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Dynamic molecular atlas of cardiac fibrosis at single-cell resolution shows CD248 in cardiac fibroblasts orchestrates interactions with immune cells

Nature Cardiovascular Research volume 4, pages 380–396 (2025)Cite this article

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Abstract

Post-injury remodeling is a complex process involving temporal specific cellular interactions in the injured tissue where the resident fibroblasts play multiple roles. Here, we performed single-cell and spatial transcriptome analysis in human and mouse infarcted hearts to dissect the molecular basis of these interactions. We identified a unique fibroblast subset with high CD248 expression, strongly associated with extracellular matrix remodeling. Genetic Cd248 deletion in fibroblasts mitigated cardiac fibrosis and dysfunction following ischemia/reperfusion. Mechanistically, CD248 stabilizes type I transforming growth factor beta receptor and thus upregulates fibroblast ACKR3 expression, leading to enhanced T cell retention. This CD248-mediated fibroblast–T cell interaction is required to sustain fibroblast activation and scar expansion. Disrupting this interaction using monoclonal antibody or chimeric antigen receptor T cell reduces T cell infiltration and consequently ameliorates cardiac fibrosis and dysfunction. Our findings reveal a CD248+ fibroblast subpopulation as a key regulator of immune–fibroblast cross–talk and a potential therapy to treat tissue fibrosis.

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Fig. 1: Characterization of CD248+ fibroblast subset after MI.
Fig. 2: CD248 deletion attenuates cardiac fibrosis and cardiac dysfunction in mouse cardiac I/R and MI models.
Fig. 3: CD248+ fibroblasts promote CD4+ T cell infiltration in the ischemic mouse hearts subjected to I/R or MI.
Fig. 4: ACKR3 in the fibroblasts mediates the increased infiltration of CD4+ T cells promoted by CD248+ fibroblasts in I/R-injured mouse hearts.
Fig. 5: CD248 inhibits the degradation of ACKR3 via stabilizing TGFβRI.
Fig. 6: CD248 monoclonal antibody treatment ameliorates cardiac fibrosis, and cardiac dysfunction accompanied with reduced cardiac T cell infiltration after ischemia injury.
Fig. 7: CD248 CAR T cell therapy ameliorates cardiac fibrosis, and cardiac dysfunction accompanied with reduced cardiac T cell infiltration after ischemia injury.
Fig. 8: Late-onset CD248 monoclonal antibody and CD248 CAR T cell therapy ameliorate preexisting cardiac fibrosis and cardiac dysfunction associated with reduced T cell infiltration after I/R.

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Data availability

All data are available in the main text or the Supplementary Information. The bulk RNA-seq raw data for Fig. 3a,b are deposited in the Gene Expression Omnibus (GEO) under accession no. GSE287283. Raw scRNA-seq data for infarcted mouse hearts (Fig. 1a–f), infarcted human hearts (Fig. 1g–i), conditional CD248 KO mouse hearts subjected to MI (Extended Data Fig. 4d–g) and spatial transcriptome data for infarcted mouse hearts (Figs. 1j,k and 3f–i) are deposited in the Genome Sequence Archive (GSA) and GSA-Human at the National Genomics Data Center, China National Center for Bioinformation, under accession numbers CRA022616, HRA010249, CRA022593 and CRA022597, respectively, and are publicly accessible at https://ngdc.cncb.ac.cn/gsa/ and https://ngdc.cncb.ac.cn/gsa-human/. Additionally, scRNA-seq data of human fibrotic lung samples are available from the GEO (GSE121611), and human fibrotic kidney scRNA-seq data and human heart spatial transcriptome data can be accessed via Zenodo (https://doi.org/10.5281/zenodo.4059315 and https://doi.org/10.5281/zenodo.6580069)8,19.

Code availability

The code used to process the data is publicly available on GitHub (https://github.com/lingjun66/CD248/).

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Acknowledgements

This work was supported by grants from the Key Project of National Natural Science Foundation of China (no. 82430013 for X.H.), National Key Research and Development Program of China (no. 2023YFA1800700 for X.H.), National Science Foundation for Distinguished Young Scholars (no. 82225004 for X.H.), National Natural Science Foundation of China (no. U22A20267 and 82030014 for Jian’an Wang, U21A20338 and 82370256 for J.C., 82370240 for C.N., and 82400296 for G.L.) and received financial support from Binjiang Institute of Zhejiang University (ZY202205SMKY002 for X.H.) and Fundamental Research Funds for the Central Universities (226-2024-00224 for G.L. and K20240127 for B.W.).

