Publications by authors named "Arata Takeuchi"

19 Publications

  • Page 1 of 1

Transdermal entry of yeast components elicits transient B cell-associated responses in skin-draining lymph nodes.

Cell Immunol 2020 09 10;355:104159. Epub 2020 Jul 10.

Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cellimm.2020.104159DOI Listing
September 2020

Essential Role of Canonical NF-κB Activity in the Development of Stromal Cell Subsets in Secondary Lymphoid Organs.

J Immunol 2018 12 5;201(12):3580-3586. Epub 2018 Nov 5.

Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan;

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 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 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.
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http://dx.doi.org/10.4049/jimmunol.1800539DOI Listing
December 2018

A Distinct Subset of Fibroblastic Stromal Cells Constitutes the Cortex-Medulla Boundary Subcompartment of the Lymph Node.

Front Immunol 2018 2;9:2196. Epub 2018 Oct 2.

Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

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 CXCL12LepR 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.
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http://dx.doi.org/10.3389/fimmu.2018.02196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176096PMC
September 2019

Visualizing the Rapid and Dynamic Elimination of Allogeneic T Cells in Secondary Lymphoid Organs.

J Immunol 2018 08 20;201(3):1062-1072. Epub 2018 Jun 20.

Department of Immunology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata 951-8510, Japan;

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 recipients. Depletion of NK cells abrogated allo-T cell reduction only in a specific combination of donor and recipient genetic backgrounds. In addition, F 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.
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http://dx.doi.org/10.4049/jimmunol.1700219DOI Listing
August 2018

Extensively re-organized systemic lymph nodes provide a feasible environment for self-reactivity in lupus-prone NZB × NZW F1 mice.

Int Immunol 2017 12;29(12):567-579

Department of Immunology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

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.
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http://dx.doi.org/10.1093/intimm/dxx066DOI Listing
December 2017

CD4 CTL, a Cytotoxic Subset of CD4 T Cells, Their Differentiation and Function.

Front Immunol 2017 23;8:194. Epub 2017 Feb 23.

Laboratory for Cell Signaling, Department of Immunology, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan; WPI Immunology Frontier Center, Osaka University, Suita, Japan.

CD4 T cells with cytotoxic activity (CD4 CTL) have been observed in various immune responses. These cells are characterized by their ability to secrete granzyme B and perforin and to kill the target cells in an MHC class II-restricted fashion. Although CD4 CTLs were once thought to be an artifact associated with long-term culturing, they have since been identified and shown to play important roles in antiviral and antitumor immunity, as well as in inflammation. Functional characterization of CD4 CTL suggests their potential significance for therapeutic purposes. However, in order to develop effective CD4 CTL therapy it is necessary to understand the differentiation and generation of these cells. Although the mechanisms regulating development of various CD4 Th subsets have been clarified in terms of the cytokine and transcription factor requirement, the CD4 CTL differentiation mechanism remains elusive. These cells are thought to be most closely related to Th1 cells secreting IFNγ and regulated by eomesodermin and/or T-bet transcription factors for their differentiation. However, our studies and those of others have identified CD4 CTLs within other CD4 T cell subsets, including naïve T cells. We have identified class I-restricted T cell-associated molecule as a marker of CD4 CTL and, by using this marker, we detected a subset of naïve T cells that have the potential to differentiate into CD4 CTL. CD4 CTL develops at sites of infections as well as inflammation. In this review, we summarize recent findings about the generation of CD4 CTL and propose a model with several differentiation pathways.
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http://dx.doi.org/10.3389/fimmu.2017.00194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321676PMC
February 2017

CRTAM determines the CD4+ cytotoxic T lymphocyte lineage.

J Exp Med 2016 Jan 22;213(1):123-38. Epub 2015 Dec 22.

