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Objective: Idiopathic inflammatory myopathies (IIM) demonstrate characteristic clinical phenotypes depending on the myositis‐specific antibody (MSAs) present. We aimed to identify common or MSA‐specific immunological pathways in different immune cell types from peripheral blood by transcriptome analysis. Methods: We recruited 33 patients with IIM who were separated into the following groups: 15 patients with active disease at onset and 18 with inactive disease under treatment. All patients were positive for MSAs: anti–melanoma differentiation‐associated gene 5 (MDA5) antibody (Ab) in 10 patients, anti‐Mi‐2 Ab in 7, and anti‐aminoacyl‐transfer RNA synthetase (ARS) Ab in 16. The patients were compared with 33 healthy controls. Twenty‐four immune cell types sorted from peripheral blood were analyzed by flow cytometry, RNA sequencing, and differentially expressed gene analysis combined with pathway analysis. Results: The frequencies of memory B cell types were significantly decreased in active patients, and the frequency of plasmablasts was prominently increased in active patients with anti‐MDA5 Ab in comparison with healthy controls. The expression of type I interferon (IFN)‐stimulated genes of all immune cell types was increased in the active, but not inactive, patients. Endoplasmic reticulum stress‐related genes in all IIM memory B cells and oxidative phosphorylation‐related genes in inactive IIM double negative B cells were also increased, suggesting prominent B cell activation in IIM. Furthermore, active patients with anti‐MDA5 Ab, anti‐Mi‐2 Ab, or anti‐ARS Ab were distinguished by IFN‐stimulated and oxidative phosphorylation‐related gene expression in plasmablasts. Conclusion: Unique gene expression patterns in patients with IIM with different disease activity levels and MSA types suggest different pathophysiologies. Especially, B cells may contribute to common and MSA‐specific immunological pathways in IIM. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is an autoimmune disease that involves multiple immune cell subsets. We analyzed immune cell subsets in human peripheral blood mononuclear cells (PBMC) in order to identify the cells that are significantly associated with SLE disease activity and treatment. The frequencies of various subsets of CD4+ T cells, B cells, monocytes and NK cells in PBMC were assessed in 30 healthy controls (HC), 30 rheumatoid arthritis (RA) patients and 26 SLE patients using flow cytometry. The correlations between subset frequencies in SLE and clinical traits including Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) were examined. Changes in subset frequencies after the treatment in SLE patients were investigated. We focused on CD25+LAG3+ T cells and investigated their characteristics, including cytokine secretion, mRNA expression and suppression capacity. We assessed correlations between CD25+LAG3+ T cells and SLEDAI by Spearman's rank correlation coefficient. CD25+LAG3+ T cells were significantly increased in SLE whereas there were few in RA and HC groups. CD25+LAG3+ T cell frequencies were significantly correlated with SLEDAI and were increased in patients with a high SLEDAI score (> 10). CD25+LAG3+ T cells produced both IL-17 and FOXP3, expressed mRNA of both FOXP3 and RORC and lacked suppressive capacity. CD25+LAG3+ T cells were associated with disease activity of SLE. CD25+LAG3+ T cells had features of both CD25+FOXP3+ regulatory T cells (CD25+ Treg) and Th17. CD25+LAG3+ T cells could be associated with the inflammatory pathophysiology of SLE. [ABSTRACT FROM AUTHOR]

Inhibitory cytokines, such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), are humoral factors involved in the suppressive function of regulatory T cells and play critical roles in maintaining immune homeostasis. However, TGF-β and IL-10 also have pleiotropic effects and induce humoral immune responses depending on conditions, and thus their therapeutic application to autoimmune diseases remains limited. Here, we show that a combination of TGF-β and IL-10, but not single cytokine, is required to suppress B cell activation induced by toll-like receptor (TLR) stimulation. In in vivo analyses, the simultaneous presence of TGF-β and IL-10 effectively suppressed TLR-mediated antigen-specific immune responses and ameliorated pathologies in imiquimod (TLR7 agonist)-induced lupus model and lupus-prone MRL/lpr mice. Intriguingly, TGF-β and IL-10 synergistically modulated transcriptional programs and suppressed cellular energetics of both glycolysis and oxidative phosphorylation via inhibition of the mammalian target of rapamycin complex 1 (mTORC1)/S6 kinase 1 (S6K1) pathway in TLR-stimulated B cells. On the other hand, enhancement of mTOR signaling and mitochondrial biosynthesis in TLR-stimulated B cells counteracted the synergistic inhibitory effects. The inhibitory cytokine synergy of TGF-β and IL-10 via suppression of energy metabolism was also observed in human TLR-stimulated B cells. There is increasing evidence supporting the importance of adequate metabolic signals in various immune cells to exert their immune function. In this study, we have shown that a previously unrecognized synergy of inhibitory cytokines regulates systemic humoral immune responses via modulating immunometabolism in B cells. Our findings indicate that inhibition of B cell metabolism mediated by two synergistic cytokines contributes to the induction of immune tolerance and could be a new therapeutic strategy for autoimmune diseases such as systemic lupus erythematosus. [ABSTRACT FROM AUTHOR]

