Rab5 expression inhibits CAR trogocytosis into tumor cells. (a) Staining strategy for detecting unengaged CARs using FLAG-tagged CARTs. FLAG-tagged 19BBz CARTs were incubated with CD19 protein at 37°C for 1 h to allow formation of CD19–CAR complexes, followed by endocytosis and potential dissociation. To track if CARs were bound to CD19 protein or not, cells were incubated with anti-FMC63 antibody, which recognizes the scFv domain of 19BBz CARs, at 37°C for indicate time point. CARs that were released from CD19 were identified as double-positive for both anti-FLAG and anti-FMC63 staining by flow cytometry. (b) High-resolution confocal imaging of the cellular localization of mCherry-CARs (red) between CARTs and GFP-expressing tumor cells (green) during the indicated time of engagement. T cell surface (F-actin, cyan) and nuclei (dark blue) are shown. The arrow indicates tumor cells exhibiting cytosolic uptake of mCherry-fused CARs. Data are representative of three independent experiments with samples from independent healthy donors. (c) High-content microscopy showing the spatial distribution of antigen and CARs in control and Rab5-CARTs during the third-round CAE assay. Control and Rab5-CARTs expressing mCherry-CARs (red) were co-cultured with K.19.GFP cells (green), then fixed, and stained with Hoechst 33342 (blue) to label nuclei and F-actin (cyan) to delineate the cell membrane. Data are representative of two independent experiments with samples from independent healthy donors.