Laboratory for Cell Signaling, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa 230-0045, Japan WPI Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan

Naive T cells differentiate into various effector T cells, including CD4(+) helper T cell subsets and CD8(+) cytotoxic T cells (CTL). Although cytotoxic CD4(+) T cells (CD4 +: CTL) also develop from naive T cells, the mechanism of development is elusive. We found that a small fraction of CD4(+) T cells that express class I-restricted T cell-associated molecule (CRTAM) upon activation possesses the characteristics of both CD4(+) and CD8(+) T cells. CRTAM(+) CD4(+) T cells secrete IFN-γ, express CTL-related genes, such as eomesodermin (Eomes), Granzyme B, and perforin, after cultivation, and exhibit cytotoxic function, suggesting that CRTAM(+) T cells are the precursor of CD4(+)CTL. Indeed, ectopic expression of CRTAM in T cells induced the production of IFN-γ, expression of CTL-related genes, and cytotoxic activity. The induction of CD4(+)CTL and IFN-γ production requires CRTAM-mediated intracellular signaling. CRTAM(+) T cells traffic to mucosal tissues and inflammatory sites and developed into CD4(+)CTL, which are involved in mediating protection against infection as well as inducing inflammatory response, depending on the circumstances, through IFN-γ secretion and cytotoxic activity. These results reveal that CRTAM is critical to instruct the differentiation of CD4(+)CTL through the induction of Eomes and CTL-related gene.
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http://dx.doi.org/10.1084/jem.20150519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710199PMC
January 2016

Bach2 maintains T cells in a naive state by suppressing effector memory-related genes.

Proc Natl Acad Sci U S A 2013 Jun 10;110(26):10735-40. Epub 2013 Jun 10.

Laboratory of Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan.

The transcriptional repressor BTB and CNC homology 2 (Bach2) is thought to be mainly expressed in B cells with specific functions such as class switch recombination and somatic hypermutation, but its function in T cells is not known. We found equal Bach2 expression in T cells and analyzed its function using Bach2-deficient (-/-) mice. Although T-cell development was normal, numbers of peripheral naive T cells were decreased, which rapidly produced Th2 cytokines after TCR stimulation. Bach2(-/-) naive T cells highly expressed genes related to effector-memory T cells such as CCR4, ST-2 and Blimp-1. Enhanced expression of these genes induced Bach2(-/-) naive T cells to migrate toward CCR4-ligand and respond to IL33. Forced expression of Bach2 restored the expression of these genes. Using Chromatin Immunoprecipitation (ChIP)-seq analysis, we identified S100 calcium binding protein a, Heme oxigenase 1, and prolyl hydroxylase 3 as Bach2 direct target genes, which are highly expressed in effector-memory T cells. These findings indicate that Bach2 suppresses effector memory-related genes to maintain the naive T-cell state and regulates generation of effector-memory T cells.
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http://dx.doi.org/10.1073/pnas.1306691110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696756PMC
June 2013

CRTAM confers late-stage activation of CD8+ T cells to regulate retention within lymph node.

J Immunol 2009 Oct 14;183(7):4220-8. Epub 2009 Sep 14.

Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan.

In vivo immune response is triggered in the lymph node, where lymphocytes for entry into, retention at, and migration to effector sites are dynamically regulated. The molecular mechanism underlying retention regulation is the key to elucidating in vivo regulation of immune response. In this study, we describe the function of the adhesion molecule class I-restricted T cell-associated molecule (CRTAM) in regulating CD8+ T cell retention within the lymph node and eventually effector function. We previously identified CRTAM as a receptor predominantly expressed on activated CD8+ T cells, and nectin-like molecule-2 (Necl2) as its ligand. In vivo function of CRTAM-Necl2 interaction was analyzed by generating CRTAM(-/-) mice. CRTAM(-/-) mice exhibited reduced protective immunity against viral infection and impaired autoimmune diabetes induction in vivo. Although Ag-specific CRTAM(-/-) CD8+ T cells showed normal CTL functions in vitro, their number in the draining lymph node was reduced. Because CRTAM+ T cells bound efficiently to Necl2-expressing CD8+ dendritic cells (DCs) that reside in T cell area of lymph node, CRTAM may induce retention by binding to CD8+ DCs at the late stage of activation before proliferation. The CRTAM-mediated late interaction with DCs induced retention of activated CD8+ T cells in an Ag-independent fashion, and this possibly resulted in effective CTL development in the draining lymph node.
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http://dx.doi.org/10.4049/jimmunol.0901248DOI Listing
October 2009

Cell type-specific regulation of ITAM-mediated NF-kappaB activation by the adaptors, CARMA1 and CARD9.

J Immunol 2008 Jul;181(2):918-30

Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama City, Kanagawa, Japan.

