Glycosylation of immunoglobulin-G secreted by murine hybridomas: Its repertoire and influence upon antibody dependent cytotoxicity mediated by natural killer lymphocytes

A panel of hybridomas was assembled to assess the effect of genetic and biological influences upon glycosylation of IgG. Glycopeptides from IgG were characterized chromatographically by size, charge and lectin affinity. Six glycosylation phenotypes were found to be common amongst all clones. Despite this heterogeneity, considerable similarity exists in glycosylation of IgG secreted by different clones. Virtually all IgG glycopeptides are core fucosylated. Secondly, the majority of the glycosylation repertoire is described by two phenotypes. Of these, the predominantly expressed one is the same for all clones. It is characterized by neutral glycopeptides of size class IV (M$\sb{\rm r}$ 2000) which are of high affinity for Lens culinaris, but of low affinity for Concanavalin A. The second most prevalent phenotype differs by glycopeptides of high affinity for Concanavalin A. Significant variability between different hybridomas exists in the relative expression of these two major phenotypes. Other differences between clones may reflect the expression of an additional site which is glycosylated differently. Furthermore, a generational variability in glycosylation is apparent upon continuous culturing of the same hybridoma. But differences in culturing conditions have limited influence upon glycosylation. Inhibitors of glycosylation (tunicamycin) and carbohydrate processing were used to probe the functional consequences of specific alterations in glycosylation of IgG secreted by the same hybridoma clone. Neither the absence of glycosylation nor the presence of an altered phenotype significantly influenced binding of the monoclonal antibody to cell surface antigen. However, cytotoxicity, assayed by chromium release, was enhanced significantly when target cells were sensitized with the altered phenotypes induced by castonospermine, N-methyldeoxynojirimycin, deoxymannojirimycin or monensin, but not by swainsonine. The enhanced cytotoxicity was mediated specifically by natural killer lymphocytes, and not by monocytes or polymorphonuclear leukocytes. Enhanced lymphocyte mediated cytotoxicity was found to be Fc receptor dependent. Furthermore, this cytotoxicity correlates with the expression of nonfucosylated phenotypes of IgG. Using a panel of monoclonal antibodies directed against several tumors, the castanospermine-induced phenotype generally was found to enhance tumoricidal activity mediated by natural killer lymphocytes. However, a functional heterogeneity amongst natural killer lymphocytes is suggested by differences in lymphocyte response to an alteration in IgG glycosylation