AFFINITY MATURATION AND ISOTYPE SWITCH IN CLONALLY RELATED ANTIERYTHROCYTE 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. Molec ular analysis of a panel of five anti-erythrocyte monoclonal antibodie s (MoAb) derived from splenocytes of unimmunized NZB mice revealed tha t these autoantibodies all had functionally rearranged genes from the V-H J558 family of immunoglobulin genes with closest homology to germl ine genes H10 and H30. Owing to clustering of nucleotide differences w ithin the CDRs, compared with the germline, it was concluded that thes e 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 whi ch are apparently clonally related. Nucleotide analysis of the light c hain cDNA indicated that bath antibodies had closest homology to germl ine gene V(kappa)24 and use J(kappa)2 gene. Determination of the funct ional affinities of the MoAb reveal that B4.13.2 (IgG2a) has a > 10-fo ld higher affinity for mouse erythrocytes when compared to 4.16.1 (IgG 1). This finding supports the view that these two autoantibodies are g enerated by an antigen-driven mechanism. The proposed mechanism would involve the selection and expansion of a small population of B-lymphoc ytes by antigen leading to isotype switch, somatic mutation and increa sed affinity. Our data also point to the possibility that some framewo rk residues may be involved in the binding to antigen.