肿瘤相关巨噬细胞诱导肝癌细胞高表达免疫抑制分子B7-H1及其增强体机制的研究

High mortality rate of human hepatocellular carcinoma (HCC) is attributed to its recurrence and metastasis after surgery. Immunotherapy based on CTL cells is a prospective treatment to eradicate the residual malignant cells after surgery, but it is impaired by immune escape of HCC. One of important mechanism of immune escape is the overexpression of B7-H1 on HCC cells since the B7-H1 can bind to its receptor PD-1 existing on CTL cells to lead them to apoptosis. However, it is not well known so far why and how the B7-H1 is expressed in HCC cells. Why is the B7-H1 overexpressed in HCC cells? Our preliminary study implied that the overexpression of B7-H1 in HCC cells might be due to inflammatory microenvironment predominated by tumor associated macrophages (TAM). We found that overexpression of B7-H1 in malignant cells was associated with macrophages infiltration in HCC tissues. In addition, The B7-H1 expression in HCC cell lines was upregulated by macrophages in a transwell coculture system. Based on these results, we put forward a novel hypothesis that TAM induces overexpression of B7-H1 in HCC cells via producing multiple inflammatory cytokines. In this project, we are going to analyze clinical samples and design cytological experiments and zoological experiments to further reveal the mechanism of B7-H1 overexpression in HCC.The anticipated results will provide new knowledge for tumor immunology. How is the B7-H1 overexpression induced by TAM? We assume that a specific enhanceosome control the induction of B7-H1 overexpression.In this project, we are also going to analyze the composition and action mode of the enhanceosome. Enhancesome is a complex located on the gene promoter region, composed of multiple transcription factors activated by signaling from the outside of the cell. The B7-H1 enhanceosome can control the B7-H1 expression more directly and specificly than the inflammatory cytokines and signaling pathways. It will be the first time to describe the B7-H1 enhanceosome.Moreover, the study will provide new targets and strategies for new medicine-design and inhibition of immune escape. Based on our preliminary study and the theory of enhanceosome, we propose our hypothesis that TAM induces the overexpression of B7-H1 on HCC cells through a specific enhanceosome mechanism. In this project, we will give an answer to why and how the B7-H1 is overexpressed in HCC cells. All the results from this project will pave a way to block immune escape and improve immunotherapy of HCC.

免疫学治疗已成为继手术、放疗和化疗后的第四种肿瘤治疗方法，而免疫抑制分子B7-H1在肝癌细胞高表达所引发的肝癌免疫逃逸，是有效免疫治疗的一大障碍。但是其高达的原因和机制至今未明。为解释B7-H1在肝癌细胞高表达的原因，本项目在前期研究工作基础上，首次提出肿瘤相关巨噬细胞诱导肝癌细胞高表达B7-H1分子,进而介导免疫逃逸的新观点，拟通过临床标本检测、细胞学和动物学实验加以科学论证，从而揭示这一炎症促肿瘤新机制，丰富肿瘤免疫学理论；为探讨B7-H1在肝癌细胞高表达的机制，本项目避繁就简，从增强体这一表达调控关键因素着手，拟首次研究肿瘤相关巨噬细胞诱导肝癌细胞高表达B7-H1的特定增强体组成和作用模式，这可为阻断B7-H1的诱导表达提供直接并特异的新靶点和新策略，是开发新型药物以抑制免疫逃逸，提高免疫治疗效果的前提和基础。

肿瘤相关巨噬细胞可以促进肿瘤的增殖和转移，还可以促进肿瘤的免疫逃逸。我们发现肝癌组织的肿瘤相关巨噬细胞浸润与肝癌细胞表达B7-H1具有相关性，而B7-H1是重要的介导肿瘤免疫逃逸的免疫负调控分子，由此提出研究假说：肝癌相关巨噬细胞诱导肝癌细胞高表达B7-H1分子介导肝癌免疫逃逸，并且巨噬细胞通过增强体机制调节肝癌细胞表达B7-H1分子。因此在本项目中我们研究肿瘤相关巨噬细胞与肝癌B7-H1表达的相关性、M1巨噬细胞对B7-H1表达的诱导作用、巨噬细胞诱导肝癌细胞表达的B7-H1介导免疫逃逸。我们进一步分析M1型巨噬细胞分泌的、诱导肝癌细胞表达B7-H1的相关细胞因子种类、M1型巨噬细胞诱导肝癌细胞表达B7-H1的相关转录因子的种类以及M1型巨噬细胞诱导肝癌细胞表达B7-H1的增强体的组成及作用模式。除此之外，我们还研究了IFN-γ诱导肝癌细胞B7-H1表达的调控机制，以及B7-H1表达的翻译后修饰调控机制。我们还延伸研究了泛素编辑酶A20基因对肝癌细胞迁移的抑制作用。 . 本项目重要的研究结果：（1）发现肝癌组织中巨噬细胞的浸润程度与肝癌细胞的B7-H1表达明显相关，并且M1样的巨噬细胞浸润也与肝癌细胞表达B7-H1具有相关性；（2）发现M1型巨噬细胞可诱导肝癌细胞高表达B7-H1，而M2型巨噬细胞不能；（3）M1巨噬细胞通过分泌IL-1、IL-6和TNF-α促进肝癌细胞高表达B7-H1；（4）M1巨噬细胞促进肝癌细胞表达B7-H1所涉及的转录因子包括IRF1, P65,和STAT3；（5）JAK1通路是介导M1巨噬细胞诱导肝癌细胞高表达B7-H1的主要信号通路；（6）M1巨噬细胞诱导肝癌细胞表达B7-H1的增强体是由P65、IRF1和STAT3转录因子组成，并且巨噬细胞诱导肝癌细胞表达B7-H1的增强体属于多模块增强体；（7）TNF-α和IFN-γ协同诱导肝癌细胞高表达B7-H1，其协同机制是TNF-α通过NF-κB通路诱导肝癌细胞高表达IFN-γ受体；（8）癌基因c-Myc抑制IFN-γ信号通路从而抑制B7-H1在肝癌细胞的诱导表达；（9）A20抑制TNF-α诱导的肝癌细胞迁移。.本项目研究目标基本完成，论证了巨噬细胞促进肝癌细胞高表达B7-H1进而促进肝癌重要机制，并提供可能的药物干预靶点。