Restriction in Vκ Gene Use and Antigen Selection in Anti-Myeloperoxidase Response in Mice

Abstract

Anti-neutrophil cytoplasmic Abs, directed primarily toward myeloperoxidase (MPO) and proteinase 3, are detected in the majority of patients with distinct forms of small vessel vasculitides and pauci-immune necrotizing glomerulonephritis. However, the origin of these autoantibodies remains unknown. We studied the V region gene use in murine anti-MPO Abs derived from Spontaneous Crescentic Glomerulonephritis/Kinjoh mice. A total of 13 anti-MPO-producing hybridomas were generated from four unimmunized mice. Ten of the 13 hybridomas (corresponding to 3 of 4 clones) expressed Vκ1C but differed in their use of VH genes. The remaining three hybridomas expressed a Vκ5 gene. Anti-MPO hybridomas from individual mice were derived from single clones as deduced by sequence similarity and splice-site identity. We found a statistically significant bias of amino acid replacement mutations to the complementarity-determining regions (CDR) in the Vκ1C-expressing hybridomas. Intriguingly, all 10 Vκ1C 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 Vκ1C L chain and compared its ability to bind MPO with its mutated counterpart. 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 plays 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 driving Ag in the murine anti-MPO Ab response.

Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis, Large Vessel Vasculitis and Kawasaki Disease in Japan

“…sister matings [51] . SCG/Kj mice also develop anti-MPO antibody [56,57] and MPO acts as a driving antigen in the anti-MPO response observed in SCG/Kj mice [58] . Hamano et al [59] investigated related traits susceptible to aberrant production of MPO-ANCA by genome-wide quantitative trait locus mapping in SCG/Kj mice.…”

Maintenance of Tolerance by Regulation of Anti-myeloperoxidase B Cells

“…12 Interbreeding produced two groups of V1C-J5 Tg mice that differed in their relative number of copies of the transgene ( Figure 1B). Mice with the larger number of transgene copies are referred to as "high copy" (HC) mice and the other group as "low copy" (LC) mice.…”

“…12 Splenocytes from Tg mice were fused with P3X-Ag8.653 or NSObcl-2 myeloma cells. After fusion, tissue culture supernatants were screened for MPO binding by ELISA.…”

“…The diversity in light-chain and heavy-chain variable region gene usage was determined using the 5Ј Rapid Amplification of cDNA Ends system (5ЈRACE; Life Technologies) as described previously. 12 The GSP1 primers and nested GSP2 primers used for the kappa light chain and the Mu heavy chain were as follows: GSP1 5Ј-TAACTGCTCACTG-GATGGTGGGAAG-3Ј, GSP1 5Ј-CAGATTCTTATCAGACAG-3Ј, GSP2 5Ј-ATGGATACAGTTGGTGCAGCATCAG-3Ј, and GSP2 5Ј-GCTCTCGCAGGAGACGAG-3Ј.…”

“…Previously, we demonstrated a restriction in V gene use in anti-MPO-producing hybridomas derived from SCG/Kj mice. 12 This restriction suggests an important role of the Ig light chain in determining specificity to MPO.…”