Barcode‐Enabled Sequencing of Plasmablast Antibody Repertoires in Rheumatoid Arthritis
Objective A hallmark of rheumatoid arthritis (RA) is the production of autoantibodies, including anti–citrullinated protein antibodies (ACPAs). Nevertheless, the specific targets of these autoantibodies remain incompletely defined. During an immune response, B cells specific for the inciting antigen...
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Published in | Arthritis & rheumatology (Hoboken, N.J.) Vol. 66; no. 10; pp. 2706 - 2715 |
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Main Authors | , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Wiley Subscription Services, Inc
01.10.2014
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Subjects | |
Online Access | Get full text |
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Summary: | Objective
A hallmark of rheumatoid arthritis (RA) is the production of autoantibodies, including anti–citrullinated protein antibodies (ACPAs). Nevertheless, the specific targets of these autoantibodies remain incompletely defined. During an immune response, B cells specific for the inciting antigen(s) are activated and differentiate into plasmablasts, which are released into the blood. We undertook this study to sequence the plasmablast antibody repertoire to define the targets of the active immune response in RA.
Methods
We developed a novel DNA barcoding method to sequence the cognate heavy‐ and light‐chain pairs of antibodies expressed by individual blood plasmablasts in RA. The method uses a universal 5′ adapter that enables full‐length sequencing of the antibodies' variable regions and recombinant expression of the paired antibody chains. The sequence data sets were bioinformatically analyzed to generate phylogenetic trees that identify clonal families of antibodies sharing heavy‐ and light‐chain VJ sequences. Representative antibodies were expressed, and their binding properties were characterized using anti–cyclic citrullinated peptide 2 (anti–CCP‐2) enzyme‐linked immunosorbent assay (ELISA) and antigen microarrays.
Results
We used our sequencing method to generate phylogenetic trees representing the antibody repertoires of peripheral blood plasmablasts from 4 individuals with anti‐CCP+ RA, and recombinantly expressed 14 antibodies that were either “singleton” antibodies or representative of clonal antibody families. Anti–CCP‐2 ELISA identified 4 ACPAs, and antigen microarray analysis identified ACPAs that differentially targeted epitopes on α‐enolase, citrullinated fibrinogen, and citrullinated histone H2B.
Conclusion
Our data provide evidence that autoantibodies targeting α‐enolase, citrullinated fibrinogen, and citrullinated histone H2B are produced by the ongoing activated B cell response in, and thus may contribute to the pathogenesis of, RA. |
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Bibliography: | Drs. Sokolove and Robinson have received consulting fees from Atreca, Inc. (more than $10,000) and own stock or stock options in Atreca. Dr. Tan and Ms Kongpachith contributed equally to this work. Dr. Tan owns stock or stock options in Atreca, Inc. Drs. Tan, Sokolove, and Robinson are listed on patents (for a method to sequence antibody repertoires) owned by Stanford University and licensed to Atreca, Inc., for which they receive royalties. ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally to this work. |
ISSN: | 2326-5191 2326-5205 |
DOI: | 10.1002/art.38754 |