FLOW CYTOMETRIC ANALYSIS FOR T CELL PROLIFERATION

描述：(申请人提供) T细胞增殖和刺激指数的计算是其中之一 在小鼠和人身上广泛使用的展示抗原的方法 特定豁免权。我们建议将“第二代”化验定义为 用[~3H]胸腺嘧啶核苷取代传统的淋巴细胞增殖试验 领悟。多参数流式细胞术将用于评估抗原特异性 通过测量胸腺嘧啶核苷类似物BrdU的掺入来检测T细胞的增殖。 除了增殖的T细胞外，表面标志物与 将对增殖细胞进行检查。将检查的表面标记将 包括CO27、CD45RO/RA和CO95。这些都是可以使用的标记， 单独或组合，以区分幼稚和记忆细胞。抗原特异性 BrdU掺入将用于鉴定增殖抗原反应 细胞，这样对其他标记的分析就可以通过 在这些细胞上设置门控。在使用SEB作为刺激的初步研究中， 流式细胞仪检测结果与上述指标呈正相关。 BrdU掺入法扩增CD_4~+T细胞及标准扩增 测定PBMC摄取[~3H]胸腺嘧啶核苷的方法。我们建议扩大使用 流式细胞术BrdU增殖法检测T细胞 对巨细胞病毒、流感和肿瘤抗原的增殖反应。 这个试点项目的目的将是将淋巴细胞增殖与 多参数非放射性BrdU参入法测定 流式细胞术和标准放射性[~3H]胸腺嘧啶核苷掺入试验 以下系统：CMV、流感和至少一个候选系统 由该联盟评估的癌症抗原(MAGE-3、HER2、CEA或 Gp100)。

Description: (provided by applicant) T cell proliferation and the calculation of a stimulation index are one of the mostly widely used methods in both mouse and man to demonstrate antigen specific immunity. We propose to define a "second generation" assay to replace traditional lymphocyte proliferation assays using [3H]thymidine uptake. Multiparameter flow cytometry will be used to assess antigen-specific T cell proliferation by measuring incorporation of BrdU, a thymidine analog. In addition to proliferating T cells, surface markers associated with proliferating cells will be examined. Surface markers to be examined will include CO27, CD45RO/RA, and CO95. These are all markers that can be used, singly or in combination, to distinguish naive from memory cells. Antigen-specific BrdU incorporation will be used to identify proliferating antigen-responsive cells, so that analysis of other markers can be perfoffi1ed by gating on these cells. In a preliminary study using SEB as a stimulus, positive correlation was observed between flow cytometric measurement of proliferating CD4+ T cells by BrdU incorporation and a standard proliferation assay measuring [3H]thymidine uptake by PBMC. We propose to extend the use of BrdU proliferation assay using flow cytometry in assessing T cell proliferative responses to CMV, flu, and tumor antigens. The aim of this pilot project will be to correlate lymphocyte proliferation assay using non-radioactive BrdU incorporation measured by multiparametric flow cytometry and a standard radioactive [3H]thymidine incorporation assay in the following systems: CMV, influenza, and at least one of the candidate cancer antigens to be evaluated by the consortium (MAGE-3, HER2, CEA, or gp100).