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包括肝细胞癌(HCC)在内的不同癌症；然而， 人肝细胞癌患者的客观疗效仅为14%左右。微生物现在被广泛接受 在癌症病理学中发挥关键作用，并针对它们来改进癌症治疗是一项活跃的研究 区域。为了研究肠道微生物区系在肝癌中的作用，建立了一种新的小鼠模型，该模型反映了 人类疾病的典型特征，并表达SV40T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区系对肝细胞癌发生发展的影响。 用含有三种抗生素的抗生素鸡尾酒(ABX)治疗癌症之前或之后的小鼠。ABX 治疗恢复了TSA CD8+T细胞功能，延缓了肝癌的发生，并在治疗上减缓了肝细胞癌的发生 成长。后基因组分析表明，ABX处理可以促进类杆菌的增殖。 补充类杆菌属成员之一的类杆菌，其作用类似于 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的关系 富含CpG的基因组DNA在肿瘤内树突状细胞中的积聚和TLR9表达的增加 巨噬细胞(MΦS)。特别是，对荷肝癌小鼠进行了五种类型的完全肠道灭菌 抗生素再接种可明显提高α-PD1抗体的治疗效果。单细胞 RNA测序(scRNA-seq)显示B.th再种群与显著抑制 Kruppel-like factor2(KLF2)和TLR9显著升高。以前的研究已经证明KLF2是 负调控TLR9表达的转录因子在M-ФS和DC中的吞噬作用及其功能 T细胞的数量。总之，这些结果表明B.th抑制KLF2的表达，KLF2的表达取消了它的抑制作用 对模式识别受体TLR9的影响。由此导致前哨MФS和DC上TLR9的增加 识别B.th来源的富含CpG的DNA，以激活TSA效应CD8+T细胞对抗肝癌。因此，这一点 研究将验证肠道类杆菌激活抗肝癌免疫反应和改善抗肝癌的假设 KLF2/TLR9/CpG分子调节DC和MФS免疫功能的免疫治疗 小路。目的1将剖析B.th调节DC和MΦS增强抗-HBs能力的分子机制。 肝细胞癌KLF2和TLR9通路的免疫和治疗作用；Aim 2将解剖细胞 B.th调节DC和M-Φ-S提高抗肝癌免疫及疗效的机制 目的3研究肠道微生物区系对人肝癌患者的治疗和免疫调节作用 至αPD-1Ab治疗。成功完成拟议的研究将使我们深入了解细胞和 B.TH激活的抗肝癌免疫反应和促进肠道微生物区系发展的分子机制 综合免疫疗法走向临床应用。

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.