Kazuyoshi Ishigaki

Autoantigen BiP-Derived HLA–DR4 Epitopes Differentially Recognized by Effector and Regulatory T Cells in Rheumatoid Arthritis

The balance between effector and regulatory CD4+ T cells plays a key role in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to examine whether the RA autoantigen BiP has epitopes for both effector and regulatory immunities.

Polygenic burdens on cell-specific pathways underlie the risk of rheumatoid arthritis

Recent evidence suggests that a substantial portion of complex disease risk alleles modify gene expression in a cell-specific manner. To identify candidate causal genes and biological pathways of immune-related complex diseases, we conducted expression quantitative trait loci (eQTL) analysis on five subsets of immune cells (CD4+ T cells, CD8+ T cells, B cells, natural killer (NK) cells and monocytes) and unfractionated peripheral blood from 105 healthy Japanese volunteers. We developed a three-step analytical pipeline comprising (i) prediction of individual gene expression using our eQTL database and public epigenomic data, (ii) gene-level association analysis and (iii) prediction of cell-specific pathway activity by integrating the direction of eQTL effects. By applying this pipeline to rheumatoid arthritis data sets, we identified candidate causal genes and a cytokine pathway (upregulation of tumor necrosis factor (TNF) in CD4+ T cells). Our approach is an efficient way to characterize the polygenic contributions and potential biological mechanisms of complex diseases.

Quantitative and qualitative characterization of expanded CD4 + T cell clones in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is an autoimmune destructive arthritis associated with CD4+ T cell-mediated immunity. Although expanded CD4+ T cell clones (ECs) has already been confirmed, the detailed characteristics of ECs have not been elucidated in RA. Using combination of a single-cell analysis and next-generation sequencing (NGS) in TCR repertoire analysis, we here revealed the detailed nature of ECs by examining peripheral blood (PB) from 5 RA patients and synovium from 1 RA patient. When we intensively investigated the single-cell transcriptome of the most expanded clones in memory CD4+ T cells (memory-mECs) in RA-PB, senescence-related transcripts were up-regulated, indicating circulating ECs were constantly stimulated. Tracking of the transcriptome shift within the same memory-mECs between PB and the synovium revealed the augmentations in senescence-related gene expression and the up-regulation of synovium-homing chemokine receptors in the synovium. Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs. Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes. Our approach may provide new insights into the pathophysiology of RA.

Transcription Factor Early Growth Response 3 Is Associated with the TGF-β1 Expression and the Regulatory Activity of CD4-Positive T Cells In Vivo

TGF-β1 is an important anti-inflammatory cytokine, and several regulatory T cell (Treg) subsets including CD4+CD25+Foxp3+ Tregs and Th3 cells have been reported to exert regulatory activity via the production of TGF-β1. However, it has not yet been elucidated which transcription factor is involved in TGF-β1 transcription. Early growth response 3 (Egr-3) is a zinc-finger transcription factor that creates and maintains T cell anergy. In this study, we found that Egr-3 induces the expression of TGF-β1 in both murine and human CD4+ T cells. Egr-3 overexpression in murine CD4+ T cells induced the production of TGF-β1 and enhanced the phosphorylation of STAT3, which is associated with TGF-β1 transcription. Moreover, Egr-3 conferred Ag-specific regulatory activity on murine CD4+ T cells. In collagen-induced arthritis and delayed-type hypersensitivity model mice, Egr-3–transduced CD4+ T cells exhibited significant regulatory activity in vivo. In particular, the suppression of delayed-type hypersensitivity depended on TGF-β1. In human tonsils, we found that CD4+CD25−CD45RO−lymphocyte activation gene 3 (LAG3)− T cells express membrane-bound TGF-β1 in an EGR3-dependent manner. Gene-expression analysis revealed that CD4+CD25−CD45RO−LAG3− T cells are quite different from conventional CD4+CD25+Foxp3+ Tregs. Intriguingly, the CD4+CD25−CD45RO−LAG3− T cells suppressed graft-versus-host disease in immunodeficient mice transplanted with human PBMCs. Our results suggest that Egr-3 is a transcription factor associated with TGF-β1 expression and in vivo regulatory activity in both mice and humans.

Immunophenotyping of rheumatoid arthritis reveals a linkage between HLA-DRB1 genotype, CXCR4 expression on memory CD4+ T cells, and disease activity

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that leads to destructive arthritis. Although the HLA class II locus is the strongest genetic risk factor for rheumatoid arthritis, the relationship between HLA class II alleles and lymphocyte activation remains unclear. We performed immunophenotyping of peripheral blood mononuclear cells on 91 HLA-DRB1-genotyped RA patients and 110 healthy donors. The frequency of memory CXCR4+CD4+ T cells, and not Th1 and Th17 cells, was significantly associated with disease severity by multiple linear regression analysis. RA patients with one or more susceptible HLA-DR haplotypes (shared epitope: SE) displayed a significantly higher frequency of memory CXCR4+CD4+ T cells. Moreover, the frequency of memory CXCR4+CD4+ T cells significantly correlated with the expression level of HLA-DR on B cells, which was elevated in RA patients with SE. In vitro analysis and transcriptomic pathway analysis suggested that the interaction between HLA-DR and T cell receptors is an important regulator of memory CXCR4+CD4+ T cells. Clinically, a higher frequency of memory CXCR4+CD4+ T cells predicted a better response to CTLA4-Ig. Memory CXCR4+CD4+ T cells may serve as a powerful biomarker for unraveling the linkage between HLA-DRB1 genotype and disease activity in RA.

