Coligation of BCR and FcγRIIB does not increase BLyS receptor expression and attenuates BCR-induced effects. (A) Proliferation and death profiles of BALB/c CD23⁺ B cells 72 hours after stimulation. B cells were loaded with CFSE and cultured with indicated stimuli (concentrations of stimuli are indicated in “B-cell subset isolation, culture, and stimulation”). 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. (B) CD23⁺ B cells were cultured with the indicated stimuli. After 24 hours, the cells were harvested and analyzed by flow cytometry for BR3 and TACI expression (BCMA expression was negligible) on live cells. Gray histograms indicate isotype control; unfilled histograms, unstimulated cell expression levels; filled black histograms, expression by stimulated cells. Median fluorescence intensities (MFI) of these populations are shown in bar graphs at bottom. (C) CD23⁺ B cells were cultured with the indicated stimuli. After 36 hours, the cells were harvested and analyzed by flow cytometry for BR3 and TACI expression (BCMA expression was negligible) on live cells. Gray histograms indicate isotype control staining; unfilled histograms, unstimulated cell expression levels; filled black histograms, stimulated cell expression levels. Populations' median fluorescence intensities (MFI) are shown in bar graphs at bottom. Plots are representative of at least 3 experiments; in all cases, n equals 3 or more. Significance levels are indicated by P values.