Stimulation of the farnesoid X receptor promotes M2 macrophage polarization.

This skewed polarization towards M2 macrophages by an FXR agonist was accompanied by increased expression of signaling molecules related to the retinoic acid receptor. Inhibition of the retinoic acid receptor suppressed FXR agonist-mediated M2 macrophage polarization, indicating that this polarization was, at least, partly dependent on the retinoic acid receptor pathway. These data demonstrate that FXR has a role in polarization toward M2 macrophages and suggest a possible therapeutic potential of FXR agonists in M2 macrophage-related conditions.


Highly sensitive detection of nucleocapsid protein from SARS-CoV-2 using a near-infrared surface plasmon resonance sensing system


Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates.

mRNA and lipid nanoparticles have emerged as powerful systems for the preparation of vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The emergence of novel variants or the necessity of cold chain logistics for approved mRNA vaccines undermines the investigation of next-generation systems that could preserve both potency and stability. However, the correlation between lipid nanoparticle composition and activity is not fully explored. Here, we screened a panel of ionizable lipids

and identified lead lipid nanoparticles with a branched-tail lipid structure. Buffer optimization allowed the determination of lyophilization conditions, where lipid nanoparticle-encapsulated mRNA encoding SARS-CoV-2 spike protein could induce robust immunogenicity in mice after 1 month of storage at 5°C and 25°C. Intramuscularly injected lipid nanoparticles distributed in conventional dendritic cells in mouse lymph nodes induced balanced T helper (Th) 1/Th2 responses against SARS-CoV-2 spike protein. In nonhuman primates, two doses of 10 or 100 μg of mRNA induced higher spike-specific binding geometric mean titers than those from a panel of SARS-CoV-2-convalescent human sera. Immunized sera broadly inhibited the viral entry receptor angiotensin-converting enzyme 2 (ACE2) from binding to the spike protein in all six strains tested, including variants of concern. These results could provide useful information for designing next-generation mRNA vaccines.


Major target for UV-induced complete loss of HIV-1 infectivity: A model study of single-stranded RNA enveloped viruses


AFF3, a susceptibility factor for autoimmune diseases, is a molecular facilitator of immunoglobulin class switch recombination.

in human B cells. These findings demonstrate that AFF3 directly regulates CSR by facilitating the recruitment of AID to the switch regions.


Markers of Memory CD8 T Cells Depicting the Effect of the BNT162b2 mRNA COVID-19 Vaccine in Japan.

BNT162b2, a nucleoside-modified mRNA vaccine for SARS-CoV-2 spike glycoprotein (S), provides approximately 95% efficacy for preventing COVID-19. However, it remains unclear how effectively memory CD8+ T cells are generated and which genetic and environmental factors affect the generation and function of memory CD8+ T cells elicited by this vaccine. Here, we investigated the frequency and functions of memory CD8+ T cells 3 weeks after the second vaccination in the Japanese population. Using a peptide-MHC pentamer, we detected an increased number of memory CD8+ T cells together with increased serum anti-S protein antibody in females compared with that in males, but the frequency of pentamer-positive cells was not positively correlated with antibody titers. Memory precursor effector cells (KLRG1-CD127+) among both CD8+ cells and pentamer+ cells and effector cells (CD38-HLA-DR+) among pentamer+ cells were more abundant in females than in males. Upon S protein-mediated stimulation of T cells, the intensity of CD107a and granzyme B expression was increased in females compared with that in males, indicating stronger memory CD8+ T cell responses in females than in males. Our studies showed that the BNT162b2 vaccine elicits increased memory CD8+ T cell proliferation and secondary CTL responses in females compared with those in males in the Japanese population. These findings provide an important basis for the distinct sex difference in cellular immune responses to mRNA vaccination and suggest that memory precursor effector cells can be one of markers to evaluate and boost cellular immunity induced by BNT162b2.


Blockade of the CXCR3/CXCL10 axis ameliorates inflammation caused by immunoproteasome dysfunction.