We analyzed the transcriptome of detailed CD4 + T cell subsets including them after abatacept treatment, and examined the difference among CD4 + T cell subsets and identified gene sets that are closely associated disease activity and abatacept treatment. Seven CD4 + T cell subsets (naive, Th1, Th17, Th1/17, nonTh1/17, Tfh and Treg) were sorted from PBMCs taken from 10 RA patients and 10 healthy controls, and three RA patients donated samples before and 6 months after abatacept treatment. Paired-end RNA sequencing was performed using HiSeq 2500. A total of 149 samples except for 12 outliers were analyzed. Overview of expression pattern of RA revealed that administration of abatacept exerts a large shift toward the expression pattern of HC. Most of differentially expressed gene (DEG) upregulated in RA (n = 1776) were downregulated with abatacept treatment (n = 1349). Inversely, most of DEG downregulated in RA (n = 1860) were upregulated with abatacept treatment (n = 1294). This DEG-based analysis revealed shared pathway changes in RA CD4 + T cell subsets. Knowledge-based pathway analysis revealed the upregulation of activation-related pathways in RA that was substantially ameliorated by abatacept. Weighted gene co-expression network analysis (WGCNA) evaluated CD4 + T cells collectively and identified a gene module that consisted of 227 genes and was correlated with DAS28-CRP (Spearman's rho = 0.46, p = 4 × 10 −9 ) and abatacept administration (Spearman's rho = −0.91, p = 5 × 10 −57 ). The most highly connected 30 genes of this module included ZAP70 and JAK3 , and pathway analysis of this module revealed dysregulation of the TCR signaling pathway network, which was ameliorated by abatacept. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

IL-10-producing regulatory T cells (IL-10-producing Tregs) are one of the regulatory T cell subsets characterized by the production of high amounts of IL-10, the lack of FOXP3 expression and the strong immunosuppressive capabilities. IL-10-producing Tregs have been primarily reported as induced populations thus far, in part because identifying naturally occurring IL-10-producing Tregs was difficult due to the lack of definitive surface markers. Lymphocyte-activation gene 3 (LAG3) is a CD4 homologue that we have identified as being expressed on IL-10 producing Tregs. In human PBMC, LAG3 combined with CD49b efficiently identifies IL-10-producing Tregs. However, naturally occurring IL-10-producing Tregs in human secondary lymphoid tissue have not been described. In this report, we identified CD4 + CD25 − LAG3 + T cells in human tonsil. This T cell subset produced high amounts of IL-10 and expressed low levels of FOXP3. Surface markers and microarray analysis revealed that this is a distinct tonsillar CD4 + T cell subset. CD4 + CD25 − LAG3 + T cells expressed interleukin 10 (IL10), PR/SET domain 1 (PRDM1), and CD274 at high levels and chemokine receptor 5 (CXCR5) at low levels. CD4 + CD25 − LAG3 + T cells suppressed antibody production more efficiently than CD4 + CD25 + T cells, and CD4 + CD25 − LAG3 + T cells induced B cell apoptosis. Moreover, analysis of humanized mice revealed that this cell subset suppressed a graft-versus-host disease (GVHD) reaction in vivo . Our study reveals the existence of naturally occurring IL-10-producing Tregs in human secondary lymphoid tissue and their function in immune regulation. [ABSTRACT FROM AUTHOR]

Objective 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. Methods The proliferation and cytokine secretion of peripheral blood mononuclear cells (PBMCs) from HLA-DR4-positive RA patients in response to BiP-derived peptides were examined by 3H-thymidine uptake and enzyme-linked immunosorbent assay. As a mouse therapeutic model, a BiP-derived peptide was administered orally to mice with collagen-induced arthritis (CIA). Results Among the peptides examined, BiP336-355 induced the strongest proliferation of PBMCs from RA patients, but not from healthy donors. The proliferation of PBMCs in response to BiP336-355 showed a correlation with clinical RA activity and serum anti-BiP/citrullinated BiP antibodies. In contrast, BiP456-475 induced interleukin-10 (IL-10) secretion from CD25-positive PBMCs obtained from RA patients and healthy donors without inducing cell proliferation, and it actively suppressed the BiP336-355-induced proliferation and proinflammatory cytokine secretion by PBMCs. Oral administration of BiP456-475 to mice with CIA reduced the severity of arthritis and T cell proliferation and increased the secretion of IL-10 from T cells as well as the number of CD4+CD25+FoxP3+ regulatory T cells. Conclusion Effector and regulatory T cells recognized different BiP epitopes. The deviated balance toward BiP-specific effector T cells in RA may be associated with disease activity; therefore, BiP-specific effector or regulatory T cells could be a target of new RA therapies. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are engaged in the maintenance of immunological self-tolerance and immune homeostasis. IL-10 has an important role in maintaining the normal immune state. Here, we show that IL-10-secreting Tregs can be delineated in normal mice as CD4+CD25-Foxp3- T cells that express lymphocyte activation gene 3 (LAG-3), an MHC-class-ll-binding CD4 homolog. Although ∼2% of the CD4+CD25-T cell population consisted of CD4+CD25-LAG3-T cells in the spleen, CD4+CD25-LAG34-T cells are enriched to ≈8% in the Peyer's patch. They are hypoproliferative upon in vitro antigenic stimulation and suppress in vivo development of colitis. Gene expression analysis reveals that CD4+CD25-LAG3+ Tregs characteristically express early growth response gene 2 (Egr-2), a key molecule for anergy induction. Retroviral gene transfer of Egr-2 converts naïve CD4+ T cells into the IL-10-secreting and LAG-3-expressing phenotype, and Egr-2+transduced CD4+ T cells exhibit antigen-specific immunosuppressive capacity in vivo. Unlike Foxp3+ natural Tregs, high-affinity interactions with selecting peptide/MHC ligands expressed in the thymus do not induce the development of CD4+CD25-LAG3+ Tregs. In contrast, the number of CD4+CD25LAG3+ Tregs is influenced by the presence of environmental microbiota. Thus, IL-b-secreting Egr-2+LAG3+CD4+ Tregs can be exploited for the control of peripheral immunity. [ABSTRACT FROM AUTHOR]