Functional Imaging of Lymphocyte Motility and Cell Interactions in Lymph Node

描述(申请人提供)：尽管淋巴细胞中信号通路的许多分子细节已经被详细地阐明，但对于免疫细胞如何迁移、相互定位和在其固有组织内传递信号等同样重要的方面，人们知之甚少。因此，迫切需要能够实时观察淋巴器官中单个细胞和分子的技术。我们已经率先使用双光子显微镜来跟踪树突状细胞、T细胞和B细胞在分离的淋巴结和活着的小鼠中的运动和细胞/细胞相互作用。这项关于功能成像的提议有三个相互关联的目标。首先，我们将在基础条件下和免疫反应开始时检查温度依赖性的基本运动特性、组织缺氧的影响以及淋巴结区的区域差异。S1P1受体对淋巴细胞转运的控制将使用特定的药理学工具，并通过直接成像出口步骤来确定淋巴细胞隔离的机制。我们引入量子点作为一种新的方法来跟踪和调制树突状细胞。其次，我们将重点研究淋巴结细胞之间的免疫调节和效应相互作用。我们引入调节性T(Treg)细胞和自然杀伤(NK)细胞作为免疫成像的新的相关细胞，并将我们的方法扩展到包括重组或转移到小鼠体内的人类免疫细胞。第三，我们建议使用[Ca~(2+)]i成像、K~+通道和PI-3-激酶基因敲除小鼠，以及特定的药理学工具来研究调节淋巴结运动和抗原反应的分子信号通路。我们预计，通过该项目获得的技术发展的进步和新的信息将为研究自身免疫性疾病和进一步了解临床使用的免疫抑制剂的机制提供手段。

DESCRIPTION (provided by applicant): Although many molecular details of the signaling pathways in lymphocytes have been elucidated with exquisite detail, less is known about the equally important aspects of how immune cells migrate, locate one another and transmit signals within their native tissues. There is thus a pressing need for techniques allowing real-time observation of single cells and molecules in lymphoid organs. We have pioneered the use of two- photon microscopy to track motility and cell/cell interactions of dendritic cells, T cells and B cells in isolated lymph nodes and in living mice. This proposal for functional imaging has three inter-related Aims. First, we will examine basic motility properties of temperature dependence, effects of tissue hypoxia and regional variations in the lymph node under basal conditions and at the start of an immune response. The control of lymphocyte trafficking by S1P1 receptors will be probed using specific pharmacological tools and by directly imaging the egress step to determine mechanisms of lymphocyte sequestration. We introduce quantum dots as a novel approach to track and modulate dendritic cells. Second, we will focus on immunoregulatory and effector interactions among cells in the lymph node. We introduce regulatory T (Treg) cells and natural killer (NK) cells as new and relevant cells for immuno-imaging, and extend our approach to include human immune cells reconstituted or transferred into mice. Third, we propose to use [Ca2+]i imaging, K+ channel and PI 3-kinase knockout mice, and specific pharmacological tools to investigate molecular signaling pathways that regulate motility and antigen responses in the lymph node. We anticipate that the advances in technology development and new information gained through this project will provide the means to investigate autoimmune diseases and further the mechanistic understanding of clinically used immunosuppressive agents.