Immunoproteasomes regulate the degradation of ubiquitin-coupled proteins and generate peptides that are preferentially presented by MHC class I. Mutations in immunoproteasome subunits lead to immunoproteasome dysfunction, which causes proteasome-associated autoinflammatory syndromes (PRAAS) characterized by nodular erythema and partial lipodystrophy. It remains unclear, however, how immunoproteasome dysfunction leads to inflammatory symptoms. Here, we established mice harboring a mutation in Psmb8 (Psmb8-KI mice) and addressed this question. Psmb8-KI mice showed higher susceptibility to imiquimod-induced skin inflammation (IMS). Blockade of IL-6 or TNF-α partially suppressed IMS in both control and Psmb8-KI mice, but there was still more residual inflammation in the Psmb8-KI mice than in the control mice. DNA microarray analysis showed that treatment of J774 cells with proteasome inhibitors increased the expression of the Cxcl9 and Cxcl10 genes. Deficiency in Cxcr3, the gene encoding the receptor of CXCL9 and CXCL10, in control mice did not change IMS susceptibility, while deficiency in Cxcr3 in Psmb8-KI mice ameliorated IMS. Taken together, these findings demonstrate that this mutation in Psmb8 leads to hyperactivation of the CXCR3 pathway, which is responsible for the increased susceptibility of Psmb8-KI mice to IMS. These data suggest the CXCR3/CXCL10 axis as a new molecular target for treating PRAAS.


Ultrasensitive detection of SARS-CoV-2 nucleocapsid protein using large gold nanoparticle-enhanced surface plasmon resonance.

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.


Genetics and animal models of familial pulmonary fibrosis.

Pulmonary fibrosis is caused by the interplay between genetic and environmental factors. Recent studies have revealed various genes associated with idiopathic pulmonary fibrosis, as well as the causative genes for familial pulmonary fibrosis. Although increased death or dysfunction of type 2 alveolar epithelial (AT2) cells has been detected in lung specimens from pulmonary fibrosis patients, it remains unclear whether and how AT2 cell death or dysfunction is responsible for the progression of pulmonary fibrosis. A recent study showed that increased AT2 cell necroptosis is the initial event in pulmonary fibrosis by analyzing patients with familial pulmonary fibrosis and an animal model that harbors the same mutation as patients. The contribution of AT2 cell necroptosis to the pathogenesis of pulmonary fibrosis has not been identified in animal model studies, which validates the effectiveness of genetic analysis of familial diseases to uncover unknown pathogeneses. Thus, further extensive genetic studies of pulmonary fibrosis along with functional studies based on genetic analysis will be crucial not only in elucidating the precise disease process but also, ultimately, in identifying novel treatment strategies for both familial and non-familial pulmonary fibrosis.


Necroptosis protects against exacerbation of acute pancreatitis.

mice exhibit weight loss, edematous pancreatitis, necrotizing pancreatitis, and acinar cell dedifferentiation in response to tissue damage. Genetic deletion of Mlkl resulted in downregulation of the antiapoptotic genes Bclxl and Cflar in association with increases in the numbers of apoptotic cells, as detected by TUNEL assay. These findings suggest that RIPK3 and MLKL-mediated necroptosis exerts protective effects in AP and caution against the use of necroptosis inhibitors for AP treatment.


Quantitative evaluation of SARS-CoV-2 inactivation using a deep ultraviolet light-emitting diode.

for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.


Distinct Roles of IL-1 and IL-18 in NLRC4-Induced Autoinflammation.

