肿瘤浸润树突状细胞高表达负性协同刺激分子B7-H4的机制及其意义

Dendritic cells (DC) are professional APC that are specialized for initiation and regulation of T cell immunity. Tumor-induced modulation of DCs function is one of the main factors responsible for tumor immune escape. DCs have been shown to infiltrate many tumors, appear to be phenotypically and functionally defective. In previous study, we showed that B7-H4 was moderately expressed in imDCs, and could be up-regulated by IL-10, and TNF-α could couteract the the up-regulatory effects of IL-10 on expression of B7-H4 in DCs in vitro. Furthermore, in Lewis lung carcinoma cell line (LLC) cell-transplanted carcinoma model, tumor infiltrated DCs expressed B7-H4 at high levels. Blockade of B7-H4 expressed in DCs highly resulted in enhanced T cell proliferation and IFN-γ production significantly. Otherwise, the high level of IL-10 and TNF-α was both detected in the tumor, which suggested that TNF-α can not couteract the effects of IL-10 on expression of B7-H4 in DCs in vivo. Interesting, we also showed that tumor-associated macrophages could induce expression of B7-H4 on the surface of lung cancer cell, in turn mediated immune escape. Here, the mechanism and significance of over-expression of B7-H4 in tumor infiltrated dendritic cells wil be investigated. We also will evaluate the possible correlation between the B7-H4 expression on TIDC and TAM, and make clear the role of B7-H4 signal in T cell mediating immune response. Correlation of B7-H4+TIDC with clinical stage of lung carcinomas will be analyzed. Blockade of B7-H4 in vivo by specific monoclonal antibodies will be used to design to immunotherapy for lung carcinomas. Our data would indicate that TAM may condition local DCs to become dysfunctional in the phenotype and reaction to some inflammatory cytokine, and that the high expression of B7-H4 may contribute to the tumor infiltrated DCs to mediate immune escape.

树突状细胞（DC）是机体功能最为强大的抗原提呈细胞，肿瘤浸润树突状细胞（TIDC）因其功能缺陷介导了免疫逃逸。本课题组前期研究发现了TIDC高表达负性协同刺激分子B7-H4，可抑制T细胞应答，且炎症因子TNF-a不能有效介导TIDC的成熟；还发现了肿瘤相关巨噬细胞（TAM）可通过分泌一些细胞因子对肿瘤微环境中B7-H4分子表达发挥调节作用。鉴此，本项目以肺癌作为模型，深入研究TIDC高表达B7-H4分子的分子机理及其意义，探讨TAM对TIDC表达B7-H4分子的调节作用及其机制，观察B7-H4信号对不同T细胞亚群功能的影响及其涉及的相关信号途径，分析B7-H4阳性TIDC在癌组织中表达的临床意义；在此基础上开展干预B7-H4信号的免疫治疗。通过以上的研究可了解TIDC介导免疫逃逸的新机制，并进一步认识B7-H4分子的功能，也将揭示肿瘤微环境中相关免疫细胞的相互作用参与了肿瘤的发展。

目前，有关负性协同刺激分子和肿瘤的关系是免疫学研究的热点，也是肿瘤免疫治疗的发展方向。解析肿瘤微环境中的关键分子和细胞作用机制，对寻找新的治疗靶点具有重要意义。本项目对肺癌肿瘤环境中负性协同刺激分子B7-H4如何介导免疫逃逸、对肺癌细胞生物学行为影响以及免疫治疗作用作了系统研究，结果如下：1、肺癌细胞中B7-H4存在不同部位的表达，发挥不同作用，胞膜上B7-H4分子主要发挥T细胞免疫调节作用，其表达受肿瘤相关巨噬细胞的调控，胞浆中B7-H4分子可直接对肺癌细胞的侵袭和转移能力发挥影响，胞核中B7-H4分子表达的出现和肺癌的分化过程有关，即在早期肺癌中较少出现胞核B7-H4的表达，而在恶性程度高的晚期肺癌细胞则大量出现B7-H4分子的表达。2、进一步研究了B7-H4阳性巨噬细胞的功能，这群细胞参与构建了肺腺癌胸膜预转移小生境的形成，在转移淋巴结中也检测到这群细胞，这群细胞可促进肺癌细胞的EMT过程，据此认为这群细胞促进了肺癌的转移。3、建立了恶性胸腔积液模型，揭示了局部接种B7-H4单抗对胸水具有良好的治疗作用。4、体内体外实验证实敲弱B7-H4抑制细胞的增值、侵袭和迁移能力，细胞生长停滞于G0/G1期，并增加细胞凋亡。5、基于B7-H4基础上还进行了拓展研究，取得了以下实验结果：①EGFR-TKI可以通过下调IL-6的分泌来阻断JAK/STAT3信号通路，进而调控STAT3表达，最终抑制PD-L1的表达，并通过PD-L1表达变化影响细胞增殖功能。②胸水中肿瘤相关巨噬细胞通过分泌IFN-γ上调A549细胞膜上PD-L1的表达，该过程需要PI3K/AKT、JAK/STAT3信号通路的参与。③因为miRNA为当前国内外的研究热点，本项目增加了对肺癌组织中有关于miRNA基因芯片分析。结果得出： miR-205在癌组织中高表达，可通过抑制Smad4的表达促进癌细胞增殖；miR-486-5p及miR-30a-5p在癌组织中低表达可抑制细胞增殖，实验证明了miR-30a-5p可通过抑制CD73的表达促进癌细胞增殖，且通过RNA-seq检测及数据分析，发现CD274与CD73存在基因表达相关性。上述研究成果对共刺激分子的生物学功能乃至肺癌的发生发展机制有了新的认识，不仅对以B7-H4分子为靶点的肺癌免疫治疗具有推动作用，还为肺癌的靶向治疗和免疫治疗相结合提供了实验依据和理论基础。