Orf virus remodels tumor immune microenvironment in melanoma mouse models revealed by single-cell RNA sequencing

Oncolytic viruses (OVs) display tumor-suppressing capacity through direct oncolysis and induction of anti-tumor immunity. Orf virus (ORFV) has shown considerable properties in inducing tumor cell pyroptosis and inflamed tumor microenvironment (TME). However, the mechanism by which ORFV reshapes tumor immune microenvironment (TIME) remains unclear. We administered a single intratumoral injection of ORFV to B16 melanoma-bearing mice and harvested tumor-infiltrating immune cells for single-cell RNA sequencing. Here, we characterized dynamic changes of TIME following ORFV challenge. Our research reveals that ORFV promotes the enrichment of activate natural killer (NK) cells and effector CD8⁺ T cells, as well as upregulates genes encoding key cytolytic factors, including Gzma , Gzmb , and Prf1 . Notably, ORFV depletes lipid-metabolizing macrophages, a change that significantly correlates with the activation of cytotoxic T cells. The decrease in lipid-metabolizing macrophages, which specifically express Trem2 , impairs APOE-TREM2 axis signaling between macrophages and T cells. Accordingly, specific blockade of TREM2 using the inhibitor IA9 recapitulated the therapeutic efficacy of ORFV in melanoma. Collectively, these data indicate that ORFV remodels the immunosuppressive TIME by amplifying cytotoxic lymphocyte activity and inhibiting the APOE-TREM2 signaling pathway between macrophages and T cells, thus providing a novel perspective on its anti-tumor mechanism.