Published papers

Regulatory T cells (Tregs) are essential for the maintenance of immune homeostasis. Studies of Treg are not only necessary for understanding the mechanism of immune homeostasis but also extremely useful for the development of treatments of various immune diseases. Forkhead box P3 (Foxp3) was identified as the master gene responsible for the immune-suppressing activity of Tregs. The promoter region and several intronic enhancers, designated conserved noncoding sequence (CNS) 0, 1, 2, and 3, at the Foxp3 gene locus have important roles in Foxp3 expression and Treg development. We demonstrated that transcription factors Nr4a and Smad2/3 are required for development of thymic Tregs and induced Tregs, respectively. In addition to transcription factors, Treg-specific DNA demethylation has been shown to be important for Treg stability. In particular, DNA demethylation of CNS2 was implicated in Treg stability, and members of the ten-eleven translocation family of demethylation factors were recently demonstrated to have important roles in 5'-C-phosphate-G-3' demethylation at CNS2. This article summarizes recent findings regarding the roles of transcription factors and epigenetic modifications in the differentiation, maintenance, and function of Tregs. This review will facilitate clinical application of Tregs to diseases in the field of ophthalmology, including uveitis and age-related macular degeneration.

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Adoptive T-cell therapy is an effective strategy for cancer immunotherapy. However, infused T cells frequently become functionally exhausted, and consequently offer a poor prognosis after transplantation into patients. Adoptive transfer of tumor antigen-specific stem cell memory T (TSCM) cells is expected to overcome this shortcoming as TSCM cells are close to naïve T cells, but are also highly proliferative, long-lived, and produce a large number of effector T cells in response to antigen stimulation. We previously reported that activated effector T cells can be converted into TSCM-like cells (iTSCM) by coculturing with OP9 cells expressing Notch ligand, Delta-like 1 (OP9-hDLL1). Here we show the methodological parameters of human CD8+ iTSCM cell generation and their application to adoptive cancer immunotherapy. Regardless of the stimulation by anti-CD3/CD28 antibodies or by antigen-presenting cells, human iTSCM cells were more efficiently induced from central memory type T cells than from effector memory T cells. During the induction phase by coculture with OP9-hDLL1 cells, interleukin (IL)-7 and IL-15 (but not IL-2 or IL-21) could efficiently generate iTSCM cells. Epstein–Barr virus-specific iTSCM cells showed much stronger antitumor potentials than conventionally activated T cells in humanized Epstein–Barr virus transformed-tumor model mice. Thus, adoptive T-cell therapy with iTSCM offers a promising therapeutic strategy for cancer immunotherapy.

© 2019, The Author(s), under exclusive licence to Springer Nature Limited. Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K. pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (T H 17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K. pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K. pneumoniae that was associated with bacterial translocation and susceptibility to T H 17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the T H 17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.

© 2018, The Author(s). Auto-inflammatory syndrome, a condition clinically distinct from rheumatoid arthritis, is characterized by systemic inflammation in tissues such as major joints, skin, and internal organs. Autonomous innate-immune activation is thought to promote this inflammation, but underlying pathological mechanisms have not been clarified nor are treatment strategies established. Here, we newly established a mouse model in which IL-1 signaling is conditionally activated in adult mice (hIL-1 cTg) and observed phenotypes similar to those seen in auto-inflammatory syndrome patients. In serum of hIL-1 cTg mice, IL-6 and IL-17 levels significantly increased, and signal transducer and activator of transcription 3 (Stat3) was activated in joints. When we crossed hIL-1 cTg with either IL-6- or IL-17-deficient mice or with Stat3 conditional knockout mice, phenotypes seen in hIL-1 cTg mice were significantly ameliorated. Thus, IL-6, IL-17 and Stat3 all represent potential therapeutic targets for this syndrome.

