Fig. 4.

Combination of LOAd viruses with CAR T cells in a killing assay. CD19 expression was analyzed in LOAd-infected lymphoma cells by flow cytometry. Bar graphs in (a) show the relative geometric mean fluorescence intensity (rMFI) compared to isotype control staining. For (b–d), CD19+ target cells Karpas422 and DG-75 cells were infected with 100 MOI of LOAd(-) or LOAd703 or left uninfected and cultured for 48 h. CAR T cells were thawed and cultured in 100 IU/ml IL-2 for 48 h. At 48 h, 5 × 10⁴ target cells were plated per well in triplicates in 96-well plates, and CAR T cells were added to achieve an effector/target cell ratio of 10:1–1:1. Forty-eight h after the co-culture setup, culture supernatants were taken and analyzed for release of IFN-γ (b), granzyme B (c) and lactate dehydrogenase (LDH) (D). Bar graphs show mean ± SD of three technical replicates. In (d), background signal from medium and CAR T cells alone was subtracted from the LDH signal, and LDH release from CAR T cells stimulated with CD3/CD28 dynabeads (Thermo Fisher) is shown as control for activation-induced cell death of CAR T cells. Statistical differences comparing all groups to each other were analyzed with one-way ANOVA followed by Tukey’s multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). In (d), the CD3/CD28 bead control was excluded from statistical analysis