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ObjectivesClinical heterogeneity is a cardinal feature of systemic sclerosis (SSc). Hallmark SSc autoantibodies are central to diagnosis and associate with distinct patterns of skin-based and organ-based complications. Understanding molecular differences between patients will benefit clinical practice and research and give insight into pathogenesis of the disease. We aimed to improve understanding of the molecular differences between key diffuse cutaneous SSc subgroups as defined by their SSc-specific autoantibodiesMethodsWe have used high-dimensional transcriptional and proteomic analysis of blood and the skin in a well-characterised cohort of SSc (n=52) and healthy controls (n=16) to understand the molecular basis of clinical diversity in SSc and explore differences between the hallmark antinuclear autoantibody (ANA) reactivities.ResultsOur data define a molecular spectrum of SSc based on skin gene expression and serum protein analysis, reflecting recognised clinical subgroups. Moreover, we show that antitopoisomerase-1 antibodies and anti-RNA polymerase III antibodies specificities associate with remarkably different longitudinal change in serum protein markers of fibrosis and divergent gene expression profiles. Overlapping and distinct disease processes are defined using individual patient pathway analysis.ConclusionsOur findings provide insight into clinical diversity and imply pathogenetic differences between ANA-based subgroups. This supports stratification of SSc cases by ANA antibody subtype in clinical trials and may explain different outcomes across ANA subgroups in trials targeting specific pathogenic mechanisms.

Background/Aims Simultaneous analysis of multiple proteins in biological fluids offers insight into the pathogenesis of SSc. Here, we report a proteomic analysis of plasma and dermal interstitial fluid in SSc compared with healthy controls (HC). Methods The prospectively collected BIOPSY cohort recruited 52 SSc patients (21 early dcSSc, 15 established dcSSc,16 lcSSc) and 16 HC. Mean baseline skin score (MRSS) for early dcSSc was 21 (sd 11.2). This analysis utilised forearm skin blister fluid obtained using the dermal suction blister method and paired simultaneous plasma samples from early dcSSc and HC at baseline. These were assayed using the Olink antibody platform(www.olink.com) and reported as normalised protein expression (NPX), corresponding to log2 (expression). T-test with FDR correction (p < 0.05) assessed statistical significance. Pathway analysis was conducted by STRING consortium 2020. Results 1,196 proteins were analysed in paired blister/plasma samples from 14 early dcSSc patients and 16 HC. 447 proteins were significantly different in the blister fluid of early dcSSc patients compared with HC (Table 1), whereas only 183 proteins in plasma. Of these, 130 proteins were simultaneously different in both blister fluid and plasma of early dcSSc including key cytokines associated with fibrosis and vasculopathy such as IL-6,VEGF-1, MCP-1, COL4A1, COMP, Thy1 and THBS4. No correlation was seen between these proteins and MRSS. 310 proteins were significantly elevated in blister fluid alone in early dcSSc patients compared to HC. These included cytokines (IL7,IL18, OSM), chemokines (CCL7,CCL18, CCL3), matricellular proteins (CYR61 and osteopontin). KEGG pathway analysis of the significantly elevated proteins in blister fluid in early dcSSc compared to HC highlighted pathways including cytokine-cytokine receptor interaction, cell adhesion molecules, MAPK signalling pathway and PI3K-AKT signalling pathway (FDR Conclusion Numerous dysregulated proteins were identified in dermal blister fluid and plasma of early dcSSc patients. Substantially more were identified in dermal blister fluid, highlighting its potential for providing detailed information on local pathologic processes. A subset of proteins were dysregulated in both plasma and blister fluid, suggesting these may reflect systemic abnormalities. Further work will utilise this cohort to integrate gene and protein expression across the full spectrum of early dcSSc, established dcSSc and lcSSc Disclosure K.E. Clark: None. C. Campochiaro: None. E. Csomor: Corporate appointments; employee of GSK. A. Taylor: Corporate appointments; employee of GSK. K. Nevin: Corporate appointments; employee of GSK. M.A. Morse: Corporate appointments; employee of GSK. V.H. Ong: None. E. Derrett-Smith: None. N. Wisniacki: Corporate appointments; employee of GSK. S. Flint: Corporate appointments; employee of GSK. C.P. Denton: Consultancies; Actelion, GlaxoSmithKline, Bayer, Sanofi, lnventiva, Boehringer Ingelheim, Roche, Bristol Myers Squibb, CSL Behring, UCB, Leadiant Biosciences, Corbus, Servier, Arxx Therapeutics.

