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To identify a panel of serum biomarkers that could specifically identify imminent cases of rheumatoid arthritis (RA) before diagnosis.Serum samples were collected at 4 time points from active component US military personnel, including 157 anti-citrullinated protein antibody (ACPA)-seropositive and 50 ACPA-seronegative RA subjects, 100 reactive arthritis (ReA) subjects, and 76 healthy controls. The cohorts were split into 2 phases, with samples tested on independent proteomic platforms for each phase. Classification models of RA diagnosis based on samples obtained within 6 months prior to diagnosis were developed both in univariate analyses and by multivariate random forest modeling of training sample sets and testing sample sets from each phase.Increases in serum analytes, including C-reactive protein levels, serum amyloid A, and soluble programmed cell death 1 (PD-1), were observed in seropositive RA subjects at the time point closest to diagnosis, up to several years before diagnosis. Only a small fraction of RA subjects had levels above the 95th percentile of healthy control levels until the time period within 6 months of diagnosis. For classification of RA diagnosis using samples obtained within 6 months prior to diagnosis, soluble PD-1 provided superior specificity compared to ReA cases (89%), with a sensitivity of 48% for RA classification. An 8-analyte model provided superior sensitivity (69%), with comparable specificity relative to ReA (82%).Our findings demonstrate that imminent RA diagnosis could be classified with high specificity, relative to healthy controls and ReA cases, using a panel of cytokines measured in serum samples collected within 6 months before actual diagnosis.

ObjectivesImmune and stromal cell communication is central in the pathogenesis of rheumatoid arthritis (RA) and psoriatic arthritis (PsA), however, the nature of these interactions in the synovial pathology of the two pathotypes can differ. Identifying immune-stromal cell crosstalk at the site of inflammation in RA and PsA is challenging. This study creates the first global transcriptomic analysis of the RA and PsA inflamed joint and investigates immune-stromal cell interactions in the pathogenesis of synovial inflammation.MethodsSingle cell transcriptomic profiling of 178 000 synovial tissue cells from five patients with PsA and four patients with RA, importantly, without prior sorting of immune and stromal cells. This approach enabled the transcriptomic analysis of the intact synovial tissue and identification of immune and stromal cell interactions. State of the art data integration and annotation techniques identified and characterised 18 stromal and 14 immune cell clusters.ResultsGlobal transcriptomic analysis of synovial cell subsets identifies actively proliferating synovial T cells and indicates that due to differential λ and κ immunoglobulin light chain usage, synovial plasma cells are potentially not derived from the local memory B cell pool. Importantly, we report distinct fibroblast and endothelial cell transcriptomes indicating abundant subpopulations in RA and PsA characterised by differential transcription factor usage. Using receptor–ligand interactions and downstream target characterisation, we identify RA-specific synovial T cell-derived transforming growth factor (TGF)-β and macrophage interleukin (IL)-1β synergy in driving the transcriptional profile of FAPα+THY1+invasive synovial fibroblasts, expanded in RA compared with PsA. In vitro characterisation of patient with RA synovial fibroblasts showed metabolic switch to glycolysis, increased adhesion intercellular adhesion molecules 1 expression and IL-6 secretion in response to combined TGF-β and IL-1β treatment. Disrupting specific immune and stromal cell interactions offers novel opportunities for targeted therapeutic intervention in RA and PsA.

ObjectivesA number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched PsA synovial fluid (SF) and blood leucocytes, with the aim of identifying cytokine production ex vivo in unstimulated lymphoid and myeloid cells.MethodsFresh SF and paired blood were either fixed or incubated with protein transport inhibitors for 6 h. Samples were stained with two CyTOF panels: a phenotyping panel and an intracellular panel, including antibodies to both T cell and myeloid cell secreted proteins. Transcriptomic analysis by gene array of key expanded cell populations and single-cell RNA-seq, and ELISA and LEGENDplex analysis of PsA SF were also performed.ResultsWe observed marked changes in the myeloid compartment of PsA SF relative to blood, with expansion of intermediate monocytes, macrophages and dendritic cell populations. Classical monocytes, intermediate monocytes and macrophages spontaneously produced significant levels of the proinflammatory mediators osteopontin and CCL2 in the absence of any in vitro stimulation. By contrast minimal spontaneous cytokine production by T cells was detected. Gene expression analysis showed the genes for osteopontin and CCL2 to be amongst those most highly upregulated by PsA monocytes/macrophages; and both proteins were elevated in PsA SF.ConclusionsUsing multiomic analyses we have generated a comprehensive cellular map of PsA SF and blood to reveal key expanded myeloid proinflammatory modules in PsA of potential pathogenic and therapeutic importance.

