Analysis of trogocytosis and ADCC in cocultures of neutrophils and myeloma cells with different CD38 expression in presence of daratumumab. Neutrophils isolated from healthy donors or patients with MM were cocultured with the indicated MM cell lines or autologous MM cells in presence of daratumumab for the evaluation of trogocytosis and ADCC. For the analysis of trogocytosis by flow cytometry, MM cells were labeled with either PKH67 or PKH26 prior to coculture. (A) Cocultures of neutrophils derived from healthy donors (n = 5) and the MOLP-8 cell line for trogocytosis and ADCC evaluation. Left: after 4 hours of coculture, PKH67+CD138– events were assessed within the CD66b+ compartment by flow cytometry for trogocytosis analysis. Right: the percentage of dead MM cells (annexin V+, 7AAD+) within the CD38me+CD66b– compartment (CD38me: anti-CD38 multi-epitope) was assessed by flow cytometry for ADCC analysis. (B) Analysis of trogocytosis (left) and ADCC (right) in cocultures of neutrophils derived from patients with MM (n = 5) and the MOLP-8 cell line following the same methodology as in panel A. (C) Evaluation of daratumumab-mediated patient neutrophil cytotoxicity on autologous myeloma cells. Neutrophils and myeloma cells from 2 patients were isolated and subsequently cocultured in absence or presence of daratumumab for 4 hours. Cytotoxicity on myeloma cells was analyzed by flow cytometry as in panel A. (D) Representative images for trogocytosis by live-cell microscopy. DiO-labeled neutrophils (green) and DiI-labeled MOLP-8 cells (red) were cocultured (Effector:Target ratio of 3:1) in the presence of 1 μg/mL daratumumab. Time-lapse acquisition during the 4-hour coculture period, with images being captured every 3 minutes, was performed on a Nikon Eclipse TE-2000 microscope (Nikon, Tokyo, Japan). (E-G) Analysis of daratumumab-induced trogocytosis (left) and ADCC (right) in cocultures of neutrophils derived from healthy donors (n = 3) and RPMI-8226, NCI-H929 or U266 cell lines, respectively, following the same methodology as in panel A. (H) Analysis of trogocytosis (left) and ADCC (right) in cocultures of neutrophils derived from healthy donors (n = 3) and the indicated cell lines following the same methodology as in panel A but using PKH26 for cell labeling. In panels A,B,E-H, data represent the mean ± standard error of the mean (SEM). Means were compared using one-way analysis of variance and Tukey post hoc tests. ∗P < .05 and ∗∗∗∗P < .0001. Whenever “basal death” is indicated, it means the death of myeloma cells in basal culture conditions (without neutrophils and drug). The remaining conditions correspond to coculture of neutrophils and myeloma cells.
Figure 1.

Analysis of trogocytosis and ADCC in cocultures of neutrophils and myeloma cells with different CD38 expression in presence of daratumumab. Neutrophils isolated from healthy donors or patients with MM were cocultured with the indicated MM cell lines or autologous MM cells in presence of daratumumab for the evaluation of trogocytosis and ADCC. For the analysis of trogocytosis by flow cytometry, MM cells were labeled with either PKH67 or PKH26 prior to coculture. (A) Cocultures of neutrophils derived from healthy donors (n = 5) and the MOLP-8 cell line for trogocytosis and ADCC evaluation. Left: after 4 hours of coculture, PKH67+CD138– events were assessed within the CD66b+ compartment by flow cytometry for trogocytosis analysis. Right: the percentage of dead MM cells (annexin V+, 7AAD+) within the CD38me+CD66b– compartment (CD38me: anti-CD38 multi-epitope) was assessed by flow cytometry for ADCC analysis. (B) Analysis of trogocytosis (left) and ADCC (right) in cocultures of neutrophils derived from patients with MM (n = 5) and the MOLP-8 cell line following the same methodology as in panel A. (C) Evaluation of daratumumab-mediated patient neutrophil cytotoxicity on autologous myeloma cells. Neutrophils and myeloma cells from 2 patients were isolated and subsequently cocultured in absence or presence of daratumumab for 4 hours. Cytotoxicity on myeloma cells was analyzed by flow cytometry as in panel A. (D) Representative images for trogocytosis by live-cell microscopy. DiO-labeled neutrophils (green) and DiI-labeled MOLP-8 cells (red) were cocultured (Effector:Target ratio of 3:1) in the presence of 1 μg/mL daratumumab. Time-lapse acquisition during the 4-hour coculture period, with images being captured every 3 minutes, was performed on a Nikon Eclipse TE-2000 microscope (Nikon, Tokyo, Japan). (E-G) Analysis of daratumumab-induced trogocytosis (left) and ADCC (right) in cocultures of neutrophils derived from healthy donors (n = 3) and RPMI-8226, NCI-H929 or U266 cell lines, respectively, following the same methodology as in panel A. (H) Analysis of trogocytosis (left) and ADCC (right) in cocultures of neutrophils derived from healthy donors (n = 3) and the indicated cell lines following the same methodology as in panel A but using PKH26 for cell labeling. In panels A,B,E-H, data represent the mean ± standard error of the mean (SEM). Means were compared using one-way analysis of variance and Tukey post hoc tests. ∗P < .05 and ∗∗∗∗P < .0001. Whenever “basal death” is indicated, it means the death of myeloma cells in basal culture conditions (without neutrophils and drug). The remaining conditions correspond to coculture of neutrophils and myeloma cells.

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