Fibrosis is observed in nearly every form of myocardial disease1. Upon injury, cardiac fibroblasts in the heart begin to remodel the myocardium by depositing excess extracellular matrix, resulting in increased stiffness and reduced compliance of the tissue. Excessive cardiac fibrosis is an important factor in the progression of various forms of cardiac disease and heart failure2. However, clinical interventions and therapies that target fibrosis remain limited3. Here we demonstrate the efficacy of redirected T cell immunotherapy to specifically target pathological cardiac fibrosis in mice. We find that cardiac fibroblasts that express a xenogeneic antigen can be effectively targeted and ablated by adoptive transfer of antigen-specific CD8+ T cells. Through expression analysis of the gene signatures of cardiac fibroblasts obtained from healthy and diseased human hearts, we identify an endogenous target of cardiac fibroblasts—fibroblast activation protein. Adoptive transfer of T cells that express a chimeric antigen receptor against fibroblast activation protein results in a significant reduction in cardiac fibrosis and restoration of function after injury in mice. These results provide proof-of-principle for the development of immunotherapeutic drugs for the treatment of cardiac disease.
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We thank J. Molkentin for sharing the PostnMCM mice with us, A. Stout for help with imaging, D. Martinez for help with image analysis, M. Scherrer-Crosbie for guidance on the echocardiogram procedure and interpretation, F. Mourkioti for sharing the mdx/mTRKO mice with us, E. Radaelli for help with immunohistochemistry and the University of Pennsylvania Diabetes Research Center (DRC) for the use of the RIA Biomarker Core (P30-DK19525). This research was supported by NIH R35 HL140018, T32 HL007843-22, F31 HL147416, the Burroughs Wellcome Fund to R.J., the Cotswold Foundation and the W. W. Smith Endowed Chair to J.A.E.
C.H.J., E.P. and S.M.A. are inventors (University of Pennsylvania, Wistar Institute) on a patent for a FAP CAR (US Utility Patent 9,365,641 issued 14 June 2016, WIPO Patent Application PCT/US2013/062717). H.A. and J.A.E. are inventors (University of Pennsylvania) on a patent for the use of CAR T therapy in heart disease (US Provisional Patent Application 62/563,323 filed 26 September 2017, WIPO Patent Application PCT/US2018/052605). C.H.J. is a scientific founder and has equity in Tmunity Therapeutics, a biotech dedicated to developing engineered T cells for therapy of cancer, infections and autoimmunity, reports grants from Novartis and Tmunity Therapeutics and is on the scientific advisory boards of Immune Design, Viracta Therapeutics, Carisma Therapeutics and Cabaletta Bio.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Jeffery D. Molkentin, Richard T. Lee and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Picro-Sirius red staining for cardiac fibrosis (red) in a coronal section of the heart of a PostnMCM;RosaOVA mouse treated with AngII/PE and tamoxifen for 1 week (left). High-powered field of the left ventricular free wall (right). Representative image of three biologically independent mice, showing similar results. Scale bars, 100 μm. b, Control and experimental hearts were measured (weight (mg)) and images captured. Representative images are shown. c, Quantification of heart weight to body weight (HW/BW) ratio of indicated genotypes and conditions. Data are mean ± s.e.m. ****P < 0.0001 (one-way ANOVA between groups, P < 0.0001; post hoc test for multiple comparisons, Tukey’s test; n = 10, 7, 6, 8 biologically independent mice from left to right).
a, Fold change and P values of cardiac-fibroblast-specific gene expression of heart samples from patients with HCM and DCM compared with samples from non-failing hearts. n = 122 (non-failing), 27 (HCM) and 89 (DCM). Differential gene-expression analyses were performed using a linear model. b, Immunohistochemistry co-staining of cardiac troponin (red) and FAP (green; left) and vimentin (green; right) in adjacent sections from the left ventricle of a patient with DCM. FAP and vimentin are expressed in the same fibroblasts (arrowheads). Representative images of two independent experiments, showing similar results. Scale bars, 100 μm.
a, Immunohistochemistry co-staining of cardiac troponin (red) and FAP (green; left) and vimentin (green; right) in adjacent sections from the left ventricle of a mouse treated with AngII/PE for 2 weeks. FAP and vimentin are expressed in the same fibroblasts (arrowheads). Representative images from two independent experiments, showing similar results (n = 7 biologically independent mice). b, Immunohistochemistry of FAP (green) in various organs or tissues after 1 week of AngII/PE treatment. Representative image of n = 3 biologically independent mice, showing similar results. c, Masson’s trichrome stain for fibrosis (blue; top, centre) and immunohistochemistry of FAP (green; bottom) in coronal heart sections of a wild-type mouse 2 weeks after continuous AngII/PE treatment. Staining and immunohistochemistry were performed on adjacent sections. Bottom insets, higher magnification of left ventricular free wall. Representative image of two independent experiments, showing similar results (n = 7 biologically independent mice). d, Immunohistochemistry of FAP (green) in mouse models of cardiac injury. DMD, Duchenne’s muscular dystrophy (mdx/mTRKO G2 mice); MI, myocardial infarction; TAC, transverse aortic constriction. Scale bars, 100 μm.
a, Immunohistochemistry of FAP (red) and GFP (green) on the left ventricular free wall of mouse heart coronal sections. Wild-type C57BL/6 mice were treated with (right) or without (left) AngII/PE for 1 week, injected with FAP–GFP CAR T cells and euthanized 1 day later. FAP–GFP CAR T cells co-localize with FAP-expressing cells (arrowheads, bottom). b–d, Picro-Sirius red staining of hearts from 21 individual mice (1–21) treated for 4 weeks with saline (b), AngII/PE (c) or AngII/PE and FAP CAR T cells (d) to assess fibrosis (red). Representative images of two independent experiments, showing similar results. Scale bars, 100 μm.
