Search

Objectives T cells adhere to enthesis fibrocartilage via integrins and intrinsically require IL-17RA-mediated signals to maintain their effector function. We analysed CD29+IL-17RA+ T cells in inflamed lesions and peripheral blood in patients with SpA and investigated their association with disease activity and therapeutic response. Methods Transcriptome analysis of synovial fluid T cells from PsA was performed using publicly available bulk cell RNA sequencing data. Blood samples were obtained from healthy controls (n = 37), RA (n = 12), IgG4-related disease (IgG4-RD; n = 12), large vessel vasculitis (LVV; n = 12) and SpA (n = 28) and were analysed by flow cytometry. Results T cells in the inflamed joints of PsA showed CD29 and IL-17RA expression. CD29+IL-17RA+ T cells showed enriched CXCR3+CD45RA+ effector cells and activation of spleen tyrosine kinase (Syk), nuclear factor κB (NF-κB) and Janus kinase–signal transducer and activator of transcription (JAK-STAT) pathways. The proportion of peripheral blood CD29+IL-17RA+ T cells was significantly increased in patients with SpA compared with patients with RA, IgG4-RD or LVV and in healthy controls. Based on the ASDAS-CRP scores, the proportion of CD29+IL-17RA+ T cells was positively correlated with disease activity in treatment-naïve patients with active SpA. Anti-IL-17 but not anti-TNF monoclonal antibodies reduced CD29+IL-17RA+ T cells. Conclusions CD29+IL-17RA+ T effector cells with enhanced Syk, NF-κB and JAK-STAT pathways were specifically increased in SpA and were correlated with disease activity, implicating a role of this newly identified T cell population in the pathogenesis. Anti-IL-17 monoclonal antibodies may be effective for patients by reducing this pathogenic T cell population.

Objectives To investigate the efficacy and safety of hydroxychloroquine (HCQ) in patients with rheumatoid arthritis (RA). Methods Patients with active RA, despite conventional synthetic disease-modifying antirheumatic drugs (csDMARDs), were recruited. HCQ was administered for 24 weeks, in addition to prior treatment. The primary end-point was the proportion of patients achieving American College of Rheumatology (ACR) 20 at Week 24, compared to that of a propensity score–matched historical control group. Results Sixty patients were enrolled and administered HCQ. We also identified 276 patients as candidates for the historical control group. Propensity score matching yielded 46 patients in each group. The proportion of patients achieving ACR20 at Week 24 was significantly higher in the HCQ group than in the control group (54.4% vs. 28.3%, P = .007). The proportion of patients achieving ACR50 and ACR70 at Week 24 were also higher in the HCQ group than in the control group (ACR50, 30.4% vs. 4.3%, P = .006; ACR70, 17.4% vs. 0%, P = .005). Neither HCQ retinopathy nor any new safety signal was observed during the study. Conclusion The addition of HCQ to csDMARDs was effective, with no new safety signal in patients with RA.

/objectives Several biological disease-modifying anti-rheumatic drugs (bDMARDs) have been widely used for the management of rheumatoid arthritis (RA). These drugs target different molecules important for the pathophysiology of RA; however, only a few studies have compared the effects of these biological drugs on cytokines and bone metabolic markers. The main aim of this study is to clarify the effects of bDMARDs with different modes of action on the cytokine and bone metabolic marker levels in patients with RA.Patients with RA who were initiated on infliximab, tocilizumab, or abatacept as the first bDMARD were prospectively enrolled in this study. Serum cytokine and bone metabolic marker levels were measured longitudinally, and changes in their levels were compared.A total of 174 patients were enrolled in this study, with 55, 70, and 49 patients in the infliximab, tocilizumab, and abatacept groups, respectively. At six months, despite the similar clinical effectiveness of the three drugs, changes in the cytokine and bone metabolic marker levels were distinct; interferon-γ and tumor necrosis factor-α levels were significantly increased with infliximab, interleukin-6 levels were increased with tocilizumab, and interleukin-1β and interleukin-8 levels were increased with abatacept treatment. Bone-specific alkaline phosphatase and osteocalcin levels increased more significantly with tocilizumab than with infliximab, while osteopontin and osteonectin levels decreased with infliximab treatment.bDMARDs with different modes of action exert different effects on the cytokine and bone metabolic marker levels in patients with RA.

To assess differences in the strength of inhibition of IL-6/STAT3 signalling induced by subcutaneously (sc) administered tocilizumab (TCZ) and sarilumab (SAR).Data were collected on patients with rheumatoid arthritis (RA) who achieved low disease activity (Clinical Disease Activity Index [CDAI]≤10) following treatment with weekly or bi-weekly administration of 162 mg sc of TCZ (TCZ qw group, n=8; TCZ q2w group, n=8), bi-weekly doses of 200 mg sc of SAR (SAR q2w group, n=7), or MTX (n=8) as a control. The clinical characteristics of each group were collected, and the serum concentrations of IL-6 and soluble IL-6 receptor (sIL-6R) were measured using ELISA. Whole blood samples from each group were stimulated with 100 ng/ml of IL-6. The proportion of phosphorylated (p)STAT3-positive CD4+ T cells was measured using phosflow cytometric analysis.The proportion of pSTAT3-positive CD4+ T cells following stimulation with 100 ng/ml of recombinant human IL-6 was significantly different among the groups (median 1.8% [0.9-3.0] vs. 7.7% [2.9-8.0] vs. 12.5% [11.4-16.6] vs. 71.5% [68.0-78.5] for the TCZ qw, SAR q2w, TCZ q2w, and MTX control groups, respectively; p0.01 for all comparisons).SAR 200 mg q2w showed significantly stronger inhibition of IL-6/STAT3 signalling than TCZ sc q2w but weaker inhibition than TCZ sc qw. The results of this study may be useful for adjusting the IL-6 blockade treatment for patients with RA.