IL-23 is considered a critical regulator of IL-17 in Th17 cells; however, its requirement for inducing IL-17 production in other human immune subsets remains incompletely understood. Mucosal associated invariant T (MAIT) cells uniformly express retinoic acid receptor-related orphan receptor gamma t (RORγt) but only a minor population have been shown to produce IL-17A. Here we show that IL-17F is the dominant IL-17 isoform produced by MAIT cells, not IL-17A. For optimal MAIT cell derived IL-17A and IL-17F production, T cell receptor (TCR) triggering, IL-18 and monocyte derived IL-12 signaling is required. Unlike Th17 cells, this process is independent of IL-23 signaling. Using an in vitro skin cell activation assay, we demonstrate that dual neutralization of both IL-17A and IL-17F resulted in greater suppression of inflammatory proteins than inhibition of IL-17A alone. Finally, we extend our findings by showing that other innate-like lymphocytes such as group 3 innate lymphoid cells (ILC3) and gamma delta (γδ) T cells are also capable of IL-23 independent IL-17A and IL-17F production. These data indicate both IL-17F and IL-17A production from MAIT cells may contribute to tissue inflammation independently of IL-23, in part explaining the therapeutic disconnect between targeting IL-17 or IL-23 in certain inflammatory diseases.

The pro-inflammatory cytokine IL-17A has been implicated in the immunopathology of inflammatory arthritis. IL-17F bears 50% homology to IL-17A and has recently been suggested to play a role in inflammation. We investigated the induction and cytokine profile of IL-17F+ CD4+ T cells, and how IL-17F may contribute to inflammation. Upon culture of healthy donor CD4+ T cells with IL-1β, IL-23, anti-CD3, and anti-CD28 mAb, both IL-17A and IL-17F-expressing cells were detected. In comparison to IL-17A+ IL-17F- CD4+ T cells, IL-17F+ IL-17A- and IL-17A+ IL-17F+ CD4+ T cells contained lower proportions of IL-10-expressing and GM-CSF-expressing cells and higher proportions of IFN-γ-expressing cells. Titration of anti-CD28 mAb revealed that strong co-stimulation increased IL-17F+ IL-17A- and IL-17A+ IL-17F+ CD4+ T cell frequencies, whereas IL-17A+ IL-17F- CD4+ T cell frequencies decreased. This was partly mediated via an IL-2-dependent mechanism. Addition of IL-17A, IL-17F, and TNF-α to synovial fibroblasts from patients with inflammatory arthritis resulted in significant production of IL-6 and IL-8, which was reduced to a larger extent by combined blockade of IL-17A and IL-17F than blockade of IL-17A alone. Our data indicate that IL-17A and IL-17F are differentially regulated upon T cell co-stimulation, and that dual blockade of IL-17A and IL-17F reduces inflammation more effectively than IL-17A blockade alone.

Autoimmune diseases resulting from MHC class II–restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA(323–339) and calcitriol targeted dLN PD-L1(hi) DCs of immunized mice and reduced their MHC class II expression. OVA(323–339)/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3(+) and IL-10(+) peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture’s vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.

