Type I mAb treatment results in modulation of CD20 from the cell surface both in vivo and in vitro. (A) hCD20 Tg B cells transferred into nondepleting γ^−/− mice as in Figure 1C were assessed 16 hours after mAb treatment for CD20 surface expression by detecting bound mAb with RPE-labeled anti–mouse Fc. Red indicates Rit m2a; blue, tositumomab; solid, background. Top histogram: CD20 expression on B cells from untreated mice labeled with Rit m2a and tositumomab ex vivo; bottom histogram, CD20 expression after treatment with Rit m2a or tositumomab in vivo. Bar chart: CD20 expression after treatment with type I and type II mAb. Bars represent mean ± SD; n ≥ 3mice, one of at least 3 experiments. *Rit m2a and 1F5 significantly differ from FGM6 and tositumomab (P < .001). (B) Surface CD20 expression on peripheral B cells in hCD20 Tg γ-chain^−/− mice after mAb treatment (250 μg); red, Rit m2a; blue, tositumomab; solid, background; black, untreated. (C) Loss of surface CD20 in vitro. Isolated splenic hCD20 Tg B cells were incubated with Rit m2a or tositumomab (10 μg/mL) before detecting surface CD20. Bars represent mean and range for 2 experiments each performed in triplicate. (D) Phagocytic potential is reduced by treatment of CD20 Tg B cells with Rit m2a. Cells were treated for 16 hours with mAbs, labeled with CFSE, incubated with bone marrow-derived macrophages, and then APC-F4/80 labeled before flow cytometry to detect double-positive macrophages (left hand panel) or confocal microscopy (right hand panel) as described in “Methods” using an HCX PL APO lambda blue 63×/1.4 oil immersion lens with 1.7× optical zoom. Sixteen-hour incubation with Rit m2a but not tositumomab caused loss of CD20 and resulted in a reduction in the number of double-positive macrophages. *P < .001; ns indicates not significant. Bars represent mean ± SEM of triplicate samples, one of 3 similar experiments. Right hand panel shows typical image of macrophages capturing and engulfing CFSE-labeled B cells coated with tositumomab.