Figure 2.

For figure legend, see page 5.Figure 2. See previous page ULBP2:7D8 and B7-H6:7D8 boost rituximab-induced ADCC. (A) Cytotoxicity against MEC2 and GRANTA-519 cells induced by single agents and by two-drug combinations of rituximab with either ULBP2:7D8 or B7-H6:7D8. MNC were used as effector cells at an E:T cell ratio of 40:1. Data points represent mean values ± SEM from at least three different experiments. Trastuzumab was employed as a control. Synergistic effects were graded into very strong synergy (+++++, CI < 0.1) strong synergy (++++, CI = 0.1 − 0.3) and synergy (+++, CI = 0.3 – 0.7). Statistically significant differences between groups treated with the single agents or the combinations are indicated (*, p < 0.05). (B) NK cells were purified by MACS technology and analyzed as effector cells (E:T cell ratio: 10:1) for combinations between rituximab and ULBP2:7D8 or B7-H6:7D8. Ramos cells served as target cells. (++++, CI = 0.1 − 0.3; +++, CI = 0.3 – 0.7; *, p < 0.05). Please note that cytotoxic effects of rituximab and B7-H6:7D8 were previously published.²⁹ (C) Isobologram analysis demonstrating synergy of immunoligands and rituximab in NK cell-mediated killing of Ramos cells. The doses of ULBP2:7D8 (left panel) and B7-H6:7D8 (right panel) resulting in 20% (ED20) lysis were plotted against equally effective doses of rituximab. The diagonal line connecting the ED20 values of the two agents (CI = 1) indicates theoretical combination doses which would be required to achieve equal effects, if additive effects were assumed (expected ED20 for CI = 1). Synergy between rituximab and the immunoligands is indicated by the experimentally determined combination doses locating below the corresponding additivity lines (antagonism would have been indicated by combination doses falling above the additivity lines). (D) Lysis of Ramos lymphoma and MEC2 CLL cells by NK cells homozygously expressing the FcγRIIIa V/V or F/F allotype at amino acid position 158 in the presence of rituximab and the immunoligands. Data points represent mean values ± SEM from triplicate determinations. (E) Cytotoxicity against Ramos cells induced by three-drug combinations of rituximab, ULBP2:7D8 and B7-H6:7D8 (Note: the data depicted in 2B and 2E were obtained in experiments performed in parallel, but were presented in different graphs for illustrative reasons).