© 2018 American Association for Cancer Research. Enhanced infiltration of regulatory T cells (Treg) into tumor tissue is detrimental to patients with cancer and is closely associated with poor prognosis as they create an immunosuppressive state that suppresses antitumor immune responses. Therefore, breaking Treg-mediated immune tolerance is important when considering cancer immunotherapy. Here, we show that the Nr4a nuclear receptors, key transcription factors maintaining Treg genetic programs, contribute to Treg-mediated suppression of antitumor immunity in the tumor microenvironment. Mice lacking Nr4a1 and Nr4a2 genes specifically in Tregs showed resistance to tumor growth in transplantation models without exhibiting any severe systemic autoimmunity. The chemotherapeutic agent camptothecin and a common cyclooxygenase-2 inhibitor were found to inhibit transcriptional activity and induction of Nr4a factors, and they synergistically exerted antitumor effects. Genetic inactivation or pharmacologic inhibition of Nr4a factors unleashed effector activities of CD8þ cytotoxic T cells and evoked potent antitumor immune responses. These findings demonstrate that inactivation of Nr4a in Tregs breaks immune tolerance toward cancer, and pharmacologic modulation of Nr4a activity may be a novel cancer treatment strategy targeting the immunosuppressive tumor microenvironment. Significance: This study reveals the role of Nr4a transcription factors in Treg-mediated tolerance to antitumor immunity, with possible therapeutic implications for developing effective anticancer therapies

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.

© 2018 The Author(s). Sustained clinical remission (CR) without drug treatment has not been achieved in patients with rheumatoid arthritis (RA). This implies a substantial difference between CR and the healthy state, but it has yet to be quantified. We report a longitudinal monitoring of the drug response at multi-omics levels in the peripheral blood of patients with RA. Our data reveal that drug treatments alter the molecular profile closer to that of HCs at the transcriptome, serum proteome, and immunophenotype level. Patient follow-up suggests that the molecular profile after drug treatments is associated with long-term stable CR. In addition, we identify molecular signatures that are resistant to drug treatments. These signatures are associated with RA independently of known disease severity indexes and are largely explained by the imbalance of neutrophils, monocytes, and lymphocytes. This high-dimensional phenotyping provides a quantitative measure of molecular remission and illustrates a multi-omics approach to understanding drug response.

© 2018 Cold Spring Harbor Laboratory Press; all rights reserved. Cytokines are key modulators of immunity. Most cytokines use the Janus kinase and signal transducers and activators of transcription (JAK-STAT) pathway to promote gene transcriptional regulation, but their signals must be attenuated by multiple mechanisms. These include the suppressors of cytokine signaling (SOCS) family of proteins, which represent a main negative regulation mechanism for the JAK-STAT pathway. Cytokine-inducible Src homology 2 (SH2)-containing protein (CIS), SOCS1, and SOCS3 proteins regulate cytokine signals that control the polarization of CD4+ T cells and the maturation of CD8+ T cells. SOCS proteins also regulate innate immune cells and are involved in tumorigenesis. This review summarizes recent progress on CIS, SOCS1, and SOCS3 in T cells and tumor immunity.

© 2018 Kimura et al. NAD(P)H:quinone oxidoreductase 1 (NQO1) protects cells against oxidative stress and toxic quinones. In this study, we found a novel role of NQO1 in suppressing Toll-like receptor (TLR)–mediated innate immune responses. NQO1-deficient macrophages selectively produced excessive amounts of IL-6, IL-12, and GM-CSF on LPS stimulation, and the deletion of NQO1 in macrophages exacerbated LPS-induced septic shock. NQO1 interacted with the nuclear IκB protein IκB-ζ, which is essential for the TLR-mediated induction of a subset of secondary response genes, including IL-6, and promoted IκB-ζ degradation in a ubiquitin-dependent manner. We demonstrated that PDLIM2, known as the ubiquitin E3 ligase, participates in NQO1-dependent IκB-ζ degradation. NQO1 augmented the association between PDLIM2 and IκB-ζ, resulting in increased IκB-ζ degradation. Collectively, this study describes a mechanism of the NQO1–PDLIM2 complex as a novel and important regulator in the innate immune signaling and suggests the therapeutic potential of NQO1 in TLR-mediated inflammation and disorders.

© 2018 The Author(s) Regulatory T (Treg) cells develop from a self-reactive, CD4-single positive (CD4SP) precursor cell pool. Thus, Treg-fated developing thymocytes are expected to possess the potential to generate pathogenic self-reactive cells. However, no such pathogenic conversion has been observed, indicating mechanisms of defense to prevent such a deleterious event. Here, we show that, after the initial developmental phase, the Nr4a family of nuclear receptors promotes the development of Treg cells by cooperating with other Treg cell developmental machineries, as well as by forming a reinforcing loop with Foxp3. Nr4a-deficient Treg-fated thymocytes survive and can elicit autoimmunity, highlighting their roles in elimination of developing Treg precursors that fail to complete their development. Our findings reveal that the defective development of Treg-fated thymocytes is a potential route for the generation of pathogenic self-reactive cells, which is normally suppressed by Nr4a factors at both developmental and cell death levels. Sekiya et al. find that developing precursors of Treg cells are a labile population compared to mature Treg cells and can convert to pathogenic self-reactive cells. They show that Nr4a family nuclear receptors prevent this pathogenic conversion of Treg precursors at the level of both development and cell death.

