No abstract

Objective. This study was undertaken to determine the molecular characteristics of clonotypic autoantibodies in the sera of patients with primary Sjögren's syndrome (SS). This characterization is hampered by the presence of mixed anti-Ro/La specificities that may conceal clonotypic species. In order to narrow clonotypic diversity, a positive selection step was performed on a peg-like determinant of Ro 60 (termed Ro 60-peg) prior to analysis of the autoantibody proteome.Methods. Monospecific anti-Ro 60-peg IgG were isolated by affinity purification from the sera of 7 patients with primary SS and anti-Ro/La and subjected to 2-dimensional gel electrophoresis and highresolution orbitrap mass spectrometric sequencing. V regions of heavy and light chains were analyzed by combined database and de novo amino acid sequencing.Results. Proteomic analysis revealed a Ro 60-peg-specific IgG1-restricted monoclonal autoantibody that was present in the sera of all patients and specified by a V H 3-23 heavy chain paired with a V 3-20 light chain. The public anti-Ro 60-peg clonotype was specified further by common mutations in the heavy-chain and light-chain complementarity-determining regions. Titers and relative affinities of clonotypic IgG did not vary over the course of the disease.Conclusion. The expression of a Ro 60-reactive public B cell clonotype in a subset of patients with primary SS as a long-lived, class-switched circulating autoantibody implies a common breach of B cell tolerance checkpoints in these patients. The unique heavy chain/light chain signature opens the possibility of tracking the development of a "forbidden" clone against a bona fide systemic autoantigen in human disease.The 60-kd Ro/SSA autoantigen (Ro 60) is a major target of humoral autoimmunity in primary Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) and has been considered a primordial autoantigen involved in the initiation of human systemic autoimmunity (1). Pathogenic maternal anti-Ro 60 autoantibodies bind cognate antigen on apoptotic cells in the fetal heart and can initiate tissue damage in congenital heart block (2). While considerable effort has been focused on Ro 60 epitope recognition, and more recently on the mapping of Ro 60 apotopes (3-5), little is known about the molecular characteristics of the anti-Ro 60 autoantibodies themselves. Analysis of the B cell receptor repertoire against a key human autoantigen such as Ro 60 will lead to a better understanding of how B cell tolerance is perturbed in systemic autoimmunity, and may lead to novel therapeutic strategies aimed at removing autoreactive B cell clones.

SummaryThe La/SSB autoantigen is a major target of long-term humoral autoimmunity in primary Sjögren's Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti-Ro60/Ro52/La responses target an NH2-terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high-resolution Orbitrap mass spectrometry to determine the clonality, isotype and V-region sequences of LaA-specific autoantibodies in seven patients with primary SS. Anti-LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope-specific affinity chromatography were analysed by combined database and de-novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa-restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3-30 heavy chain paired with IGKV3-15 light chain and the second by an IGHV3-43/IGKV3-20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity-determining regions, consistent with a common breach of B cell tolerance followed by antigendriven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein-RNA complexes are mediated by public sets of autoreactive B cell clonotypes.