IL-21单独或联合IL-15/IL-2对G-CSF动员人外周血单个核细胞体外增殖和抗肿瘤活性作用研究

Effects of IL-21 Alone or in Combination with IL-15/IL-2 on Proliferation and Anti-tumor Activity of G-CSF-Mobilized Peripheral Blood Mononuclear Cells In Vitro

本研究探讨IL-21单独或联合IL-15/IL-2对G-CSF动员的人外周血单个核细胞(G-PBMNC)体外增殖和抗瘤活性的影响,评价IL-21及相关细胞因子组合在肿瘤免疫治疗方面的可行性。应用IL-21单独或联合IL-15/IL-2体外培养G-PBMNC,用CCK-8法进行细胞增殖分析;以白血病细胞株K562为靶细胞,用CFSE/PI流式双染法检测其对肿瘤的杀伤作用;用流式细胞术分析免疫细胞表型。结果显示:培养72小时后单独IL-21因子组对靶细胞杀伤活性与IL-2结果相近;当效靶比为25∶1时IL21+IL15/IL21+IL15+IL2组与IL21+IL2组相比杀伤能力有显著增强(p<0.05);效靶比50∶1时,细胞因子联合应用组均显著高于细胞因子单独应用组(p<0.05),以IL21+IL15+IL2诱导效果最佳。复苏冻存的G-PBMNC处理结果与新鲜的G-PBMNC结果基本一致。细胞因子组和对照组相比CD3、CD4和CD8抗原表达有所增加,以IL21+IL15组的CD4、CD3-56+和CD3+56+抗原表达增加最为显著(p<0.05)。结论:IL-21可增强G-PBMNC的体外杀伤活性,联合IL-15可更显著提高G-PBMNC的抗瘤活性。

This study was purposed to investigate the proliferation and antitumor activity of rhG-CSF-mobilized peripheral blood mononuclear cells （G-PBMNCs） activated by interleukin 21 （IL-21） alone or in combination with interleukin 15（ IL-15 ）/interleukin 2 （IL-2） and to evaluate the feasibility and value of tumor immunotherapy with cytokine combinations. G-PBMNCs were activated by IL-21 alone or in combination with IL-15/IL-2 in vitro, and the proliferation of the activated G-PBMNCs was analyzed by CCK-8 assay. The cytotoxicity of the activated G-PBMNC to the K562 cells was studied by the test principle which is based on target cell labeling with 5-（6）-carboxy-fluorescein succinimidyl ester （CFSE） and subsequent DNA-labeling with propidium iodide （PI） for identification of target cells with compromised cell membranes. The phenotypes of the activated G-PBMNCs were assayed by flow cytometry. The results showed that the cytotoxicity of IL-21 group had no difference from which of IL-2 group. When G-PBMNCs were exposed to the combinations of IL21 ＋ IL15/IL21 ＋ IL15 ＋IL2, the cytotoxicity was significantly enhanced at E： T ratio of 25： 1, as compared with combination of IL21 ＋ IL2 （p 〈 0.05 ）. The cytotoxicity of the cytokines combinations was significantly higher than that in cytokine used alone at E： T ratio of 50 ： 1 （p 〈 0. 05 ）. The cryopreservative and resuscitative G-PBMNCs showed the same result with the fresh G-PBMNCs in cytotoxicity test. The proportions of CD3^＋ and CD8^＋ T cells were increased when G-PBMNCs were incubated with cytokines for 72 hours. CD4, CD3^- 56 ^＋ and CD3 ^＋ 56 ^＋ counts were significantly elevated when G-PBMNCs were exposed to IL21 ＋ IL15 （p 〈 0.05 ）. It is concluded that IL-21 alone enhance the antitumor activity of G-PBMNCs, which further strengthens when IL-21 combinated with IL-15.