CD248-targeted BBIR-T cell therapy against late-activated fibroblasts in cardiac repair after myocardial infarction

Figures

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  Fig. 1. Identification of cardiac fibroblast subpopulation F-Act. a UMAP clustering of 13441 cardiac interstitial cells (left) and fibroblasts (right) isolated from C57BL/6 J wild-type (WT) mice at 7 and 14 days post-sham and post-MI surgery. Each dot represents a single cell. The expression of known marker genes annotated cell type. b UMAP and violin plots depict Postn, Acta2, and Comp expression in fibroblasts. c Triple IF staining for periostin (green) and αSMA (red) in the peri-infarct zone of the hearts of C57BL/6 J WT mice 0, 7, 14, 21, and 28 days after MI surgery. Nuclei were stained blue. Scale bar = 100 μm. Quantification of the number of periostin+αSMA+ F-Myo and periostin+αSMA- F-Act in each high-power field (HPF). n = 5 mice at each time point after MI. d Flow cytometry of fibroblasts isolated from hearts of C57BL/6 J WT and Pdgfra-CRE:tdTomato mice. Total cardiac fibroblasts were lineage traced by pdgfrα. F-Myo and F-Act were marked with antibodies against periostin-AF488 and αSMA-AF647. Quantification of the pdgfrα+ fibroblast ratio in live cells, periostin+ fibroblasts ratio in pdgfrα+ fibroblasts, periostin+ αSMA+ F-Myo ratio in pdgfrα+ fibroblasts, and periostin+ αSMA-…

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  Fig. 2. Screening for the characteristic surface antigen of F-Act. a Heatmap of genes highly expressed in the F-Act and encoding membrane proteins. b UMAP depicts Cd248 expression in mouse cardiac interstitial cells (left) and fibroblasts (right). c UMAP depicts Cd248 expression in human heart muscle in the HPA.

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  Fig. 3. Identification of CD248 as the target antigen for CAR-T cells against F-Act. a Triple IF staining for periostin (green) and CD248 (red) in the peri-infarct zone of the hearts of C57BL/6J WT mice 0, 7, 14, 21, and 28 days after MI surgery. Nuclei were stained blue. Scale bar = 100 μm. Quantification of the periostin+CD248+ cell to DAPI ratio in each HPF. n = 5 mice at each time point after MI. Triple IF staining of periostin, αSMA, COMP (stained green), and CD248 (stained red) in the hearts of MI mouses (b) and a patient with ischemic cardiomyopathy (c). Nuclei were stained blue. Scale bar =100 μm. IF staining of mouse heart was repeated in heart sections from 5 independent mice, while IF of human heart was repeated 5 times in heart sections from one patient with ischemic cardiomyopathy, yielding similar results. d Flow cytometry of cells isolated from hearts of C57BL/6 J WT mouses and Pdgfra-CRE:tdTomato mice. Total cardiac fibroblasts were lineage traced by pdgfrα. Quantification of the CD248+ cell ratio in live cells, pdgfrα+ fibroblast ratio in CD248+ cells, CD248+ cell ratio in pdgfrα+ fibroblasts, periostin+CD248+ cell ratio in pdgfrα+ fibroblasts, CD248+ cell ratio in…

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  Fig. 4. Construction and functional verification of the BBIR-T cell system targeting CD248. a Schematic diagram of the structure of the BBIR-T system. Created in BioRender. Haiting, C. (2025) https://BioRender.com/l46n352. b Schematic images of dcAv. BBIR-28-137ζ and CD19. CAR-137ζ sequence. c Western blotting shows exogenous CD3ζ expression in HEK-293T cells transduced with lentivirus (LV)-dcAv.BBIR and LV-anti-CD19.CAR. The samples derive from the same experiment and that gels/blots were processed in parallel. Quantifying the relative protein expression of CD3ζ to GAPDH. n = 4 cell culture wells of each group. d Flow cytometry of human peripheral T cells transduced with LV-dcAv.BBIR and LV-anti-CD19.CAR. Quantifying GFP+ cells ratio to total T cells and biotin+ cells ratio to GFP+ cells. n = 5 cell culture wells of each group. e Western blotting by BF shows CD248 expression in NIH-3T3 cells with or without Cd248 overexpression(OE) or knockout(KO). The samples were derived from the same experiment and that gels/blots were processed in parallel. Quantifying the relative protein expression of CD248 to GAPDH. n = 4 cell culture wells of each group. f Triple IF staining of periostin, αSMA,…

