Selective blockade of B7-H1/CD80 interaction by anti–B7-H1 mAbý clone 43H12. (A) 293T cells transfected with mock or mouse B7-H1–encoding plasmids were stained with 1 μg/mL anti–B7-H1 monoclonal antibody (mAb) clone 43H12 (black histogram) or control rat immunoglobulin (IgG; gray histogram) followed by fluorescein isothiocyanate (FITC)–conjugated anti–rat IgG. Binding of 43H12 to B7-H1 was analyzed by flow cytometry. (B) ELISA plate was coated with 2 μg/mL mouse B7-H1-Fc (●), mouse CD80-Fc (○), mouse B7-DC-Fc (□), mouse B7-H3-Fc (▴), or mouse B7-H4-Fc (♦) fusion proteins. Indicated doses of 43H12 were added into wells and its binding with the coated proteins were detected by horseradish peroxidase (HRP)–conjugated anti–rat IgG antobody (Ab). Average ± SD of optical density (O.D.) from triplicate wells are shown. (C) 293T cells transfected with plasmids encoding mock (gray histogram) or B7-H1 (black histogram) were incubated with 2 μg/mL biotin-conjugated CD80-Fc (left panels) or PD-1-Fc (right panels) fusion proteins in the presence of 2 μg/mL 43H12, 10B5, or control rat IgG. The staining of fusion proteins were detected by streptavidin-PE in flow cytometry. (D) 293T cells transfected with plasmids encoding B7-H1 were stained with CD80-Fc (○) or PD-1-Fc (●) fusion proteins in the presence of indicated doses of 43H12. Percentage of positively stained cells was assessed by flow cytometry. (E) T cells isolated from CD80-knockout (KO) mice were stimulated with anti-CD3 mAb together with immobilized B7-H1-Fc (■) or control human Fc (□) in the presence of soluble 43H12 or control rat IgG. Proliferation of the culture cells were assessed by 3H-thymidine incorporation. All experiments were repeated at least 3 times and representative data are shown.
