 mice per genotype, pooled from two independent experiments with similar trends. (H) Representative flow cytometry histograms of IFNγ expression by live activated (CD4⁺GFP⁺tdT⁺) T_reg and (CD4⁺GFP^-tdT⁺) ex-T_reg cells in lymph nodes and spleen from Foxp3^{GFP-Fth∆/∆-tdT} and control Foxp3^GFP-tdT mice (left panels) and corresponding quantification of the percentage of IFNγ expressing (CD4⁺GFP⁺tdT⁺) T_reg and (CD4⁺GFP^-tdT⁺) ex-T_reg cells (right panels). Expression of IFNγ was induced upon Phorbol-12-myristate-13-acetate (PMA) and Ionomycin re-activation in vitro. Data from N = 3 wells per genotype, in one experiment, representative of two independent experiments with similar trend. (I) Representative flow cytometry dot plots (left panels) and corresponding quantification (right panel) of the percentage of (CD4⁺GFP⁺tdT⁺) T_reg and (CD4⁺GFP^-tdT⁺) ex-T_reg cells expressing Ki67 and CD71 in the lymph nodes Foxp3^{GFP-Fth∆/∆-tdT} and control Foxp3^GFP-tdT mice. Data from N = 6 mice per genotype, pooled from two independent experiments, with similar trend. Data information: Data in (B–D) represented as mean ± SD. Circles correspond to mean values. Data in (E–I) circles correspond to individual mice and red bars to mean values. (H, I) represented as mean ± SD. P values in (B–D, H, I) calculated using Two-way ANOVA analysis with Sidak’s multiple comparisons test and in (E–G) with unpaired t test with Welch’s correction. NS not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.

Figure 4. FTH enforces T_reg cell lineage stability via a cell-autonomous mechanism.

(A) Schematic representation of bone marrow (BM) chimeric mice used for flow cytometry analyzed 6 weeks after reconstitution. For reconstitution BM cells from C57BL/6 CD45.1⁺ mice were mixed with BM cells from congenic C57BL/6 CD45.2⁺ Foxp3^{GFP-Fth∆/∆-tdT} or control Foxp3^GFP-tdT mice at a 1:1 ratio and injected (i.v.; tail vein, 200 µL) into congenic C57BL/6-recipient Rag2-deficient (Rag2^−/−) female mice, 2 h after irradiation (600 Gys). (B) Representative flow cytometry dot plots and (C) Percentage of CD45.1⁺ vs. CD45.2⁺ CD4⁺ T cells, CD8⁺ T cells and CD4⁺Foxp3⁺ T_reg cells in the lymph nodes of mixed BM chimeric mice, from (A). Data from N = 10 mice per genotype. (D–G) Representative flow cytometry dot plots (D) and corresponding quantification (E, F) of the percentage and number of (CD45.2⁺CD4⁺GFP⁺tdT⁺) T_reg cells (E), (CD45.2⁺CD4⁺GFP⁻tdT⁺) ex-T_reg cells (F) and relative proportion of ex-T_reg cells over total CD45.2⁺CD4⁺tdT⁺ cells in lymph nodes (LN) (G) of mixed BM chimeric mice (as in A). Data from N = 9–10 mice per genotype. (H) Schematic representation of (CD45.2⁺CD4⁺GFP⁺tdT⁺) T_reg cells and (CD45.2⁺CD4⁺GFP^-tdT⁺) ex-T_reg cells FACS-sorting from the lymph nodes of mixed BM chimeric mice, used for RNA sequencing analysis. (I, J) Volcano plot representations of RNA sequencing data of genes overexpressed (red) or under-expressed (blue) in T_reg cells (I) and ex-T_reg cells (J) from lymph nodes of BM chimeric mice (as in H). Data from N = 3–4 mice per genotype. P values determined by Benjamini and Hochberg adjusted probabilities. (K) Euler plot of differentially regulated genes in (CD45.2⁺CD4⁺GFP⁺) T_reg cells sorted from the Foxp3^{GFP-Fth∆/∆} or control Foxp3^GFP T_reg cells (non-chimeric; blue; from analysis described in Fig. 2A,B) or from the mixed BM chimeric mice (chimeric; red; from analysis illustrated in H–J). (L) Dumbbell plot, showing the adjusted P value of the 134 overlapping differentially regulated genes (from analysis described in K). Gray bars connecting dots represent the difference in P values for the differentially regulated genes (from analysis described in K). (M) Functional enrichment analysis of the overlapping genes (N = 134) (from analysis described in K), considering five different functional categories: biological processes (Bio. Proc.); KEGG (Kyoto Encyclopedia of Genes and Genomes) database; reactome (Reac.); transcription factors (TF); and WikiPathways (WP). Data were analyzed using g:SCS multiple testing correction method with a significance threshold of 0.05. Data information: Data in (C) are represented as mean ± SD. Data in (E–G) are represented as mean, circles correspond to individual mice and red bars are mean values. P value in (C) was determined by two-way ANOVA with Sidak’s multiple comparisons test and in (E–G) by unpaired t test with Welch’s correction. *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure.

