Figure 1.

(A) Simultaneous binding of rituximab (200 nM) and the immunoligands ULBP2:7D8 (10 µM) and B7-H6:7D8 (25 µM) to Ramos cells was analyzed by 2-color flow cytometry. On the basis of pilot tests, the molecules were applied at distinct concentration ratios for compensation of different binding and cytotoxic activities (data not shown). Secondary antibodies conjugated to APC and FITC were used for detection of rituximab and the immunoligands, respectively. (B) Dose-dependent binding of rituximab (left panel) and ULBP2:7D8 (right panel) when either applied alone or in combination. As a control an isotype matched antibody was combined with the similar constructed immunoligand (ImLig) binding HER2 (MFI, mean fluorescence intensity). (C) Impact of B7-H6:7D8 on binding of rituximab (left) or vice versa (right). Data points indicate mean values ± SEM obtained in three independent experiments.