Functional heterogeneity of CAR-T cells revealed by multimodal imaging of activation dynamics

Abstract Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable efficacy in B cell malignancies, yet the determinants of functional heterogeneity remain incompletely understood. Here we develop an integrated multimodal imaging platform to investigate CAR-T cell activation and cytotoxic dynamics at single-cell resolution, combining time-lapse confocal microscopy, holotomography, fluorescence lifetime imaging (FLIM), structured illumination microscopy (SIM), and stimulated emission depletion (STED) microscopy. We show that CAR-T cell activation is rapid and associated with biophysical remodeling of the immunological synapse, including increased membrane refractive index and prolonged eGFP fluorescence lifetime consistent with CAR protein crowding. We further identify bidirectional membrane protein transfer between effector and target cells via extracellular vesicles and trogocytosis. Quantitative live-cell imaging reveals reduced motility and enhanced clustering in healthy donor-derived freshly analyzed CAR-T cells compared with patients-derived cryopreserved counterparts, correlating with faster activation and increased cytotoxic potency. Together, these findings provide mechanistic insight into CAR-T cell activation and highlight advanced microscopy as a tool to optimize CAR-T design and monitoring.