Fig. 3.

CD22^−/− B cells respond to BLyS survival signals but undergo BCR-induced cell death. (A) BLyS enhances unstimulated CD22^−/− B-cell survival. Purified splenic B cells from wild-type and CD22^−/− mice were cultured with medium alone, with anti-IgM antibody (10 μg ml⁻¹) or with CD40 mAb (2 μg ml⁻¹). BLyS (5 ng ml⁻¹, filled bars) was added to the indicated cultures. Values represent the mean percentage (±SEM) of viable cells in triplicate cultures as determined by cell size and granularity (forward versus side light scatter) and 7-AAD exclusion in flow cytometry assays. (B) BLyS enhances CD22^−/− B-cell survival but does not reverse BCR-induced cell death. Purified splenic B cells from wild-type (WT, triangles) and CD22^−/− (circles) mice were cultured with anti-IgM antibody at the concentrations indicated (open symbols) or with anti-IgM antibody plus BLyS (5 ng ml⁻¹, closed symbols). Cellular ³H-thymidine incorporation during the final 18 h of 3-day cultures was quantified. Values represent mean counts per minute (±SEM) from triplicate wells in a representative experiment. (C) CD22^−/− B cells survive and proliferate with BLyS but die following BCR stimulation. B cells from wild-type (thin line) or CD22^−/− (thick line) mice were stained with CFSE and cultured for 3 days in medium alone, with anti-IgM antibody (10 μg ml⁻¹) or with CD40 mAb (2 μg ml⁻¹). BLyS (5 ng ml⁻¹) was added to the cultures as indicated (right panels). Cell viability and proliferation was determined by flow cytometry analysis based on forward versus side light scatter and CFSE dilution. (A–C) Data are representative of three independent experiments. Significant differences between sample means for each genotype are indicated; *P ≤ 0.05; **P ≤ 0.01.