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ObjectivesThe treatments for rheumatoid arthritis (RA) have been greatly improved, and the tight control of disease activity yields superior clinical outcomes. This study aimed to elucidate the accompanying changes in hip destruction following the implementation of a treat-to-target strategy for patients with RA.MethodsWe extracted 190 hips over two periods, i.e. the early period (1998–2003) and the late period (2013–19), with 103 and 87 hips, respectively. The observed rheumatic changes, such as inward migration, upward migration, and femoral head collapse, were quantitatively evaluated, while osteoarthritic changes, such as the formation of a capital drop, were investigated from radiographs before primary total hip arthroplasty.ResultsA comparison of the two periods’ data showed that the degree of inward migration (−3.44 vs. −7.45 mm; P ConclusionsThe degree of inward and upward migration representative of rheumatic changes reduced, whereas the frequency of capital drops as osteoarthritic changes increased during the late period.

G protein-coupled receptor kinase 5 (GRK5) regulates inflammatory responses via the nuclear factor-kappa B (NF-κB) pathway. This study investigated the functional involvement of GRK5 in the pathogenesis of inflammatory arthritis. Immunohistochemically, rheumatoid arthritis (RA) synovium had a significantly higher proportion of GRK5-positive cells in the synovial lining layer than healthy control synovium. Gene expression and NF-κB activation in lipopolysaccharide-stimulated human SW982 synovial cells were significantly suppressed by silencing of the GRK5 gene. Similarly, GRK5 kinase activity inhibition in human primary RA synovial cells attenuated gene expressions of inflammatory factors. In a murine model of collagen antibody-induced arthritis, arthritis scores and serum IL6 production of GRK5 knockout (GRK5-/-) mice were significantly lower than those of wild-type mice. Histologically, the degree of synovitis and cartilage degeneration in GRK5-/- mice was significantly lower than in wild-type mice. In in vitro analyses using activated murine macrophages and fibroblast-like synoviocytes, gene expression of inflammatory factors and p65 nuclear translocation were significantly lower in GRK5-/- mice compared to wild-type mice. In conclusion, our results suggested that GRK5 is deeply involved in the pathogenesis of inflammatory arthritis, therefore, GRK5 inhibition could be a potential therapeutic target for types of inflammatory arthritis such as RA.

Adaptive immunity plays central roles in the pathogenesis of rheumatoid arthritis (RA), as it is regarded as an autoimmune disease. Clinical investigations revealed infiltrations of B cells in the synovium, especially those with ectopic lymphoid neogenesis, associate with disease severity. While some B cells in the synovium differentiate into plasma cells producing autoantibodies such as anti-citrullinated protein antibody, others differentiate into effector B cells producing proinflammatory cytokines and expressing RANKL. Synovial B cells might also be important as antigen-presenting cells. Synovial T cells are implicated in the induction of antibody production as well as local inflammation. In the former, a recently identified CD4 T cell subset, peripheral helper T (Tph), which is characterized by the expression of PD-1 and production of CXCL13 and IL-21, is implicated, while the latter might be mediated by Th1-like CD4 T cell subsets that can produce multiple proinflammatory cytokines, including IFN-γ, TNF-α, and GM-CSF, and express cytotoxic molecules, such as perforin, granzymes and granulysin. CD8 T cells in the synovium are able to produce large amount of IFN-γ. However, the involvement of those lymphocytes in the pathogenesis of RA still awaits verification. Their antigen-specificity also needs to be clarified.

Autoreactive CD4 T cells are thought to play pivotal roles in the pathogenesis of rheumatoid arthritis (RA). Recently, a subset of CD4 T cells that express high levels of programmed death-1 (PD-1) but are distinct from follicular helper T cells have been identified in the joints of RA patients and named peripheral helper T (Tph) cells. Because PD-1 is expressed on T cells chronically stimulated with the Ags, we tested a hypothesis that Tph cells are the pathogenic autoreactive CD4 T cells in RA. We found that human Tph cells in RA joints produce proinflammatory effector cytokines, including IFN-γ, TNF-α, and GM-CSF, in addition to B cell–helping cytokines, such as IL-21 and CXCL13. Flow cytometric analysis showed different bias of TCR Vβ usage between PD-1high Tph cells and PD-1low/neg CD4 T cells, including Th1 cells, in the joint or memory CD4 T cells in the peripheral blood, whereas there was little difference between the latter two subsets. In line with this, deep sequencing of TCR demonstrated an overlap of expanded clones between peripheral blood memory CD4 T cells and PD-1low/neg CD4 T cells but not Tph cells in the joint. Interestingly, Tph cells preferentially exhibited autologous MLR in vitro, which required recognition of self–MHC class II and was pronounced by blocking PD-1 signaling. Taken together, these results suggest that Tph cells are the pathogenic autoreactive CD4 T cells in RA, which expand locally in the joints and are regulated by PD-1 signaling.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder affecting systemic synovial tissues, leading to the destruction of multiple joints. Its etiology is still unknown, but T-cell-mediated autoimmunity has been thought to play critical roles, which is supported by experimental as well as clinical observations. Therefore, efforts have been made to elucidate the functions and antigen specificity of pathogenic autoreactive T cells, which could be a therapeutic target for disease treatment. Historically, T-helper (Th)1 and Th17 cells are hypothesized to be pathogenic T cells in RA joints; however, lines of evidence do not fully support this hypothesis, showing polyfunctionality of the T cells. Recent progress in single-cell analysis technology has led to the discovery of a novel helper T-cell subset, peripheral helper T cells, and attracted attention to the previously unappreciated T-cell subsets, such as cytotoxic CD4 and CD8 T cells, in RA joints. It also enables a comprehensive view of T-cell clonality and function. Furthermore, the antigen specificity of the expanded T-cell clones can be determined. Despite such progress, which T-cell subset drives inflammation is yet known.