Background/Aims The major antinuclear autoantibodies of systemic sclerosis (SSc) associate with different skin score trajectories and risk of internal organ manifestations. To elucidate molecular differences between ANA-defined subgroups, we utilised the prospective BIOPSY cohort of well-characterised SSc patients. Methods The prospectively collected BIOPSY cohort recruited 52 SSc patients (21 early dcSSc, 15 established dcSSc, 16 lcSSc) and 16 healthy controls (HC). 36 (69%) of the SSc patients are female. Mean disease duration in the early dcSSc cohort was 24 months (sd 12 months), and in established dcSSc was 11.3 years. ANA frequency in BIOPSY reflected the overall dcSSc population: anti-topoisomerase-1 (ATA) n = 14 (27%), anti-RNA pol III (ARA) n = 12 (23%) and other n = 26 (50%). Mean baseline skin score (MRSS) for early dcSSc was 21 (sd 11.2). At a group level mRSS peak was 21.9 (11.8) at 3 months and fell to 19.1(10.5) at 12 months. Serum biomarkers of ECM turnover and fibrosis were measured three monthly and genome-wide transcriptomic profiling of whole skin and whole blood performed by RNA-Seq. Statistical analysis used RStudio with ANOVA, and Tukey post-hoc test. Differential gene expression used the Bioconductor limma software, with standard thresholds for significance. Results At baseline, there were differences in soluble markers between clinical SSc sugroups and HC but not for major ANA subgroups. However, we found clear differences in early dcSSc analysed by major ANA subset for longitudinal change in serum markers of fibrosis and in whole skin gene expression, suggesting a mechanistic basis for the distinct clinical phenotypes associated with hallmark ANAs. During follow-up, significant differences were observed in HA, TIMP1, and PIIINP at 6 and 12 months (p < 0.05), with stable levels in ATA+ patients compared to progressively increased levels in the other subgroups. There were 564 significantly differentially expressed genes in skin between early dcSSc and HC. Unsupervised clustering differentiated patients with ARA and ATA positivity with early dcSSc. 54 genes were significantly differentially expressed in skin between ATA and ARA patients. Whilst 179 genes were differentially expressed in whole blood between early dcSSc compared with HC, no genes could significantly differentiate ATA from ARA. Functional analysis using HALLMARK pathway analysis identified both shared pathways associated with SSc across ANA groups (e.g. TGF beta signaling, IL6 JAK STAT3 signalling, inflammatory response), and pathways only upregulated in patients with ATA (e.g. Wnt beta catenin signaling, Notch signaling), and ARA (e.g. interferon gamma response, adipogenesis). Conclusion We have found significant differences in skin gene expression and longitudinal change in serum markers by autoantibody specificity in dcSSc. Our findings have implications for SSc pathogenesis and support stratification by ANA subgroup in clinical studies. Disclosure K.E. Clark: None. C. Campochiaro: None. E. Csomor: Corporate appointments; employee of GSK. A. Taylor: Corporate appointments; employee of GSK. K. Nevin: Corporate appointments; employee of GSK. N. Galwey: Corporate appointments; employee of GSK. M.A. Morse: Corporate appointments; employee of GSK. V.H. Ong: None. E. Derrett-Smith: None. N. Wisniacki: Corporate appointments; employee of GSK. S. Flint: Corporate appointments; employee of GSK. C.P. Denton: Consultancies; Actelion, GlaxoSmithKline, Bayer, Sanofi, lnventiva, Boehringer Ingelheim, Roche, Bristol Myers Squibb, CSL Behring, UCB, Leadiant Biosciences, Corbus, Servier, Arxx Therapeutics.

