Abstract
Primary immune thrombocytopenia (ITP) is an acquired form of thrombocytopenia caused by IgG anti-platelet autoantibodies and represents an organ-specific autoimmune disorder. Although the glycoprotein (GP)IIb/IIIa and GPIb/IX have been shown to be targets for autoantibodies, the antigen specificity of autoantibodies is not fully elucidated. To identify the characteristics of IgG B-cell receptor (BCR) repertoires in ITP, we took advantage of adaptor-ligation PCR and high-throughput DNA sequencing methods for analyzing the clone-based repertoires of IgG-expressing peripheral blood B cells. A total of 2,009,943 in-frame and 315,469 unique reads for IGH (immunoglobulin heavy) were obtained from twenty blood samples. Comparison of the IGHV repertoires between patients and controls revealed an increased usage of IGHV4–28 in ITP patients. One hundred eighty-six distinct IGHV4–28-carrying sequences were identified in ITP patients and the majority of these clones used an IGHJ4 segment. The IGHV4–28/IGHJ4-carrying B-cell clones were found in all ITP patients. Oligoclonal expansions of IGHV4–28/IGHJ4-carrying B cells were accompanied by multiple related clones with single amino substitution in the CDR3 region suggesting somatic hypermutation. Taken together, the expansion of IGHV4–28/IGHJ4-carrying IgG-expressing B cells in ITP may be the result of certain antigenic pressure and may provide a clue for the immune pathophysiology of ITP.
Highlights
The principal pathophysiology of immune thrombocytopenia (ITP) is an IgG-mediated autoimmune disease, the B-cell receptor (BCR) repertoires associated with this disorder are largely unknown
Taking advantage of this novel method, we investigated the repertoires of IgG-BCRs of peripheral blood B cells from ITP patients in order to identify the characteristics of IgG-BCR repertoires in this disorder, and were able to find the oligoclonal expansions of IGHV4–28/IGHJ4-carrying IgG-expressing B cells with small clonal sizes
Fourteen other clones had CDR3 regions with a single amino acid substitution compared to that of clone #33. This oligoclonal expansion of particular B cells with IGHV4–28/IGHJ4-carrying IgG-BCR and the presence of multiple related clones with a single amino substitution in the CDR3 region were observed in two other patients AHK07G and AHK02G (Supplementary Tables 5 and 6). These findings strongly suggest that the expansion of B cells with IGHV4–28/IGHJ4-carrying IgG BCR may be the result of antigenic pressure leading to somatic hypermutation of CDR3
Summary
The principal pathophysiology of ITP is an IgG-mediated autoimmune disease, the B-cell receptor (BCR) repertoires associated with this disorder are largely unknown. High-throughput sequencing of BCR genes have revealed the landscape and longitudinal changes of B-cell repertoires and have identified clonal expansions[11,12,13,14,15,16,17,18]. Somatic hypermutation among antibody subclasses can be disclosed by this method. Taking advantage of this novel method, we investigated the repertoires of IgG-BCRs of peripheral blood B cells from ITP patients in order to identify the characteristics of IgG-BCR repertoires in this disorder, and were able to find the oligoclonal expansions of IGHV4–28/IGHJ4-carrying IgG-expressing B cells with small clonal sizes
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