CX₃CR1⁺macrophages interact with HSCs to promote HCC through CD8⁺T-cell suppression

Background and Aims: HSCs contribute to HCC progression by regulating multiple factors. However, the entire immunoregulatory functions of HSCs are still obscure. Here, we aim to investigate whether HSCs impose CX3CR1+ macrophages to protumorigenic properties in the peritumoral area. Approach and Results: In single-cell RNA-sequencing analysis of patients with HCC, a infpopulation of macrophages specifically expressed Arg1 and Cx3cr1 in the peritumoral area and were highly enriched with retinol metabolism-related genes. Flow cytometry analysis showed significantly increased frequencies of CD14+CD11b+HLA-DR- macrophages with CX3CR1 in the HCC adjacent region where α-smooth muscle actin-expressing activated hepatic stellate cells (aHSCs) showed colocalized expression of CX3CL1. Accordingly, in tumor-bearing mice, Cx3cl1 mRNA expression was notably increased in aHSCs within the adjacent HCC, where infiltration of CX3CR1+Ly6C+ macrophages was mostly observed with decreased CD8+ T cells. In adoptive transfer and in vitro coculture of myeloid cells, we demonstrated that CX3CR1+Ly6C+ macrophages migrated and highly expressed arginase-1 by interacting with retinoid-enriched aHSCs in the adjacent HCC. Direct treatment of retinoids or coculturing with retinol-storing mouse aHSCs or human LX-2 cells significantly increased arginase-1 expression in CX3CR1+Ly6C+ macrophages and human blood CD14+ cells, leading to the suppression of CD8+ T-cell proliferation. Moreover, genetic deficiency of CX3CR1 in myeloid cells or pharmacological inhibition of retinol metabolism remarkably attenuated HCC development. Conclusions: We showed that CX3CR1+Ly6C+ macrophages migrate and interact with aHSCs in the peritumoral region where retinoids induce arginase-1 expression in CX3CR1+Ly6C+ macrophages, infsequently depriving CD8+ T cells of arginine and promoting HCC.