Background Elevated B lymphocyte stimulator (BLyS) levels in patients with systemic lupus erythematosus (SLE) correlate positively with disease activity; BLyS expression is directly linked to interferon (IFN) pathway activation. This post hoc meta-analysis of BLISS-52 and BLISS-76 explored the relationship between baseline BLyS mRNA/protein levels and/or type 1 IFN-inducible gene signature (IFN-1) and responses to the BLyS-targeting monoclonal antibody belimumab in SLE. Methods In BLISS-52 and BLISS-76, patients with autoantibody-positive SLE and a SELENA-SLEDAI score ≥ 6 and receiving stable standard SLE therapy were randomised to intravenous belimumab 10 mg/kg or placebo, plus standard of care (SoC), for 52 or 76 weeks. For this post hoc meta-analysis, patients with an appropriate mRNA sample were stratified by BLyS mRNA expression (tertiles: high/medium/low; revised quantiles: high/low), IFN-1 mRNA expression (high/low) and BLyS protein level (high/low). Co-primary endpoints were correlation between baseline BLyS and IFN-1 mRNA levels and SLE Responder Index (SRI)4 response at week 52 within BLyS/IFN-1 subgroups. Secondary endpoints included time to first severe SELENA-SLEDAI Flare Index (SFI) flare. Results Of 554 patients included in this analysis, 281 had received belimumab and 273 had received placebo. Baseline BLyS and IFN-1 mRNA levels were highly correlated (Spearman’s rank correlation coefficient 0.7799; 95% confidence interval [CI] 0.7451, 0.8106; p p = 0.0153), high BLyS mRNA quantile (OR 1.58; 95% CI 1.02, 2.44; p = 0.0402), high IFN-1 mRNA (OR 1.58; 95% CI: 1.08, 2.31; p = 0.0186) and high BLyS protein (OR 3.57; 95% CI 1.63, 7.83; p = 0.0015) subgroups only. The risk of severe SFI flare was significantly lower with belimumab than placebo in the high BLyS mRNA quantile (hazard ratio [HR] 0.59; 95% CI 0.36, 0.97; p = 0.0371) and high BLyS protein (HR 0.39; 95% CI 0.19, 0.79; p = 0.0090) subgroups. Conclusions This post hoc meta-analysis demonstrated a tendency towards improved response to add-on intravenous belimumab 10 mg/kg versus SoC alone in patients with high baseline BLyS protein and IFN-1 mRNA levels and medium/high BLyS mRNA levels.

Background The striking heterogeneity in skin fibrosis within systemic sclerosis (SSc) is likely to reflect differences between pro- and anti-fibrotic pathways and underlie the spontaneous regression of skin fibrosis observed in late stage diffuse SSc (dcSSc). We studied potential serum markers of profibrotic activity in SSc, aiming to understanding their relationship with progression of fibrosis as measured by the modified Rodnan skin score (MRSS) to improve stratification of patients likely to respond to fibrosis-targeted therapies Methods The BIOPSY cohort are well characterised patients recruited prospectively from across the scleroderma spectrum. Serum and plasma samples were collected from 67 participants were included in the analysis (21 early dcSSc ( Results Our results confirmed markers of collagen synthesis were significantly different between the patient subgroups (Pro-C6,Pro-C3,PIIINP), while markers of collagen degradation were not significantly altered (C3M,C6M,C4M2,C7M) (Table 1). Consistent with previous reports, the C3 fibrotic index (Pro-C3:C3M) and the ELF test also showed differentiation across subgroups. Across all tests, this difference was most significant between the early dcSSc subgroup compared with the other subgroups. There was significant upregulation of IL-6, MCP-1, and oncostatin M in SSc compared to HC. There were significant correlations between several candidate profibrotic serum markers and MRSS: Pro-C3, Pro C6, PIIINP, and IL6 (all p < 0.01). Conclusion Our results show the utility of extended patient cohorts to delineate fundamental biology in SSc. We identify key pro-fibrotic molecular markers upregulated in SSc and correlated these to extent of skin fibrosis. Markers of collagen III and collagen VI synthesis are particularly raised, especially in the early stages of the disease. However, markers of collagen degradation did not significantly differ between SSc subgroups and HCs. These promising cross-sectional data suggest that therapies targeting drivers of fibrosis are most likely to show benefit for skin in early dcSSc. This will be further explored longitudinally in the BIOPSY cohort. Disclosures K.E.N. Clark: None. C. Campochiaro: None. K. Nevin: Other; GSK employee. E. Csomor: Other; GSK employee. N. Galwey: Other; GSK employee. M. Morse: Other; GSK employee. N. Wisniacki: Other; GSK employee. S. Flint: Other; GSK employee. V.H. Ong: None. E. Derrett-Smith: None. C.P. Denton: Grants/research support; Actelion, GlaxoSmithKline, Bayer, Sanofi, lnventiva, Boehringer Ingelheim, Roche, Bristol Myers Squibb, CSL Behring, UCB, Leadiant Biosciences.

