Live-Cell Microscopy for quantitative analysis of CAR T Cell fratricide and immune synapse.

Chimeric antigen receptor (CAR) T cell therapy has redefined cancer immunotherapy, offering remarkable efficacy against hematologic malignancies and solid tumors. However, CAR T cell therapy targeting T cell malignancies is limited by fratricide, self-directed cytotoxicity caused by shared antigen expression on both therapeutic and endogenous T cells. This process impairs CAR T cell expansion, viability, and overall efficacy. In this study, we present a live cell microscopy-based methodology to visualize and quantify CAR T cell fratricide and immune synapse. Through fluorescent labeling, time-lapse imaging and automated analysis, we track dynamic T cell interactions, cytotoxic events, and synaptic structures. This protocol enables precise measurement of fratricide events kinetics, serial killing behavior, and immune synapse morphology, thereby offering valuable insights into CAR design optimization and underlying mechanisms. This imaging-based approach complements conventional assays by providing temporally and spatially resolved data, thereby enhancing our understanding of CAR T cell function and cytotoxic regulation in the context of fratricide.