Abstract
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.
Highlights
Determining the preferred antigenic epitopes of T cells holds immense therapeutic promise with respect to infectious diseases, autoimmune diseases, and cancer
Nuclear factor of activated T cells-based reporter systems have been quite useful for identification and isolation of T cells that are activated by a stimulus, including antigens [24,25,26,27,28]
Taking advantage of the Nuclear factor of activated T cells (NFAT)-based reporter system, in this report we established an assay method that allows for the testing of antigen reactivity of multiple T cell receptor (TCR) concurrently by flow cytometry
Summary
Determining the preferred antigenic epitopes of T cells holds immense therapeutic promise with respect to infectious diseases, autoimmune diseases, and cancer. The discovery of T cell antigens can inform the development of personalized therapies for autoimmune diseases and cancer, especially. The determination of antigens along with MHC restriction has led to studies investigating the use of drugs and antibodies to block the expression or antigen presentation of disease-associated peptide-MHC complexes in autoimmune diseases [11, 12]. Another context in which antigen discovery is important is the emerging field of chimeric antigen receptor (CAR) T cell therapy, which has produced very favorable results in clinical trials [13]. One of the challenges limiting the advancement of these therapies is the availability of techniques that facilitate rapid and costeffective discovery of disease-specific antigens
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