外泌体介导DC耐受/T细胞耗竭参与放疗远位效应的免疫负调控机制及干预策略研究

Local radiotherapy combined with immunotherapy can enhance the abscopal effect of cancer treatment, but the efficacy is still not satisfactory. Our previous studies suggest that radiotherapy can increase the release of exosomes from tumors and result in the increase of immunosuppressive molecules such as indoleamine 2, 3-dioxygenase(IDO) and PD-1. The inhibitory molecules of IDO, PD-1/PD-L1 and others play an important role in dendritic cell (DC) tolerance and T cell exhaustion. It is still poor understood if radiotherapy could induce DC tolerance/T cell exhaustion involving in the negative regulation of tumor immunity through exosomes. In this project, the abscopal effect of radiotherapy, the changes of immunosuppressive cells and the inhibitory molecules will be observed in the tumor-bearing mice. treated with local radiotherapy. And the exosomes released from tumor cells and the changes of miRNA expression profile induced by irradiation treatment will be detected. The mechanism of negative immunoregulation of exosomes and their miRNA-targeted immunosuppressive molecules involving in regulating the function of DC and T cell will be analyzed. Further, the exosomes released by miRNA-modified DCs were applied to study the anti-tumor immune mechanisms of DC/T cells in the abscopal effect. The investigation on the mechanism about how the negative immunoregulation was induced by radiotherapy against tumor in this project is helpful for the comprehension of the biological behavior of tumors. It has important theoretical significance and strong clinical application value for radiotherapy combined with other novel immune treatment strategy against metastatic tumors.

局部放疗联合免疫疗法可增强肿瘤治疗的远位效应，然而疗效仍不尽人意。我们的前期研究结果提示放疗可增加肿瘤释放外泌体并引起机体IDO、PD-1等免疫抑制分子升高。IDO，PD-1/PD-L1等抑制分子对树突状细胞（DC）耐受、T细胞耗竭起重要作用。放疗是否可通过外泌体诱导DC耐受/T细胞耗竭参与肿瘤免疫的负调控目前尚未见报道。本项目通过局部放疗处理荷瘤小鼠，观察放疗的远位效应及远位效应小鼠免疫抑制细胞和抑制分子的变化；检测辐射后肿瘤细胞释放的外泌体及其miRNA表达谱的变化，分析外泌体及其miRNA靶向免疫抑制分子调节DC/T细胞免疫功能参与的免疫负调控机制。进而应用miRNA修饰DC后释放的外泌体对远位效应中DC/T细胞抗肿瘤免疫机制进行研究。本研究将深入探讨放疗诱导的免疫负调节机制，对全面认识肿瘤的生物学行为及放疗联合新的免疫干预方案治疗转移性肿瘤具有重要的理论意义和较强的临床应用价值。

放射治疗(RT)是治疗癌症的重要手段，局部放疗联合免疫疗法可增强肿瘤治疗效果并有可能通过诱导远位效应消除肿瘤的转移和复发，然而目前联合治疗疗效仍不尽人意且放疗诱发机体免疫功能变化的机制尚未完全明了。研究发现RT 可以诱导肿瘤细胞释放更多的外泌体，外泌体（exosomes）作为新兴的细胞间沟通媒介，正被越来越多地关注和探究。那么 RT 是否通过外泌体传递信号实现对机体的免疫系统进行调控，这些调控又是如何进行的，是否可以应用体外修饰改变外泌体成分进行抗肿瘤治疗目前还知之甚少。本项目采用X线处理肿瘤细胞，检测肿瘤细胞释放的外泌体对肿瘤生长及免疫细胞生物学功能的影响，并应用生物信息学方法分析了放疗前后患者差异表达miRNA相关调控网络及免疫细胞浸润的相关情况；进而选择了放疗前后有差异表达且与HIF-1ɑ/PD-L1/TIM-3免疫抑制分子有结合位点的mir-212-3p进行深入研究，mir-212-3p的表达对小鼠黑色素瘤细胞B16-F10放疗敏感性以及DC-T细胞抗肿瘤免疫应答的调控作用。研究结果表明：辐射肿瘤细胞衍生的外泌体可以抑制肿瘤细胞的侵袭、迁移，且通过增强促进DC细胞的成熟，改善T细胞耗竭，增强T细胞的活化促进原位和远位肿瘤消退，提示X线处理后肿瘤细胞释放的外泌体具有一定的激活抗肿瘤应答能力；放疗前后患者的miRNA发生了一定的变化，这些变化可以通过基因调控影响患者免疫细胞浸润，并影响患者预后；mir-212-3p通过调控HIF-1ɑ、PD-L1、TIM3的表达，影响了黑色素瘤细胞的增殖、侵袭、迁移和凋亡，并且可以提高小鼠黑色素瘤B16-F10的放疗敏感性，高表达mir-212-3p的肿瘤细胞放疗后释放的外泌体可以促进DC成熟、降低抑制分子PD-L1的表达，从而刺激T细胞分化，改善T细胞耗竭状态，促进细胞毒性T淋巴细胞的抗肿瘤免疫应答。