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.

We used a DNA barcoding method to enable high-throughput sequencing of the cognate heavy- and light-chain pairs of expressed antibodies. We used this approach to elucidate the plasmablast antibody response to influenza vaccination. We show that >75% of the rationally selected plasmablast antibodies bind and neutralize influenza, and that antibodies from clonal families, defined by sharing both heavy chain VJ and light chain VJ sequence usage, do so most effectively. Vaccine-induced heavy chain VJ regions contained on average >20 nucleotide mutations as compared to their predicted germline gene sequences, and some vaccine-induced antibodies exhibited higher binding affinities for hemagglutinins derived from prior years’ seasonal influenza as compared to their affinities for the immunization strains. Our results show that influenza vaccination induces the recall of memory B cells that express antibodies that previously underwent affinity maturation against prior years’ seasonal influenza, suggesting that ‘original antigenic sin’ shapes the antibody response to influenza vaccination.

Objective The disease process in rheumatoid arthritis (RA) starts years before clinical diagnosis, and elevated disease-specific autoantibodies can be detected in this period. Early responses to known or novel autoantigens likely drive the eventual production of pathogenic autoimmunity. Importantly, the presence of disease-specific autoantibodies can identify individuals who are at high-risk for future RA onset but are currently without arthritis. The goal of the current studies is to characterize plasmablasts in these individuals. Methods We investigated the antibody-secreting plasmablasts of a well characterized cohort of individuals at-risk for RA based on serum RA-related autoantibody positivity (Ab+) in comparison to patients with early (<1 yr) seropositive RA and healthy controls. The plasmablast antibody repertoires of at-risk subjects were analyzed using DNA barcode-based methods with paired heavy- and light-chain gene sequencing. Cells were single-cell sorted prior to sequentially adding cell- and plate-specific DNA barcodes, followed by next-generation sequencing. Results Total plasmablast levels were similar in Ab+ individuals (1%) and controls (0.4–1.6%). However, increased frequencies of IgA+ vs. IgG+ plasmablasts were observed in Ab+ individuals (39% IgA+, 37% IgG+ plasmablasts) as compared to other groups (1–9% IgA+, 71–87% IgG+ plasmablasts). Paired antibody sequences from Ab+ subjects revealed cross-isotype clonal families and similar sequence characteristics between the IgA and IgG plasmablast repertoires. Ab+ individuals also demonstrated elevated serum levels of IgA isotype anti-CCP3 antibodies. Conclusion The IgA plasmablast dominance in these Ab+ individuals suggests that a subset of RA-related autoantibodies may arise from mucosal immune responses and be involved in early disease pathogenesis in individuals who are at-risk for developing RA.

Anti-cancer immunotherapy is encountering its own checkpoint. Responses are dramatic and long lasting but occur in a subset of tumors and are largely dependent upon the pre-existing immune contexture of individual cancers. Available data suggest that three landscapes best define the cancer microenvironment: immune-active, immune-deserted and immune-excluded. This trichotomy is observable across most solid tumors (although the frequency of each landscape varies depending on tumor tissue of origin) and is associated with cancer prognosis and response to checkpoint inhibitor therapy (CIT). Various gene signatures (e.g. Immunological Constant of Rejection - ICR and Tumor Inflammation Signature - TIS) that delineate these landscapes have been described by different groups. In an effort to explain the mechanisms of cancer immune responsiveness or resistance to CIT, several models have been proposed that are loosely associated with the three landscapes. Here, we propose a strategy to integrate compelling data from various paradigms into a “Theory of Everything”. Founded upon this unified theory, we also propose the creation of a task force led by the Society for Immunotherapy of Cancer (SITC) aimed at systematically addressing salient questions relevant to cancer immune responsiveness and immune evasion. This multidisciplinary effort will encompass aspects of genetics, tumor cell biology, and immunology that are pertinent to the understanding of this multifaceted problem.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0355-5) contains supplementary material, which is available to authorized users.

Our findings suggest that ACPA+ and RF+ B cells are imprinted with distinct transcriptional programs, which suggests that these autoantibodies associated with increased inflammation in RA arise from 2 different molecular mechanisms.

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.

Infection by Staphylococcus aureus is on the rise, and there is need for a better understanding of host immune responses that combat S. aureus. Here we use DNA barcoding to enable deep sequencing of the paired heavy- and light-chain immunoglobulin genes expressed by individual plasmablasts derived from S. aureus-infected humans. Bioinformatic analysis of the antibody repertoires revealed clonal families of heavy-chain sequences and enabled rational selection of antibodies for recombinant expression. Of the ten recombinant antibodies produced, seven bound to S. aureus, of which four promoted opsonophagocytosis of S. aureus. Five of the antibodies bound to known S. aureus cell-surface antigens, including fibronectin binding protein A. Fibronectin binding protein A-specific antibodies were isolated from two independent S. aureus infected patients and mediated neutrophil killing of S. aureus in in vitro assays. Thus, our DNA barcoding approach enabled efficient identification of antibodies involved in protective host antibody responses against S. aureus.

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.