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.

No abstract

In December 2018, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.99 days. ABSTRACTRationale: Accumulating evidence implicates inflammation in pulmonary arterial hypertension (PAH) and therapies targeting immunity are under investigation, though it remains unknown if distinct immune phenotypes exist.Objective: Identify PAH immune phenotypes based on unsupervised analysis of blood proteomic profiles. Methods and Results:In a prospective observational study of Group 1 PAH patients evaluated at Stanford University (discovery cohort, n=281) and University of Sheffield (validation cohort, n=104) between 2008-2014, we measured a circulating proteomic panel of 48 cytokines, chemokines, and factors using multiplex immunoassay. Unsupervised machine learning (consensus clustering) was applied in both cohorts independently to classify patients into proteomic immune clusters, without guidance from clinical features. To identify central proteins in each cluster, we performed partial correlation network analysis. Clinical characteristics and outcomes were subsequently compared across clusters. Four PAH clusters with distinct proteomic immune profiles were identified in the discovery cohort. Cluster 2 (n=109) had low cytokine levels similar to controls. Other clusters had unique sets of upregulated proteins central to immune networks-cluster 1 (n=58)(TRAIL, CCL5, CCL7, CCL4, MIF), cluster 3 (n=77)(IL-12, IL-17, IL-10, IL-7, VEGF), and cluster 4 (n=37)(IL-8, IL-4, PDGF-, IL-6, CCL11). Demographics, PAH etiologies, comorbidities, and medications were similar across clusters. Non-invasive and hemodynamic surrogates of clinical risk identified cluster 1 as high-risk and cluster 3 as low-risk groups. Five-year transplant-free survival rates were unfavorable for cluster 1 (47.6%, CI 35.4-64.1%) and favorable for cluster 3 (82.4%, CI 72.0-94.3%)(across-cluster p<0.001). Findings were replicated in the validation cohort, where machine learning classified four immune clusters with comparable proteomic, clinical, and prognostic features.Conclusions: Blood cytokine profiles distinguish PAH immune phenotypes with differing clinical risk that are independent of World Health Organization Group 1 subtypes. These phenotypes could inform mechanistic studies of disease pathobiology and provide a framework to examine patient responses to emerging therapies targeting immunity. Nonstandard Abbreviations and Acronyms: CI confidence interval 95% EC pulmonary artery endothelial cell IQR interquartile range 25-75% K cluster number in unsupervised consensus clustering MFI median fluorescence intensity mPAP mean pulmonary arterial pressure NT-proBNP N-terminal pro b-type natriuretic peptide PAH pulmonary arterial hypertension PVDOMICS Pulmonary Vascular Disease Phenomics Program PVR pulmonary vascular resistance REVEAL Registry to Evaluate Early and Long-term PAH Disease Management SMC pulmonary artery smooth muscle cell SQL structured query language TAPSE tricuspid annular pla...

Human rotaviruses (RVs) are the leading cause of severe diarrhea in young children worldwide. The molecular mechanisms underlying the rapid induction of heterotypic protective immunity to RV, which provides the basis for the efficacy of licensed monovalent RV vaccines, have remained unknown for more than 30 years. We used RV-specific single cell-sorted intestinal B cells from human adults, barcode-based deep sequencing of antibody repertoires, monoclonal antibody expression, and serologic and functional characterization to demonstrate that infection-induced heterotypic immunoglobulins (Igs) primarily directed to VP5*, the stalk region of the RV attachment protein, VP4, are able to mediate heterotypic protective immunity. Heterotypic protective Igs against VP7, the capsid glycoprotein, and VP8*, the cell-binding region of VP4, are also generated after infection; however, our data suggest that homotypic anti-VP7 and non-neutralizing VP8* responses occur more commonly in people. These results indicate that humans can circumvent the extensive serotypic diversity of circulating RV strains by generating frequent heterotypic neutralizing antibody responses to VP7, VP8*, and most often, to VP5* after natural infection. These findings further suggest that recombinant VP5* may represent a useful target for the development of an improved, third-generation, broadly effective RV vaccine and warrants more direct examination.

Toll-like receptor 9 (TLR9) promiscuously binds self-and microbial DNA, but only microbial DNA elicits an inflammatory response. How TLR9 discriminates between self-and foreign DNA is unclear, but inappropriate localization of TLR9 permits response to self-DNA, suggesting that TLR9 localization and trafficking are critical components. The molecular mechanisms controlling the movement of TLR9 may provide new insight into the recognition of DNA in normal and in pathological conditions such as autoimmune systemic lupus erythematosus. We have shown earlier that TLR9 is retained in the endoplasmic reticulum (ER) and it moves to endolysosomes to recognize CpG DNA. Other studies have suggested that TLR9 bypasses the Golgi complex to access endolysosomes. Here, we show that TLR9 translocates from ER to endolysosomes through the Golgi complex and that Golgi export is required for optimal TLR9 signaling. In all, 6-13% of TLR9 constitutively exits the ER, moves through the Golgi complex and resides in lysosomal-associated membrane protein-1-positive vesicles. TLR9 bound to CpG DNA had glycan modifications indicative of Golgi processing confirming that TLR9 travels through the Golgi complex to access CpG DNA in endolysosomes. Together, these data support a model where TLR9 uses traditional secretory pathways and does not bypass the Golgi complex. Keywords: brefeldin A; CpG DNA; endolysosome; Golgi; TLR9; trafficking Toll-like receptors (TLRs) are innate immune receptors important for host defense against pathogens. 1 Although many TLRs are present at the cell surface, those dedicated to recognition of various nucleic acid structures are retained in intracellular compartments. Nucleic acid structures recognized by TLRs include double-stranded RNA by TLR3, 2 single-stranded RNA by TLR7 and 8, 3 and CpG DNA by TLR9. 4,5 It is noted that, these structures are also present in selfnucleic acids.Recognition of DNA by TLR9 occurs in endolysosomes, but TLR9 resides predominantly in the endoplasmic reticulum (ER) in resting cells. 5-9 Endolysosomal localization is important for TLR9 response to CpG DNA, as TLR9 binds some DNA preferentially at low pH, endosomal acidification inhibitors block TLR9 signaling and TLR9 undergoes a conformational change upon CpG DNA binding in endolysosomes. [10][11][12] CpG DNAs with different sequence and secondary structure can elicit either production of type I interferon-a or maturation of plasmacytoid dendritic cells depending on the precise location of interaction with TLR9. Those CpG DNAs that induce interferon-a production accumulate in early endosomes, whereas those that induce maturation accumulate in lysosomes. 13 The mechanism regulating TLR9 translocation from the ER to these different compartments remains unclear.Intracellular localization of TLRs 3, 7, 8 and 9 may be a key factor to prevent recognition of self-nucleic acids. Self-nucleic acids are poorly endocytosed and rapidly degraded in the extracellular milieu. However, self-DNA is recognized by TLR9 when it is presented by chromatin-s...

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.

No abstract