Journal: Molecular Therapy Oncology
Article Title: Enhanced solid tumor cell targeting by a neoepitope-encoding oncolytic measles virus combined with CAR therapy
doi: 10.1016/j.omton.2025.201043
Figure Lengend Snippet: CAR-T cells targeting the MICB-derived octapeptide show CAR-dependent activation and efficiently kill tumor cells (A) Schematic representation of scFv of both 8-VHVL CAR and 8-VLVH CAR targeting the octapeptide VLQSQRTD used in this study. (B) Expression of CAR molecules on T cells. Left panel shows representative plots of flow cytometry data of primary T cells, 8-VHVL CAR-T and 8-VLVH CAR-T. Right panel shows expression of both experimental CARs (8-VHVL and 8-VLVH) as well as CD4 and CD19 CAR on T cells. Black bars represent CAR expression detected via anti-mouse F(abʹ) 2 antibody, and green bars represent EGFP signal; data from n = 18 independent experiments are depicted as mean (SEM). (C) CAR-T cells were cocultured with Mac-1 cells stably transduced to express indicated transgenes. Tumor cells were labeled with CellTrace Violet, and killing was assessed via PI staining. Killing is depicted as ratio of PI+ cells in cocultures and independently cultured Mac-1 cells. Data from n ≥ 6 experiments are depicted as mean (SEM). (D) CD137 expression was measured on CAR-T cells after incubation with Mac-1 for 24 h. Data are shown from EGFP+ CAR-T cells and are depicted from n = 6 independent experiments as mean (SEM). (E) CAR-T cells were cocultured with T cells from the same donor transduced to express indicated transgenes. T cells used as target cells were labeled with CellTrace Violet, and killing was assessed via PI staining. Only EGFP+ target T cells were analyzed for PI staining. Killing is depicted as the ratio of PI+ cells in cocultures and independently cultured target T cells. Data from n = 9 independent experiments are shown as mean (SEM). (F) CD137 expression was measured on CAR-T cells after incubation with transduced T cells as target cells for 24 h. Data are shown from EGFP+ CAR-T cells and are depicted from n = 6 independent experiments as mean (SEM). (G) LAG-3 expression was measured on CAR-T cells after incubation with transduced T cells as target cells for 72 h. Data are shown from EGFP+ CAR-T cells from n = 4 experiments as mean (SEM); statistical analysis of (C) through (G) was performed by two-way ANOVA followed by Dunnett’s multiple-comparisons test, and experimental groups were compared to CD19 CAR. ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, and ∗∗∗∗ = p ≤ 0.0001.
Article Snippet: For evaluation of CAR-T cell activation, staining with CD137 (CD137 Antibody, PE-Vio770, anti-human, REAfinity) (Miltenyi Biotech, Bergisch Gladbach, Germany) was carried out after 24 h of co-incubation.
Techniques: Derivative Assay, Activation Assay, Expressing, Flow Cytometry, Stable Transfection, Labeling, Staining, Cell Culture, Incubation
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