Fig. 1

Representative CAR good and poor IS formed on the lipid bilayer. (a) Diagram of a lipid bilayer model to study GD2-CAR IS formation and GD2-CAR-T cell activation using idiotypic antibody (clone, 1A7) against the GD2-CAR to trigger CAR signaling on the glass-supported planar lipid bilayer system. (b) Schematic representation of three-dimensional (3D) confocal image reconstitutuin. Briefly, primary GD2-CAR-T cells were stimulated on the SLB containing biotinylated fluorescently labeled anti-GD2-CAR (yellow), fixed, permeabilized, and then stained with phalloidin (blue), anti-F-actin (green), and perforin (red). An individual cell was imaged under the 3D Olympus confocal setting, and then the reconstitution of these 3D confocal images . (c & d) Comparison of good CAR IS (c) and poor CAR IS (d) is illustrated by these reconstituted 3D confocal images. The x-y focal plane represents the lipid bilayer surface. The Z focal plane represents the CAR T cell position on the top of lipid bilayer. Scale bars, 5μm. Notably, stronger F-actin accumulation, lytic granule polarization, and CAR molecular accumulation is associated with optimal CAR IS