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We read with interest the paper from Jilani et al 1 in which rituximab treatment appeared to down-modulate CD20 expression through a combination of internalization and RNA regulation. The result is unexpected because previous studies had shown that CD20 is not modulated by monoclonal antibody (mAb) treatment, 2-4 even in vivo. 5 The study by Jilani et al used an anti-mouse immunoglobulin polyclonal antibody (Ab) that binds to the mouse V regions in rituximab. This reagent will bind to rituximab, while the chimeric antibody is coated onto CD20 ϩ cells. We are concerned that the apparent loss of binding reported may be due to factors other than loss of CD20 expression-specifically blocking by normal immunoglobulin.As part of our ongoing CD20 studies, we have developed a mouse anti-idiotype (Id) mAb (2A4) specific for the V regions of rituximab and its parent Ab 2B8 (M.S.C. and M.C.B., manuscript submitted, March 2004). This reagent binds rituximab or 2B8 while it is coated onto CD20 ϩ cells ( Figure 1A). Using this highly specific reagent we can find no evidence that CD20 is down-modulated on malignant B cells in either the presence or absence of plasma ( Figure 1B). Using the anti-Id mAb (MB2A4), together with a human Fc␥-specific mAb, we examined the ability of rituximab and its murine counterpart 2B8 to bind to CD20 on Raji cells and fresh B-cell chronic lymphocytic leukemia (B-CLL) cells with and without plasma. In these experiments, cells were incubated for 2 hours at 37°C as described by Jilani et al, before the cells were washed twice by centrifugation and stained for surface CD20 mAb. Interestingly, when using fluorescein isothiocyanate (FITC)-conjugated anti-Fc␥ mAb, our

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