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The PD-1–PD-L1 pathway maintains an immunosuppressive environment essential for neonatal heart regeneration

Nature Cardiovascular Research volume 3, pages 389–402 (2024)Cite this article

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Abstract

The adult mouse heart responds to injury by scarring with consequent loss of contractile function, whereas the neonatal heart possesses the ability to regenerate. Activation of the immune system is among the first events upon tissue injury. It has been shown that immune response kinetics differ between regeneration and pathological remodeling, yet the underlying mechanisms of the distinct immune reactions during tissue healing remain unclear. Here we show that the immunomodulatory PD-1–PD-L1 pathway is highly active in regenerative neonatal hearts but rapidly silenced later in life. Deletion of the PD-1 receptor or inactivation of its ligand PD-L1 prevented regeneration of neonatal hearts after injury. Disruption of the pathway during neonatal cardiac injury led to increased inflammation and aberrant T cell activation, which ultimately impaired cardiac regeneration. Our findings reveal an immunomodulatory and cardioprotective role for the PD-1–PD-L1 pathway in heart regeneration and offer potential avenues for the control of adult tissue regeneration.

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Fig. 1: PD-1 and PD-L1 are highly enriched in regenerative hearts.
Fig. 2: PD-1 and PD-L1 are required for neonatal heart regeneration.
Fig. 3: Absence of PD-1 exacerbates the inflammatory response to MI in neonatal hearts.
Fig. 4: PD-1+ DN T cells infiltrate hearts upon P2-MI and are increased in the absence of PD-1.
Fig. 5: DN T cells become aberrantly activated in the absence of PD-1.

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Data availability

All data are available in the main text or the supplementary materials. All sequencing data generated from this study have been deposited in the NCBI Gene Expression Omnibus under accession no. GSE207290. Data used in this study include mm10/GRCm38, GSE207290, GSE123868, GSE153480 and GSE95755.

Code availability

All code used in this study is freely available on GitHub (https://github.com/CodeUTSW/Code_for_Paper/tree/main).

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Acknowledgements

We thank S. Bezprozvannaya at the University of Texas Southwestern Medical Center (UTSW) for technical help; J. Cabrera (UTSW) for assistance on graphic design; A. Mobley and the Flow Cytometry Core at UTSW for assistance with CyTOF; the Moody Foundation Flow Cytometry Facility for assistance with cell sorting; J. Xu and Y. Jung Kim from the Children’s Research Institute for performing the Illumina sequencing; L. Straub from the Touchstone Diabetes Center at UTSW for experimental advice and scientific discussions; K. Chen for support on the analysis of CyTOF data; L. Pathira Kankanamge and S. Miriyala from Boston Children’s Hospital for assistance with scCODA single-cell and spatial transcriptome analysis, respectively; B. Li from the Lyda Hill Department of Bioinformatics; Z. Zhong from the Department of Immunology for scientific discussions; and K. O’Donnell from Department of Molecular Biology at UTSW for scientific discussions and reagents. S.V.A. is supported by a Postdoctoral Fellowship from the American Heart Association (no. 916883) and M.C. is supported by grants from the National Institutes of Health (K99HL153683 and R00HL1536833). This work was supported by grants from the National Institutes of Health (HL130253 and HL157281, to E.N.O. and R.B.-D.), the Robert A. Welch Foundation (1-0025, to E.N.O.) and the Leducq Foundation Transatlantic Network of Excellence (20CVD04, to E.N.O.).

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  1. These authors contributed equally: Stephanie Vargas Aguilar, Miao Cui.

Authors and Affiliations

  1. Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Stephanie Vargas Aguilar, Miao Cui, Wei Tan, Efrain Sanchez-Ortiz, Rhonda Bassel-Duby, Ning Liu & Eric N. Olson

  2. Department of Cardiology, Boston Children’s Hospital and Department of Genetics, Harvard Medical School, Boston, MA, USA

    Miao Cui

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Contributions

S.V.A., M.C. and E.N.O. were responsible for conceptualization. S.V.A., M.C., W.T. and E.S.-O. were responsible for the methodology. S.V.A. and M.C. were responsible for investigation. S.V.A. and M.C. were responsible for visualization. E.N.O., R.B.-D., S.V.A. and M.C. were responsible for funding acquisition. E.N.O., N.L. and R.B.-D. were responsible for supervision. S.V.A., M.C., N.L., E.N.O. and R.B.-D. were responsible for writing.

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Correspondence to Eric N. Olson.

