Surface IgM of Chronic Lymphocytic Leukemia Cells Displays Unusual Glycans Indicative of Antigen Engagement In Vivo.

Abstract 55

Several lines of evidence support the idea that surface immunoglobulin M (sIgM) plays a key role in determining the clinical behavior of chronic lymphocytic leukemia (CLL). For example, the presence of somatic mutations in immunoglobulin variable region genes is a strong prognostic marker with unmutated CLL (U-CLL) associated with a poor outcome relative to mutated CLL (M-CLL). U-CLL also generally express higher levels of sIgM and retain the ability to signal via this receptor. In this study, we used surface biotinylation to analyse sIgM in CLL and discovered that it exists in two forms with differing mobility on SDS-PAGE. Treatment with glycosidases revealed that these forms were due to different N-glycosylation patterns in the μ constant region. One form is similar to that of normal B cells in bearing mature complex glycans common to most cell surface glycoproteins. The other is an immature mannosylated form more characteristic of endoplasmic reticulum (ER)-located μ chains. CLL cells expressed variable proportions of the immature mannosylated form and quantitative analysis demonstrated that, on average, the proportion of mannosylated sIgM was approximately 2-fold higher (p=0.006) in U-CLL compared to M-CLL. Although normal B cells isolated from blood expressed only the mature form of sIgM, in vitro treatment with anti-μ resulted in upregulation of the immature form, suggesting that glycan modification is a consequence of antigen exposure. Consistent with this, in vitro incubation of CLL cells was associated with increased expression of the mature form of sIgM. Phosphotyrosine analysis demonstrated that both forms of sIgM were able to signal following sIgM engagement in vitro. Taken together, these findings support the concept that CLL cells are continuously exposed to antigen in vivo. This process leads to a change in the N-glycosylation pattern of the re-expressed sIgM so that a mannosylated form predominates, especially in U-CLL. Strikingly, expression of mannosylated sIgM is also characteristic of follicular lymphoma, where it is constitutively displayed via N-glycosylation sites in the Ig variable region (Radcliffe et al. J Biol Chem. 2007; 282, 7405-15). Persistent mannosylation of sIgM appears to be a feature common to several B-cell malignancies, suggesting a role in pathogenesis.