Gerner Marlene C, Niederstaetter Laura, Ziegler Liesa, Bileck Andrea, Slany Astrid, Janker Lukas, Schmidt Ralf L J, Gerner Christopher, Del Favero Giorgia, Schmetterer Klaus G
Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria.
Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Vienna, Austria.
Front Pharmacol. 2019 Jul 10;10:727. doi: 10.3389/fphar.2019.00727. eCollection 2019.
While genetic traits and epigenetic modifications mainly encode cell type-specific effector functions, the eventual outcome is also prone to modulation by post-transcriptional regulation mechanisms. T cells are a powerful model for the investigation of such modulatory effects, as common precursor cells may differentiate either to helper CD4 T cells or cytotoxic CD8 cells, which elicit distinct functionalities upon TCR-stimulation. Human primary CD4 and CD8 T cells were purified from three individual donors and activated with anti-CD3/CD28 antibodies. Associated proteome alterations were analyzed by high-resolution mass spectrometry using a label-free shotgun approach. Metabolic activation was indicated by upregulation of enzymes related to glycolysis, NADH production, fatty acid synthesis, and uptake as well as amino acid and iron uptake. Besides various inflammatory effector molecules, the mitochondrial proteins CLUH, TFAM, and TOMM34 were found specifically induced in CD4 T cells. Investigation of overrepresented conserved transcription binding sites by the oPOSSUM software suggested interferon type I inducer IRF1 to cause many of the observed proteome alterations in CD4 T cells. RT qPCR demonstrated the specific induction of in CD4 T cells only. While the interferon regulatory factor IRF4 was found induced in both T cell subtypes at protein and mRNA level, IRF9 and the type I interferon-induced proteins IFIT1, IFIT3, and MX1 were only found induced in CD4 T cells. As oxidative stress enhances mitochondrial DNA-dependent type I interferon responses, the present data suggested that mitochondrial activities regulate those cell type-specific signaling pathways. Indeed, we detected mitochondrial superoxide formation predominantly in CD4 T cells FACS analysis with MitoSOX™ and confirmed this observation by live cell imaging with confocal microscopy. As interferon signaling regulates important features such as resistance regarding immune checkpoint blockade therapy, the present data may identify potential new targets for the efficient control of highly relevant immune cell properties.
虽然遗传特征和表观遗传修饰主要编码细胞类型特异性效应功能，但最终结果也容易受到转录后调控机制的调节。T细胞是研究此类调节作用的有力模型，因为常见的前体细胞可分化为辅助性CD4 T细胞或细胞毒性CD8细胞，它们在TCR刺激后引发不同的功能。从三名个体供体中纯化出人原代CD4和CD8 T细胞，并用抗CD3/CD28抗体激活。使用无标记鸟枪法通过高分辨率质谱分析相关蛋白质组变化。糖酵解、NADH产生、脂肪酸合成和摄取以及氨基酸和铁摄取相关酶的上调表明代谢激活。除了各种炎症效应分子外，还发现线粒体蛋白CLUH、TFAM和TOMM34在CD4 T细胞中特异性诱导。通过oPOSSUM软件对过度表达的保守转录结合位点进行研究表明，I型干扰素诱导剂IRF1导致CD4 T细胞中许多观察到的蛋白质组变化。RT qPCR仅在CD4 T细胞中证明了 的特异性诱导。虽然在两种T细胞亚型的蛋白质和mRNA水平上都发现干扰素调节因子IRF4被诱导，但IRF9和I型干扰素诱导蛋白IFIT1、IFIT3和MX1仅在CD4 T细胞中被发现诱导。由于氧化应激增强线粒体DNA依赖性I型干扰素反应，目前的数据表明线粒体活动调节那些细胞类型特异性信号通路。事实上，我们使用MitoSOX™通过FACS分析在CD4 T细胞中主要检测到线粒体超氧化物形成，并用共聚焦显微镜进行活细胞成像证实了这一观察结果。由于干扰素信号调节重要特征，如对免疫检查点阻断疗法的抗性，目前的数据可能会确定有效控制高度相关免疫细胞特性的潜在新靶点。
