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Summary: Several pre-clinical models reveal that chronic chimeric antigen receptor (CAR) stimulation drives a dysfunctional state that mimics in vivo failure. In this protocol, we describe steps to induce T cell dysfunction by persistent and long-term stimulation of CAR-engineered T cells using antigen-expressing cancer cells in suspension cultures. We first described a validated method for manufacturing of CAR T cells, followed by a detailed method for chronic stimulation of CAR T cells and a strategy to evaluate these cells during the process of chronic stimulation.For complete details on the use and execution of this protocol, please refer to Singh et al. (2020).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.