FLOW CYTOMETRIC ANALYSIS FOR T CELL PROLIFERATION

说明：（申请人提供） T细胞增殖和刺激指数的计算是本发明的一个方面。 在小鼠和人中最广泛使用的方法来证明抗原 特异性免疫我们建议定义“第二代”检测， 取代使用[3 H]胸苷的传统淋巴细胞增殖试验 摄取。多参数流式细胞术将用于评估抗原特异性 通过测量BrdU（一种胸苷类似物）掺入的T细胞增殖。 除了增殖的T细胞外，与T细胞增殖相关的表面标志物 将检查增殖细胞。待检查的表面标记将 包括CO27、CD 45 RO/RA和CO 95。这些都是可以使用的标记， 单独或组合，以区分幼稚细胞和记忆细胞。抗原特异 BrdU掺入将用于鉴定增殖的抗原应答性细胞。 细胞，以便可以通过以下方法进行其他标记物的分析： 门控这些细胞在一项使用SEB作为刺激的初步研究中， 流式细胞术测量的细胞凋亡率与细胞凋亡率呈正相关。 通过BrdU掺入增殖CD 4 + T细胞和标准增殖 测定PBMC摄取[3 H]胸苷的试验。我们建议扩大使用 流式细胞术检测BrdU对T细胞增殖的影响 对CMV、流感和肿瘤抗原的增殖反应。 该试点项目的目的是将淋巴细胞增殖与 使用多参数测量的非放射性BrdU掺入的测定 流式细胞术和标准放射性[3 H]胸苷掺入试验， 以下系统：CMV、流感和至少一种候选系统 由联合体评估的癌抗原（法师-3，HER 2，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).