Background Race and ethnic differences affect the disease characteristics and clinical outcomes in many tumors, including acute myeloid leukemia (Byrne et al. AJCO, 2011). While earlier population-based studies reported no significant impact of race on survival outcomes in myelodysplastic syndromes (MDS) (Ma et al. Cancer, 2007), this study had a limited number of patients, short duration of follow-up including patients diagnosed from 2001-2003 prior to the approval and routine use of hypomethylating agents (HMA). We hypothesized that there are racial differences in the clinical characteristics and outcomes of patients with MDS. We thus analyzed the differences in disease characteristics and survival outcomes based on race and ethnic background in patients with MDS over 13 years. Methods We used the Surveillance, Epidemiology, and End Results (SEER) database to identify adult patients diagnosed with MDS between 2004 and 2016. Disease characteristics and patient outcomes were analyzed among three groups based on race/ethnicity: non-Hispanic white (NHW), non-Hispanic black (NHB) and Hispanics, Fisher exact test and chi-square test were used to compare categorical variables. SEER*Stat version 8.3.6 was used to calculate the incidence rate (IR) and incidence rate ratio (IRR) for NHW, NHB, and Hispanics. Univariate survival analysis was estimated using the Kaplan-Meier method, and groups were compared using a log-rank test. Multivariable survival analyses were performed using the Cox-proportional regression model after adjusting for age, gender, insurance status, histologic risk classification, marital status, and treatment with chemotherapy. Cause-specific survival (CSS) was calculated from the date of diagnosis to the date of MDS-related death and compared amongst the three groups. Results A total of 52,031 patients with MDS were included in this study; 83.4% were NHW, 8.5% were NHB, and 8.2% Hispanics. The incidence was 7.81, 6.46, and 5.17 per 100,000 among NHW, NHB, and Hispanic patients, respectively. Compared to NHW, NHB and Hispanic patients had a significantly lower incidence rate among the overall population (p Conclusions Myelodysplastic syndromes have significant differences in age at presentation, disease risk, and survival outcomes based on racial and ethnic backgrounds. To our knowledge, this study represents the largest population-based analysis with the longest follow-up, specifically looking at such differences in patients with MDS. Further studies are warranted that incorporate treatment patterns and genomic data in order to identify factors that may account for these differences. Disclosures Wang: Celgene/BMS: Research Funding. Patel:France Foundation: Honoraria; Agios: Consultancy; DAVA Pharmaceuticals: Honoraria; Celgene: Consultancy, Speakers Bureau. Zeidan:Astellas: Consultancy, Honoraria; Taiho: Consultancy, Honoraria; Cardinal Health: Consultancy, Honoraria; Otsuka: Consultancy, Honoraria; MedImmune/Astrazeneca: Research Funding; Astex: Research Funding; CCITLA: Other; Leukemia and Lymphoma Society: Other; Epizyme: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Cardiff Oncology: Consultancy, Honoraria, Other; Ionis: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria; ADC Therapeutics: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BeyondSpring: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria; Celgene / BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Aprea: Research Funding; Agios: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria.

The inflammatory CD40–CD40L pathway is implicated in various autoimmune diseases, but the activity status of this pathway in various stages of rheumatoid arthritis (RA) progression is unknown. In this study, we used gene signatures of CD40L stimulation derived from human immature dendritic cells and naive B cells to assess the expression of CD40-downstream genes in synovial tissues from anti-citrullinated protein Ab–positive arthralgia, undifferentiated arthritis (UA), early RA, and established RA cohorts in comparison with healthy donors. Interestingly, the expression of CD40LG and active full-length CD40 was increased in the disease tissues, whereas that of a dominant-negative CD40 isoform was decreased. Gene set variation analysis revealed that CD40L-responsive genes in immature dendritic cells and naive B cells were significantly enriched in synovial tissues from UA, early RA, and established RA patients. Additionally, CD40L-induced naive B cell genes were also significantly enriched in synovial tissues from arthralgia patients. In our efforts to characterize downstream mediators of CD40L signaling, we have identified GPR120 and KDM6B as novel components of the pathway. In conclusion, our data suggest that therapeutic CD40–CD40L blocking agents may prove efficacious not only in early and established RA, but also in inhibiting the progression of the disease from arthralgia or UA to RA.

Background The requirement for IL-23 in driving IL-17A and IL-17F production in humans is incompletely understood. Preclinical data support IL-17F, together with IL-17A, as a key driver of chronic tissue inflammation.1 MAIT cells, an innate-like T cell population, uniformly express RORγt but only a minority have been shown to produce IL-17A.2 Dysregulation in frequency and function of MAIT cells has been associated with IL-17A-mediated inflammatory diseases, including PsA and AS.3,4 IL-17F production in MAIT cells remains largely unexplored. Objectives To explore the importance of IL-23 signalling in MAIT cell-derived IL-17A and IL-17F production, examine the presence of MAIT cells in psoriatic lesional skin and assess the contribution of MAIT cell-derived IL-17A and IL-17F using in vitro models of skin inflammation. Methods IL-17A and IL-17F production by MAIT cells was assessed by flow cytometry, ELISA, qPCR and CyTOF upon activation by anti-CD3/CD28 or fixed E. coli via MR1–presented riboflavin metabolites, with or without disease-relevant recombinant cytokines or an IL-23-neutralising antibody. RNAscope was utilised to observe MAIT cells in psoriatic lesional skin. MAIT cell supernatant, generated from FACS sorted activated MAIT cells, was cultured with normal human dermal fibroblasts (NHDFs) in the presence of IL-17-isoform-specific antibodies, including bimekizumab, a monoclonal antibody that potently and selectively neutralises both IL-17A and IL-17F. Results Optimal MAIT cell IL-17A and IL-17F production occurred upon T cell receptor triggering with IL-12 and IL-18, independently of IL-23. IL-17F expression was greater at both gene and protein levels than IL-17A. The kinetics and threshold of activation for IL-17A and IL-17F suggest tighter regulation compared with other inflammatory cytokines, including IFNγ and TNF. Optimal MAIT cell IL-17A and IL-17F production requires monocytes, which contribute to IL-12 production upon IL-18 stimulation. MAIT cells were abundant in psoriatic lesional skin. NHDFs cultured with supernatant generated from activated MAIT cells produced inflammatory mediators IL-6, IL-8 and CCL2, which were reduced upon inhibition of either IL–17A or IL-17F, with optimal suppression achieved following dual neutralisation with bimekizumab. Conclusion IL-17A and IL-17F can be produced from MAIT cells independently of IL-23, and contribute to inflammatory responses in NHDFs. These results support dual neutralisation of IL-17A and IL-17F as a complete and targeted approach to supress IL–17–driven inflammatory responses. Reference [1] Glatt Ann Rheum Dis 2018;77:523–32, 2Dusseaux Blood 2011;117:1250–59, 3Menon Arthritis Rheumatol 2014;66:1272–81, 4Gracey Ann Rheum Dis 2016;75:2124–32 Acknowledgement Funded by UCB Pharma. The authors would like to acknowledge Alexandra Webster MSc, of iMed Comms, an Ashfield Company, for editorial support that was funded by UCB Pharma in accordance with Good Publication Practice (GPP3) guidelines. The authors acknowledge Alvaro Arjona, PhD, of UCB Pharma, for publication and editorial support. Disclosure of Interests Suzanne Cole Employee of: UCB Pharma, Catherine Simpson Employee of: UCB Pharma, Remi Okoye Shareholder of: UCB Pharma, Employee of: UCB Pharma, Meryn Griffiths Consultant for: UCB Pharma, Employee of: UCB Pharma, Dominique Baeten Shareholder of: UCB Pharma, Employee of: UCB Pharma, Stevan Shaw Employee of: UCB Pharma, Ash Maroof Shareholder of: UCB Pharma, Employee of: UCB Pharma

