Objective:Liver cancer is a deadly malignancy associated with high mortality and morbidity. Less than 20% of patients with advanced liver cancer respond to a single anti-PD-1 treatment. The high heterogeneity of neutrophils in the tumor immune microenvironment in liver cancer may contribute to resistance to immune checkpoint blockade (ICB). However, the underlying mechanism remains largely unknown.Methods:We established an orthotopic liver cancer model by using transposable elements to integrate the oncogenesMycandKrasG12Dinto the genome in liver cells from conditional Trp53 null/null mice (pTMK/Trp53−/−). Flow cytometry and immunohistochemistry were used to assess the changes in immune cells in the tumor microenvironment. Anex vivococulture assay was performed to test the inhibitory effects of tumor-associated neutrophils (TANs) on CD8+T cells. The roles of neutrophils, T cells, and NK cells were validated through antibody-mediated depletion. The efficacy of the combination of neutrophil depletion and ICB was evaluated.Results:Orthotropic pTMK/Trp53−/−mouse liver tumors displayed a moderate response to anti-Ly6G treatment but not PD-1 blockade. Depletion of neutrophils increased the infiltration of CD8+T cells and decreased the number of exhausted T cells in the tumor microenvironment. Furthermore, depletion of either CD8+T or NK cells abrogated the antitumor efficacy of anti-Ly6G treatment. Moreover, the combination of anti-Ly6G with anti-PD-L1 enhanced the infiltration of cytotoxic CD8+T cells and thereafter resulted in a significantly greater decrease in tumor burden.Conclusions:Our data suggest that TANs may contribute to the resistance of liver cancer to ICB, and combining TAN depletion with T cell immunotherapy synergistically increases antitumor efficacy.