Figure 3

Daratumumab induces phagocytosis in AML. (A) Cell proliferation analysis using BD BrdU flow assay. Left, representative flow cytometry plots, right quantification for OCI-AML-3 cells mono- or co-cultured with MS-5 or HUVEC after treatment with 0.1 µg/ml daratumumab or IgG₁ control for 4 days, statistical analyses are given below (n = 3 independent experiments). (B) Quantification of apoptotic cells by Annexin V assay of OCI-AML-3 cells mono- or co-cultured with MS-5 or HUVEC after treatment with 0.1 µg/ml daratumumab or IgG₁ control for 4 days (n = 3 independent experiments). (C) Quantification of complement-dependent-cytotoxicity (CDC). Calcein stained OCI-AML-3 cells were incubated in medium supplemented with 10% normal human serum and treated with increasing dosages of daratumumab or IgG₁ control for 1 h. (D) Quantification of antibody-dependent cell-mediated cytotoxicity (ADCC). Calcein stained OCI-AML-3 cells were incubated with effector cells and treated with increasing dosages of daratumumab or IgG₁ control. For CDC and ADCC, calcein-fluorescence was measured in supernatant by spectrophotometry. Lysis was calculated by using detergent as maximum and medium as minimum control (n = 3 independent experiments). (E–G) Quantification of antibody-dependent phagocytosis (ADCP). Macrophages were cultured with calcein stained OCI-AML-3 (n = 3 independent experiments) (E) as well as primary AML cells (n = 5 different AML donors, each primary sample was tested in triplicate) (F) and treated with increasing dosages of daratumumab or IgG₁ control. CD16⁺ calcein⁺ phagocyting macrophages were measured by flow-cytometry. Each dot represents the mean of triplicates. (G) Representative flow cytometry plot of one calcein stained primary AML sample co-cultured with macrophages showing the fraction of AML-phagocyting macrophages. (H) Tracking mitochondrial transfer in co-culture was performed by staining mitochondria with MitoTracker™. Left, MFI of MitoTracker™ in MOLM-13 cells after co-culture with MitoTracker™ stained HUVEC and treatment with 0.1 µg/ml daratumumab or IgG₁ control for 24 h or mono-culture of unstained MOLM-13 as control. Right, MFI of MitoTracker™ in HUVEC after co-culture with MitoTracker™ stained MOLM-13 cells and treating with 0.1 µg/ml daratumumab or IgG₁ control for 24 h or mono-culture of unstained HUVEC as control. Below, representative histogram of MFI of MitoTracker™, gated on MOLM-13 cells (left) or HUVEC (right) at the indicated culture conditions and treatment with 0.1 µg/ml daratumumab or IgG₁ control. (I) Indicated AML cells were co-cultured with either HUVEC (top) or MS-5 cells (bottom) for 24 h, sorted by flow cytometry and mRNA expression levels were measured by rt-PCR for either human mitochondrial DNA (hmtDNA) after HUVEC co-culture or mouse mitochondrial DNA (mmtDNA) after MS-5 co-culture, relative to ncDNA. Data are shown as mean ± SEM. n.s., not significant, *p < 0.05****p < 0.0001 as determined by unpaired student´s t-test (A, B, E, H, Mann–Whitney-U Test (C, D, I), and Wilcoxon signed-rank test (F). See also Supplementary Fig. S3-5.