肿瘤诱导的髓系来源抑制细胞中IDO表达相关机制研究

摘要

Objective: To set up an in vitro co-culture system of tumor cells and normal human CD33+ myeloid cells to simulate the induction process of Myeloid-derived suppressor cells (MDSCs)，to observe the expression and immunosuppression of Indoleamine 2, 3-dioxygenase (IDO) in MDSCs, and to explore the related mechanism of IDO expression in MDSCs.Methods: CD33+ cells were collected from healthy donors by a magnetic cell sorting system.CD33+ cells were cultured alone as the control group.CD33 + cells were co-cultured with MDA-MB-231 using Transwell plates as the co-culture group.Two days later, CD33+ cells were harvested to investigate the phenotypes.The expression and activation of IDO and STAT3 mRNA were determined using real-time PCR and Western blot, respectively.MDSCs-induced T cell apoptosis was detected using flow cytometry, and 1-MT was used to investigate the role of IDO in MDSCs.Results: At two days after the co-culture of breast cancer cell line MDA-MB-231 and normal human CD33+ cells, a population of Lin-CD33+CD13+ CD14-CD15-MDSCs appeared, which could induce autoallergic T cell apoptosis and produce high expression of IDO, stat3 and p-STAT3 protein.JAK2-STAT3 inhibitors JSI-124 treated MDSCs showed a lower level of IDO expression.In addition, IDO inhibitors 1-MT significantly reduced the MDSC-induced T cell apoptosis.Conclusion: The co-culture of tumor cells and normal myeloid cells can induce MDSCs with specific phenotypes and immunosuppressive activities.Upregulation of IDO expression resulting from the increase of STAT3 phosphorylation may be one of the major mechanisms of the MDSCs-induced immunosuppression.%目的:建立正常CD33+细胞与肿瘤细胞共培养系统,模拟肿瘤局部髓系来源抑制细胞的诱导过程,观察吲哚胺2,3双加氧酶在MDSCs中表达及其免疫抑制作用,并探讨MDSCs中IDO表达相关机制.方法:免疫磁珠分选健康供者CD33+细胞,体外与乳腺癌细胞系MDA-MB-231共孵育诱导MDSCs,以单独培养CD33+细胞为对照组,培养2天后收获实验组MDSCs及对照组CD33+细胞,流式细胞仪检测细胞表型;实时定量PCR和Western Blotting方法检测IDO和STAT3的表达与活化情况;Annexin V-FITC的方法检测MDSCs对T细胞促凋亡作用,并观察IDO在MDSCs诱导T细胞凋亡中的作用.结果:正常CD33+细胞与MDA-MB-231共孵育2天后,出现一群表型为Lin-CD33+13+CD14-CD15-的MDSCs,体外诱导的MDSCs中IDOmRNA及蛋白表达增加、STAT3蛋白磷酸化增强,经过STAT3磷酸化抑制剂JSI-124预处理后,MDSCs中IDO蛋白表达明显降低并可诱导高百分比T细胞凋亡,经IDO抑制剂1-MT处理后T细胞凋亡显著降低.结论:体外将乳腺癌细胞系与正常CD33+细胞共孵育可以诱导出具有独特表型特征和免疫抑制活性的MDSCs,而STAT3磷酸化导致的IDO表达升高可能是MDSCs抑制T细胞功能的主要机制之一.