Figure 5. FTH is a T_reg cell-autonomous cytoprotectant.

(A) Schematic representation of flow cytometry analyses of (CD4⁺GFP⁺tdT⁺) T_reg cells, isolated from the lymph nodes of lymphopenic Rag2^−/− mice, 6 weeks after adoptive transfer of T_reg cells sorted from Foxp3^{GFP-Fth∆/∆-tdT} vs. control Foxp3^GFP-tdT mice. (B) Representative flow cytometry dot plots of (CD4⁺GFP⁺tdT⁺) T_reg cells and (CD4⁺GFP^-tdT⁺) ex-T_reg cells, analyzed, as illustrated in (A). (C) Number (CD4⁺GFP⁺tdT⁺) T_reg cells and (CD4⁺GFP^-tdT⁺) ex-T_reg (left panel), as well as the relative proportion of ex-T_reg cells over total CD4⁺tdT⁺ cells (right panel), 6 weeks after adoptive transfer into Rag2^-/- mice, as illustrated in (A). Data from N = 4 per genotype, in one out of two independent experiments with similar trend. (D) Schematic representation of the experimental approach (left panel) and number (Nbr.) of mitochondria in (CD4⁺GFP⁺) T_reg cells sorted from lymph nodes of the Foxp3^{GFP-Fth∆/∆} or control Foxp3^GFP mice (right panel), quantified by genomic quantitative PCR. Data from N = 3 mice per genotype in one experiment. (E) Schematic representation of the experimental approach used to quantify mitochondrial membrane potential in splenic mouse (CD4⁺GFP⁺) T_reg cells (left panel). Representative flow cytometry histograms of tetramethylrhodamine ethyl ester (TMRE) staining (middle panels). Mean fluorescence intensity (MFI) of TMRE (right panel). Data from N = 4–5 mice per genotype, pooled from two independent experiments, with similar trend. (F) Schematic representation of experimental approach (left panel) used to quantify oxygen consumption rate (OCR) in live splenic (CD4⁺GFP⁺) T_reg cells (right panel). Data pooled from N = 3 mice per genotype, represented as mean ± SD (N = 3–5 technical replicates) in one out of three independent experiments, with similar trend. Oligomycin (Oligo.), carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP), Antimycin A/Rotenone (A/A+Rot.). (G) Quantification of basal respiration, ATP production and spare respiratory capacity, from data represented in (F). (H) Extracellular acidification rate (ECAR) in live splenic (CD4⁺GFP⁺) T_reg cells represented as mean ± SD (N = 3–5 technical replicates) in one out of three independent experiments, with similar trend. (I) Schematic representation of the experimental approach (left panel), used to quantify the ratio of α-ketoglutarate to isocitrate and α-ketoglutarate to glutamate (right panels) in live splenic (CD4⁺GFP⁺) T_reg cells. Data from N = 4 mice per genotype in one out of two independent experiments with similar trend. Data information: Data in (C–E) circles correspond to individual value and red bars to mean values. Data are presented as mean ± SD. Data in (F, H) are presented as mean ± SD, circles correspond to technical replicates. Data in (G) are presented as mean ± SD of technical replicates. Data in (I) are presented as mean ± SD, circles correspond to individual values. P values in (C (left panel), G) were calculated using two-way ANOVA with Sidak’s multiple comparison test, P values in (F, H) were calculated using two-way ANOVA with Bonferroni’s multiple comparisons test. P values in (C, right panel), D, E, I) were calculated using unpaired t test with Welch’s correction. NS not significant (P > 0.05), *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure.

