白血病微泡在T细胞耗竭中的作用及机制研究

Adoptive T cellular therapy, especially chimeric antigen receptor T cell (CAR-T), is promising in the therapy of leukemia and other cancers. However, in the context of chronic antigen exposure in chronic viral infections and cancer, T cells could become exhausted/dysfunctional. These exhausted T cells exhibit defective proliferative capacities and cytokine production, but are not totally inert and may exert lytic functions. Importantly, exhausted T cells upregulate multiple inhibitory receptors/immune checkpoints, such as CTLA-4 and PD-1. Immune checkpoint blockades with CTLA-4 and/or PD-1 mAbs successfully reinvigorate tumor-infiltrating T lymphocytes and provide persistent clinical benefits to a large number of patients with advanced cancer. However, multiple publications have demonstrated that T cell exhaustion could not be solved by PD-1 inhibitor once for all. A latest paper published in Science demonstrated that after blockade of PD-1, re-invigorated T cell became re-exhausted if antigen remained high, and failed to become antigen clearance. Epigenetic fate inflexibility may limit T cell re-invigorated. . The discovery that cancer cells generate large membrane-enclosed packets of epigenetic information, known as microvesicles (MVs), that can be transferred to other cells and influence their behavior. This is largely because, in addition to growth factors and cytokines, MVs contain a variety of components from their parental cells, particularly RNA transcripts and micro-RNAs. We found that MV derived from leukemia cells could induce T cell exhaustion in vitro. The aim of this work was to demonstrate that MV could epigenetic silence T cell activation pathways such as NF-κB via horizontal transferring miRNAs.We will further confirm T cell exhaustion could be induced by different MVs in this subject. Besides, the mechanism of T cell dysfunction induced by MVs could be explained as down regulated NF-κB by miR-146b within the leukemia MV. The success of this project will clarify that leukemia cells derived MV is a key regulatory factor of T cell exhasution. Not only can MV be expected to become a new indicator but a new interference target which provides guidance for the individual patient treatment. As our understanding of the mechanisms supporting tumor-induced T-cell dysfunction improves based upon preclinical and clinical studies, we expect that novel mechanism discovery will emerge to improve the clinical outcome of patients with advanced cancers.

T细胞耗竭是肿瘤免疫逃逸的重要机制，也是免疫细胞治疗的桎梏所在。目前靶向免疫检查点(PD-1等)并不能完全激活耗竭的T细胞，其根源在于未能重塑T细胞内被表观沉默的活化信号。微泡(MV)是一种在细胞间传递表观因子的新型交流方式，申请人前期发现白血病来源MV能够诱导T细胞耗竭，结合诱导前后T细胞的表达谱测序结果分析，我们推测：白血病MV能够通过传递miRNA等表观遗传学因子沉默T细胞活化通路从而诱导耗竭。本课题拟通过体内外实验进一步验证MV能够诱导不同亚型T细胞以及CAR-T细胞耗竭；重点证明MV诱导耗竭的机制在于通过传递miR-146b等因子，沉默T细胞内NF-kappaB等活化相关通路。本课题的完成，将有助于发现肿瘤免疫逃逸的全新机制；肿瘤MV的数目及内容物有望成为评判T细胞耗竭重要指标，其本身极有可能成为重要的干预靶点；同时为增强CAR-T、PD-1等治疗手段的疗效提供重要的策略选择。

T细胞耗竭是目前肿瘤免疫细胞治疗的桎梏所在。靶向免疫检查点(PD-1等)的治疗策略响应率较低，不能完全激活耗竭的T细胞，其根源在于未能重塑T细胞内被表观沉默的活化信号。微泡(MV)是一种在细胞间传递表观调控因子的新型交流方式，申请人前期发现白血病来源MV能够诱导T细胞耗竭，本课题在前期工作基础上发现，MV诱导正常外周血单个核细胞(PBMC)恶变时间在14天左右，但是在去除一部分T细胞后细胞恶变时间缩短，而增加T细胞数量恶变时间不变；PD-1等分子检测结果提示MV在诱导正常PBMC过程出现了T细胞耗竭。经MV处理后，T细胞在增殖、活化、细胞因子分泌等方面，均出现先升高再降低的趋势；PCR显示细胞因子IL-2、IFN-γ水平与上清中细胞因子相同趋势，而PD-1、Tim-3、CTLA-4等水平在各类MV诱导后呈现持续升高的趋势。在小鼠模型中，MV注射后T细胞表面PD-1、Tim-3的表达较对照组明显升高；且小鼠脾脏明显增大；脾脏及肝脏PCR提示处理组PD-1、Tim-3、CTLA-4和LAG-3表达水平升高；而注射MV的小鼠停止注射后，流式检测发现T细胞表面PD-1、Tim-3表达水平较前降低，且脾脏回缩。机制方面，MV与T细胞共培养72h后，T细胞内BCR-ABL1、miR-182,、miR-92a表达明显升高，共聚焦显微镜观察发现MV与T细胞发生融合；转录组测序发现，T细胞发生耗竭前后涉及细胞粘附(CXCL16、CXCL5)、炎症活化(NF-κB、NFAT 等)、脂质代谢(LGALS1)等大量基因改变，体外实验证明MV诱导后T细胞中NF-KB信号活性下降。本课题的完成，有助于解析肿瘤细胞诱导T细胞耗竭的全新机制；肿瘤MV的数目及内容物有望成为评判T细胞耗竭重要指标，其本身极有可能成为重要的干预靶点，为增强CAR-T，PD-1等免疫治疗手段疗效提供重要的策略选择。同时，申请人在上述工作基础上探讨了已有CAR-T细胞及新型CAR-T的构建工作。本项目共形成SCI论文6篇，其中大于5分3篇，包括Leukemia杂志2篇，均标注受本项目资助。