【摘要】 目的比较不同细胞因子组合联合抗CD3/CD28磁珠体外诱导CD8~+中央记忆性T细胞(Tcm)体生成的作用,为CD8~+ Tcm过继性免疫疗法的临床应用提供实验依据。方法分离健康供者外周血单个核细胞(PBMC),应用免疫磁珠法分离纯化初始CD8~+ T细胞,CD3/CD28抗体偶联磁珠刺激48 h,同时根据细胞因子种类和组合进行分组:白细胞介素2(IL-2)、 IL-7/IL-15、 IL-7/IL-15/IL-21、 IL-7/IL-15/IL-21/IL-23。采用全自动血球计数仪进行细胞计数;5, 6-羧基荧光素二乙酸琥珀酰亚胺酯(CFSE)染色检测不同细胞因子组合诱导CD8~+ T细胞的增殖水平,流式细胞术检测不同细胞因子组合诱导的CD8~+ T细胞免疫记忆表型,细胞凋亡及细胞γ干扰素(IFN-γ)、穿孔素以及颗粒酶B水平,Western blot法检测不同细胞因子组合诱导的CD8~+ T细胞的B细胞淋巴瘤因子2(Bcl2)蛋白表达。结果与其他组合相比,IL-7/IL-15/IL-21/IL-23处理CD8~+ T细胞后,促进T细胞增殖,CD62L~+/CD45RA~-的CD8~+中央记忆性T细胞亚群的表达显著增加。同时,IL-7/IL-15/IL-21/IL-23处理组显著降低细胞的IFN-γ、穿孔素以及颗粒酶B的分泌水平,细胞的凋亡水平也显著降低。结论 IL-7/IL-15/IL-21/IL-23联合刺激有效诱导初始CD8~+ T细胞向CD8~+ Tcm分化,有助于优化过继免疫治疗中CD8~+ Tcm的体外扩增策略。更多还原

【Abstract】 Objective To compare the effect of different cytokine combinations combined with anti-CD3/CD28 beads in vitro inducing the generation of central memory T cell(Tcm). Methods Peripheral blood mononuclear cells(PBMCs) were isolated from healthy donors. Na6 ve CD8~+ T cells were purified using immunomagnetic beads and stimulated with CD3/CD28 antibody for 48 hours. Cells were treated with different cytokine combinations as follows: Interleukin-2(IL-2), IL-7/IL-15, IL-7/IL-15/IL-21, and IL-7/IL-15/IL-21/IL-23. The automatic blood cell counting instrument was used for cell counting. 5, 6-carboxyfluorescein diacetate succinimidyl ester(CFSE)was employed to test the cell proliferation and flow cytometry was adopted to measure the immune memory phenotype, apoptosis and intracellular factor expression of CD8~+ T cells induced by different cytokine combinations. The expression of Bcl-2 was determined by Western blot analysis. Results Unlike other cytokine combinations, IL-7/IL-15/IL-21/IL-23 promoted the proliferation of CD8~+ T cells and significantly increased the expression of CD8~+CD62L~+CD45RA~- central memory T cell subsets. At the same time, IL-7/IL-15/IL-21/IL-23 treatment significantly reduced the secretion levels of IFN-γ, perforin, and granzyme B. The level of cell apoptosis was also significantly decreased. Conclusion The cytokines combination of IL-7/IL-15/IL-21/IL-23 can effectively induce the differentiation of na6 ve CD8~+T cells to CD8~+ Tcm cells in vitro, which provides a new strategy for the generation of human CD8~+ Tcm in immunotherapy.更多还原