Abstract
To assess the potency of low-affinity anti-red blood cell (RBC) autoantibodies in the induction of anemia, we generated an immunoglobulin (Ig)G2a class-switch variant of a 4C8 IgM anti-mouse RBC autoantibody, and compared its pathogenic potential with that of its IgM isotype and a high-affinity 34-3C IgG2a autoantibody. The RBC-binding activity of the 4C8 IgG2a variant was barely detectable, at least 1,000 times lower than that of its IgM isotype, having a high-binding avidity, and that of the 34-3C IgG2a monoclonal antibody (mAb). This low-affinity feature of the 4C8 mAb was consistent with the lack of detection of opsonized RBCs in the circulating blood from the 4C8 IgG2a-injected mice. However, the 4C8 IgG2a variant was highly pathogenic, as potent as its IgM isotype and the 34-3C IgG2a mAb, due to its capacity to interact with Fc receptors involved in erythrophagocytosis. In addition, our results indicated that the pentameric form of the low-affinity IgM isotype, by promoting the binding and agglutination of RBCs, is critical for its pathogenic activity. Demonstration of the remarkably high pathogenic potency of low-affinity autoantibodies, if combined with appropriate heavy chain effector functions, highlights the critical role of the Ig heavy chain constant regions, but the relatively minor role of autoantigen-binding affinities, in autoimmune hemolytic anemia.
Highlights
Incubation of mouse RBCs with 0.1 ng of the 4C8 IgM or 34-3C IgG2a monoclonal antibody (mAb) resulted in substantial binding, whereas Ͼ250 ng of the IgG2a switch variant was required to show significant binding, with a maximal binding at 1 g (Fig. 1)
It should be mentioned that the staining intensity obtained by the 4C8 IgG2a variant was far lower than that observed with the 4C8 IgM or the 34-3C IgG2a mAb
These results indicated that the 4C8 mAb has a low binding affinity, while its binding activity was markedly promoted by the pentameric high-avidity IgM isotype
Summary
The VDJH4C8-C␥2a plasmid containing the complete 4C8 IgG heavy chain gene of the IgG2a isotype was constructed using the following DNA fragments: the rearranged VDJ region isolated from cDNA encoding the variable region of the heavy chain of the 4C8 mAb [16], the promoter region isolated from pSV-V1 [17], the heavy chain enhancer region isolated from pSVE2-neo [18], and the C␥2a region derived from the genomic clone, pIgH10 [19]. MAbs. Hybridomas secreting the 4C8 IgM and 34-3C IgG2a anti–mouse RBC mAbs were derived from unmanipulated NZB mice [2, 4]. The 4C8 IgG2a class-switch variant was obtained by transfecting 4C8 heavy chain loss mutant cells by electroporation with the VDJH4C8-C␥2a plasmid together with a pSVE2-neo plasmid containing the neomycin-resistant gene. The purity of IgG and IgM was Ͼ90% as documented by SDS-PAGE
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