添加细胞因子优化人外周血γδ T细胞的体外扩增培养体系  

Optimization of in vitro amplification of peripheral blood γδ T cells with cytokines

作　　者：王淑莉[1] 陈慧[1] 陆必超 张建民[1] 何维[1] 

机构地区：[1]中国医学科学院北京协和医学院基础医学研究所医学分子生物学国家重点实验室,北京100005

出　　处：《基础医学与临床》2017年第6期752-757,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(81673010);国家自然科学基金青年项目(31500725);中国医学科学院基本科研业务项目(2016ZX310180-5)

摘　　要：目的从添加细胞因子角度,优化用于肿瘤过继免疫治疗的γδ T细胞体外扩增培养方案。方法在固相化抗TCRγδ抗体加IL-2扩增人外周血γδ T细胞的体系基础上,添加其他的γ链受体家族细胞因子,包括IL-7、IL-15或IL-21,单独或联合使用、长期使用或短时添加,通过流式细胞术检测γδ T细胞纯度、细胞增殖及细胞毒活性相关分子的表达,计算γδ T细胞扩增效率;乳酸脱氢酶法检测γδ T细胞对人Burkitt's淋巴瘤细胞系Daudi的细胞毒活性。结果单独使用IL-7或IL-21不能有效扩增γδ T细胞,但单独使用IL-15可以使γδ T细胞纯度、扩增效率及细胞毒活性均达到与单独使用IL-2相当的水平;IL-2与IL-15联合使用,可促进γδ T细胞表达CD69,从而显著提高其扩增效率(P<0.05);而IL-2与IL-21联合使用,可通过促进γδ T细胞颗粒酶A的表达,增强其对Daudi细胞的细胞毒活性(P<0.001);IL-2、IL-15和IL-21联合使用可以显著提高γδ T细胞的细胞毒活性,但是会影响其扩增效率;而仅在效应前短时间添加IL-21,同样可以有效增强γδ T细胞对肿瘤细胞的细胞毒活性(P<0.05)。结论在用固相化TCRγδ抗体加IL-2体外扩增培养γδ T细胞体系中,使用IL-15,而在回输效应前短时间添加IL-21,显著提高γδ T细胞扩增效率及对肿瘤细胞的细胞毒活性,此为目前最优化的γδ T细胞体外扩增培养方案。Objective To optimize in vitro amplification of human γδ T cells with cytokines for tumor adoptive im- munotherapy. Methods On the basis of the immobilized anti-TCR γδ T antibody plus 1L-2 system, other γ chain receptor family cytokines, including IL-7, IL-15 and IL-21, were tested to amplify human peripheral blood γδ T cells either alone or in diversity combination. The percentage of γδ T cells was measured by flow cytometry, and the proliferation efficiency of γδ T cells was calculated. The expression of proliferation-or cytotoxicity-related molecules on γδ T cells was examined by flow cytometry in order to explore the relevant mechanisms. The cytotoxicity of γδ T cells to Daudi cells was detected by lactate dehydrogenase. Results IL-15 alone but not IL-7 or IL-21 increases the γδ T cell purity, amplification efficiency and cytotoxicity to reach comparable levels to those of IL-2. IL-2 plus IL-15 up-regulates the expression of CD69 on γδ T cells and significantly increases their amplification efficiency （P 〈0. 05 ）. IL-2 plus IL-21 enhanced the cytotoxicity of γδ T cells against Daudi cells by increasing the expression of granzyme A （P 〈0. 001 ）. The combination of IL-2, IL-15 and IL-21 significantly improves tyrotoxicity of γδ T cells but reduces their amplification efficiency. In addition, when IL-21 was applied for a short time, it also enhanced the cytotoxicity of γδ T cells （P 〈 0. 05）. Conclusions The combination of IL-2 and IL-15 as well as a short time addition of IL-21 is the best cytokine recipe to amplify human peripheral blood γδ T cells in vitro with immobilized anti-TCR γδ T antibody, which can increase both the proliferation efficiency and the cytotoxicity to tumor cells of γδ T cells.

关 键 词：γδ T细胞 体外扩增 白细胞介素-15 白细胞介素-21 

分 类 号：R730.51[医药卫生—肿瘤]