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  Fig. 5. In vitro killing effect of BBIR-T cell therapy on CD248-positive fibroblasts. a-d BBIR-T cells were cocultured with BF-labelled NIH-3T3 cells with or without Cd248 OE or KO. The E:T ratio =2:1, 5:1, 10:1, and 20:1. ELISA determined the secretion of IFN-γ (a) and IL-2 (b) in the supernatant 24 h after coculture. n = 5 cell culture wells of each group. Data are the mean ± SEM and p-values are displayed in the bar charts, two-way ANOVA followed by Dunnett’s multiple comparison test. c Cell lysates were analyzed at 24 h after coculture by CCK-8 cell viability assay. d Cytotoxicity was measured by LDH activity in the supernatant 24 h after coculture. c, d Biological replicates (n = 5 cell culture wells) were conducted for each E:T ratio across different target cells. P-values less than 0.05 are displayed in the line charts, two-way ANOVA followed by Dunnett’s multiple comparison test. e–h Different effector cells cocultured with Cd248-OE NIH-3T3 cells. E: T = 10:1. ELISA was used to determine the secretion of IFN-γ (e) and IL-2 (f) in the supernatant 24 h after coculture. g Cell lysates were analysed at 24 h after coculture by CCK-8 cell viability assay. h Cytotoxicity was measured by…

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  Fig. 6. Three-time injections of BBIR-T cells improved the cardiac function of MI mice. a Schematic diagram of the number of BBIR-T-cell injections. Pdgfra-CRE:tdTomato mice were intramyocardially injected with biotinylated anti-CD248 F(ab’)2 immediately after MI surgery to label the F-Act. A total of 106 BBIR-T cells were injected into the tail vein every three days beginning 14 days after MI. Mouse hearts were obtained for flow cytometry detection three days after BBIR-T cells injection. Created in BioRender. Haiting, C. (2025) https://BioRender.com/m61n139. b Flow cytometry of fibroblasts isolated from hearts of C57BL/6J WT and Pdgfra-CRE:tdTomato mice. Total cardiac fibroblasts were lineage traced by pdgfrα. F-Act was marked with antibodies against CD248-AF647. Quantifying the pdgfrα+ fibroblast ratio in live cells and the CD248+ cell ratio in pdgfrα+ fibroblasts. n = 5 mice at each group. Data are mean ± SEM and p-values are displayed in the bar charts, two-way ANOVA followed by Šídák multiple comparison test, one-way ANOVA followed by Tukey’s multiple comparison test. c, d Schematic diagram of the animal experiments. Mice were divided into 6 groups: sham, MI + BF, MI + BF +…

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  Fig. 7. The BBIR-T cell therapy could inhibit fibrosis expansion and improve cardiac remodeling. a Flow cytometry of fibroblasts isolated from hearts of C57BL/6 J WT and Pdgfra-CRE:tdTomato mouses. Typical images show CD248+ F-Act versus the pdgfrα+ fibroblasts ratio. Quantifying the pdgfrα+ fibroblast ratio in live cells and the CD248+ F-Act ratio in pdgfrα+ fibroblasts. n = 7 mice in the MI + BF + BBIR-T group and n = 5 mice in other groups. b Representative full-view images of heart HE and Picro-Sirius Red staining, magnifying the PIZ. Scale bars = 2.5 mm and 100 µm. Quantifying LV wall thickness (mm), infarct size (mm2) and PIZ size (mm2). Calculating the ratio of infarct size or PIZ size to LV area. n = 7 mice of each group. c Serum BNP levels were detected by ELISA. The number of mice (n) in each group was labelled in the bottom of the bar chart. d–f HW/BW ratio, HW/TL ratio and lung wet/dry weight ratio were shown. The number of mice in each group was labelled in the bottom of the bar charts. g Double staining of fluorescent WGA-FITC and DAPI. Scale bar = 50 μm. Quantify each group’s cardiomyocyte CSA (μm2). n = 5 mice of each group. a–g Data are mean ± SEM and p-values are…

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  Fig. 8. The BBIR-T cell therapy did not aggravate inflammation response and tissue damage. Luminex assay was performed using the Bio-Plex 200 system with standard controls to detect the concentrations of IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL12p7, and CXCL1 in heart tissue lysates (a) and serum (b). n = 6 mice of each group. P-values are displayed in the violin plots, one-way ANOVA followed with Dunnett’s comparison test. c Representative images of HE staining of the liver, spleen, lung, and kidney. Scale bar = 100 μm. Each experiment was repeated 5 times independently with similar results. Source data are provided as a Source Data file.