© 2018, Springer-Verlag GmbH Germany, part of Springer Nature. Inflammatory responses play a multifaceted role in regulating both disability and recovery after ischemic brain injury. In the acute phase of ischemic stroke, resident microglia elicit rapid inflammatory responses by the ischemic milieu. After disruption of the blood-brain barrier, peripheral-derived neutrophils and mononuclear phagocytes infiltrate into the ischemic brain. These infiltrating myeloid cells are activated by the endogenous alarming molecules released from dying brain cells. Inflammation after ischemic stroke thus typically consists of sterile inflammation triggered by innate immunity, which exacerbates the pathologies of ischemic stroke and worsens neurological prognosis. Infiltrating immune cells sustain the post-ischemic inflammation for several days; after this period, however, these cells take on a repairing function, phagocytosing inflammatory mediators and cellular debris. This time-specific polarization of immune cells in the ischemic brain is a potential novel therapeutic target. In this review, we summarize the current understanding of the phase-dependent role of innate myeloid cells in ischemic stroke and discuss the cellular and molecular mechanisms of their inflammatory or repairing polarization from a therapeutic perspective.

© 2018, The Author(s), under exclusive licence to Springer Nature Limited. Crosstalk between the autonomic nervous system and the immune system by means of the sympathetic and parasympathetic pathways is a critical process in host defence. Activation of the sympathetic nervous system results in the release of catecholamines as well as neuropeptide Y (NPY). Here, we investigated whether phagocytes are capable of the de novo production of NPY, as has been described for catecholamines. We show that the synthesis of NPY and its Y1 receptor (Y1R) is increased in phagocytes in lungs following severe influenza virus infection. The genetic deletion of Npy or Y1r specifically in phagocytes greatly improves the pathology of severe influenza virus infection, which is characterized by excessive virus replication and pulmonary inflammation. Mechanistically, it is the induction of suppressor of cytokine signalling 3 (SOCS3) via NPY–Y1R activation that is responsible for impaired antiviral response and promoting pro-inflammatory cytokine production, thereby enhancing the pathology of influenza virus infection. Thus, direct regulation of the NPY–Y1R–SOCS3 pathway on phagocytes may act as a fine-tuner of an innate immune response to virus infection, which could be a therapeutic target for lethal influenza virus infection.

© 2018 The Author(s) T helper 17 (Th17) cell development is programmed by the orphan nuclear receptor RORγt, but the underlying mechanism is not well understood. Nuclear receptor-mediated transcriptional activation depends on coactivators. Here, we show that steroid receptor coactivator-3 (SRC-3) critically regulates Th17 cell differentiation. Reduced incidence of experimental autoimmune encephalitis (EAE) associated with decreased Th17 cell generation in vivo was observed in mice with SRC-3 deletion specifically in T cells. In vitro, SRC-3 deficiency did not affect TGF-β/IL-6-induced Th17 cell generation but severely impaired pathogenic Th17 differentiation induced by IL-1/IL-6/IL-23. Microarray analysis revealed that SRC-3 not only regulates IL-17A but also IL-1R1 expression. SRC-3 bound to Il17a and Il1r1 loci in a RORγt-dependent manner and was required for recruitment of the p300 acetyltransferase. Thus, SRC-3 is critical for RORγt-dependent gene expression in Th17 cell-driven autoimmune diseases. The unique transcriptional programs in IL-1-induced inflammatory Th17 cells have remained unclear. Tanaka et al. demonstrate that SRC-3 in CD4+ T helper cells functions as a specific coactivator of RORγt, a master transcription factor of Th17 cells, by regulating IL-1-IL1R1 signaling.

