FIGURE 4.

Natural killer (NK) cells enhance benralizumab-induced eosinophil (Eos) depletion by macrophages (Mac). Human macrophages were derived from peripheral blood monocytes after 7 days stimulation with 50 ng·mL⁻¹ macrophage colony-stimulating factor. Primary NK cells and eosinophils were isolated from autologous healthy donors, and each cell type was labelled individually. Eosinophils were pre-treated with 10 nM of indicated antibodies, then labelled with far-red lipid membrane dye and co-incubated with green-labelled macrophages and/or red-labelled NK cells in the presence of PO-PRO-1. Cells were mixed for 6 h and analysed by flow cytometry or confocal microscopy for live cell imaging. a) Left: representative image depicting increased phagocytic activity of macrophages (engulfment of six eosinophils) in the presence of NK cells upon benralizumab treatment (interacting eosinophils are delineated by white dashed lines); right: quantification of eosinophils engulfed by individual macrophages post-treatment with the indicated antibodies via live imaging (n=5). Scale bar: 2 µm. b) Quantification of eosinophil death (macrophage-engulfed and PO-PRO-1⁺ eosinophils) by live imaging, as percentage of total far-red-labelled eosinophils (n=3), in the presence of macrophages and/or NK cells. c) Changes in caspase activation assessed by flow cytometry using CellEvent Caspase-3/7 (Casp3/7) reagent and represented as percentage of total CD66b⁺ Siglec-8⁺ eosinophils (n=3). d) Representative image of significant immune synapses between NK cells and macrophages. A representative highly activated macrophage is delineated by the green dashed line. Scale bar: 2 µm. a–c) Data are mean±sem. Two-way ANOVA with Tukey's multiple comparisons. *: p<0.05; ***: p<0.001; ****: p<0.0001, benralizumab versus parent fucosylated anti-interleukin-5 receptor α (PF anti-IL-5Rα). ⁺: p<0.05; ⁺⁺⁺: p<0.001, Eos+Mac+NK versus Eos+Mac. Statistical values are presented in supplementary table S2.