Zfp335 establishes eTreg lineage and neonatal immune tolerance by targeting Hadha-mediated fatty acid oxidation

Abstract

Regulatory T cells (Tregs) are instrumental in maintaining immune tolerance and preventing destructive autoimmunity, but how heterogeneous Treg populations are established remains largely unknown. Here, we show that Zfp335 deletion in Tregs failed to differentiate into effector Tregs (eTregs) and lose Treg-suppressive function and that KO mice exhibited early-onset lethal autoimmune inflammation with unrestricted activation of conventional T cells. Single-cell RNA-Seq analyses revealed that Zfp335-deficient Tregs lacked a eTreg population and showed dramatic accumulation of a dysfunctional Treg subset. Mechanistically, Zfp335-deficient Tregs displayed reduced oxidative phosphorylation and dysfunctional mitochondrial activity. Further studies revealed that Zfp335 controlled eTreg differentiation by regulating fatty acid oxidation (FAO) through direct targeting of the FAO enzyme Hadha. Importantly, we demonstrate a positive correlation between ZNF335 and HADHA expression in human eTregs. Our findings reveal that Zfp335 controls FAO-driven eTreg differentiation to establish immune tolerance.

Keywords: Adaptive immunity; Autoimmune diseases; Autoimmunity; Immunology; T cells.

Figure 1. Spontaneous multiorgan immunopathology after Treg-specific ablation of Zfp335.

(A) ZFP335 expression in naive CD4⁺ T cells (Tcons), Tregs, and Tregs stimulated (stim) with anti-CD3/anti-CD28 Abs and IL-2 for 2 days. (B) MFI of ZFP335 in A. (C) Representative images of 3-week-old WT (Foxp3^Cre) and KO (Foxp3^Cre Zfp335^fl/fl) mice. (D) Survival curves for WT and KO mice (n = 27). (E) ELISA quantification of dsDNA-specific IgG in serum of WT (n = 10) and KO (n = 9) mice. (F) H&E staining of sections of the indicated organs from WT and KO mice (original magnification, ×20). (G) Representative images of spleens and peripheral LNs from WT and KO mice. (H) Frequencies and numbers of splenic CD4⁺ and CD8⁺ T cells from WT and KO mice (n = 3). (I–K) Frequencies of CD44⁺CD62L^– cells (I), IFN-γ⁺ cells (J), and IL-17⁺ (K) in splenic CD4⁺ and CD8⁺ T cells (n = 3–4). (L) Representative FACS plots of PD-1 and CXCR5 expression in splenic CD4⁺ T cells. (M) Number of PD-1⁺CXCR5⁺ cells in splenic CD4⁺ T cells (n = 3). (N) Representative FACS plots of GL7 and Fas expression in splenic CD19⁺ B cells. (O) Number of GL7⁺Fas⁺ GC B cells (n = 3). Data are representative of 3 independent experiments and are shown as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test (B), log-rank (Mantel-Cox) test (D), and 2-sided, unpaired t test (E, H–K, M, and O).

Figure 2. Impaired Treg signatures in Zfp335-deficient mice.

(A and B) Representative FACS plots (A) and frequencies (B) of CD25⁺Foxp3⁺ Tregs in the thymus and spleen of 1-week-old WT and KO mice (n = 3). (C and D) Representative FACS plots (C) and frequencies (D) of CD25⁺Foxp3⁺ Tregs in the thymus and spleen of 3-week-old WT and KO mice (n = 3). (E) GSEA plots depict the identified WT and KO Treg gene sets associated with Th cell signatures. NES, normalized enrichment score. (F and G) Heatmaps of the DEGs associated with Treg (F) and Th1, Th2, and Th17 cell (G) differentiation between WT and KO groups. Heatmap colors represent the z score values relative to the control. (H and I) Representative FACS plots of Foxp3 expression (H) and MFI of Foxp3 (I) in Tregs from 3-week-old WT and KO mice (n = 3–4). (J) Frequencies of IFN-γ⁺ and IL-4⁺ Tregs in WT and KO mice (n = 3). (K) Frequencies of T-bet⁺ and GATA-3⁺ Tregs in WT and KO mice (n = 3). Data are representative of 3 independent experiments and are shown as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, and ****P ≤ 0.0001, by 2-sided, unpaired t test.

Figure 3. Zfp335 deficiency impairs the suppressive function of Tregs.

