Citrullinated Protein Antibodies in Rheumatoid Arthritis
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ARTHRITIS & RHEUMATOLOGY Vol. 70, No. 12, December 2018, pp 1946–1958 DOI 10.1002/art.40587 © 2018, American College of Rheumatology Affinity Maturation Drives Epitope Spreading and Generation of Proinflammatory Anti–Citrullinated Protein Antibodies in Rheumatoid Arthritis Serra E. Elliott, Sarah Kongpachith, Nithya Lingampalli, Julia Z. Adamska, Bryan J. Cannon, Rong Mao, Lisa K. Blum, and William H. Robinson Objective. Rheumatoid arthritis (RA) is character- were observed (P < 0.01). Shared complementarity-deter- ized by the presence of anti–citrullinated protein antibod- mining region 3 sequence motifs were identified across ies (ACPAs); nevertheless, the origin, specificity, and subjects. A subset of the plasmablast lineages included functional properties of ACPAs remain poorly under- members derived from later time points with divergent stood. The aim of this study was to characterize the evolu- somatic hypermutations that encoded antibodies that tion of ACPAs by sequencing the plasmablast antibody bind an expanded set of citrullinated antigens. Further- repertoire at serial time points in patients with estab- more, these recombinant, differentially mutated plas- lished RA. mablast antibodies formed immune complexes that Methods. Blood samples were obtained at up to 4 stimulated higher macrophage production of tumor serial time points from 8 individuals with established RA necrosis factor (TNF) compared to antibodies represent- who were positive for ACPAs by the anti–cyclic citrulli- ing earlier time point–derived lineage members that were nated peptide test. CD19+CD3ÀIgDÀCD14ÀCD20À less mutated. CD27+CD38++ plasmablasts were isolated by single-cell Conclusion. These findings demonstrate that sorting and costained with citrullinated peptide established RA is characterized by a persistent IgA tetramers to identify ACPA-expressing plasmablasts. ACPA response that exhibits ongoing affinity matura- Cell-specific oligonucleotide barcodes were utilized, fol- tion. This observation suggests the presence of a per- lowed by large-scale sequencing and bioinformatics sistent mucosal antigen that continually promotes the analysis, to obtain error-corrected, paired heavy- and production of IgA plasmablasts and their affinity mat- light-chain antibody gene sequences for each B cell. uration and epitope spreading, thus leading to the gen- Results. Bioinformatics analysis revealed 170 eration of ACPAs that bind additional citrullinated persistent plasmablast lineages in the RA blood, of antigens and more potently stimulate macrophage pro- which 19% included multiple isotypes. Among IgG- duction of TNF. and IgA-expressing plasmablasts, significantly more IgA-expressing than IgG-expressing persistent lineages Rheumatoid arthritis (RA) is characterized by the Supported by the NIH (National Institute of Allergy and generation of autoantibodies, including rheumatoid factor Infectious Diseases grant 5T32-AI-07290-30 to Dr. Elliott and grants and anti–citrullinated protein antibodies (ACPAs) (1). In U19-AI-11049103, U19-AI-110491, and U01-AI-101981 to Dr. Robin- son and National Institute of Arthritis and Musculoskeletal and Skin the present study, we sequenced the plasmablast antibody Diseases grant R01-AR-063676 to Dr. Robinson). repertoire over serial time points in patients with estab- Serra E. Elliott, PhD, Sarah Kongpachith, Nithya Lingampalli, lished RA to gain insights into the evolution of ACPAs, Julia Z. Adamska, BS, Bryan J. Cannon, MSc, Rong Mao, PhD, Lisa K. Blum, PhD, William H. Robinson, MD, PhD: Stanford University, and to further define the role of ACPAs in promoting the Stanford, California, and VA Palo Alto Health Care System, Palo Alto, pathogenesis of RA. California. ACPAs target citrullinated epitopes arising from Dr. Robinson is a founder, member of the Board of Direc- tors, consultant for, and owner of equity in Atreca, Inc. posttranslational modifications of arginine to citrulline by Address correspondence to William H. Robinson, MD, PhD, peptidylarginine deiminase (PAD) (2). Epitope spreading Division of Immunology and Rheumatology, CCSR 4135, 269 Cam- of ACPAs precedes the onset of clinical symptoms in RA pus Drive, Stanford, CA 94305. E-mail: [email protected]. Submitted for publication November 22, 2017; accepted in (3). Previous studies have demonstrated that recombinant revised form June 12, 2018. murine ACPAs can increase the severity of arthritis in 1946 EPITOPE SPREADING IN RHEUMATOID ARTHRITIS 1947 mice (4,5). A therapeutic strategy of B cell depletion using lineages in the RA blood that persisted across serial time rituximab provides clinical benefit in patients with sero- points and revealed shared heavy-chain (HC) and light- positive