Summary Background B cells produce alloantibodies and activate alloreactive T cells, negatively affecting kidney transplant survival. By contrast, regulatory B cells are associated with transplant tolerance. Immunotherapies are needed that inhibit B-cell effector function, including antibody secretion, while sparing regulators and minimising infection risk. B lymphocyte stimulator (BLyS) is a cytokine that promotes B-cell activation and has not previously been targeted in kidney transplant recipients. We aimed to determine the safety and activity of an anti-BLyS antibody, belimumab, in addition to standard-of-care immunosuppression in adult kidney transplant recipients. We used an experimental medicine study design with multiple secondary and exploratory endpoints to gain further insight into the effect of belimumab on the generation of de-novo IgG and on the regulatory B-cell compartment. Methods We undertook a double-blind, randomised, placebo-controlled phase 2 trial of belimumab, in addition to standard-of-care immunosuppression (basiliximab, mycophenolate mofetil, tacrolimus, and prednisolone) at two centres, Addenbrooke's Hospital, Cambridge, UK, and Guy's and St Thomas' Hospital, London, UK. Participants were eligible if they were aged 18–75 years and receiving a kidney transplant and were planned to receive standard-of-care immunosuppression. Participants were randomly assigned (1:1) to receive either intravenous belimumab 10 mg per kg bodyweight or placebo, given at day 0, 14, and 28, and then every 4 weeks for a total of seven infusions. The co-primary endpoints were safety and change in the concentration of naive B cells from baseline to week 24, both of which were analysed in all patients who received a transplant and at least one dose of drug or placebo (the modified intention-to-treat [mITT] population). This trial has been completed and is registered with ClinicalTrials.gov, NCT01536379, and EudraCT, 2011–006215–56. Findings Between Sept 13, 2013, and Feb 8, 2015, of 303 patients assessed for eligibility, 28 kidney transplant recipients were randomly assigned to receive belimumab (n=14) or placebo (n=14). 25 patients (12 [86%] patients assigned to the belimumab group and 13 [93%] patients assigned to the placebo group) received a transplant and were included in the mITT population. We observed similar proportions of adverse events in the belimumab and placebo groups, including serious infections (one [8%] of 12 in the belimumab group and five [38%] of 13 in the placebo group during the 6-month on-treatment phase; and none in the belimumab group and two [15%] in the placebo group during the 6-month follow-up). In the on-treatment phase, one patient in the placebo group died because of fatal myocardial infarction and acute cardiac failure. The co-primary endpoint of a reduction in naive B cells from baseline to week 24 was not met. Treatment with belimumab did not significantly reduce the number of naive B cells from baseline to week 24 (adjusted mean difference between the belimumab and placebo treatment groups −34·4 cells per μL, 95% CI −109·5 to 40·7). Interpretation Belimumab might be a useful adjunct to standard-of-care immunosuppression in renal transplantation, with no major increased risk of infection and potential beneficial effects on humoral alloimmunity. Funding GlaxoSmithKline.

B cells are important in the pathogenesis of many, and perhaps all, immune-mediated diseases. Each B cell expresses a single B cell receptor (BCR)1, and the diverse range of BCRs expressed by the total B cell population of an individual is termed the 'BCR repertoire'. Our understanding of the BCR repertoire in the context of immune-mediated diseases is incomplete, and defining this could provide new insights into pathogenesis and therapy. Here, we compared the BCR repertoire in systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, Crohn's disease, Behçet's disease, eosinophilic granulomatosis with polyangiitis, and immunoglobulin A (IgA) vasculitis by analysing BCR clonality, use of immunoglobulin heavy-chain variable region (IGHV) genes and-in particular-isotype use. An increase in clonality in systemic lupus erythematosus and Crohn's disease that was dominated by the IgA isotype, together with skewed use of the IGHV genes in these and other diseases, suggested a microbial contribution to pathogenesis. Different immunosuppressive treatments had specific and distinct effects on the repertoire; B cells that persisted after treatment with rituximab were predominately isotype-switched and clonally expanded, whereas the inverse was true for B cells that persisted after treatment with mycophenolate mofetil. Our comparative analysis of the BCR repertoire in immune-mediated disease reveals a complex B cell architecture, providing a platform for understanding pathological mechanisms and designing treatment strategies.