Figure 6. FTH regulates mitochondrial function and cytosine methylation in T_reg cells.

(A) Schematic representation of mixed bone marrow (BM) chimeric mice from which lymph node CD45.2⁺CD4⁺tdT⁺ cells (i.e., GFP⁺ T_reg cells and GFP⁻ ex-T_reg cells) were sorted for genome-wide EM-seq analyses. (B) Principal component analysis (PCA) of the methylome of lymph node T_reg and ex-T_reg cells in BM chimeric mice generated, as illustrated in (A). Data from N = 4 mice per genotype is represented as individual circles in one experiment. (C) Unsupervised heatmap representation of genome-wide EM-seq analyzes (i.e., 5-hmC) in CD45.2⁺CD4⁺tdT⁺ cells sorted from the mixed BM chimeric mice illustrated in (A). Statistical analysis for multiple testing correction was performed with Sliding Linear Model (SLIM). (D) The relative percentage of total methylated regions (i.e., hyper- and hypomethylated regions) (q < 0.01 and methylation difference >=10%) according to different genome regions (promoter, exon, intron and intergenic). (E) The number of hyper- and hypomethylation events (10% change in methylation and a q value of 1%) per chromosome, shown as a percent of the differential sites. (F) Schematic representation of the Foxp3 in enhancer regions CNS1 and CNS2. (G) Relative quantification of the methylation rate of CpG sequences in the Foxp3 CNS1 (left panel) and CNS2 (right panel) from CD45.2⁺CD4⁺tdT⁺ cells sorted from the lymph nodes of BM chimeric mice, illustrated in (A). Data from N = 4 mice per genotype in one experiment. (H) TET activity in nuclear extracts from HEK293T cells transiently transfected with human TET3-flag, FTH-flag, or FTH^mut-flag cDNAs. Data shows technical replicates, pooled from four independent experiments. (I) FTH-flag, FTH^mut-flag, TET3-flag, Lamin A/C and GAPDH protein expression, detected by western blot in nuclear and cytosol extracts from HEK293T cells transfected as described in (H). Relative quantification of TET3-flag, normalized to Lamin A/C (bottom left panel), and FTH-flag, normalized to GAPDH (bottom right panel). Data from one experiment, representative of three independent experiments with similar trend. Data information: Data in (G) are presented as mean ± SD, circles correspond to individual mice and red bars to mean values. Data in (H, I) are presented as mean ± SD, circles correspond to technical replicates. P values in (G) were calculated using Two-way ANOVA with Sidak’s multiple comparison test, and in (H, I) using one-way ANOVA using with Sidak’s multiple comparison test. NS not significant (P > 0.05), *P < 0.05; **P < 0.01, ***P < 0.001. Source data are available online for this figure.

Figure 7. FTH is required to sustain Foxp3 transcription and expression.