Activating NK cell receptors transduce signals through ITAM-containing adaptors, including FcRgamma and DAP12. Although the caspase recruitment domain (CARD)9-Bcl10 complex is essential for FcRgamma/DAP12-mediated NF-kappaB activation in myeloid cells, its involvement in NK cell receptor signaling is unknown. Herein we show that the deficiency of CARMA1 or Bcl10, but not CARD9, resulted in severe impairment of cytokine/chemokine production mediated by activating NK cell receptors due to a selective defect in NF-kappaB activation, whereas cytotoxicity mediated by the same receptors did not require CARMA1-Bcl10-mediated signaling. IkappaB kinase (IKK) activation by direct protein kinase C (PKC) stimulation with PMA plus ionomycin (P/I) was abrogated in CARMA1-deficient NK cells, similar to T and B lymphocytes, whereas CARD9-deficient dendritic cells (DCs) exhibited normal P/I-induced IKK activation. Surprisingly, CARMA1 deficiency also abrogated P/I-induced IKK activation in DCs, indicating that CARMA1 is essential for PKC-mediated NF-kappaB activation in all cell types, although the PKC-CARMA1 axis is not used downstream of myeloid ITAM receptors. Consistently, PKC inhibition abrogated ITAM receptor-mediated activation only in NK cells but not in DCs, suggesting PKC-CARMA1-independent, CARD9-dependent ITAM receptor signaling in myeloid cells. Conversely, the overexpression of CARD9 in CARMA1-deficient cells failed to restore the PKC-mediated NF-kappaB activation. Thus, NF-kappaB activation signaling through ITAM receptors is regulated by a cell type-specific mechanism depending on the usage of adaptors CARMA1 and CARD9, which determines the PKC dependence of the signaling.
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http://dx.doi.org/10.4049/jimmunol.181.2.918DOI Listing
July 2008

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function.

Eur J Immunol 2007 Nov;37(11):3197-207

Department of Immunochemistry, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.
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http://dx.doi.org/10.1002/eji.200737052DOI Listing
November 2007

The adaptor protein CARD9 is essential for the activation of myeloid cells through ITAM-associated and Toll-like receptors.

Nat Immunol 2007 Jun 7;8(6):619-29. Epub 2007 May 7.

Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan.

Immunoreceptor tyrosine-based activation motifs (ITAMs) are crucial in antigen receptor signaling in acquired immunity. Although receptors associated with the ITAM-bearing adaptors FcRgamma and DAP12 on myeloid cells have been suggested to activate innate immune responses, the mechanism coupling those receptors to 'downstream' signaling events is unclear. The CARMA1-Bcl-10-MALT1 complex is critical for the activation of transcription factor NF-kappaB in lymphocytes but has an unclear function in myeloid cells. Here we report that deletion of the gene encoding the Bcl-10 adaptor-binding partner CARD9 resulted in impaired myeloid cell activation of NF-kappaB signaling by several ITAM-associated receptors. Moreover, CARD9 was required for Toll-like receptor-induced activation of dendritic cells through the activation of mitogen-activated protein kinases. Although Bcl10-/- and Card9-/- mice had similar signaling impairment in myeloid cells, Card11-/- (CARMA1-deficient) myeloid cell responses were normal, and although Card11-/- lymphocytes were defective in antigen receptor-mediated activation, Card9-/- lymphocytes were not. Thus, the activation of lymphoid and myeloid cells through ITAM-associated receptors or Toll-like receptors is regulated by CARMA1-Bcl-10 and CARD9-Bcl-10, respectively.
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http://dx.doi.org/10.1038/ni1466DOI Listing
June 2007

A critical role for the innate immune signaling molecule IRAK-4 in T cell activation.

Science 2006 Mar;311(5769):1927-32

Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan.

IRAK-4 is a protein kinase that is pivotal in mediating signals for innate immune responses. Here, we report that IRAK-4 signaling is also essential for eliciting adaptive immune responses. Thus, in the absence of IRAK-4, in vivo T cell responses were significantly impaired. Upon T cell receptor stimulation, IRAK-4 is recruited to T cell lipid rafts, where it induces downstream signals, including protein kinase C activation through the association with Zap70. This signaling pathway was found to be required for optimal activation of nuclear factor kappaB. Our findings suggest that T cells use this critical regulator of innate immunity for the development of acquired immunity, suggesting that IRAK-4 may be involved in direct signal cross talk between the two systems.
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http://dx.doi.org/10.1126/science.1124256DOI Listing
March 2006

DNA polymerase theta contributes to the generation of C/G mutations during somatic hypermutation of Ig genes.