PURPOSE OF REVIEW: Antineutrophil cytoplasmic antibodies (ANCAs) remain central to our current understanding of the pathogenesis of ANCA-associated vasculitis (AAV), and this review considers recent developments in the context of four key questions: are there targets for ANCA beyond myeloperoxidase (MPO) and proteinase 3 (PR3); are all ANCA pathogenic; how are ANCAs generated; and how do ANCA cause disease? RECENT FINDINGS: B-cell epitope mapping raises the possibility that only a subset of ANCA may be pathogenic. Anti-lysosomal-associated membrane protein 2 autoantibodies have recently emerged as a novel form of ANCA and can be found in anti-MPO and anti-PR3 negative disease. These also provide recent evidence for molecular mimicry in the pathogenesis of AAV, but a definitive proof in human AAV remains elusive. Neutrophil extracellular traps may represent an important mechanism by which MPO and PR3 are taken up by dendritic cells for presentation to the adaptive immune system, and the role of the alternative pathway of complement in AAV has recently been emphasized, with therapeutic implications. SUMMARY: Our current understanding of the pathogenesis of AAV not only reinforces the central role of neutrophils but also provides a sound rationale for B-cell and complement-directed therapies.

Primary immune thrombocytopenia is an autoimmune disorder in which platelet destruction is a consequence of both B- and T-cell dysregulation. Flow cytometry was used to further characterize the B- and T-cell compartments in a cross-sectional cohort of 26 immune thrombocytopenia patients including antiplatelet antibody positive (n=14) and negative (n=12) patients exposed to a range of therapies, and a cohort of matched healthy volunteers. Markers for B-cell activating factor and its receptors, relevant B-cell activation markers (CD95 and CD21) and markers for CD4(+) T-cell subsets, including circulating T-follicular helper-like cells, were included. Our results indicate that an expanded population of CD95(+) naive B cells correlated with disease activity in immune thrombocytopenia patients regardless of treatment status. A population of CD21-naive B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in naive B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia.

Objective: To describe the impact of pandemic (H1N1) 2009 influenza (nH1N1) on Indigenous people in the Top End of the Northern Territory at community, hospital and intensive care unit (ICU) levels. Design, setting and participants: We analysed influenza notifications for the Top End from 1 June to 31 August 2009, as well as data on patients admitted through Top End emergency departments with an influenza-like illness. In addition, data on patients with nH1N1 who were admitted to Royal Darwin Hospital (RDH) and the RDH ICU were prospectively collected and analysed. Main outcome measures: Age-adjusted notification rates for nH1N1 cases, Top End hospital admission rates for patients with nH1N1 and RDH ICU admission rates for patients with nH1N1, stratified by Indigenous status. Results: There were 918 nH1N1 notifications during the study period. The age-adjusted hospital admission rate for nH1N1 was 82 per 100 000 (95% CI, 68–95) estimated resident population (ERP) overall, with a markedly higher rate in the Indigenous population compared with the non-Indigenous population (269 per 100 000 versus 29 per 100 000 ERP; adjusted incidence rate ratio, 12 [95% CI, 7.8–18]). Independent predictors of ICU admission compared with hospitalisation were hypoxia (adjusted odds ratio [aOR], 4.5; CI, 1.5–13.1) and chest x-ray infiltrates (aOR, 4.3; CI, 1.5–12.6) on hospital admission. Conclusions: Pandemic (H1N1) 2009 influenza had a disproportionate impact on Indigenous Australians in the Top End, with hospitalisation rates higher than those reported elsewhere in Australia and overseas. These findings have implications for planning hospital and ICU capacity during an influenza pandemic in regions with large Indigenous populations. They also confirm the need to improve health and living

A small but increasing number of gene expression based biomarkers are becoming available for routine clinical use, principally in oncology and transplantation. These underscore the potential of gene expression arrays and RNA sequencing for biomarker development, but this potential has not yet been fully realized and most candidates do not progress beyond the initial report. The first part of this review examines the process of gene expression- based biomarker development, highlighting how systematic biases and confounding can significantly skew study outcomes. Adequate validation in an independent cohort remains the single best means of protecting against these concerns. The second part considers gene-expression based biomarkers in Systemic Lupus Erythematosus (SLE) and systemic vasculitis. The type 1 interferon inducible gene signature remains by far the most studied in autoimmune rheumatic disease. While initially presented as an objective, blood-based biomarker of active SLE, subsequent research has shown that it is not specific to SLE and that its association with disease activity is considerably more nuanced than first thought. Nonetheless, it is currently under evaluation in ongoing trials of anti-interferon therapy. Other candidate markers of note include a prognostic CD8+ T-cell gene signature validated in SLE and ANCA-associated vasculitis, and a disease activity biomarker for SLE derived from modules of tightly correlated genes.

