Analysis of B-cell activation by FcRH1 ligation. (A) Concomitant FcRH1 ligation enhances BCR-induced calcium flux. Daudi B cells were labeled with the calcium indicator dye Fluo-4 and, a reference dye, SNARF-1, and calcium levels were evaluated by flow cytometry before and after stimulation. Thin black line indicates streptavidin crosslinker alone (20 μg/mL). Thick black line indicates biotinylated F(ab′)₂ fragments of goat anti–human μ HC (2 μg/mL) plus streptavidin. Thick gray line indicates biotinylated F(ab′)₂ fragments of goat anti–human μ HC (2 μg/mL), biotinylated Fab fragments of anti-FcRH1 (5A3; 1 μg/mL), and streptavidin. (B) Ligation induced FcRH1 tyrosine phosphorylation. HA-tagged FcRH1 overexpressing mouse IIA1.6 cells were serum-starved for 2 hours before incubation with biotinylated Fab fragments of anti-FcRH1 mAb plus streptavidin (20 μg/mL). Cell lysate (200 μg) was immunoprecipitated with anti-HA antibody, and the immunoprecipitates were immunoblotted with either anti-phosphotyrosine antibody (anti-pTyr) or anti-FcRH1 antibody. (C) FcRH1 ligation induces DNA synthesis. Purified tonsillar B cells were incubated in 96-well plates (10⁵/well) for 72 hours in the presence or absence of varying concentrations of biotinylated Fab fragments of anti-FcRH1 or control mAbs plus streptavidin (20 μg/mL). Cells were pulsed for an additional 16 hours with ³H-thymidine (1 μCi/well [0.037 MBq/well]) before measuring ³H-thymidine incorporation. (D) FcRH1 coligation enhances BCR-induced B-cell proliferation. Tonsillar B cells were incubated in 96-well plates (10⁵/well) for 72 hours in the presence or absence of anti-μ HC antibody (DA4.4; 1 μg/mL), biotinylated Fab fragments of anti-FcRH1 mAbs (3 μg/mL) plus streptavidin (20 μg/mL), or the combination of both antibodies. Cells were analyzed as in panel C.