Pretreating JeKo-1 xenograft mice with tafasitamab reduces early activation and apoptosis of CART19. (A) CD19⁺ JeKo-1 were cocultured with CART19 in the presence of different concentrations of tafasitamab (10-400 μg/mL) or IgG isotype for 24 hours, and CD25, CD69, granzyme B, and HLA-DR were assessed by flow cytometry (mean ± SEM, ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001, 1-way ANOVA; n = 3, 2 replicates). (B) JeKo-1 were cocultured with CART19 in the presence of different concentrations of tafasitamab (10-400 μg/mL) or isotype IgG control for 24 hours. The expression of PD-1, CTLA-4, and LAG-3 in CD3⁺ T cells was analyzed by flow cytometry (mean ± SEM, ∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001, 1-way ANOVA; n = 3, 2 replicates). (C) JeKo-1 were cocultured with CART19 in the presence of different concentrations of tafasitamab (10-400 μg/mL) or IgG isotype control for 1 hour. Apoptotic T cells were analyzed by flow cytometry. Apoptotic T cells were defined as CD3⁺ annexin V⁺ 7-AAD⁻. (∗P < .05, 1-way ANOVA; n = 3, 2 replicates). (D-E) Experimental schema and BLI analysis. NSG mice were inoculated with luciferase⁺ JeKo-1 on day −14, and tumor burden was assessed using BLI on day −6. Mice were randomized according to their tumor burden to receive PBS vehicle control (IP, 7 mice, group 1) or 10 mg/kg per day of tafasitamab (IP, 8 mice, group 2). On day 0, all mice received 1.0 × 10⁶ CART19; 24 hours after CART19 cell infusion, 3 mice from each group were euthanized, and the spleens were harvested. There were no significant differences in tumor burden on days −8 or −1 in either group (mean ± SEM, t test). (F) Flow cytometric analysis of CD69 and HLA-DR in human CD3⁺ T cells in splenocytes. Human CD3⁺ T cells were determined using murine CD45⁻, human CD45⁺, and human CD3⁺ (mean ± SEM, ∗P < .05, t test; n = 3 per group). (G) Flow cytometric analysis of apoptotic CD3⁺ T cells in the splenocytes. Apoptotic T cells were defined as CD3⁺ annexin V⁺ 7-AAD⁻ (mean ± SEM, ∗P < .05, t test; n = 3 per group).