(A) Representative histogram of CTV dilution of naive CD4⁺ T cells stimulated with anti-mCD3 Ab and antigen-presenting cells (APCs) in the presence of WT or KO Tregs for 60 hours. (B) Frequencies of each cell division (n = 4). (C) Frequencies of initial body weights of recipient mice 25 days after transfer with 5 × 10⁵ Tcons alone, or together with 2.5 × 10⁵ WT (Tcon+WT Treg) or KO (Tcon+KO Treg) Tregs. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (Tcon+WT Treg vs. Tcon+KO Treg); ^#P ≤ 0.05 and ^##P ≤ 0.01 (Tcon vs. Tcon+WT Treg). (D–F) Representative images of colons (D), histology images of colon sections (E), and images of spleens (F) from recipient mice (original magnification, ×8). (G) Numbers of CD44⁺CD62L^– splenic CD4⁺ T cells (n = 3). (H and I) Representative FACS plots of IFN-γ expression (H) and frequencies of IFN-γ⁺ (I) in splenic CD4⁺ T cells. (J and K) Representative FACS plots of IL-17 expression (J) and frequencies of IL-17⁺ (K) in splenic CD4⁺ T cells (n = 4). (L) Schematic diagram of Treg transfer assay and images of recipient mice and spleens and LNs 19 days after the transfer. (M) Frequencies of CD44⁺CD62L^– cells in CD4⁺ and CD8⁺ T cells in spleens (left) and LNs (right) from recipient mice (n = 3–4). Data are representative of 2 independent experiments and are shown as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001; ^#P ≤ 0.05 and ^##P ≤ 0.01. Statistical significance was determined by 2-sided, unpaired t test (B and C) and 1-way ANOVA with Tukey’s multiple-comparison test (G, I, K, and M).

Figure 4. Single-cell transcriptomics delineates distinct Treg populations.

(A) Schematic diagram of the experimental design, scRNA-Seq, data analysis, and validation. Tregs from 7-day-old WT and KO mice were sorted and subjected to scRNA-Seq by 10x Genomics. (B) Heatmap showing the relative expression of marker genes across different immune cell types. (C) Violin plots showing the Treg marker genes in each Treg cluster. (D) UMAP projections of Treg clusters, color-coded by cluster. (E) Violin plots showing the Treg signature genes specifically expressed in each Treg cluster. (F) Pseudotime plot shows the progression of 4 Treg populations reconstructed by monocle2 using scRNA-Seq data. (G) UMAP projections and percentage of Treg clusters in WT and KO mice. (H) Representative FACS plots of ICOS and CXCR3 expression in splenic Tregs from 8-day-old WT and KO mice. (I and J) Frequencies (I) and numbers (J) of ICOS⁺CXCR3⁺ (left) and ICOS⁺CXCR3^– (right) splenic Tregs (n = 3). Gene expression in B, C, and E is represented as the expression of normalized log₂ (count +1). Data are representative of 3 independent experiments and are shown as the mean ± SEM (H–J). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 2-sided, unpaired t tests (I and J).

Figure 5. Zfp335 targets metabolism pathways of Tregs.

(A) Hallmark pathways significantly enriched in rTreg, dysTregs, and eTregs based on RRA scores (see Methods). (B) Heatmap shows representative gene expression in PI3K/AKT/MTOR signaling and OXPHOS pathways. (C) CD4⁺CD25⁺CD44^–ICOS^– rTregs sorted from ER^Cre and ER^Cre Zfp335^fl/fl mice were activated with anti-mCD3/anti-CD28 Abs and IL-2. After a 12-hour stimulation, Seahorse analysis of the OCR in these cells was performed. Oligomycin (Oligo), FCCP, and rotenone plus antimycin A (Rot+AA) were injected as indicated. (D) Quantification of basal respiration, maximal respiration, and spare respiratory capacity in C (n = 3). (E) Representative FACS plots of MitoTracker Deep Red, TMRE, and MitoSox staining in ER^Cre and ER^Cre Zfp335^fl/fl rTregs activated with anti-mCD3/anti-CD28 Abs and IL-2 for 5 days. (F) MitoTracker Deep Red, TMRE, and MitoSox MFI in E (n = 3). Data are representative of 2 or 3 independent experiments and are shown as the mean ± the SEM. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, by 2-sided, unpaired t test.

Figure 6. Zfp335 facilitates eTreg differentiation via direct targeting of Hadha.

