Figure 2.

Dara induces primary NK cell fratricide, which occurs via non-traditional ADCC between NK cells in the absence of tumor targets. A, The expression of cleaved (C)-CASP3 and C-PARP1 were detected by immunoblotting after NK cells were treated for 16 h with various doses of Dara as indicated. B, NK cells were treated for 16 h with 10 μg/mL of Dara, then stained with an anti-Annexin V antibody and SYTOX Blue viability dye. NK cell apoptosis was analyzed by flow cytometry (n=5). Each line in the panel on the right indicates survival of Dara-treated vs. untreated NK cells from the same individual donors. C, NK cells were treated for 4 h with 10 μg/mL of Dara, then expression of CD107a, an NK degranulation marker, was analyzed by flow cytometry (n=4). Each line in the panel on the right indicates Dara-treated vs. untreated NK cells from the same individual donors. D, Dara-mediated NK cell fratricide (both effector cells and target cells are NK cells) was performed using a standard 4-h ⁵¹Cr-release assay (n=3). E, A 4-h flow cytometry-based cytotoxicity assay was performed (Effector/Target is 1:1; n=9). NK cells serving as target cells were labeled with CFSE and pre-treated with or without Dara or Elo, an anti-CS1 monoclonal antibody, for 30 min. Effector NK cells (effector) were pre-treated with F(ab)₂ fragments of Dara or Elo for 30 min to block binding of intact Dara or Elo to CD38 or CS1, respectively, thus preventing fratricide among the effector cells. Target cells were gated from CFSE⁺ events, and cell death was detected by flow cytometric analysis via staining with an anti-Annexin V antibody and SYTOX Blue viability dye. Each line in the panel on the right compares target alone vs. target+effector group from the same individual donors. F, Expression of cleaved(C)-CASP3 and C-PARP-1 were detected by immunoblotting after 16 h treatment with 10 μg/mL of intact Dara or Dara enzyme-digested by an IgG-specific protease (IdeZ). G, NK cells were treated for 16 h with 10 μg/mL of Dara or enzyme-digested Dara, then apoptosis was analyzed (n=9) as described in (B). H, Schematic detailing mechanism for Dara-induced NK cell fratricide. CASP3, Caspase 3; Dara, daratumumab; Elo, elotuzumab; Error bars, S.D; N.S., not significant; *, P< 0.05; ***, P< 0.001.