Background Rheumatoid Arthritis (RA) patients with established disease can have autoantibodies reactive to a broad array of citrullinated antigens. Autoantibodies reactive against several citrullinated proteins can develop 10-15 years before the clinical onset of disease. Objectives We aimed to identify common patterns of citrullinated peptide reactivities that emerge among subjects during progression from a pre-disease state through diagnosis of RA. Methods 500 subjects with RA (based on ICD9-CM code) were identified from the Defense Medical Surveillance System. For each subject, up to four serum samples were obtained from the Department of Defense (DoD) serum repository: 1 from earliest time point before diagnosis (9.2 ± 2.0 years before, mean ± SD); one proximal to (immediately prior to or after) disease diagnosis (127 ± 125 days before diagnosis); plus 2 intermediate time points. A discovery subset of serum samples from 88 RA subjects confirmed to be positive for anti-citrullinated protein antibodies (ACPA) at the diagnosis-proximal time point (>5 U/mL for cyclic citrullinated peptide (CCP)-II test) was assessed for IgG antibodies to 36 antigens (24 citrullinated-peptide antigens among 14 proteins and 12 non-citrullinated antigens among 9 proteins) using a custom Luminex-based assay. Subjects testing above the upper limit of detection for the CCP-II test (300 U/mL) were considered ACPA-high (n=28), with the remainder considered moderate (n=60). Results For the group of ACPA-high subjects at the diagnosis-proximal time point, average IgG autoantibody binding to 9 citrullinated peptide antigens (from 7 proteins: clusterin, enolase, fibrinogen A, fibrinogen B, fibronectin, filaggrin, vimentin) showed significant increases from the earliest through diagnosis-proximal time points at the population level (FDR Conclusion Novel patterns of citrullinated peptide autoantibody reactivities that begin to emerge on average about 6 years before diagnosis of RA have been identified in samples from the US DoD serum repository. Evaluation of specific anti-citrullinated peptide autoantibodies could potentially provide sensitive, patient-tailored biomarkers to monitor disease trajectories as at-risk individuals progress to clinically-defined RA. Disclosure of Interests: Matthew J Loza Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, Sunil Nagpal Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, Renee Laird: None declared, Suzanne Cole Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, Frederic Baribaud Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, Ian Anderson Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, Navin Rao Shareholder of: Johnson & Johnson, Employee of: Janssen Research & Development, LLC, Mark Riddle: None declared, Chad Porter: None declared

