CAR is enriched at sites of microvilli close contacts in synapses with cognate antigen, but not following engagement of endogenous TCR. (A–C) Three examples of anti-HER2 CAR T cells interacting with HER2+ SKBR3 are shown from an oblique view of the synapse interface (A, Imaris normal shading mode, scale bar = 2 μm), en face synapse view (B, Imaris normal shading mode, scale bar = 2 μm), and a single z-slice (C, scale bar = 1 μm). The SKBR3 target cell location is marked by dashed magenta lines (A and C). Human CD8⁺ T cell expressing anti-HER2 CAR (4D5) was labeled with antibodies to MYC (CAR)-Alexa488 and CD45-Alexa647. (D) Schematic of synaptic contact mapping method. Lipid bilayer on glass support is loaded with Qdot 605, which is a red-fluorescent probe that occupies ∼16 nm in height. Areas where the cell makes a close contact (<16 nm distance) appear as holes in the QD605 signal. (E) CAR T cell synapse imaged by TIRF showing CAR-mEmerald, MV projections (as seen by holes in QD605 signal), and overlay. Human CD8⁺ T cell expressing anti-HER2 CAR (mut4D5) interacting with lipid bilayer loaded with 625 ng HER2 + ICAM + QD605. Scale bar = 2 μm. (F) Line scans from E show anti-correlation of CAR-mEmerald and QD605, indicating enrichment of CARs within MV contacts. (G) OT-I mouse T cells were retrovirally transduced with anti-HER2 CAR (mutCD45). Synapse shown was formed on lipid bilayer loaded with pMHC:SL8 + ICAM + QD605. TIRF imaging of CAR-mEmerald, QD605, and overlay are shown. Scale bar = 2 μm. (H) Line scans from G showing the lack of enrichment in CAR signal within QD605 holes, indicating that CAR microclusters do not accumulate in MV close contacts in absence of the CAR’s cognate antigen.