Masaru Takeshita

Number of Circulating Follicular Helper 2 T Cells Correlates With IgG4 and Interleukin-4 Levels and Plasmablast Numbers in IgG4-Related Disease.

To elucidate the pathologic role of follicular helper T (Tfh) cells and their subsets in active, untreated IgG4‐related disease.

Serum proteomic analysis identifies interleukin 16 as a biomarker for clinical response during early treatment of rheumatoid arthritis

OBJECTIVES To conduct a comprehensive quantitative proteomics analysis of novel serum protein biomarkers based on synovitis status associated with matrix metalloproteinase-3 (MMP-3) and to determine the clinical significance of these biomarkers in rheumatoid arthritis (RA). METHODS Patients with untreated RA (n=28), primary Sjogrens syndrome (pSS) (n=30), and healthy controls (HCs) (n=30) were enrolled for the screening assay. A total of 1128 serum proteins were analyzed using the SOMAscan™ assay. Serum levels of MMP-3 and interleukin (IL)-16 were measured using a latex turbidimetric immunoassay and ELISA at baseline and 12weeks after treatment with methotrexate (MTX) for MTX-naïve RA patients (n=28) or with the biologics tocilizumab (TCZ) (n=7), abatacept (ABT) (n=11) or infliximab (n=22) for MTX-inadequate response (IR) RA patients. Correlation analysis was conducted using Spearmans rank correlation method. RESULTS Proteomics showed that serum IL-16 levels were most positively correlated with those of MMP-3 (ρ=0.51, p<0.01) and were significantly increased in patients with untreated active RA compared to HCs (p<0.01) or those with pSS (p<0.01). IL-16 levels decreased following treatment in both the MTX-naïve and MTX-IR groups. Regarding clinical response, fluctuations in IL-16 levels were positively associated with changes in clinical indicators, particularly the Clinical Disease Activity Index (ρ=0.89, p<0.01) in the TCZ and ABT-treated group. However, no similar correlation was noted in MMP-3 and acute phase reactants in any groups. CONCLUSIONS IL-16 was a more effective clinical parameter than MMP-3, C-reactive protein, or erythrocyte sedimentation rate in both MTX-naive and MTX-IR RA patients. IL-16 might be a useful biomarker for evaluating clinical response in RA patients.

Polarization diversity of human CD4 + stem cell memory T cells

T cells are considered to develop through three stages, from naïve T (Tn) into central memory T (Tcm) and finally into effector memory T (Tem). Among the subsets of Tn, stem cell memory T (Tscm) were recently found to be the least developed memory subset. While this subset was revealed to possess self-reproducibility and multipotentiality, little is known about the relationship between development and polarity. We conducted transcriptome analysis of human CD4(+) T subsets and found that Tscm was a clearly distinct subset, located between Tn and Tcm. Surface antigen analysis and differentiation assay showed that the flexibility of polarity and the cytokine production progressively changed as the differentiation of CD4(+) T cells advanced. Interestingly, we found that most cells of the CD45RO(-)CCR7(+)CCR6(+) subset, hitherto considered the naïve precursor of Th17, were in fact Tscm. These findings may advance our understanding of the highly heterogeneous human helper T cells.

Multiomic disease signatures converge to cytotoxic CD8 T cells in primary Sjögren's syndrome

Objectives Multiomics study was conducted to elucidate the crucial molecular mechanisms of primary Sjögren’s syndrome (SS) pathology. Methods We 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. Results Our 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. Conclusions Our 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.

Infliximab and etanercept have distinct actions but similar effects on cytokine profiles in rheumatoid arthritis

OBJECTIVE Pro-inflammatory cytokines, especially TNFα, play a central role in the pathogenesis of rheumatoid arthritis (RA). The available TNF inhibitors are only slightly different from one another in terms of clinical efficacy, at least at the group level, but their structures and modes of action are not identical. Infliximab (IFX) and etanercept (ETN) differ in their ability to induce antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity, and in their ability to bind TNFβ. The purpose of our study was to elucidate the different cytokine pathways through which these two drugs enact their clinical efficacy. METHODS Serum from 44 RA patients treated with IFX and 24 patients treated with ETN was studied. All patients had been given these biologics at identical dosages and intervals for one year. The concentrations of 11 inflammatory cytokines and their receptors (IL-1β, IL-2, IL-6, IL-6R, IL-8, IL-10, IL-12, TNFα, TNFβ, IFNγ, and GM-CSF) were measured at weeks 0, 22, and 54 using a high-sensitivity electro-chemiluminescence assay. Cytokine profiles were analyzed along with clinical efficacy. RESULTS IL-6 was significantly decreased in the ETN+MTX and IFX+MTX groups, although not in the ETN-only group; this change was consistent with changes in disease activity. IFNγ was gradually increased only in the non-remission subgroup of the IFX group, and not at all in the ETN group. TNFβ increased after starting IFX regardless of clinical efficacy. CONCLUSION IL-6 inhibition is a pathway affected by both IFX and ETN. In addition, IFNγ increase is a distinctive feature of the inefficacy of IFX.