(A) Venn diagram shows the genes shared among the upregulated genes in eTregs and rTregs (WT vs. KO) and genes associated with metabolic pathways. (B) Heatmaps of the indicated genes between ER^Cre and ER^Cre Zfp335^fl/fl Tregs. (C) ChIP-Seq peaks of Zfp335-bound regions in the indicated genes in WT Tregs compared with input. (D and E) CD4⁺YFP⁺CD44^–ICOS^– Tregs were isolated from 8-day-old WT and KO mice. WT Tregs were transfected with mock, while KO Tregs were transfected with mock, Ndufa4, Hadha, and Actr2 for 4 days. Representative FACS plots (D) and frequencies (E) of ICOS⁺ Tregs in different groups (n = 3). (F and G) Representative FACS plots (F) and MFI of MitoTracker Deep Red (G) in the indicated groups (n = 3). (H) Frequencies of each cell division of Tcons alone or in the presence of WT eTregs transfected with mock and KO eTregs transfected with mock or Hadha. (I–L) CD4⁺CD25⁺CD44^–ICOS^– rTregs sorted from ER^Cre and ER^Cre Zfp335^fl/fl mice were activated by anti-CD3/anti-CD28 Abs and IL-2 in the presence or absence of 30 mM malate. Two days later, the cells were collected and subjected to FACS analysis. (I) Representative FACS plots of ICOS⁺ Tregs in different groups. (J) Frequencies of ICOS⁺ Tregs (n = 3). (K and L) Representative FACS plots (K) and MFI of MitoTracker Deep Red (L) (n = 3). Data are representative of 2 or 3 independent experiments and are shown as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test.

Figure 7. Zfp335-mediated generation of eTregs depends on FAO.

(A) Schematic diagram of in vitro ETO treatment assay. CD4⁺CD25⁺CD44^–ICOS^– rTregs sorted from ER^Cre and ER^Cre Zfp335^fl/fl mice were activated by anti-CD3/anti-CD28 Abs and IL-2 in the presence or absence of 40 μM ETO. Two days later, the cells were collected and analyzed by FACS. (B) Representative FACS plots of ICOS⁺ Tregs in the different groups. (C) Frequencies of ICOS⁺ Tregs (n = 3). (D) Quantification of neutral lipid droplets was evaluated by BODIPY-493/503, and representative FACS plots of BODIPY-493/503 staining are shown. (E) MFI of BODIPY-493/503 (n = 3–4). (F) Representative FACS plots of MitoTracker Deep Red staining. (G) MFI of MitoTracker Deep Red (n = 3). (H) Schematic diagram of the classic pathway of FAO-driven OXPHOS. (I–N) CD4⁺CD25⁺CD44^–ICOS^– rTregs sorted from ER^Cre and ER^Cre Zfp335^fl/fl mice were activated by anti-CD3/anti-CD28 Abs and IL-2 in the presence or absence of 50 μM oleic acid (OA). Two days later, the cells were collected and analyzed by FACS. (I) Representative FACS plots of ICOS⁺ Tregs in the different groups. (J) Frequencies of ICOS⁺ Tregs (n = 3). (K) Quantification of neutral lipid droplets by BODIPY-493/503. (L) MFI of BODIPY-493/503 (n = 3–4). (M) Representative FACS plots of MitoTracker Deep Red staining. (N) MFI of MitoTracker Deep Red (n = 3). Data represent 2 independent experiments and are shown as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 1-way ANOVA with Tukey’s multiple-comparison test.

Figure 8. Human ZNF335 expression in eTregs from HDs and patients with autoimmune diseases.

(A and B) ZNF335 expression in Tcons, Tregs, and Tregs from HD PBMCs (n = 17) stimulated with anti-CD3/anti-CD28 Abs and IL-2 for 2 days. Representative FACS plots (A) and MFI (B). (C and D) ZNF335 expression in rTreg and eTregs from PBMCs of HDs. Representative FACS plots (C) and MFI (D). (E) Representative FACS plots of CD4⁺CD127^–CD25⁺Foxp3⁺ICOS⁺ cells from HDs (n = 20), patients with SLE (n = 15), patients with RA (n = 8), and patients with SS (n = 6). (F) Frequencies of CD4⁺CD127^–CD25⁺Foxp3⁺ICOS⁺ cells in HDs and patients with SLE, RA, or SS. (G) PBMCs from peripheral blood of HDs and patients with SLE, RA, or SS were stimulated with anti-CD3/anti-CD28 Abs and IL-2. Two days later, ZNF335 and HADHA expression and MitoTracker Deep Red staining were examined. (H and I) Representative FACS plots of ZNF335 expression (H) and MFI of ZNF335 (I) in Tregs from HDs and patients with SLE, RA, or SS. (J and K) Representative FACS plots (J) and MFI of HADHA expression (K) in Tregs from HDs and patients with SLE, RA, or SS. (L and M) Representative FACS plots (L) and MFI of MitoTracker Deep Red staining (M) in Tregs from HDs and patients with SLE, RA, or SS. Data are shown as the mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, by 2-sided, unpaired t test (D) and Kruskal-Wallis test with the 2-stage step-up procedure of the Benjamini, Krieger, and Yekutieli multiple-comparison test (B, F, I, K, and M).