Background:Immune checkpoint blockade with agents targeting CTLA4 and PD-1/PD-L1 alone or in combination has demonstrated exceptional efficacy in multiple cancer types by “unleashing” the cytotoxic action of quiescent, tumor-infiltrating T cells. However, the therapeutic action of these immunotherapies goes hand in hand with the loss of immune tolerance and appearance of immune-related adverse events such as colitis, arthralgia and inflammatory arthritis in responsive patients. Therefore, immune checkpoint molecules have been proposed as targets for the treatment of autoimmune diseases.Objectives:Herein, we interrogate the potential of BTLA/HVEM axis as a target for restoring immune homeostasis in rheumatoid arthritis (RA), Systemic Lupus Erythematosus (SLE) and Sjogren’s Syndrome (SjS) by examining their expression patterns in autoimmune disease tissues.Methods:Message and protein expression of BTLA and HVEM were examined in RA and SLE synovial tissues, SLE cutaneous lesions, SjS salivary glands and peripheral blood samples of autoimmune disease by RNA sequencing and flow cytometry.Results:Tissue dysregulation of the BTLA-HVEM axis was observed: Increased BTLA RNA level in RA synovium, SLE-affected skin, and SjS salivary gland samples, whereas HVEM level was affected only in the RA synovium when compared to unaffected tissues. Detailed immunophenotyping of B, T, and myeloid cell populations in RA, SLE, SjS and healthy control PBMCs revealed differential modulation of the BTLA+ or HVEM+ immune cell subsets in a disease-context dependent manner. SjS patients showed an overall decrease in memory B cells and most of the BTLA+ B cell subsets while a decrease in HVEM+ B cells was observed only in SLE PBMC samples and not RA and SLE samples. Immunophenotyping with a T cell panel exhibited decreased BTLA and HVEM expression on T cell subsets in SjS and SLE but not in RA patients. In addition, protein levels of HVEM were differentially decreased in SLE myeloid cell subsets. Finally, we demonstrate tissue-specific surface expression patterns of BTLA in RA and SLE samples: higher surface BTLA levels on RA and SLE PBMC B cells than matched tissue-derived B cells.Conclusion:Our results demonstrate a dysregulation of the BTLA/HVEM axis in either lesional tissue or peripheral blood in an autoimmune disease context-dependent manner. These results also indicate the potential of targeting BTLA-HVEM axis for the treatment of multiple autoimmune diseases.Disclosure of Interests:Sunil Nagpal Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Suzanne Cole Shareholder of: Janssen Research & Development employee, Employee of: Janssen Research & Development employee, Achilleas Floudas: None declared, Mihir Wechalekar Grant/research support from: Grant from Janssen Research & Development, Qingxuan Song Shareholder of: Employee of Janssen Research, Employee of: Employee of Janssen Research, Tom Gordon: None declared, Roberto Caricchio Grant/research support from: Financial grant from Janssen Research & Development, Douglas Veale: None declared, Ursula Fearon: None declared, Navin Rao Shareholder of: Janssen Pharmaceuticals, Employee of: Janssen Pharmaceuticals, Ling-Yang Hao Shareholder of: Employee of Janssen Research, Employee of: Employee of Janssen Research

Background Plasmablasts and plasma cells play a key role in many autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). This study was undertaken to evaluate the potential of targeting CD38 as a plasma cell/plasmablast depletion mechanism by daratumumab in the treatment of patients with RA and SLE. Methods RNA-sequencing analysis of synovial biopsies from various stages of RA disease progression, flow cytometry analysis of peripheral blood mononuclear cells (PBMC) from patients with RA or SLE and healthy donors, immunohistochemistry assessment (IHC) of synovial biopsies from patients with early RA, and ex vivo immune cell depletion assays using daratumumab (an anti-CD38 monoclonal antibody) were used to assess CD38 as a therapeutic target. Results We demonstrated that the plasma cell/plasmablast-related genes CD38, XBP1, IRF4, PRDM1, IGJ and TNFSF13B are significantly up-regulated in synovial biopsies from patients with arthralgia, undifferentiated arthritis (UA), early RA and established RA as compared to healthy controls and control patients with osteoarthritis. In addition, the highest CD38 expression was observed on plasma cells and plasmablasts compared to natural killer (NK) cells, classical dendritic cells (DCs), plasmacytoid DCs (pDCs) and T cells, in blood from healthy controls and patients with SLE and RA. Furthermore, IHC showed CD38 staining in the same region as CD3 and CD138 staining in synovial tissue biopsies from patients with early RA. Most importantly, our data show for the first time that daratumumab effectively depletes plasma cells/plasmablasts in PBMC from patients with SLE and RA in a dose-dependent manner ex vivo. Conclusion These results indicate that CD38 may be a potential target for RA disease interception and daratumumab should be evaluated clinically for the treatment of both RA and SLE. Electronic supplementary material The online version of this article (10.1186/s13075-018-1578-z) contains supplementary material, which is available to authorized users.

Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections. IMPORTANCE Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of proinflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival compared to that of the wild-type mice despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells reduces immunopathogenesis after influenza infections.

Maternal immunity to human cytomegalovirus (HCMV) prior to conception is ~70% protective against congenital transmission and in utero infection of HCMV. Both functional antibodies capable of neutralizing virus and effective T-cells are believed to be important for the protection. Previous HCMV vaccines have rarely been shown able to induce neutralizing antibody titers comparable to those seen in naturally infected HCMV seropositive subjects. Recent studies link a glycoprotein H (gH) complex to receptor-mediated viral entry of endothelial/epithelial cells and leukocytes. This pentameric gH complex, composed of five proteins (gH, gL, UL128, UL130 and UL131 proteins), is notably missing in all HCMV vaccine previously evaluated in clinic. Here we showed that a HCMV virus, with restored expression of the pentameric gH complex, can induce 10-fold higher neutralizing antibody titers than an attenuated AD169 virus or a recombinant glycoprotein B vaccine in multiple animal species in which viral replication is not expected. Encouragingly, the peak neutralizing titers post vaccination in rabbits and monkeys were within 2-4-fold of the levels determined in HCMV seropositive subjects. Functional antibodies by vaccination could further be improved when formulated with a novel adjuvant, and the titers of the antiviral antibodies were sustained in rabbits for over a year after vaccination. These results indicate that the pentameric gH complex is associated with greatly improved functional antibodies following vaccination, and support a vaccine concept based on a nonreplicating whole HCMV with the pentameric gH-associated epithelial tropism restored.

Congenital human cytomegalovirus (HCMV) infection occurs in ~0.64% of infants born each year in the United States and is the leading nongenetic cause of childhood neurodevelopmental disabilities. No licensed HCMV vaccine is currently available. Natural immunity to HCMV in women before pregnancy is associated with a reduced risk of fetal infection, suggesting that a vaccine is feasible if it can reproduce immune responses elicited by natural infection. On the basis of this premise, we developed a whole-virus vaccine candidate from the live attenuated AD169 strain, with genetic modifications to improve its immunogenicity and attenuation. We first restored the expression of the pentameric gH/gL/pUL128-131 protein complex, a major target for neutralizing antibodies in natural immunity. We then incorporated a chemically controlled protein stabilization switch in the virus, enabling us to regulate viral replication with a synthetic compound named Shield-1. The virus replicated as efficiently as its parental virus in the presence of Shield-1 but failed to produce progeny upon removal of the compound. The vaccine was immunogenic in multiple animal species and induced durable neutralizing antibodies, as well as CD4+ and CD8+ T cells, to multiple viral antigens in nonhuman primates.

Following the disappointing outcome of the phase IIb test-of-concept step study in which Merck's adenovirus type 5 (Ad5) HIV-1 clade B gag/pol/nef vaccine failed to demonstrate efficacy in HIV high-risk individuals, an extensive review of the trial and preclinical studies which supported the trial is ongoing. One point of interest is how well preclinical nonhuman primate immunogenicity studies predicted what was observed in humans. Here we compare the HIV-1-specific cellular immune responses elicited in nonhuman primates and human clinical trial subjects to several HIV-1 vaccine candidates. We find that although rhesus macaques are immunologically more responsive to vaccination than humans, the hierarchy in potency of single-modality prime–boost regimens using several vector approaches (adenovirus, DNA, and pox vectors) was well predicted. Vaccine approaches using complex formulations such as novel adjuvants (DNA + CRL1005) or mixed-modality prime–boost (DNA/Ad5; Ad5/ALVAC) did not correlate as well between rhesus macaques and humans. Although the immunogenicity of the vaccines and vaccine regimens evaluated were not all accurately predicted, testing in rhesus macaques generally offers an indispensable tool for ranking the immunological potential of HIV-1 vaccine candidates.

Autoantibodies play a significant role in the progression and pathogenesis of many autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). No studies have analyzed the expression of plasma cell/plasmablast genes during RA disease progression and the potential of an anti-CD38 antibody in depleting plasma cells and plasmablasts for efficacy in autoimmunity. Therefore, we interrogated the rationale of daratumumab, an anti-CD38 monoclonal antibody, as a potential therapeutic in RA and SLE. RNA-Seq analysis of synovial biopsies from various stages of RA disease development shows that plasma cell/plasmablast-related genes CD38, XBP1, IRF4, PRDM1, IGJ and TNFSF13B are significantly up-regulated in synovial biopsies from arthralgia, undifferentiated arthritis, early RA and established RA compared to healthy and osteoarthritis controls. In addition, flow cytometry analysis reveals highest CD38 expression on plasma cells and plasmablasts compared to natural killer cells, classical dendritic cell (DC), plasmacytoid DC, and T cells, in peripheral blood of healthy control, SLE and RA donors. We show that IGJ expression mRNA strongly correlates with the number of plasma cell/plasmablast in SLE patient PBMCs. Most importantly, IGJ mRNA down-regulation correlates with daratumumab-mediated depletion of these cells ex vivo. The data indicates a potential use of IGJ mRNA as a surrogate pharmacodynamic biomarker in clinical testing of daratumumab. Taken together, our data provides rationale for daratumumab in the treatment of RA and SLE.

