Spatiotemporal control of reactive oxygen species in T cells

 说明(申请人提供)：活性氧物种(ROS)在不同的细胞位置产生--通过氧化酶和线粒体的细胞器位置--并在离产生地点只有几纳米的地方发挥作用。关于细胞如何调节活性氧的产生来控制信号转导，人们知之甚少。这一应用的目的是对ROS的局部细胞间调节及其对免疫过程的控制进行详细的定量分析。观察到线粒体在T细胞激活过程中的迁移在很大程度上归因于局部的ATP需求；然而，我们对细胞内活跃的氧化还原信号调节和细胞内过氧化氢的定量建模的发现使我们假设T细胞通过产生ROS来协调线粒体向免疫突触(IS)的移动来控制T细胞激活信号。这项研究的长期理论基础是，通过了解ROS是如何在信号传递过程中被用来氧化蛋白质的，靶向线粒体功能的方法可以适当地应用于增强或抑制自身或抗原肽的呈递。该项目将为细胞相互作用研究提供新的实验平台和分析工具，并结合对T细胞反应性的定量洞察 代谢表型的功能。首先，我们将开发一个高通量的采集平台，用于监测T细胞/抗原提呈细胞的结合。其次，来自氧化刺激的单细胞频率响应特征将与T细胞的激活和IS特征有关。最后，我们 将对TCR连接过程中的区域氧化进行建模，以测试在IS形成过程中发生的线粒体运动在pMHC：TCR界面附近创建氧化带的假设。这些技术将被用来确定局部ROS来源对T细胞信号的贡献，并研究ROS生成和钙之间的时空关系。提出的研究具有创新性，因为它融合了新的建模方法和微流控平台的技术发展，以解决在T细胞信号转导过程中分析局部氧化的挑战。这项工作的结果有望从根本上促进我们对细胞如何利用空间上不同的ROS来源来调节受体启动的信号的理解。这一知识将对最终通过更多与疾病诊断和治疗的生物学相关指标重新定义细胞内氧化产生重大影响。

 DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) are produced in distinct cellular locations - by the organelle location of oxidases and mitochondria - and exert their effects only nanometers from the site of production. Little is known about how cells regulate production of reactive oxygen species to control signal transduction. The objective of this application is a detailed quantitative analysis of the local intercellular regulation of ROS ad its control over immunological processes. Observations of mitochondrial migration during T cell activation have been largely attributed to local ATP demands; however our findings of active redox signal regulation and quantitative modeling of intracellular H2O2 leads us to hypothesize that T cells coordinate mitochondrial movement to the immunological synapse (IS) to control T cell activation signaling via ROS production. The long-term rationale for this research is that by understanding how ROS is used to oxidize proteins during signaling, methods of targeting mitochondrial function can be appropriately applied to augment or suppress self- or antigenic peptide presentation. This project will yield new experimental platforms and analytical tools for cellular interaction studies in conjunction with quantitative insight of T cell responsiveness as a function of metabolic phenotype. First, we will develop a high-throughput acquisition platform for monitoring T cell/antigen-presenting cell engagement. Secondly, single cell frequency response signatures from oxidative stimuli will be related to T cell activation and IS features. Finally, we will model regional oxidation during TCR ligation to test the hypothesis that the mitochondrial movement that occurs during IS formation creates oxidized zones proximal to the pMHC:TCR interface. These technologies will be used to determine the contributions of localized ROS sources to T cell signaling and investigate spatiotemporal relationships between ROS generation and calcium. The proposed research is innovative because it merges the technological developments of new modeling methods and microfluidic platforms to address the challenge of analyzing local oxidation during T cell signaling. The outcomes of this work are expected to fundamentally advance our understanding of how cells use spatially distinct ROS sources to regulate receptor-initiated signaling. This knowledge will have large impact in ultimately redefining intracellular oxidation by more biologically relevant metrics for diagnosis and treatment of diseases.