Background To further improve rheumatoid arthritis (RA) treatment, it is necessary to understand each RA patient’s satisfaction and to identify the factors affecting their satisfaction. Despite the rise in medical costs for RA, little is known about the factors that influence patient satisfaction with the cost of treatment in RA patients. Methods This is a multicenter observational study of Japanese RA patients from the FRANK Registry with data analyzed from March 2017 to August 2020. We collected data on demographic characteristics, clinical data, quality of life which was evaluated using the EuroQol 5-dimensional questionnaire (EQ5D), and patient satisfaction. The four categories of patient satisfaction were evaluated individually (i.e., cost, treatment efficacy, activities of daily living [ADL], and global treatment satisfaction). We analyzed the factors that affected each patient’s satisfaction, such as age, sex, EQ5D, disease duration, disease activity, and treatment. Results This study included 2235 RA outpatients (406 males, 1829 females). In RA patients, “very satisfied” and “satisfied” were given for nearly half of each satisfaction aspect (cost 49%; efficacy 72%; ADL 58%; global treatment 66%) at the time of the initial registration. To investigate the factors influencing each satisfaction, multivariate analysis has revealed that the use of b/tsDMARDs increased satisfaction of treatment effect (odds ratio [OR] 0.66) and ADL (OR 0.78) but decreased cost satisfaction (OR 2.21). Age (50–64 years; OR 0.91; 65–74 years, 0.55: ≥ 75 years, 0.35), female (OR 0.81), and history of musculoskeletal surgery (OR 0.60) all increased cost satisfaction. Patients with lower disease activity and higher EQ5D scores had higher levels of satisfaction in all areas. Conclusions In this study, patient satisfaction in terms of cost, treatment effect, ADL, and overall treatment was generally higher, but some patients were dissatisfied. The cost of satisfaction increased with age and a history of musculoskeletal surgery, while it decreased with a lower EQ5D score and the use of b/tsDMARDs.

BackgroundRheumatoid arthritis (RA) is a prototypical autoantibody-driven autoimmune disease in which T-B interactions play a critical role. Recent comprehensive analysis suggests that PD-1+CD8+ T cells as well as two distinct IL-21-producing PD-1+CD4+ T cell subsets, follicular helper T (Tfh) and peripheral helper T (Tph) cells, are involved in the pathogenesis of RA. Herein, we aimed to clarify a generation mechanism of IL-21-producing CD8+ T cells in humans, and to characterize this novel subset in patients with RA.MethodsCD8+ T cells in the peripheral blood (PB) and synovial fluid (SF) of healthy control (HC) and patients with RA were subject to the analysis of IL-21 mRNA and protein. We evaluated the surface marker, cytokine and transcription profiles of IL-21-producing CD8+ T cells in HCPB, RAPB and RASF.ResultsIL-21-producing CD8+ T cells were enriched in the CD45RA-(memory) PD-1+, especially PD-1hi subpopulation, and IL-12 and IL-21 synergistically induced IL-21 production by naïve CD8+ T cells. Memory PD-1hiCD8+ T cells in HCPB facilitated plasmablast differentiation and IgG production in an IL-21-dependent manner. In addition, PD-1hiCD8+ T cells in RASF and RAPB produced large amounts of IL-21 and were characterized by high levels of CD28, ICOS, CD69, HLA-DR, and CCR2 but not CXCR5. Furthermore, PD-1hiCD8+ T cells expressed high levels of transcripts of MAF and PRDM1, a feature observed in Tph cells.ConclusionsIdentification of IL-21-producing PD-1hiCD8+ T cells expands our knowledge of T cell subsets with B helper functions in RA. Selective targeting of these subsets could pave an avenue for the development of novel treatment strategies for this disease.