Objective To clarify relevant proteins and clinical characteristics of a phenotype of IgG4-related disease (IgG4-RD) with lymphadenopathy. Methods We enrolled patients newly diagnosed with IgG4-RD in our department between January 2000 and June 2018 and performed proteomic analysis to measure serum concentrations of 1305 proteins. We extracted proteins overexpressed in patients with IgG4-RD with lymphadenopathy by comparing between those with lymphadenopathy, those without lymphadenopathy and healthy controls. We further reviewed all the patients with IgG4-RD in our institution and investigated the characteristics and prognosis of the patients with IgG4-RD with lymphadenopathy. Results Eighty-five patients with IgG4-RD were enrolled, of which, 55% had lymphadenopathy. Proteomic analysis in 31 patients with IgG4-RD and 6 healthy controls revealed that eotaxin-3 was a potential serum biomarker in the patients with lymphadenopathy versus those without lymphadenopathy and healthy controls. A cohort of 85 patients with IgG4-RD demonstrated that patients with lymphadenopathy showed a significantly higher serum IgG4, IgG4:IgG ratio, IgG4-RD responder index and eosinophilia (P Conclusion Lymphadenopathy in IgG4-RD represents a phenotype associated with high disease activities, eosinophilia and relapsing disease. Eotaxin-3 is a novel biomarker related to IgG4-RD with lymphadenopathy.

Background Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disease that affects small- to medium-sized blood vessels. Despite treatments having been improved, patients often experience disease relapses. It remains unclear how the immune cells involve in the development of vasculitis and how they fluctuate over the course of treatment. In this study, we aimed to identify the immune subsets and serum cytokines associated with disease relapse by comprehensive immuno-phenotyping in AAV patients. Methods We reviewed consecutive patients (n = 29) from Keio University Hospital who had been newly diagnosed with AAV from January 2015 to February 2019 and chronologically followed until 52 weeks. Numbers of circulating T cells, B cells, monocytes, and granulocytes were analyzed by flow cytometry (FACS). Serum levels of cytokines were measured by electrochemiluminescence enzyme immunoassay. Clinical information was obtained from patients’ records and association with time-course changes in immuno-phenotypes and serum levels of cytokines were assessed. Results Comprehensive immuno-phenotyping data from 161 samples from 29 AAV patients at diagnosis; at weeks 4, 12, 24, and 52 of treatment; and at time of major relapse were examined. FACS analysis from patients with relapse revealed that CD14++ CD16+ intermediate monocytes and plasma cells concomitantly changed associated with disease relapse, which were independent from treatment regimen, ANCA status, or disease phenotype. In particular, the number of CD14++ CD16+ intermediate monocytes at relapse was significantly higher than that in remission or in healthy controls. Serum cytokine measurement revealed that changes of monocyte-derived proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α were associated with disease status. Conclusions Chronological changes in CD14++ CD16+ intermediate monocyte counts can be a marker of disease relapse in AAV patients.

Interleukin (IL)-21-producing T peripheral helper (Tph) cells are thought to contribute to extra-follicular B cell activation and play a pathogenic role in autoimmune diseases. In this study, we investigated the relationship between Tph cells and interferons (IFNs) in several autoimmune diseases because our previous study demonstrated that type I IFNs promote the differentiation of IL-21-producing Tph-like cells. The frequency of Tph cells in the blood as well as serum IFN-α2a and IFN-λ1 were markedly elevated in patients with active systemic lupus erythematosus (SLE) compared to other autoimmune diseases or healthy controls. Notably, the frequency of Tph cells was positively correlated with the SLE disease activity index, serum IFN-α and serum IFN-λ1 in SLE patients. Additionally, we found that type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) promote the differentiation of programmed cell death-1 (PD-1)+ CXCR5 −CD4+ T cells and enhance the secretion of IL-21, IFN-γ and CXCL13. IFN-λ1, like IFN-α, up-regulated the mRNA expression of IL21, IFNG, CXCL13, CD244, SLAMF7, GZMB, PRF1, CCR5 and PRDM1, whereas it down-regulated that of CXCR5 and BCL6, reflecting a Tph-related gene expression pattern. IFN-α in combination with IFN-λ1, IFN-λ2 or IFN-λ3 significantly increased the differentiation of PD-1+CXCR5− Tph-like cells and the secretion of Tph-related cytokines as compared with each IFN alone, suggesting a cooperative interaction. From these findings, it is highly probable that type III IFNs in addition to type I IFNs play a key role in the differentiation of Tph cells and that high levels of IFN-α and IFN-λ1 trigger the differentiation and expansion of Tph cells in SLE.

ObjectivesMultiple studies suggest that interleukin (IL)-21 plays a pivotal role in the differentiation of B cells and activation of cytotoxic T cells and is involved in the pathogenesis of IgG4-related disease (IgG4-RD). T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is a new marker of T follicular helper (Tfh) cells, yet its significance remains unknown. The objective of this study was to investigate whether TIGIT expression could detect high IL-21-producing peripheral Tfh populations and their association with disease activity in IgG4-RD.MethodsTIGIT expression in peripheral CD4+T cell subsets was comprehensively analyzed by multi-color flow cytometry. Single cell mapping was performed by t-SNE method, and IL-21 production was compared in TIGIT+ and TIGIT-T cells. The effect of OX40 signal on cytokine expression was analyzed by RNA-sequencing. Clinical significance of TIGIT+ and TIGIT- peripheral T cells was analyzed in active patients with IgG4-RD, both at baseline and after 12 weeks of glucocorticoid treatment.ResultsUnbiased single cell mapping revealed two high IL-21-producing peripheral T cell populations; TIGIT+ Tfh and TIGIT-T helper cells. OX40 signal was associated with high IL-21 production in TIGIT+ Tfh and TIGIT-T helper cells. IL-21 production in Tfh cells correlated with the proportion of TIGIT+ cells in Tfh cells, serum IgG4 level, and scores of disease activity. Furthermore, the skewing toward peripheral TIGIT+ Tfh cells, particularly TIGIT+Tfh2 subset correlated with disease activity and was corrected by glucocorticoid treatment in IgG4-RD.ConclusionsOX40 is associated with high IL-21 production in peripheral TIGIT+ Tfh cells, and the increase in peripheral TIGIT+ Tfh cells reflects disease activity in IgG4-RD.