(A) FTH protein detected by western blot in whole-cell extracts from HEK293T cells infected with recombinant lentiviruses coding shRNAs targeting FTH (FTH⁴²⁹ and FTH⁴³²) or control (Ctrl.) recombinant lentiviruses non-targeting shRNA. (B) Mean fluorescence intensity (MFI) of FOXP3 expression, detected by flow cytometry in human (CD4⁺CD45RA⁺CD25⁺) T_reg cells infected with the same recombinant lentiviruses as in (A). Data from N = 6 samples per experimental group. (C) Schematic representation of the experimental approach (left panel) used to monitor GFP transgene expression in the mesenteric LN (MLN) of (CD4⁺GFP⁺Nrp1⁺) tT_reg cells and (CD4⁺GFP⁺Nrp1⁻) pT_reg cells. Representative flow cytometry histogram (middle panel) and quantification of relative GFP expression (right panel), shown as mean fluorescence intensity (MFI). Data from N = 8 mice per genotype, pooled from two independent experiments with similar trend. (D) Schematic representation of the experimental approach (left panel) used to monitor Foxp3 expression by flow cytometry in mouse spleen and MLN (CD4⁺Foxp3⁺) T_reg cells. Representative flow cytometry staining of Foxp3 (middle panel). Relative quantification of Foxp3 expression (right panel), represented as mean of fluorescence intensity (MFI). Data from N = 9 mice per genotype, pooled from two to three independent experiments with similar trend. (E) Schematic representation of the experimental approach (left panel) used to monitor Foxp3 expression in (CD45.2⁺CD4⁺GFP⁺tdT⁺) T_reg cells isolated from the spleen and LN of BM chimeras. Representative flow cytometry of Foxp3 staining (middle panel). Relative quantification of Foxp3 expression (right panel), shown as mean fluorescence intensity (MFI). Data in (E) from N = 5–6 mice per genotype, representative of two independent experiments with similar trend. (F) Schematic representation of the experimental approach (left panel) used to monitor GFP expression in the spleen and LN of CD45.2⁺CD4⁺tdT⁺ cells (T_reg+ ex-T_reg) from BM chimeras. Representative flow cytometry of GFP (F) staining (middle panel). Relative quantification of GFP expression (right panel), represented as mean fluorescence intensity (MFI). Data from N = 10 mice per genotype, pooled from two independent experiments with similar trend. (G) Schematic representation of the experimental approach (left panel), where (CD4⁺tdT⁺) cells were sorted from the spleen and LN for qRT-PCR (G, left panel). Relative expression of Foxp3 (right panel). (H) Gfp, Fth, and tdT mRNA expression normalized to Arbp0 of cells sorted as in (G). Data in (G, H) from N = 3–4 mice per genotype from one experiment. Data information: Data in (B) are presented as mean ± SD, circles correspond to individual wells and red bars to mean values. Data in (C–H) are presented as mean ± SD, circles correspond to individual mice and red bars to mean values. P values in (B) were calculated using the Fiedman test with Dunn’s multiple comparison test, in (C–H) using two-way ANOVA with Sidak’s multiple comparison test. NS not significant (P > 0.05), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.

Figure 8. FTH expression in T_reg cells controls the pathologic outcome of experimental immune-driven inflammatory conditions.

(A) Schematic representation of the induction experimental autoimmune encephalomyelitis (EAE) in response to MOG_35-55 immunization. (B) EAE incidence (percentage) and (C) EAE severity in MOG_35-55 immunized mice. Data from N = 18–20 mice per genotype, pooled from three independent experiments, with similar trend. (D) Experimental approach (right panel), representative flow cytometry dot plots (middle panel) and corresponding quantification (left panel) of the relative percentage of activated T_H1 (CD3⁺CD4⁺IFN-γ⁺), T_H17 (CD3⁺CD4⁺IL-17A⁺); and double positive IFN-γ⁺IL-17A⁺ T_H cells in the spinal cord, 22 days after MOG_35-55 immunization. Data from N = 3–4 mice per genotype. (E) Survival (left panel) and number of circulating Plasmodium chabaudi chabaudi (Pcc)-infected red blood cells (iRBC) per µL of whole blood (i.e., parasite burden) (right panel). N = 9 mice per genotype, pooled from two independent experiments, with similar trend. (F, G) Representative flow cytometry dot plot (left panels) and corresponding percentage and cell numbers (right panels) of splenic (CD4⁺Foxp3-GFP⁺) T_reg cells (F) and IFNγ⁺CD4⁺ activated T_H cells (G), 7 days after Pcc infection. Data from N = 8–9 mice per genotype, pooled from two independent experiments, with similar trend. (H) Relative tumor (B16-F10-luc2) size, 13–19 days after inoculation (2 × 10⁵ cells). Data from N = 7–11 mice per genotype, pooled from 3 independent experiments, with similar trend. (I, J) Representative flow cytometry dot plots (left panels) and corresponding percentage (right panels) of live tumor-infiltrating (CD4⁺Foxp3⁺) T_reg cells (I) and (CD4⁺Foxp3^-CD25⁺) effector T_H cells (J), 3 weeks after tumor inoculation. N = 7 mice per genotype, pooled from three independent experiments, with similar trend). Data information: Circles in (D, F, G, I, J) correspond to individual mice and red bars to mean values. Data in (C, H) are presented as mean ± SEM. Data in (E, right panel) are presented as mean ± SD. P values in (C), (E, right panel), and (H) were determined using Holm–Sidak method (multiple t tests), with alpha = 0.05 under the assumption that both genotypes have similar SEM, in (B, E) by log-rank (Mantel–Cox) test, and in (D, F, G, I, J) by unpaired t test with Welch’s correction. NS not significant (P > 0.05); *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure.

