Language：English   Publishing type：Research paper (scientific journal)  

Immune responses to non-pathogenic yeasts induced within the draining lymph node remain to be understood. In this study, we have investigated the changes in lymphocytes and their activity in skin-draining lymph nodes in response to transdermally injected zymosan (component of the yeast cell wall). Zymosan elicited the transient increase of B cell number and activation status without affecting the capacity for proliferation. The increased B cell content in the regional lymph nodes was likely due to the reduction of B cell egress from the tissue and in part the increase of homing from the circulation. Zymosan also upregulated the inflammatory cytokines, such as IL-1β, IL-6, IL-12, and IFNγ, regulatory cytokines IL-10 and TGFβ, and lymphoid chemokine CXCL13. Among these, the expression of IL-12 and IL-10 was markedly high in B cells. Altogether, these findings demonstrate a unique B cell-associated response to non-pathogenic yeast component in the draining lymph nodes. This will provide insights into the clinical and healthcare applications of non-pathogenic beneficial microbes.

DOI： 10.1016/j.cellimm.2020.104159

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Organized tissue structure in the secondary lymphoid organs (SLOs) tightly depends on the development of fibroblastic stromal cells (FSCs) of mesenchymal origin; however, the mechanisms of this relationship are poorly understood. In this study, we specifically inactivated the canonical NF-κB pathway in FSCs in vivo by conditionally inducing IκBα mutant in a Ccl19-IκBSR mouse system in which NF-κB activity is likely to be suppressed in fetal FSC progenitors. Given that NF-κB activation in fetal FSCs is essential for SLO development, the animals were expected to lack SLOs. However, all SLOs were preserved in Ccl19-IκBSR mice. Instead, the T cell area was severely disturbed by the lack of CCL21-expressing FSCs, whereas the follicles and associated FSC networks were formed. Fate mapping revealed that IκBSR-expressing cells constituted only a small fraction of stromal compartment outside the follicles. Taken together, our findings indicate an essential role of the canonical NF-κB pathway activity in the development of three FSC subsets common to SLOs and suggest transient or stochastic CCL19 expression in FSC progenitors and a compensatory differentiation program of follicular FSCs.

DOI： 10.4049/jimmunol.1800539

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Allogeneic organ transplants are rejected by the recipient immune system within several days or weeks. However, the rejection process of allogeneic T (allo-T) cells is poorly understood. In this study, using fluorescence-based monitoring and two-photon live imaging in mouse adoptive transfer system, we visualized the fate of allo-T cells in the in vivo environment and showed rapid elimination in secondary lymphoid organs (SLOs). Although i.v. transferred allo-T cells efficiently entered host SLOs, including lymph nodes and the spleen, ∼70% of the cells had disappeared within 24 h. At early time points, allo-T cells robustly migrated in the T cell area, whereas after 8 h, the numbers of arrested cells and cell fragments were dramatically elevated. Apoptotic breakdown of allo-T cells released a large amount of cell debris, which was efficiently phagocytosed and cleared by CD8+ dendritic cells. Rapid elimination of allo-T cells was also observed in nu/nu recipients. Depletion of NK cells abrogated allo-T cell reduction only in a specific combination of donor and recipient genetic backgrounds. In addition, F1 hybrid transfer experiments showed that allo-T cell killing was independent of the missing-self signature typically recognized by NK cells. These suggest the presence of a unique and previously uncharacterized modality of allorecognition by the host immune system. Taken together, our findings reveal an extremely efficient and dynamic process of allogeneic lymphocyte elimination in SLOs, which could not be recapitulated in vitro and is distinct from the rejection of solid organ and bone marrow transplants.