© The Japanese Society for Immunology. 2018. T helper type 1 (Th1) cells form one of the most stable CD4 T-cell subsets, and direct conversion of fully differentiated Th1 to regulatory T (Treg) cells has been poorly investigated. Here, we established a culture method for inducing Foxp3 from Th1 cells of mice and humans. This is achieved simply by resting Th1 cells without T-cell receptor ligation before stimulation in the presence of transforming growth factor-beta (TGF-ß). We named the resulting Th1-derived Foxp3+ cells Th1reg cells. Mouse Th1reg cells showed an inducible Treg-like phenotype and suppressive ability both in vitro and in vivo. Th1reg cells could also be induced from in vivo-developed mouse Th1 cells. Unexpectedly, the resting process enabled Foxp3 expression not through epigenetic changes at the locus, but through metabolic change resulting from reduced mammalian target of rapamycin complex 1 (mTORC1) activity. mTORC1 suppressed TGF-ß-induced phosphorylation of Smad2/3 in Th1 cells, which was restored in rested cells. Our study warrants future research aiming at development of immunotherapy with Th1reg cells.

© 2019 The Author(s). Background: Ultrasonography (US) can directly demonstrate joint inflammation, including grayscale (GS) signs of synovial hypertrophy and power Doppler (PD) techniques to demonstrate increased blood flow and vascularization. Recently, echogenicity, especially hypoechoic synovium, has also been associated with local inflammatory activity. However, only a few studies have demonstrated correlation between histopathologic and immunopathologic evaluation and US findings. The aim of this study was to clarify whether joint US findings including synovial hypertrophy, vascularity, and echogenicity can accurately characterize synovial pathophysiology in patients with active rheumatoid arthritis (RA). Methods: A total of 44 patients with RA were included, both treated (n = 25) and untreated (n = 19) and scheduled for US examination of the knee joint with synovial fluid (SF) aspiration and two treated patients also underwent synovial biopsy. US images were quantitatively analyzed using grayscale assessment of synovial hypertrophy and PD for vascularity and echogenicity. Levels of nine SF cytokines and growth factors were also measured. Results: Both US synovial hypertrophy and PD vascularity significantly correlated with SF inflammatory cytokine levels such as IL-6, IL-8, IL-1β and IL-10 in untreated patients. Angiogenic factors, including vascular endothelial growth factor (VEGF), only correlated with PD vascularity. In the treated patients, the associations between synovial hypertrophy and any cytokines were diminished, although synovial vascularity and echogenicity correlated with IL-6 and VEGF (p < 0.05). Histopathologic analysis revealed that hypoechogenicity of the synovium correlated with marked infiltration of lymphocytes and hypervascularity. Conclusions: We demonstrated the pathophysiological origins of US findings in the joint. The degree of US vascularity of the synovium correlated with local inflammatory cytokine levels and angiogenetic factors in patients with active RA. Synovial echogenicity, and not hypertrophy, correlated with inflammation, especially in treated patients with RA.

© 2018 Elsevier Inc. Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis. Tadokoro et al. show that Spred1 negatively regulates HSC self-renewal in a manner supported by ROCK activity and that Spred1 safeguards HSC homeostasis under high-fat diet (HFD) conditions by regulating HSC self-renewal. The gut microbiota dysbiosis induced by HFD disrupts the fine-tuning of Spred1-mediated signals that govern HSC homeostasis.

© 2018 Gao et al. STAT3 is a master regulator of the immune responses. Here we show that M. tuberculosis-infected stat3fl/fllysm cre mice, defective for STAT3 in myeloid cells, contained lower bacterial load in lungs and spleens, reduced granuloma extension but higher levels of pulmonary neutrophils. STAT3-deficient macrophages showed no improved control of intracellular mycobacterial growth. Instead, protection associated to elevated ability of stat3fl/fllysm cre antigen-presenting cells (APCs) to release IL-6 and IL-23 and to stimulate IL-17 secretion by mycobacteria-specific T cells. The increased IL-17 secretion accounted for the improved control of infection since neutralization of IL-17 receptor A in stat3fl/fllysm cre mice hampered bacterial control. APCs lacking SOCS3, which inhibits STAT3 activation via several cytokine receptors, were poor inducers of priming and of the IL-17 production by mycobacteria-specific T cells. In agreement, socs3fl/flcd11c cre mice deficient of SOCS3 in DCs showed increased susceptibility to M. tuberculosis infection. While STAT3 in APCs hampered IL-17 responses, STAT3 in mycobacteria-specific T cells was critical for IL-17 secretion, while SOCS3 in T cells impeded IL-17 secretion. Altogether, STAT3 signalling in myeloid cells is deleterious in the control of infection with M. tuberculosis.