Specific loading of VSV viruses on CR2/3-CAR promoted the CAR signaling, metabolic fitness, and functional activity of CAR-T cells. (A and B) Transcriptome comparison between CR2/3–B7H3–CARTVSV cells and B7H3–CAR-TVSV cells at tumor antigen-free condition (n = 3 biological replicates). Significantly enriched terms of KEGG-annotated (A) and GO-annotated (B) are shown. (C and D) Flow cytometry analysis for functional activity of CAR-T cells by measuring the expression of granzyme B, IFNγ, CD107a, and TNFα after loading of VSVΔ51 (MOI = 10) for indicated time from 1 to 4 h. (C) Representative images of flow cytometry analysis. (D) Quantitative data of flow cytometry analysis (n = 3 biological replicates). (E) Immunoblotting analysis for CAR-mediated proximal signaling. The phosphorylation of ZAP70, LAT, and PLCγ of CAR-T cells were measured after VSVΔ51 loading (MOI = 10) at indicated times from 0 to 30 min. A representative experiment is shown, repeated twice. (F) Ca2+ influx was measured by Fluo-4 fluorescence probe in CAR-T cells. CAR-T cells were loaded with VSVΔ51 (MOI = 10), and then the MFI of Fluo-4 was measured by flow cytometry in real time. A representative experiment is shown, repeated twice. (G and H) Glycolytic capacity and oxidative phosphorylation capacity of CAR-T were measured by Seahorse XF extracellular flux analyzer. ECAR (G) or OCR (H) of CAR-T cells was measured after CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 8 h (n = 4 technical replicates). The data depicted are one representative of three independent experiments with similar results. (I) Measurement of glucose uptake in CAR-T cells. CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 8 h, then were stained with 2-NBDG, a fluorescent derivative of glucose (n = 3 biological replicates). (J) Measurement of mitochondria mass in CAR-T cells. CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 8 h, then were stained with MitoTracker Deep Red (n = 3 biological replicates). (K) Ki67 expression of CAR-T cells was measured via flow cytometry. CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 12 h (n = 3 biological replicates). Data represent the mean ± SD from three independent experiments (D and I–K) and one representative of three independent experiments (G and H). Statistics by unpaired two-tailed Student’s t test (D and G–K). *, P < 0.05; **, P < 0.01; N.S., no significance. GO, gene ontology. Source data are available for this figure: SourceData F4.
Figure 4.

Specific loading of VSV viruses on CR2/3-CAR promoted the CAR signaling, metabolic fitness, and functional activity of CAR-T cells. (A and B) Transcriptome comparison between CR2/3–B7H3–CARTVSV cells and B7H3–CAR-TVSV cells at tumor antigen-free condition (n = 3 biological replicates). Significantly enriched terms of KEGG-annotated (A) and GO-annotated (B) are shown. (C and D) Flow cytometry analysis for functional activity of CAR-T cells by measuring the expression of granzyme B, IFNγ, CD107a, and TNFα after loading of VSVΔ51 (MOI = 10) for indicated time from 1 to 4 h. (C) Representative images of flow cytometry analysis. (D) Quantitative data of flow cytometry analysis (n = 3 biological replicates). (E) Immunoblotting analysis for CAR-mediated proximal signaling. The phosphorylation of ZAP70, LAT, and PLCγ of CAR-T cells were measured after VSVΔ51 loading (MOI = 10) at indicated times from 0 to 30 min. A representative experiment is shown, repeated twice. (F) Ca2+ influx was measured by Fluo-4 fluorescence probe in CAR-T cells. CAR-T cells were loaded with VSVΔ51 (MOI = 10), and then the MFI of Fluo-4 was measured by flow cytometry in real time. A representative experiment is shown, repeated twice. (G and H) Glycolytic capacity and oxidative phosphorylation capacity of CAR-T were measured by Seahorse XF extracellular flux analyzer. ECAR (G) or OCR (H) of CAR-T cells was measured after CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 8 h (n = 4 technical replicates). The data depicted are one representative of three independent experiments with similar results. (I) Measurement of glucose uptake in CAR-T cells. CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 8 h, then were stained with 2-NBDG, a fluorescent derivative of glucose (n = 3 biological replicates). (J) Measurement of mitochondria mass in CAR-T cells. CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 8 h, then were stained with MitoTracker Deep Red (n = 3 biological replicates). (K) Ki67 expression of CAR-T cells was measured via flow cytometry. CAR-T cells were loaded with VSVΔ51 (MOI = 10) for 12 h (n = 3 biological replicates). Data represent the mean ± SD from three independent experiments (D and I–K) and one representative of three independent experiments (G and H). Statistics by unpaired two-tailed Student’s t test (D and G–K). *, P < 0.05; **, P < 0.01; N.S., no significance. GO, gene ontology. Source data are available for this figure: SourceData F4.

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