DOI： 10.4049/jimmunol.1700219

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The spatiotemporal regulation of immune responses in the lymph node (LN) depends on its sophisticated tissue architecture, consisting of several subcompartments supported by distinct fibroblastic stromal cells (FSCs). However, the intricate details of stromal structures and associated FSC subsets are not fully understood. Using several gene reporter mice, we sought to discover unrecognized stromal structures and FSCs in the LN. The four previously identified FSC subsets in the cortex are clearly distinguished by the expression pattern of reporters including PDGFRβ, CCL21-ser, and CXCL12. Herein, we identified a unique FSC subset expressing both CCL21-ser and CXCL12 in the deep cortex periphery (DCP) that is characterized by preferential B cell localization. This subset was clearly different from CXCL12highLepRhigh FSCs in the medullary cord, which harbors plasma cells. B cell localization in the DCP was controlled chiefly by CCL21-ser and, to a lesser extent, CXCL12. Moreover, the optimal development of the DCP as well as medulla requires B cells. Together, our findings suggest the presence of a unique microenvironment in the cortex-medulla boundary and offer an advanced view of the multi-layered stromal framework constructed by distinct FSC subsets in the LN.

DOI： 10.3389/fimmu.2018.02196

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

Lymphadenopathy is a frequently observed symptom in systemic lupus erythematosus, although the immunological role of lymph nodes (LNs) in systemic autoimmunity remains largely unknown. Here, we performed comprehensive and systematic analyses of LNs in lupus-prone NZB × NZW F1 (BWF1) mice, demonstrating extensive tissue re-organization of the systemic LNs with follicular expansion, hyper germinal center (GC) formation, atrophy of the paracortical T-cell area and expansion of the medulla in aged BWF1 mice bearing glomerulonephritis. The proportion of B cells was significantly increased in these reactive LNs but not in the spleen, and lymphocyte subsets involved in antibody production, i.e. GC B cells, follicular helper T cells and plasma cells, were elevated. Draining LNs of the affected organs, such as the renal and cervical nodes, showed enhanced tissue re-organization and accumulation of effector lymphocytes, suggesting the presence of a positive feedback loop of regional responses. LN cells isolated from disease-bearing animals produced anti-DNA antibody, indicating activation of autoreactive lymphocytes in situ. The substantial development of disease and LN alterations in mice that received a splenectomy at a young age points to the importance of other secondary lymphoid organs, most likely LNs, for the progression of autoimmune responses independent of the spleen. Taken together, our findings highlight the value of taking LN alterations and activities into consideration for understanding the pathogenesis of systemic autoimmunity.

DOI： 10.1093/intimm/dxx066

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Antigen-specific adhesion between T cells and antigen-presenting cells (APC) during the formation of the immunological synapse (IS) is mediated by LFA-1 and ICAM-1. Here, LFA-1-ICAM-1 interactions were measured at the single-molecule level on supported lipid bilayers. High-affinity binding was detected at low frequencies in the inner peripheral supramolecular activation cluster (SMAC) zone that contained high levels of activated Rap1 and kindlin-3. Rap1 was essential for T cell attachment, whereas deficiencies of ste20-like kinases, Mst1/Mst2, diminished high-affinity binding and abrogated central SMAC (cSMAC) formation with mis-localized kindlin-3 and vesicle transport regulators involved in T cell receptor recycling/releasing machineries, resulting in impaired T cell-APC interactions. We found that NDR1 kinase, activated by the Rap1 signaling cascade through RAPL and Mst1/Mst2, associated with and recruited kindlin-3 to the IS, which was required for high-affinity LFA-1/ICAM-1 binding and cSMAC formation. Our findings reveal crucial roles for Rap1 signaling via NDR1 for recruitment of kindlin-3 and IS organization.

