Fig. 4.

CD22^−/− B cells consume endogenous BLyS and express BLyS receptors. (A) In vivo B-cell depletion leads to increased serum BLyS levels. C57BL/6 mice were given B-cell-depleting CD20 mAb (n = 4, open squares) or control mAb (n = 4, closed circles) at time 0, with serum harvested at the indicated time points. Serum BLyS concentrations were determined by ELISA. (B) Serum BLyS levels are equivalent in wild-type, CD22^−/− and CD22Δ1-2 mice. Bars represent mean (±SEM) BLyS concentrations in 8-week-old mice as determined by ELISA. (C) Spleen B cells from CD22^−/− and CD22Δ1-2 mice express all three BLyS receptors. Relative BR3, TACI and BCMA transcript levels for wild-type, CD22^−/− and CD22Δ1-2 B cells were compared by real-time PCR analysis with endogenous CD20 transcripts used as the internal standard. Transcript levels in wild-type mice were normalized to a value of 1 (dashed line) with relative mean (±SEM) fold-differences shown for CD22^−/− and CD22Δ1-2 B-cell transcripts (n = 3). (A–C) Significant differences between sample means are indicated; *P ≤ 0.05; **P ≤ 0.01. (D) Activated CD22^−/− and CD22Δ1-2 B cells up-regulate BR3 expression. Spleen B cells from wild-type, CD22^−/− and CD22Δ1-2 mice were cultured for 17 h in medium alone (thin line) or with anti-IgM antibody (10 μg ml⁻¹, thick line). Viable cells were assessed for cell surface BR3 expression by immunofluorescence staining with flow cytometry analysis. Histograms represent the results of three independent experiments.