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Author notes

  1. These authors contributed equally: Guohua Li, Cheng Ni, Jiacheng Wang.

Authors and Affiliations

  1. Department of Cardiology, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China

    Guohua Li, Cheng Ni, Jiacheng Wang, Feimu Zhang, Zaiyang Fu, Lingjun Wang, Biqing Wang, Ye Liu, Jing Zhao, Mo Li, Hao Lin, Fei Liao, Shuchang Ye, Yu Zhang, Jiayue Cai, Zhiwei Zhong, Yanna Shi, Junhua He, Yang Xu, Jinghai Chen, Wei Zhu, Jian’an Wang & Xinyang Hu

  2. State Key Laboratory of Transvascular Implantation Devices, Hangzhou, China

    Guohua Li, Cheng Ni, Jiacheng Wang, Feimu Zhang, Zaiyang Fu, Lingjun Wang, Biqing Wang, Ye Liu, Jing Zhao, Mo Li, Hao Lin, Fei Liao, Shuchang Ye, Yu Zhang, Jiayue Cai, Zhiwei Zhong, Yanna Shi, Junhua He, Yang Xu, Jinghai Chen, Wei Zhu, Jian’an Wang & Xinyang Hu

  3. Heart Regeneration and Repair Key Laboratory of Zhejiang Province, Hangzhou, China

    Guohua Li, Cheng Ni, Jiacheng Wang, Feimu Zhang, Zaiyang Fu, Lingjun Wang, Biqing Wang, Ye Liu, Jing Zhao, Mo Li, Hao Lin, Fei Liao, Shuchang Ye, Yu Zhang, Jiayue Cai, Zhiwei Zhong, Yanna Shi, Junhua He, Yang Xu, Jinghai Chen, Wei Zhu, Jian’an Wang & Xinyang Hu

  4. Liangzhu Laboratory, Zhejiang University, Hangzhou, China

    Shaohui Shi & Xushen Xiong

  5. Programme in Cardiovascular and Metabolic Diseases, Duke-NUS Medical School, Singapore, Singapore

    Yibin Wang

  6. Research Center for Life Science and Human Health, Binjiang Institute of Zhejiang University, Hangzhou, China

    Jian’an Wang & Xinyang Hu

Authors
  1. Guohua Li
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  2. Cheng Ni
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Contributions

G.L., C.N., Jiacheng Wang and F.Z. performed mouse in vivo surgeries and downstream experiments. G.L. isolated NMCFs, and G.L., F.L. and S.Y. performed downstream in vitro experiments. G.L. isolated spleen T cells, and performed all CAR T cell therapy-associated experiments. G.L., H.L. and J.Z. performed Sirius red staining and analyzed images. Y.Z., J. Cai, Y.S. and Z.Z. performed immunofluorescence staining and analyzed images. Z.F., L.W., M.L. and S.S. performed scRNA-seq and spatial transcriptomic analysis. B.W., Y.L. and J.H. analyzed the data. X.X., Y.X., J. Chen, W.Z. and Y.W. contributed to the experimental design and manuscript modification. Jian’an Wang and X.H. designed the experiments, supervised the study and drafted and revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Xinyang Hu.

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Nature Cardiovascular Research thanks Joel G. Rurik, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Global view of cardiac fibroblast single-cell RNA sequencing from human myocardial infarction heart.

a, UMAP dimensionality reduction plots of all 6989 sequenced cardiac fibroblasts from different cardiac areas of a MI patient. b, Enrichment of each fibroblast subset in human heart from infarct zone or remote area. c, Heatmap of top marker genes for each fibroblast subtype in UMAP. d, Venn diagram analysis of mouse cardiac F9 subset and human F5 subset. e, Quantification score of top 100 differentially expressed genes in mouse heart CD248+ fibroblast subset across different human heart fibroblast subsets. f, Immunofluorescence analysis of CD248 (red) expression in vimentin-marked cardiac fibroblasts from normal and infarcted human heart. g, Western-blot analysis of CD248 expression (by the Abcam antibody, no. 67273) in I/R-injured mouse hearts at different timepoints (sham, 3 days, 7 days, 14 days and 28 days post I/R). h, Different cell types were FACS-sorted from infarcted heart to analyze CD248 expression (by the Abcam antibody, no. 67273) by western-blot at 28 days post-infarction. i, Co-immunofluorescence staining of CD248 (red) with cardiomyocytes (Troponin I, green), endothelial cells (CD31, green), fibroblasts (Vimentin, green), T cells (CD3, green) and macrophage (CD68, green) in mouse heart 28 days post-infarction respectively. Scale bar = 20 μm.

