Genetic analysis of autoantibodies in idiopathic thrombocytopenic purpura reveals evidence of clonal expansion and somatic mutation

Abstract

Although idiopathic thrombocytopenic purpura (ITP) is the most common autoimmune hematologic disorder, little is known about the associated autoantibodies on a molecular level. Consequently, diagnostic assays and therapy for ITP lack specificity. To avoid technical limitations imposed by B-cell immortalization methods, we used repertoire cloning (Fab/phage display) to clone platelet autoantibodies and examine the relation between immunoglobulin (Ig) gene usage, clonality, and antigen specificity. Phage display libraries were constructed from splenocytes from 2 patients with chronic ITP, and competitive cell-surface selection was used to isolate several dozen unique IgG platelet-specific autoantibodies. Platelet-reactive Fabs in both patients were associated almost exclusively with rearrangements of a single Ig heavy-chain variable-region gene (VH3-30), despite an apparent diversity of antigen specificities. Comparative analysis of platelet-reactive Fab Ig gene rearrangements from each patient suggested that they evolved from a restricted number of B-cell clones through somatic mutation with high replacement-to-silent mutation ratios. Although VH3-30–encoded heavy chains were found with light chains encoded by several different Ig genes, molecular repairing experiments showed exquisite restriction on the specific heavy- and light-chain pairings that permitted platelet reactivity. Together, these data suggest that the development of platelet-reactive antibodies associated with ITP is driven by an encounter with diverse platelet antigens through the clonal expansion of B cells using genetically restricted and highly specific combinations of heavy- and light-chain gene products. The extraordinarily high usage of the VH3-30 heavy-chain gene in these patients has implications for the pathogenesis, diagnosis, and management of chronic ITP.