The chimeric antigen receptor (CAR) has been extensively exploited in cancer immunotherapy. In spite of the success of CAR T cells in clinical applications, the molecular mechanism underlying CAR-T cell activation remains unclear. Key questions remain: how are CARs activated by tumor antigens? How do activated CARs transduce signaling to downstream pathways? Here we introduce a microscopy-based method for studying CAR signaling. We use an antigen-coated supported lipid bilayer to activate CARs and combine it with TIRF microscopy to visualize the initial activation process of CAR T cells. This enables monitoring CAR signaling at high spatial and temporal resolutions.
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