Neutrophils form neutrophil extracellular traps (NETs), which are involved in the pathogenesis of ANCA-associated vasculitis (AAV). Recent reports suggest that platelets stimulated via toll-like receptor (TLR) pathways can induce NETs formation. However, the mechanism underlying the involvement of platelets in NETs formation in AAV is unknown. We investigated the role of platelets in the pathogenesis of AAV. Platelets from AAV patients and healthy controls (HCs) were co-cultured with peripheral neutrophils, and NETs formation was visualized and quantified. The expression levels of TLRs on platelets were examined by flow cytometry. Platelets were treated with a TLR agonist, platelet-derived humoral factor, CXCL4 (platelet factor 4: PF4), and/or anti-CXCL4 antibody to investigate the effects of TLR–CXCL4 signaling on NETs formation. Platelets from AAV significantly upregulated NETs formation in vitro. Flow cytometric analysis revealed that the proportion of TLR9 positive platelets was significantly higher in AAV than HCs. CXCL4 released from TLR9 agonist-stimulated platelets was significantly enhanced in AAV, which subsequently increased NETs formation. Further, neutralizing anti-CXCL4 antibody significantly inhibited NETs formation enhanced by platelets from AAV. TLR9 signaling and CXCL4 release underlie the key role that platelets play in NETs formation in the pathogenesis of AAV.

Objectives Large vessel vasculitis (LVV) is characterised by a high relapse rate. Because accurate assessment of the LVV disease status can be difficult, an accurate prognostic marker for initial risk stratification is required. We conducted a comprehensive longitudinal investigation of next‐generation RNA‐sequencing data for patients with LVV to explore useful biomarkers associated with clinical characteristics. Methods Key molecular pathways relevant to LVV pathogenesis were identified by examining the whole blood RNA from patients with LVV and healthy controls (HCs). The data were examined by pathway analysis and weighted gene correlation network analysis (WGCNA) to identify functional gene sets that were differentially expressed between LVV patients and HCs, and associated with clinical features. We then compared the expression of the selected genes during week 0, week 6, remission and relapse. Results The whole‐transcriptome gene expression data for 108 samples obtained from LVV patients (n = 27) and HCs (n = 12) were compared. The pathway analysis and WGCNA revealed that molecular pathway related to interleukin (IL)‐1 was significantly upregulated in LVV patients compared with HCs, which correlated with the positron emission tomography vascular activity score, a disease‐extent score based on the distribution of affected arteries. Further analysis revealed that the expression levels of genes in the IL‐1 signalling pathway remained high after conventional treatment and were associated with disease relapse. Conclusion Upregulation of the IL‐1 signalling pathway was a characteristic of LVV patients and was associated with the extent of disease and a poor prognosis., In this study, we found that interleukin (IL)‐1 signalling was upregulated in large vessel vasculitis (LVV) and correlated with the disease‐extent score. IL‐1 signalling remained upregulated even after conventional treatment and was associated with disease relapse. Targeting IL‐1 signalling may be a promising therapeutic approach for LVV.

Background Milk fat globule epidermal growth factor (MFG-E8) is related secreted protein which links phosphatidylserine on apoptotic cells and integrin αvβ3/5 on phagocytes. To clarify the clinical significance of MFG-E8 in SLE, we analyzed the correlation between expression level of MFG-E8 in circulating phagocytic leukocytes and clinical parameters of patients. Methods The study was conducted under a multi-center, prospective cohort design. Patients with one or both BILAG A or B, or SLEDAI- 2 K ≥ 4 with clinical symptoms were defined as the active SLE group. Expression of MFG-E8 on monocytes and concentration in serum were measured by FACS and ELISA, respectively. Results 96 subjects were enrolled. The absolute number and proportion of MFG-E8-positive monocytes to total monocytes were significantly higher in the active SLE group (p Conclusions The proportion of MFG-E8-positive monocytes to total monocytes in peripheral blood was positively associated with disease activity in SLE and may be a novel biomarker of disease activity.

Objectives Tocilizumab (TCZ) is a humanised anti‐interleukin (IL)‐6 receptor (IL‐6R) monoclonal antibody that is a promising agent to treat various autoimmune diseases. However, the mechanism of TCZ efficacy is unclear. This study aims to elucidate the relationship between Tregs and IL‐6R blockade in autoimmunity‐mediated renal disease based on a TCZ‐treated cohort of patients with anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV) and in an experimental model of crescentic glomerulonephritis (cGN). Methods We examined multiple serum levels of cytokines and chemokines and peripheral blood mononuclear cells in patients with AAV who received TCZ monotherapy and achieved drug‐free remission. Moreover, we investigated the mechanistic role of IL‐6R blockade in accelerated cGN model to analyse the local sites of inflammation. Results Serum chemokines CCL22 and CCL17, in addition to the CCR4+Foxp3+ Treg population, increased in patients who demonstrated drug‐free remission after the cessation of TCZ. In the cGN model, IL‐6R blockade ameliorated the disease, elevated CCL22/17 in CD206+CD11b+CD11c+ kidney M2‐like type macrophages, and increased the migration of Tregs into the kidney and regional lymph nodes. The local administration of CCL22 in the kidney facilitated Treg accumulation and reduced glomerular crescent formation. Conclusions This study revealed a new mechanism whereby effector Tregs migrate into the inflammatory kidney via the CCL22/17–CCR4 axis that is facilitated by M2‐like type macrophages that are induced by IL‐6R blockade., Our study has revealed a new remission mechanism whereby effector Tregs migrate into the crescentic glomerulonephritis via the CCL22/17–CCR4 axis that is facilitated by M2‐like type macrophages that are induced by IL‐6R blockade.

