BCR and FcγRIIB coaggregation are required for the attenuation of BLyS receptor expression. (A) CD23+ B cells were cultured with biotinylated anti-IgM, biotinylated anti-FcγRIIB, or both, and cross-linked with avidin as described in “B-cell subset isolation, culture, and stimulation.” After 24 hours, the cells were harvested and analyzed by FACS for BR3 and TACI expression on live cells. Fluorescence histograms are shown at the top, and MFIs are shown in bar graphs at the bottom. (B) CD23+ B cells from BALB/c or C57BL/6 mice were cultured with the indicated stimuli. Proliferation and death profiles of CD23+ B cells from BALB/c or C57BL/6 mice are shown at 72 hours after stimulation. B cells were loaded with CFSE and cultured with indicated stimuli. One minute before analysis, TO-PRO-3 was added as a vital dye. CFSE profiles of live (TO-PRO-3−) cells are shown at right and F(ab′)2 anti-IgM histograms shaded and overlaid to highlight effects of anti-IgD reagents. (C) CD23+ B cells from BALB/c or C57BL/6 mice were analyzed by FACS for BLyS binding or anti-BLyS receptor staining on live cells at 24 hours after stimulation. Gray histograms indicate isotype control staining; unfilled histograms, unstimulated cell levels; filled black histograms, stimulated cell levels. Median fluorescence intensities (MFI) of populations are shown in bar graphs at the bottom. Plots are representative of at least 3 experiments; in all cases, n equals 3 or more. Significance levels are indicated by P values.