TGF-β3 Inhibits Antibody Production by Human B Cells

TGF-β is a pleotropic cytokine involved in various biological processes. Of the three isoforms of TGF-β, TGF-β1 has long been recognized as an important inhibitory cytokine in the immune system and has been reported to inhibit B cell function in both mice and humans. Recently, it has been suggested that TGF-β3 may play an important role in the regulation of immune system in mice. Murine CD4+CD25-LAG3+ regulatory T cells suppress B cell function through the production of TGF-β3, and it has been reported that TGF-β3 is therapeutic in a mouse model of systemic lupus erythematosus. The effect of TGF-β3 on human B cells has not been reported, and we herein examined the effect of TGF-β3 on human B cells. TGF-β3 suppressed B cell survival, proliferation, differentiation into plasmablasts, and antibody secretion. Although the suppression of human B cells by TGF-β1 has long been recognized, the precise mechanism for the suppression of B cell function by TGF-β1 remains elusive; therefore, we examined the effect of TGF-β1 and β3 on pathways important in B cell activation and differentiation. TGF-β1 and TGF-β3 inhibited some of the key molecules of the cell cycle, as well as transcription factors important in B cell differentiation into antibody secreting cells such as IRF4, Blimp-1, and XBP1. TGF-β1 and β3 also inhibited B cell receptor signaling. Our results suggest that TGF-β3 modifying therapy might be therapeutic in autoimmune diseases with B cell dysregulation in humans.

Cardiopulmonary arrest after severe anaphylactic reaction to second infusion of infliximab in a patient with ankylosing spondylitis.

To the Editor: Infliximab is an anti-tumor necrosis factor-α (TNF-α) chimeric monoclonal antibody widely used to treat chronic inflammatory diseases such as rheumatoid arthritis, Crohn’s disease, ankylosing spondylitis (AS), and psoriasis. Infusion reaction to infliximab occurs in approximately 5% of patients1. Although severe anaphylactic reactions including hypotension, arrhythmia, and bronchospasm have been reported, there is no report of cardiopulmonary arrest strongly connected with the infliximab infusion. We describe a patient with AS who developed cardiopulmonary arrest after an anaphylactic reaction to the second infliximab infusion. A 37-year-old man with no history of allergy or coronary artery event received infliximab infusion for AS at our hospital. His symptoms were refractory to nonsteroidal antiinflammatory drugs, and he had been treated with … Address correspondence to Dr. Fujio; E-mail: kfujio-tky{at}umin.ac.jp

Splicing variant of WDFY4 augments MDA5 signalling and the risk of clinically amyopathic dermatomyositis

Objectives Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of rare autoimmune diseases in which both genetic and environmental factors play important roles. To identify genetic factors of IIM including polymyositis, dermatomyositis (DM) and clinically amyopathic DM (CADM), we performed the first genome-wide association study for IIM in an Asian population. Methods We genotyped and tested 496 819 single nucleotide polymorphism for association using 576 patients with IIM and 6270 control subjects. We also examined the causal mechanism of disease-associated variants by in silico analyses using publicly available data sets as well as by in in vitro analyses using reporter assays and apoptosis assays. Results We identified a variant in WDFY4 that was significantly associated with CADM (rs7919656; OR=3.87; P=1.5×10−8). This variant had a cis-splicing quantitative trait locus (QTL) effect for a truncated WDFY4isoform (tr-WDFY4), with higher expression in the risk allele. Transexpression QTL analysis of this variant showed a positive correlation with the expression of NF-κB associated genes. Furthermore, we demonstrated that both WDFY4 and tr-WDFY4 interacted with pattern recognition receptors such as TLR3, TLR4, TLR9 and MDA5 and augmented the NF-κB activation by these receptors. WDFY4 isoforms also enhanced MDA5-induced apoptosis to a greater extent in the tr-WDFY4-transfected cells. Conclusions As CADM is characterised by the appearance of anti-MDA5 autoantibodies and severe lung inflammation, the WDFY4 variant may play a critical role in the pathogenesis of CADM.

