食管癌组织中IDO1的表达及犬尿酸对间皮素CAR-T细胞功能的影响

Expression of IDO1 and effects of kynurenine on the function of mesothelin-CAR-T cells

目的:探讨食管癌组织中吲哚加双氧酶1(IDO1)的表达,及其代谢产物犬尿酸对间皮素嵌合抗原受体T细胞(CAR-T)功能的影响。方法:利用癌症基因组图谱(TCGA)数据库中数据分析食管癌组织中IDO1的表达对患者生存的影响;采用密度梯度离心法获得健康供者外周血单个核细胞,并用细胞免疫磁珠分选出CD8+T细胞;采用病毒感染的方法制备间皮素CAR-T细胞,用200μmol/L犬尿酸处理48 h后,采用流式细胞术检测细胞表面标志物的表达和细胞内因子分泌的水平,检测其对KYSE510细胞的杀伤能力。以未用犬尿酸处理的间皮素CAR-T细胞作对照。结果:高表达IDO1的食管癌患者预后较差。与对照组相比,犬尿酸处理组间皮素CAR-T细胞表面抑制性分子PD-1表达上调(P <0. 05),而激活性分子CD28表达下降(P <0. 05),效应因子IFN-γ和IL-2分泌减少(P <0. 05);与KYSE510细胞共孵育后,间皮素CAR-T细胞CD107a表达降低,杀伤能力下降(P <0. 05)。结论:犬尿酸可以抑制间皮素CAR-T细胞的功能。

Aim:To investigate the expression of IDO1 and the effects of IDO1 metabolite kynurenine on the function of mesothelin-CAR-T cells.Methods:The expression of IDO1 in esophagus cancer and its effects on patients′survival were analyzed using TCGA database.The peripheral blood mononuclear cells of healthy donors were isolated by density gradient centrifugation,and then the CD8^+T cells were sorted using immunomagnetic beads.Mesothelin-CAR-T cells were prepared by virus infection.After being treated by 200μmol/L kynurenine for 48 hours,the surface markers and cytokine production of mesothelin-CAR-T cells were determined by flow cytometry.Mesothelin-CAR-T cells were co-incubated with esophagus cancer KYSE510 cells,and flow cytometry was used to detect the effects of kynurenine on the killing function of mesothelin-CAR-T cells.The cells not treated were the control.Results:Esophagus cancer patients with overexpressed IDO1 had a poor prognosis.Compared with control group,kynurenine could induce the up-regulation of the immune checkpoint PD-1 on mesothelin-CAR-T cells(P<0.05),and down-regulation expression of activated marker CD28(P<0.05).Moreover,kynurenine could also inhibit the cytokines IFN-γand IL-2 secretion of mesothelin-CAR-T cells(P<0.05).After co-incubation with tumor cells,kynurenine inhibited the killing function of mesothelin-CAR-T cells(P<0.05).Conclusion:Kynurenine can inhibit the function of mesothelin-CAR-T cells.