Efficacy of B-cell depletion therapy highlights the antibody-independent effector functions of B cells in rheumatoid arthritis (RA). Given type 1 helper T (Th1) cells abundant in synovial fluid (SF) of RA, we have determined whether Th1 cells could generate novel effector B cells. Microarray and qPCR analysis identified CXCL9/10 transcripts as highly expressed genes upon BCR/CD40/IFN-γ stimulation. Activated Th1 cells promoted the generation of CXCL9/10-producing T-bet

BackgroundFine specificity of anti-citrullinated protein antibodies (ACPAs), in which cross-reactivity exists, varies among patients with rheumatoid arthritis (RA), but it is unclear whether the mechanism of ACPA production is same or different among individuals. Since avidity of serum antibody reflects the direction of immune response, we compared the levels of avidity and cross-reactivity between various ACPAs in a cohort of RA patient.MethodsSera from 180 RA patients positive for anti-cyclic citrullinated peptide (CCP) 2 antibody were screened for positivity of antibodies against CCP1, and citrullnated fibrinogen (cFib), enolase (cEno), and vimentin (cVim) peptides. Avidity of the four ACPAs, and some autoantibodies and antibodies against foreign antigens was determined by an elution assay using sodium thiocyanate solution. Cross-reactivity between different ACPAs was estimated by measuring the inhibition of binding by competitor peptides. ResultsThe prevalence of anti-CCP1, anti-cFib, anti-cEno, and anti-cVim antibodies in the anti-CCP2-positive RA cohort were 37.7%, 38.3%, 15.6%, and 23.9%, respectively. The avidity of ACPAs, except for anti-cVim antibody, was significantly lower than that of antibodies against foreign antigens, while there was a large variety in the avidity of other autoantibodies. At individual levels, the avidity of anti-cVim was significantly higher than that of other ACPAs, and there was a significant correlation in the avidity of anti-CCP and anti-cFib antibodies. Substantial extent of cross-reactivity was seen between different ACPAs, which also showed a fixed hierarchy.ConclusionThe fixed hierarchy in the avidity and cross-reactivity between different ACPAs suggests that the mechanism underlying ACPA production is common to all RA patients. Presence of a dominant antigen that induces whole ACPA response is speculated.

Abatacept may exert its clinical effect on rheumatoid arthritis (RA) by suppressing anti-cyclic citrullinated peptide (CCP) antibody production. This study was undertaken to test this hypothesis by examining the changes of disease activity of RA and anti-CCP antibody levels over time after starting abatacept. Sixty Japanese RA patients who started abatacept were included in this multicenter, prospective observational study. Simple Disease Activity Index (SDAI) and anti-CCP antibody levels were evaluated at 12, 24, and 52 weeks. The mean SDAI score significantly decreased within 12 weeks after starting abatacept and was maintained thereafter. On the contrary, the mean anti-CCP antibody levels did not change until 52 weeks. At the individual level, there were substantial changes of anti-CCP antibody levels, but these were not correlated with the changes of disease activity at any time points. Thus, abatacept reduces the disease activity of RA independently of modulating anti-CCP antibody production.

To investigate the roles of P2X7 receptors (P2RX7) in the pathogenesis of experimental autoimmune uveoretinitis (EAU). Experimental. Either wild-type (P2rx7 +/+ ) or P2rx7-deficient (P2rx7 −∕− ) mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 1-20. Severity of EAU was evaluated clinically and histopathologically. The induction of IRBP-specific proliferation and cytokines in draining lymph nodes was assessed by enzyme-linked immunosorbent assays (ELISA). The frequency of activation markers was examined by flow cytometry. Furthermore, inhibitory roles of systemic administration of Brilliant Blue G (BBG), an antagonist for P2RX7, in EAU were also assessed in the wild-type mice. The severity of EAU in P2rx7 −∕− mice was reduced as compared with that in P2rx7 +/+ mice, both clinically and histopathologically. IRBP-specific proliferation in P2rx7 −∕− on day 16 was slightly decreased compared to that in P2rx7 +/+ mice. The induction of IRBP-specific interferon (IFN)-γ and interleukin (IL)-17 in P2rx7 −∕− mice on day 16 was lower than that in P2rx7 +/+ mice. The up-regulation of surface expression of activation markers such as CD25, CD44, and CD69 in response to TCR stimulation in P2rx7 −∕− mice was decreased as compared with that in P2rx7 +/+ mice. Furthermore, neutralization of P2RX7 in vivo by BBG suppressed EAU clinically and histopathologically. IRBP-specific IFN-γ and IL-17 induction in BBG-treated mice was significantly lower than that in vehicle-treated mice. The results suggest that P2RX7 is a novel preventative therapeutic target for uveitis as it suppresses the effector functions of both Th1 and Th17 cell responses.

