Synergistic innate-adaptive immunity by NKG2D-specific CAR-macrophages drives durable remission in hepatocellular carcinoma

Abstract Background Hepatocellular carcinoma (HCC) immunotherapy is limited by antigenic heterogeneity and an immunosuppressive microenvironment. This study engineered chimeric antigen receptor macrophages (CAR-Ms) targeting stress-inducible NKG2D ligands (NKG2DLs), broadly overexpressed in HCC, to enhance phagocytic clearance and remodel immunity. Methods NKG2DL expression in HCC and association with survival were analyzed. CAR-Ms were constructed by fusing the NKG2D extracellular domain to FcγRI signaling. In vitro assays assessed phagocytosis, cytokine secretion, signaling, and T cell interactions. Therapeutic efficacy was evaluated in immunocompetent mice bearing subcutaneous, orthotopic, or metastatic HCC models, with or without anti-PD-L1. Tumor progression, immunity, and survival were analyzed via bioluminescence imaging, flow cytometry, histopathology, and serum biochemistry. Statistics analyses were performed using t-tests, ANOVA, and log-rank tests. Results NKG2DLs were significantly upregulated in human HCC and correlated with poor prognosis. CAR-Ms selectively engulfed NKG2DL⁺ tumor cells, polarized to an M1 phenotype, and activated PI3K-AKT and cGAS-STING pathways, driving phagocytosis and pro-inflammatory cytokines secretion. They enhanced T cell chemokines (Cxcl10, Ccl5) and antigen presentation, boosting T cell recruitment and activation in vitro. In subcutaneous models, CAR-Ms suppressed tumor growth, reprogrammed tumor-associated myeloid cells toward M1, and induced durable immune memory (100% tumor rejection upon rechallenge), with T cell activation. In orthotopic models, CAR-M monotherapy induced complete regression by week 5 and 100% survival, with elevated CD8⁺ T cells and CAR-M specifically homing to liver tumors. CAR-Ms suppressed metastasis in peritoneal/pulmonary models. Combining CAR-Ms with PD-L1 blockade accelerated tumor clearance and survival versus monotherapies, enhancing T cell cytotoxicity. Safety assessments showed no significant organ toxicity based on histopathology and serum biochemistry. Conclusions NKG2D-directed CAR-Ms eliminate HCC through integrated innate phagocytosis, adaptive immune activation, and myeloid reprogramming, overcoming key therapeutic barriers. Combination with anti-PD-L1 enhances therapeutic efficacy by leveraging innate-adaptive crosstalk, providing a promising approach for HCC immunotherapy.