The NLRC4 inflammasome assembles in response to detection of bacterial invasion, and NLRC4 activation leads to the production of IL-1 and IL-18 together with pyroptosis-mediated cell death. Missense activating mutations in cause autoinflammatory disorders whose symptoms are distinctly dependent on the site of mutation and other aspects of the genetic background. To determine the involvement of IL-1 and IL-18 in the inflammation induced by mutation, we depleted IL-1 , IL-18, or both cytokines in Nlrc4-transgenic mice in which mutant is expressed under the MHC class II promoter (Nlrc4-H443P-Tg mice). The deletion of the or gene in Nlrc4-H443P-Tg mice reduced the neutrophil numbers in the spleen, and mice with deletion of both genes had an equivalent number of neutrophils compared to wild-type mice. Deletion of ameliorated but did not eliminate bone marrow hyperplasia, while mice deficient in showed no bone marrow hyperplasia. In contrast, tail bone deformity remained in the presence of deficiency, but deficiency completely abolished bone deformity. The decreased bone density in Nlrc4-H443P-Tg mice was counteracted by but not deficiency. Our results demonstrate the distinct effects of IL-1 and IL-18 on NLRC4-induced inflammation among tissues, which suggests that blockers for each cytokine should be utilized depending on the site of inflammation.


A homozygous SFTPA1 mutation drives necroptosis of type II alveolar epithelial cells in patients with idiopathic pulmonary fibrosis.

promotes necroptosis of AEII cells through JNK-mediated up-regulation of Ripk3, highlighting the necroptosis pathway as a therapeutic target for IPF.


Dysregulation of immunoproteasomes in autoinflammatory syndromes.

Immunoproteasomes degrade ubiquitin-coupled proteins and play a role in creating peptides for presentation by MHC class I proteins. Studies of gene-deficient mice, in which each immunoproteasomal subunit was affected, have demonstrated that dysfunction of immunoproteasomes leads to immunodeficiency, i.e. reduced expression of MHC class I and attenuation of CD8 T-cell responses. Recent studies, however, have uncovered a new type of autoinflammatory syndrome characterized by fever, nodular erythema and progressive partial lipodystrophy that is caused by genetic mutations in immunoproteasome subunits. These mutations disturbed the assembly of immunoproteasomes, which led to reduced proteasomal activity and thus accumulation of ubiquitin-coupled proteins. Those findings suggest that immunoproteasomes function as anti-inflammatory machinery in humans. The discovery of a new type of autoinflammatory syndrome caused by dysregulated immunoproteasomes provides novel insights into the important roles of immunoproteasomes in inflammation as well as the spectrum of autoinflammatory diseases.


The Significance of MicroRNA-449a and Its Potential Target HDAC1 in Patients With Colorectal Cancer.

miR-449a level might be a prognostic indicator for colorectal cancer and miR-449a might regulate HDAC1 expression.


Regulation of membrane phospholipid asymmetry by Notch-mediated flippase expression controls the number of intraepithelial TCRαβ+CD8αα+ T cells.

Intestinal intraepithelial lymphocytes (IELs) expressing CD8αα on αβ T cells (TCRαβ+CD8αα+ IELs) have suppressive capabilities in enterocolitis, but the mechanism that maintains homeostasis and cell number is not fully understood. Here, we demonstrated that the number of TCRαβ+CD8αα+ IELs was severely reduced in mice lacking recombination signal binding protein for immunoglobulin kappa J region (Rbpj) or Notch1 and Notch2 in T cells. Rbpj-deficient TCRαβ+CD8αα+ IELs expressed low levels of Atp8a2, which encodes a protein with flippase activity that regulates phospholipid asymmetry of plasma membrane such as flipping phosphatidylserine in the inner leaflet of plasma membrane. Rbpj-deficient TCRαβ+CD8αα+ IELs cannot maintain phosphatidylserine in the inner leaflet of the plasma membrane. Furthermore, depletion of intestinal macrophages restored TCRαβ+CD8αα+ IELs in Rbpj-deficient mice, suggesting that exposure of phosphatidylserine on the plasma membrane in Rbpj-deficient TCRαβ+CD8αα+ IELs acts as an "eat-me" signal. Together, these results revealed that Notch-Atp8a2 is a fundamental regulator for IELs and highlighted that membrane phospholipid asymmetry controlled by Notch-mediated flippase expression is a critical determinant in setting or balancing the number of TCRαβ+CD8αα+ IELs.