Immune checkpoint blockade with therapeutic anti-cytotoxic T lymphocyte-associated antigen (CTLA)-4 (Ipilimumab) and anti-programmed death (PD)-1 (Nivolumab and Pembrolizumab) antibodies alone or in combination has shown remarkable efficacy in multiple cancer types, concomitant with immune-related adverse events, including arthralgia and inflammatory arthritis (IA) in some patients. Herein, using Nivolumab (anti-PD-1 antagonist)-responsive genes along with transcriptomics of synovial tissue from multiple stages of rheumatoid arthritis (RA) disease progression, we have interrogated the activity status of PD-1 pathway during RA development. We demonstrate that the expression of PD-1 was increased in early and established RA synovial tissue compared to normal and OA synovium, whereas that of its ligands, programmed death ligand-1 (PD-L1) and PD-L2, was increased at all the stages of RA disease progression, namely arthralgia, IA/undifferentiated arthritis, early RA and established RA. Further, we show that RA patients expressed PD-1 on a majority of synovial tissue infiltrating CD4+ and CD8+ T cells. Moreover, enrichment of Nivolumab gene signature was observed in IA and RA, indicating that the PD-1 pathway was downregulated during RA disease progression. Furthermore, serum soluble (s) PD-1 levels were increased in autoantibody positive early RA patients. Interestingly, most of the early RA synovium tissue sections showed negative PD-L1 staining by immunohistochemistry. Therefore, downregulation in PD-1 inhibitory signaling in RA could be attributed to increased serum sPD-1 and decreased synovial tissue PD-L1 levels. Taken together, these data suggest that agonistic PD1 antibody-based therapeutics may show efficacy in RA treatment and interception.

The fish immune system is quite different from the mammalian system because the anterior kidney forms the main site for hematopoiesis in this species. Using transcription factor-specific Abs derived from the murine system, together with anti-trout Ig Abs and Percoll gradient separation, we analyzed B cells from trout kidney sections and compared them to those from spleen and blood. For this study, immune cells were separated by Percoll gradients, and the resulting subpopulations were defined based on expression of B cell-specific transcription factors Pax-5 and B lymphocyte-induced maturation protein-1, as well as proliferative and Ig-secreting properties. Comparison of kidney, blood, and spleen B cell subsets suggest that 1) the anterior kidney contains mostly proliferating B cell precursors and plasma cells; 2) posterior kidney houses significant populations of (partially) activated B cells and plasmablasts; and 3) trout blood contains resting, non-Ig-secreting cells and lacks plasma cells. After LPS induction of resting B cells in vitro, the kidney and spleen have a high capacity for the generation of plasma cells, whereas the blood has virtually none. Our results indicate that trout B cell subsets are profoundly different among blood, anterior kidney, posterior kidney, and spleen. We hypothesize that developing B cells mature in the anterior side of the kidney and then migrate to sites of activation, either the spleen or the posterior kidney. Lastly, our data support the notion that the trout kidney is a complex, multifunctional immune organ with the potential to support both hemopoiesis as well as humoral immune activation.