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Nature Cardiovascular Research thanks Sikander Hayat, Douglas Mann, Javid Moslehi, Nadia Rosenthal and Han Zhu for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 PD-1 and PD-L1 are expressed in the neonatal heart.

a, Expression of PD-L1 and PD-L2 in P1 mouse hearts at steady state, as measured by qPCR. Fold change expression normalized to PD-L1 is shown. n = 3 mice per group. p = 0.0026 (PD-L1 vs. PD-L2). b, t-SNE visualization of cardiac cell types identified by CyTOF represented as a composite of all samples combined. c, Median intensity of marker genes for each cardiac cell type (scaled across individual columns) in combined samples, as measured by CyTOF. d, t-SNE plots depicting PD-L1, CD45, and CD31 expression in cardiac cell types from hearts at d3 after MI or sham performed at P2 (P2-MI or P2-sham) or at P8 (P8-MI or P8-sham), as measured by CyTOF. e, Immunofluorescence of PD-L1 (red), MOMA-2 (green) and DAPI (blue) on heart sections at d1 after MI performed at P1 (P1-MI). Sections were collected at 200μm below the ligation. Scale bar, 50μm. f, Immunofluorescence of PD-L1 (red), PECAM-1 (green) and DAPI (blue) on heart sections at d1 after P8-sham. Scale bar, 50μm. g, Violin plots of PD-L1 expression in individual cells in the conditions depicted, measured by CyTOF. These values represent the raw data used to create the heat map in Fig. 1d. The boxplots contain the 25th (Q1) to 75th (Q3) percentile of the dataset (IQR, interquartile range), and the center line denotes the median value. Whiskers mark the minimum (Q1-1.5*IQR) and maximum (Q3 + 1.5*IQR) values. Statistics were calculated using a two-sided Wilcoxon test and are shown only for selected comparisons. h, PD-L1 (left) and PD-1 (right) expression in cardiomyocytes, fibroblasts, immune cells, and endothelial cells from hearts collected at d3 after MI performed at P1 (P1-MI) or at P56 (P56-MI), measured by RNA Sequencing. Data have been previously published as GSE95755. Expression is shown as count per million reads (CPM). n = 4 biologically independent samples. **p < 0.01 by two-tailed Student’s t-test, compared to the expression in corresponding cell type from P1 hearts. p = 0.0027 (PD-L1 Immune cell: P1 vs. P56), p = 0.0085 (PD-L1 Endothelial cell: P1 vs. P56), p = 0.0038 (PD-1 Immune cell: P1 vs. P56). i, t-SNE plots depicting CD3 (upper panel) and PD-1 (lower panel) expression, as measured by CyTOF. t-SNE plots are composites of all samples combined. Red circle denotes CD3+ cells. j, Absolute counts of PD-1+ T cells and PD-1+ macrophages/monocytes/neutrophils in hearts at d3 after P2-MI (n = 7 mice per group) or P8-MI (n = 9 mice per group), as measured by flow cytometry. p = 9.4 ×10−8 (P2-MI PD-1+ T cells vs. P2-MI PD-1+ macrophages/monocytes/neutrophils), p = 1.74 ×10−7 (P2-MI PD-1+ T cells vs. P8-MI PD-1+ T cells). Results are shown as mean ± s.d (h) or mean ± s.e.m (a, j); nsp > 0.05, **p < 0.01, ****p < 0.0001 by two-tailed Student’s t-test (a) and by two-way ANOVA with Fisher’s LSD test on multiple comparisons (j).

Source data

Extended Data Fig. 2 Gating strategies used for flow cytometry analyses throughout the study.

a, Gating strategy used to detect depicted innate immune cells in the heart. Tissue-resident macrophages, TRMs. b, Gating strategy used to detect depicted immune cells in the spleen. c, Gating strategy used to detect depicted T cell populations in the heart.

Extended Data Fig. 3 Ablation of the PD-1/PD-L1 pathway impairs neonatal heart regeneration.