T Cells and Antitumor Immunity by Notch Signaling.

T cells and discuss issues and implications for its use in antitumor immunity.


Slc3a2 Mediates Branched-Chain Amino-Acid-Dependent Maintenance of Regulatory T Cells.

Foxp3(+) regulatory T (Treg) cells, which suppress immune responses, are highly proliferative in vivo. However, it remains unclear how the active replication of Treg cells is maintained in vivo. Here, we show that branched-chain amino acids (BCAAs), including isoleucine, are required for maintenance of the proliferative state of Treg cells via the amino acid transporter Slc3a2-dependent metabolic reprogramming. Mice fed BCAA-reduced diets showed decreased numbers of Foxp3(+) Treg cells with defective in vivo proliferative capacity. Mice lacking Slc3a2 specifically in Foxp3(+) Treg cells showed impaired in vivo replication and decreased numbers of Treg cells. Slc3a2-deficient Treg cells showed impaired isoleucine-induced activation of the mTORC1 pathway and an altered metabolic state. Slc3a2 mutant mice did not show an isoleucine-induced increase of Treg cells in vivo and exhibited multi-organ inflammation. Taken together, these findings demonstrate that BCAA controls Treg cell maintenance via Slc3a2-dependent metabolic regulation.


SIRP(+) dendritic cells regulate homeostasis of fibroblastic reticular cells via TNF receptor ligands in the adult spleen.

In secondary lymphoid organs, development and homeostasis of stromal cells such as podoplanin (Pdpn)-positive fibroblastic reticular cells (FRCs) are regulated by hematopoietic cells, but the cellular and molecular mechanisms of such regulation have remained unclear. Here we show that ablation of either signal regulatory protein (SIRP), an Ig superfamily protein, or its ligand CD47 in conventional dendritic cells (cDCs) markedly reduced the number of CD4(+) cDCs as well as that of Pdpn(+) FRCs and T cells in the adult mouse spleen. Such ablation also impaired the survival of FRCs as well as the production by CD4(+) cDCs of tumor necrosis factor receptor (TNFR) ligands, including TNF-, which was shown to promote the proliferation and survival of Pdpn(+) FRCs. CD4(+) cDCs thus regulate the steady-state homeostasis of FRCs in the adult spleen via the production of TNFR ligands, with the CD47-SIRP interaction in cDCs likely being indispensable for such regulation.

Proceedings of the National Academy of Sciences of the United States of America


MicroRNA-449a deficiency promotes colon carcinogenesis.

MicroRNAs have broad roles in tumorigenesis and cell differentiation through regulation of target genes. Notch signaling also controls cell differentiation and tumorigenesis. However, the mechanisms through which Notch mediates microRNA expression are still unclear. In this study, we aimed to identify microRNAs regulated by Notch signaling. Our analysis found that microRNA-449a (miR-449a) was indirectly regulated by Notch signaling. Although miR-449a-deficient mice did not show any Notch-dependent defects in immune cell development, treatment of miR-449a-deficient mice with azoxymethane (AOM) or dextran sodium sulfate (DSS) increased the numbers and sizes of colon tumors. These effects were associated with an increase in intestinal epithelial cell proliferation following AOM/DSS treatment. In patients with colon cancer, miR-449a expression was inversely correlated with disease-free survival and histological scores and was positively correlated with the expression of MLH1 for which loss-of function mutations have been shown to be involved in colon cancer. Colon tissues of miR-449a-deficient mice showed reduced Mlh1 expression compared with those of wild-type mice. Thus, these data suggested that miR-449a acted as a key regulator of colon tumorigenesis by controlling the proliferation of intestinal epithelial cells. Additionally, activation of miR-449a may represent an effective therapeutic strategy and prognostic marker in colon cancer.


