title: 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 (V(H)3-30), 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 V(H)3-30-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 V(H)3-30 heavy-chain gene in these patients has implications for the pathogenesis, diagnosis, and management of chronic ITP.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, 19104, USA.
authors.forename: Jessica H
authors.initials: JH
authors.lastname: Roark
authors.forename: James B
authors.initials: JB
authors.lastname: Bussel
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Cines
authors.forename: Don L
authors.initials: DL
authors.lastname: Siegel
mesh_terms: D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D001323:Autoantibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D001792:Blood Platelets / Q000276:immunology*; D003001:Cloning, Molecular; D005260:Female; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology; D010980:Platelet Membrane Glycoproteins; D016553:Purpura, Thrombocytopenic, Idiopathic / Q000235:genetics* / Q000276:immunology*; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013154:Spleen / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; C032193:glycoprotein receptor GPIb-IX