Protective Mechanisms Against Pandemic Respiratory Virus (Resource D)

项目D：用多参数流式细胞仪分析细胞激活状态 首页--期刊主要分类--期刊细介绍--期刊题录与期刊详细文摘内容 概述：以前的免疫细胞表型在很大程度上依赖于基于 曲面标记。近二十年来，利用流式细胞术进行的免疫学研究取得了进展 完全依赖于这项直截了当和被接受的技术。然而，细胞激活通常会首先导致 细胞内的变化，通过与信号转导相关的连续蛋白质修饰， 以及后来细胞表面的变化。因此，能够检测和准确测量这些 细胞表型的细胞内变化是一个重要的技术前沿。 通过细胞内FACS分析测量磷酸化分子水平提供了一种 在单个细胞水平上评估细胞激活的定量方法。诺兰实验室已经 最近开发的这种方法使用单抗和多克隆抗体来测量血清中 用流式细胞术检测免疫细胞中关键信号分子的磷酸化形式(1，2)。这 这项技术已被应用于分析成熟T淋巴细胞亚群B细胞的激活状态 NK细胞和巨噬细胞，揭示了细胞之间以前不为人知的异质性(1)和 揭示了来自特定表面受体的信号的贡献以及 由它们的并发连接启动的信号级联的整合(2)。 在这里，我们建议进一步开发这项技术，以便应用于具体的研究 U19应用的项目。用于FACS单细胞激活状态分析的新工具将是 发展起来的。这些工具将用于比较分析流感的激活表型 针对自然甲型流感产生的特异性T细胞(方案1和方案2)和B细胞(方案3) 感染和应对两种形式的三价流感疫苗。试剂和分析方法 在此组件中开发的也将适用于分析NK细胞在应对 流感感染目标(项目4)。通过这种方式，我们将直接与具体的研究项目合作 全面了解流感的免疫反应和疫苗接种方法 这种病原体。 此外，我们将把这项技术的发展扩展到新的检测方法，以探测激活状态 人类外周血中存在的抗原提呈细胞(APC)类型。信号转导途径 与T细胞和B细胞相比，APC的特性稍差，尤其是这些受体 这是APC独有的，但这是目前正在积极调查的一个领域。我们假定APC 信号配置文件将反映宿主的免疫状态(与适当的疫苗接种有关 策略)或病原体的存在，增加了在这里开发快速分析格式的可能性， 或为临床用途进行修改，可能会产生对生物防御努力具有重要价值的信息。我们的龙 学期目标是开发用于免疫复杂群体的快速表型的工具和方法 多维细胞分析，并将基于流式细胞仪的检测数据与临床相关结果联系起来。

PROJECT D: CELL ACTIVATION STATE ANALYSIS BY MULTI-PARAMETER FACS Mellins, E., P.I., Nolan, G., co-Pl. Overview: Previous immune cell phenotyping has relied heavily on subset identification on the basis of surface markers. The last two decades of immunological research using Flow Cytometry has advanced solely on this straightforward and accepted technology. However, cell activation typically results first in intracellular changes, through the sequential protein modification associated with signal transduction, and later in changes at the cell surface. Thus, the ability to detect and accurately measure these intracellular changes in cellular phenotype represents an important technological frontier. Measuring levels of phosphorylated molecules by intracellular FACS analysis provides a quantitative approach to assessing cell activation at the single cell level. The Nolan laboratory has recently developed this approach using monoclonal and polyclonal antibodies to measure levels of phosphorylated forms of key signaling molecules in immune cells by Flow Cytometry (1,2). This technology has been applied to the analysis of activation states of mature T lymphocyte subsets, B cells NK cells and macrophages, revealing previously unappreciated heterogeneity among cells (1) and shedding light on the contributions of signals from particular surface receptors as well as on the integration of signaling cascades initiated by their concurrent ligation (2). Here we propose to further develop this technology for application to the specific research projects of the U19 application. New tools for single cell activation state analysis by FACS will be developed. These tools will be utilized for comparative analysis of the activation phenotypes of influenza specific T cells (project 1 and 2), and B cells (project 3) generated in response to natural influenza A infection and in response to two forms of trivalent influenza immunization. The reagents and assays developed in this component will also be applicable to the analysis of NK cell activation in response to influenza-infected targets (project 4). In this way, we will work directly with the specific research projects to generate a comprehensive view of the immune response to influenza and to vaccine approaches to this pathogen. In addition, we will extend this technology development to new assays to probe activation states of antigen presenting cell (APC) types present in human peripheral blood. Signal transduction pathways in APCs are somewhat less well characterized than in T cells and B cells, particularly for those receptors that are unique to APCs, but this is an area of active current investigation. We postulate that APC signaling profiles will reflect the immune status of the host (with implications for appropriate vaccination strategies) or the presence of a pathogen, raising the possibility that rapid assay formats developed here, or modified for clinical utility, may yield information of significant value to biodefense efforts. Our long term goal is to develop tools and approaches for rapid phenotyping of complex populations of immune cells in multiple dimensions and to link data from FACS-based assays to clinically relevant outcomes.