Probing the mechanistic basis for T cell fate decisions (R01)

探讨T细胞命运决定的机制基础（R01）

基本信息

批准号：
8702920
负责人：
Michael S Kuhns
金额：
$ 37.88万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-10 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8702920
关键词：
Address Antigen-Presenting Cells Antigens Architecture Attenuated Autoantigens Biochemical Biological Assay Biological Models CD28 gene CD3 Antigens CD4 Positive T Lymphocytes CD80 gene Cancer Vaccines Cell division Cells Cellular biology Complex Data Development Dimerization Disabled Persons Energy Transfer Enzymes Event Fluorescence Microscopy Goals Health Human Image Immune Immune response Immunity Immunologic Surveillance In Vitro Individual Infection Knowledge Life Ligands Macromolecular Complexes Mediating Membrane Memory Molecular Molecular Biology Techniques Molecular Target Peptide/MHC Complex Phosphotransferases Positioning Attribute Process Publishing Reagent Receptor Signaling Recruitment Activity Reporting Side Signal Transduction Structure Sum Surface Surface Antigens Surveys System T-Cell Development T-Cell Receptor T-Lymphocyte Tertiary Protein Structure Testing Transplantation Vaccines Work base biophysical properties cytokine design extracellular handicapping condition in vivo insight mouse model mutant novel pathogen prevent research study response sensor spatial relationship

项目摘要

DESCRIPTION (provided by applicant): The TCR-CD3 complex, CD4, and CD28 are vital checkpoint molecules that allow T cells to survey for antigens and activation induced molecules on the surfaces of antigen presenting cells (APCs). They then transfer this antigen- and APC-specific information to the T cell's intracellular signaling apparatus. Ultimately, this informatio directs the T cell fate decisions that drive development, activation, differentiation, and the execution of effector functions. The fundamental importance of these molecules to immune surveillance and human health has resulted in several studies regarding their structure and function over the past 25+ years. As a result, much is now known about their individual structures, their interactions with their respective ligands in isolation, and the signaling cascads that result from these interactions. But, we have yet to determine how they fit and work together as components of the molecular machinery that drives T cell fate decisions and this has prevented us from understanding how this molecular machinery, as a whole, executes its functions. Further, this lack of knowledge has handicapped our ability to strategically target this molecular machinery with reagents designed to either enhance responses to vaccines, tumor, or pathogens, or to attenuate responses to transplants or auto-antigens. Our goal is to deconstruct, understand, and ultimately manipulate the form and function of this complex macromolecular machine. To this end, we will: 1) determine how antigen-specific information is relayed across the membrane by the TCR to the intracellular signaling domains of the CD3 subunits; 2) determine how these signaling subunits are positioned in close proximity with the enzymes that modify them; and 3) determine how the surfaces that stabilize the architecture of this higher order machinery influence CD4+ T cell fate decisions in vivo. We have developed a novel experimental platform that combines classic biochemical and molecular biology techniques with modern polycistronic retroviral systems, kinase- based dimerization assays, and live cell fluorescent video imaging (including the use of total internal reflection fluorescence microscopy (TIRFM) and Forster Resonance Energy Transfer (FRET)), to take a highly controlled reductionist approach to addressing Aim 1 and 2. In addition, we are building novel mouse model systems to accomplish Aim 3. Specifically we will study the surfaces that we have already identified as mediating TCR- CD3 complex stability and TCR dimerization to determine how these interactions, which are at the core of the higher-order macromolecular machinery that transfers pMHC-specific information from the outside to the inside of a cell, influence T cell fate decisions in vivo. Altogether, these experiments will yield important insights into the molecular mechanisms that underlie CD4+ T cell fate decisions and identify potential targets for the development of translational immune-modulating reagents.

描述（由申请人提供）：TCR-CD3复合物，CD4和CD28是重要的检查点分子，可让T细胞调查抗原和激活诱导抗原存在细胞表面上的分子（APC）。然后，他们将此抗原和APC特异性信息传递到T细胞的细胞内信号传导设备。最终，此信息将指导T细胞命运的决策，这些决策推动了效应子功能的开发，激活，分化和执行。这些分子对免疫监测和人类健康的基本重要性导致了过去25年以上的几项有关其结构和功能的研究。结果，现在已经知道了他们的各个结构，与各自的配体的相互作用，以及这些相互作用导致的信号传导cascads。但是，我们尚未确定它们如何作为分子机械的组成部分的拟合和合作，从而驱动T细胞命运决策，这使我们无法理解该分子机械（整体上）如何执行其功能。此外，这种缺乏知识使我们有能力从战略上瞄准这一点具有旨在增强对疫苗，肿瘤或病原体的反应的试剂的分子机械，或者减弱对移植或自动抗原的反应。我们的目标是解构，理解和最终操纵这款复杂的大分子机器的形式和功能。为此，我们将：1）确定如何通过TCR在整个膜上中继到CD3亚基的细胞内信号传导结构域的抗原特异性信息； 2）确定如何将这些信号亚基定位在与修改它们的酶紧邻的位置； 3）确定稳定这种高阶机械结构的表面如何影响体内CD4+ T细胞命运的决策。我们已经开发了一个新型的实验平台，该平台将经典的生物化学和分子生物学技术与现代多种逆转录病毒系统，基于激酶的二聚化测定和实时细胞荧光视频成像（包括使用总内部反射荧光显微镜（TIRFM）（TIRFM）和Forster Conconcons Encors（FRET）以及高度对方法进行重新构建，还可以重新构建，将其除外。实现目标3的新型鼠标模型系统。具体而言，我们将研究我们已经确定的表面介导TCR- CD3复杂稳定性和TCR二聚化，以确定这些相互作用是如何在高阶的大分子机械上核心，这些相互作用的核心是传输的pmhc特异性信息，从体内决定。总的来说，这些实验将对CD4+ T细胞命运决定的分子机制产生重要的见解，并确定潜在的靶标，以开发转化免疫调节试剂。