Objectives The regulation system for oxidative stress in systemic sclerosis (SSc) remains unclear. This study aimed to clarify the possible involvement of ataxia telangiectasia mutated (ATM), which plays a key role in DNA repair and redox balance, in the pathogenesis of SSc. Methods Thirty patients with SSc and 15 healthy controls were enrolled. Expression of ATM and phosphorylated ATM (pATM), an activated form of ATM, in phagocytes in whole blood samples was analysed by FACS. Correlations between expression levels of ATM/pATM and clinical parameters of SSc patients were statistically analysed. Peripheral monocytes were cultured with an ATM-specific inhibitor (KU55933), and reactive oxygen species production in the cells was measured. Results Expression level of pATM in peripheral monocytes and neutrophils from SSc patients was significantly lower than those in healthy controls (P = 0.04 and P Conclusion Our data revealed that decreased ATM activation in monocytes was associated with SSc-interstitial lung disease and that impaired ATM activation in monocytes may contribute to the disease process of SSc via uncontrolled reactive oxygen species production.

Background Large vessel vasculitis (LVV) is a type of vasculitis characterized by granulomatous inflammation of medium- and large-sized arteries. Clinical assessment of acute phase reactants has been conventionally used to diagnose and monitor diseases; however, accurate assessment of vascular disease activity status can be difficult. In this study, we investigated comprehensive immuno-phenotyping to explore useful biomarkers associated with clinical characteristics. Methods Consecutive patients with newly diagnosed LVV who visited our institution between May 2016 and May 2019 were enrolled. The number of circulating T cells, B cells, natural killer cells, dendritic cells, monocytes, and granulocytes was examined and chronologically followed. Baseline and time-course changes in immuno-phenotyping associated with disease activity were assessed. Results Comprehensive immuno-phenotyping data from 90 samples from each of 20 patients with LVV were compared with those from healthy controls (HCs). The number of helper T (Th), follicular helper T (Tfh), CD8+ T, CD14++ CD16+ monocytes, and neutrophils were higher in patients with giant cell arteritis (GCA) and/or Takayasu arteritis (TAK) than in HCs. Among them, the number of CD8+ T and CD8+ Tem were higher in patients with TAK than in GCA. Notably, memory CD4+ and CD8+ T cells in patients with TAK remained high even in the remission phase. Further analysis revealed that the number of Th1, Th17, and Tfh cells was associated with disease relapse in GCA and TAK and that the number of CD8+ T cells was associated with relapse in TAK. Th1, Th17, and Tfh cells decreased after treatment with biologic agents, while CD8+ T cells did not. Conclusions Our results from peripheral immuno-phenotyping analysis indicate that the numbers of Th and Tfh cells changed along with the disease condition in both GCA and TAK, while that of CD8+ T cells did not, especially in TAK. Treatment with biologic agents decreased the proportion of Th and Tfh cells, but not CD8+ T cells, in the patients. Chronological immuno-phenotyping data explained the difference in therapeutic response, such as reactivities against biologics, between GCA and TAK.

Sustained clinical remission (CR) without drug treatment has not been achieved in patients with rheumatoid arthritis (RA). This implies a substantial difference between CR and the healthy state, but it has yet to be quantified. We report a longitudinal monitoring of the drug response at multi-omics levels in the peripheral blood of patients with RA. Our data reveal that drug treatments alter the molecular profile closer to that of HCs at the transcriptome, serum proteome, and immunophenotype level. Patient follow-up suggests that the molecular profile after drug treatments is associated with long-term stable CR. In addition, we identify molecular signatures that are resistant to drug treatments. These signatures are associated with RA independently of known disease severity indexes and are largely explained by the imbalance of neutrophils, monocytes, and lymphocytes. This high-dimensional phenotyping provides a quantitative measure of molecular remission and illustrates a multi-omics approach to understanding drug response., Little information is available on molecular changes in response to treatment of rheumatoid arthritis (RA). Here the authors report a multi-omics study collecting patients' transcriptome, proteome, and immunophenotype data to help understand the impact of drug treatments on RA molecular phenotypes.

A causal relationship between protracted exposure to low-dose rate radiation and health effects remains unclear despite extensive international studies of nuclear workers. One potential reason is that radiation epidemiological studies that adjust for tobacco smoking, which heavily influences mortality, have been limited. In the present study, we examined radiation-related cancer risk by directly assessing the possible confounding effect of smoking, using data from two questionnaire surveys performed among Japanese nuclear workers in 1997 and 2003. Mortality follow-up was carried out for 71 733 male respondents for an average of 8.2 years during the observation period of 1999-2010. The mean cumulative dose was 25.5 mSv at the end of the follow-up period. Estimates of excess relative risk per Sv (ERRs/Sv) were obtained by Poisson regression. By adjusting for smoking directly on the basis of a linear dose-response model, we quantified the confounding effects of smoking on radiation risks. Statistically significant ERRs/Sv were found for all causes, all diseases, all non-cancer diseases, and liver cancer: 0.97 (90% confidence interval: 0.23, 1.78), 1.32 (0.40, 2.34), 1.87 (0.47, 3.49), and 4.78 (0.09, 11.68), respectively, without adjustment for smoking. However, the ERRs/Sv were no longer statistically significant after adjustment for smoking: 0.45 (-0.22, 1.19), 0.77 (-0.08, 1.72), 1.28 (-0.03, 2.79), and 3.89 (-0.46, 10.34), respectively. The ERRs/Sv for all cancers excluding leukaemia and lung cancer were not significant before adjustment for smoking, but declined after adjustment for smoking. The present study demonstrates that in this cohort of workers, smoking heavily distorts radiation risk estimates of mortality. The possibility of confounding by smoking depends on how strongly smoking is correlated with radiation exposure. If a correlation between smoking and radiation dose is suggested, smoking is an important confounder when assessing the radiation and health risks.