a-c, Ejection fraction (a), left ventricular end-diastolic volume (b) and left ventricular end-systolic volume (c) of IgG- or anti-PD-L1-treated mice at 1, 2, and 3 weeks after P3-MI. IgG sham: n = 6, 5, and 5 mice at 1, 2, 3 weeks, respectively. anti-PD-L1 sham: n = 6 mice. IgG MI: n = 5, 6, and 6 mice at 1, 2, 3 weeks, respectively. anti-PD-L1 MI: n = 7 mice (a, b c). p = 0.00052 (1 week), p = 0.000008 (2 weeks), p = 0.000009 (3 weeks) (a). p = 0.004638 (2 weeks), p = 0.000823 (3 weeks) (b). p = 0.002964 (2 weeks), p = 0.000145 (3 weeks) (c). d-f, Ejection fraction (d), left ventricular end-diastolic volume (e) and left ventricular end-systolic volume (f) of Pd-1+/− and Pd-1−/− mice at 1, 2, and 3 weeks after P2-MI. Pd-1+/− sham: n = 5, 5, and 4 mice at 1, 2, 3 weeks, respectively. Pd-1−/− sham: n = 5 mice. Pd-1+/− MI: n = 4 mice. Pd-1−/− MI: n = 6, 5, and 5 mice at 1, 2, 3 weeks, respectively (d, e, f). p = 0.000703 (1 week), p = 0.001559 (2 weeks), p = 0.021383 (3 weeks) (d). p = 0.01633 (2 weeks), p = 0.019505 (3 weeks) (e). p = 0.004214 (2 weeks), p = 0.042845 (3 weeks) (f). The same cohort of mice was measured every week. Results are shown as mean ± s.e.m; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by two-tailed Student’s t-test.

Source data

Extended Data Fig. 4 Ablation of PD-1 promotes cardiomyocyte apoptosis post-MI.

a,b, Immunofluorescence of TUNEL (magenta), cardiac troponin T (cTnT, green) and DAPI (blue) on heart sections from Pd-1+/− (a) and Pd-1−/− (b) mice at d7 after MI performed at P2. Sections were collected at 200μm below the ligation. Scale bar, 100μm. Four Pd-1+/− biological replicates and three Pd-1-/- biological replicates are shown. Replicate, rep.

Extended Data Fig. 5 Ablation of PD-1 impairs cardiomyocyte proliferation post-MI.

a,b, Immunofluorescence of phosphorylated histone H3 (pH3, red), cardiac troponin T (cTnT, green) and DAPI (blue) on heart sections from Pd-1+/- (a) and Pd-1-/- (b) mice at d7 after MI performed at P2. Sections were collected at 200μm below the ligation. Scale bar, 100μm. Four Pd-1+/- biological replicates and three PD-1-/- biological replicates are shown. Replicate, rep.

Extended Data Fig. 6 PD-1 is required for neonatal heart regeneration.

a, Illustration showing time points of antibody injection, MI or sham and sample collection. b, c, Fractional shortening (b) and ejection fraction (c) of IgG-treated or anti-PD-1 antibody (anti-PD-1)-treated mice at 1, 2 and 3 weeks post-MI or sham. The same cohort of mice was measured every week. IgG/ anti-PD-1 sham: n = 5 mice. IgG MI: n = 8, 7, and 7 mice at 1, 2, 3 weeks, respectively. anti-PD-1 MI: n = 8 mice (b c). p = 0.0022 (1 week), p = 0.0175 (2 weeks), p = 0.0175 (3 weeks) (b). p = 0.0014 (1 week), p = 0.0227 (2 weeks), p = 0.0227 (3 weeks) (c). d, Immunofluorescence of TUNEL (magenta), cardiac troponin T (cTnT, green) and DAPI (blue) on heart sections from IgG-treated or anti-PD-1-treated mice at d7 post-MI with infarct and remote zones depicted. Sections were collected at 200μm below the ligation. Scale bar, 100μm. e, Quantification of TUNEL+ cells in (d). p = 0.002577 (Infarct: IgG vs. anti-PD-1). f, Immunofluorescence of pH3 (red), cardiac troponin T (cTnT, green) and DAPI (blue) in hearts from IgG-treated or anti-PD-1-treated mice at d7 post-MI. Sections were collected at 200μm below the ligation, and pictures were taken at the border zone of the injury. Scale bar, 100μm. g, Quantification of pH3+ cardiomyocytes from (f). p = 0.0395 (IgG vs. anti-PD-1). n = 6 mice per group (3 images counted per mouse) (e, g). Results are shown as mean ± s.e.m; *p < 0.05, **p < 0.01 by two-tailed Student’s t-test (e, g) or two-tailed Student’s t-test with correction for multiple comparisons using the Holm-Šidák method (b, c).

Source data

Extended Data Fig. 7 Identification of immune cell types by scRNA-seq.

a, Violin plots showing distribution of numbers of genes (nFeature), numbers of RNA molecules (nCount) and percentage of mitochondrial DNA (percent.mt) that were detected in Pd-1+/- or Pd-1-/- hearts at d7 after MI or sham performed at P2. Sham, Sh. b, UMAP plot of scRNA-seq data showing identified immune cell types in combined samples. c, Heat map showing the expression of top 10 marker genes of each cell type from (b) in combined samples, as measured by scRNA-seq. d, UMAP plot showing macrophage subclusters separated by sample. e, Heat map showing expression of the pro-inflammatory cytokines Il6, Il18, Il23a and Nos2, separated by cell type, genotype, and condition. Sham, Sh.

