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）在协调对感染性病原体的适应性免疫反应中发挥关键作用 以及癌细胞。T细胞表达T细胞抗原受体（TCR），其特异性地响应于MHC-1。 来源于病原体或癌细胞突变自身蛋白的相关抗原肽（pMHC）。TCR后 与这样的激动剂pMHC接合，胞内信号转导增强，最终导致新的基因转录 T细胞活化所需的程序。在体内稳态中，幼稚T细胞需要较短的持续时间， TCR与自身pMHC接合以产生其存活所需的紧张性信号传导事件， 体内平衡然而，这些紧张性信号不会导致细胞激活，因为这将导致 免疫病理学诱导紧张性信号的配体和激动剂之间的半衰期差异不大。 动态校对被认为是理解精细特异性的概念框架， TCR信号传导途径区分配体。尽管在理解上取得了很大进展， 膜近端TCR信号传导和配体识别，强直性存活信号如何定性或 与激活信号在数量上的不同并不完全已知。根据初步数据，我们假设 与自身pMHC和激动剂pMHC相互作用引起的TCR信号传导事件不同， 反馈调节机制叠加在近端和远端的动力学校对上， TCR。我们建议确定这种反馈调节的机制及其对配体的影响 通过将计算建模、生物化学、小鼠模型和单分子 活细胞和重组系统的实验。我们将着重于两个具体目标。在目标1中，我们定义 负反馈回路以及它们在哪里起作用以调节配体辨别。我们的初步模型 研究已经预测，TCR近端而非远端的负反馈对于抑制 噪音和对自身pMHC的不适当反应。我们将探讨3个近端阴性 反馈回路协同计算和实验研究预计将确定源，节点 以及这些负反馈回路对配体识别的影响。在目标2中，我们将确定 LAT冷凝物形成的机制及其在正调控中的作用 反馈我们的模型研究表明，正反馈调节远离受体，但仍然 响应于TCR-pMHC停留时间的时间对于对激动剂刺激的稳健响应是重要的。我们 初步的实验数据显示，LAT是TCR信号传导的关键调节因子，在细胞内形成离散的缩合物， 针对个体TCR-pMHC结合事件的应答。通过将基于统计物理的建模与 实验中，我们将剖析LAT冷凝成核的机制及其对TCR-pMHC的依赖性 结合停留时间，以及LAT缩合在通过SOS催化的正反馈调节中的作用 Ras激活。本项目中建议的工作将在项目1、3和4中进行桥梁研究。

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.