Imaging CAR T cell:target interactions. (A) Time-lapse of anti-CD45-Alexa647-stained CAR T cell interacting with CFSE-labelled HER2+ SKBR3 tumor cell by LLS imaging shows stable cell:cell interaction. Maximum intensity projection from Imaris is shown. Scale bar = 3 μm. (B) Additional example of a side-view of CAR T cell synapse with HER2+ SKBR3 target. CAR T cell was labeled and imaged as in Fig. 3, A–C but with CAR intensity based off endogenous mEmerald tag alone, without additional anti-MYC labeling. CAR localization without anti-MYC labeling shows the same enrichment of CAR in synaptic projections. Displayed in nomal shading mode in Imaris. Scale bar = 2 μm. (C) The percentage of MV close contacts occupied by receptor was not significantly different between the OT-I TCR and the LA CAR murine T cell synapses with cognate antigen (SL8:pMHC and 625 ng/well HER2). n = 4 cells, 55–133 close contacts were averaged for each cell. (D–J) Additional examples of cells prepared and analyzed by line scan as in Fig. 3, F–I. (K) 2D patch analysis of CAR in the TCR-stimulated synapse using a bin size of 300 nm—chosen in order to compare to clustering analysis in Fig. 1, F and G. No CAR intensity exceeded 3 SD above mean.