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. 2016 Jul 15;5(9):e1211220. doi: 10.1080/2162402X.2016.1211220

Figure 4.

Figure 4.

Enhancement of primary NK cell effector functions by bispecific immunoligands and triplebodies. Cytotox assays. Primary NK cells were purified from peripheral blood mononuclear cells (PBMC) of healthy donors by negative selection and were cultured with IL-2 (200 U/mL) and IL-15 (10 ng/mL) overnight before the experiments on the following day. For all experiments, each N represents independent healthy donor. (A, Upper panel) Purified NK cells were co-incubated with DiR labeled CD19+ (MEC1) and CD19− (HL60) cell lines at indicated effector to target (E:T) ratios either alone (•) or in presence of 10 nM ULBP2-aCD19 (▪) or ULBP2-aCD19-aCD19 (▴) immunoligand for 3 h and dead target cells were measured by 7-AAD staining on FACS. (A, lower lane) Purified NK cells were co-incubated with DiR labeled CD19+CD33+ cell lines (BV173 and SEM) at indicated effector to target (E:T) ratios either alone (•) or in presence of 100 nM ULBP2-aCD19 (▪), ULBP2-aCD33 (▾) or ULBP2-aCD19-aCD33 (▴) immunoligand for 3 h and dead target cells were measured by 7-AAD staining on FACS. For simplicity, selected statistical significances are shown in comparison with “No construct” group (*p < 0.05; **p < 0.01). Error bars indicate SEM (MEC1 (N = 4), HL60 (N = 3), BV173 (N = 5) and SEM (N = 4)). (B) Degranulation assay. Purified NK cells were co-incubated with MEC1 and SEM cells at E:T ratio of 2.5:1 either alone or in presence of indicated immunoligand for 6 h and CD107a/LAMP-1 staining within NK cells (stained and gated with anti-CD56 and anti-NKp46 antibodies) were measured to determine degranulated NK cell population. Error bars indicate SEM and ** represents p < 0.01; *** represents p < 0.001 (MEC1 (N = 3) and SEM (N = 5)) (C) ELISA-based IFNγ assay. Left: Purified NK cells were co-incubated with MEC1 cells at E:T ratio of 1:1 either alone or in presence of 10 nM immunoligand for 24 h and supernatant was collected for IFNγ detection by ELISA. IFNγ secretion by MEC1 cells (with or without immunoligand) was carefully controlled and was found to be negative (data not shown). Experiments were conducted with two independent NK donors and one example is shown where error bars indicate SEM of duplicates. (C, right) Purified NK cells were cultured in plate pre-coated with indicated immunoligands for 48 h and supernatant was collected for IFNγ detection by ELISA. Experiments were conducted with three independent NK cell donors and one example is shown where error bars indicate SEM of duplicates.