Alteration of matrix metalloproteinase-3 O-glycan structure as a biomarker for disease activity of rheumatoid arthritis

BackgroundNearly all secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. In rheumatoid arthritis (RA), C-reactive protein (CRP), and matrix metalloproteinase-3 (MMP-3) are widely used as serologic biomarkers, but they lack sufficient specificity or precision. We performed comparative glycosylation profiling of MMP-3 using a recently developed antibody-overlay lectin microarray technology that allows semicomprehensive and quantitative analysis of specific protein glycosylation to develop an RA-specific disease activity biomarker.MethodsSerum was taken from patients with RA (n = 24) whose disease activity was scored using composite measures, and MMP-3 was immunoprecipitated and subjected to lectin microarray analysis. A disease activity index (DAI) based on lectin signal was developed and validated using another cohort (n = 60). Synovial fluid MMP-3 in patients with RA and patients with osteoarthritis (OA) was also analyzed.ResultsIntense signals were observed on a sialic acid-binding lectin (Agrocybe cylindracea galectin [ACG]) and O-glycan-binding lectins (Jacalin, Agaricus bisporus agglutinin [ABA], and Amaranthus caudatus agglutinin [ACA]) by applying subnanogram levels of serum MMP-3. ACG, ABA, and ACA revealed differences in MMP-3 quantity, and Jacalin revealed differences in MMP-3 quality. The resultant index, ACG/Jacalin, correlated well with disease activity. Further validation using another cohort confirmed that this index correlated well with several DAIs and their components, and reflected DAI changes following RA treatment, with correlations greater than those for MMP-3 and CRP. Furthermore, MMP-3, which generated a high ACG/Jacalin score, accumulated in synovial fluid of patients with RA but not in that of patients with OA. Sialidase digestion revealed that the difference in quality was derived from O-glycan α-2,6-sialylation.ConclusionsThis is the first report of a glycoprotein biomarker using glycan change at a local lesion to assess disease activity in autoimmune diseases. Differences in the degree of serum MMP-3 α-2,6-sialylation may be a useful index for estimating disease activity.

SAT0652 Clinical and immunological significance of radiographic thymic alterations in patients with rheumatoid arthritis

Background The thymus, a primary lymphoid organ, plays a crucial role in immune system homeostasis [1,2]. Although several small-scale studies of the association between radiographic thymus alterations and serological features have been reported in systemic autoimmune diseases, information in patients with rheumatoid arthritis (RA) is limited. Objectives We conducted a large-scale cross-sectional analysis of radiographic thymus alterations and their association with clinical and immunological features in patients with RA. Methods RA patients were randomly selected from all patients who visited our department and underwent chest CT scan between January 2013 and December 2015. Patients with thymoma or thymic cyst and those aged less than 30 years were excluded. Thymic enlargement and thymus attenuation score in axial images of CT scans were quantitatively interpreted. We defined thymic enlargement as a thickness of more than 13 mm and graded the score by a four-point scale (score 0–3) according to previous studies [3,4]. Associations with immunophenotyping data of peripheral blood by flow cytometry and clinical and serological information were statistically analyzed in some available patients. Results 387 RA patients were enrolled. 78% were women and mean age was 65.2±12.3 years. Thymic enlargement was found in 76 (19.6%) patients. Thymus attenuation (score ≥2) was found in 154 (39.8%) patients. These findings were more frequent than in undiagnosed controls (11.3% (P=0.078) and 22.5% (P=0.017)). Importantly, radiographic thymus alterations in these RA patients, especially thymus attenuation score, were significantly associated with serological features such as serum level of CRP, ESR, IgG, RF-positivity or ACPA-positivity (P=0.0003, 0.001, 0.0009, 0.005, and 0.0009 respectively). When we investigated the association with the proportion of 68 peripheral blood subpopulations in 83 RA patients, thymic enlargement was significantly associated with the proportions of CD45RA+CCR7+ naïve CD4+T cell or CD45RO+CCR7-effector memory CD4+T cell (P=0.04 and 0.009). Furthermore, thymus attenuation score was also significantly associated with the proportions of CD4+ naïve T cell, CD4+effector memory T cell, CXCR5-CXCR3+CXCR6+CD4+Th1/Th17 cell, CD45RO-CCR7+CD95+CD4+ stem cell memory T cell, and CD19+ B cell (P=0.03, 0.02, 0.04, 0.04, and 0.037 respectively). Conclusions Radiographic thymus alterations are frequent in RA patients and may reflect immunological features of autoantibody production or T cell differentiation and function. References Gorozny JJ, et al. Trends Immunol 2001;22:251–255. Seddon B, et al. Immunol Today 2000;21:94–99. Naidich P, et al. Lippincott-Raven 1999:57–73. Ackman JB, et al. Radiology 2013;268(1):245–253. Disclosure of Interest None declared

