The role of positive and negative regulation on ligand discrimination by the TCR signaling pathway

摘要--项目2 T淋巴细胞(T细胞)在协调对感染病原体的适应性免疫反应中起着关键作用 以及癌细胞。T细胞表达T细胞抗原受体(TCR)，该受体对MHC- 相关抗原肽(PMHC)来源于癌细胞的病原体或突变的自身蛋白。在TCR上 与这种激动剂pMHC结合后，细胞内的信号转导随之发生，最终导致新的基因转录。 T细胞激活所需的程序。在体内的动态平衡状态下，幼稚T细胞所需的持续时间较短 TCR与自身pMHC结合，产生生存和生存所需的紧张性信号事件 自平衡。然而，这些紧张性信号不会导致细胞激活，因为这会导致 免疫病理学。产生紧张性信号的配体和激动剂之间的半衰期差别并不大。 动态校对被认为是理解细微特异性的概念框架 TCR信号通路区分不同的配体。尽管在理解上取得了很大的进步 膜-近端TCR信号和配体识别，紧张性生存信号如何定性或 与激活信号在数量上的不同尚不完全清楚。根据初步数据，我们假设 与自身pMHC和激动剂pMHC相互作用产生的TCR信号事件不同是因为 反馈调节机制叠加在从近端和远端的动态校对上 TCR。我们建议确定这种反馈调节的潜在机制及其对配体的影响 通过将计算模型、生物化学、小鼠模型和单分子结合起来进行区分 在活细胞和重组系统中的实验。我们将重点关注两个具体目标。在目标1中，我们将定义 负反馈环路以及它们在哪里起作用来调节配基歧视。我们的初步模型 研究预测，TCR近端但不远端的负反馈对抑制很重要 噪音和对自我pMHC的不适当反应。我们将探讨3个近端负波的参与 反馈循环。协同的计算和实验研究有望确定来源、节点 以及这些负反馈循环对配基歧视的影响。在目标2中，我们将确定 LAT凝析油形成的机制及其在正向调节中的作用 反馈。我们的模型研究表明，正反馈调节在受体的远端，但仍然 对TCR-pMHC驻留时间的反应，对于激动剂刺激的强大反应是重要的。我们的 初步实验数据显示，作为TCR信号的关键调节因子，LAT在体内形成离散的凝聚体 对单个TCR-pMHC结合事件的响应。通过将基于统计物理的建模与 实验中，我们将剖析LAT凝结成核的机制及其对TCR-pMHC的依赖 结合停留时间，以及LAT缩合在SOS催化的正反馈调节中的作用 RAS激活。本项目中提议的工作将在项目1、项目3和项目4中进行研究。

ABSTRACT - PROJECT 2 T lymphocytes (T cells) play a key role in orchestrating an adaptive immune response to infectious pathogens as well as cancer cells. T cells express T cell antigen receptors (TCRs) that respond specifically to MHC- associated antigenic peptides (pMHC) derived from pathogens or mutant self-proteins of cancer cells. Upon TCR engagement with such agonist pMHC, intracellular signaling ensues, ultimately leading to new gene transcription programs required for T cell activation. In the homeostatic state in vivo, naïve T cells require shorter duration TCR engagement with self-pMHC to produce tonic signaling events that are required for their survival and homoeostasis. However, these tonic signals do not lead to cell activation as that would result in immunopathology. The half-life differences between ligands that induce tonic signals and agonists are not large. Kinetic proofreading is considered to be the conceptual framework for understanding the fine specificity with which the TCR signaling pathway discriminates between ligands. In spite of much progress in understanding membrane-proximal TCR signaling and ligand discrimination, how the tonic survival signals qualitatively or quantitatively differ from activation signals is not completely known. Based on preliminary data, we hypothesize that TCR signaling events resulting from interactions with self-pMHC and agonist-pMHC differ because of feedback regulatory mechanisms superimposed on kinetic proofreading both proximally and distally from the TCR. We propose to determine the mechanisms underlying such feedback regulation and their impact on ligand discrimination by bringing together computational modeling, biochemistry, mouse models, and single molecule experiments in live cells and reconstituted systems. We will focus on two specific aims. In Aim 1, we will define negative feedback loops and where they act to regulate ligand discrimination. Our preliminary modeling studies have predicted that that negative feedback, proximal but not distal to the TCR, is important for dampening noise and inappropriate responses to self-pMHC. We will explore the involvement of 3 proximal negative feedback loops. Synergistic computational and experimental studies are expected to identify the sources, nodes of action, and impact of these negative feedback loops on ligand discrimination. In Aim 2, we will determine the mechanisms underlying the formation of the LAT condensate and its role in positive regulatory feedback. Our modeling studies suggest that positive feedback regulation distal from the receptor, but still responsive to TCR-pMHC dwell time, is important for a robust response to stimulation by agonists. Our preliminary experimental data reveal that LAT, a key regulator of TCR signaling, forms discrete condensates in response to individual TCR-pMHC binding events. By combining statistical physics-based modeling with experiments, we will dissect the mechanism of LAT condensation nucleation and its dependence on TCR-pMHC binding dwell time, and the role of LAT condensation in mediating positive feedback regulation via SOS-catalyzed Ras activation. The work proposed in this project bridges studies to be conducted in Projects 1, 3 and 4.