摘要Background:Tenascin-C(TNC)is an extracellular matrix(ECM)protein involved in tissue damage and fibrosis.Chimeric antigen receptor(CAR)cell therapy is a novel therapeutic approach that has attracted increasing attention in recent years.Here,we engineered CAR-macrophages targeting TNC(TNC-CAR-Ms)and explored the underlying mechanism through which TNC-CAR-Ms treat liver fibrosis.Methods:The role of TNC in liver fibrosis was studied in established Tnc knockout(KO)and littermate control mice.A TNC-targeted single-chain variable fragment(scFv)was designed to generate TNC-CAR-Ms and evaluate their biological function.The phagocytosis and killing effects of TNC-CAR-Ms were tested in vitro,while the anti-fibrotic efficacy and safety of TNC-CAR-Ms were evaluated in vivo.The underlying mechanism through which TNC-CAR-Ms treat liver fibrosis was investigated by Western blotting,flow cytometry,and RNA sequencing.Results:TNC expression was significantly upregulated in the liver and activated hepatic stellate cells(HSCs)in carbon tetrachloride(CCl4)-treated mice.Animal studies showed that Tnc KO protects mice from CCl4-induced liver damage and fibrosis.Upon demonstrating their ability to engulf and kill activated HSCs,we intravenously administered TNC-CAR-Ms to fibrotic mice and found that TNC-CAR-Ms significantly reduced liver fibrosis.Mechanistically,TNC-CAR-Ms specifically migrated to liver tissues,potently reduced TNC expression,and decreased the activity of the Toll-like receptor 4(TLR4)/nuclear factor kappa-B(NF-κB)and integrin/focal adhesion kinase(FAK)signaling pathway.In addition,TNC-CAR-Ms significantly modified the hepatic immune microenvironment,characterized mainly by an increase in the numbers of M2-polarized macrophages and CD8+T cells in the liver.Finally,in CCl4-treated mice,the depletion of CD8+T cells with an anti-CD8α antibody significantly impaired the antifibrotic effect of TNC-CAR-Ms.Conclusions:Our proof-of-concept study demonstrates the therapeutic potential of TNC-CAR-Ms in alleviating liver fibrosis and may inform the development of future therapeutic strategies for the treatment of a range of liver diseases with a fibrotic phenotype.