IL-7/IL-15/IL-21/IL-23体外联合刺激有效促进人CD8+中央记忆性T细胞生成

IL-7/IL-15/IL-21/IL-23 effectively promote the generation of human CD8+central memory T cells in vitro

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。

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.