Fig 1.
Human Ab1-10 mAb recognizes M2e on animal cells.
(A) 293FT cells (left) and M2-293FT cells (right) were cell surface stained with the human anti-M2 Ab1-10 mAb (upper panel) and the mouse anti-M2 14C2 mAb (lower panel) and analyzed by flow cytometry. Shaded histograms represent unstained cells. The binding of the isotype controls (dotted line) and anti-M2e specific mAbs (black line) is shown. (B) Uninfected A549 (left) and influenza infected (right) A549-H1N1 cells were cell surface stained with the human anti-M2 Ab1-10 mAb. The binding of secondary Ab alone (grey histogram) and the binding of Ab1-10 mAb (black line histogram) is shown.
Fig 2.
The anti-M2e Ab1-10 mAb induces freshly isolated NK cell-mediated ADCC.
(A) Effectors (freshly isolated NK cells) and targets (293FT and M2-293FT cells) were co-incubated at different ratios either in the presence or absence of Ab1-10 mAb (Ab). The percentage of specific lysis is shown. Error bars represent the standard error of the mean (SEM) from five independent experiments with freshly isolated NK cells from five donors (* P<0.05, ** P<0.01). (B) Influenza infected A549 cells were co-cultured with human NK cells from healthy donors in the absence or presence of Ab1-10 mAb (Ab). The percentage of lysis is shown. Error bars represent the SEM from four independent experiments with freshly isolated NK cells from four donors (* P<0.05).
Fig 3.
Cytokine release by freshly isolated NK cells in the presence of Ab1-10 mAb.
Freshly isolated NK cells were either left untreated or co-cultured with both 293FT cells and M2-293FT cells in the presence and absence of Ab1-10 mAb (Ab). Culture supernatants were harvested and tested for the secretion of human cytokines and chemokines using flow-cytometric bead analysis. The values on the y-axis correspond to the concentrations of TNF-α, IFN-γ, MIP-1α, MIP-1β and RANTES in pg/ml. Graph bars represent the average ± SEM. Data shown are from five independent experiments with freshly isolated NK cells from five donors (* P<0.05, ** P<0.01).
Fig 4.
The anti-M2e Ab1-10 mAb induces cytokine-preactivated NK cell-mediated ADCC.
293FT or M2-293FT cells were used as targets and control (cultured with low concentrations of IL-15) NK cells (A) and cytokine-preactivated (pretreated with IL-12, IL-15 and IL-18) NK cells (B) were used as effector cells in an ADCC assay. Effector and target cells were co-incubated at different ratios either in the presence or absence of Ab1-10 mAb (Ab). The percentage of specific lysis is shown. Error bars represent the SEM from four independent experiments with NK cells from four donors.
Fig 5.
Cytokine release by cytokine-preactivated NK cells in the presence of Ab1-10 mAb.
Control (cultured with low concentrations of IL-15) NK cells and cytokine-preactivated (pretreated with IL-12, IL-15 and IL-18) NK cells were co-cultured with both 293FT cells and M2-293FT cells in the presence and absence of Ab1-10 mAb (Ab). The cell culture supernatants were harvested and subjected to cytometric bead analysis for cytokine release measurements. The values on the y-axis correspond to the concentrations of TNF-α, MIP-1α, MIP-1β and RANTES in pg/ml. Graph bars represent the average ± SEM. Data shown are from independent experiments five independent experiments with NK cells from five donors (* P<0.05, ** P<0.01 ****P<0.0001).