Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation Reporter-based Detection System

• Published in: Bio-protocol 2021-01, Vol.11 (2), p.e3883-e3883
• Main Authors: Landry, Laurie G, Mann, Sarah E, Anderson, Amanda M, Nakayama, Maki
• Format: Article
• Language: English
• Subjects: Biology; Methods
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/BioProtoc.3883

Immune tolerance and response are both largely driven by the interactions between the major histocompatibility complex (MHC) expressed by antigen presenting cells (APCs), T-cell receptors (TCRs) on T-cells, and their cognate antigens. Disordered interactions cause the pathogenesis of autoimmune diseases such as type 1 diabetes. Therefore, the identification of antigenic epitopes of autoreactive T-cells leads to important advances in therapeutics and biomarkers. Next-generation sequencing methods allow for the rapid identification of thousands of TCR clonotypes from single T-cells, and thus there is a need to determine cognate antigens for identified TCRs. This protocol describes a reporter system of T-cell activation where the fluorescent reporter protein ZsGreen-1 is driven by nuclear factor of activated T-cells (NFAT) signaling and read by flow cytometry. Reporter T-cells also constitutively express additional pairs of fluorescent proteins as identifiers, allowing for multiplexing of up to eight different reporter T-cell lines simultaneously, each expressing a different TCR of interest and distinguishable by flow cytometry. Once TCR expression cell lines are made they can be used indefinitely for making new T-cell lines with just one transduction step. This multiplexing system permits screening numbers of TCR-antigen interactions that would otherwise be impractical, can be used in a variety of contexts ( , screening individual antigens or antigen pools), and can be applied to study any T-cell-MHC-antigen trimolecular interaction.