Fig 2: BAFF-R-IgG4mut treatment inhibits the elevation of BAFF serum levels during lupus development. Serum BAFF levels in BXSB mice after different treatments were measured by ELISA. Data are presented as mean ± S.E.M. of five mice in each group. *Statistically different when BAFF-R-IgG4mut group is compared with PBS or IgG4 group by Student’s t-test for independent samples (P < 0.05).

Fig 3: Characterization of BAFF-R-IgG4mut fusion protein. (A) SDS-PAGE analysis. A and B both are loaded with purified BAFF-R-IgG4mut. (B) Western blot analysis with anti-BAFF-R mAb. 1, negative control; 2, BAFF-R-IgG4mut fusion protein; M, molecular weight marker. (C) In vitro bioactivity assay. Purified mouse splenic B cells were stimulated with 5 µg/ml (left side) or 10 µg/ml (right side) of anti-IgM in the presence of 2 ng/ml of BAFF and indicated doses of BAFF-R-IgG4mut fusion protein. B-cell proliferation after 3 days was measured by 3H-thymidine incorporation in triplicates. B-cell thymidine incorporation by anti-IgM stimulation alone was labelled as a flat line in each side. BAFF-R-IgG4mut alone did not stimulate B-cell proliferation (data not shown).

Fig 4: BAFF-R-IgG4mut significantly suppresses B-cell expansion in the spleen and the peripheral blood of BXSB mice. (A) At 12 weeks of age, the percentages of viable B (B220+CD5-) and T (B220-CD5+) cells in the peripheral blood (upper panels) and the spleen (lower panels) of mice in each group were determined by flow cytometry. (B) The ratios of B (B220+CD5-) or T (B220-CD5+) cells versus the non-T non-B population (B220-CD5-) in the peripheral blood (upper panels) and the spleen (lower panels) of mice in each group were presented as bar graphs over time. Control groups, open bar; BAFF-R-IgG4mut groups, solid bar. Statistical significance was calculated by Student’s t-test.

Fig 5: BAFF-R-IgG4mut prevents the development of lupus nephritis. (A) Eight weeks old male BXSB mice (15 mice per group) were injected intraperitoneally with PBS or IgG4 as control, or 100 µg BAFF-R-IgG4mut three times a week for 5 weeks. The percentage of mice in each group that have developed proteinuria (>100 mg/dl) is shown. BAFF-R-IgG4mut treatment significantly delayed the onset of proteinuria by log-rank test (P < 0.01). (B) Serum levels of circulating anti-dsDNA antibodies are shown as A490 values from an ELISA. (C) BAFF-R-IgG4mut treatment significantly delayed host death by log-rank test (P < 0.01). Kaplan-Meier survival plot of treated animals is shown.