Multicolor Flow Users Network Study

描述：(申请人提供)稀有的身份证明 外周血或组织中抗原特异性T细胞群 疫苗接种可以通过提高化验的灵敏度或 通过减少测量的细胞群体的分母，其中 抗原特异性细胞被定位。减少这一分母的战略 可以通过使用流式细胞术和选择多个表面或 细胞内标记物，以准确定义感兴趣的T细胞亚群。在……里面 此外，抗原特异性T细胞效应器功能的定义将 可能需要多个细胞内和表面标记。因此，我们认为 这种多参数流式细胞术(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数量和 免疫后诱导的T细胞功能。这个项目的总体目标是 是召集一个调查工作组，帮助制定 使用MFC所需的工具、试剂和专业知识 可用于艾滋病毒疫苗研究的实用方法。该字段有一个 将解决的一些重要路障。其中包括基本的 没有标准硬件配置的激光器、滤光片和 探测器(PMT或光电倍增管)，有限商业软件，用于 收集和分析数据，在两个表面上都没有达成一致 标记、细胞内标记、荧光团或染色的组合 应用于未来对抗原特异性T细胞群体的评估。 最后，迄今为止开发的技术还没有被严格地应用于 疫苗研究，因此在以下情况下的表现特征 记忆群体可能很小是未知的。 这项建议的具体目标是：1)开发T细胞锚定系统 稀有抗原特异性CD_4~+和CD_8~+的表型分析 并定义这一系统的性能特征。这个 个体之间的可变性，以及检测间的可变性将是 用新鲜和冷冻保存的淋巴细胞测定。2)进行 通过发送SELECTED的样本对我们的验证小组进行多中心试验 将志愿者接种到四个具有多参数流动能力的中心。我们 还将确定样本是否应现场刺激、固定和 邮寄，或者是否可以邮寄，然后在现场刺激。3)申请 这种方法可以确定接种疫苗后的CD4和CD8T细胞反应， 并特别强调反应细胞的Th1/Th2表型。 我们已经开始了一系列使用乙肝疫苗和gp120的佐剂试验。 在上述每个目标中开发的小组将应用于这些研究 为了确定之前在抗体结果中看到的差异是否可以 在T细胞表型和抗原特异性水平上被确定。

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.