RA (6), and its efficacy is associated with mild chain (LC) third complementarity-determining region reductions in circulating ACPA levels (7). A mucosal drive (CDR3) motifs across subjects. Using antigen microarrays has been postulated in the pathogenesis of RA (8). Cigar- and enzyme-linked immunosorbent assays (ELISAs), we ette smoking represents a risk factor for ACPA+ RA (9), demonstrated that, compared to plasmablast lineage mem- and ACPAs are detectable in the sputum and/or serum bers derived from earlier time points, a subset of lineage from subjects with early RA and individuals at risk for members from later time points that were differentially developing RA (10). mutated encoded antibodies targeting additional citrulli- ACPAs may contribute to the pathogenesis of RA nated antigens. In vitro macrophage stimulation assays by stimulating immune effector cells, including macro- revealed that these later time point–derived, affinity- phages (11), which produce tumor necrosis factor (TNF) matured, differentially mutated recombinant plasmablast and other cytokines (12). Macrophages can be activated antibodies generated ICs that more potently stimulated by proinflammatory cytokines, immune complexes (ICs), macrophage production of TNF, a mechanism that may be and Toll-like receptor (TLR) agonists (13). Studies from postulated to promote the pathogenesis of RA. our laboratory and others have shown that ICs composed of RA blood–derived ACPAs and citrullinated proteins PATIENTS AND METHODS stimulate macrophages, both from the blood (14) and from the synovial fluid (15), to produce TNF. These Human blood samples. After obtaining written informed ACPA ICs stimulate macrophages via Fcc receptor type II consent from all potential study subjects under protocols c – approved by the Stanford University Institutional Review Board, (Fc RII) (15 18) and TLR-4 (16,17). Nevertheless, the blood samples were collected in heparin tubes at serial time role of ACPAs and the mechanisms by which affinity mat- points, at intervals of ≥2 months apart, from 8 anti-CCP+ uration and the evolving ACPA repertoire contribute to patients with RA (Table 1). All subjects were recruited from the the development and persistence of RA remain unclear. VA Palo Alto Healthcare System (Palo Alto, California). In this study, we utilized our cell-barcoding anti- Patients with RA met the American College of Rheumatology 1987 and 2010 classification criteria (22,23). Monocyte-derived body repertoire sequencing method (19) to investigate the macrophages were generated from blood samples that were evolution of the ACPA B cell response in RA. Previous obtained from the Stanford Blood Center. Peripheral blood studies utilizing this approach have focused on the blood mononuclear cells (PBMCs) were isolated by density-gradient IgG repertoire at a single time point in patients with estab- centrifugation with Ficoll-Paque PLUS (GE Healthcare Life lished RA (19) or the IgA and IgG repertoire at a single Sciences). Single-cell sorting of plasmablasts. CD19+CD3ÀIgDÀ time point in subjects prior to RA development or in those CD14ÀCD20ÀCD27+CD38++ plasmablasts were single-cell with early RA (20). In this study, at up to 4 serial time sorted using a BD FACSAria (BD Biosciences) as described points, we sequenced plasmablasts, which have been shown previously (19,24) (for more details see Supplementary Meth- to produce ACPAs (19,21), from the blood of 8 individuals ods, available on the Arthritis & Rheumatology web site at with established RA who were positive for ACPAs by the http://onlinelibrary.wiley.com/doi/10.1002/art.40587/abstract). – + For staining of PBMCs, we used fluorophore-conjugated antibod- anti cyclic citrullinated peptide test (anti-CCP ). Our find- ies against CD19 (HIB19; BioLegend), CD3 (UCHT1; BD Bio- ings from bioinformatics analysis revealed plasmablast sciences), IgD (IA6-2; BD Biosciences), CD14 (MφP9; BD Table 1. Demographic and clinical characteristics of the study subjects with rheumatoid arthritis Number of tender and swollen joints* Age at T1, years Sex† T1 T2 T3 T4 Subject 1 63 F 18 14 (4 mos.) 7 (10 mos.) – Subject 2 61 M 5 2 (3 mos.) 3 (6 mos.) – Subject 3 60 M 4 10 (3 mos.) 22 (8 mos.) 18 (10 mos.) Subject 4 67 M 0 0 (4 mos.) –– Subject 5 76 M 0 10 (7 mos.) –– Subject 6 66 M 0 4 (12 mos.) –– Subject 7 83 M 10 1 (11 mos.) 12 (13 mos.) – Subject 8 74 M 5 4 (10 mos.) –– * The number of tender and swollen joints was summed at each time point (T1–T4), and the number of months (mos.) from initial sampling is indicated in parentheses. † The sex distribution at the VA Palo Alto Health Care System, where the samples were obtained, is predomi- nantly male. 1948 ELLIOTT ET AL Biosciences), CD20 (L27; BD Biosciences), CD27 (CLB-27/1; Life all antibodies were expressed on the human IgG1 Fc domain.