Figure EV1. FTH expression in T_REG cells alters systemic iron metabolism.

(A) Schematic representation of experimental approach (left panel) and relative quantification of Fth mRNA, by qRT-PCR, normalized to Arbp0 mRNA (right panel), of (CD4⁺ GFP⁺) T_reg cells sorted from mesenteric lymph nodes (MLN). Data from N = 3 mice per genotype. (B) Schematic representation of experimental approach used for relative quantification of intracellular Fe²⁺ in (CD4⁺GFP⁺) T_reg cells in mesenteric lymph nodes (MLN), using the FeRhoNox™-1 probe (left panel). Representative flow cytometry histograms (middle panel) and relative quantification (right panel) of mean fluorescence of intracellular Fe²⁺ intensity (MFI) N = 3 mice per genotype. (C) Schematic representation of experimental approach (left panel), representative flow cytometry dot plots (middle panel) and corresponding quantification of percentage (%) and cell number (Nbr.) (right panels) of live splenic follicular (CD4⁺GFP⁺CXCR5⁺PD1⁺) FT_reg cells. Data from N = 4 mice per genotype, from one experiment. (D, E) Representative flow cytometry dot plots (left panels) and number (right panel) of live activated (CD4⁺CD44^highCD62L^low) and (CD8⁺CD44^highCD62L^low) T cells in the spleen (D) and MLN (E). Data from N = 6–8 mice per genotype, pooled from four independent experiments, with similar trend. (F, G) Representative flow cytometry dot plots (left panels) and percentage (%) (right panel) of live activated (CD3⁺CD4⁺Foxp3⁻IFN-γ⁺; T_H1) T_H1 and (CD3⁺CD8⁺Foxp3⁻IFN-γ⁺) T_C in the spleen (F) and MLN (G). Data representative of N = 5 mice per genotype, pooled from two independent experiments, with similar trend. Data information: Data in (A–G) represented as mean ± SD, circles in (A, C–G) correspond to individual mice and red bars to mean values. P values in (A–C) calculated using unpaired t test with Welch’s correction, and in (D–G) using two-way ANOVA with Sidak’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure.

Figure EV2. FTH expression in T_REG cells prevents systemic cellular inflammation.

(A) Schematic representation of experimental approach (left panel) for (CD4⁺CD25⁺GFP⁺) T_reg cell sorting from the mesenteric lymph nodes (MLN) and coculture with conventional activated T cells (αCD3/28 + IL-2) to evaluate suppressive function of T_reg cells. Representative flow cytometry proliferation histograms (Cell Tracer Violet) of in vitro suppression assay of mouse T_N by different ratios of T_reg cells (middle panel). Inhibition of T_N cell proliferation quantified as percentage of undivided cells (right panel). Data from 1 out of 3 representative experiments, with similar trend. (B) Representative images of H&E-stained liver, lung, kidney, colon, and pancreas from N = 3–4 mice per genotype at 27–31 weeks after birth. (C) Schematic representation of the experimental approach (left panel) used to generate the Heatmap (right panel) of individual genes associated with T_H effector function programs, differentially expressed in (CD4⁺GFP⁺) T_reg cells sorted from Foxp3^GFP-FthΔ/Δ vs. Foxp3^GFP mice (same experiment as Fig. 2A,B). (D) Schematic representation of the experimental approach used (left panel) to evaluate the percentage (right panel) of (CD4⁺Foxp3⁺CD44^highCD62L^low) activated T_reg cells in the MLN and spleen. Data from N = 7–8 mice per genotype, pooled from three independent experiments, with similar trend. (E) Schematic representation of the experimental approach used (top panel), representative flow cytometry dot plots (bottom left panel) and percentage (bottom right panel) of splenic (CD4⁺Foxp3⁺) IFNγ-secreting T_reg. Data from N = 5 mice per genotype, pooled from two independent experiments, with similar trend. Data information: Data in (A, D, E) represented as mean ± SD. Circles in (A) represent individual wells, and red bars are mean values. Circles in (D, E) represent individual mice, and red bars are mean values. P values in (A, D) calculated using Two-way ANOVA with Sidak’s multiple comparison test and in (E) using unpaired t test with Welch’s correction. NS not significant (P > 0.05), *P < 0.05; ***P < 0.001. Source data are available online for this figure.

