Role of complement in B-cell depletion. (A) Various concentrations (2-50 μg/mL) of Rit m2a and 1F5 or their K322A mutant forms were bound to hCD20 Tg B cells before the addition of normal rat serum as a source of complement for 30 minutes at 37°C. Cell lysis was then determined by flow cytometry. Bars represent the mean and range for duplicate samples and are representative of 3 independent experiments. (B) hCD20 Tg (T) or non-Tg (NT) BALB/c mouse splenocytes were labeled with high or low levels of CFSE, respectively, pooled and injected into WT or C1q^−/− recipient mice; 24 hours later, mice were injected with various mAbs (100 μg, intravenously), and 16 hours after that splenocytes were labeled with anti-CD19 mAb and were assessed by flow cytometry for the relative proportion of surviving hCD20 Tg B cells expressed as the target:nontarget ratio. Bars represent mean and range for duplicate samples. Transgenic hCD20 BALB/c (C) or C57Bl/6 (D) mice received 250 μg of antihuman CD20 mAb (rituximab, Rit m2a, 1F5) or mAb lacking complement activity (K322A mutation) intravenously on day 0. The numbers of circulating B cells was then assessed from peripheral blood by CD19 and B220 staining and flow cytometry. The results are expressed as percentage of B cells observed at time 0, n ≥ 4.