A case of severe bilateral lupus chorioretinopathy without major organ involvement

Dear Editor, Here we report a very rare case of severe bilateral lupus chorioretinopathy without other vital organ damage. A 65-year-old woman with systemic lupus erythematosus (SLE) continued to attend our hospital regularly since the diagnosis had been made at the age of 56 based on the presence of butterfly rash, photosensitivity, arthritis and positive antinuclear antibodies (ANA). Although she suffered from photosensitive eruptions every summer, she was generally in a stable condition, taking low-dose glucocorticoids. She began to feel general malaise and lost weight in autumn 2007. In July 2008, she suddenly experienced impaired eyesight in both eyes and almost 2 weeks later, she was diagnosed with bilateral serous retinal detachment and was hospitalized for close examination. Physical examination revealed that she had a normal blood pressure of 120/70 mmHg, and normal heart and lung sounds. She was neurologically intact and her intelligence level was maintained. She had a malar butterfly rash and photosensitive rash over her chest and arms, and polyarthritis. The major laboratory data revealed ANA at 1 : 320 with a speckled pattern and low complement level, represented by total hemolytic complement (CH50) at 16 U/mL (normal range: 30–47). In contrast, blood count, urinalysis and spinal fluid findings were normal and antiphospholipid antibodies were negative. The Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) was 16 (arthritis, low complement, skin rash, visual disturbance). Ophthalmologic investigation was further performed. No abnormal findings were observed in the conjunctivae, anterior and posterior sclera, anterior segment, lens and vitreous body. Visual acuity (VA) was 6/60 for the right and 12/60 for the left, accompanied by altered color vision. Visual field test was normal. Fundoscopy revealed a normal vasculature with no findings of exudates, cottonwool patch or hemorrhage. Mild papilledema was observed in the right, but the optic disc was normal in the left eye. Serous retinal detachment was not rhegmatogenous, and was observed extensively on the temporal side of the optic disc in the right eye, extending from below the disc to the proximal part of the superior temporal arcade, and the temporal margin involved the macular lesion. In the left eye, it was located temporal to the optic disc, and the macula was slightly involved (Fig. 1a). Fluorescein angiography (FAG) revealed multiple spots of leakage in the area and along the rim of the detached retina (Fig. 1b). Indocyanine green angiography (IA) revealed leakage and subretinal pooling of the dye in the late phase (Fig. 1c). Optical coherence tomography (OCT) revealed fluid collection above the retinal pigment epithelium (RPE) (Fig. 1d). These findings were consistent with lupus retinochoriopathy. However, as similar findings are also seen in Vogt-Koyanagi-Harada disease (VKH), we had to make a differential diagnosis. Our patient did not have inner ear or meningeal symptoms, nor did she have vitritis or iridocyclitis, which often accompanies posterior uveitis in VKH. Furthermore, she did not show the disease-characteristic human leukocyte antigen DR4 or DR53; thus we thought that a diagnosis of VKH was inappropriate for her. As she had been treated with glucocorticoids for a long period, central serous chorioretinopathy (CSC) was another differential diagnosis. However, the number of leakage spots in our patient was too numerous for CSC, in which only one or several spots are generally observed. Finally, taking into consideration the increased lupus activity, we reached a diagnosis of lupus chorioretinopathy. Methylprednisolone (MPSL) pulse was started, followed by high-dose oral prednisolone (PSL) at 45 mg/ day. The VA as well as color vision recovered completely (6/60–6/6 in the right and 12/60–6/6 in the left) and the SLEDAI also improved (16–0). Together with the improvement of fundus and FAG findings, International Journal of Rheumatic Diseases 2012; 15: e153–e155

HLA-DRB1 Shared Epitope Alleles and Disease Activity Are Correlated with Reduced T Cell Receptor Repertoire Diversity in CD4+ T Cells in Rheumatoid Arthritis

Objective. Shared epitope (SE) alleles are the most significant genetic susceptibility locus in rheumatoid arthritis (RA); however, their target populations in CD4+ T cells are not well elucidated. We analyzed the association between SE alleles and the T cell receptor (TCR) repertoire diversity of naive and memory CD4+ T cells using next-generation sequencing (NGS). Methods. The TCR beta chains in naive and memory CD4+ T cells from the peripheral blood of 22 patients with RA and 18 age- and sex-matched healthy donors (HD) were analyzed by NGS. The Renyi entropy was used to evaluate TCR repertoire diversity and its correlations with SE alleles and other variables were examined. Serum cytokine levels were measured by multiplex ELISA. Results. The TCR repertoire diversity in memory CD4+ T cells was reduced in SE allele-positive patients with RA compared with HD, and showed a significant negative correlation with the SE allele dosage in RA. The TCR repertoire diversity of naive and memory T cells was also negatively correlated with disease activity, and the SE allele dosage and disease activity were independently associated with reduced TCR repertoire diversity. TCR repertoire diversity showed a significant positive correlation with the serum interleukin 2 levels. Conclusion. SE alleles and disease activity were negatively correlated with the TCR repertoire diversity of CD4+ T cells in RA. Considering the pivotal role of CD4+ T cells in RA, restoring the altered TCR repertoire diversity will provide a potential RA therapeutic target.