We investigated the correlation between the increased proportion of peripheral B cell subsets and clinical and immunological features in primary Sjogren's syndrome (pSS). We found that the proportion of CD19+ B cells was significantly increased in pSS as compared with HC and was correlated with serum IgG levels. Moreover, in vitro IgG production by CD19+ B cells was significantly increased in pSS and was positively and significantly correlated with serum IgG levels. FACS analysis revealed that the proportions of peripherally CD38highIgD+ B cells and CD38highIgD- B cells were significantly increased in pSS. In addition, the proportion of CD38highIgD+ B cells positively correlated with ESSDAI scores and serum levels of IgG, anti-Ro/SSA and anti-La/SSB antibodies while that of CD38highIgD- B cells showed no correlation with these parameters. Our data suggest that increased proportion of CD38highIgD+ B cells in pSS is involved in IgG overproduction including autoantibodies, and correlates with disease progression.

ObjectivesMultiomics study was conducted to elucidate the crucial molecular mechanisms of primary Sjögren’s syndrome (SS) pathology.MethodsWe generated multiple data set from well-defined patients with SS, which includes whole-blood transcriptomes, serum proteomes and peripheral immunophenotyping. Based on our newly generated data, we performed an extensive bioinformatic investigation.ResultsOur integrative analysis identified SS gene signatures (SGS) dysregulated in widespread omics layers, including epigenomes, mRNAs and proteins. SGS predominantly involved the interferon signature and ADAMs substrates. Besides, SGS was significantly overlapped with SS-causing genes indicated by a genome-wide association study and expression trait loci analyses. Combining the molecular signatures with immunophenotypic profiles revealed that cytotoxic CD8 ­T cells­ were associated with SGS. Further, we observed the activation of SGS in cytotoxic CD8 T cells isolated from patients with SS.ConclusionsOur multiomics investigation identified gene signatures deeply associated with SS pathology and showed the involvement of cytotoxic CD8 T cells. These integrative relations across multiple layers will facilitate our understanding of SS at the system level.

ObjectivesTo elucidate the association between clinical characteristics and immuno-phenotypes in patients with ANCA-associated vasculitis (AAV).MethodsPeripheral blood from 36 patients with active AAV and 18 healthy controls was examined for numbers of circulating T cells, B cells, NK cells, dendritic cells, monocytes and granulocytes using flow cytometry. These immuno-phenotyping data were subjected to cluster analysis and principal components analysis to divide AAV patients into subgroups. Associated organ involvement or therapeutic prognosis were assessed for each subgroup.ResultsAAV patients had higher proportions of plasma cells, plasmablasts, activated T cells, CD14++ CD16+ monocytes, eosinophils and neutrophils than healthy controls. Immuno-phenotyping findings were similar between patients with microscopic polyangiitis, granulomatosis with polyangiitis and eosinophilic granulomatosis with polyangiitis. Cluster analysis indicated that AAV patients could be divided into three subgroups according to peripheral immune cell numbers: antibody production-related (n = 9), cytotoxic activity-related (n = 4) and neutrocytosis/lymphocytopenia-related (n = 23). The antibody production-related or cytotoxic activity-related group was associated with CNS involvement, and the neutrocytosis/lymphocytopenia-related group was associated with high incidence of kidney involvement. Incidence of severe infection was markedly higher in the neutrocytosis/lymphocytopenia-related group than the other two groups. Incidence of disease relapse was comparable among the three groups.ConclusionPatients with active AAV can be divided into three subgroups based on immuno-phenotyping. These results may provide a hint to understanding disease pathophysiology and prognosis, and determining appropriate treatment.

Background Primary Sjogren’s syndrome (pSS) is an idiopathic autoimmune disease whose major clinical manifestations are xerostomia and keratoconjunctive sicca, and is often accompanied with hypergammaglobulinemia. Several lines of evidence suggest that a focal lymphocytic infiltrate of the exocrine glands is responsible for lesion formation, IgG production and subsequent dysfunction of the glands. It is well known that B cell activating factor (BAFF) and its receptor, BR3, are deeply involved in the pathogenesis of pSS, and hence BAFF and BR3 are promising targets to treat the disease. We have reported that the abnormally elevated expression of BR3 in monocytes is associated with overproduction of inflammatory cytokines, such as IL-6, by monocytes of pSS patients. Our in vitro experiments suggest that BAFF-stimulated monocytes contribute to IgG overproduction by pSS B cells. We also found that the population of a specific subset of B cells overexpressing BR3 was abnormally increased in pSS patients and that the proportion of the subset was positively and significantly correlated with serum levels of autoantibodies and total IgG as well as ESSDAI, one of the indicators of disease activity of pSS. Our results collectively suggest that the elevated expression of BR3 in pSS monocytes results in B cell activation and subsequent overproduction of IgG. However, the relationship between the abnormalities of peripheral cells and clinical manifestations of pSS has not been fully understood. Objectives To elucidate possible roles of abnormal monocytes in the development of clinical manifestations of pSS. Methods The expression level of BR3 in peripheral monocytes of patients with pSS (n = 65) and healthy controls (HC: n = 38) was analyzed by FACS. Peripheral B cells were cultured with BAFF-stimulated monocytes in the presence or absence of an anti-IL-6 receptor antibody, and the proportion of CD38highIgDlow cells among CD19+ cells, and IgG production were analyzed by FACS and ELISA, respectively. The serological data and focus scores by lip biopsy of the patients were collected by clinical records. Results FACS analysis revealed that the expression level of BR3 in pSS monocytes was significantly higher than that of HC (p Conclusion Our data strongly suggest that accelerated signal transduction through a BAFF-BR3 axis in monocytes is involved in the pathogenesis of pSS and associates with clinical manifestations of the disease. These results also suggest that BAFF and the BAFF signaling pathways through BR3 are the possible therapeutic targets for pSS. Disclosure of Interests Keiko Yoshimoto: None declared, Katsuya Suzuki: None declared, Yumi Ikeda: None declared, Eriko Takei: None declared, Tsutomu Takeuchi Grant/research support from: Astellas Pharma Inc, Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Takeda Pharmaceutical Co., Ltd., AbbVie GK, Asahikasei Pharma Corp., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Eisai Co., Ltd., AYUMI Pharmaceutical Corporation, Nipponkayaku Co. Ltd., Novartis Pharma K.K., Grant/research support from: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Grant/research support from: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Abbivie GK, Nipponkayaku Co.Ltd, Janssen Pharmaceutical K.K., Astellas Pharma Inc., Taiho Pharmaceutical Co. Ltd., Chugai Pharmaceutical Co. Ltd., Taisho Toyama Pharmaceutical Co. Ltd., GlaxoSmithKline K.K., UCB Japan Co. Ltd., Consultant for: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Asahi Kasei Medical K.K., AbbVie GK, Daiichi Sankyo Co., Ltd., Bristol Myers Squibb, and Nipponkayaku Co. Ltd., Speakers bureau: Astellas Pharma Inc., Bristol Myers Squibb, Chugai Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahi Kasei Pharma Corp., Taisho Toyama Pharmaceutical Co., Ltd., SymBio Pharmaceuticals Ltd., Janssen Pharmaceutical K.K., Celltrion Inc., Nipponkayaku Co. Ltd., and UCB Japan, Speakers bureau: AbbVie, Asahi Kasei, Astellas, AstraZeneca, AYUMI, Bristol-Myers Squibb, Chugai, Daiichi Sankyo, Eisai, Eli Lilly Japan, Janssen, Mitsubishi Tanabe, Nippon Kayaku, Novartis, Pfizer Japan Inc, Taiho, Taisho Toyama, Takeda, Teijin, Speakers bureau: AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co. Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Astellas Pharma Inc, Diaichi Sankyo Co. Ltd., Eisai Co. Ltd., Sanofi K.K., Teijin Pharma Ltd., Takeda Pharmaceutical Co. Ltd., Novartis Pharma K.K.

