Pretreating tumor cells with tafasitamab does not affect the antitumor activity of CART19. (A) Cytotoxicity assay of CART19 with tafasitamab-pretreated CD19⁺ cell line JeKo-1. JeKo-1 were treated with either the isotype IgG control or increasing doses of tafasitamab (10-400 μg/mL) overnight. The next day, tafasitamab was removed from the JeKo-1 by centrifugation. CART19 were cocultured at different effector-to-target ratios (E:T) with tafasitamab-pretreated luciferase⁺ JeKo-1. At 24 hours, cell death was assessed by luminescence, relative to that of the control. CART19 were able to induce cell death in all CD19⁺ cell lines, regardless of tafasitamab treatment (mean ± SEM, 2-way ANOVA; n = 3, 2 replicates). (B) CART19 cell antigen-specific proliferation assay after stimulation with tafasitamab-pretreated JeKo-1. The CD19⁺ cell line JeKo-1 was treated with different doses of tafasitamab (10-400 μg/mL) or an isotype IgG control overnight. Before coculture, tafasitamab or the isotype control was removed from JeKo-1 by centrifugation. On day 5 of culture, the absolute number of CD3⁺ T cells was quantified by volumetric flow cytometry (1-way ANOVA; n = 3, 2 replicates). (C) CART19 CD107a degranulation and intracellular cytokine assays after stimulation with tafasitamab-pretreated JeKo-1. CD19⁺ cell line JeKo-1 was treated with different doses of tafasitamab (10-400 μg/mL) or isotype IgG control overnight. Before coculture, tafasitamab or isotype control was removed from JeKo-1 by simple centrifugation. The levels of CD107a and intracellular cytokines were assessed by flow cytometry. The negative gate was determined based on the fluorescence minus one (FMO) control (mean ± SEM, 1-way ANOVA; n = 3, 2 replicates).