Antigen receptor inputs: linking structural, molecular, and cellular responses

描述(由申请人提供)：本项目的首要目标是了解调节细胞激活的分子机制。通过对抗原的特定识别来激活淋巴细胞带来的好处和风险之间存在着狭隘的平衡，因此受到严格的监管。我们知道，一些信号用于调节或终止激活，而另一些信号则导致细胞无反应或死亡。不同结果的调控过程背后的分子机制尚不清楚，而且在很大程度上还不确定。该方案由五个高度互动的项目组成，涉及来自大学五个不同系的六名调查人员。这些研究人员带来了创新的技术和精辟的想法来解决淋巴细胞激活及其调节的问题。这五个项目涵盖了导致T细胞激活或无反应的各种状态的一系列刺激、提示和结果，从抗原识别的初始事件到转录调节。这些项目涵盖了从纳米级分子相互作用到全细胞和动物模型的生物规模。该程序涉及以下方面：1)TCR空间组织对T细胞反应的影响，2)改变免疫突触和T细胞无能的分子结构，3)Sprouty 1作为一种新的T细胞激活抑制物，4)TFII-I调节T细胞中的钙信号，5)TCR调节核因子-kB和共刺激信号。这五个项目支持了解调节细胞激活的分子机制的总体目标。该项目是在一个核心调查小组多年互动的基础上发展起来的，这些调查人员在过去两年里加入了新的同事。调查人员之间的互动正在蓬勃发展。从详细的项目说明中可以明显看出小组成员之间的协同程度。我们想了解T细胞对抗原的反应机制。了解这些机制将导致开启或关闭有益或有害的免疫反应的方法。 项目1：TCR空间组织对T细胞反应的影响(PI[Scheck，Jonathan]) 项目1描述(由申请人提供)：越来越多的证据表明，T细胞受体的聚集和空间模式对T细胞反应至关重要。集群和模式的变化可以将细胞的响应性从低到高，甚至改变为无反应。我们的目标是研究T细胞受体聚集和模式的遗传学和生物物理学。基因筛查将被用来识别对激活的T细胞上发现的T细胞受体的增强聚集至关重要的途径。我们将使用shRNA筛选来关注已知的影响T细胞受体聚集的途径-糖基化以及胆固醇的作用及其与细胞骨架的相互作用。其次，我们将分析筛选出的基因在TCR配体、可溶性多肽-MHC-LG复合体与T细胞的结合以及体外和体内T细胞反应中的影响。此外，我们还将设计量子点，将TCR配体、MHC/肽或抗CD3呈现给幼稚和激活的T细胞。这些量子点将被用来探测TCR在初始和激活的T细胞上的横向组织。结合量子点的闪烁将被定量分析，以跟踪受体组织随时间的变化。 与其他项目的协同作用：项目2的一个具体目标是分析激活的CD4细胞中TCR的组织和Smac的形成。项目1‘S在TCR群的遗传控制方面的工作将与该项目直接相关。此外，本项目中用于表征TCR组织的纳米探针和方法将被用于探索TCR在初始或激活的CD4+细胞上的纳米组织。在项目3中，量子点将被用来表征体内无能诱导过程中TCR聚集的变化。量子点和基因操作还将与德西德里奥博士的项目4合作，用来表征缺乏TFII-I或携带突变形式的蛋白质的细胞和克隆的T细胞受体状态。在设计用于TCR聚集性遗传控制的选择/筛选试验方面，已经与项目5进行了很强的交互作用。此外，使用在特定目标1中开发的技术，我们将分析在项目5中确定的基因对TCR聚类的影响。 许多免疫反应，例如对寄生虫、肿瘤和一些病毒的反应，往往是迟钝的，因为T细胞对抗原的反应是关闭的，而不是通过激活和攻击它们的目标。了解最有可能触发T细胞的T细胞抗原受体的排列，将导致开发出操纵T细胞表面以产生强烈的、集中的反应的方法，或者，为了抑制反应，重新排列受体以限制自身免疫反应。

DESCRIPTION (provided by applicant): The overarching goal of this Program is to understand the molecular mechanisms that regulate cell activation. Activation of lymphocytes through specific recognition of antigen poses narrowly balanced benefits and risks, and hence is subject to tight regulation. We know that some signals serve to modulate or terminate activation, while other signals induce cell unresponsiveness or death. The molecular mechanisms underlying the regulatory processes with different outcomes are unknown and largely undefined. The Program consists of five highly interactive projects involving six investigators, from five different departments of the University. These investigators bring innovative technology and incisive ideas to bear on the problem of lymphocyte activation and its regulation. The five projects cover a range of stimuli, cues and outcomes that result in various states of T cell activation or unresponsiveness, from the initial events of antigen recognition to the regulation of transcription. The projects cover a biological scale from nano-scale molecular interactions to whole cell and animal models. The program addresses the following areas: 1) Influence of TCR spatial organization on T cell responses, 2) Altered molecular architecture at the Immunological Synapse and T cell anergy, 3) Sprouty 1 as a novel inhibitor of T cell activation, 4) Regulation of calcium signaling in T cells by TFII-I, 5) Regulation of NF-kB by TCR and costimulatory signaling. These five projects support the overall goal of understanding the molecular mechanisms regulating cell activation. The program has developed out of years of interaction among a core group of investigators who in