Summary: We recently found that a unique subset of innate-like γδ T cells develops from the DN2a stage of the fetal thymus independently of the zinc-finger transcription factor B cell leukemia/lymphoma 11b (Bcl11b). Herein, we characterize these Bcl11b-independent γδ T cells in the periphery as CD5−NK1.1+ and Granzyme B+, and we show that they are capable of producing interferon (IFN)-γ upon T cell receptor stimulation without Ca2+ influx. In wild-type mice, these cells were sparse in lymphoid tissues but abundant in non-lymphoid tissues, such as the liver. Bcl11b-independent CD5−NK1.1+ γδ T cells appeared and contributed to early protection before Bcl11b-dependent CD5+NK1.1− γδ T cells following Listeria monocytogenes infection, resembling their sequential appearance during development in the thymus. : Bcl11b is essential for transition from the DN2a to the DN2b stage in the thymus. Hatano et al. find that CD5−NK1.1+ γδ T cells develop from the DN2a stage in a Bcl11b-independent manner and participate in host defense at an early stage after bacterial infection in periphery. Keywords: innate immunity, γδ T cell, Bcl11b, DN2a, IFN-γ, Granzyme, IL-17A, host defense, bacteria, Listeria monocytogenes

Background Recent studies show that innate lymphoid cells (ILCs) contribute to the development of chronic inflammation and autoimmune disease. In this study, we assessed the ILC function in the development of rheumatoid arthritis (RA). Methods In a mouse model of collagen-induced arthritis (CIA), we identified and purified the ILC subsets in peripheral blood (PB), local lymph nodes (LNs), and joints by fluorescence-activated cell sorting and used quantitative PCR to assess the expression levels of representative cytokines. We also correlated the frequencies of each ILC subset in synovial fluid (SF) with clinical parameters in RA patients. Results In the CIA model, the proportion of CCR6+ ILC3s to total ILCs in joints with active inflammation significantly increased relative to non-arthritic joints (median 29.6% vs 16.7%, p = 0.035). CCR6+ ILC3s from mice with arthritis expressed significantly higher levels of IL-17A and IL-22 mRNA than did comparable cells from control mice (p

T cell development and homeostasis requires IL-7R α-chain (IL-7Rα) signaling. Tyrosine Y449 of the IL-7Rα is essential to activate STAT5 and PI3K, whereas PI3K recruitment requires IL-7Rα methionine M452. How IL-7Rα activates and regulates both signaling pathways differentially remains unclear. To characterize differential signaling, we established two lines of IL-7Rα mutant mice: IL-7R–Y449F mice and IL-7R–M452L mice. IL-7R–Y449F mice showed decreased PI3K and STAT5 signals, whereas IL-7R–M452L mice showed decreased PI3K but significantly increased STAT5 signaling, owing to a competition between PI3K and STAT5 signaling through Y449 of IL-7Rα. The number of T, B, and mature innate lymphoid cells were markedly reduced in IL-7R–Y449F mice, whereas IL-7R–M452L mice showed impaired early T cell development and memory precursor effector T cell maintenance with the downregulation of transcription factor T cell factor–1. Peripheral T cell numbers increased in IL-7R–M452L mice with enhanced survival and homeostatic proliferation. Furthermore, although wild type and IL-7R–Y449F mice showed comparable Th1/Th2 differentiation, IL-7R–M452L mice exhibited impaired Th17 differentiation. We conclude that PI3K competes with STAT5 under IL-7Rα and maintains an appropriate signal balance for modulating T cell development and homeostasis. To our knowledge, this study provides a new insight into complex regulation of IL-7Rα signaling, which supports immune development and responses.

Table S1. List of antibodies used in flow cytometry (DOCX 23 kb)

Objectives: To assess long-term outcomes for seven biological disease-modifying antirheumatic drugs (bDMARDs) and one Janus kinase (JAK)-inhibitor in rheumatoid arthritis. Methods: We retrospectively analyzed data from 801 rheumatoid arthritis patients visiting our rheumatology clinic between 2003 and 2017. We determined drug survival rates, drug discontinuation, and switching rates in these patients. Results: Among the drugs administered to naïve subjects, the drug-survival rate was highest for tocilizumab, at 77.8% after 6 years, whereas the rates for golimumab, etanercept, abatacept, infliximab, and adalimumab, were 61.5%, 48.9%, 41.6%, 34.5%, and 34.4%, respectively. Switching drugs led to decreased survival rates. The discontinuation rates for all drugs due to adverse events and poor efficacy increased rapidly in the first 2 years and the first 6 months, respectively. Conclusions: This report is a long-term analysis of a large cohort of rheumatoid arthritis patients from a single rheumatology clinic in Japan. We conclude that to maximize the survival rate of antirheumatic drugs, it is important to maintain their effectiveness over long periods of time by appropriate drug choices and optimizing dosage before switching drugs.

