Proteomic analysis of humoral Ro/La autoimmunity

Abstract

Burnet’s forbidden Clone Hypothesis, a corollary of Clonal Selection Theory, posits that the emergence of forbidden (self-reactive) clones of lymphocytes is causal for autoimmune disease. Modern formulations suggest a stochastic appearance of these clones via inherited and somatic mutations that enable a forbidden clone to bypass tolerogenic checkpoints. However, 50 years after Clonal Selection Theory there is limited direct evidence for forbidden clones reactive with authentic autoantigens in human systemic autoimmune disease. When humoral responses again the classical Ro/SSA autoantigens are considered, identification of Ro-specific clonotypes in whole sera is hampered by the supposed diversity and polyclonality of anti-Ro/La responses; termed the ‘clonotypic conundrum’. We have developed a novel proteomic approach that utilises epitope-selection combined with high resolution sequencing of variable (V)-regions to assess clonality, V-region usage and mutational status of anti-Ro52/Ro60 autoantibodies concealed in sera from lupus and Sjo¨gren’s syndrome patients. Remarkably, humoral responses to immunodominant epitopes of Ro52 and Ro60 are far more restricted than believed previously, share public B cell clonotypes, are heavily mutated, and are linked in some patients by the same germline clonotypes with unique heavy and light chain pairing signatures. These findings force a re-evaluation and perhaps re-introduction of the concept of forbidden clones in human systemic autoimmunity, a term that has been partly lost from the lexicon of autoimmunology. Clonal restriction of circulating pathogenic autoantibodies to a few dominant clonotypes that are public in unrelated patients has important diagnostic and therapeutic implications.