红桂木凝集素扩增移植诱导的记忆干性T细胞抗肝癌研究

The memory stem T cells (Tscm) have self-renewal, long survival time, and strong anti-tumor function and are increasingly considered to be ideal cell populations for adoptive immunotherapy in the future. However, the Content of Tscm in the peripheral blood is very low, which hinders its clinical application. Therefore, how to promote Tscm production through external interventions and explore its in vitro large-scale amplification and activity maintenance are key scientific issues to be solved by the project. Our previous study found that mice can produce specific Tscm cells in response to skin xenografts, while the use of Artocarpus lingnanensis lectin (ALL) can effectively induce the expansion of Tscm cells and kill tumor cells.. In this project, we will construct mice allografted with liver cancer cells to obtain hepatoma-specific Tscm cells, establish techniques for in vitro amplification of Tscm using lectin in combination with cytokines, and study amplification mechanisms to create in vitro expanded Tscm cell platforms; We will study the differences in gene expression and epigenetic profiles of Tscm proliferation and differentiation status, and explore the mechanism of Tscm maintenance. In addition, we will study the effect of ALL on Tscm cell proliferation and its anti-liver cancer effect and explore its mechanism of action. . The study of this project will clarify that ALL promotes Tscm cell expansion and its molecular mechanism, and lays a foundation for the future development of ALL in adoptive immunotherapy of Tscm cells.

记忆干性T细胞(Tscm)具有自我更新、存活时间长和极强的抗肿瘤功能而被认为是未来过继性免疫治疗理想的细胞群。但Tscm在体内含量极低阻碍了其临床应用，如何通过外界干预促进Tscm生成，探索其体外大量扩增与活性维持是项目拟解决的关键科学问题。我们前期发现小鼠体内能产生应答异体移植瘤的特异Tscm，采用红桂木凝集素（ALL）能有效体外诱导Tscm扩增及抑制肿瘤细胞。本项目将构建肝癌异种移植小鼠以获取肝癌特异性Tscm细胞，探索ALL体外扩增Tscm最适条件及其影响扩增的信号通路作用机制，创建体外扩增Tscm技术平台；研究Tscm增殖与分化状态下转录表达谱、表观遗传学谱的分子差异，发现与Tscm干性维持关系密切的调节分子；研究ALL体内对Tscm增殖的影响及其抗肝癌效果的作用机制。本项目的开展将明确ALL促进Tscm扩增及其分子机制，为今后开发ALL在Tscm过继性免疫治疗中的应用奠定基础。

本项目围绕红桂木凝集素（ALL）扩增淋巴细胞特别是记忆性干T细胞（Tscm）发挥抗肝癌作用在体内外展开了研究，主要结果如下：①分离纯化了ALL，电泳与质谱分析证实ALL分别由2条133个和117个氨基酸多肽组成的四聚体。②当ALL凝集活性在1×1011~12时，ALL联合IL2明显促进部分细胞亚群增殖，最适浓度为7.5μg/mL ALL +12.5ng/mL IL-2；当ALL凝集活性在1×1013~14时，刺激的最适浓度为0.25到0.5μg/mL ALL +12.5ng/mL IL-2。③体外实验显示ALL与IL2联合较二者单独用药显著扩增人外周血T、Tscm、DC细胞，促进鼠脾Tscm、DC、Tcm和B淋巴细胞的增殖。④ALL促进淋巴细胞增殖的机制与其促进细胞增殖相关因子MCM2、PCNA表达，促进BCL2、抑制caspase8和FAS等凋亡相关因子表达以及抑制淋巴细胞衰竭相关蛋白LAG-3、TIM-3、CTLA4的表达有关。⑤发现 ALL自身对肝癌细胞无抑制作用，但是经ALL活化的淋巴细胞体外对肝癌细胞有杀伤作用，可能与ALL能促进T淋巴细胞活化分子CD69、CD25的表达，分泌释放LDH、穿孔素（PRF1）、IL-2、TNF-α和GrB有关。⑥给荷瘤肝癌小鼠静脉注射ALL活化的淋巴细胞进行过继免疫治疗，肿瘤生长受到抑制。荷瘤小鼠接受过继免疫治疗后，其外周血中T幼稚干细胞（CD44lowCD62Lhigh）、CD95+T干性细胞、T、B、NK细胞比例明显升高， DC、MΦ细胞有一定程度升高；鼠脾脏T幼稚干细胞、CD95+T细胞比例升高；肿瘤组织中T、NK等肿瘤浸润性淋巴细胞比例增高。⑦给荷瘤肝癌小鼠体内直接注射一定剂量的ALL能明显抑制肿瘤生长。发现ALL治疗后，荷瘤小鼠外周血中T、NK、效应记忆性T细胞(Tem)亚群的比例升高；同时，ALL治疗后小鼠肿瘤组织中的Tscm浸润性淋巴细胞比例提高。⑧ALL治疗诱导荷肝癌小鼠分泌IL-2、TNF-α和GrB 细胞因子。本项目研究提示ALL通过调节细胞增殖、凋亡和细胞衰竭相关因子促进外周血幼稚性或记忆性性干T细胞、CD3+T、DC等细胞增殖，促进Tem水平增高，诱导肿瘤淋巴细胞侵润，促进淋巴细胞分泌细胞因子发挥抗肿瘤作用，项目的研究为开发利用ALL在肿瘤免疫治疗的应用奠定良好理论基础。