Table S2. Clinical characteristics of patients with RA. TCZ, Tocilizumab. ETN, Etanercept. MZR, Mizoribine. IGU, Iguratimod. (DOCX 16 kb)

Efficacy of B-cell depletion therapy highlights the antibody-independent effector functions of B cells in rheumatoid arthritis (RA). Given type 1 helper T (Th1) cells abundant in synovial fluid (SF) of RA, we have determined whether Th1 cells could generate novel effector B cells. Microarray and qPCR analysis identified CXCL9/10 transcripts as highly expressed genes upon BCR/CD40/IFN-γ stimulation. Activated Th1 cells promoted the generation of CXCL9/10-producing T-bet+ B cells. Expression of CXCL9/10 was most pronounced in CXCR3+ switched memory B cells. Compared with peripheral blood, SFRA enriched highly activated Th1 cells that coexisted with abundant CXCL9/10-producing T-bet+ B cells. Intriguingly, anti-IFN-γ antibody and JAK inhibitors significantly abrogated the generation of CXCL9/10-producing T-bet+ B cells. B cell derived CXCL9/10 significantly facilitated the migration of CD4+ T cells. These findings suggest that Th1 cells generate the novel CXCL9/10-producing T-bet+ effector B cells that could be an ideal pathogenic B cell target for RA therapy.

Interleukin 21 (IL-21) is a pleiotropic common cytokine receptor γ chain cytokine that promotes the effector functions of NK cells and CD8+ T cells and inhibits CD8+ T cell exhaustion during chronic infection. We found that the absolute number of short-lived effector CD8+ T cells (SLECs) (KLRG1high CD127low) decreased significantly in IL-21 receptor-deficient (IL-21R-/-) mice during Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection. Early effector CD8+ T cells (EECs) (KLRG1low CD127low) were normally generated in IL-21R-/- mice after infection. Exhausted CD8+ T cells (PD-1high KLRG1low) were also normally generated in IL-21R-/- mice after infection. Mixed bone marrow (BM) chimera and transfer experiments showed that IL-21R on CD8+ T cells was essential for the proliferation of EECs, allowing them to differentiate into SLECs after BCG infection. On the other hand, the number of SLECs increased significantly after infection with recombinant BCG (rBCG) that secreted an antigen 85B (Ag85B)-IL-21 fusion protein (rBCG-Ag85B-IL-21), but the number of exhausted CD8+ T cells did not change after rBCG-Ag85B-IL-21 infection. These results suggest that IL-21 signaling drives the differentiation of SLECs from EECs but does not inhibit the exhaustion of CD8+ T cells following BCG infection in mice.

Generation of neoantigens by citrullination is implicated in the production of anti–citrullinated protein Abs in rheumatoid arthritis, but citrullination is also a physiological process. To verify whether citrullin-specific B cells are immunologically ignorant or tolerant in normal conditions, transgenic (Tg) mice expressing IgM with the V region of an anti–cyclic citrullinated peptide (CCP) mAb cloned from a rheumatoid arthritis patient were generated. CCP-specific B cells developed in the anti-CCP IgM Tg mice with an alteration of bone marrow B cell fractions, and the number of mature B cells decreased compared with wild-type or the control anti–influenza nucleoprotein–specific IgM Tg mice. In addition, B cells in anti-CCP IgM Tg mice are functionally anergic. Thus, tolerance is induced in CCP-specific B cells in vivo, suggesting that the immune systems are naturally exposed to citrullinated Ags, and anti-CCP Ab production requires additional steps beyond the generation of neoantigens by citrullination.

Interleukin 21 (IL-21) is a pleiotropic common cytokine receptor γ chain cytokine that promotes the effector functions of NK cells and CD8+ T cells and inhibits CD8+ T cell exhaustion during chronic infection. We found that the absolute number of short-lived effector CD8+ T cells (SLECs) (KLRG1high CD127low) decreased significantly in IL-21 receptor-deficient (IL-21R−/−) mice during Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection.

Establishing effective central tolerance requires the promiscuous expression of tissue-restricted antigens by medullary thymic epithelial cells. However, whether central tolerance also extends to post-translationally modified proteins is not clear. Here we show a mouse model of autoimmunity in which disease development is dependent on post-translational modification (PTM) of the tissue-restricted self-antigen collagen type II. T cells specific for the non-modified antigen undergo efficient central tolerance. By contrast, PTM-reactive T cells escape thymic selection, though the PTM variant constitutes the dominant form in the periphery. This finding implies that the PTM protein is absent in the thymus, or present at concentrations insufficient to induce negative selection of developing thymocytes and explains the lower level of tolerance induction against the PTM antigen. As the majority of self-antigens are post-translationally modified, these data raise the possibility that T cells specific for other self-antigens naturally subjected to PTM may escape central tolerance induction by a similar mechanism., Post-translational modifications are associated with autoimmune diseases but definitive evidence of their contribution to escape from central tolerance mechanisms is needed. Here, the authors show that T cells specific for post-translational modifications of type II collagen escape intrathymic tolerance induction in a mouse model of rheumatoid arthritis.

