Potential role of liver-resident CD3⁺ macrophages in HBV clearance in a mouse hepatitis B model

Background & Aims: Chronic HBV infection usually causes cirrhosis and hepatocellular carcinoma. Comparative investigations of acute and chronic HBV cases would help determine the immune responses crucial for viral clearance. Methods: A fast-cleared HBV mouse model was established in Alb-Cre mice via hydrodynamic injection of HBV plasmid, while persistent HBV model mice were generated via recombinant covalently closed circular DNA-adeno-associated virus 8 infection. The single-cell transcriptomes of CD45⁺ intrahepatic non-parenchymal cells from these mice were conducted. Multiplexed immunohistochemistry and flow cytometry were used to confirm the findings from single-cell transcriptomes. Transwell, coculture, and adoptive transfer experiments were performed to study the generation and functions of macrophages. Results: Twenty-four clusters of immune cells were identified. Myeloid cells, including granulocytes, monocytes, and dendritic cells, are activated early in HBV fast-cleared mice. Significantly, a cluster of CD3⁺ macrophages was found in the viral clearance phase, which was confirmed in liver tissue from five acute patients with HBV. These cells highly expressed CXCL1, tumor necrosis factor alpha, and HBsAg-specific T cell receptors. The transwell assay revealed that CD3⁺ macrophages originate from macrophages (n = 6). T cells and anti-HBsAg antibodies are indispensable for their differentiation, which was further confirmed in T- and/or B-cell-deficient mice. Interestingly, these CD3⁺ macrophages capable of killing peptide-loaded hepatocytes and engulfing IgG-coated beads were persistently detectable in the mouse liver for 10 weeks after HBV clearance. The expression levels of CD5L and Bcl2, two classical antiapoptotic proteins, increased (p <0.001), suggesting that the CD3⁺ macrophages are long-term resident populations. Finally, adoptive transfer of CD3⁺ macrophages accelerated HBV clearance in mice (n = 5, p <0.01). Conclusions: We identified long-term polyfunctional CD3⁺ macrophages residing in HBV fast-cleared livers that could help elucidate the immune responses involved in eliminating HBV. Impact and implications: The liver is a special organ with unique immune characteristics and tolerance to foodborne antigens. Chronic infections can develop in newborns after exposure to HBV; however, acute infections usually occur in adults, indicating that immune cells in the liver tissue microenvironment can also effectively fight against the virus. Nevertheless, the mechanisms involved in acute HBV infection have rarely been studied. In this study, we identified a macrophage population with both T cell and macrophage characteristics in the livers of acute HBV model mice and revealed that these macrophages play important roles in HBV clearance. Moreover, we confirmed that this population is derived from macrophages in the presence of virus-specific T cells and antibodies. This finding highlights the complexity of antiviral immune responses in liver microenvironments.