Long-term Ro60 humoral autoimmunity in primary Sjögren's syndrome is maintained by rapid clonal turnover.

Long-term humoral autoimmunity to RNA-protein autoantigens is considered a hallmark of systemic autoimmune diseases. We use high resolution Orbitrap mass spectrometric autoantibody sequencing to track the evolution of a Ro60-specific public clonotypic autoantibody in 4 patients with primary Sjögren's syndrome. This clonotype is specified by a VH3-23/VK3-20 heavy and light chain pairing. Despite apparent stability by conventional immunoassay, analysis of V-region molecular signatures of clonotypes purified from serum samples collected retrospectively over 7years revealed sequential clonal replacement. Prospective longitudinal studies confirmed clonotype loss and replacement at approximately three-monthly intervals. Levels of secreted anti-Ro60 clonotypes fluctuated markedly over time, despite minimal changes in clonal affinity. Our novel findings indicate a relentless turnover of short-lived clonotypic variants, masquerading as long-lived Ro60 humoral autoimmunity.

Pathogenicity and proteomic signatures of autoantibodies to Ro and La.

Ro/SSA and La/SSB comprise a linked set of autoantigens that are clinically important members of the extractable nuclear antigen family and key translational biomarkers for lupus and primary Sjögren's syndrome. Autoantibodies directed against the Ro60 and La polypeptide components of the Ro/La ribonucleoprotein complex, and the structurally unrelated Ro52 protein, mediate tissue damage in the neonatal lupus syndrome, a model of passively acquired autoimmunity in humans in which the most serious manifestation is congenital heart block (CHB). Recent studies have concentrated on two distinct pathogenic mechanisms by which maternal anti-Ro/La autoantibodies can cause CHB: by forming immune complexes with apoptotic cells in developing fetal heart; and/or by acting as functional autoantibodies that cross-react with and inhibit calcium channels. Although the precise role of the individual autoantibodies is yet to be settled, maternal anti-Ro60 and anti-Ro52 remain the most likely culprits. This article will discuss the molecular pathways that culminate in the development of CHB, including the recent discovery of ß2 glycoprotein I as a protective factor, and present a proteomic approach based on direct mass spectrometric sequencing, which may give a more representative snapshot of the idiotype repertoire of these autoantibodies than genomic-based technologies.

Secreted human Ro52 autoantibody proteomes express a restricted set of public clonotypes.

Long-lived secreted autoantibody responses in systemic autoimmunity are generally regarded to be polyclonal and to express a diverse B-cell repertoire. Here, we have used a proteomic approach based on de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against a prototypic systemic autoantigen, Ro52 (TRIM21). Remarkably, anti-Ro52 autoantibodies from patients with primary Sjögren's syndrome, systemic lupus erythematosus, systemic sclerosis or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain paired with one of two closely-related heavy chains; and were public in unrelated patients. Targeted mass spectrometry using these uniquely mutated V region peptides as surrogates detected anti-Ro52 autoantibodies in human sera with high sensitivity and specificity compared with traditional ELISA. Mass spectrometry-based detection of specific autoantibody motifs provides a powerful new tool for analysis of humoral autoimmunity.

Molecular signature of a public clonotypic autoantibody in primary Sjögren's syndrome: a "forbidden" clone in systemic autoimmunity.

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 high-resolution 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 IgG1κ-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(κ) 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.