Mechanistic studies of gut microbiota-mediated immune activation against hepatocellular cancer

摘要 抗PD-1抗体已被批准用于治疗不同的癌症，包括肝细胞癌（HCC）;然而， 在人HCC患者中的客观治疗反应仅为约14%。微生物现在被广泛接受 在癌症病理学中发挥关键作用，并针对它们来改善癌症治疗是一项积极的研究， 区为了研究肠道微生物群在HCC中的作用，建立了一种新的小鼠模型，其反映了HCC中肠道微生物群的作用。 在人类疾病中的典型特征，并表达SV 40 T抗原（TAg）作为可追踪的肿瘤特异性抗原 （TSA）。该模型用于研究肠道微生物群对HCC起始和进展的影响， 前或后恶性小鼠与抗生素鸡尾酒（ABX），其中包含三种类型的抗生素。ABX 治疗恢复了TSA CD 8 + T细胞功能，延缓了肝癌发生，并在治疗上减缓了HCC 增长宏基因组分析表明ABX处理介导了类杆菌的富集。 多形拟杆菌（Bacteroides thetaiotaomicron，B.th）的作用与 ABX在抑制肿瘤生长和激活抗肿瘤免疫反应中的作用，与肿瘤内 肿瘤内树突状细胞（DC）中富含CpG的基因组DNA的积累和TLR 9表达的增加 和巨噬细胞（MΦ）。特别是，用五种类型的 抗生素治疗后，B.th再增殖显著提高了α PD 1 Ab的治疗效果。单细胞 RNA测序（scRNA-seq）显示，B.th再增殖与显著抑制 Kruppel样因子2（KLF 2）和TLR 9显著增加。以前的研究表明，KLF 2是 一种转录因子，负性控制TLR 9的表达、巨噬细胞和树突状细胞中的吞噬作用和功能 的T细胞。总之，这些结果意味着B.th抑制KLF 2表达，这消除了其抑制作用。 对模式识别受体（PRR）TLR 9的作用。结果增加了前哨M细胞和DC上的TLR 9， 识别肿瘤中源自B th的富含CpG的DNA以激活TSA效应CD 8 + T细胞对抗HCC。因此， 一项研究将验证肠道拟杆菌激活抗HCC免疫应答并提高抗HCC免疫应答的假设。 通过KLF 2/TLR 9/CpG分子调节DC和M β细胞免疫功能的免疫治疗 途径。目的1将剖析调节DC和MΦ以提高抗肿瘤活性的分子机制。B.th 通过KLF 2和TLR 9途径的HCC免疫和治疗效果;目的2将剖析细胞免疫和治疗效应。 B.th调节DC和MΦ以提高抗HCC免疫力和治疗效果的机制;以及 目的3将研究人类HCC患者应答中肠道微生物群的治疗和免疫调节作用 αPD-1 Ab治疗。成功完成拟议的研究将提供深入了解细胞和 B. th激活的抗HCC免疫应答的分子机制和先进的肠道微生物群- 整合免疫疗法的临床应用。

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 microbiota- integrated immunotherapy to clinical application.