Fig 1: DuoBody-CD3x5T4 can induce bystander kill which is dependent on IFNGR1 expression.(A) Parental and 5T4 KO MDA-MB-231 tumor cells were mixed in different ratios, as indicated, and incubated with purified T cells (E:T ratio = 4:1, n = 2 donors) and DuoBody-CD3x5T4 for 72 h. T cell–mediated cytotoxicity of 5T4+ (left panel) and 5T4- (right panel) tumor cells was determined by flow cytometry, showing a representative donor of three donors tested. (B, C, D) Parental (5T4+) MDA-MB-231 tumor cells were cocultured with purified T cells (E:T = 4:1, n = 2 donors) and incubated with 10 µg/ml DuoBody-CD3x5T4 or bsIgG1-CD3xctrl for 72 h. As a positive control, T cells were incubated with anti-CD3/CD28 beads (but without tumor cells) for 72 h. (B) The supernatant (either with or without T cells) was transferred to MDA-MB-231 5T4 KO cells and incubated for 72 h (B). As negative control, MDA-MB-231 5T4 KO cells were incubated with fresh T cells and indicated antibodies for 72 h. (C, D) T cell–mediated cytotoxicity (C) and Fas expression (D) of MDA-MB-231 5T4 KO cells were determined by flow cytometry. A representative donor of two donors tested is shown. (E) 5T4, Fas, IFNGR1, and PD-L1 expression on MDA-MB-231 parental, 5T4/Fas KO, and 5T4/IFNGR1 KO cells was measured by flow cytometry with or without prior overnight incubation with IFN? (100 ng/ml). (F, G, H, I) Parental and 5T4/Fas KO or 5T4/IFNGR1 MDA-MB-231 tumor cells were mixed in different ratios, as indicated, and incubated with purified T cells (E:T ratio = 4:1, n = 4–6) and 1 µg/ml DuoBody-CD3x5T4 for 72 h, after which T cell–mediated cytotoxicity (F), CD4+ (G) and CD8+ (H) T-cell activation, and IFN? production (I) were analyzed. The predicted black line in (F) refers to the outcome of the assay when no bystander killing is expected, for example, if 50% of tumor cells are 5T4+, only 50% of tumor cells will be killed.
Fig 2: Fas and IFNGR1 expression contribute to the induction of T cell–mediated cytotoxicity by DuoBody-CD3x5T4.(A) 5T4 and Fas expression on MDA-MB-231 parental and Fas KO cells was measured by quantitative flow cytometry with or without prior overnight incubation with IFN? (100 ng/ml). (B, C, D, E) MDA-MB-231 parental and Fas KO cells were incubated with purified CD4+ (B, D) or CD8+ (C, E) T cells (E:T ratio = 4:1, n = 5 donors) and DuoBody-CD3x5T4 for 72 h. T-cell activation (B, C) and T cell–mediated cytotoxicity (D, E) were analyzed. (B, C) Activation of T cells from five donors at 1 µg/ml DuoBody-CD3x5T4 (*P = 0.05, paired t test). (D, E) The left panels show dose-dependent T cell–mediated cytotoxicity in a representative experiment, and the right panels show tumor cell viability at 1 µg/ml DuoBody-CD3x5T4 from five donors (*P = 0.05 and **P = 0.01, paired t test). (F) 5T4, IFNGR1 and Fas expression on MDA-MB-231 parental and IFNGR1 KO cells was measured by flow cytometry. (G, H) MDA-MB-231 parental and IFNGR1 KO cells were incubated with purified T cells (E:T ratio = 4:1, n = 4 donors) and DuoBody-CD3x5T4 for 72 h. (G) The left panel shows dose-dependent T cell–mediated cytotoxicity in a representative experiment, and the right panel shows tumor cell viability at 1 µg/ml DuoBody-CD3x5T4 from four donors (***P = 0.005, paired t test). (H) T-cell activation at 1 µg/ml DuoBody-CD3x5T4 from four donors.
Fig 3: Fas contributes to the induction of T cell–mediated cytotoxicity by DuoBody-CD3x5T4.(A) MDA-MB-231 parental and Fas KO cells were incubated with purified CD4+ or CD8+ T cells (E:T ratio = 4:1, n = 5 donors) and DuoBody-CD3x5T4 for 72 h. T-cell activation was analyzed by measuring up-regulation of CD69. (B) 5T4 and Fas expression on NCI-H1299 parental and Fas KO cells was measured by flow cytometry with or without incubation with IFN? (100 ng/ml). (C, D) NCI-H1299 parental and Fas KO cells were incubated with purified CD4+ (top panels) or CD8+ (bottom panels) T cells (E:T ratio = 4:1, n = 5–6 donors tested) and DuoBody-CD3x5T4 for 72 h. (C) The left panels show dose-dependent T cell–mediated cytotoxicity, and the right panels show T cell–mediated cytotoxicity at 1 µg/ml DuoBody-CD3x5T4. A paired t test was used to compare T cell–mediated cytotoxicity at 1 µg/ml, with *P = 0.05 and **P = 0.01. (D) The left panels show dose-dependent T-cell activation, and the right panels show T-cell activation at 5 µg/ml DuoBody-CD3x5T4. (E) MDA-MB-231 parental and IFNGR1 KO cells were incubated with purified T cells (E:T ratio = 4:1, n = 4 donors) and DuoBody-CD3x5T4 for 72 h, and T-cell activation was analyzed measuring up-regulation of CD69. A representative donor of four donors tested is shown.
Fig 4: DuoBody-CD3x5T4 can induce bystander kill which is dependent on IFNGR1 expression.(A, B) Parental (5T4+) MDA-MB-231 tumor cells were cocultured with purified T cells (E:T = 4:1, n = 2 donors) and incubated with 10 µg/ml DuoBody-CD3x5T4 or bsIgG1-CD3xctrl for 72 h. As a positive control, T cells were incubated with anti-CD3/CD28 beads (but without tumor cells) for 72 h. The supernatant (either with or without T cells) was transferred to MDA-MB-231 5T4 KO cells and incubated for 72 h. As negative control, MDA-MB-231 5T4 KO cells were incubated with fresh T cells and indicated antibodies for 72 h. (A, B) T cell–mediated cytotoxicity of MDA-MB-231 parental cells (A) and T-cell activation (B) were determined by flow cytometry. (C, D, E) Parental and 5T4, 5T4/Fas, or 5T4/IFNGR1 KO MDA-MB-231 tumor cells were mixed in different ratios, as indicated, and incubated with purified T cells (E:T ratio = 4:1) and DuoBody-CD3x5T4 for 72 h, after which T cell–mediated cytotoxicity (C, three representative donors of 4 [5T4/IFNGR1 KO] or six [5T4 and 5T4/Fas KO] analyzed), CD8+ T-cell activation (D, a representative donor of 4 [5T4/IFNGR1 KO] or six [5T4 and 5T4/Fas KO] analyzed), and IFN? production (E, one representative donor of 4 [5T4/IFNGR1 KO] or six [5T4 and 5T4/Fas KO] analyzed) were determined.
Supplier Page from Thermo Fisher Scientific for FAS Antibody PE