CD47 Regulation of Leukocyte Integrins during Leukocyte Trafficking

 描述(申请人提供)：炎症是心血管疾病和相关病理生理过程的重要因素，包括中风、高血压、动脉粥样硬化、糖尿病代谢综合征。效应T细胞亚群的激活和募集到组织中是一个高度调控的过程，依赖于T细胞亚群和血管内皮细胞之间的粘附性相互作用。因此，我们的总体目标是更好地了解调节CD4+T细胞亚群募集到免疫介导性炎症部位的机制。根据我们令人兴奋的初步数据和最近发表的文章，我们假设CD47通过调节高亲和力构象的表达在白细胞β1和β2整合素黏附功能中发挥重要作用。由于其对LFA-1和VLA-4整合素黏附功能的影响以及其在免疫细胞动态平衡中的作用，CD47及其配体Sirpα在炎症反应的多个步骤(停滞和迁移)中发挥关键作用。CD47调控T细胞LFA-1和VLA-4黏附功能的分子机制和潜在的结构基础，以及CD47在T效应细胞功能中的作用。我们提出了互补和特征良好的体外细胞培养系统和新的细胞飞行时间质谱仪技术来剖析CD47在人和小鼠免疫细胞中调节整合素功能的生化和分子信号通路。具体地说，Special Aim 1将使用最近发展的单细胞质谱仪(CyTOF质谱仪)和标准生化分析来询问CD47-/-T细胞的细胞内信号通路，以确定导致CD47-/-白细胞整合素活性受损的缺陷。使用CyTOF可以在单个样本中测量多达40个细胞表面分子、细胞内信号分子和介体(细胞因子)，以便在单个细胞水平上提供更多关于细胞表型的信息。具体目标2将讨论在T细胞募集过程中，反式CD47L配体、信号调节蛋白α和信号调节蛋白γ以及分泌的细胞外基质蛋白血栓反应蛋白-1是否影响CD47对LFA-1和VLA4功能的顺式调节。在具体目标3中，我们建议确定CD47中与β2整合素相互作用的残基，这可能为设计新的和创新的治疗靶点(S)提供指导，并确定破坏这些相互作用的生物试剂。这些方法和我们与知名研究人员的合作将对CD47在T细胞运输中整合素激活和黏附功能的调节产生新的见解。

 DESCRIPTION (provided by applicant): Inflammation is a significant contributor to cardiovascular disease and related pathophysiological processes including stroke, hypertension, atherosclerosis, diabetes-metabolic syndrome. The activation and recruitment of effector T cell subsets into tissues is a highly regulated process that is dependent upon adhesive interactions between T cell subsets and the endothelium lining the vascularture. Therefore, our overall goal is to gain a better understanding of the mechanisms that regulate CD4+ T cell subset recruitment to sites of immune-mediated inflammation. Based on our exciting preliminary data and recent publication, we hypothesize that CD47 plays an important role in leukocyte β1 and β2 integrin adhesive functions by regulating the expression of high affinity conformations. By virtue of its effect on LFA-1 and VLA-4 integrin adhesive function and its well-documented role in immune cell homeostasis, CD47 and its ligand SIRPα play a critical role in multiple steps (arrest and migration) in the inflammatory response. The specific aims will determine the molecular mechanisms and underlying structural basis of CD47 regulation of T cell LFA-1 and VLA-4 adhesive function and its role in T effector cell functions. We propose complementary and well-characterized in vitro cell culture systems and novel CyTOF mass spectroscopy technology to dissect the biochemical and molecular signaling pathways used by CD47 in human and murine immune cells to regulate integrin function. Specifically, Specific Aim 1 will employ recently developed single cell mass spectrometry (CyTOF mass spectrometry) and standard biochemical assays to interrogate intracellular signaling pathways in CD47-/- T cells to identify defects that cause impaired integrin activation in CD47-/- leukocytes. Up to 40 cell-surface molecules, intracellular signaling molecules and mediators (cytokines) can be measured in a single sample using CyTOF to provide much more information about cell phenotypes at a single-cell level. Specific Aim 2 will address whether in trans CD47 ligands, Signal Regulatory Protein (SIRP)α, and SIRPγ, and the secreted extracellular matrix protein, thrombospondin-1, affect CD47 in cis regulation of LFA-1 and VLA4 function during T cell recruitment. In Specific Aim 3 we propose to determine the residues in CD47 that interact with β2 integrins, which may provide guidance for designing novel and innovative therapeutic target(s), and identify biological reagents that impair these interactions. These approaches and our collaborations with established investigators will generate novel insights into CD47 regulation of integrin activation and adhesive functions in T cell trafficking.