不同浓度IL-2对体外诱导肽特异性细胞毒性T淋巴细胞培养体系的影响  

The influence of different concentrations of IL-2 on the cultivation system for peptide-specific CTL induction in vitro

作　　者：李桉琪[1,2] 祁元明[3] 周哲骏 高艳锋[3] 张震[1] 张毅[1,3,4] LI Anqi QI Yuanming ZHOU Zhejun GAO Yanfeng ZHANG Zhen ZHANG Yi(Biotherapy Center, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China School of Life Sciences, Zhengzhou University, Zhengzhou 450001, Henan Province, China Department of Oncology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China Engineering and Technology Research Center for Tumor Immunotherapy of Henan Province, Zhengzhou 450052, Henan Province, China)

机构地区：[1]郑州大学第一附属医院生物治疗中心,河南郑州450052 [2]郑州大学第一附属医院肿瘤科,河南郑州450052 [3]郑州大学生命科学学院,河南郑州450001 [4]河南省肿瘤免疫治疗工程技术研究中心,河南郑州450052

出　　处：《中国癌症杂志》2016年第9期756-762,共7页China Oncology

基　　金：国家自然科学基金面上项目(81171986);国家自然科学基金国际(地区)合作与交流项目(81261120402)

摘　　要：背景与目的：细胞毒性T淋巴细胞（cytotoxic T lymphocyte，CTL）介导的特异性细胞免疫在抗肿瘤免疫过程中发挥着主要作用。该研究探讨不同浓度IL-2（50、200和1 000 U/mL）对体外诱导表位肽特异性CTL培养体系培养的细胞亚群比例和功能的影响，以及高剂量IL-2是否会诱导该体系中的调节性T细胞（regulatory cell，Treg）的富集。方法：选取HLA-A2超型的肿瘤患者和健康供者各10例，取外周血分离外周血单核细胞，使用环氧合酶-2（Cox-2）来源的CTL表位肽P321（ILIGETIKI）与不同浓度IL-2体外诱导肽特异性CTL。运用流式细胞仪检测细胞的增殖能力、CD4^＋T淋巴细胞和CD8^＋T淋巴细胞亚群的比例、Treg细胞亚群的比例，以及CD8^＋T淋巴细胞分泌穿孔素（perforin）、颗粒酶B（granzyme-B）、干扰素IFN-γ的能力。使用Elispot实验检测实验组细胞分泌IFN-γ的能力。结果：高浓度的IL-2有利于细胞的增殖。肿瘤患者组的CD4^＋T淋巴细胞比例高于健康供者组，而CD8^＋T淋巴细胞比例较健康供者组低；不同浓度IL-2对CD4^＋T淋巴细胞、CD8^＋T淋巴细胞和Treg细胞亚群比例以及CD8^＋T淋巴细胞分泌穿孔素、颗粒酶B、IFN-γ的能力没有影响。随着IL-2浓度的增高，Elispot实验出现的阳性斑点数越多。结论：不同浓度IL-2条件对体外诱导表位肽特异性CTL培养体系培养出的细胞亚群比例和功能没有影响，在50-1 000 U/mL IL-2浓度范围内，高剂量的IL-2不会诱导该体系中的Treg的富集。但高浓度的IL-2可以提高细胞的增殖能力，并且促进细胞分泌IFN-γ，而高浓度的IL-2可以使培养体系中存在的NK细胞或NKT细胞能够非特异性产生IFN-γ，从而对Elispot实验产生干扰。因此，在体外诱导肽特异性CTL时选用50 U/mL的IL-2浓度可以很好地维持T细胞的增殖和存活，并且最大程度地降低对Elispot实验的干扰。Background and purpose： Cytotoxic T lymphocyte （CTL） plays a vital role in the process of antitumor immunology. The aim of this study was to investigate whether changes in concentration of IL-2 （50, 200 and 1 000 U/mL） would affect the sub-population and cytotoxic function of cells cultivated by peptide-specific CTL induction system in vitro and also observe whether using the concentration of IL-2 at a range of 50-1 000 U/mL is beneficial to regulatory cells （Tregs） enrichment. Methods： Peripheral blood from 10 healthy donors and 10 cancer patients that were HLA-A2 positive, were collected in the study. HLA-A2 restricted CTL epitope P321 （ILIGETIKI） derived from COX-2 pulsed with different concentrations of IL-2 were used to induce peptides-specific CTL in vitro. Flow cytometry was performed to analyze the proliferative capability, the proportion of different T-cell subsets, and secretion of perforin, granzyme B and IFN-γ. IFN-γ secretion was assessed by ELISpot assay. Results： High concentration of IL-2 increased the proliferative activity. The percentage of CD4^＋ T cells of cancer patient group was significantly higher than that of healthy donor group, while the percentage of CD8^＋ T cells of cancer patient group was significantly lower than that of healthy donor group. And there was no significant difference in the percentages of CD4^＋ T cells, CD8^＋ T cells and Tregs among groups with different IL-2 concentrations. No difference was seen in cytokine （perforin, granzyme B, IFN-γ） secretion capacity of CD8^＋ T cells. ELISpot study revealed that high-dose IL-2 resulted in the increasing of IFN-γ secretion. Conclusion： The sub-population and the function of cells cultured by peptide-specific CTL induction system in vitro are not affected by different concentrations of IL-2. Furthermore, high concentrations of IL-2 （50-1 000 U/mL） do not provide the enrichment for Tregs. Higher concentration of IL-2 is likely to cause high secretion of IFN-γ in ELISpot assay. In order

关 键 词：IL-2 细胞毒性T淋巴细胞 多肽疫苗 调节性T细胞 细胞毒活性 

分 类 号：R73-35[医药卫生—肿瘤]