Background Traditional markers associated with erosive disease in RA do not reliably predict radiographic progression. There are limited data regarding the role of bone biomarkers in erosive disease in early RA treated with conventional DMARDs and no longitudinal data on osteoclast-associated receptor (OSCAR) with therapy. Objectives To determine whether bone biomarkers associated with osteoclast activation (RANKL, OSCAR and Dkk-1) or inhibition (OPG) are associated with erosion progression over 3 years, independent of disease activity in an inception cohort of RA receiving treat-to-target combination DMARD therapy without oral corticosteroids. Methods Dual RF and anti-CCP positive patients with early RA ( Results Mean (SD) baseline DAS28ESR was 5.55 (1.14), 76% were females, 70% were current/past smokers and mean duration of symptoms prior to diagnosis was 18 (12) weeks. Median (IQR) total SvH score was 2 (7); 24% had erosive disease. With treatment, there was a reduction in DAS28 at 1 year to 3.14 (1.31), significant reduction in RANKL, slight increase in OPG and no significant change in Dkk-1 and OSCAR. DAS28 remission, assessed over 3 years, was less likely to occur both in the presence of detectable RANKL (p=0.01) and/or in the presence of higher average Dkk-1 levels (p=0.007). Baseline erosion scores were higher in the presence of RANKL (p=0.046) and lower OSCAR (p=0.021). There was a trend towards higher erosion scores in the presence of lower OPG levels (p=0.16). Unexpectedly, those with higher mean OPG over the first 12 months had a higher annual increase in erosion scores over 3 years (p=0.018), which may reflect the dynamic processes of bone inflammation. Conclusions Conventional DMARD treatment results in reduction of RANKL, but not OPG, OSCAR or Dkk-1 levels. Higher RANKL levels, lower OSCAR levels and higher mean OPG were associated with erosions. Failure of bone biomarkers to respond to treatment may warrant escalation of therapy to prevent erosion progression. Bone biomarkers may also provide an explanation for the apparent dissociation between disease activity and bone damage in RA. Disclosure of Interest M. D. Wechalekar: None declared, S. Lester: None declared, S. Nagpal Employee of: Johnson & Johnson, S. Cole Employee of: Johnson & Johnson, A. Das Employee of: Johnson & Johnson, P. Hissaria: None declared, T. Crotti: None declared, L. Spargo: None declared, J. Walker: None declared, M. Smith: None declared, S. Proudman: None declared

T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1. Trial Registration ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00849680","term_id":"NCT00849680"}}NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.

Background: Allogeneic hematopoietic stem cell transplantation (allo SCT) has two potential advantages over autologous SCT: a tumor-free graft and graft-versus-myeloma (GVM) effect. Allo SCT’s potential to induce long term remission has, however, been offset by high rates of transplant-related non-relapse mortality (TRM). Reduced-intensity conditioning (RIC) regimens for allo SCT are associated with lower TRM without compromising the GVM effect. Methods: We retrospectively analyzed our experience in 69 patients (30 females and 39 males) with heavily pretreated, relapsed myeloma, who received allo SCT at our institution between1985 and 2007. Eighteen patients received myeloablative regimens (MA), while 51 received RIC regimens. MA regimens were TBI-based in 5 patients, high-dose busulfan-containing in 6 patients and high-dose melphalan containing (180–200 mg/m2) in 7 patients. RIC regimens were a combination of fludarabine (90–120 mg/m2) and melphalan (100–140 mg/m2). Median age of patients at allo SCT in both groups was 51 years. Median interval from diagnosis to allo SCT was 35.4 months in MA group, and 34.2 months in RIC group. Eight (44%) patients in MA group and 36 (70%) patients in RIC group had prior autologous SCT. Six patients (33%) in the MA group and 11 (25%) in the RIC group received allo SCT from unrelated donors (p=0.3). Median number of prior treatment regimens were 5 (range 1–10) in both groups. Stem cell source was peripheral blood in 3 patients in MA group and 41 patients in the RIC group (p=0.0001). Results: Median follow-up in surviving patients was 27 months (3–98). All patients achieved engraftment. Cumulative TRM at 1 year was 56% in the MA group and 25% in the RIC group (p=0.03). Overall response rates in evaluable patients were 69% (CR=15%, PR= 54%) in MA group, and 79% (CR=23%, PR=56%) in the RIC group (p=0.47). Disease progression at 2 years was seen in 8 patients (44%) in the MA group and 25 patients (49%) in the RIC group (p=0.78). Median progression-free survival (PFS) in MA vs. RIC groups was 4.1 and 6.8 months, respectively (p=0.003) and median overall survival (OS) ) in MA vs. RIC group was 5.3 and 13.9 months, respectively (p=0.001). Cumulative Incidence of grade II–IV acute graft-vs.-host (GVHD) in MA vs. RIC groups disease was 33 vs. 27% (p=0.76); cumulative incidence of chronic GVHD in MA vs. RIC group was 54% vs. 47% (p=0.41) in evaluable patients. At the time of this analysis, 13 patients (25%) were still alive in RIC group, 7 of whom (14%) were in remission for up to 6 years post allo SCT. The most common causes of death were recurrent disease (30 patients; 43%), acute or chronic GVHD (16 patients; 23%) and opportunistic infections (5 patients: 7%). Conclusions: Allo SCT after RIC regimens is associated with longer PFS and OS and lower TRM. There was no increase in the risk of relapse, or acute or chronic GVHD. These regimens can safely replace MA regimens and may offer greater benefit if utilized earlier in the course of disease. Figure Figure