We investigated the effects of bendamustine on B cell functions and explored potential clinical applications of the drugs to autoimmune diseases. Proliferation of Ramos cells, a human B cell line, was significantly inhibited by 25-100μM of bendamustine in a dose-dependent manner. Concordantly, IgM secretion from Ramos cells was significantly inhibited at these concentrations by up to 70%. Interestingly, however, the production and secretion of interleukin-10 (IL-10) were dramatically (at least >10-fold) increased by bendamustine at growth inhibitory concentrations. Exploration of the molecular mechanism of IL-10 production revealed that bendamustine enhanced the phosphorylation of p38 MAP kinase. Further, Sp1 was identified as a downstream transcription factor, and the inhibition of p38 MAP kinase and Sp1 with their inhibitors led to the abrogation of bendamustine-induced IL-10 production and the DNA binding of Sp1. Importantly, when PBMC from healthy donors were cultured with bendamustine at the concentration of 30μM, under the stimulation with an anti-IgM antibody, an anti-CD40 antibody, recombinant human IL-21 (rhIL-21) and recombinant human soluble BAFF (rhsBAFF), IL-10 production by B cells (CD20+CD4-CD8-CD14-) among peripheral blood mononuclear cell (PBMC) was significantly enhanced by adding bendamustine. These results collectively suggest that the p38 MAP kinase-Sp1 pathway plays a crucial role in bendamustine-induced IL-10 production by B cells. Our findings suggest a novel therapeutic possibility for autoimmune diseases through the upregulation of IL-10 which has an anti-inflammatory effects.

Background B cell activating factor belonging to TNF family (BAFF) is well known as a factor which regulates proliferation, differentiation and survival of B cells, and plays a pivotal role in the pathogenesis of primary Sjogren’s syndrome (pSS). In our previous study, we found that BAFF significantly enhanced IL-6 production by pSS monocytes and the amount of IL-6 produced by BAFF-stimulated monocytes was positively and significantly correlated with the expression level of a BAFF receptor (BR3). These data collectively suggest that the BAFF signalling through BR3 is involved in activation of monocytes to promote production of inflammatory cytokines, such as IL-6. Matrix metalloproteinase-9 (MMP-9) is well known as one of the enzymes involved in degradation of extracellular matrix (ECM) and mainly produced by activated T cells and monocytes. It has been reported that the concentration of MMP-9 in saliva was significantly higher in pSS patients as compared to healthy controls (HC). Therefore, it is conceivable that MMP-9 is involved in the pathogenesis of pSS through degradation of ECM of salivary glands, which consequently results in decrease in saliva, one of the clinical manifestations of pSS. Objectives To explored the relationship between BAFF and MMP-9 in the pathogenesis of pSS. Methods Peripheral monocytes from pSS patients (n=37) and HC (n=19) were prepared by using CD14 +microbeads and cultured in vitro in the presence or absence of recombinant human soluble BAFF (rhsBAFF) for 96 hours. The amounts of IL-6 and MMP-9 in the culture supernatants were measured by ELISA. Signal transduction pathways were investigated by exposing rhsBAFF-stimulated pSS monocytes to several inhibitors against NF-κB (BAY11–7082 and BAY11–7085) and PI3 kinase (LY294002). FACS analysis of whole blood samples was performed to investigate the expression levels of BR3 and MMP-9 in monocytes. The expression level of MMP-9 was also analysed by quantitative RT-PCR (qPCR). Serum levels of BAFF and MMP-9 were measured by an electrochemiluminescence assay. Results Serum levels of BAFF and MMP-9 in pSS patients were significantly higher than those of HC, and the levels showed positive and significant correlation. FACS analysis of whole blood samples demonstrated that MMP-9 was mainly expressed in monocytes and that the expression level was significantly higher in pSS than in HC. ELISA and qPCR revealed that stimulation of pSS moncytes with rhsBAFF drastically enhanced the expression of MMP-9 as compared to normal monocytes. Remarkably, the amount of MMP-9 produced by the cells was positively and significantly correlated with the expression level of BR3 in pSS monocytes, suggesting that BAFF-signalling is involved in the production of MMP-9 by the cells. Moreover, the elevated production of MMP-9 was significantly suppressed by specific inhibitors against NF-κB and PI3 kinase in a dose dependent manner. Conclusions The present study suggests that BAFF stimulates monocytes through BR3 to promote MMP9 production and may contribute to ECM degradation. Our study also suggests that NF-κB and PI3 kinase are involved in the pathway. Disclosure of Interest None declared