Figure EV3. FTH expression in T_REG cells prevents T_reg transdifferentiation into inflammatory T_reg cells.

(A) Representative flow cytometry dot plots of GFP and tdT expression in circulating CD4⁺ cells (same experiment as Fig. 3A–D). Numbers in quadrants correspond to percentages of positive cells at the indicated weeks after birth. (B) Relative quantification of Fth mRNA, by qRT-PCR, normalized to Arbp0 mRNA, in (CD4⁺GFP⁺tdT⁺) T_reg and (CD4⁺GFP^-tdT⁺) ex-T_reg cells sorted from the lymph nodes (LN). Data from N = 5–7 mice per genotype, pooled from two experiments with similar trend. (C) Mean fluorescence intensity (MFI) of IFNγ in activated (CD4⁺GFP⁺tdT⁺) T_reg cells and (CD4⁺GFP^-tdT⁺) ex-T_reg from the lymph nodes (LN) and spleen in the same experiment as (Fig. 3H). Data from N = 3 wells per genotype in one experiment, representative of 2 independent experiments with similar trend. (D) Representative flow cytometry dot plots (left panel) and corresponding percentage of Ki67⁺CD71⁺ (right panel) among splenic (CD4⁺GFP⁺TdT⁺) T_reg cells and (CD4⁺GFP^-TdT⁺) ex-T_reg cells. Data from N = 6 mice per genotype, pooled from two independent experiments, with similar trend. (E) Mean fluorescence intensity (MFI) of CD71 expression in Ki67⁺ T_reg and ex-T_reg cells from the lymph nodes (LN) and spleen, from the same experiments as in (D). (F) Representative flow cytometry dot plots and (G) corresponding percentages of CD45.1⁺ and CD45.2⁺ double negative (DN), double positive (DP) thymocytes, T_H cells, cytotoxic T cells and (CD4⁺Foxp3⁺) T_reg cells in the thymus from the same BM chimeric mice illustrated in (Fig. 4A–G). Data from N = 11–12 mice per genotype, pooled from two independent experiments with similar trend. Data information: Data in (B–E, G) are presented as mean ± SD, circles in (B, D, E) correspond to individual mice or individual wells (C) and red bars are mean values. P values in Panel (B–E, G) were calculated using two-way ANOVA with Sidak’s multiple comparison test. NS not significant (P > 0.05), *P < 0.05; **P < 0.01; ***P < 0.001. Source data are available online for this figure.

Figure EV4. FTH acts in a non-cell-autonomous manner to prevent T_REG cells from transdifferentiating into inflammatory T_reg cells.

