Figure 2.

GM7 CAR T Cells Exhibit Antigen-Specific Reactivity Against Cell Surface MISIIR In Vitro

MISIIR-deficient OC cell line C30 was transduced to stably overexpress human MISIIR. (A–C) MISIIR expression on engineered C30.MISIIR was detected by (A) quantitative real-time PCR, (B) western blot analysis showing a predicted ~65-kb band, and (C) flow cytometry using an anti-flag antibody. For functional assays, co-cultures were performed at an E:T ratio of 1:1. (D) Antigen-specific IFN-γ production by MISIIR CAR T cells as detected by ELISA from 24-h co-culture supernatants. (E) Real-time cytotoxicity assays performed using xCELLigence technology in C30 and C30.MISIIR target cells. (F) Intracellular cytokine staining assay after 5 h of co-culture with target cells, showing IL-2, TNF-α, and IFN-γ production by GM7-27Z CAR T cells. Degranulation marker CD107a was also measured. Mean ± SD is represented for all experiments. Significance was determined by two-way ANOVA comparison and Tukey’s multiple comparison test as compared to the UTD group. *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figures S2 and S3.