Standardized protocol for the evaluation of chimeric antigen receptor (CAR)-modified cell immunological synapse quality using the glass-supported planar lipid bilayer.

Chimeric antigen receptor (CAR)-modified cell therapy is an effective therapy that harnesses the power of the human immune system by re-engineering immune cells that specifically kill tumor cells with tumor antigen specificity. Key to the effective elimination of tumor cells is the establishment of the immunological synapse (IS) between CAR-modified immune cells and their susceptible tumors. For functional activity, CAR-modified cells must form an effective IS to kill tumor cells specifically. The formation of the CAR-specific IS requires the coordination of many cellular processes including reorganization of the cytoskeletal structure, polarization of lytic granules, accumulation of tumor antigen, and phosphorylation of key signaling molecules within the IS. Visualization and assessment of the CAR IS quality can reveal much about the molecular mechanisms that underlie the efficacy of various CAR-modified immune cells. This chapter provides a standardized method of assessing the IS quality by quantifying the tumor antigen (defining the CAR IS formation), cytoskeleton (key component of CAR IS structure), and various molecules of interest involved in the IS formation (key molecular mechanism signatures of CAR IS function) using immunofluorescence on the glass-supported planar lipid bilayer, with a focus on tumor antigen only in this study. We provide specific insights and helpful tips for reagent and sample preparation, assay design, and machine learning (ML)-based data analysis. The protocol described in this chapter will provide a valuable tool to visualize and assess the IS quality of various CAR-modified immune cells.