谷氨酰胺缺乏诱导CD8^(+)T细胞铁死亡促进肿瘤生长

Glutamine deficiency promotes tumor growth by inducing ferroptosis in CD8^(+)T cells

目的探究谷氨酰胺(glutamine,Gln)对CD8^(+)T细胞抗肿瘤免疫反应的影响及机制。方法TCGA数据库分析肿瘤Gln代谢与浸润CD8^(+)T细胞数量及功能的关系。利用CRISPR/Cas9敲低MC38细胞GLS表达,构建小鼠肿瘤模型。流式细胞术检测肿瘤增殖和凋亡,分析肿瘤浸润免疫细胞数量和功能。用Gln缺乏的培养基、完全培养基或添加GSH、RSL3的培养基分别处理CD8^(+)T细胞,检测GSH含量,细胞凋亡,GPX4和Lipid-ROS及效应功能蛋白表达,并进行RNA-seq分析。结果肿瘤Gln代谢与肿瘤浸润CD8^(+)T细胞数量和功能负相关。敲低MC38细胞GLS表达抑制了C57BL/6肿瘤的生长及Ki67的表达,促进了Caspase3表达,提高了肿瘤浸润免疫细胞的数量,抑制了肿瘤浸润CD8^(+)T细胞PD-1、TIM-3和LAG-3表达,并提高了CD8^(+)T细胞CD137、CD107a、IFN-γ及TNF-α表达。RNA-seq结果显示,Gln缺乏后CD8^(+)T细胞铁死亡相关基因TFRC、HMOX1、CYBB、SLC7A11表达上调。Gln缺乏后CD8^(+)T细胞死亡增多、CD137、CD107a、IFN-γ、GSH和GPX4表达下调,Lipid-ROS表达上调;补充GSH后CD8^(+)T细胞GPX4上调,Lipid-ROS下调,CD8^(+)T细胞分泌的IFN-γ增多。结论Gln缺乏诱导CD8^(+)T细胞铁死亡抑制其效应功能。

This study was performed to explore the impact of glutamine(Gln)on the anti-tumor immune response of CD8^(+)T cells and its mechanism.TCGA database was used to analysis the relationship between tumor Gln metabolism and the quantity and functionality of infiltrating CD8^(+)T cells.CRISPR/Cas9 was employed to knock down GLS expression in mouse MC38 cells,and a mouse tumor model was established.Flow cytometry was conducted to assess tumor proliferation,apoptosis,and the quantity and functionality of tumor-infiltrating immune cells.Lymphocytes isolated from health individuals were treated with Gln-deficient media,complete media or media supplemented with GSH,RSL3 in vitro.Then the apoptosis,the expression levels of GPX4,Lipid-ROS,and effector function protein of CD8^(+)T cells were detected by flow cytometry.Furthermore,RNA-seq was performed to analyze the differential gene expression on the Gln-depleted CD8^(+)T cells.Data showed that tumor Gln metabolism was inversely associated with the quantity and functionality of tumor-infiltrating CD8^(+)T cells.Low expression of GLS in MC38 cells could inhibit C57BL/6 tumor growth,decrease Ki-67 expression,promote casepase-3 expression,increase the amount of tumor-infiltrating immune cells,suppress PD-1,TIM-3,and LAG-3 expression,and enhance CD137,CD107a,IFN-γ and TNF-α expression in tumor-infiltrating CD8^(+)T cells.RNA-seq results indicated an upregulation of ferroptosis genes TFRC,HMOX1,CYBB and SLC7A11 in CD8^(+)T cells following glutamine deficiency.Gln deficiency led to lower CD137,CD107a,IFN-γ,GSH,GPX4 expression,increased Lipid-ROS level,and caused cell death in CD8^(+)T cells.Supplementation of GSH upregulated GPX4 expression,downregulated Lipid-ROS level,and increased IFN-γ secretion in CD8^(+)T cells.In conclusion,Gln deficiency inhibits the effector function of CD8^(+)T cells by inducing ferroptosis,and promotes tumor growth.