Cytometric assessment of antigen-specific T cell signaling.

T cell receptor (TCR) signaling strength influences critical T cell characteristics including cytotoxic capacity, differentiation, memory formation, and exhaustion. Naturally occurring or engineered single-nucleotide variants (SNVs) and gene deletions which modulate T cell signaling pathways can significantly impact the potency of T cell cytolytic activity, including in the setting of T cell-based immunotherapies such as tumor-infiltrating lymphocyte (TIL) therapy and chimeric antigen receptor T cell (CAR T) therapy. Thus, studying T cell signaling represents a valuable component of the engineering and preclinical testing process for cell therapies and immune checkpoint blockade (ICB). Flow cytometry is a powerful experimental and diagnostic tool which enables rapid, quantitative analysis of T cell signaling responses by phosphoprotein detection with fluorophore-labeled antibodies. However, many approaches that have been developed to induce and study T cell signaling rely on supraphysiological and non-specific stimulation methods, such as crosslinking of T cell CD3 and CD28 or treatment with chemical stimulating agents such as ionomycin and phorbol 12-myristate 13-acetate (PMA). These assays bypass the endogenous TCR machinery and limit the conclusions which can be drawn regarding the physiological relevance of the T cell responses measured. Here, we present a simple, efficient, and scalable workflow to assess physiological T cell signaling responses to antigen-specific stimulation with cognate peptide-MHC expressing target cells using flow cytometry. With minimal modifications, this approach can be successfully applied to the study of chimeric antigen receptor (CAR) signaling or signaling in other immune cell subtypes such as B cells, using analogous antigen-matched co-culture systems.