AB0048 Development of The New Rheumatoid Arthritis Disease Activity Marker Using Glycosylation Change of MMP-3

Background It is known that almost all secreted proteins are glycosylated, that glycosylation patterns are influenced by cellular differentiation, and that serum glycoproteins exhibiting disease-associated glycosylation changes have potential to be biomarkers [1]. In rheumatoid arthritis (RA), C-reactive protein (CRP) and matrix metalloproteinase-3 (MMP-3) are widely used as serologic disease activity markers, but they lack sufficient specificity or precision. Thus, development of a RA-specific fine disease activity marker is needed. Objectives We focused on an existing marker, MMP-3, and examined the association between its glycosylation pattern and RA disease activity. Methods Serum was taken from RA patients (n=24) whose disease activity was scored using composite measures, and MMP-3 immunoprecipitated and subjected to recently developed antibody-overlay lectin microarray analysis. A disease activity index was developed based on lectin signal, and validated using another cohort, which included 60 serum samples from 30 RA patients, before and after treatment. Synovial fluid MMP-3 in RA and osteoarthritis (OA) patients was also analysed. Results Intense signals were observed on the O-glycan binding lectins (ABA, ACA, and Jacalin) and a sialic acid binding lectin (ACG) by applying sub-nanogram levels of serum MMP-3. Jacalin and ABA, ACA, and ACG revealed differences in MMP-3 quality and quantity, respectively. The resultant index, ACG/Jacalin, correlated with disease activity. This correlation was validated using second cohort (r2=0.341, p<0.0001), and the correlation was greater than those of MMP-3 and CRP. In addition, the change in ACG/Jacalin and disease activity before and after treatment also correlated. Furthermore, we confirmed that MMP-3, which generated a high ACG/Jacalin score, accumulated in RA but not OA patient synovial fluid. Conclusions We succeeded in development a new RA specific and sensitive disease activity marker using glycosylation change of MMP-3. The difference in quality but not quantity of MMP-3 may be a useful index for estimating RA disease activity. References Kuno A, et al. Methods Mol Biol. 2014;1200:265–285. Acknowledgement We especially thank Mr. Atsushi Kuno and Hisashi Narimatsu, the members of Research Center for Medical Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST) for their support and cooperation, and Ms. Harumi Kondo and Ms. Yuki Otomo for their assistance. Disclosure of Interest M. Takeshita: None declared, K. Suzuki: None declared, T. Takeuchi Grant/research support from: AbbVie GK, Asahikasei Pharma Corp., Astellas Pharma, Astra Zeneca K.K., Bristol–Myers K.K., Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co. Ltd., Eisai Co. Ltd., Eli Lilly Japan K.K., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co. Ltd., SymBio Pharmaceuticals Ltd., Takeda Pharmaceutical Co. Ltd., Teijin Pharma Ltd., Taisho Toyama Pharmaceutical Co. Ltd., and UCB Japan Co. Ltd., Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., Asahi Kasei Medical K.K., Abbivie GK, Daiichi Sankyo Co. Ltd., Bristol–Myers K.K., Nipponkayaku Co. Ltd, Speakers bureau: AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co. Ltd., Eisai Co. Ltd., Janssen Pharmaceutical K.K., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Takeda Pharmaceutical Co. Ltd., Astellas Pharma, Diaichi Sankyo Co. Ltd., Celtrion, Nipponkayaku Co. Ltd

FRI0609 Ultrasonography is a Useful Modality with Ease Access Reflecting Local Molecular Pathophysiology of Inflammatory Joint in Rheumatoid Arthritis

