Specific ablation of CD4⁺ T-cells promotes heart regeneration in juvenile mice

Figures

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  Figure 1. CD4+ but not CD8+ T-cells are responsive to neonatal myocardial injury. (A) Schematic diagram showing the experimental design for B-F. (B) Immunostaining on frozen sections for CD3+ (red) cells with nuclear DAPI counterstain (blue) showing infiltration of T-cells in the infarcted (CI) and normal (N) myocardium at day 1 post CI to the P3 heart, scale bar: 100 μm. Square denotes an enlarged image on the right, scale bar: 50 μm. Flow cytometric analysis showing (C) %CD3+CD4+ or (D) %CD3+CD8+ cells among total PI-CD45+ cells at days 7 and 14 after CI to the P3 or P8 heart, respectively. Quantifications of (E) C or (F) D in terms of absolute number of cells harvested per mg heart tissue at days 1, 7 and 14 after CI to the P3 or P8 heart, respectively. (G) Quantification of CD3+CD4+hCD2- conventional T-cells per mg heart tissue harvested at day 7 after CI to the P3 or P8 heart of the Foxp3hCD2 mice. (E-G) Data are presented as mean±S.E.M., *P<0.05, **P<0.01, n=4 per group.

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  Figure 2. Ablation of CD4+ but not CD8+ T-cells reactivates neonatal heart regeneration after cryoinfarction. (A) Schematic diagram showing the experimental design. (B) Images of scar tissues, scale bars: 2000 μm; and Masson's trichrome staining showing representative serial cross sections of fibrotic tissues in blue, scale bars: 1000 μm. (C) Quantification of fibrotic tissue coverage based on (B). Immunostaining on frozen sections for (D) COLA1+ (red) and cTnT+ (green) cells within the infarct zone, scale bars: 100 μm; or (F) pH3+ (red) or Ki67+ (red) and cTnT+ (green) cells within the border zone, scale bars: 50 μm. (F) Arrows indicate cardiomyocytes positive for pH3 or Ki67 and squares denote magnified images on the right. Quantification of absolute number of (E) %cTnT+ coverage, (G) pH3+cTnT+ or (H) Ki67+cTnT+ cardiomyocytes per mm2 area. (C, E, G, H) Data are presented as mean±S.E.M., *P<0.05, **P<0.01, n=4-5 per group.

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  Figure 3. Ablation of CD4+ T-cells reactivates neonatal heart regeneration after apical resection. (A) Schematic diagram showing the experimental design. (B) Masson's trichrome staining showing representative serial cross sections of fibrotic tissues in blue, scale bars: 1000 um; and magnified images, scale bars: 200 um. (C) Quantification of scarring based on (B). Immunostaining on frozen sections for (D) COLA1+ (red) and cTnT+ (green) cells within the infarct zone, scale bars: 100 μm; or (F) pH3+ (red) or Ki67+ (red) and cTnT+ (green) cells within the border zone, scale bars: 50 μm. (F) Arrows indicate cardiomyocytes positive for pH3 or Ki67 and squares denote magnified images on the right. Quantification of absolute number of (E) %cTnT+ coverage, (G) pH3+cTnT+ or (H) Ki67+cTnT+ cardiomyocytes per mm2 area. (C, E, G, H) Data are presented as mean±S.E.M., *P<0.05, **P<0.01, n=4-6 per group.

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  Figure 4. Single-cell transcriptomics reveal the lack of a pro-fibrotic CD4+ T-cell subset in the regenerating neonatal heart after injury. (A) Biaxial scatter plots by t-SNE analysis showing single-cell transcriptomic clustering of CD3+ T-cells purified at day 7 post CI to P3 and P8 hearts, respectively, by flow cytometry. Cells are subgrouped into specific subsets (S1-6). (B) Analysis of subset-specific genes showing the top 3-5 most significantly upregulated pathways determined by GO functional annotations in terms of biological processes (Table S1). (C) Upregulated genes in (B) are selectively displayed by a heatmap. (D) t-SNE plots showing specific genes expressed by cells that represent each of the six subsets: Ccr7+ memory cells, Foxp3+ Treg, Il17a+Rorc+ Th17 cells, Fabp4+Sparc+ pro-fibrotic cells, Pf4+ chemotactic cells and Mki67+ proliferating cells. (E) Scattered plots showing selected most significantly upregulated genes in S4 cells of the P3 and P8 hearts after CI that regulate fibroblast activation and fibrosis.

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  Figure 5. Cytokines of Th1 and Th17 cells directly regulate the survival of neonatal cardiomyocytes. (A) Gene expression levels of pro- and anti-inflammatory cytokines of the infarct tissues at day 7 post CI to the P8 heart as determined by qRT-PCR, data are presented as mean±S.E.M., *P<0.05, **P<0.01, n=5 per group. (B) Quantification of the absolute number of total cardiomyocytes derived from the P1 heart after cultured with PBS (solvent control), 50 ng/ml TNF-α, 50 ng/ml IFN-γ or 100 ng/ml IL-17A for 3 days. (C) Immunocytochemistry for cTnT+ (red) and pH3+ (green) or cCASP3+ (green) cells after cultured with PBS (solvent control), 50 ng/ml TNF-α, 50 ng/ml IFN-γ or 100 ng/ml IL-17A for 1 day, scale bars: 50 μm. White arrows indicate proliferating or apoptotic cTnT+ cells. (D, E) Quantification of (D) %pH3+cTnT+ or (E) cCASP3+cTnT+ cardiomyocytes among total cTnT+ cardiomyocytes based on (C). (B, D, E) Data are presented as mean±S.E.M., n = 3 independent experiments with a total of ~20,000 cardiomyocytes counted, *P<0.05, **P<0.01.

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  Figure 6. Ablation of CD4+ T-cells contributes to skewed polarization of the myocardial macrophages away from the pro-fibrotic M2-like signature after injury. (A) Schematic diagram showing the experimental design. (B) Flow cytometric analysis showing M1- or M2-like macrophages among total PI-F4/80+ macrophages at day 7 after CI to the P8 heart. Quantifications of (B) in terms of (C) %F4/80+ cells and (D) %Ly6Chi M1- or (E) %Ly6CloCD206+ M2-like cells among total F4/80+ cells harvested from the non-depleted (control) or CD4+ T-cell depleted (GK1.5) hearts, respectively. (F) qRT-PCR showing M2-like macrophage-associated genes of total F4/80+ cells purified from the control and GK1.5-treated hearts, respectively, by flow cytometry. (C-F) Data are presented as mean±S.E.M., *P<0.05, n=4-5 per group.

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  Figure 7. Ablation of CD4+ T-cells did not reactivate adult heart repair after myocardial infarction. (A) Schematic diagram showing the experimental design. (B) Images of scar tissues, scale bars: 1000 μm; and Sirius red/fast green staining showing representative serial cross sections of fibrotic tissues, scale bars: 1000 μm. (C) Quantification of fibrotic tissue coverage based on (B). Data are presented as mean±S.E.M., *P<0.05, n=6-7 per group.