This study was conducted to evaluate the period from symptom onset to diagnosis of ankylosing spondylitis (AS) in Japanese patients and to examine possible reasons for delayed diagnosis.Seventy-two consecutive patients with AS were studied. Diagnostic delay was defined as the gap between the first spondyloarthropathic symptom and diagnosis of AS according to the modified New York criteria.The mean patient ages at disease onset and diagnosis were 25.6 ± 11.3 and 33.3 ± 13.2 years old, respectively, resulting in diagnostic delay of 6.7 years. The number of medical institutions to which patients were referred before diagnosis was 2.4, and orthopedic surgeons were most commonly visited (62%). Non-specific low back pain or lumbar spondylitis (33%) and degenerative arthritis (28%) were the primary diagnoses preceding that of AS. Absence of articular symptoms significantly correlated with diagnostic delay. The patients with disease onset on year 2000 or later had significantly shorter periods until diagnosis than those before 2000 (3.6 vs. 7.5 years).The present study showed a marked diagnostic delay among Japanese patients with AS. Although it has been improved, continuing medical education focusing on inflammatory back pain in adolescent is required for early diagnosis of AS.

Objective To establish a method to culture synovial tissue explants from patients with rheumatoid arthritis (RA). Methods Synovial tissue explants obtained from 10 patients with RA were cultured at the air–liquid interface or were submerged in culture medium. As a control, synovial explants were engrafted subcutaneously into SCID mice. The synovial explants were harvested at different time points, and histologic or flow cytometric analysis was performed. Cytokine levels in the culture supernatants were measured by enzyme-linked immunosorbent assay. Infliximab was added to the air–liquid interface culture to evaluate the effect of tumor necrosis factor α blockade on inflammatory cytokine production. Results The histologic features of RA synovitis, including a hyperplastic lining layer and the presence of cellular infiltrate in the sublining layer, were maintained in synovial tissue explants in air–liquid interface culture. In synovial grafts harvested from SCID-HuRAg mice, the cellular infiltrate was well maintained in the sublining, but the lining layer was lost. Viable CD4+ T cells and macrophages were abundant after air–liquid interface culture but were virtually absent after submerged culture. Furthermore, synovial tissue explants in air–liquid interface culture produced interleukin-6 (IL-6) and IL-8 for a prolonged period of time. The addition of infliximab effectively reduced cytokine production. Conclusion RA synovial explants can be maintained for weeks using an air–liquid interface culture. This simple culture system might be useful for analyzing the pathogenesis of RA synovitis and for developing antirheumatic drugs.

Background Nonmyeloablative allogeneic stem cell transplantation (SCT) is an option for the treatment of metastatic renal cancer. Mature donor T cells cause graft versus-host disease (GVHD) although they are also the main mediators of the beneficial graft-versus-tumor (GVT) activity associated with this treatment. Hence, the segregation of GVT activity from GVHD is an important challenge in managing the clinical course of treatment. We previously reported a series of studies regarding the allograft tolerance induced by allogeneic spleen cells (with bone marrow cells) and cyclophosphamide in mice. Methods The aim of the present study was to modify the cyclophosphamide-using cell therapy to reduce the risk of GVHD while preserving the antitumor activity against RENCA, a murine carcinogen-induced renal cell carcinoma with recipient lymphocyte infusion (RLI). Results Regarding the in vivo antitumor effect, there was a significant difference between RLI and no lymphocyte infusion after the cyclophosphamide treatment, whereas the histologic findings of the small intestine showed that the cyclophosphamide-using cell therapy with RLI decreased the risk of GVHD as compared with donor lymphocyte infusion. In addition, the acquired immunity against RENCA was clearly observed in RLI-treated mice. Conclusions Our results show that RLI during cyclophosphamide-using nonmyeloablative cell therapy can dissociate GVT effects from GVHD by reducing the risk of GVHD. We considered that this was the first report to provide the evidence of nonmyeloablative allogeneic SCT with RLI for the treatment of renal cell carcinoma which never induce complete chimerism.

