Effective anti-PD-1 therapy reduces neutrophil infiltration via T cell–macrophage chemokine axis in hepatocellular carcinoma

Objective: Immune checkpoint inhibitor (ICI) therapies, such as anti-PD-1 treatment, have achieved great success in various cancers, including hepatocellular carcinoma. Tumor-infiltrating lymphocytes have major roles in the mechanism of action for ICIs, but numerous other components of the tumor microenvironment also contribute to the effectiveness of immunotherapy. However, little is known about the changes in the tumor microenvironment after ICI therapy, which may sustainably affect tumor progression. Methods: Integrated single-cell RNA sequencing data were collected for hepatocellular carcinoma (HCC) patient samples and analyzed to characterize immune cell alterations within the tumor microenvironment. Three different orthotopic allograft HCC models were established for ICI therapy and microenvironment component detection by using flow cytometry and multiple ELISA assays. Transwell assays were combined with flow cytometry to evaluate neutrophil migration. In vitro coculture systems were employed to investigate communication between different cell types. Multiplex immunochemistry analysis was conducted for human clinical samples to confirm the findings. Results: Our results indicate that neutrophil infiltration was significantly reduced after effective ICI therapy, but not in patients with failed immunotherapy. Mechanistically, we found that effective anti-PD-1 therapy activates T cells, which secrete interferon-γ and other cytokines that act on tumor-associated macrophages. This T cell–macrophage crosstalk suppresses tumor-associated neutrophil-related chemokines secreted by macrophages. Consequently, neutrophil infiltration into the tumor microenvironment is diminished. Conclusion: Our findings show that immunotherapy reinvigorates T cells, but also remodels the myeloid compartment and reduces neutrophil recruitment, potentially alleviating neutrophil-mediated immunosuppression, thereby providing potential myeloid-targeted combinatorial strategies for improving checkpoint blockade responses.