Polyreactive binding of antibodies generated by polyclonal B cell activation. II. Crossreactive and monospecific antibodies can be generated from an identical Ig rearrangement by differential glycosylation.

Abstract

In this work the authors tested the hypothesis that differential glycosylation may be one of the mechanisms by which B cells could be able to produce monospecific or polyreactive antibodies flexibly. The same genetic information will be used in both situations. To prove this hypothesis the authors used mice transgenic for the SP6 (mu + kappa) hybridoma cell line producing monoclonal antibodies (MoAbs) with specificity for the hapten 2,4,6-trinitrophenyl (TNP). In these animals most cells in the periphery express exclusively the transgene SP6. To obtain polyreactive antibodies (Abs) spleen cells from transgenic animals were stimulated in vitro with lipopolysaccharide (LPS). The authors used LPS derived B cell blasts to produce a hybridoma cell collection. A high proportion of the monoclonal antibodies were found to bind to TNP and to crossreact with unrelated antigens. Endogenous immunoglobulins (Igs) were not responsible for crossreactivity since the crossreactive clones only expressed the transgene products. This was demonstrated by polymerase chain reaction (PCR) amplification of cDNA and by sequencing analysis of the PCR products. The nucleotide sequences of the expressed mono- and crossreactive genes were identical to the sequences of the rearranged V(H) and V(K) SP6 which undoubtedly demonstrates that crossreactive Igs are derived from the same rearrangement and also that no mutations in the V(H) or V(K) or in the CDR3 could account for the observed crossreactivity. It is also shown here that the crossreactive antibodies bear the idiotype Id 20-5 characteristic of SP6 binding Abs. Crossreactive monoclonal antibodies were found to be slightly more glycosylated than the TNP-monospecific Abs. Furthermore, binding to TNP was not influenced by treatment with tunicamycin, an inhibitor of glycosylation, while, in the same molecule, other types of binding were considerably reduced. This supports the hypothesis of the importance of glycosylation in polyreactivity.