Proc Natl Acad Sci U S A 2005 Sep 19;102(39):13986-91. Epub 2005 Sep 19.

Laboratories for Antigen Receptor Diversity, Cell Signaling, and Developmental Genetics, Research Center for Allergy and Immunology, RIKEN Yokohama Institute, Yokohama, Kanagawa 230-0045, Japan.

Somatic hypermutation of Ig variable region genes is initiated by activation-induced cytidine deaminase; however, the activity of multiple DNA polymerases is required to ultimately introduce mutations. DNA polymerase eta (Poleta) has been implicated in mutations at A/T, but polymerases involved in C/G mutations have not been identified. We have generated mutant mice expressing DNA polymerase (Pol) specifically devoid of polymerase activity. Compared with WT mice, Polq-inactive (Polq, the gene encoding Pol) mice exhibited a reduced level of serum IgM and IgG1. The mutant mice mounted relatively normal primary and secondary immune responses to a T-dependent antigen, but the production of high-affinity specific antibodies was partially impaired. Analysis of the J(H)4 intronic sequences revealed a slight reduction in the overall mutation frequency in Polq-inactive mice. Remarkably, although mutations at A/T were unaffected, mutations at C/G were significantly decreased, indicating an important, albeit not exclusive, role for Pol activity. The reduction of C/G mutations was particularly focused on the intrinsic somatic hypermutation hotspots and both transitions and transversions were similarly reduced. These findings, together with the recent observation that Pol efficiently catalyzes the bypass of abasic sites, lead us to propose that Pol introduces mutations at C/G by replicating over abasic sites generated via uracil-DNA glycosylase.
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http://dx.doi.org/10.1073/pnas.0505636102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1236561PMC
September 2005

Heterotypic interaction of CRTAM with Necl2 induces cell adhesion on activated NK cells and CD8+ T cells.

Int Immunol 2005 Sep 9;17(9):1227-37. Epub 2005 Aug 9.

Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan.

NK cells and CD8+ T cells exhibit cytotoxicity and cytokine production upon recognizing target cells through cell-cell interaction. We screened the molecules involved in the recognition and regulation of these cells using cDNA subtraction between naive and activated NK cells. We identified class I-restricted T cell-associated molecule (CRTAM), a two Ig domain-bearing surface receptor, as a molecule rapidly and transiently expressed on NK cells and CD8+ T cells upon activation. CRTAM is expressed as a dimer on the cell surface, and its expression is transcriptionally regulated. Using an expression-cloning system, we then further identified Nectin-like (Necl) molecule 2, a three Ig domain-containing receptor, as a ligand of CRTAM. While Necl2 mediates homotypic interaction, CRTAM interacts with Necl2 but not with CRTAM itself. The heterotypic CRTAM-Necl2 interaction has a higher affinity than the homotypic Necl2 interaction. Although there was no clear alteration in the cytotoxic function of the NK cells and CD8+ T cells against the Necl2-expressing target cells, T cells expressing CRTAM tightly bound to Necl2-expressing cells. CRTAM+ cells did not induce homotypic aggregation but they did exert strong heterotypic binding with Necl2+ cells, which was inhibited by the addition of the CRTAM-Ig fusion protein. These results suggest that the heterotypic interaction between CRTAM and Necl2 plays an important role in the adhesion, interaction or migration of NK cells and CD8+ T cells upon stimulation.
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http://dx.doi.org/10.1093/intimm/dxh299DOI Listing
September 2005

Defective function of GABA-containing synaptic vesicles in mice lacking the AP-3B clathrin adaptor.

J Cell Biol 2004 Oct 18;167(2):293-302. Epub 2004 Oct 18.

RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan.

AP-3 is a member of the adaptor protein (AP) complex family that regulates the vesicular transport of cargo proteins in the secretory and endocytic pathways. There are two isoforms of AP-3: the ubiquitously expressed AP-3A and the neuron-specific AP-3B. Although the physiological role of AP-3A has recently been elucidated, that of AP-3B remains unsolved. To address this question, we generated mice lacking mu3B, a subunit of AP-3B. mu3B-/- mice suffered from spontaneous epileptic seizures. Morphological abnormalities were observed at synapses in these mice. Biochemical studies demonstrated the impairment of gamma-aminobutyric acid (GABA) release because of, at least in part, the reduction of vesicular GABA transporter in mu3B-/- mice. This facilitated the induction of long-term potentiation in the hippocampus and the abnormal propagation of neuronal excitability via the temporoammonic pathway. Thus, AP-3B plays a critical role in the normal formation and function of a subset of synaptic vesicles. This work adds a new aspect to the pathogenesis of epilepsy.
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http://dx.doi.org/10.1083/jcb.200405032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172536PMC
October 2004

Overexpression of human acyl-CoA thioesterase upregulates peroxisome biogenesis.