Extended Data Fig. 8 Ablation of PD-1 causes local increased immune infiltration in the heart post-MI.

a,b, a, Quantification of spatial transcriptomic data shown in Fig. 3h. Baseline cell abundance was computed by averaging cell abundance across all spatial positions. EndoEC, endocardial cell; Mono, monocyte; Macro, macrophage; DC-like, dendric cell-like cells; FB, fibroblast. b, c, Immunofluorescence of MOMA-2 (green), cardiac troponin T (cTnT, red) and DAPI (blue) on heart sections from Pd-1+/- (b) and Pd-1-/- (c) mice at d7 after MI performed at P2. Absence of cTnT staining indicates damaged myocardium. Sections were collected at 200μm below the ligation. Scale bar, 100μm. Three Pd-1+/- and three Pd-1-/- biological replicates are shown. Replicate, rep. d, Quantification of MOMA-2+ cells from (b) and (c). n = 3 mice per group. p = 0.033 (Pd-1+/- vs. Pd-1-/-). Results are shown as mean ± s.e.m; *p < 0.05, by two-tailed Student’s t-test.

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Extended Data Fig. 9 TCRγδ+ PD-1+ DN T cells accumulate in hearts injured at P2.

a, Relative composition of T cell populations depicted in Fig. 4a. Double-positive, DP. Double-negative, DN. Plotted values are shown for CD8+, CD4+ and DN T cells. b, t-SNE plots depicting CD4, CD8 and PD-1 expression in T cells from hearts at d3 after MI or sham performed at P2 (P2-MI or P2-sham) or P8 (P8-MI or P8-sham), as measured by CyTOF. c, Expression of TCRγδ, TCRβ and CD90 in DN, CD4+, CD8+ T cells from hearts at d3 after P2-MI, as measured by flow cytometry. d, Percentage of DN T cells among all T cells in spleens at steady state at 2 and 8 days of age, as measured by flow cytometry. n = 5 mice per group. p = 0.0010 (P2 vs. P8). Results are shown as mean ± s.e.m; **p < 0.01 by two-tailed Student’s t-test.

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Extended Data Fig. 10 Neonatal DN T cells become aberrantly activated upon disruption of the PD-1/PD-L1 axis during MI.

a,b, Absolute counts of T cell populations (a) and neutrophils (b) in hearts of IgG- or αPD-L1-treated mice, at steady state and at d5 after MI performed at P3 (P3-MI), as measured by flow cytometry. n = 6 mice per group. p = 0.0495 (DN: IgG vs. αPD-L1) (a), p = 0.0004 (IgG vs. αPD-L1) (b). c, Heat map showing expression of marker genes in double-negative (DN), double-positive (DP), CD8+ and CD4+ T cell populations, as measured by scRNA-seq. d, Expression of DN T cell marker genes [log2(fold change) >0.6, p-adj <0.01] in DN, DP, CD8+ and CD4+ T cell populations (left) and their enriched gene ontology (GO) terms (right), as measured by scRNA-seq. A complete gene list is provided in Supplementary Data 3. Red shaded area denotes p-value (-Log10 (p-value)) by two-sided Wilcoxon Rank Sum test. e, Volcano plot depicting differentially expressed genes between Pd-1-/- DN T cells and Pd-1+/- DN T cells upon MI. p-value < 0.01, two-sided Wilcoxon Rank Sum test. A complete gene list is provided in Supplementary Data 4. f, Gene ontology (GO) terms enriched in genes upregulated in Pd-1-/- MI DN T cells vs Pd-1+/- MI DN T cells. Red shaded area denotes p-value (-Log10 (p-value)) by two-sided Wilcoxon Rank Sum test. g, NK1.1 expression in DN T cells in the heart of Pd-1+/- and Pd-1-/- mice at d5 after MI performed at P2 (P2-MI), as measured by flow cytometry. Results are shown as mean ± s.e.m; *p < 0.05, ***p < 0.001 by two-way ANOVA with Šidák correction on multiple comparisons (a) or two-tailed Student’s t-test (b).

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Vargas Aguilar, S., Cui, M., Tan, W. et al. The PD-1–PD-L1 pathway maintains an immunosuppressive environment essential for neonatal heart regeneration. Nat Cardiovasc Res 3, 389–402 (2024). https://doi.org/10.1038/s44161-024-00447-7

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