Notch Balances Th17 and Induced Regulatory T Cell Functions in Dendritic Cells by Regulating Aldh1a2 Expression.

Dendritic cells (DCs) are important for adaptive immune responses through the activation of T cells. The molecular interplay between DCs and T cells determines the magnitude of T cell responses or outcomes of functional differentiation of T cells. In this study, we demonstrated that DCs in mice that are Rbpj deficient in CD11c(+) cells (Rbpj(-/-) mice) promoted the differentiation of IL-17A-producing Th17 cells. Rbpj-deficient DCs expressed little Aldh1a2 protein that is required for generating retinoic acid. Those DCs exhibited a reduced ability for differentiating regulatory T cells induced by TGF-. Rbpj protein directly regulated Aldh1a2 transcription by binding to its promoter region. The overexpression of Aldh1a2 in Rbpj-deficient DCs negated their Th17-promoting ability. Transfer of naive CD4(+) T cells into Rag1-deficient Rbpj(-/-) mice enhanced colitis with increased Th17 and reduced induced regulatory T cells (iTreg) compared with control Rag1-deficient mice. The cotransfer of iTreg and naive CD4(+) T cells into Rag1-deficient Rbpj(-/-) mice improved colitis compared with transfer of naive CD4(+) T cell alone. Furthermore, cotransfer of DCs from Rbpj(-/-) mice that overexpressed Aldh1a2 or Notch-stimulated DCs together with naive CD4(+) T cells into Rbpj(-/-)Rag1-deficient mice led to reduced colitis with increased iTreg numbers. Therefore, our studies identify Notch signaling in DCs as a crucial balancer of Th17/iTreg, which depends on the direct regulation of Aldh1a2 transcription in DCs.


Survival of mature T cells depends on signaling through HOIP.

T cell development in the thymus is controlled by a multistep process. The NF-B pathway regulates T cell development as well as T cell activation at multiple differentiation stages. The linear ubiquitin chain assembly complex (LUBAC) is composed of Sharpin, HOIL-1L and HOIP, and it is crucial for regulating the NF-B and cell death pathways. However, little is known about the roles of LUBAC in T-cell development and activation. Here, we show that in T-HOIP(linear) mice lacking the ubiquitin ligase activity of LUBAC, thymic CD4(+) or CD8(+) T cell numbers were markedly reduced with severe defects in NKT cell development. HOIP(linear) CD4(+) T cells failed to phosphorylate IB and JNK through T cell receptor-mediated stimulation. Mature CD4(+) and CD8(+) T cells in T-HOIP(linear) mice underwent apoptosis more rapidly than control T cells, and it was accompanied by lower CD127 expression on CD4(+)CD24(low) and CD8(+)CD24(low) T cells in the thymus. The enforced expression of CD127 in T-HOIP(linear) thymocytes rescued the development of mature CD8(+) T cells. Collectively, our results showed that LUBAC ligase activity is key for the survival of mature T cells, and suggest multiple roles of the NF-B and cell death pathways in activating or maintaining T cell-mediated adaptive immune responses.


Transmission of survival signals through Delta-like 1 on activated CD4(+) T cells.

Notch expressed on CD4(+) T cells transduces signals that mediate their effector functions and survival. Although Notch signaling is known to be cis-inhibited by Notch ligands expressed on the same cells, the role of Notch ligands on T cells remains unclear. In this report we demonstrate that the CD4(+) T cell Notch ligand Dll1 transduces signals required for their survival. Co-transfer of CD4(+) T cells from Dll1(-/-) and control mice into recipient mice followed by immunization revealed a rapid decline of CD4(+) T cells from Dll1(-/-) mice compared with control cells. Dll1(-/-) mice exhibited lower clinical scores of experimental autoimmune encephalitis than control mice. The expression of Notch target genes in CD4(+) T cells from Dll1(-/-) mice was not affected, suggesting that Dll1 deficiency in T cells does not affect cis Notch signaling. Overexpression of the intracellular domain of Dll1 in Dll1-deficient CD4(+) T cells partially rescued impaired survival. Our data demonstrate that Dll1 is an independent regulator of Notch-signaling important for the survival of activated CD4(+) T cells, and provide new insight into the physiological roles of Notch ligands as well as a regulatory mechanism important for maintaining adaptive immune responses.


