[摘要] 目的：探讨γc 家族细胞因子对体外培养T细胞表型的影响，为过继免疫治疗体外细胞制备提供实验依据。方法：采用健康人外周静脉血单个核细胞（PBMC），应用尼龙毛柱法、CD3 磁珠阳性分选法、CD3 磁珠阴性分选法和自然沉降法等4 种方法分选T细胞，比较各种方法获取的T细胞纯度、回收率和细胞增殖。用CD3/CD28 免疫磁珠激活CD3+T细胞，γc 家族细胞因子分为IL-2 组和IL-7、IL-15、IL-21 混合组，比较两组T细胞扩增倍数和表型的差异。结果：CD3 磁珠阴性分选的T细胞纯度显著高于尼龙毛柱分选、CD3 磁珠阳性分选、自然沉降分选[ （94.06±1.07）% vs（86.74±1.06）%、（89.61±1.40）%、（88.48±1.86）%，P<0.05]，自然沉降分选的T 细胞回收率显著高于其余3 种分选法[ （60.29±1.53）% vs（45.03±2.79）%、（20.15±3.41）%、（42.98±2.82）%，P<0.05)，综合比较效果以自然沉降法为最优选择。IL-2 组T 细胞扩增倍数显著高于混合组[ （262.6±143.2）vs（73.0±25.8）倍，P<0.05]。混合组的早期记忆T细胞占总T细胞的比例、Tscm和Tscm-like、Tcm所占比例均显著高于IL-2 组[ （55.6±1.82）% vs（39.6±1.52）%，（16.6±1.82）% vs（9.8±1.30）%，（39.0±1.58）% vs（29.2±1.79）%；均P<0.05]。结论：自然沉降法从PBMC中分选T 细胞具有低成本、高纯度、高回收率的优势。IL-7、IL-15 和IL-21 混合培养有利于早期记忆T细胞的生成，为肿瘤过继免疫治疗的体外细胞制备提供了实验依据。

[Abstract] Objective：To explore the impact of γ-chain (γc) family cytokines (IL-2, IL-7, IL-15, IL-21) on T cell phenotypes in ex vivo culture to provide experimental evidence for ex vivo cell preparation in adoptive immunotherapy. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers; nylon column sorting, CD3+ magnetic beads sorting,CD3- magnetic beads sorting and natural sedimentation were used to sort T cells from PBMCs. The purity, recovery rate and viability of T cells sorted by the above methods were compared. The CD3/CD28 magnetic beads-activated CD3+T cells were cultured in AIMV medium with IL-2 or mixed cytokines (IL-7, IL-15, IL-21). The expansion fold and phenotypes of T cells in ex vivo culture were detected by flow cytometry. Results：The purity of T cells sorted by CD3- magnetic beads sorting was significantly higher than that sorted by nylon column, CD3+ magnetic beads sorting and natural sedimentation ([94.06 ± 1.07]% vs [86.74 ± 1.06]%, [89.61 ± 1.40]%, [88.48 ±1.86]%, P<0.05); The recovery rate of T cells sorted by natural sedimentation was significantly higher than that by other three methods ([60.29±1.53]% vs [45.03±2.79]%, [20.15±3.41]%, [42.98±2.82]%, P<0.05). Comprehensively, the natural sedimentation method is the best option. The ex vivo expansion fold of T cells in IL-2 group was significantly higher than that in mixed group ([262.6±143.2] times vs [73.0±25.8] times, P<0.05). The proportions of early memory T cells, Tscm+Tscm-like and Tcm in the mixed group were significantly higher than those in the IL-2 group ([55.6±1.82]% vs [39.6±1.52]%,[16.6±1.82]% vs [9.8±1.30]%, [39.0±1.58]% vs [29.2±1.79]%; all P <0.05). Conclusion：Natural sedimentation sorting has advantages of low cost, high recovery and purity. Mixed cytokines of IL-7, IL-15 and IL-21 are beneficial for production of early memory T cells. This study provides an experimental data of ex vivo T cell preparation for cancer adoptive immunotherapy.

国家自然科学基金资助项目（No.81372405，No.81772670）