Results from echocardiogram analyses of C57BL/6 mice treated for 4 weeks with saline, AngII/PE or AngII/PE and FAP CAR T cells (n = 12, 10 and 7 biologically independent mice, respectively). Data are mean ± s.e.m. CO, cardiac output; E/E′, ratio of mitral peak velocity of early filling (E) to early diastolic mitral annular velocity (E′); EDV, end diastolic volume; ESV, end systolic volume; FS, fractional shortening; HR, heart rate; IVSd, interventricular septal end diastole; IVSs, interventricular septal end systole; LVIDd, left ventricular internal diameter end diastole; LVIDs, left ventricular internal diameter end systole; LVLd, left ventricular endocardial length (diastole); LVLs, left ventricular endocardial length (systole); LVAEpid, left ventricular epicardial area (diastole); LVAENDd, left ventricular endocardial area (diastole); LVAENDs, left ventricular endocardial area (systole); MV E, early ventricular filling velocity; SV, stroke volume.
a, Masson’s trichrome stain (blue; left, centre) and FAP immunohistochemistry (green; right) on adjacent heart coronal sections 1 week after commencement of continuous AngII/PE treatment. FAP expression is present in interstitial, but not perivascular, fibroblasts (white arrowheads). Image is centred on the vessel shown in Fig. 2c. Representative images of two independent experiments, showing similar results. b, Picro-Sirius red staining of perivascular fibrosis (black arrowheads, red) in heart coronal sections from mice treated for 4 weeks with saline, AngII/PE or AngII/PE and FAP CAR T cells. Representative images of two independent experiments with similar results. c, H&E staining of various tissue sections from mice treated for 4 weeks with saline, AngII/PE or AngII/PE and FAP CAR T cells. Representative images of two independent experiments, showing similar results. Scale bars, 100 μm.
Serum cytokine levels in mice treated with either AngII/PE or AngII/PE and FAP CAR T cells over 12 weeks. FAP CAR T cells were injected at 1 and 2 weeks as indicated. Levels were assessed at 10 days, 2 weeks, 4 weeks and 12 weeks. Basal levels were determined by the average cytokine levels of three untreated mice. INFγ and IL-4 were below the limit of detection in all conditions. *P = 0.019, #P = 0.035, †P = 0.045; two-tailed unpaired Student’s t-test; n = 3 biologically independent mice per condition.
Extended Data Fig. 8 Cardiotoxicity, inflammation and immune assessments after FAP CAR T cell transfer.
a, Volcano plot showing the differential expression of genes known to be modified in cardiotoxicity in the hearts of mice treated with either AngII/PE and FAP CAR T cells or a saline control for 4 weeks. Statistically significant changes are marked to indicate whether genes are expected to increase (orange) or decrease (blue) in the setting of cardiotoxicity. b, Differential expression of the same conditions in a at 8 weeks. a, b, Two-sided Welch’s t-test; n = 3 biologically independent mice per condition. c, Volcano plot showing the differential expression of 1,659 immune- and inflammation-related genes from hearts of mice treated with AngII/PE and FAP CAR T cells or AngII/PE for 4 weeks. In total, 22 genes were differentially expressed between the conditions. n = 3 mice per condition. d, Photomicrographs and quantification (mean ± s.e.m.) of immune cell (arrowheads) residency of the left ventricle at 4 weeks after either AngII/PE or AngII/PE and FAP CAR T cell treatment. Two-tailed unpaired Student’s t-test; n = 3 or 4 biologically independent mice, respectively. Scale bars, 100 μm.
a, Kaplan–Meier survival curve of mice treated with either AngII/PE or AngII/PE and FAP CAR T cells for 12 weeks. b, Body weight measurements at 12 weeks. Two-tailed unpaired Student’s t-test. c, H&E of sections of heart and organs or tissues at 12 weeks from a mouse treated with AngII/PE and FAP CAR T cells. Representative images of n = 3 independent mice, showing similar results. Scale bars, 100 μm. d, Photomicrographs and H&E sections of a healing wound over 8 days in mice treated with either FAP CAR or control T cells immediately and 3 days after wounding. Scale bars, 1 mm (wounds) and 250 μm (H&E sections). e, f, Quantification of wound area (e) and measurements of body weight (f). g, Serum levels of amylase at day 8 to test pancreatic toxicity. Data are mean ± s.e.m.
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Aghajanian, H., Kimura, T., Rurik, J.G. et al. Targeting cardiac fibrosis with engineered T cells. Nature 573, 430–433 (2019). https://doi.org/10.1038/s41586-019-1546-z
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