DEVELOPMENT OF NOVEL T CELL ASSAYS

尽管在过去的七年里，在定量和定性分析方面都取得了巨大的进步 人类抗原特异性T细胞反应--基于单细胞的分析假设比以往任何时候都要大 显着性--在全面鉴定T细胞所需的各种化验方法中仍然存在明显的漏洞 免疫反应。这项提案旨在填补其中的三个漏洞。在目标1中，我们将开发一种新的检测方法来 在单个细胞水平上检测抗原特异性T细胞的裂解功能，使用借鉴自 ELISPOT试验。在目标2中，我们建议利用多参数流动的新发展。 细胞学仪器和染色技术将允许同时分析更多的T细胞 更广泛的效应器功能阵列的特异性和/或同时分析 1和2型细胞因子。在目标3中，我们建议映射黄热病病毒的T细胞表位，这将 显著提高项目1中提出的研究的分辨率，并开发新的技术，基于 在重组VSV载体上，用于在基因水平上映射T细胞对较大病原体的反应或 天花/牛痘等疫苗。这些技术都将显著改进对疫苗诱导的分析 并将更好地理解疫苗诱导免疫的相关因素 保护。

Although the last seven years have seen tremendous advances in both quantitative and qualitative analyses of human antigen-specific T cell responses--with single cell-based assays assuming ever greater prominence--significant holes remain in the arsenal of assays that are needed to fully characterize T cell immune responses. This proposal aims to fill three of these holes. In Aim 1, we will develop a new assay to detect the lytic function of antigen-specific T cells at the single cell level, using principles borrowed from the ELISpot assay. In Aim 2, we propose to take advantage of new developments in multiparameter flow cytometry instrumentation and dye technology that will permit both simultaneous analysis of more T cell specificities and/or simultaneous analysis of a broader array of effector functions such as production of type 1 and type 2 cytokines. In Aim 3, we propose to both map T cell epitopes from Yellow Fever Virus that will significantly enhance the resolution of studies proposed in Project 1, and to develop new techniques, based on recombinant VSV vectors, for mappping at the gene level T cell responses to larger pathogens or vaccines such as variola/vaccinia. These techniques will all permit significantly improved analysis of vaccineinduced immunty and will provide better understandings of the correlates of vaccine-induced immune protection.