The comparison of different CAR-T/OV combination strategies. (A and B) Comparison of the viral loading amount between CAR-T^{VSV spin-infected} and CR2/3–CAR-T^{VSV loading}. (A) Flow cytometry analysis for viral loading efficiency by measuring the surface VSV-G expression on CAR-T cells. The representative flow cytometry plots are shown. (B) The ratio of VSV-G–positive CAR-T cells was quantified. For CAR-T^{VSV spin-infected} group, B7H3–CAR-T cells adopted a virus transduction protocol (centrifugation 2,000 g for 2 h) to promote the infection of VSV on CAR-T cells at a MOI of 1, 3, 10, and 30. For CR2/3–CAR-T^{VSV loading} group, CR2/3–B7H3–CAR-T cells were specifically loaded with VSVΔ51 (MOI = 10) in 4°C for 1 h. Then the viral loading was evaluated by surface VSV-G expression on CAR-T cells (n = 3 biological replicates). (C) Depiction of the gating strategy of flow cytometry for analyzing intra-tumoral CAR-T cells. The representative flow cytometry plots are shown for gating the exhaustion markers, including LAG-3, PD-1, and TIM-3, or gating the functional markers, including granzyme B, TNFα, and IFNγ. (D–G) Flow cytometry for analyzing the T exhaustion and functional activity of intra-tumoral CAR-T cells. Flow cytometry analysis of for analyzing the T exhaustion and functional activity of intra-tumoral CAR-T cells in Fig. 6 H. The ratio of PD-1⁺ TIM-3⁺ LAG⁺CAR-T (D), Ki67 expression (E), IFNγ expression (F), and granzyme B expression (G) of intra-tumoral CAR-T cells were analyzed. Left panel: Representative flow cytometry plots. Right panel: The quantitative analysis of the ratio of PD-1 and TIM3-positive CAR-T (n = 4 biological replicates). Data represent the mean ± SD from three independent experiments (B) or one representative from two independent experiments (D–G). Statistics by one-way ANOVA with Tukey’s multiple comparison test (D–G). *, P < 0.05; **, P < 0.01; N.S., no significance.