Yoshiaki Kassai

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.

Enhanced IgG4 production by follicular helper 2 T cells and the involvement of follicular helper 1 T cells in the pathogenesis of IgG4-related disease

BackgroundThe aim of this study was to elucidate the function of circulating follicular helper T (Tfh) cell subsets in helping B cells in patients with active, untreated IgG4-related disease (IgG4-RD) and determine their relationship with disease activity.MethodsSeventeen consecutive patients with active, untreated IgG4-RD, 20 with primary Sjögren syndrome (pSS), 5 with multicentric Castleman’s disease (MCD), and 12 healthy controls (HC) were enrolled. Tfh cell subset function was evaluated by co-culture with naïve B cells in vitro. Activated Tfh cell subsets were defined as a CCR7lowPD-1high subset among Tfh cell subsets. Disease activity was evaluated by IgG4-RD responder index (IgG4-RD RI) score.ResultsThe number of Tfh2 cells was significantly higher in IgG4-RD compared to pSS, MCD, or HC, and correlated with serum IgG4 level or the number of plasmablasts. In vitro, Tfh2 cells more efficiently induced the differentiation of naïve B cells into plasmablasts compared to Tfh1 or Tfh17 cells. Of note, while IgG production in culture supernatants of Tfh2 cells was comparable between IgG4-RD and HC, IgG4 production was significantly higher with Tfh2 cells from patients with IgG4-RD than in those from HC. Accordingly, the IgG4:IgG ratio in culture supernatants was also significantly higher with Tfh2 cells from IgG4-RD compared to HC. Moreover, the number of activated Tfh2 cells was higher in IgG4-RD compared to pSS, MCD, or HC, and strongly correlated with IgG4-RD RI score in the baseline active phase. Particularly, the number of activated Tfh2 cells was associated with the number of affected organs and serum IgG4 level. Importantly, the number of activated Tfh2 cells was decreased after glucocorticoid treatment and paralleled disease improvement. Moreover, the number of activated Tfh1 cells was also increased in IgG4-RD compared to pSS, MCD, or HC, correlating with IgG4-RD RI score, but not with serum IgG4 level.ConclusionsTfh2 cells, but not Tfh1 or Tfh17 cells, induce the differentiation of naïve B cells into plasmablasts and enhanced production of IgG4 in patients with active, untreated IgG4-RD. Furthermore, activated Tfh2 cells reflect disease activity, suggesting the involvement of this T cell subset in the pathogenesis of IgG4-RD. Interestingly, the number of activated Tfh1 cells was also increased in IgG4-RD, correlating with disease activity but not with serum IgG4 level, suggesting the involvement of Tfh1 cells but not in the process of IgG4 production in patients with IgG4-RD.

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.

Notch-mediated conversion of activated T cells into stem cell memory-like T cells for adoptive immunotherapy

Adoptive T-cell immunotherapy is a promising approach to cancer therapy. Stem cell memory T (TSCM) cells have been proposed as a class of long-lived and highly proliferative memory T cells. CD8+ TSCM cells can be generated in vitro from naive CD8+ T cells via Wnt signalling; however, methods do not yet exist for inducing TSCM cells from activated or memory T cells. Here, we show a strategy for generating TSCM-like cells in vitro (iTSCM cells) from activated CD4+ and CD8+ T cells in mice and humans by coculturing with stromal cells that express a Notch ligand. iTSCM cells lose PD-1 and CTLA-4 expression, and produce a large number of tumour-specific effector cells after restimulation. This method could therefore be used to generate antigen-specific effector T cells for adoptive immunotherapy.

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.

Identification of definitive serum biomarkers associated with disease activity in primary Sjögren’s syndrome

BackgroundIn this study, we sought to identify definitive biomarkers associated with disease activity in primary Sjögren’s syndrome (pSS).MethodsSerum protein concentrations in pSS patients and healthy controls (HCs) were comprehensively screened using high-throughput proteomic analysis, and differentially expressed proteins were extracted. Correlation between differentially expressed proteins and European League Against Rheumatism Sjögren’s Syndrome Disease Activity Index (ESSDAI) scores was analyzed and disease activity-associated biomarkers were identified. These biomarkers were validated by enzyme-linked immunosorbent assay (ELISA) in a separate pSS cohort.ResultsThe serum concentrations of 1100 proteins were compared between 30 pSS patients and 30 HCs, with 82 differentially expressed proteins identified as pSS-associated proteins. Of these 82 proteins, 9 were identified as disease activity-associated biomarkers. These nine biomarkers underwent validation by ELISA in a separate pSS validation cohort (n = 58), with five proteins (CXCL13, TNF-R2, CD48, B-cell activating factor (BAFF), and PD-L2) subsequently being confirmed as candidate biomarkers. Of these five candidate biomarkers, CXCL13 exhibited the most significant correlation with the lymphadenopathy, glandular, and pulmonary domains of the ESSDAI. CXCL13, TNF-R2 and CD48 exhibited a positive correlation with the biological domain of the ESSDAI. TNF-R2 exhibited the most negative correlation with uptake in the submandibular gland on technetium 99m-pertechnetate salivary gland scintigraphy.ConclusionsOur approach successfully identified serum biomarkers associated with disease activity in pSS patients. These markers might be potential therapeutic targets in pSS patients.