The CD30 ligand (CD30L)/CD30 axis plays a critical role in Th1 and Th17 cell differentiation. However, the role in the pathogenesis of central nervous system autoimmunity remains unknown. Here we show the resistance for experimental autoimmune encephalomyelitis (EAE) with markedly reduced induction of antigen-specific Th1 and Th17 cells in CD30L knockout mice. Bone marrow chimera experiments indicated that CD30L on bone marrow-derived cells were critical for the development of EAE and that CD30L reverse signaling in CD4 T cells was dispensable for the pathogenic Th17 cell differentiation at the induction phase. Adoptive transfer experiment revealed an additional role for CD30L in the environment at the effector phase. In vivo neutralization of CD30L by soluble murine CD30-Immunoglobulin fusion protein before disease onset or even after disease onset significantly ameliorated the clinical symptoms. These results indicate that CD30L/CD30 signaling is critically involved in antigen-specific CD4 T cell responses at both the induction and effector phase, thus could be a new target molecule for the treatment of central nervous system autoimmunity.

Since the presence of IgM antibodies is a hallmark of ongoing immune response, we aimed to identify immunologically active rheumatoid arthritis (RA) patients by detecting IgM anti-citrullinated protein antibody (ACPA) levels.IgM ACPA levels were determined in the serum of 176 RA patients by enzyme-linked immunosorbent assay, in which parameters of reactivity against citrullinated and non-citrullinated peptides were compared to ensure the specificity. Influence of IgM rheumatoid factor (RF) on IgM ACPA detection was examined by removing IgG, using protein G-conjugated beads, or by purifying ACPA, using citrullinated peptide-conjugated beads.Although IgM specific for citrullinated proteins was detected in some patients (11%), IgM molecules reactive to both citrullinated and non-citrullinated peptides were detected in a substantial number of patient samples (12%). IgM ACPA-positive reactions were associated with the presence of IgG ACPA and IgM RF. Surprisingly, protein G-mediated removal of IgG from the serum eliminated positivity for IgM ACPA, suggesting that IgG ACPA-IgM RF complex was being detected. This assumption was confirmed by the detection of IgM RF in the eluate of protein G beads and citrullinated peptide-conjugated beads.In an attempt to detect IgM ACPA, we mostly revealed false positive reactions due to the presence of IgM molecules, which were not specific for citrullinated proteins, and IgG ACPA-IgM RF immune complex. The latter complex had been proposed to play a role in the pathogenesis of RA, and here, for the first time, we have demonstrated its presence in the sera of RA patients.

To investigate the roles of P2X7 receptors (P2RX7) in the pathogenesis of experimental autoimmune uveoretinitis (EAU).Experimental.Either wild-type (P2rx7The severity of EAU in P2rx7The results suggest that P2RX7 is a novel preventative therapeutic target for uveitis as it suppresses the effector functions of both Th1 and Th17 cell responses.

To retrospectively evaluate the long-term results of cementless total hip arthroplasty (THA) in patients with rheumatoid arthritis (RA) and postoperative patient mortality after THA.This study included 191 hips in 149 RA patients who underwent cementless THA between 1998 and 2005. Mean age at surgery was 54.2 years, and mean follow-up was 12.6 years. Implant and patient survivorships were determined using the Kaplan-Meier method, and the associated influencing factors were determined.Implant survivals at 17 years were 99.5% for stems, 93.9% for cups, and 90.8% for liners. Among the liners used, THAs with highly cross-linked polyethylene showed better survivals compared with those with conventional polyethylene and alumina-bearing surface (93.4%, 90.9%, and 52.2%, respectively). A total of 64 deaths occurred; 45 patients died within 10 years and 19 patients died between 10 and 17 years. Malignancy (25.0%) was the leading cause of death, followed by pneumonia (20.8%) and sepsis (20.8%). The patient survival rate was 36.9% at 17 years after THA. Multivariate analysis exhibited that older age at operation and greater dose of concomitant corticosteroid resulted in shorter patient survivals.Cementless THA worked well in patients with RA. Mortality remained high among RA patients who needed THA.

Background Klebsiella pneumoniae frequently causes life-threatening infection in children. Interleukin 17A (IL-17A) is known to be involved in protection against K. pneumoniae infection through activation of neutrophils. Methods and results We found that IL-17A-producing γδ T cells existed more frequently in younger mice on examination of IL-17A-producing lymphocytes in the lung of naive mice at various ages. We hence compared the protective role of IL-17A-producing γδ T cells against pulmonary K. pneumoniae infection in young (3 weeks old) and adult (8-12 weeks old) mice. IL-17A-deficient mice were susceptible to K. pneumonia regardless of age. Cγ-, Vγ4/6-, or Vδ1-deficient mice were susceptible to K. pneumonia at young age, while interleukin 23p19 (IL-23p19)-deficient mice were susceptible at adult age. IL-17A-producing Vγ1-Vγ4- γδ T cells expressing canonical Vγ6/Vδ1 genes were dominant over IL-17A-producing Vγ4+ γδ T cells in the lungs of young mice after infection. The IL-17A-producing Vγ1-Vγ4- γδ T cells expressed an activation marker, CD69, and proliferated in an IL-23-independent manner, while the IL-17A-producing Vγ4+ γδ T cells expressing IL-23 receptor but no CD69 proliferated in IL-23-dependent manner. Conclusions These results suggest that 2 types of IL-17A-producing γδ T cells are activated for host defense against K. pneumoniae infection by IL-23-dependent or independent mechanism.

