Severe hypochromic microcytic anemia caused by a congenital defect of the iron transport pathway in erythroid cells.

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 immunoglobu-lin 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 Figure 1. Lack of CD20 modulation on malignant B cells following rituximab treatment in vitro in the presence or absence of plasma. (A) Specificity of the mouse anti-rituximab idiotype (anti-Id) mAb, MB2A4. Following binding of various mouse/human chimeric mAbs (10 ␮g/mL) to EHRB or Raji cells for 15 minutes at room temperature (mAb [target]; 2B8 [mouse Ab against CD20]; rituximab [CD20]; chAT80 [CD20]; chWR17 [CD37]; chAT13/5 [CD38]; and chLOB7-4/7-6 [CD40]), cells were washed twice in phosphate-buffered saline/bovine serum albumin (BSA)/Azide before being stained with FITC-labeled mAb MB2A4 (10 ␮g/mL) for 15 minutes at room temperature and being analyzed by flow cytometry. As can be seen, binding of 2A4 was observed only on cells coated with rituximab or its parent mAb 2B8, demonstrating both the ability …