Generation of TCR-engineered reference cell samples to control T-cell assay performance

In vitro cellular assays analyzing antigen-specific T cells are characterized by their high complexity and require controlled conditions to lower experimental variations. Without standard cellular reagents, it is difficult to compare results over time and across institutions. To overcome this problem, a simple and robust technology was developed to generate TCR-engineered reference samples (TERS) containing defined numbers of antigen-specific T cells. Utilization of TERS enables performance control of three main T-cell assays: MHC-peptide multimer staining, IFN-γ ELISpot and cytokine flow cytometry. TERS continuously deliver stable results and can be stored for longer periods of time. Here, an optimized manufacturing protocol, based on the electroporation of stable T-cell receptor in vitro-transcribed mRNA, is provided for versatile in-house production of TERS. Included are a guideline to optimize the electroporation settings on locally available electroporation devices and a step-by-step protocol for the production process.