DOI： 10.1128/MCB.00424-16

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

Regulation of thymocyte trafficking plays an important role during thymic selection, but our understanding of the molecular mechanisms underlying these processes is limited. In this study, we demonstrated that class III semaphorin E (sema3e), a guidance molecule during neural and vascular development, directly inhibited Rap1 activation and LFA-1-dependent adhesion through the GTPase-activating protein activity of plexin D1. Sema3e inhibited Rap1 activation of thymocytes in response to chemokines and TCR stimulation, LFA-mediated adhesion, and T cell-APC interactions. Immunological synapse (IS) formation in mature thymocytes on supported lipid bilayers was also attenuated by sema3e. Impaired IS formation was associated with reduced Rap1 activation on the contact surface and cell periphery. Moreover, a significant increase of CD4(+) thymocytes was detected in the medulla of mice with T cell lineage-specific deletion of plexin D1. Two-photon live imaging of thymic explants and slices revealed enhanced Rap1 activation and migration of CD69(+) double-positive and single-positive cells with plexin D1 deficiency. Our results demonstrate that sema3e/plexin D1 modulates IS formation and Ag-scanning activities of thymocytes within thymic tissues.

DOI： 10.4049/jimmunol.1502121

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Mammalian ste-20 like kinase Mst1 plays important roles during apoptosis, proliferation, cell polarity, and migration. Here, we report a novel role of Mst1 for cytotoxic T-cell responses and tumor suppression. The defect of Mst1 caused decreased levels of FoxO, and promoted cytotoxicity in vitro. Mst1(-/-) cytotoxic T cells also exhibited enhanced T-bet expression that was associated with elevated expression levels of IFN and granzyme B. Moreover, Mst1(-/-) cytotoxic T cells suppressed tumor growth in vivo. The data suggest that Mst1 inhibits cytotoxicity via T-bet suppression by FoxO1 and FoxO3a. Thus, Mst1 is a potential therapeutic target for tumor immunotherapy.

DOI： 10.1002/1873-3468.12045

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC ADVANCEMENT SCIENCE  

In lymphocytes, the kinase Mst1 is required for the proper organization of integrins in the plasma membrane at the leading edge of migrating cells, which is critical for lymphocyte trafficking. We found a functional link between the small G protein Rab13 and Mst1 in lymphocyte adhesion and migration. In response to stimulation of T lymphocytes with chemokine, Mst1 promoted phosphorylation of the guanine nucleotide exchange factor DENND1C (differentially expressed in normal and neoplastic cells domain 1C), which activated Rab13. Active Rab13 associated with Mst1 to facilitate the delivery of the integrin LFA-1 (lymphocyte function-associated antigen 1) to the leading edge of lymphocytes. Delivery of LFA-1 involved the recruitment of myosin Va along actin filaments, which extended as a result of the localization of the actin regulatory protein VASP to the cell periphery through phosphorylation of VASP at Ser(157) by Mst1. Inhibition of Rab13 function reduced the adhesion and migration of lymphocytes on ICAM-1 (intercellular adhesion molecule-1), the ligand for LFA-1, and inhibited the formation of a ring-like arrangement of LFA-1 at the contact sites between T cells and antigen-presenting cells. The lymphoid tissues of Rab13-deficient mice had reduced numbers of lymphocytes because of the defective trafficking capability of these cells. These results suggest that Rab13 acts with Mst1 to regulate the spatial distribution of LFA-1 and the motility and trafficking of lymphocytes.

DOI： 10.1126/scisignal.2005199

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals. (C) 2010 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.theriogenology.2010.01.024

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Language：Japanese   Publisher：Japanese Pharmacological Society  

For a long time ago, people have believed that good medicine tastes bitter to the mouth; however, whether the bitter taste itself has therapeutic effects is less studied. Generally, bitter taste is recognized by Type-2 bitter-taste receptors (TAS2Rs) belonging to G-protein coupled receptors and TAS2Rs are localized on taste bud cells of the tongue. Growing evidence suggests that TAS2Rs are expressed not only in the taste bud cells but also in other cells including airway smooth muscle cells, intestinal tuft cells and immune cells. In this study, we show that bitter taste substance-TAS2R axis regulates neutrophil migration. By gene expression analysis, we found that neutrophils express TAS2R126, TAS2R135 and TAS2R143. Next, we observed the effect of TAS2R126/135/143 agonists on neutrophil migration. Although TAS2R135 agonists did not affect neutrophil migration, TAS2R126/143 agonists significantly enhanced CXCL2-induced neutrophil migration. The enhancing effects were not observed in a TAS2R126/143 deficient neutrophil-like cell line. In addition, TAS2R126/143 agonist also promotes neutrophil infiltration into zymosan-injected abdominal cavity. These results suggest that TAS2R126/143 signaling facilitates neutrophil-mediated immune responses and may be targets to promote host defense against infection.