Background Milk fat globule epidermal growth factor 8 (MFG-E8) is an apoptosis-related secreted protein. It has been reported that MFG-E8 deficient or excess mice developed a systemic lupus erythematosus (SLE)-like autoimmune disease due to impaired clearance of apoptotic cells,1 suggenting that abnormal expression of MFG-E8 is involved in the pathogenesis of SLE. Since elevated serum MFG-E8 level has been found in SLE patients,2 it may possibly provide the classification of the disease by pathological conditions.3 However, the assosiation with MFG-E8 expression and clinical features of SLE patients is not fully understood. Objectives To clarify the clinical significance of MFG-E8 in SLE, we analysed the correlation between the expression level of MFG-E8 in circulating phagocytic leukocytes and clinical parameters of the patients. Methods A multi-centre, exploratory and prospective SLE cohort was established. Among SLE patients who visited our division from May 2015 to March 2017 and satisfied the 1997 revised criteria, patients with one or both of BILAG A or B, or with SLEDAI-2K≥4 and clinical symptoms were defined as active SLE. These patients were then matched with randomly selected patients by age, gender, history of nephritis, and daily glucocorticoid dose as an inactive SLE group. Age and sex matched healthy controls (HC) were also recruited. The expression level of MFG-E8 in monocytes and its concentration in serum of the patients were measured by FACS and ELISA, respectively. The clinical parameters of the patients were collected from their clinical records. Results A total of 108 cases were enrolled, consisting of 36 active (mean age: 44.2±18.6, female: 80.6%, nephritis: 69.7%), 38 inactive SLE and 24 HC cases. The absolute number and the proportion of MFG-E8 positive-monocytes to total monocytes were significantly higher in the active SLE group (p Conclusions Our study indicate that the proportion of MFG-E8-positive monocytes to total monocyte in peripheral blood was positively associated with disease activity of SLE and may be a novel mechanistic biomarker to determine the disease activity. References [1] Science2004;304(5674):1147–50. [2] J Leuko Biol. 2008;83:1300–7. [3] Lupus2014;23:386–94. Disclosure of Interest None declared

Background Ataxia-telangiectasia mutated (ATM) is a protein kinase associated with ataxia-telangiectasia (AT), which is an autosomal recessive disorder due to defective functional activity of ATM. Telangiectasia, seen in AT, is also well known as one of the major characteristics of systemic sclerosis (SSc). ATM plays an important role not only in DNA damage repairing system, but also in the process of regulation of oxidative stress. 1 Moreover, it has been reported that oxidative stress may contribute to disease process of SSc 2 3 . Based on these background, we hypothesised that ATM may play a substantial role in the pathogenesis of SSc. However, the possible association between ATM activity and SSc development is not fully understood. Objectives To clarify the role of ATM in the pathogenesis of SSc, we demonstrated the expression and activation level of ATM in circulating immune cells and analysed the association with clinical characteristics of the patients. Methods Whole blood samples were collected from twenty-four patients with SSc and 12 healthy controls (HC). Expression levels of total ATM and active phosphorylated ATM (pATM) were examined in each immune cell subset (neutrophil, monocyte, T cell, B cell and NK cell) by mean fluorescence intensity (MFI) using flow cytometer. Each MFI level of ATM and pATM was compared between patients with SSc and HC, and was analysed the correlation with clinical characteristics of SSc patients, retrospectively collected from patients’ records. Results The expression level of pATM was significantly lower in monocytes, neutrophils, and T cells in SSc as compared with HC (1887 ±128 vs 2386±181, p=0.03; 10265±861 vs 16087±1218, p=0.0004; 1326±73 vs 1675±103, p=0.009; respectively), whereas no significant difference in total ATM level was observed in each cell subset between two groups. Notably, the expression levels of pATM in monocytes of the patients with interstitial lung disease (ILD) was lower than that of the patients without ILD (1663 ±136 vs 1999±96, p=0.05). Furthermore, there was a tendency of correlation between pATM level in monocyte and parameters of pulmonary function test, such as forced vital capacity. No significant differences and/or correlation between pATM expression level in other cell subsets and clinical parameters of the patients, such as SSc subtype, SSc-specific autoantibodies, presence of pulmonary arterial hypertension, gastrointestinal involvement, digital tip ulcer, pitting scar and modified Rodnan skin score were observed. Conclusions In SSc, phosphorylated level of ATM in monocytes, neutrophils, and T cells was significantly lower than that of HC. Importantly, we found that ATM activation was impaired in monocyte of the SSc patients with ILD. These results collectively suggest that the loss of ATM activation in monocytes may contribute to the disease process of SSc, and is possibly due to DNA damage and oxidative stress. References [1] Guo Z, et al. Science. 2010;330:517–521. [2] Sambo P, et al. J Invest Dermatol. 1999;112:78–84. [3] Gabrielli A, et al. Open Rheumatol J. 2012;6:87–95. Disclosure of Interest None declared