Clinical characterization of patients with RA included in the study. (DOCX 18 kb)

Analysis of osteoclast differentiation using RAW-Venus1. (A) Differentiation into Venus positive pre-osteoclasts and osteoclasts were induced by RANKL in a dose-dependent manner. TNF-α exerted synergistic effects on RANKL-induced osteoclast differentiation (magnification × 20). (B) Both anti-RANKL Ab and OPG strongly reduced the number of Venus positive cells, indicated the inhibition of osteoclast differentiation (magnification × 20). (C) Venus positive cells were tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (magnification × 40). Cells were stained using a commercial TRAP assay (Sigma-Aldrich, St Louis, MO, USA). (A-C) Cells were cultured at 4.5 × 104 cells/ml for 3 days in a 96-well plate. No stim No stimulation. (PDF 304 kb)

Phenotypic analysis of RANKL+ and RANKL− effector memory B cells. Purified switched-memory B cells from HC were stimulated with BCR/CD40 and IFN-γ for 48 hours. RANKL+ and RANKL− cells were analyzed for expression of CD20, CD21, CD95, CD11c, CCR1 and CCR5 using respective Abs (all from BioLegend). Representative data are shown (n = 3–4). (PDF 177 kb)

Glucocorticoids are steroid hormones with strong anti-inflammatory and immunosuppressive effects that are produced in a diurnal fashion. Although glucocorticoids have the potential to induce interleukin-7 receptor (IL-7R) expression in T cells, whether they control T cell homeostasis and responses at physiological concentrations remains unclear. We found that glucocorticoid receptor signaling induces IL-7R expression in mouse T cells by binding to an enhancer of the IL-7Ra locus, with a peak at midnight and a trough at midday. This diurnal induction of IL-7R supported the survival of T cells and their redistribution between lymph nodes, spleen, and blood by controlling expression of the chemokine receptor CXCR4. In mice, T cell accumulation in the spleen at night enhanced immune responses against soluble antigens and systemic bacterial infection. Our results reveal the immunoenhancing role of glucocorticoids in adaptive immunity and provide insight into how immune function is regulated by the diurnal rhythm., ステロイドが免疫力を高める --免疫の新たな昼夜サイクルを解明--. 京都大学プレスリリース. 2018-01-24.

Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is used for the treatment of bladder cancer. The recruitment of neutrophlis to the bladder after BCG instillation exerts anti-tumor activity against bladder tumor. We have recently demonstrated that interleukin (IL)-17A produced by γδ T cells played a role in the recruitment of neutrophlis to the bladder after BCG instillation. IL-15 is known to play an important role in neutrophil migration during inflammation. We previously constructed a recombinant BCG strain expressing the fusion protein of IL-15 and Ag85B (BCG-IL-15) for prevention of Mycobacterium tuberculosis infection. Here we compared the efficacy of the BCG-IL-15 in protection against bladder cancer with that of rBCG-Ag85B (BCG). Six-week-old female C57BL/6 mice were inoculated with MB49 bladder tumor cells in the bladder and subsequently intravesically inoculated with BCG or BCG-IL-15. BCG-IL-15 treatment significantly prolonged survival of mice inoculated with bladder cancer cells compared with BCG treatment. Infiltration of neutrophils was significantly elevated in BCGB-IL-15 treated mice accompanied by increased chemokines (MIP-2 and MIP-1α) in the bladder. Thus, BCG-IL-15 exerted additive effect on Infiltration of neutrophils in the bladder. BCG-IL-15 may be a promising drug for non-muscle invasive bladder cancer.

CD30, a member of the tumor necrosis factor receptor superfamily, is expressed preferentially by effector or memory helper T cells. Here we show that experimental autoimmune encephalomyelitis (EAE) is ameliorated with markedly reduced induction of antigen-specific Th1 and Th17 cells in CD30 knockout mice. Passive EAE indicated that CD30 on non-hematopoietic parenchymal cell is not required and mixed bone marrow chimera experiments revealed that CD30 signaling on CD4 T cells amplified the development of antigen-specific and encephalitogenic CD4 T cells. Thus, CD30 expression on CD4 T cells is critically involved in the pathogenesis of central nervous system autoimmunity.