DOI： 10.1254/jpssuppl.94.0_2-y-g3-3

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Language：Japanese   Publisher：The Medical Society of Kansai Medical University  

Cytotoxic CD8 T lymphocytes are the major cells of the immune response to viral infection and tumor rejection. Differentiated CTLs have a large number of modified lysosomes including perforin and granzyme B.

Mammalian ste-20 like kinase Mst1 is abundantly expressed on lymphocytes and plays important roles during apoptosis, proliferation, cell polarity, and migration.

Here, we report the new role of Mst1 for cytotoxic T-cell responses and tumor reduction.

<i>Mst1</i>^–/– cytotoxic T cells also showed enhanced T-bet expression associated with elevated expression levels of IFNγ and granzyme B.

In addition, <i>Mst1</i>^–/– cytotoxic T cells suppressed tumor growth and prolonged overall survival of tumor bearing mice <i>in vivo</i>.<i></i>

Thus, Mst1 is a potential therapeutic target for tumor immunotherapy.

DOI： 10.5361/jkmu.68.9

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Other Link： https://www.jstage.jst.go.jp/article/jkmu/68/0/68_9/_pdf

Language：English   Publisher：(NPO)日本免疫学会  

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Language：Japanese   Publisher：(一社)日本繁殖生物学会  

DOI： 10.14882/jrds.101.0.580.0

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Language：Japanese   Publisher：近畿大学先端技術総合研究所  

マウス胚性幹細胞（mouse embryonic stem cell;mES 細胞）は、各系譜の細胞へと分化可能な分化多能性を有している（1）。この特徴を用いて、様々な分化誘導実験が行なわれてきた。生殖細胞の分化誘導もまた、発生メカニズムの解明に繋がるとして研究の対象となってきた。しかし、受精能を獲得した精子様の細胞は得られたが、同様な卵母細胞様の細胞は得られていない。そこで本実験では、卵巣表面に存在する卵巣上皮細胞（ovarian surface epithelium cells;OSECs）がmES細胞から生殖細胞への分化に関与するかどうか、分子生物学的手法及び免疫化学的手法を用いて未分化マーカー遺伝子であるOct4 遺伝子、生殖細胞関連遺伝子であるMouse vasa homolog（Mvh）、Stella、Fragilis 遺伝子について解析した。その結果、胚様体（Embryoid Body;EB）をOSECs の馴化培養液で接着培養を行った場合、通常の接着培養と比べ、Oct4 遺伝子の発現が維持された。今回の実験系より得られた結果から、OSECs の分泌する液性因子が、mES 細胞から生殖細胞への分化を助長する傾向にあることが示された。

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Language：Japanese   Publisher：近畿大学先端技術総合研究所  

胚性幹細胞（Embryonic Stem Cell ; ES 細胞）は自己複製能と様々な細胞へと分化することの出来る分化多能性を有している細胞である。近年、このES 細胞を用いた再生修復医療の可能性が注目されている。しかし、受精卵からES 細胞を作出するため、移植後に免疫拒絶反応を示すことが示唆されており、ES 細胞を用いた再生修復医療の1 つの課題となっている。そこで、片親性の雌性単為発生胚と雄性発生胚からES 細胞を樹立し、片親性ES 細胞の分化多能性を観察した。

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Language：Japanese   Publisher：(一社)日本繁殖生物学会  

DOI： 10.14882/jrds.100.0.20108.0

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Language：Japanese   Publisher：(一社)日本生殖医学会  

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DOI： 10.14882/jrds.99.0.158.0

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DOI： 10.14882/jrds.99.0.152.0

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