Protective Mechanisms Against Pandemic Respiratory Virus (Resource D)

项目D：通过多参数流式细胞仪分析细胞活化状态 Mellins，E.，私家侦探，诺兰，G.，co-Pl. 概述：以前的免疫细胞表型分析严重依赖于基于以下的亚群鉴定： 表面标记。在过去的二十年中，使用流式细胞术的免疫学研究取得了进展 仅仅依靠这种简单明了的技术。然而，细胞活化通常首先导致 通过与信号转导相关的连续蛋白质修饰， 以及随后细胞表面的变化。因此，检测和准确测量这些的能力 细胞表型的细胞内变化代表了重要的技术前沿。 通过细胞内FACS分析测量磷酸化分子的水平提供了一种方法， 在单细胞水平上评估细胞活化的定量方法。诺兰实验室 最近开发了这种方法，使用单克隆和多克隆抗体来测量 通过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 该技术已被应用于分析成熟T淋巴细胞亚群、B细胞 NK细胞和巨噬细胞，揭示了细胞之间以前未被认识到的异质性（1）， 揭示了来自特定表面受体的信号的贡献以及 由它们的同时连接引发的信号级联的整合（2）。 在此，我们建议进一步发展这项技术，将其应用到具体的研究中 U19的应用程序。通过FACS进行单细胞活化状态分析的新工具将是 开发这些工具将用于流感病毒激活表型的比较分析， 特异性T细胞（项目1和2）和响应天然甲型流感产生的B细胞（项目3 感染和对两种形式的三价流感免疫的应答。所述试剂和测定 本部分中开发的方法也适用于分析NK细胞活化， 流感感染目标（项目4）。这样，我们将直接与具体的研究项目 全面了解对流感的免疫反应和疫苗接种方法， 这个病原体。 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 人外周血中存在的抗原呈递细胞（APC）类型。信号转导途径 在APC中的表达比T细胞和B细胞中的表达更少，特别是对于那些受体 这是APC所独有的，但这是目前正在积极研究的一个领域。我们假设APC 信号传导谱将反映宿主的免疫状态（暗示适当的疫苗接种 策略）或病原体的存在，提高了这里开发的快速测定格式的可能性， 或为临床应用而修改，可以产生对生物防御努力具有重要价值的信息。我们漫长 长期目标是开发工具和方法，用于对复杂的免疫细胞群体进行快速表型分析。 细胞在多个维度，并从基于FACS的分析数据链接到临床相关的结果。

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.