Resolution of elevated circulating regulatory T cells by corticosteroids in patients with IgG4-related dacryoadenitis and sialoadenitis

Dear Editor, Immunoglobulin G4-related disease (IgG4-RD) is characterized by elevated serum IgG4 concentrations and tissue infiltration by IgG4-positive plasmacytes. Patients with autoimmune pancreatitis (AIP) are reported to have increased numbers of circulating CD4+CD25 regulatory T cells (Tregs). In addition, large numbers of Tregs have been found in affected tissues in patients with IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) and autoimmune pancreatocholangitis. Regarding treatment for IgG4-DS, corticosteroids were considered effective and are first-line therapy. However, the impact of corticosteroids on the frequency of Tregs in IgG4-DS patients remains unclear. Here, we report two cases of IgG4-DS in which the percentage of circulating Tregs in CD4+ T cells was analyzed by flow cytometry during treatment with corticosteroids. This study was approved by the ethics committee of our institution and informed consent had been obtained from all individual patients.

THU0007 Enhanced IGG4 Production by Follicular Helper Type 2 T Cells in IGG4-Related Disease

Background IgG4-related disease (IgG4-RD) is characterized by elevated serum IgG4 and increased circulating plasmablasts1. The high degree of somatic hypermutation and emergence of distinct plasmablast clones at disease relapse suggests the de novo recruitment of naïve B cells into T-cell–dependent responses1. Follicular helper T (Tfh) cells play a major role in T-cell–dependent B cell responses, and their counterparts and subsets (Tfh1, Tfh2 or Tfh17 cells) have only been recognized in peripheral blood2. We previously reported that Tfh2 cells were increased in IgG4-RD3, although the functional role of Tfh2 cells remains to be elucidated. Objectives To elucidate the function of Tfh2 cells and involvement in the pathogenesis of active, untreated IgG4-RD. Methods Active, untreated IgG4-RD (n=17) were compared to healthy controls (HC) (n=12), patients with primary Sjögren syndrome (pSS) (n=20) and multicentric Castlemans disease (MCD) (n=5). Tfh2 cells (CD3+CD4+CD45RA-CXCR5+CXCR3-CCR6-cells), activated Tfh2 cells (CCR7loPD-1hi subset in Tfh2 cells) and plasmablasts (CD19+CD20-CD27+CD38+cells) were detected by flow cytometry. The function of Tfh2 cells was evaluated by co-culture with autologous naïve B cells in vitro. Disease activity was evaluated by IgG4-RD Responder Index (IgG4-RD RI) score. Results The percentage of Tfh2 cells was significantly increased in IgG4-RD compared to pSS, MCD or HC, and correlated with serum IgG4 levels (ρ=0.7377, P =0.0011) or the percentage of plasmablasts (ρ=0.6127, P=0.0104). In vitro, Tfh2 cells induced a greater percentage of differentiation of naïve B cells into plasmablasts in IgG4-RD and HC compared to Tfh1 and Tfh17 cells. Of note, while IgG production in culture supernatants of Tfh2 cells were comparable between IgG4-RD and HC, IgG4 production was significantly higher in IgG4-RD compared to HC. Accordingly, IgG4/IgG ratio in culture supernatants was also significantly higher only with Tfh2 cells from IgG4-RD. In addition, the percentage of activated Tfh2 cells was significantly increased in IgG4-RD (8.0 ± 1.6%) compared to pSS (1.4±0.2%, P<0.0001), MCD (1.6±0.5%, P<0.0001) or HC (1.2±0.2%, P<0.0001), and correlated strongly with IgG4-RD RI score (ρ=0.7984, P=0.0002). In particular, the percentage of activated Tfh2 cells associated with the number of affected organs (ρ=0.8152, P=0.0001), and decreased with glucocorticoid treatment in parallel with disease improvement. Of note, the percentage of activated Tfh2 cells was re-elevated at disease relapse. Conclusions Tfh2 cells, but not Tfh1 or Tfh17 cells, induce the differentiation of naïve B cells into plasmablasts and IgG4 production in patients with active, untreated IgG4-RD. This increase in activated Tfh2 cells is linked to disease activity, suggesting that Tfh2 cells play a pivotal role in the pathogenesis of IgG4-RD. References J Allergy Clin Immunol. 2014;134:679–87. Immunity 2011;34:108–21. Arthritis Rheumatol. 2015;67:2476–81. Acknowledgement We thank the patients and healthy individuals who participated in this study. We thank Mr. Noriyasu Seki, Mr. Humitsugu Yamane, Mrs. Yoshiko Yogiashi and Ms. Yuki Otomo for their technical assistance. Disclosure of Interest None declared

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