ABSTRACT Anti-PD-1 antibody has been approved to treat distinct cancers including hepatocellular cancer (HCC); however, the objective therapeutic response in human HCC patients is only about 14%. Microbes are now widely accepted to play critical roles in cancer pathology, and targeting them to improve cancer treatment is an active research area. To investigate the role of gut microbiota in HCC, a novel murine model was created which reflects the typical features in human disease and expresses SV40 T antigen (TAg) as a trackable tumor specific antigen (TSA). This model was used to study the influence of gut microbiota on HCC initiation and progression by treating pre- or post-malignant mice with an antibiotic cocktail (ABX) that contains three types of antibiotics. ABX treatment restored TSA CD8+ T-cell function, retarded hepatocarcinogenesis, and therapeutically slowed HCC growth. Metagenomic assay demonstrated ABX treatment mediated an enrichment of Bacteroides. Supplementation of Bacteroides thetaiotaomicron (B.th), one member of genus Bacteroides, acted similarly to ABX in suppressing tumor growth and activating anti-tumor immune response, associating with the intratumoral accumulation of CpG-rich genomic DNAs and increased expression of TLR9 in intratumoral dendritic cells (DCs) and macrophages (MΦs). In particular, complete gut sterilization of HCC-bearing mice with five types of antibiotics followed by B.th repopulation markedly improved the therapeutic efficacy of αPD1 Abs. Single cell RNA sequencing (scRNA-seq) revealed that B.th repopulation was associated with significant suppression of Kruppel-like factor 2 (KLF2) and significant increase of TLR9. Previous studies have demonstrated that KLF2 is a transcription factor which negatively controls expression of TLR9, phagocytosis in MФs and DCs, and function of T cells. Together, these results imply that B.th suppresses KLF2 expression, which abrogates its suppressive effect on TLR9, a pattern recognition receptor (PRR). The resultant increased TLR9 on sentinel MФs and DCs recognizes B.th-derived CpG-rich DNAs in tumors to activate TSA effector CD8+ T cells against HCC. Thus, this study will test the hypothesis that gut Bacteroides activate anti-HCC immune responses and improve anti-HCC immunotherapy by modulating immunological function of DCs and MФs via KLF2/TLR9/CpG molecular pathways. Aim 1 will dissect the molecular mechanisms by which B.th modulates DCs and MΦs to improve anti- HCC immunity and therapeutic effect through KLF2 and TLR9 pathways; aim 2 will dissect the cellular mechanisms by which B.th modulates DCs and MΦs to improve anti-HCC immunity and therapeutic effect; and aim 3 will investigate therapeutic and immune regulatory effect of gut microbiota in human HCC patient response to αPD-1 Ab treatment. Successful completion of the proposed studies will provide insight into the cellular and molecular mechanisms underlying B.th-activated anti-HCC immune response and advance gut ...