These findings demonstrate that established RA is characterized by a persistent IgA ACPA response that exhibits ongoing affinity maturation. This observation suggests the presence of a persistent mucosal antigen that continually promotes the production of IgA plasmablasts and their affinity maturation and epitope spreading, thus leading to the generation of ACPAs that bind additional citrullinated antigens and more potently stimulate macrophage production of TNF.

Objective. Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). While epitope spreading of the serum ACPA response is believed to contribute to RA pathogenesis, little is understood regarding how this phenomenon occurs. This study was undertaken to analyze the antibody repertoires of individuals with RA to gain insight into the mechanisms leading to epitope spreading of the serum ACPA response in RA.Methods. Plasmablasts from the blood of 6 RA patients were stained with citrullinated peptide tetramers to identify ACPA-producing B cells by flow cytometry. Plasmablasts were single-cell sorted and sequenced to obtain antibody repertoires. Sixty-nine antibodies were recombinantly expressed, and their anticitrulline reactivities were characterized using a cyclic citrullinated peptide enzyme-linked immuosorbent assay and synovial antigen arrays. Thirty-six mutated antibodies designed either to represent ancestral antibodies or to test paratope residues critical for binding, as determined from molecular modeling studies, were also tested for anticitrulline reactivities.Results. Clonally related monoclonal ACPAs and their shared ancestral antibodies each exhibited differential reactivity against citrullinated antigens. Molecular modeling identified residues within the complementarity-determining region loops and framework regions predicted to be important for citrullinated antigen binding. Affinity maturation resulted in mutations of these key residues, which conferred binding to different citrullinated epitopes and/or increased polyreactivity to citrullinated epitopes.Conclusion. These results demonstrate that the different somatic hypermutations accumulated by clonally related B cells during affinity maturation alter the antibody paratope to mediate epitope spreading and polyreactivity of the ACPA response in RA, suggesting that these may be key properties that likely contribute to the pathogenicity of ACPAs.

Lyme disease (Borrelia burgdorferi infection) is increasingly recognized as a significant source of morbidity worldwide. Here, we show that blood plasmablasts and CD27− memory B cells are elevated in untreated Lyme disease, with higher plasmablast levels associated with more rapid resolution of clinical symptoms. Stronger serum reactivity to surface proteins and peptides from B. burgdorferi was also associated with faster resolution of clinical symptoms. Through molecular identifier-enabled antibody heavy-chain sequencing of bulk B cells and single-cell paired-chain antibody sequencing of blood plasmablasts, we characterized immunoglobulin gene usage patterns specific to B. burgdorferi infection. Recombinantly expressed antibodies from expanded lineages bound B. burgdorferi antigens, confirming that these clones are driven by the infection. Furthermore, recombinant sequence-derived antibodies were functional, inhibiting growth of B. burgdorferi in vitro. Elevations and clonal expansion of blood plasmablasts were associated with rapid return to health, while poor plasmablast responses were associated with a longer duration of symptoms following treatment. Plasmablasts induced by B. burgdorferi infection showed preferential antibody gene segment usage, while bulk sequencing of total B cells revealed convergent CDR3 motifs specific to B. burgdorferi-infected patients. Our results show that robust plasmablast responses encoding Bb-static antibodies are associated with more rapid resolution of Lyme disease, and these antibodies could provide the basis for next-generation therapeutics for Lyme disease.