Background We reported that BAFF robustly increases IL-6 production by peripheral monocytes of patients with primary Sjogren’s syndrome (pSS) and that the BAFF-induced IL-6 production and serum IgG levels of the patients were positively correlated with the expression level of a BAFF receptor (BR3) in pSS monocytes. Moreover, we found that IgG production by pSS B cells in vitro was significantly enhanced by BAFF-stimulated monocytes. These results collectively suggest that BAFF signalling via BR3 is involved in IgG overproduction and that is a possible therapeutic target to treat autoimmune diseases, such as pSS. We successfully discovered a pyrroloprymidine derivative, BIK-13, which inhibits BAFF binding to BR3 by our original high throughput screening. Objectives To explored the possibility of BIK-13 as a drug to treat autoimmune diseases. Methods Human PBMC were stimulated in vitro with a mixture of soluble BAFF (sBAFF), recombinant human IL-21, and anti-IgM and anti-CD40 antibodies (‘multiple stimulation’) to differentiate B cells into plasma blasts and/or plasma cells. BIK-13 was added to the culture and the differentiation of B cells was monitored by the expression levels of CD19/CD38/IgD and Activation-induced cytidine deaminase (AID) analysed by FACS and quantitative RT-PCR, respectively. The amounts of IL-6, IL-10 and IgG in the culture supernatants were measured by ELISA. BIK-13 was administered intraperitoneally to MRL/lpr mice and serum levels of an anti-dsDNA antibody, IL-6 and IL-10 were measured by ELISA. The proportion of B cells among splenic lymphocytes of the mice was analysed by FACS. Production of cytokines in vitro by stimulated splenic lymphocytes was measured by ELISA. Infiltration of lymphocytes into organs was analysed by immunohistochemistry. Results FACS analysis of multiple-stimulated human PBMC indicated that differentiation of B cells into plasma blasts and/or plasma cells was inhibited by BIK-13 in a dose dependent manner. Interestingly, increased IgG, IL-6 and IL-10 production by the stimulated PBMC were concomitantly and significantly suppressed by BIK-13. In addition, the expression level of AID in the cells was also suppressed by BIK-13, suggesting that an IgG class switching was impaired. Administration of BIK-13 to MRL/lpr mice for 16 weeks decreased the serum levels of an anti-dsDNA antibody, IL-6 and IL-10 as compared to the control. Notably, immunohistochemical analysis revealed that infiltration of B cells into salivary and lacrimal glands was remarkably suppressed in BIK-13-treated mice. Moreover, the proportion of B cells among splenic lymphocytes was also decreased in BIK-13-treated mice as compared to the control. In addition, production of IL-6 and IL-10 by activated splenic lymphocytes of the BIK-13-treated mice was also remarkably suppressed as compared to control mice. Conclusions Our data collectively suggest that BIK-13, a low molecular weight BAFF-signalling inhibitor, suppresses B cell activation/differentiation in vitro and consequently inhibits production of IgG and cytokines, such as IL-6 and IL-10, by the cells. The compound inhibited infiltration of B cells into organs of model mice of autoimmune diseases. These data suggest that the compound is a promising drug candidate to treat autoimmune diseases, such as pSS. Disclosure of Interest K. Yoshimoto: None declared, N. Seki Employee of: Mitsubishi Tanabe Pharma Corporation, K. Suzuki: None declared, K. Sugahara Employee of: Mitsubishi Tanabe Pharma Corporation, K. Chiba Employee of: Mitsubishi Tanabe Pharma Corporation, T. Takeuchi Grant/research support from: Mitsubishi Tanabe Pharma Corporation

Background Systemic sclerosis (SSc) is characterised by autoantibody production, microvascular injury and systemic excessive fibrosis1). Genetic and environmental factors are thought to be important for the trigger of development of the disease, however, direct connexion between these factors and pathogenesis of SSc is not yet elucidated23. Recent reports showed that environmental toxic pollutants, such as dioxins, play a significant role in the disturbance of immune system and the trigger of autoimmunity through binding aryl hydrocarbon receptor (AhR)4)5). However, little is known about the association between AhR and pathogenesis of SSc yet. Objectives To elucidate the association between AhR and the clinical characteristics of SSc. Methods Twenty-one patients with SSc who fulfilled 2013 ACR/EULAR classification criteria and 10 healthy controls (HC) were analysed. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood by using gradient centrifugation and total RNA was prepared from PBMCs. Expression of Ahr mRNA was detected by quantitative polymerase chain reaction and standardised by mRNA level of 18S ribosomal RNA in each sample. Level of Ahr mRNA in the cells was compared between SSc and HC and also between SSc patients with and without various clinical features. Results The proportion of diffuse cutaneous subset (dcSSc) was 33%. Mean disease duration was 9±9 years. The positive proportion of anticentromere antibodies, anti-topoisomerase I antibodies, anti-U1 ribonucleoprotein antibody, anti-RNA polymerase III antibody and antinuclear antibody positive without SSc-specific antibodies among 21 patients were 33.3%, 33.3%, 4.8%, 9.5% and 9.5%, respectively. Antinuclear antibody was negative in 2 (9.5%) patients. Ahr mRNA expression level was tended to be higher in SSc compared to HC (1.7±1.1 versus 1.2±0.6, p=0.1). Notably, the expression level of AhR mRNA in dc SSc was tended to be higher than that of limited cutaneous SSc (p=0.15), whereas no significant difference was detected between with or without SSc related autoantibodies, vasculopathy such as pulmonary arterial hypertension or digital tip ulcer. Importantly, the expression level of Ahr mRNA was significantly higher in patients with interstitial lung disease (ILD) (n=14) than those without (n=7) (2.0±1.1 versus 1.0±0.4, p Conclusions Expression level of Ahr mRNA was higher in patients with SSc, especially in SSc patients with ILD. In addition, AhR expression level was correlated with a parameter of pulmonary function test, DLCO% predicted. These results collectively suggest that AhR possibly plays an important role in the disease process of ILD in SSc. References [1] Nat Rev Rheumatol2013;9(8):451–64. [2] Nat. Rev. Rheumatol2014;10:671–681. [3] Semin. Immunopathol2015;37:463–473. [4] Annu Rev Immunol2014;32:403–32. [5] Nature. 2008;453(7191):65–71. Disclosure of Interest None declared