Exp Cell Res 2004 Jul;297(1):127-41

Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan.

The biological functions of human acyl-CoA thioesterase III (ACTEIII/PTE-1), initially identified as an HIV-1 Nef binding protein, have remained unclear. We report herein that the stable overexpression of ACTEIII/PTE-1 in human and murine T-cell lines resulted in an increase in both peroxisome number and lipid droplet formation in a manner dependent on the amount of the protein. Peroxisome proliferation was evidenced by immunofluorescence staining for catalase, a peroxisome marker protein, as well as by direct peroxisome enumeration on electron micrographs. Consistently, the amount of catalase was elevated as the amount of ACTEIII/PTE-1 was increased. ACTEIII/PTE-1 mutants with reduced enzymatic activity or with the defect in peroxisome localization did not induce peroxisome proliferation, indicating that peroxisome proliferation was mediated by metabolites generated by ACTEIII/PTE-1 within peroxisomes. Finally, thymocytes isolated from a T-cell-specific ACTEIII/PTE-1 transgenic mouse as well as human and murine cell lines of lymphoid and non-lymphoid origins exhibited a similar proliferation of peroxisomes. Thus, ACTEIII/PTE-1 may be involved in the metabolic regulation of peroxisome proliferation.
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http://dx.doi.org/10.1016/j.yexcr.2004.02.029DOI Listing
July 2004

NFAM1, an immunoreceptor tyrosine-based activation motif-bearing molecule that regulates B cell development and signaling.

Proc Natl Acad Sci U S A 2004 May 13;101(21):8126-31. Epub 2004 May 13.

Department of Molecular Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan.

A functional cDNA cloning system was developed by using a retrovirus library encoding CD8-chimeric proteins and a nuclear factor of activated T cells (NFAT)-GFP reporter cell line to identify molecules inducing NFAT activation. By using this strategy, NFAT activating molecule 1 (NFAM1) was cloned as an immunoreceptor tyrosine-based activation motif (ITAM)-bearing cell surface molecule belonging to the Ig superfamily and is predominantly expressed in spleen B and T cells. NFAM1 crosslinking induced ITAM phosphorylation, ZAP-70/Syk recruitment, NFAT activation, and cytokine production. In vivo overexpression of NFAM1 in bone marrow chimeras and transgenic mice induced severe impairment of early B cell development in an ITAM-dependent manner. In NFAM1-expressing B cells, B cell antigen receptor stimulation induced NFAM1 translocation to lipid raft, and NFAM1 co-crosslinking augmented B cell antigen receptor signaling. The results suggest that NFAM1 modulates B cell signaling through its ITAM, which regulates B cell development.
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http://dx.doi.org/10.1073/pnas.0401119101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC419568PMC
May 2004

Cutting edge: negative regulation of immune synapse formation by anchoring lipid raft to cytoskeleton through Cbp-EBP50-ERM assembly.

J Immunol 2002 Jan;168(2):541-4

Department of Molecular Genetics and General Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan.

Ag recognition by T lymphocytes induces immune synapse formation and recruitment of signaling molecules into a lipid raft. Cbp/PAG is a Csk-associated membrane adapter protein exclusively localized in a lipid raft. We identified NHERF/EBP50 as a Cbp-binding molecule, which connects the membrane raft and cytoskeleton by binding to both Cbp through its PDZ domain and ezrin-radixin-moesin through the C terminus. Overexpression of Cbp reduced the mobility of the raft on the cell surface of unstimulated T cells and prevented synapse formation and subsequent T cell activation, whereas a mutant incapable of EBP50 binding restored both synapse formation and activation. These results suggest that anchoring of lipid raft to the cytoskeleton through Cbp-EBP50-ezrin-radixin-moesin assembly regulates membrane dynamism for synapse formation and T cell activation.
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http://dx.doi.org/10.4049/jimmunol.168.2.541DOI Listing
January 2002