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

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.

Proinsulin-Reactive CD4 T Cells in the Islets of Type 1 Diabetes Organ Donors

“…These TCR clonotypes were selected for analysis because they were detected from multiple cells or have a specific V-gene motif such as TRAV13-1, TRAV26-1, or TRAV38-2, which are preferentially used by CD4 T cells specific to amino acids 9-23 of insulin B chain ( 10 , 26 , 39 ). We expressed each TCR clonotype in a 5KC murine T-hybridoma cell line, which has an added activation reporter driven by the production of nuclear factor of activated T cells (NFAT) ( 38 ), to test the response to 99 peptide pools containing 12-mer to 15-mer of peptides derived from preproinsulin ( Supplementary Table 2 ). To detect responses against peptides presented by any possible HLA molecules expressed by a given donor, autologous EBV-transformed B cells were used.…”

“…TCR sequences were identified as described previously ( 10 ). TCR transductants were generated using a recently published protocol ( 38 ). Briefly, 5KC T-hybridoma cells ( 56 ) were transduced with a NFAT-driven fluorescent reporter, ZsGreen-1, along with the human CD4 gene with two amino acid mutations at positions 40 (glutamine to tyrosine) and 45 (threonine to tryptophan) that increase binding to MHC molecules ( 57 ) (the retroviral vector is available from addgene, plasmid ID 162745) using a standard spinfection protocol with viral supernatant produced from phoenix-eco cells (ATCC CRL-3214) ( 58 ).…”

“…Immortalized cell lines expressing TCR clonotypes of interest promoted feasibility of directly evaluating reactivity to specific peptide-MHC complexes. Hence, we recently developed a multiplex assay system that can simultaneously assess reactivity of a number of TCR transductant cell lines ( 38 ). Multiplex efforts spare samples and reagents necessary for the assay, thereby allowing analysis of reactivity to hundreds of peptides within a relatively short period of time.…”

A T‐cell reporter platform for high‐throughput and reliable investigation of TCR function and biology

“…Hence, a cell line that provides TCR function close to primary T cells would enable more standardised testing as well as simplify the whole process because of cell lines’ easy handling and almost unlimited proliferative capacity. The urgent need for such a cell line is highlighted by various publications that proposed different cellular platforms for TCR testing 43–47 …”

“…The urgent need for such a cell line is highlighted by various publications that proposed different cellular platforms for TCR testing. [43][44][45][46][47] Here, we propose an advanced Jurkat E6.1based TCR signal reporter system that is unbiased by endogenous TCR expression. Our study, which analysed 59 different human TCRs, isto our knowledgethe first to comprehensively compare TCR function in a cell line with primary human T cells.…”