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

描述（由申请人提供）：该计划的首要目标是了解调节细胞活化的分子机制。通过抗原的特异性识别激活淋巴细胞带来了狭义平衡的益处和风险，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，而其他信号诱导细胞无反应或死亡。具有不同结果的调节过程的分子机制是未知的，并且在很大程度上不确定。该计划包括五个高度互动的项目，涉及六名调查人员，来自大学的五个不同部门。这些研究人员带来了创新的技术和深刻的思想，以承担淋巴细胞活化及其调节的问题。这五个项目涵盖了一系列刺激，线索和结果，导致T细胞活化或无反应的各种状态，从抗原识别的初始事件到转录的调节。这些项目涵盖了从纳米级分子相互作用到整个细胞和动物模型的生物学规模。该计划涉及以下领域：1）TCR空间组织对T细胞应答的影响，2）免疫突触和T细胞无反应性的分子结构改变，3）Sprouty 1作为T细胞活化的新型抑制剂，4）TFII-I对T细胞中钙信号的调节，5）TCR和共刺激信号对NF-κ B的调节。这五个项目支持理解调节细胞活化的分子机制的总体目标。该计划是在一个核心调查小组多年互动的基础上发展起来的，在过去的两年里，新同事加入了这个小组。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协同作用的程度。我们希望了解T细胞对抗原反应的机制。了解这些机制将导致出现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细胞应答的影响（PI [Schneck，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细胞应答至关重要。聚类和模式的变化可以将细胞反应性从低改变为高，甚至改变为无反应。我们的目的是研究T细胞受体聚集和模式的遗传学和生物物理学。遗传筛选将用于识别对活化T细胞上发现的T细胞受体的增强聚类重要的途径。我们将使用shRNA筛选来关注已知影响T细胞受体聚集的途径-糖基化和胆固醇的作用及其与细胞骨架的相互作用。其次，我们将分析在筛选中鉴定的基因在TCR配体、可溶性肽-MHC-Ig复合物与T细胞的结合以及体外和体内T细胞应答中的影响。此外，我们还将设计量子点，将TCR配体，MHC/肽或抗CD 3呈递给初始和活化的T细胞。量子点将用于探测初始和活化T细胞上TCR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活化的CD 4细胞中的TCR组织和SMAC形成。项目1关于TCR聚集的遗传控制的工作将与该项目直接相关。此外，该项目的用于表征TCR组织的纳米探针和方法将用于探测初始或活化的CD 4+细胞上TCR的纳米组织。在项目3中，量子点将用于表征体内诱导无反应性期间TCR聚集的变化。量子点和遗传操作也将与Desiderio博士的项目4合作，用于表征缺乏TFII-I或携带突变形式的蛋白质的细胞和克隆中的T细胞受体状态。在设计用于TCR聚集的遗传控制的选择/筛选测定中，已经与项目5有很强的相互作用。此外，使用特定目标1中开发的技术，我们将分析项目5中鉴定的基因对TCR聚类的影响。 许多免疫反应，例如对寄生虫、肿瘤和某些病毒的反应，往往是迟钝的，因为T细胞对抗原的反应是关闭的，而不是激活和攻击它们的靶点。了解最有可能触发T细胞的T细胞抗原受体的排列，将导致开发操纵T细胞表面以产生强烈的集中反应的方法，或者抑制反应，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.