Measuring Chimeric Antigen Receptor T Cells (CAR T Cells) Activation by Coupling Intracellular Cytokine Staining with Flow Cytometry

Chimeric antigen receptor T cells (CAR T cells) therapy has revolutionarily changed the landscape of immunotherapy and been approved by the U. S Food and Drug Administration (FDA) since 2017 for several blood malignancies. To translate novel CAR T cells into clinical applications, it is essential to evaluate their antigen specificity, cytotoxic capacity, and off-target effects in vitro. A commonly used criteria to assess CAR T cell functionality involves detecting cytokine secretion following their engagement with target antigens. This chapter describes a method of combining intracellular cytokine staining and multi-color flow cytometry to measure CAR T cells activation following antigen stimulation.