Loading of VSVΔ51 induced CAR clustering and synapse formation on CR2/3–CAR-T cells. (A) Super-resolution imaging analysis of CAR clustering in CAR-T. B7H3–CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 30 min, then the CAR micro-clusters were detected using anti-G4S Ab. Scale bar, 5 μm. (B) Super-resolution imaging analysis of co-localization of mcherry-CR2/3-CAR and GFP-CR2/3-CAR in CR2/3–CAR-T cells. CR2/3–B7H3–CAR-T cells were co-transfected with mcherry-CR2/3-CAR and GFP-CR2/3-CAR and loaded with VSVΔ51 (MOI = 10) for 30 min, then the co-localization of CAR was measured. Scale bar, 5 μm. (C–F) TIRFM analysis of IS of B7H3–CAR-T cells on stimulatory recombinant human B7H3 protein coated on glass slides. (C) Representative images for lamp1 (green), pZAP70 (red), and F-actin (white) at IS. (D) Quantified fluorescent data of synapse area and MFI of lamp1 and pZAP70 are shown (n = 30 cells from three independent experiments). (E) Representative images for VSV-G (green), CAR (red), and F-actin (white) at IS. (F) Quantified fluorescent data of viral protein VSV-G are shown (n = 30 cells from three independent experiments). Scale bar, 10 μm. (G) Scheme of loading of OV VSVΔ51 on CR2/3-CAR moiety, which pre-activates CAR-T cells and induces more efficient formation of IS. Data represent the mean ± SD from three independent experiments (D and F). A representative experiment is shown, repeated at least twice (A–C and E). Statistics by one-way ANOVA with Tukey’s post hoc test (D and F). *, P < 0.05; **, P < 0.01; N.S., no significance.