Source data

Extended Data Fig. 2 Global knock-out of CD248 attenuates cardiac dysfunction in mice subjected to I/R or MI.

a, Genotyping of CD248 knock-out (KO) mice. b, Representative pulse-wave Doppler (left) and tissue Doppler (right) images of WT and CD248 KO mice. c, Quantification of baseline mitral E/e’ ratio, isovolumic relaxation time (IVRT), and left ventricular myocardial performance index (LV MPI) in WT and CD248 KO mice. d, e, Flow cytometry analysis of the proportion (d) and number (e) of PDGFRα+ fibroblasts in WT and CD248 KO mouse hearts. f, Biochemical analysis of hepatic function, renal function, and blood lipid levels by analyzing serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB), total protein (TP), total bilirubin (TBIL), uric acid (UA), urea nitrogen (BUN), creatinine (CREA), triglyceride (TG), cholesterol (CHOL), high density lipoprotein (HDL), and low density lipoprotein (LDL) in WT and CD248 KO mice. g, Schematic of experiment for I/R in CD248 KO mice. h, Representative M-mode echocardiogram images of mice from WT+Sham, CD248 KO+Sham, WT + I/R, CD248 KO + I/R at 4 days and 28 days post-I/R injury. i, LVESV and LVEDV measurement in animals of WT+Sham, CD248 KO+Sham, WT + I/R, and CD248 KO + I/R groups at 4 days and 28 days post I/R. n = 5 animals in CD248 KO group, and n = 6 animals in the other groups. j, Schematic of experiment for MI in CD248 KO mice. k. Representative M-mode echocardiogram images of mice from WT+Sham, CD248 KO+Sham, WT + I/R, CD248 KO + I/R at 4 days and 28 days post-MI injury. l, LVESV and LVEDV measurement in animals of WT+Sham (n = 6), CD248 KO+Sham (n = 6), WT + MI (n = 9), and CD248 KO + MI (n = 9) groups at 4 days and 28 days post MI. Data are shown as mean ± s.e.m.; unpaired two-tailed Student’s t-test (c-f) and two-way ANOVA (i and l).

Source data

Extended Data Fig. 3 Specific deletion of CD248 in cardiac fibroblasts improved cardiac function in mice subjected to I/R injury.

a, Genotyping of CD248flox-Postn-CreER mice. b, Scheme of in vivo I/R experiment conducted on CD248fl/fl and CD248Postn-creER mice. c, Western-blot analysis of CD248 deletion efficiency (by the Proteintech antibody, no. 60170) driven by Postn-CreER in mice with or without I/R injury. d, UMAP of fibroblast subpopulations of infarcted CD248Postn-CreER and CD248fl/fl mouse hearts 14 days post-myocardial infarction. e, Top expressed marker genes of each fibroblast subpopulation. f, Proportion of each fibroblast subpopulation in the infarcted CD248fl/fl and CD248Postn-CreER mouse hearts 14 days post-myocardial infarction. g, z-score of CD248 in UAMP. h, Representative M-mode echocardiogram images of animals from CD248fl/fl+Sham, CD248Postn-CreER+Sham, CD248fl/fl+I/R, CD248Postn-CreER+I/R groups at 4 days and 28 days post-I/R. i, LVESV and LVEDV measurement of animals from CD248fl/fl+Sham (n = 6), CD248Postn-CreER+Sham (n = 6), CD248fl/fl+I/R (n = 7), CD248Postn-CreER+I/R (n = 7) groups at 4 days and 28 days post I/R. Data are shown as mean ± s.e.m.; Statistical analysis was performed using two-way ANOVA.

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Extended Data Fig. 4 CD248+ fibroblasts were involved in cardiac inflammation regulation post ischemia injuries, in which ACKR3 played an important role.

a, Heatmap of up-regulated and down-regulated genes related with inflammation between CD248+ and CD248- fibroblasts isolated from infarcted mouses heart at 14 days post-infarction. b-e, Immunofluorescence analysis of the infiltration of CD68+ macrophage (b) and CD3+ T cells (d) in hearts of CD248 KO and WT mice 14 days after I/R injury, with statistical analysis respectively shown in c and e. n = 5 in each group. Scale bar = 20 μm. f, g. Representative dot plots showing cardiac T cells in WT and CD248 KO mice at one week post-I/R (f) or MI (g) injuries. h, ACKR3 mRNA level detected by RT-PCR in NMCFs transfected with sh-CD248 and OE-CD248 lentivirus. n = 4 in each group. i, ACKR3 protein expression in NMCFs with sh-CD248 treatment compared with scramble treatment, among which CD248 expression analysis was performed using the Abcam antibody (no. 67273). Statistical analysis was plotted in j. n = 3 in each group. Data are shown as mean ± s.e.m.; unpaired two-tailed Student’s t-test (c, e and j) and one-way ANOVA (h).

