Imaging CAR-T Synapse as a Quality Control for CAR Engineering

The chimeric antigen receptor (CAR) evolves as a powerful tool to reprogram T cells for targeted killing. CAR-T therapy succeeded in treating certain types of blood cancers, and its application is now expanding towards solid tumors, autoimmune diseases, viral infection, and fibrosis. These require the design of a large number of new CARs that target a variety of antigens. Here we described two methods as a quality control for validating newly developed CARs: (1) the cell-cell conjugation assay as a reflection of efficient binding of CAR to antigen in the cellular context and (2) CD45 exclusion in the synapse as an indication of CAR signaling potential. These assays examine prerequisites for a functional CAR-T and reveal causes for ineffective CAR-T activation.