Loading of VSV virus particles enhanced the specific cytotoxicity against CAR antigen-positive tumor cells. (A–C) Coculture experiments of GSC-1 tumor spheroids and CAR-T cells. (A) Cytotoxic effect of CR2/3–B7H3–CAR-TVSV against 3D spheroids of GSC-1 cells was measured by Calcein/PI staining. Representative images are shown. Scale bar, 100 μm. (B) PI-positive cells was quantified via flow cytometer analysis (n = 3 biological replicates). (C) Quantitative analysis of cytokines IFNγ, granzyme B, and TNFα released by CAR-T cells in coculture with GSC-1 cells for indicated time points via ELISA (n = 4 technical replicates). The data depicted are one representative of three independent experiments with similar results. (D) Illustration for producing VSVΔG*G and VSVΔG*S recombinant viruses. HEK-293T cells were transfected with plasmid carrying G protein of VSV (pCAG-VSV-G) or spike protein of SARS-CoV-2 (pCAG-SARS-CoV2-S), respectively. Then the cells were infected with VSVΔG*G pseudovirus for packing new VSVΔG*G or VSVΔG*S, respectively. (E–G) Cytotoxic measurement of CAR-T cells at different ratios (CAR-T:Tumor). B7H3–CAR-T or CR2/3–B7H3–CAR-T cells loaded with the replicative deficient viral strain VSVΔG*G or VSVΔG*S. Then CAR-T cells were cocultured with GSC-1 (E), U87 (F), and LN229 (G) at indicated ratios from 0.1:1 to 3:1 (CAR-T:Tumor) for 24 h. The cytotoxicity of CAR-T cells was measured by LDH release assay (n = 3 biological replicates). (H) Verification of the KO efficiency of B7H3 in U87 cells. U87 cells were infected with lentivirus carrying the sgRNA-targeting B7H3. After the puromycin (2 µg/ml) resistance selection for 2 wk, the B7H3 expression level of U87 cells were measured by flow cytometry. (I) Cytotoxic activities of CAR-T cells preloaded with VSVΔG*G to U87-B7H3-KO (U87-sgB7H3) cells. CAR-T and U87-B7H3-KO cells were cocultured at indicated ratio from 0.1:1 to 3:1 (CAR-T:Tumor) for 24 h (n = 3 biological replicates). Data represent the mean ± SD from three independent experiments (B, E–G, and I). Statistics by one-way ANOVA with Tukey’s post hoc test (B and C) or two-way ANOVA with Bonferroni post hoc test (E–G and I). *, P < 0.05; **, P < 0.01; N.S., no significance.
Figure 3.

Loading of VSV virus particles enhanced the specific cytotoxicity against CAR antigen-positive tumor cells. (A–C) Coculture experiments of GSC-1 tumor spheroids and CAR-T cells. (A) Cytotoxic effect of CR2/3–B7H3–CAR-TVSV against 3D spheroids of GSC-1 cells was measured by Calcein/PI staining. Representative images are shown. Scale bar, 100 μm. (B) PI-positive cells was quantified via flow cytometer analysis (n = 3 biological replicates). (C) Quantitative analysis of cytokines IFNγ, granzyme B, and TNFα released by CAR-T cells in coculture with GSC-1 cells for indicated time points via ELISA (n = 4 technical replicates). The data depicted are one representative of three independent experiments with similar results. (D) Illustration for producing VSVΔG*G and VSVΔG*S recombinant viruses. HEK-293T cells were transfected with plasmid carrying G protein of VSV (pCAG-VSV-G) or spike protein of SARS-CoV-2 (pCAG-SARS-CoV2-S), respectively. Then the cells were infected with VSVΔG*G pseudovirus for packing new VSVΔG*G or VSVΔG*S, respectively. (E–G) Cytotoxic measurement of CAR-T cells at different ratios (CAR-T:Tumor). B7H3–CAR-T or CR2/3–B7H3–CAR-T cells loaded with the replicative deficient viral strain VSVΔG*G or VSVΔG*S. Then CAR-T cells were cocultured with GSC-1 (E), U87 (F), and LN229 (G) at indicated ratios from 0.1:1 to 3:1 (CAR-T:Tumor) for 24 h. The cytotoxicity of CAR-T cells was measured by LDH release assay (n = 3 biological replicates). (H) Verification of the KO efficiency of B7H3 in U87 cells. U87 cells were infected with lentivirus carrying the sgRNA-targeting B7H3. After the puromycin (2 µg/ml) resistance selection for 2 wk, the B7H3 expression level of U87 cells were measured by flow cytometry. (I) Cytotoxic activities of CAR-T cells preloaded with VSVΔG*G to U87-B7H3-KO (U87-sgB7H3) cells. CAR-T and U87-B7H3-KO cells were cocultured at indicated ratio from 0.1:1 to 3:1 (CAR-T:Tumor) for 24 h (n = 3 biological replicates). Data represent the mean ± SD from three independent experiments (B, E–G, and I). Statistics by one-way ANOVA with Tukey’s post hoc test (B and C) or two-way ANOVA with Bonferroni post hoc test (E–G and I). *, P < 0.05; **, P < 0.01; N.S., no significance.

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