Multicolor Flow Users Network Study

多色流用户网络研究

• 批准号: 6496162
• 负责人: DAVID M ASMUTH
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6496162
• 关键词: AIDS vaccines; HIV envelope protein gp120; clinical research; cytotoxic T lymphocyte; flow cytometry; helper T lymphocyte; hepatitis vaccine; human tissue; immunologic assay /test; method development

DESCRIPTION: (Provided by Applicant) The identification of rare antigen-specific T cell populations in peripheral blood or tissues following vaccination can be achieved either by increasing the sensitivity of an assay or by reducing the denominator of the measured cell population in which the antigen-specific cells are located. A strategy that reduces this denominator can be achieved by using flow cytometry and choosing multiple surface or intracellular markers to accurately define the T cell subsets of interest. In addition, the definition of effector function of antigen-specific T cells will likely require multiple intracellular and surface markers. Thus, we believe that multiparameter flow cytometry (MFC), which may employ up to 11 phenotypic or functional markers, will be a useful tool for the study of the quantity and function of T cells induced after vaccination. The overall goal of this project is to bring together a working group of investigators to help to develop the tools, reagents and expertise needed to make the use of MFC a reliable and practical approach that can be used in HIV vaccine research. The field has a number of important roadblocks that will be addressed. These include the basic problem that there is no standard hardware configuration of lasers, filters and detectors (PMTs or photomultiplier tubes), limited commercial software for collecting and analyzing the data, and no agreement on either the surface markers, intracellular markers, fluorophores, or combinations of stains that should be used for future assessment of antigen-specific T cell populations. Lastly, the techniques developed to date have not been rigorously applied to vaccine studies, and thus the performance characteristics in settings in which the memory population may be small is unknown. The specific AIMS of this proposal are: 1) To develop a T cell anchored system of phenotyping for the analysis of rare antigen-specific CD4+ and CD8+ populations, and to define the performance characteristics of this system. The variability between individuals, and the inter-assay variability will be determined using both fresh and cryopreserved lymphocytes. 2) To conduct a multicenter trial of our validated panel by sending specimens from chosen vaccinated volunteers to four centers with multiparameter flow capability. We will also determine whether the samples should be stimulated on site, fixed and mailed, or whether they can be mailed, and then stimulated on site. 3) To apply this methodology to determine the CD4 and CD8 T cell responses to vaccination, with a special emphasis given to the Th1/Th2 phenotype of the responding cells. We have begun a series of adjuvant trials using hepatitis B vaccine and gp120. The panel developed in each of the AIMS above will be applied to these studies to determine whether the differences previously seen in antibody outcomes can be determined at the T cell phenotype and antigen-specific level.

描述：（申请人提供）鉴定稀有 外周血或组织中的抗原特异性T细胞群 接种可以通过增加测定的灵敏度或 通过减小测量的细胞群的分母， 定位抗原特异性细胞。一个能降低这个分母的策略 可以通过使用流式细胞术并选择多个表面或 细胞内标记物来准确地定义感兴趣的T细胞亚群。在 此外，抗原特异性T细胞的效应子功能的定义将 可能需要多种细胞内和表面标记。因此我们认为 多参数流式细胞术（MFC）可以使用多达11种表型， 或功能标记，将是一个有用的工具，研究的数量和 接种后诱导的T细胞功能。本项目的总体目标 是召集一个调查工作组， 工具，试剂和专业知识，使MFC的使用是一个可靠的， 这是一种可用于艾滋病毒疫苗研究的实用方法。所述字段具有一 一些重要的障碍将得到解决。其中包括基本的 问题是没有激光器、滤波器和 探测器（光电倍增管或光电倍增管），有限的商业软件， 收集和分析数据，表面上没有达成协议， 标记物、细胞内标记物、荧光团或 应用于抗原特异性T细胞群的未来评估。 最后，迄今为止开发的技术尚未严格应用于 疫苗研究，因此，在设置中的性能特征， 存储器数量可能很小是未知的。 本课题的具体目标是：1）开发T细胞锚定系统 用于分析罕见抗原特异性CD 4+和CD 8 + 人口，并确定该系统的性能特征。的 个体之间的变异性，以及试验间变异性 使用新鲜和冷冻保存的淋巴细胞测定。2)进行 我们的验证面板的多中心试验，通过发送样本从选择 接种疫苗的志愿者到四个具有多参数流动能力的中心。我们 还将确定是否应在现场刺激样品，固定样品， 邮寄，或者是否可以邮寄，然后现场刺激。3)申请 该方法用于确定CD 4和CD 8 T细胞对疫苗接种的应答， 特别强调应答细胞的Th 1/Th 2表型。 我们已经开始了一系列使用B型肝炎疫苗和gp 120的辅助试验。 在上述每一个大西洋、印度洋、地中海和南海区域开发的小组将应用于这些研究 以确定先前在抗体结果中观察到的差异是否可以 可以在T细胞表型和抗原特异性水平上确定。