Internalization of B Cell Receptors in Human EU12 μHC+ Immature B Cells Specifically Alters Downstream Signaling Events
BCR internalization reduces Ca2+ influx upon restimulation. (a) Schematic representation of different stimulation conditions for EU12 μHC+, Daudi, and Ramos cells with goat anti-IgM antibody F(ab′)2 fragments. (b) EU12 μHC+, Daudi, and Ramos cells were prepared as outlined in (a). EU12 μHC+, Daudi, and Ramos cells were either directly stimulated with goat F(ab′)2 anti-IgM antibodies (10 μg/mL or 20 μg/mL) or pretreated with goat anti-IgM antibody F(ab′)2 fragments (2 μg/mL) for 30 min to internalize BCRs, washed to remove excessive anti-IgM antibodies, recovered for 4 h, and then restimulated with goat anti-IgM antibody F(ab′)2 fragments (10 μg/mL or 20 μg/mL). Baseline fluorescence was established with the mock experiments without addition of stimulatory antibodies. Arrows indicate the time points when antibodies were added. Results shown are representative of three independent experiments.