DEVELOPMENT OF NOVEL T CELL ASSAYS

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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.