BackgroundPreeclampsia (PE) is a life‐threatening hypertensive disorder of pregnancy associated with autoantibodies, termed AT 1‐AA, that activate the AT 1 angiotensin receptor. Although the pathogenic nature of these autoantibodies has been extensively studied, little is known about the molecular cause of their generation.Methods and ResultsHere we show that tissue transglutaminase (TG2), an enzyme that conducts posttranslational modification of target proteins, directly modified the 7‐amino acid (7‐aa) epitope peptide that localizes to the second extracellular loop of the AT 1 receptor. These findings led us to further discover that plasma transglutaminase activity was induced and contributed to the production of AT 1‐AA and disease development in an experimental model of PE induced by injection of LIGHT, a tumor necrosis factor superfamily member. Key features of PE were regenerated by adoptive transfer of purified IgG from LIGHT‐injected pregnant mice and blocked by the 7‐amino acid epitope peptide. Translating our mouse research to humans, we found that plasma transglutaminase activity was significantly elevated in PE patients and was positively correlated with AT 1‐AA levels and PE features.ConclusionsOverall, we provide compelling mouse and human evidence that elevated transglutaminase underlies AT 1‐AA production in PE and highlight novel pathogenic biomarkers and innovative therapeutic possibilities for the disease.

BackgroundThe pathogenesis of essential hypertension is multifactorial with different underlying mechanisms contributing to disease. We have recently shown that TNF superfamily member 14 LIGHT (an acronym for homologous to lymphotoxins, exhibits inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes, also known as TNFSF14) induces hypertension when injected into mice. Research reported here was undertaken to examine the role of transglutaminase (TGase) in LIGHT‐induced hypertension.Methods and ResultsInitial experiments showed that plasma and kidney TGase activity was induced by LIGHT infusion (13.91±2.92 versus 6.75±1.92 mU/mL and 19.86±3.55 versus 12.00±0.97 mU/10 μg) and was accompanied with hypertension (169±7.16 versus 117.17±11.57 mm Hg at day 14) and renal impairment (proteinuria, 61.33±23.21 versus 20.38±9.01 μg/mg; osmolality, 879.57±93.02 versus 1407.2±308.04 mmol/kg). The increase in renal TGase activity corresponded to an increase in RNA for the tissue TGase isoform, termed TG2. Pharmacologically, we showed that LIGHT‐induced hypertension and renal impairment did not occur in the presence of cystamine, a well‐known competitive inhibitor of TGase activity. Genetically, we showed that LIGHT‐mediated induction of TGase, along with hypertension and renal impairment, was dependent on interleukin‐6 and endothelial hypoxia inducible factor‐1α. We also demonstrated that interleukin‐6, endothelial hypoxia inducible factor‐1α, and TGase are required for LIGHT‐induced production of angiotensin receptor agonistic autoantibodies.ConclusionsThus, LIGHT‐induced hypertension, renal impairment, and production of angiotensin receptor agonistic autoantibodies require TGase, most likely the TG2 isoform. Our findings establish TGase as a critical link between inflammation, hypertension, and autoimmunity.

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The presence of maternal autoantibodies has been previously associated with pre-eclampsia, although the composition of the antibody repertoire in pre-eclampsia has not been well characterized. Given this, we applied bacterial display peptide libraries to identify peptides that preferentially react with plasma antibodies from patients with pre-eclampsia (n=15) versus healthy-outcome pregnancies (n=18). Screening using fluorescence activated cell sorting identified 38 peptides that preferentially bind to antibodies from individuals with pre-eclampsia. These pre-eclampsia specific peptides possessed similar motifs of RG/SG/−WWG/S, RWWG/S, or WGWGXXR/K distinct from the angiotensin II type 1 receptor epitope AFHYESQ. Seven library-isolated peptides and a cell-surface displayed type 1 receptor epitope were used to construct a diagnostic algorithm with a training set of 18 new pre-eclamptic and 22 healthy-outcome samples from geographically distinct cohorts. Cross-validation within the training group resulted in averaged areas underneath a receiver operating characteristic curve of 0.78 and 0.72 with and without the known receptor epitope, respectively. In a small validation set (pre-eclamptic = 12, healthy = 8), the algorithm consisting only of library-isolated peptides correctly classified 10 pre-eclamptic and 6 healthy, using a predefined cutoff that achieved 61% sensitivity (95%CI: 36–83%) at 95% specificity (95%CI: 77–100%) in training set (n=40) cross-validation. Our results indicate that antibodies with specificities other than anti-angiotensin II type 1 receptor are prevalent in pre-eclampsia patients and may be useful as diagnostic biomarkers.