达沙替尼通过调控转录因子Eomes/T-bet增强Vγ9Vδ2T细胞对AML细胞的杀伤功能及机制研究

Vγ9Vδ2T(γδ)T cells were proved to be able to kill a variety of tumor cells, and the mechanism of its anti-tumor effect has attracted many researchers. Eomes/T-bet are important transcription factors involved in the differentiation and development of CD8+ cytotoxic T cells and NK cells, but whether it is involved in the mechanism of γδ T cells remains unclear. It has been reported that both participate in the regulation of γδ T cell activation and secretion of IFN-γ.We previously proved that dasatinib could promote the proliferation of γδ T cells and induce cytotoxicity to AML cells in vitro (published in <leukemia>).Our recent pre-experiment further showed that the expression level of transcription factor Eomes and T-bet has increased during this process.Therefore we hypothesis that dasatinib may enhance γδ T cell proliferation and the cytotoxic effect to AML cells through regulating the expression of Eomes and T-bet. Our study aims to explore the mechanism of how dasatinib regulate the activity of Eomes and T-bet，and upstream and downstream signal transduction pathway in depth by knockdown technology in vitro, as well as humanized NOD/SCID AML mice in vivo experiment. Our study is expected to improve the understanding of the anti-tumor biological behavior of γδ T cells, and also provide a more solid theoretical basis for the guidance of γδ T cells clinical application to cure AML and optimize treatment strategies.

Vγ9Vδ2（γδ）T细胞已证实可杀伤多种肿瘤细胞，有关其肿瘤杀伤机制是近年来研究热点。Eomes和T-bet是参与CD8+T细胞、NK细胞等分化发育和功能调控的重要转录因子，在γδT细胞中作用尚不清楚，有报道可参与γδT细胞活化及IFN- γ分泌的调控。我们前期证明达沙替尼体外诱导促进γδT细胞增殖及对急性髓系白血病（AML）细胞毒作用（《leukemia》），近期预实验发现诱导过程伴随核转录因子Eomes及T-bet上调。由此提出假说，达沙替尼通过调控Eomes/T-bet表达，增强γδT细胞增殖及杀伤AML细胞作用。本研究拟通过体外培养结合knock down技术研究机制，结合人源化AML小鼠模型体内实验，系统阐明达沙替尼对Eomes/T-bet的调控作用及上下游信号转导分子机制，有望完善对γδT细胞抗肿瘤生物学行为的认识，为指导γδT细胞临床应用治疗AML及优化治疗策略提供实验依据

根据实验内容，课题组首先将Vγ9Vδ2T细胞常规诱导培养及达沙替尼诱导培养组，培养12天，并比较常规诱导培养组与达沙替尼诱导培养组的Vγ9Vδ2T细胞Ras，Raf，MEK，ERK的蛋白的表达情况。数据显示，第12天常规诱导培养组与达沙替尼诱导培养组的Vγ9Vδ2T细胞Ras和Raf的蛋白水平无明显差异，而MEK和ERK的蛋白表达水平在达沙替尼诱导培养组中显著升高，且ERK蛋白的上调更为明显。表明达沙替尼可能通过诱导Vγ9Vδ2T细胞的MEK和ERK途径发挥作用，其中ERK通路可能发挥主要作用。课题组进一步研究了达沙替尼增强Vγ9Vδ2T细胞的ERK信号转导通路对转录因子Eomes和T-bet的调控作用。使用ERK抑制剂PD98059阻断Vγ9Vδ2 T细胞（与达沙替尼加药同步）12天，比较阻断与非阻断组 Vγ9Vδ2T细胞中转录因子Eomes和T-bet的转录水平。数据显示，第12天阻断组Vγ9Vδ2T细胞中Eomes的转录水平较非阻断组显著降低。同时，T-bet的mRNA水平在阻断组Vγ9Vδ2T细胞中也明显下调。课题组深入研究了导致患者Vγ9Vδ2T细胞功能失调的机制，结果发现，AML患者的Vγ9Vδ2T细胞在体外呈现较低的IL-21R表达，导致IL-21介导的AML患者Vγ9Vδ2T细胞的扩增效力较低；IL-21激活患者Vγ9Vδ2T细胞中STAT1的能力降低和而诱导Tim-3的能力增加，通过Tim-3阻断可以显着改善IL-21介导的Vγ9Vδ2T细胞增殖以及增加STAT1磷酸化，为AML免疫治疗策略改进提供新的思路和方法，这部分工作已经完成，并已经发表SCI论文。