Background Number of cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1 play important roles in the pathogenesis of rheumatoid arthritis (RA). Biologics have become an effective therapeutic agent, although definition of “effect” largely relies on subjective evaluation by the physician and patient. Ultrasonography (US) is a useful modality that can directly visualize joint inflammation such as synovial thickening and vascularization and has been reported as a useful tool for objective evaluation of RA activity. However, association with pathophysiology of RA is poorly unknown and requires further evaluation. Objectives To clarify the association of joint US findings and cytokine levels in synovial fluid (SF). Methods This was a cross-sectional study. Thirty-six RA patients and 24 osteoarthritis (OA) patients were enrolled. SF aspiration was performed in 28 RA patients and the followings were measured; IL-6, IL-8, TNF-α, IL-1β, IL-10, IL-17A, IL-12/23p40, Fractalkine, VEGF, MMP-3, Granzyme A and Granzyme B. The knee joint US scans were assessed at the suprapatellar pouch, lateral and medial artcular recesses. Gray scale ultrasound (GSUS) and power Doppler ultrasound (PDUS) were analyzed by 2 experienced rheumatologists blinded to clinical information. US findings were graded on a semi-quantitative scale from 0 to 3 and mean GSUS and PDUS scores were calculated in three areas of the knee joint aforementioned. Results Median age, disease duration and DAS28-ESR were 63 years, 5 years and 5.33 respectively. Mean GSUS and PDUS were 2.37 and 1.78. The intraclass correlation coefficient values for inter-observer agreement of GSUS and PDUS were good and notably better in PDUS (GSUS; 0.716, PDUS; 0.909). IL-6, IL-8, TNF-α, IL-1β, IL-10, IL-17A, IL-12/23p40, MMP-3, Granzyme A and B in RASF were significantly higher compared to OASF. US findings especially PDUS of RA knee joint well correlated with SF IL-6, IL-8, IL-1β and IL-10. (GSUS; range of ρ=0.40 to 0.44, p<0.05, PDUS range of ρ=0.49 to 0.59, p<0.01, summarized in Table.1 and Figure 1.) Additionally PDUS correlated with VEGF involved in vasculogenesis (ρ=0.40, p=0.03), fractalkine expressed in RA synovial endothelial cells (ρ=0.42, p=0.03) and SF total cell count (ρ=0.64, p<0.01) while GSUS associated with SF MMP-3. (ρ=0.55, p<0.01) Conclusions US findings especially PDUS of RA affected joint well correlated with RA SF cytokines and cell number. Our results suggest that US is a useful modality with ease access reflecting local molecular pathophysiology of inflammatory joint in RA. References Naredo E et al. Arthritis Rheum 2008; 58: 2248–56 Schmidt WA et al. Ann Rheum Dis 2004; 63: 988–94. Nicola J et al. PLoS One. 2010 Sep 1;5(9). Disclosure of Interest Y. Kondo: None declared, K. Suzuki Grant/research support from: Eisai Co.Ltd. and Bristol-Myers Squibb Company, Y. Inoue: None declared, M. Takeshita: None declared, R. Morita: None declared, Y. Kasai Employee of: Takeda Pharmaceutical Company Limited, T. Miyazaki Employee of: Takeda Pharmaceutical Company Limited, Y. Niki: None declared, H. Hanaoka Consultant for: Astrazeneca, Y. Kaneko Consultant for: Abbvie, Paid instructor for: Eisai Pharmaceutical, Chugai Pharmaceutical, Astellas Pharmaceutical, Mitsubishi Tanabe Pharma Corporation, Pfizer, Speakers bureau: Abbvie, Eisai Pharmaceutical, Chugai Pharmaceutical, Bristol Myers Squibb, Astellas Pharmaceutical, Mitsubishi Tanabe Pharma Corporation, Pfizer, Janssen, UCB, H. Yasuoka Consultant for: Abbvie, K. Yamaoka: None declared, A. Yoshimura Consultant for: Pfizer, Speakers bureau: Pfizer, Chugai Pharma, Mitsubishi-Tanabe Pharma, Takeda Industrial Pharma, GlaxoSmithKline, Nippon Shinyaku, Eli Lilly, Janssen Pharma, Eisai Pharma, Astellas Pharma and Actelion Pharmaceuticals, T. Takeuchi Grant/research support from: Astellas Pharma, Bristol–Myers K.K., Chugai Pharmaceutical Co, Ltd., Daiichi Sankyo Co., Ltd., Eisai Co., Ltd., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., Santen Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Ltd., Teijin Pharma Ltd., AbbVie GK, Asahikasei Pharma Corp., and Taisho Toyama Pharmaceutical Co., Ltd.,SymBio Pharmaceuticals Ltd., Consultant for: Astra Zeneca K.K., Eli Lilly Japan K.K., Novartis Pharma K.K., Mitsubishi Tanabe Pharma Co., and Asahi Kasei Medical K.K.,abbivie GK, Daiichi Sankyo Co.,Ltd., Bristol–Myers K.K., Nipponkayaku Co.Ltd., Paid instructor for: Mitsubishi Tanabe Pharma Co., Eisai Co., Ltd.,Abbivie GK, Speakers bureau: AbbVie GK., Bristol–Myers K.K., Chugai Pharmaceutical Co,. Ltd., Eisai Co., Ltd., Janssen Pharmaceutical K.K., Mitsubishi Tanabe Pharma Co., Pfizer Japan Inc., and Takeda Pharmaceutical Co., Ltd., Astellas Pharma, and Diaichi Sankyo Co.,Ltd., Celtrion, Nipponkayaku Co.Ltd