The association between anti–lysosome-associated membrane protein 2 (LAMP-2) autoantibodies and vasculitis is not new; it dates back to work by Kain et al. [1][1] in 1995 that identified LAMP-2 as a target of antineutrophil cytoplasmic antibodies (ANCA) in 14 of 16 patients with a pauci-immune

Convergent lines of evidence suggest that immune mechanisms are important for pathogenesis of schizophrenia. Preliminary evidence from immune biomarker studies suggest differences in cytokines, immune cell counts and expression of immune genes between patients with schizophrenia and healthy volunteers. It is increasingly clear that peripheral immune cells express many supposedly “brain-specific” genes, and classical neurotransmitters, like acetylcholine and dopamine, are not only expressed but also crucially important for immune system signalling. We reasoned that changes in the immune system that contribute to pathology may be measurable in peripheral blood, providing easily accessible biomarkers of psychotic states. Using multi-parameter flow cytometry, we quantified the proportions of 73 immune cell subsets in patients with chronic schizophrenia, first episode psychosis or healthy controls. When compared with healthy controls, patients with chronic schizophrenia had increased relative numbers of NK cells, naive B cells and classical monocytes, and decreased numbers of DCs, HLA-DR + regulatory T cells (Tregs), and CD4 + memory T cells. Importantly, decreased relative numbers of HLA-DR + Tregs and CD4 + memory T cells were associated with greater psychotic symptom severity within the patient group. Additionally, we found significantly increased dopamine receptor 3 (DRD3) gene expression in CD4 + T cells of patients with schizophrenia, and this was significantly correlated with reduced Treg numbers. With further development, these measurements may prove useful as peripherally accessible biomarkers of disease state or severity.

Diagnosis of the autoimmune disease type 1 diabetes (T1D) is preceded by the appearance of circulating autoantibodies to pancreatic islets. However, almost nothing is known about events leading to this islet autoimmunity. Previous epidemiological and genetic data have associated viral infections and antiviral type I interferon (IFN) immune response genes with T1D. Here, we first used DNA microarray analysis to identify IFN-β–inducible genes in vitro and then used this set of genes to define an IFN-inducible transcriptional signature in peripheral blood mononuclear cells from a group of active systemic lupus erythematosus patients (n = 25). Using this predefined set of 225 IFN signature genes, we investigated the expression of the signature in cohorts of healthy controls (n = 87), patients with T1D (n = 64), and a large longitudinal birth cohort of children genetically predisposed to T1D (n = 109; 454 microarrayed samples). Expression of the IFN signature was increased in genetically predisposed children before the development of autoantibodies (P = 0.0012) but not in patients with established T1D. Upregulation of IFN-inducible genes was transient, temporally associated with a recent history of upper respiratory tract infections (P = 0.0064), and marked by increased expression of SIGLEC-1 (CD169), a lectin-like receptor expressed on CD14+ monocytes. DNA variation in IFN-inducible genes altered T1D risk (P = 0.007), as exemplified by IFIH1, one of the genes in our IFN signature for which increased expression is a known risk factor for disease. These findings identify transient increased expression of type I IFN genes in preclinical diabetes as a risk factor for autoimmunity in children with a genetic predisposition to T1D.

Antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) commonly results in glomerulonephritis, in which neutrophils and monocytes have important roles. The heterodimer calprotectin (S100A8/S100A9, mrp8/14) is a Toll-like receptor-4 ligand found in neutrophils and monocytes and is elevated in inflammatory conditions. By immunohistochemistry of renal biopsies, patients with focal or crescentic glomerular lesions were found to have the highest expression of calprotectin and those with sclerotic the least. Serum levels of calprotectin as measured by ELISA were elevated in patients with active AAV and the levels decreased but did not normalize during remission, suggesting subclinical inflammation. Calprotectin levels in patients with limited systemic disease increased following treatment withdrawal and were significantly elevated in patients who relapsed compared with those who did not. As assessed by flow cytometry, patients with AAV had higher monocyte and neutrophil cell surface calprotectin expression than healthy controls, but this was not associated with augmented mRNA expression in CD14(+) monocytes or CD16(+) neutrophils. Thus, serum calprotectin is a potential disease biomarker in patients with AAV, and may have a role in disease pathogenesis.

