Author: Rhianna Lindop
Lindop, Rhianna, 2013 PROTEOMIC CHARACTERISATION OF AN IMMUNODOMINANT 60-kDa Ro/SSA AUTOANTIBODY IN PRIMARY SJOGREN'S SYNDROME, Flinders University, School of Medicine
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The Ro (SSA)/La (SSB) ribonucleoprotein complex is a frequent target of long-lived humoral autoimmunity in primary Sjogren's syndrome (SS), systemic lupus erythematosus and the neonatal lupus syndrome. High-titer IgG autoantibodies to Ro60 are a serological hallmark of primary SS and have been proposed as a key event in the initiation of human systemic autoimmunity. Whilst most research on Ro60 has been focused on mapping of Ro60 epitopes, little is known about the molecular characteristics of the anti-Ro60 autoantibodies themselves. Evidence for the existence of forbidden (autoreactive) B cell clones and secreted clonotypic autoantibodies in human systemic autoimmune diseases is limited. This thesis exploits the recent finding of an immunodominant epitope present on Ro60 (termed Ro60peg) to characterise a Ro60-specific autoantibody proteome in patients with primary SS. A proteomic approach based on high resolution Orbitrap mass spectrometry (MS) was utilised to determine the clonality, isotype, and variable-region sequences of human autoantibodies directed Ro60peg in 7 patients with primary SS. Anti-Ro60peg IgGs purified from polyclonal sera by epitope-specific chromatography were analysed by 2-dimensional gel electrophoresis followed by combined database and de novo mass spectrometric sequencing. Remarkably, anti-Ro60peg-specific autoantibody responses comprised an IgG1 kappa restricted monoclonal species that was shared (public) across unrelated patients and specified by a VH3-23 heavy (H) chain paired with a VK3-20 light (L) chain. The public anti-Ro60peg clonotype was specified further by common mutations in the H chain and L chain complementarity determining regions. Further studies using high resolution Orbitrap MS were performed to track the evolution of the VH3-23/ VK3-20 Ro60peg public clonotypic autoantibody in 4 patients with primary SS. Direct sequencing of variable-region molecular signatures of clonotypes purified from serial serum samples collected retrospectively over 10 years revealed sequential clonal replacement. Prospective longitudinal studies confirmed clonotypic loss and replacement at approximately 3 monthly intervals. Levels of secreted anti-Ro60 clonotypes fluctuated markedly over time, despite minimal changes in affinity. In conclusion, the mass spectrometric sequencing of a secreted anti-Ro60 autoantibody proteome has revealed a class-switched clonotypic autoantibody that is common (public) to different patients and specified by a unique H and L chain pairing. The expression of this somatically mutated public B cell clonotype implies a common breach of B cell tolerance checkpoints in patients with primary SS followed by antigen-driven clonal selection. These findings provide evidence in humans for Burnet's 'forbidden' clone hypothesis. The application of proteomic technology to track the evolution of the secreted anti-Rop60peg autoantibody proteome in serial serum samples demonstrates a dynamic process of repeated clonal turnover that masquerades as long-lived Ro60 humoral autoimmunity.
Keywords: autoimmunity,autoantobodies,Sjogren's syndrome,B cells
Subject: Clinical Immunology thesis, Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Prof. Tom Gordon