OP0115 Increased T Follicular Helper Subset 2 Related to Increased IGG4 and Plasmablasts Through IL-4 in IGG4-Related Disease

Background Immunoglobulin (Ig) G4-related disease (IgG4-RD) is characterized by elevated serum IgG4 and tissue infiltration of IgG4+ plasma cells1. B cells producing excessive IgG4 is one of the diagnostic immunological feature of IgG4-RD. T follicular helper cells (Tfh) is the distinct subset of CD4+T cells that has been recognized to help B cell differentiation. Recently, Tfh subset: Tfh1, Tfh2, and Tfh17 was newly recognized based on different expression pattern of CXCR3 and CCR6. The skewed Tfh subset was reported to correlate with disease activity in autoimmune diseases. However, the role of Tfh and its subsets in IgG4-RD remains unclear. Objectives Elucidate the role of Tfh and its subsets in active, untreated IgG4-RD. Methods Peripheral blood from 15 active, untreated, biopsy-proven IgG4-RD patients, 24 primary Sjogrens Syndrome (pSS), 12 allergic rhinitis (AR), and 23 healthy controls (HC) were evaluated. Tfh was defined as CD3+CD4+CXCR5+CD45RA- and was subdivided into three subsets (Tfh1, Tfh2, and Tfh17) based on the expression of CXCR3 and CCR6. CD19+CD20-CD27+CD38+ cells were defined as plasmablasts. Results IgG4-RD patients had significantly increased proportion of Tfh compared to AR and HC. Among Tfh subsets, Tfh2 was specifically increased in IgG4-RD compared to pSS, AR and HC, whereas Tfh1 and Tfh17 resulted in non-statistical difference. Increased Tfh2 strongly correlated with serum IgG4, IgG4/IgG ratio and proportion of plasmablast in IgG4-RD (ρ =0.892, p<0.0001, ρ =0.802, p<0.0001 and ρ =0.674, p=0.006). In order to investigate the mechanism of increased cell subsets and IgG4, cytokines reported to be involved in Tfh function were measured. As a result, serum IL-4 positively correlated with IgG4 and IgG4/IgG ratio but not with IL-10, IL-21, and IL-33. Moreover, Tfh2 and plasmablast also correlated with serum IL-4. Interestingly, plasmablasts and IgG4 decreased after treatment with glucocorticoids (GCs), whereas no obvious change was observed with Tfh2. Conclusions Our results suggest that Tfh2 plays a crucial role in IgG4 production and generation of plasmablasts in IgG4-RD. In addition, IL-4 seemed to be the key cytokine for increased Tfh2, plasmablast and IgG4. More interestingly, IgG4 and plasmablast but not Tfh2 decreased after treatment with GCs suggesting Tfh2 as the underlying pathological T cell subset and as a potential therapeutic target for IgG4-RD. References Umehara H, Okazaki K, Masaki Y, Kawano M, Yamamoto M, Saeki T, et al. Comprehensive diagnostic criteria for IgG4-related disease (IgG4-RD), 2011. Mod Rheumatol 2012;22:21-30. Acknowledgements We thank all the patients and healthy individuals that participated this study. We specially thank Ms. Yoshiko Yogiashi and Ms. Yuki Ootomo for their technical assistance. Disclosure of Interest None declared