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Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System

Research output: Contribution to journalArticle

Sarah E. Mann, Zhicheng Zhou, Laurie G. Landry, Amanda M. Anderson, Aimon K. Alkanani, Jeremy Fischer, Mark Peakman, Roberto Mallone, Kristen Campbell, Aaron W. Michels, Maki Nakayama

Original languageEnglish
Article number633
JournalFrontiers in Immunology
Publication statusPublished - 9 Apr 2020

King's Authors


Recent advancements in single cell sequencing technologies allow for identification of numerous immune-receptors expressed by T cells such as tumor-specific and autoimmune T cells. Determining antigen specificity of those cells holds immense therapeutic promise. Therefore, the purpose of this study was to develop a method that can efficiently test antigen reactivity of multiple T cell receptors (TCRs) with limited cost, time, and labor. Nuclear factor of activated T cells (NFAT) is a transcription factor involved in producing cytokines and is often utilized as a reporter system for T cell activation. Using a NFAT-based fluorescent reporter system, we generated T-hybridoma cell lines that express intensely fluorescent proteins in response to antigen stimulation and constitutively express additional fluorescent proteins, which serve as identifiers of each T-hybridoma expressing a unique TCR. This allows for the combination of multiple T-hybridoma lines within a single reaction. Sensitivity to stimulation is not decreased by adding fluorescent proteins or multiplexing T cells. In multiplexed reactions, response by one cell line does not induce response in others, thus preserving specificity. This multiplex assay system will be a useful tool for antigen discovery research in a variety of contexts, including using combinatorial peptide libraries to determine T cell epitopes.

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