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Extended Data Fig. 5 Knocking-down CD248 reduced TGF-βRI expression and inhibited the activation of canonical and non-canonical signaling down-streaming of TGF-β receptor.

a, Western-blot analysis of canonical and non-canonical TGF-β signaling pathways (p-Smad2/Smad2, p-Smad3/Smad3, p-ERK/ERK, p-p38/p38) in WT and CD248 KO mouse hearts with or without I/R injury. Statistical analysis was shown in b. n = 6 in each group. c, Western-blot analysis of TGF-βRI and its downstream canonical and non-canonical signaling (p-Smad2/Smad2, p-Smad3/Smad3, p-ERK/ERK, p-p38/p38) in cardiac fibroblasts transfected with scramble or OE-CD248 lentivirus, and statistical analysis was shown in d. n = 3 in each group. e, RT-PCR analysis of TGF-βRI and TGF-βRII mRNA level in CD248-overexpression (OE-CD248) versus scramble-treated cardiac fibroblasts, n = 4 in each group. f, TGF-βRI protein level detected in cardiac fibroblasts treated with Scramble+DMSO, sh-CD248 + DMSO, sh-CD248+baflomycin and sh-CD248 + MG132 separately. Statistical analysis was shown in g. n = 6 in each group. h, Combination analysis of TGF-βRI and CD248 by co-immunoprecipitation in cultured cardiac fibroblasts. CD248 expression analysis in a and f was conducted using the Abcam antibody (no. 67273), while analysis in c and h was performed using the Proteintech antibody (no. 60170). Data are shown as mean ± s.e.m.; unpaired two-tailed Student’s t-test (d and e) and two-way ANOVA (b and g).

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Extended Data Fig. 6 CD248 monoclonal antibody reduced cardiac T cell infiltration and attenuated cardiac dysfunction in mice subjected to I/R injury.

a, RT-PCR analysis of CD248 mRNA level in the mouse heart, lung, aorta, liver, kidney, spleen and intestinal tract at baseline and 3 days, 7 days, and 14 days post myocardial infarction, with CD248 mRNA level in sham-operated mouse hearts as normalization. n = 5-6 animals in each group. b, Binding affinity of IgG78 with CD248 protein as reflected by OD value at different concentration. c, Flow cytometry analysis of the binding specificity of IgG78 with CD248 using CD248 knockout and CD248 overexpressed adult mouse cardiac fibroblasts. d, Representative M-mode echocardiogram images of mice from sham+IgG, sham+IgG78, I/R+IgG, I/R + IgG78 at 4 days and 28 days post I/R injury. e, LVESV and LVEDV measurement in mice from Sham+IgG, Sham+IgG78, I/R+IgG, I/R + IgG78 groups at 4 days and 28 days post I/R injury. n = 6 animals in each group. f-i, Immunofluorescence analysis of CD3+ T cell (f) and CD68+ macrophage (h) infiltration in I/R-injured mouse hearts with IgG or IgG78 treatment; and statistical analysis were respectively shown in g and i. n = 5 animals in each group. Scale bar = 20 μm. j, Representative dot plots showing cardiac T cells infiltration in I/R-injured mouse hearts after three times of IgG or IgG78 treatment. k, Western-blot analysis of ACKR3 expression in TGF-β-stimulated cardiac fibroblasts with IgG or IgG78 treatment; and statistical analysis were shown in l. Data are shown as mean ± s.e.m.; unpaired two-tailed Student’s t-test (g and i) and two-way ANOVA (e and l).

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Extended Data Fig. 7 CD248 CAR T construction strategy and in vivo detection.