Background Belimumab is a B-lymphocyte stimulator (BLyS)-specific inhibitor indicated for the treatment of adult patients with active, autoantibody-positive, systemic lupus erythematosus. Belimumab has also been shown to reduce anti-phospholipase A2 receptor (PLA2R) autoantibodies and proteinuria in a mechanistic study in primary membranous nephropathy (PMN) 1 . Here we report an interim analysis of B and T cell phenotypes in PMN subjects treated with belimumab to further elucidate the mechanism of action in autoantibody mediated disease. Objectives To characterize changes in B and T lymphocyte phenotypes in PMN patients treated with belimumab, as a comparison with known pharmacodynamic (PD) effects in SLE 2 , and to assess correlation with anti-PLA2R autoantibody. Methods 14 anti-PLA2R autoantibody positive PMN patients received 10mg/kg iv belimumab every 4 wks (or 2 wks) for up to 100 wks. 11 of these completed at least 28 wks treatment. Whole blood was collected at baseline, Wks 8, 16 and 28 for analysis of B and T-cell phenotypes by flow cytometry. Serum levels of anti-PLA2R antibodies were measured by ELISA (Euroimmun). Statistical analysis to identify PD-induced changes in lymphocyte subsets was carried out by paired t-test (2 tailed) against baseline. Correlations between anti-PLA2R antibody and B and T-cell subsets, either at a specific time point or in the linearised trend over time, were calculated using Pearson9s correlation coefficient (r). Results There was a significant reduction (-63%, p=0.002) in absolute numbers of naive B cells (CD19+ CD27-) at Wk 8 which was sustained through to Wk 28. There was a significant increase in absolute numbers of memory B cells (CD19+ CD27+) at Wk 8 (70%, p=0.023) which increased further through to Wk 28. Plasmablasts (or SLE subset, CD19lo CD38hi CD27hi) showed a trend at Wk 8 to decrease (-64%, p=0.20) but with high variance and low cell numbers. In addition, the expression (mean fluorescence intensity) of BAFF-R (B-cell activating factor receptor) on B-memory cells increased through Wk 28, while expression of TACI (transmembrane activator and calcium modulator and cyclophylin ligand interactor) decreased. Activated cells as identified by surface CD95 expression, decreased as a proportion of B-memory cells through Wk 28. No clear trend was seen in T-regulatory or T-activated cells. There was a significant negative correlation between decrease in absolute numbers of naive B-cells and immunological efficacy defined as level of anti-PLA2R antibody remaining at Wk 28 (r=-0.87, p=0.0005). Correlations were also seen between antibody and changes in proportion of B-memory CD95+ cells. Conclusions This mechanistic trial of belimumab in PMN has enabled a novel analysis of PD endpoints, confirming but also extending the PD effects previously reported in studies of SLE. Changes in B-cell subsets were associated with improvements in levels of autoantibody in PMN which in turn correlates with proteinuria. Further analysis of longer term anti-PLA2R antibody kinetics as related to early changes in B and T cell phenotypes may also be informative. References Willcocks L, Barrett C, Brenchley P et al. Nephrology Dialysis Transplantation. 2015; 30 (Supplement 3): iii32–iii33. Stohl W, Hiepe F, Latinis KM et al. Arthritis & Rheumatism. 2012;64(7):2328–2337. Acknowledgement This study was funded by GSK (study sponsor) (BEL116472, NCT01610492). Disclosure of Interest C. Barrett Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, R. Henderson Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, N. Galwey Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, S. Flint Grant/research support from: GlaxoSmithKline, A. Gibson Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline, C. Savage Shareholder of: GlaxoSmithKline, Employee of: GlaxoSmithKline