the last two years have been joined by new colleagues. Interactions between investigators are flourishing. The extent of synergy between group members is apparent from the detailed project descriptions. We want to understand the mechanisms of T cell responses to antigen. Understanding these mechanisms will lead to ways of turning up, or turning off immune responses that are helpful or harmful. PROJECT 1: The Influence of TCR spatial organization on T cell responses (PI [Schneck, Jonathan]) PROJECT 1 DESCRIPTION (provided by applicant): There is growing evidence that the clustering and spatial pattern of T cell receptors is critical for T cell responses. Changes in clustering and pattern can alter cell responsiveness from low to high, or even to unresponsive. We aim to investigate the genetics and the biophysics of T cell receptor clustering and pattern. Genetic screens will be used to identify pathways that are important for the enhanced clustering of T cell receptors found on activated T cells. We will use an shRNA screen to focus on pathways known to affect T cell receptor clustering -glycosylation and the role of cholesterol and its interaction with the cytoskeleton. Second we will analyze the impact of genes identified in the screen in binding of a TCR ligand, soluble peptide-MHC-lg complexes to T cells and in vitro and in vivo T cell responses. In addition we will engineer quantum dots that will present TCR ligands, MHC/peptide or anti-CD3 to naive and activated T cells. The quantum dots will be used to probe the lateral organization of TCR on naive and activated T cells. Blinking of bound quantum dots will be analyzed quantitatively to follow changes in receptor organization with time. Synergies with other projects: A specific aim of Project 2, is to analyze the TCR organization and SMAC formation in activated CD4 cells. Project 1's work on the genetic control of TCR clustering will be directly relevant to that project. Also, the nanoprobes and methods for characterizing TCR organization of this project will be used to probe the nano-organization of TCR on naive or activated CD4+ cells. In Project 3 quantum dots will be used to characterize changes in TCR clustering during induction of anergy in vivo. Quantum dots and genetic manipulation will also be used in collaboration with Dr. Desiderio's Project 4 to characterize T cell receptor states in cells and clones lacking TFII-I or carrying a mutated form of the protein. There has already been strong interaction with Project 5 in designing selection/screening assays for genetic control of TCR clustering. In addition using the techniques developed in Specific Aim 1 we will analyze the effects of the genes identified in Project 5 on TCR clustering. Many immune responses, for example responses to parasites, to tumors and to some viruses, are often blunted because T cells respond to antigen by turning off, rather than by activating and attacking their targets. Understanding the arrangement of T cell antigen receptors that is most likely to trigger T cells, will lead to development of methods to manipulate the T cell surface to produce a strong, focused response, or, to suppress response, rearranging the receptors to limit autoimmune responses.