Regulation of monocyte cell fate by blood vessels mediated by Notch signalling.

A population of monocytes, known as Ly6C(lo) monocytes, patrol blood vessels by crawling along the vascular endothelium. Here we show that endothelial cells control their origin through Notch signalling. Using combinations of conditional genetic deletion strategies and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6C(hi) monocytes into Ly6C(lo) monocytes in vivo and in vitro, thereby regulating monocyte cell fate under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1) expressed by a population of endothelial cells that constitute distinct vascular niches in the bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed myeloid cell fate regulation.


Differentiation of preadipocytes and mature adipocytes requires PSMB8.

The differentiation of adipocytes is tightly regulated by a variety of intrinsic molecules and also by extrinsic molecules produced by adjacent cells. Dysfunction of adipocyte differentiation causes lipodystrophy, which impairs glucose and lipid homeostasis. Although dysfunction of immunoproteasomes causes partial lipodystrophy, the detailed molecular mechanisms remain to be determined. Here, we demonstrate that Psmb8, a catalytic subunit for immunoproteasomes, directly regulates the differentiation of preadipocytes and additionally the differentiation of preadipocytes to mature adipocytes. Psmb8(-/-) mice exhibited slower weight gain than wild-type mice, and this was accompanied by reduced adipose tissue volume and smaller size of mature adipocytes compared with controls. Blockade of Psmb8 activity in 3T3-L1 cells disturbed the differentiation to mature adipocytes. Psmb8(-/-) mice had fewer preadipocyte precursors, fewer preadipocytes and a reduced ability to differentiate preadipocytes toward mature adipocytes. Our data demonstrate that Psmb8-mediated immunoproteasome activity is a direct regulator of the differentiation of preadipocytes and their ultimate maturation.


Treatment with DHA/EPA ameliorates atopic dermatitis-like skin disease by blocking LTB4 production.

Atopic dermatitis (AD) is caused by both dysregulated immune responses and an impaired skin barrier. Although leukotriene B4 (LTB4) is involved in tissue inflammation that occurs in several disorders, including AD, therapeutic strategies based on LTB4 inhibition have not been explored. Here we demonstrate that progression of an AD-like skin disease in NC/Nga mice is inhibited when docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) is administered together with FK506. Treatment with DHA/EPA and FK506 decreases the clinical score of dermatitis in NC/Nga mice and lowers local LTB4 concentrations. The treatment also suppressed the infiltration of T cells, B cells, eosinophils and neutrophils, and promoted reduced serum IgE levels. Secretion of IL-13 and IL-17A in CD4(+) T cells was lower in DHA/EPA- and FK506-treated mice than in mice treated with FK506 alone. The inhibition of disease progression induced by DHA/EPA was reversed by local injection of LTB4, suggesting that the therapeutic effect of DHA/EPA is LTB4-dependent. Our results demonstrate that treatment of AD with DHA/EPA is effective for allergic skin inflammation and acts by suppressing LTB4 production. J. Med. Invest. 63: 187-191, August, 2016.

The Journal of Medical Investigation : JMI


An interlaboratory comparison of dosimetry for a multi-institutional radiobiological research project: Observations, problems, solutions and lessons learned.

For the dosimetrically non-compliant laboratories, the relatively high rates of dosing errors were problematic, potentially compromising the quality of ongoing radiobiological research. This dosimetry effort proved to be instructive in establishing rigorous reviews of basic dosimetry protocols ensuring that dosing errors were minimized.

International Journal of Radiation Biology


Notch 1 and Notch 2 synergistically regulate the differentiation and function of invariant NKT cells.

