Modulating to boost anti-PD-1 immunotherapy in HCC.

[BACKGROUND] The gut microbiota is increasingly recognized as a critical external regulator along the gut-liver axis, influencing hepatocarcinogenesis and modulating responses to immunotherapy. However, the specific microbial determinants, underlying mechanisms, and potential clinical applications remain incompletely elucidated.

[METHODS] Building on the observed association between gut microbiota and anti-programmed cell death protein-1 (PD-1) immunotherapeutic efficacy in patients with hepatocellular carcinoma (HCC), we leveraged a suite of clinically relevant murine HCC models to comprehensively characterize tumor-associated microbial signatures using 16S ribosomal RNA gene sequencing. By precisely manipulating microbial composition through a non-hepatotoxic antibiotic cocktail 3 (ABX-3), targeted microbial supplementation, human fecal microbiota transplant (FMT), and controlled ) repopulation following gut sterilization with ABX-5, we demonstrated a direct causal relationship between microbiota modulation and intrahepatic immune activation. Single-cell RNA sequencing of hepatic non-parenchymal cells, together with functional validation experiments, was performed to elucidate the underlying immune mechanisms.

[RESULTS] -enriched gut microbiota derived from anti-PD-1-responsive patients with HCC significantly suppressed tumor growth in murine HCC models. In parallel, within our murine HCC system, ABX-3 administration, implemented as both a preventive and therapeutic intervention, attenuated tumor initiation and progression by selectively enriching within the gut microbial community. Functionally, ABX-3 enhanced the capacity of tumor antigen-specific T-cell receptor-I T cells to mount robust immune responses, culminating in targeted tumor regression following antigen-specific immunization. Among the species, emerged as a critical mediator that potentiated αPD-1 immunotherapy in HCC by relieving Krüppel-like factor 2 (KLF2)-dependent suppression in dendritic cells (DCs). Mechanistically, the KLF2-toll-like receptor 9 (TLR9) signaling axis in DCs governed the activation of antigen-specific CD8 T cells, thereby amplifying antitumor immunity within the HCC microenvironment.

[CONCLUSIONS] is identified as a key immunomodulatory species that enhances anti-PD-1 efficacy by reprogramming DCs through the KLF2/TLR9 signaling pathway. These findings reveal a novel microbiota-informed strategy to improve immunotherapeutic outcomes in HCC.