a, Schematic diagram of Pan T isolation, anti-CD3/CD28 microbead activation, and retroviral vector transduction to construct CAR T cell. b, Construct maps encoding mouse CD248 CAR and mouse PSCA CAR. c, Flow cytometry analysis showing the expression level of CAR in PSCA CAR T or CD248 CAR T cells, with statistical analysis shown in d. e, CAR T cell subsets analysis by flow cytometry. f, g. Cytotoxic T cell activity as determined by CD248 expression (f) and CD248+ proportion (g) of fibroblasts using flow cytometry, in which the CD248-overexpressed fibroblasts as targets. T: E ratio, target-to-effector ratio. n = 3 independent experiments in each group. h, Representative CFSE images of the hearts from T cell or CD248 CAR T cell-transfused I/R-injured mice 48 hours after cell transfusion, and statistical analysis of fluorescence intensity were plotted in i. n = 3 animals in each group. j, Immunofluorescence analysis of PSCA CAR T and CD248 CAR T infiltration by GFP staining in I/R-injured mouse hearts 72 hours post-CAR T cell injection. k, Representative dot plots showing cardiac PSCA or CD248 CAR T cells infiltration in I/R-injured mouse hearts 72 hours after CAR T cell injection. l, Quantification of the number of PSCA or CD248 CAR T cells in the I/R-injured mouse hearts at 72 hours post-CAR T cell injection. m, Assessment of the activation status of cardiac infiltrated CAR T cells by CD69 detection using flow cytometry, with statistical analysis shown in n. o, p, Flow cytometry analysis of PSCA or CD248 CAR T cell infiltration (o) and activation (p) in the lung tissues of mice subjected to cardiac I/R injury at 72 hours post-CAR T cell injection. Data are shown as mean ± s.e.m.; unpaired two-tailed Student’s t-test (i, l, n, o and p), one-way ANOVA (d and e) and two-way ANOVA (g).

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Extended Data Fig. 8 Assessment of cardiac function, cardiac T cell infiltration, and safety evaluation after CD248 CAR T cell therapy in mice.

a, Representative M-mode echocardiogram images of animals from Sham, I/R, I/R + PCSA CAR T, I/R + CD248 CAR T groups at 7 days and 28 days post I/R injury. b, LVESV and LVEDV measurement in animals from Sham, I/R, I/R + PCSA CAR T, I/R + CD248 CAR T groups at 7 days and 28 days post I/R. n = 8 animals in I/R + PSCA CAR T group, and n = 9 animals in the other groups. c, Representative dot plots showing cardiac T cells infiltration in I/R-injured mouse hearts one week after PSCA CAR T or CD248 CAR T treatment. d, Safety evaluation of CD248 CAR T cell therapy by serum detection of IL-6, IL-10, MCP-1, IFN-γ, TNF-α, IL-1β, IL-10, and IL12p7 using cytometric bead arrays in mice subjected to I/R. n = 6 in each group. e, Representative images of H&E staining of the liver, lung, spleen in animals from Sham, I/R and I/R + CD248 CAR T groups. Data are shown as mean ± s.e.m.; unpaired two-tailed Student’s t-test (d) and two-way ANOVA (b).

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Extended Data Fig. 9 Analysis of CD248+ fibroblast in mouse pulmonary and renal fibrosis samples and single-cell RNA sequencing database on human pulmonary and renal fibrosis.

a, b, Immunofluorescence analysis of CD248 (red) positive fibroblasts (Vimentin, green) in mouse bleomycin-treated lungs (a) and unilateral ureteral obstruction-subjected kidneys (b). Scale bar = 20 μm. c, UMAP of pulmonary fibroblast subpopulation in patients with pulmonary fibrosis. d, CD248 expression analysis across all lung fibroblast subsets. e, f, GO (e) and KEGG (f) signaling enrichment analysis from CD248+ F5 lung fibroblast subset. g, UMAP of renal fibroblast subpopulation in patients with chronic kidney disease. h, CD248 expression analysis across all renal fibroblast subsets. i, j, GO (i) and KEGG (j) signaling enrichment analysis from CD248+ F6 kidney fibroblast subset.

Extended Data Fig. 10 Gating strategies used for flow cytometry analysis and cell sorting.

a, Gating strategy to sort cardiac fibroblasts (CD45-CD31-PDGFR-α+ ) from infarcted mouse hearts for single-cell RNA sequencing (Fig. 1a-f) and CD248 expression analysis (Fig. 1n-p). b, Gating strategy to sort cardiac fibroblasts (CD45-CD31-PDGFR-α+ ) from ischemia/reperfusion-injured mouse hearts for ACKR3 expression analysis (Fig. 4d, e). c, Gating strategy for flow cytometry analysis of the proportion of macrophages, neutrophils, T cells and B cells in ischemia/reperfusion-injured mouse hearts (Fig. 3c). d, Gating strategy for flow cytometry analysis of CD4+ /CD8+ and TH1/TH2/TH17 T cell number in the ischemic mouse hearts (Figs. 3d, e, 4n, 6h, and 7g).

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Li, G., Ni, C., Wang, J. et al. Dynamic molecular atlas of cardiac fibrosis at single-cell resolution shows CD248 in cardiac fibroblasts orchestrates interactions with immune cells. Nat Cardiovasc Res 4, 380–396 (2025). https://doi.org/10.1038/s44161-025-00617-1

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