Invariant natural killer T cells are a distinct subset of T cells that exert Janus-like functions. Moreover, Notch signaling is known to have critical roles in the development and functions of T cells. However, it is not known whether Notch signaling contributes to the development or functions of invariant natural killer T cells. Here, we found that CD4-specific gene ablation of Notch 1 and Notch 2 (N1N2(-/-)) increased the number of invariant natural killer T cells in the thymus but decreased them in the liver. N1N2(-/-) mice showed impaired thymic maturation of invariant natural killer T cells from the NK1.1(-)CD44(+) to the NK1.1(+)CD44(+) stage, resulting in accumulation of NK1.1(-)CD44(+) invariant natural killer T cells in the thymus. Upon activation, hepatic invariant natural killer T cells from N1N2(-/-) mice produced lower cytokine levels and increased apoptosis versus wild-type invariant natural killer T cells. Furthermore, Notch 1/Notch 2-deficient, but not wild type, invariant natural killer T cells failed to promote antibody-induced arthritis in CD1d(-/-) mice. Unlike N1N2(-/-) mice, RBP-j(lox) (/) (lox) CD4-Cre mice showed similar percentages and numbers of thymic invariant natural killer T cells to wild-type mice but had defects in their homeostasis, maturation, and cytokine production in the liver. Taken together, our data indicate distinct effects of Notch signaling on invariant natural killer T cells in the thymus and liver, which are at least partly independent of RBP-j in the thymus.


CD98 Heavy Chain Is a Potent Positive Regulator of CD4+ T Cell Proliferation and Interferon- Production In Vivo

Upon their recognition of antigens presented by the MHC, T cell proliferation is vital for clonal expansion and the acquisition of effector functions, which are essential for mounting adaptive immune responses. The CD98 heavy chain (CD98hc, Slc3a2) plays a crucial role in the proliferation of both CD4+ and CD8+ T cells, although it is unclear if CD98hc directly regulates the T cell effector functions that are not linked with T cell proliferation in vivo. Here, we demonstrate that CD98hc is required for both CD4+ T cell proliferation and Th1 functional differentiation. T cell-specific deletion of CD98hc did not affect T cell development in the thymus. CD98hc-deficient CD4+ T cells proliferated in vivo more slowly as compared with control T cells. C57BL/6 mice lacking CD98hc in their CD4+ T cells could not control Leishmania major infections due to lowered IFN- production, even with massive CD4+ T cell proliferation. CD98hc-deficient CD4+ T cells exhibited lower IFN- production compared with wild-type T cells, even when comparing IFN- expression in cells that underwent the same number of cell divisions. Therefore, these data indicate that CD98hc is required for CD4+ T cell expansion and functional Th1 differentiation in vivo, and suggest that CD98hc might be a good target for treating Th1-mediated immune disorders.


Notch controls the survival of memory CD4+ T cells by regulating glucose uptake.

CD4+ T cells differentiate into memory T cells that protect the host from subsequent infection. In contrast, autoreactive memory CD4+ T cells harm the body by persisting in the tissues. The underlying pathways controlling the maintenance of memory CD4+ T cells remain undefined. We show here that memory CD4+ T cell survival is impaired in the absence of the Notch signaling protein known as recombination signal binding protein for immunoglobulin J region (Rbpj). Treatment of mice with a Notch inhibitor reduced memory CD4+ T cell numbers and prevented the recurrent induction of experimental autoimmune encephalomyelitis. Rbpj-deficient CD4+ memory T cells exhibit reduced glucose uptake due to impaired AKT phosphorylation, resulting in low Glut1 expression. Treating mice with pyruvic acid, which bypasses glucose uptake and supplies the metabolite downstream of glucose uptake, inhibited the decrease of autoimmune memory CD4+ T cells in the absence of Notch signaling, suggesting memory CD4+ T cell survival relies on glucose metabolism. Together, these data define a central role for Notch signaling in maintaining memory CD4+ T cells through the regulation of glucose uptake.