(A) Schematic representation of the experimental approach used for flow cytometry analysis from the lymph nodes of BM chimeric mice (same experiment as Fig. 4A). (B, C) Representative flow cytometry dot plots (B), quantification of percentage (left panel) and number (right panel) (C) of live activated (CD45.2⁺CD4⁺CD44^highCD62L^low) and (CD45.2⁺CD8⁺CD44^highCD62L^low) cells in the lymph nodes of BM chimeric mice from (A). Data in (C) from n = 11–12 mice per genotype, pooled from 2 independent experiments, with similar trend. (D) Schematic representation of cell sorting for adoptive transfers in the experiment illustrated in Fig. 5A. (E) Representative flow cytometry dot plots of (CD4⁺GFP⁺tdT⁺) T_reg cells and relative level of Fth mRNA expression in (CD4⁺GFP⁺tdT⁺) T_reg cells (right panel) used for adoptive transfers in the experiment illustrated in Fig. 5A. (F) Schematic representation of experimental approach used for in vitro generation of induced T_reg (iT_reg) cells and conventional T_H (T_CONV) cells from sorted naive T_H (T_N) cells, activated with anti-CD3 and anti-CD28 mAb plus IL-2 and TGFβ. (G) Representative flow cytometry dot plots of iT_reg and T_CONV cells, generated in (F). (H) Percentage (%) and (I) Number (Nbr.) of Foxp3⁺ T_CONV and iT_reg cells, generated as described in (F). N = 2–5 independent experiments with similar trend. Each experiment corresponds to the average of different wells. (J) Representative flow cytometry carboxyfluorescein succinimidyl ester (CFSE) staining (left panel) and quantification of percentage (%) (right panel) of proliferating (CD4⁺Foxp3⁺) iT_reg cells, generated as described in (F). Data from 3 to 6 technical replicates in 1 out of 3 independent experiments, with similar trend. Data information: Data in (C, E) are presented as mean ± SD, circles correspond to individual mice and red bars are mean values. Circles in (H–J) correspond to individual wells and red bars are mean values. P values in panels (C, H, I) were calculated using two-way ANOVA with Sidak’s multiple comparison test. P values in (E) were calculated using Mann–Whitney test. NS not significant, ***P < 0.001. Source data are available online for this figure.

Figure EV5. FTH regulates mitochondrial energy metabolism and CpG methylation in T_reg cells.

(A) Oxygen consumption rate (OCR) in live splenic (CD4⁺GFP⁺) T_reg cells and (CD4⁺GFP^-) T_CONV cells from Foxp3^GFP mice. (B) Quantification of spare respiratory capacity, from data represented in (A). (C) Oxygen consumption rate (OCR) in live splenic (CD4⁺GFP⁺) T_reg cells and (CD4⁺GFP^-) T_CONV cells from Foxp3^{GFP-Fth∆/∆} mice. (D) Quantification of spare respiratory capacity, from data represented in (C). Data in (A–D) pooled from N = 3 mice per genotype, represented as mean ± SD. N = 3–5 technical replicates in 1 out of 3 independent experiments, with similar trend. Oligomycin (Oligo.), carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP), Antimycin A/Rotenone (A/A+Rot.). (E) Schematic representation of sorting of splenic (CD4⁺GFP⁺) T_reg cells used for targeted metabolomics (left panel). Quantification of intermediate metabolites from targeted metabolomics analyzes of splenic T_reg cells (right panel). Data from N = 3–4 mice per genotype in one experiment representative of 3 independent experiments with similar trend. (F) Schematic representation of the experimental approach used for flow cytometry analysis of tumor-infiltrating cells (left panel), representative flow cytometry dot plots (middle panel) and corresponding percentage and number (right panel) of live tumor-infiltrating (CD4⁺IFNγ⁺) T_H cells (CD8⁺IFNγ⁺) T_C cells, 3 weeks after tumor inoculation (2 × 10⁵ B16 cells). Data from N = 6 mice per genotype, pooled from two independent experiments, with similar trend. (G) Schematic representation of the experimental approach used for flow cytometry analysis of tumor-infiltrating cells (left panel), representative flow cytometry dot plots (middle panel) and corresponding percentage and number (right panels) of live tumor-infiltrating (CD8⁺GrzmB⁺) T cells, 3 weeks after tumor inoculation (2 × 10⁵ B16 cells). Data from N = 6 mice per genotype, pooled from two independent experiments, with similar trend. Data information: Circles and triangles in (A, C) correspond to mean values, circles, and triangles in (B, D) correspond to individual wells, and circles in (E–G) correspond to individual mice, and red bars are mean values. P values in (A, C) calculated using two-way ANOVA with Bonferroni’s (A, C) or Sidak´s (E, F) multiple comparisons test, in (B, D, G) using unpaired t test with Welch’s correction. NS, not significant (P > 0.05); *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available online for this figure.