FIGURE 5.

Sandy-5 and 5A8 stimulate trimeric mouse BAFF. A, hBAFFR:Fas reporter cells were exposed to titrated amounts of size-fractionated FLAG-mBAFF trimers in the presence of a fixed concentration (1 μg/ml) of the indicated anti-mBAFF or anti-EDA (EctoD3) mAbs. After 16 h of culture, cell viability was monitored with the phenazine methosulfate/3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Antibodies whose combined presence with FLAG-mouse BAFF caused more cell death than FLAG-mouse BAFF with the control EctoD3 antibody were considered to be activating antibodies. B, primary mouse B splenocytes were cultured for 72 h in the presence of the indicated concentrations of size-fractionated trimeric FLAG-mBAFF with or without inhibitors or activators at 1 μg/ml. Cell viability was determined by FACS as described under “Experimental Procedures.” Error bars represent S.E. of triplicate measures. The experiment was performed twice (one in monoplicate) with similar results. C and D, wild type mice were treated weekly during a 6-week period with i.p. injections of control (EctoD1) or anti-mBAFF (Sandy-5 or 5A8) antibodies at 2 mg/kg. At the end of the treatment period (day 42), lymphocytes of the spleen and lymph nodes were analyzed by FACS to determine ratios of mature B cells to T cells in the spleen (C) and of B cells to T cells in lymph nodes (D). Error bars represent S.E. One-way analysis of variance was performed: *, p < 0.05; **, p < 0.01; ns, not significant.