Stable and functional regulatory T cell attenuates fibrotic remodeling in heart failure

Figures

• 

  Fig. 1. Characterization and immunosuppressive function of S/F-iTreg. (a) Schematic illustration of the induction of S/F-iTreg from magnetic Separators for cell separation-isolated CD4+ conventional T cell from BALB/c mice under CD28-blockade conditions. (b) Flow cytometry analysis of S/F-iTreg showing expression of CD4, Foxp3, and CTLA-4. (c) Enzyme-linked immunosorbent assay analysis of inflammatory and anti-inflammatory cytokines in culture supernatants from the Tconv, Treg, Tconv + DC, and Treg + DC groups. (d) Suppression assay using CFSE-labeled Tconv co-cultured with S/F-iTreg at varying ratios. Data are presented as mean ± standard error of the mean. Statistical analyses were performed using a one-way analysis of variance with Tukey's multiple comparisons (c, d). ∗p < 0.05, ∗∗p < 0.01 vs. Tconv only (1:0) (d). CFSE, carboxyfluorescein succinimidyl ester; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; DC, dendritic cell; S/F-iTreg, stable and functional in vitro-induced regulatory T cell; Tconv, conventional T cell; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha; Treg, regulatory T cell.

• 

  Fig. 2. Cardiac function after S/F-iTreg administration in the TAC model. (a) Schematic of the TAC procedure and systemic S/F-iTreg administration in the in vivo experimental timeline. (b) Representative echocardiographic images before TAC and at 4 weeks post-TAC in each group. (c) Time-course changes in left ventricular diameter in diastole, left ventricular diameter in systole, and ejection fraction over 4 weeks (n = 7 for each group). Data are presented as mean ± standard error of the mean. Statistical analyses were performed using a mixed-effects model that included factors for group, time, and their interaction. Post hoc comparisons were conducted using the Tukey-Kramer method. ∗p < 0.05 vs. Control and Tconv groups at 4 weeks; NS, not significant vs. Control and Tconv groups. S/F-iTreg, stable and functional in vitro-induced regulatory T cell; TAC, transverse aortic constriction; Treg, regulatory T cell; Tconv, conventional T cell.

• 

  Fig. 3. Myocardial fibrosis and tissue distribution of S/F-iTreg after systemic administration. (a) Picro-Sirius Red staining of the heart at 4 weeks after TAC in each group. From left to right: representative whole-section image (scale bar, 500 μm), magnified view of fibrotic area (scale bar, 100 μm), and color-segmented image highlighting the quantified fibrotic region in yellow (scale bar, 500 μm) (b) Quantification of fibrotic area in each group (n = 7 for each group). (c) Early fibrotic/inflammatory marker assessment at day 9 after TAC. Representative western blots of LOX in left ventricular tissue, with densitometric quantification normalized to GAPDH (n = 4 per group). (d) Representative immunofluorescence images showing PKH26-labeled Treg (red) in the heart, lung, liver, and spleen 2 days after systemic administration. Nuclei were stained with Hoechst (blue), and F-actin with phalloidin (green). Scale bar, 50 μm. Data are presented as mean ± standard error of the mean. Statistical analysis was performed using a one-way analysis of variance with Tukey's multiple comparisons. LOX, lysyl oxidase; TAC, transverse aortic constriction; Tconv, conventional T cell; Treg, regulatory T cell.

• 

  Fig. 4. Transcriptomic profiling of left ventricular myocardium after S/F-iTreg administration in the TAC model. (a) Experimental setup of the animal study for RNA sequencing (n = 3 for each group). (b) Differential gene expression analysis comparing the Treg and Control groups. Left: MA plot showing significantly DEGs filtered by fold change (fold change > ±1.5) and false discovery rate-adjusted p-value (<0.05). Right: Heatmap illustrating expression patterns of DEGs across biological replicates (Treg vs Control; n = 3 per group). (c) Volcano plot of canonical pathways enriched in DEGs, based on Ingenuity Pathway Analysis. The x-axis represents the z-score, the y-axis shows –log10(p-value), and the circle size indicates the number of genes in each pathway (Treg vs Control; n = 3 per group). DEGs, differentially expressed genes; PDGF, platelet-derived growth factor; S/F-iTreg, stable and functional in vitro-induced regulatory T cell; TAC, transverse aortic constriction; Treg, regulatory T cell.

• 

  Fig. 5. GSEA of left ventricular transcriptomes following S/F-iTreg administration. (a) GSEA of hallmark and featured gene sets associated with immune modulation and fibrosis. Enrichment plots of representative gene sets are shown (Treg vs Control; n = 3 per group). (b) Dot plot visualizing individual leading-edge genes from selected enriched pathways. The Dot color indicates log2 fold change; dot size reflects statistical significance (–log10 q-value) (Treg vs Control; n = 3 per group). GSEA, gene set enrichment analysis; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; S/F-iTreg, stable and functional in vitro-induced regulatory T cell; TGF-β, transforming growth factor-beta; TNF-α, tumor necrosis factor-alpha; Treg, regulatory T cell.

• 

  Fig. 6. Transcriptomic changes specific to S/F-iTreg therapy compared with Tconv. (a) Differential gene expression analysis comparing the Treg and Tconv groups. Left: MA plot showing significantly DEGs filtered by fold change (fold change > ±1.5) and false discovery rate-adjusted p-value (<0.05). Right: Heatmap illustrating expression patterns of DEGs across biological replicates (Treg vs Tconv; n = 3 per group). (b) GSEA of hallmark and featured gene sets associated with immune modulation and fibrosis. Enrichment plots of representative gene sets are shown (Treg vs Tconv; n = 3 per group). (c) Evaluation of Transcriptome Similarities using RRHO2. To assess the global concordance of transcriptomic changes under different control conditions (i.e., Control or Tconv condition), genes were ranked according to the Wald statistic obtained from DESeq2 differential expression analysis for each comparison, reflecting both the magnitude and direction of expression changes. RRHO2 analysis was performed using ranked gene lists restricted to genes detected in both comparisons. DEGs, differentially expressed genes; GSEA, gene set enrichment analysis; RRHO2, rank–rank hypergeometric overlap; TAC,…