BACKGROUND: Hypothetically, psychotic disorders could be caused or conditioned by immunological mechanisms. If so, one might expect there to be peripheral immune system phenotypes that are measurable in blood cells as biomarkers of psychotic states. METHODS: We used multi-parameter flow cytometry of venous blood to quantify and determine the activation state of 73 immune cell subsets for 18 patients with chronic schizophrenia (17 treated with clozapine), and 18 healthy volunteers matched for age, sex, BMI and smoking. We used multivariate methods (partial least squares) to reduce dimensionality and define populations of differentially co-expressed cell counts in the cases compared to controls. RESULTS: Schizophrenia cases had increased relative numbers of NK cells, naïve B cells, CXCR5+ memory T cells and classical monocytes; and decreased numbers of dendritic cells (DC), HLA-DR+ regulatory T-cells (Tregs), and CD4+ memory T cells. Likewise, within the patient group, more severe negative and cognitive symptoms were associated with decreased relative numbers of dendritic cells, HLA-DR+ Tregs, and CD4+ memory T cells. Motivated by the importance of central nervous system dopamine signalling for psychosis, we measured dopamine receptor gene expression in separated CD4+ cells. Expression of the dopamine D3 (DRD3) receptor was significantly increased in clozapine-treated schizophrenia and covaried significantly with differentiated T cell classes in the CD4+ lineage. CONCLUSIONS: Peripheral immune cell populations and dopaminergic signalling are disrupted in clozapine-treated schizophrenia. Immuno-phenotypes may provide peripherally accessible and mechanistically specific biomarkers of residual cognitive and negative symptoms in this treatment-resistant subgroup of patients.

Background Reactive oxygen species (ROS), generated via the phagocyte NADPH oxidase cytochrome b558, are essential for effective immune responses to common and serious pathogens. The phagocyte NADPH oxidase is a multisubunit protein complex and deficiency of either the membrane bound or cytoplasmic components leads to chronic granulomatous disease, a serious and often fatal illness characterised by recurrent infections and autoimmunity. Moreover, abnormal generation of ROS has been implicated in the pathogenesis of multigenic autoimmune diseases such as systemic lupus erythematosus. Eros (essential for reactive oxygen species), encoded by bc017643 , is a novel transmembrane protein that is highly expressed in the immune system and highly conserved in evolution but has no previously identified function. Eros is an orthologue of the plant protein Ycf4, necessary for expression of proteins of the photosynthetic photosystem 1 complex, an NADPH oxio-reductase complex. We elucidated its role in infection in mice. Methods ROS are essential for host defence against the serious bacterial pathogen Salmonella enterica serovar Typhimurium. We screened individual knockout mice (Wellcome Trust Knockout mouse project) for susceptibility to salmonella infection. Having identified mice deficient in Eros as being highly susceptible to salmonella, we used ex-vivo approaches including reactive oxygen burst assays and western blot, to characterise their defect further. Findings We found that Eros was essential for host defence to infection. Eros was crucial for generating reactive oxygen species through regulation of the essential NADPH oxidase components, gp91 and p22. Eros-deficient mice expressed almost no gp91 and p22 in neutrophils and macrophages secondary to accelerated degradation in the absence of Eros. As a result Eros-deficient mice died rapidly after infection with salmonella or listeria. Eros also regulated the ROS-dependent formation of neutrophil extracellular traps and melanoma metastases. Interpretation We have found a a key role for Eros in regulating host defence. The finding that Eros-deficient mice lack gp91 and p22 at the protein, though not mRNA, level shows how these key components of the reatcive oxygen burst are protected from degradation and furthers our understanding of reactive oxygen burst biology. Eros is highly conserved between mouse and man so it is likely that it also has a crucial role in human immunity. Funding Wellcome Trust, Academy of Medical Sciences starter grant.

Background Calprotectin, an endogenous toll-like receptor 4 (TLR 4) agonist that is expressed in neutrophils, monocytes, and infiltrating macrophages, promotes endothelial activation and transcription of pro-inflammatory cytokines. We investigated calprotectin in renal biopsy samples and serum of patients with anti-neutrophil cytoplasmic antibody associated vasculitis (AAV) and in mice deficient in calprotectin (cal−/−), and we assessed the interaction of calprotectin with macrophages and endothelial cells in vitro. Methods We examined renal biopsy samples with immunohistochemistry. Serum calprotectin levels were measured with ELISA, and cell surface expression with flow cytometry. Accelerated nephrotoxic nephritis experiments were performed on both wild-type (WT) and cal−/− mice. Macrophages were isolated from WT, TLR4−/−, and cal−/− mice, and kidney endothelial cells from WT mice, and stimulated with calprotectin and supernatants harvested. Phagocytosis with opsonised beads was compared between WT and cal−/− macrophages. Findings Patients with active AAV glomerular lesions demonstrated the most calprotectin positivity in renal biopsy samples, sclerotic lesions the least (p Interpretation Serum calprotectin is a potential biomarker in AAV, and may predict relapse. Calprotectin contributes to pathogenesis by promoting leukocyte and endothelial cell activation in a positive feedback loop. Funding UK Medical Research Council.