针对CD4膜蛋白的CAR-T细胞抗T细胞淋巴瘤的活性研究

An experimental study of CD4 targeted chimeric antigen receptor modified T cell with antilymphoma activity

【摘要】目的探讨针对CD4膜蛋白的CAR—T细胞对CD4＋T细胞淋巴瘤细胞的靶向特异性杀伤作用。方法采用重组DNA技术构建含4．1BB共刺激分子的第二代针对CD4的CAR慢病毒载体，应用293T细胞包装慢病毒，采用流式细胞术检测T细胞的转染效率及T细胞亚群动态变化，采用流式细胞术微球法检测培养上清中IFN-γ浓度。结果①构建的慢病毒载体转染激活的T细胞后CAR膜蛋白阳性率达到50．O％～70．0％。T细胞激活后部分CD8＋T细胞弱表达（dim）CD4膜蛋白。T细胞转染针对CD4的CAR慢病毒后CD4＋T细胞、CD8＋CD4dimT细胞逐渐被清除。②CAR-T细胞、对照组T细胞（空载体转染的T细胞）以8：1效靶比分别与CD4＋人T细胞淋巴瘤细胞株KARPAS299细胞共培养24h，杀伤效率分别为（96．9±2．1）％和（11．2±3．1）％，前者明显高于后者（t=7．137，P=0．028）。③CAR-T细胞单独培养，与转染慢病毒载体表达人CD4的K562细胞（K562-CD4细胞）、K562细胞共培养后上清中IFN—γ浓度分别为（1785±268）、（15648±2168）、（1978±354）pg／ml，CAR-T细胞与K562．CD4细胞共培养上清IFN-γ浓度明显高于其他两组，差异有统计学意义（P〈O．01）。结论CD4特异性CAR—T细胞效应细胞免疫表型为CD8＋CD4＋T细胞，在体外具有杀伤正常CD4＋T细胞和CD4＋T细胞淋巴瘤细胞的活性，对于CD4dimT细胞也有较好的清除活性。

Objective To study the specific killing effect of CD4 membrane protein targeted chimeric antigen receptor modified T （CAR-T） cell. Methods The second generation CD4 targeted chimeric antigen receptor containing 4-1BB costimulation domain was insert into lentiviral vector through recombinant DNA technology. Lentivirus was prepared and packaged by 293T cells with four plasmids. Beads activated T ceils were transduced with lentivirus and the transduction efficiency was checked with Protein L and flow cytometry. T cell subsets and IFN-γ concentrations were detected with probe-tagged antibody and cytometric bead assay. Results （1） The transduction efficiency of activated T cells with prepared lentivirus were 50.0%-70.0%. A subset of CD8＋T cell acquired dim expression of CD4 membrane protein after activation. CD4＋T cell and CD8＋CD4dim T cell were gradually killed by CD4 targeted CAR-T post lentivirus transduction. （2）The kill efficacy of CD4 targeted CAR-T cell and control T cell toward KARPAS 299 T cell at an E：T ratio of 8：1 for 24 h was （96.9±2.1）% and （11.2±3.1）%, CAR-T cell has a higher killing efficacy than control T cell （t= 7.137, P= 0.028）. The IFN-γ concentrations in culture supematant of CAR-T cell with K562-CD4 cell, CAR-T cell with K562 cell and CAR-T cell alone were （15 648±2 168）, （1 978±354） and （1 785±268） pg/ml, CAR-T cell cocultured with K562-CD4 cell produced more IFN-7 than the other two controls （P〈0.01）. Conclusions CD4 targeted CAR-T has an immunophenotype of CD8＋CD4 T cell. CD4 targeted CAR-T cell has killing efficacy toward normal CD4＋ T cell and CD4＋T lymphoma cell. CD4 targeted CAR-T cell also has a killing efficacy toward CD4dim target cell.