Affinity maturation and isotype switch in clonally related anti-erythrocyte autoantibodies

The NZB mouse is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. Molecular analysis of a panel of five anti-erythrocyte monoclonal antibodies (MoAb) derived from splenocytes of unimmunized NZB mice revealed that these autoantibodies all had functionally rearranged genes from the VH J558 family of immunoglobulin genes with closest homology to germline genes H10 and H30. Owing to clustering of nucleotide differences within the CDRs, compared with the germline, it was concluded that these antibodies were most likely generated by an antigen-driven mechanism. We report here further molecular analysis of two (4.16.1 and B4.13.2) of the panel of five anti-mouse erythrocyte producing hybridomas which are apparently clonally related. Nucleotide analysis of the light chain cDNA indicated that both antibodies had closest homology to germline gene V kappa 24 and use J kappa 2 gene. Determination of the functional affinities of the MoAb reveal that B4.13.2 (IgG2a) has a > 10-fold higher affinity for mouse erythrocytes when compared to 4.16.1 (IgG1). This finding supports the view that these two autoantibodies are generated by an antigen-driven mechanism. The proposed mechanism would involve the selection and expansion of a small population of B-lymphocytes by antigen leading to isotype switch, somatic mutation and increased affinity. Our data also point to the possibility that some framework residues may be involved in the binding to antigen.