基因修饰的白血病细胞外泌体靶向结合活化的CD4+T细胞抗白血病免疫效应及其机制的研究

Leukemia cell-derived exosome (LEX) loaded leukemia cell antigens, Although LEX can induce a certain anti-leukemic immune, its efficiency is low. Previous studies shown that LEX-targeted dendritic cells (DC-LEX) can induce stronger anti-leukemia immune than LEX alone. However, production, purifying and storing of DCs remain a barrier and limit its broad clinical utility. Our previous studies have showed that dendritic cell-derived exosome(DEX)-targeted activated CD4+ T cells( T-DEX) could induce more stronger antitumor immune compared to DEX-trageted DC. T cells are easy to be separated and expanded in vitro. Thus, LEX-based T cells vaccines have a potential applicability in immunotherapy for leukemia. However, our recent preliminary study shown that LEX-targeted activated CD4+ T cells (T-LEX)could not induce expected anti-leukemia immune as T-DEX does. Meanwhile, it was found that expression of MHC-II and co-stimulator of LEX was significantly lower than that of DEX, We hypothesis that it may be the key factors for T-LEX could not induce strong anti-leukemia immune like T-DEX. In this study, we aim at creating a gene-modified LEX with expression of high level of MHC-II and costimulatory molecules through their parent leukemia cell transferred with lentiviral vectors carrying IFN-gama and costimulatory molecules. The effects and mechanism of anti-leukemic immunities of this novel LEX-targeted activated CD4+ T cells will be studied in vitro and in animal model. The final goal of this study is to develop a highly immunogeneic, exosome-based T cell vaccines for immunotherapy of leukemia.

白血病细胞外泌体(LEX)能诱导抗白血病免疫，但效率低。LEX靶向结合树突状细胞（DC-LEX）能诱导较LEX更强的抗白血病免疫，但DC细胞培养及储存等过程复杂难以满足临床实际。前期研究显示，DC外泌体（DEX）结合活化的CD4+T细胞（T-DEX）能诱导较其结合的DC（DC-DEX）更强的抗肿瘤免疫，而T细胞易于分离和体外扩增，因此，基于LEX的T细胞疫苗有更重要的临床应用前景。但预实验显示，LEX结合活化的CD4+T细胞（T-LEX）并没有诱导出较强的抗白血病效应。同时发现LEX的MHC-II及共刺激分子的表达明显低于DEX。推测这可能是T-LEX未能有效诱导抗白血病免疫的关键。本项目拟采用基因修饰将IFN-gama和共刺激分子导入白血病细胞，以获得高表达MHC-II及共刺激分子的LEX并对其结合活化的CD4+T细胞的抗白血病效应及机制进行研究，以开发高效的抗白血病疫苗。

背景和目的：前期研究证实活化CD4+T细胞可以摄取DC外泌体（DEXO），DEXO负载p/MHC I和共刺激分子。靶向结合DEXO的CD4+ T细胞可诱导高效的抗肿瘤免疫。我们前期研究同样显示，CD4+ T细胞可摄取白血病细胞外泌体（LEX）。然而，LEX靶向结合的CD4+ T细胞所诱导的抗白血病免疫较弱，主要原因是LEX低表达甚至不表达共刺激分子。为此。本研究拟应用携带共刺激分子基因的慢病毒载体转染白血病细胞，从而获得过表达共刺激分子的白血病细胞，通过分离其上清获得高表达CD80和CD86外泌体（LEX-CD8086），应用LEX-CD8086体外靶向结合CD4+ T细胞，并对其靶向结合的CD4+ T细胞（CD4 TLEX-CD8086）的抗白血病免疫效应进行系统研究。方法：构建CD80和CD86的慢病毒载体，转染L1210白血病细胞株，获得过表达CD80和CD86的L1210白血病细胞株，从其上清液中分离获得LEX-CD8086；利用细胞和分子技术研究生物学特征；研究LEX-CD8086靶向结合CD4+T细胞时效动力学；研究其对CD4+T细胞增殖、分化的影响；应用体外细胞毒实验、小鼠动物模型研究D4 TLEX-CD8086抗白血病免疫效应。结果： （1）成功构建了过表达CD80和CD86的L1210稳转株，并获得高表达共刺激分子的LEX-CD8086；(2) LEX-CD8086能够在体外靶向结合活化的CD4+ T细胞，在体外共培养过程中，LEX-CD8086能促进CD4+ T细胞增殖以及其Th1细胞因子分泌；（3）体外细胞毒研究显示，CD4 TLEX-CD8086能诱导高效的抗原特异性抗白血病免疫效应；（4）动物模型研究显示，CD4+ TLEX-CD8086不仅能诱导高效的预防性抗肿瘤免疫保护，同时也能够发挥较强的治疗性抗肿瘤免疫效应。结论：LEX-CD8086能够在体外有效地靶向结合活化的CD4+ T细胞，CD4 TLEX-CD8086可以高效地诱导肿瘤抗原特异性的抗肿瘤免疫。共刺激分子基因修饰的LEX有望开发成为一种高效的抗白血病疫苗。