Barcode‐Enabled Sequencing of Plasmablast Antibody Repertoires in Rheumatoid Arthritis

Abstract

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. In this study we 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 datasets 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 characterized using CCP2 ELISA and antigen microarrays. Results We used our sequencing method to generate phylogenetic trees representing the antibody repertoires of peripheral blood plasmablasts of 4 individuals with anti-CCP+ RA, and recombinantly expressed 14 antibodies that were either “singleton” antibodies or representative of clonal antibody families. CCP2 ELISA identified four ACPAs, and antigen microarray analysis identified ACPAs that differentially targeted epitopes on α-enolase, citrullinated fibrinogen, and citrullinated histone 2B. Conclusions Our data provide evidence that autoantibodies targeting α-enolase, citrullinated fibrinogen, and citrullinated histone 2B are produced by the ongoing activated B cell response in, and thus may contribute to the pathogenesis of, RA.

Antibody repertoire analysis in polygenic autoimmune diseases

“…Switching occurred primarily to IgG and IgA 114. Evidence for class‐switched autoreactive B‐cells in RA is also supported by other studies,105, 109 where ~25% of synovial IgG+ B‐cells were reactive to citrullinated autoantigens in ACPA‐positive RA patients,109 and production of ACPAs by RA peripheral blood IgG+ plasmablasts was found in ACPA+ RA patients, but not ACPA− RA or psoriatic arthritis patients 94. Tan et al .…”

“…Autoantibody production in patients with RA is well described. Rheumatoid factor and anti‐citrullinated protein antibodies (ACPA)94, 95 are the most well studied. Infiltrating lymphocytes have been identified in the majority of synovial tissue samples in patients with RA 96, 97.…”

“…In about 10% of patients, these manifest as large follicle‐like structures, known as synovial germinal centres 98, 99. Furthermore, studies have shown that the proportion of peripheral blood plasmablasts is positively correlated with disease activity in RA,94, 100 and these have been found to produce ACPAs 94, 95. In a manner redolent of SLE and MS, IGHV gene polymorphisms associate with susceptibility to RA, most notably in IGHV1‐69 101, 102.…”

“…examined B‐cell specificities in RA using DNA barcoding and sequencing of heavy‐ and light‐chains of antibodies expressed by plasmablasts. The antigen‐specificities of antibodies encoded by IgG+ plasmablasts from patients with RA were tested using microarrays from recombinantly expressed antibodies 94. They showed that representative antibodies from patients with RA were able to bind cyclic citrullinated peptides from epitopes on α ‐enolase, citrullinated fibrinogen and citrullinated histone H2B in an ongoing B‐cell response in RA.…”

Неугодная Иммунология

“…Y. Tan et al [118] методом ДНК-штрих-кодирования (DNA barcoding method) на примере от-ветов на подтипы вируса гриппа серотипа H3N2 пока-зали, что вакцинация индуцирует ответы со стороны В-клеток памяти, продуцировавших высокоаффинные антитела в отношении подтипов вирусов предыдущих сезонных вспышек болезни. Они считают, что для уклонения от антигенного импринтинга необходимо проводить вакцинацию с учетом иммунологической истории индивидуума (immune history of individuals).…”