Restriction in V kappa gene use and antigen selection in anti-myeloperoxidase response in mice

Anti-neutrophil cytoplasmic Abs, directed primarily toward myeloperoxidase (MPO) and proteinase 3, are detected in the majority of patients with disti nct forms of small vessel vasculitides and pauci-immune necrotizing glomeru lonephritis. However, the origin of these autoantibodies remains unknown, W e studied the V region gene use in murine anti-MPO Abs derived from Spontan eous Crescentic Glomerulonephritis/Kinjoh mice. A total of 13 anti-MPO-prod ucing hybridomas were generated from four unimmunized mice. Ten of the 13 h ybridomas (corresponding to 3 of 4 clones) expressed V kappa 1C but differe d in their use of V-H genes. The remaining three hybridomas expressed a V k appa 5 gene. Anti-MPO hybridomas from individual mice were derived from sin gle clones as deduced by sequence similarity and splice-site identity. We f ound a statistically significant bias of amino acid replacement mutations t o the complementarity-determining regions (CDR) in the V kappa 1C-expressin g hybridomas. Intriguingly, all 10 V kappa 1C hybridomas share a lysine to glutamate mutation in the CDR1, To determine the effects of somatic V gene mutations on binding to MPO, we generated an anti-MPO Ab with an unmutated V kappa 1C L chain and compared its ability to bind MPO with its mutated co unterpart. The mutated hybridoma-derived Ab has a 4.75-fold higher avidity for MPO than the unmutated Ab, These results suggest that: 1) the L chain p lays a dominant role in determining Ab specificity to MPO, 2) the anti-MPO Ab response is oligoclonal, consistent with Ag selection, and 3) MPO is a d riving Ag in the murine anti-MPO Ab response.