An inherited mutation in NLRC4 causes autoinflammation in human and mice.

Autoinflammatory syndromes cause sterile inflammation in the absence of any signs of autoimmune responses. Familial cold autoinflammatory syndrome (FCAS) is characterized by intermittent episodes of rash, arthralgia, and fever after exposure to cold stimuli. We have identified a missense mutation in the NLRC4 gene in patients with FCAS. NLRC4 has been known as a crucial sensor for several Gram-negative intracellular bacteria. The mutation in NLRC4 in FCAS patients promoted the formation of NLRC4-containing inflammasomes that cleave procaspase-1 and increase production of IL-1. Transgenic mice that expressed mutant Nlrc4 under the invariant chain promoter developed dermatitis and arthritis. Inflammation within tissues depended on IL-1-mediated production of IL-17A from neutrophils but not from T cells. Our findings reveal a previously unrecognized link between NLRC4 and a hereditary autoinflammatory disease and highlight the importance of NLRC4 not only in the innate immune response to bacterial infections but also in the genesis of inflammatory diseases.


Differentiation of CD11c+ CX3CR1+ cells in the small intestine requires Notch signaling.

The gastrointestinal tract comes into direct contact with environmental agents, including bacteria, viruses, and foods. Intestine-specific subsets of immune cells maintain gut homeostasis by continuously sampling luminal antigens and maintaining immune tolerance. CD11c(+)CX3CR1(+) cells sample luminal antigens in the small intestine and contribute to the trafficking of bacteria to lymph nodes under dysbiotic conditions. The molecular mechanisms crucial for the differentiation of CD11c(+)CX3CR1(+) cells remain unclear. Here we demonstrate that the Notch1- or Notch2-Rbpj axis is essential for the development of CD11c(+)CX3CR1(+) cells. In mice in which Rbpj or Notch1 and Notch2 were deleted from CD11c(+) cells, there was a deficit of CD11c(+)CX3CR1(+) cells and an accumulation of CD11c(low)CX3CR1(+) cells. The CD11c(low)CX3CR1(+) cells could not differentiate to CD11c(+)CX3CR1(+) cells, suggesting that CD11c(low)CX3CR1(+) cells represent a lineage distinct from CD11c(+)CX3CR1(+) cells. These data indicate that Notch signaling is essential for lineage fixation of intestinal CD11c(+)CX3CR1(+) cells.

Proceedings of the National Academy of Sciences of the United States of America


Abrogation of Rbpj attenuates experimental autoimmune uveoretinitis by inhibiting IL-22-producing CD4+ T cells.

Experimental autoimmune uveoretinitis (EAU) is an organ-specific T cell-mediated disease induced by immunizing mice with interphotoreceptor retinoid binding protein (IRBP). Autoaggressive CD4(+) T cells are the major pathogenic population for EAU. We investigated the contribution of Notch signaling in T cells to EAU pathogenesis because Notch signaling regulates various aspects of CD4(+) T cell functions. Rbpj is required for Notch signaling, and Rbpj deficiency in T cells inhibited EAU disease severity. The amelioration of EAU in T cell-specific Rbpj-deficient mice correlated with low levels of IL-22 production from CD4(+) T cells, although IRBP-specific CD4(+) T cell proliferation and Th17 differentiation were unaffected. Administration of recombinant IL-22 during the late phase, but not the early phase, of EAU increased EAU clinical scores in T cell-specific Rbpj-deficient mice. Notch inhibition in mice immunized with IRBP with a -secretase inhibitor (GSI) suppressed EAU progression, even when GSI was administered as late as 13 days after IRBP immunization. Our data demonstrate that Rbpj/Notch-mediated IL-22 production in T cells has a key pathological role in the late phase of EAU, and suggest that Notch blockade might be a useful therapeutic approach for treating EAU.


CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4(+) T cells.

CD4(+) T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4(+) T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4(+) T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4(+)CD25(-) T cells into Rag2(-/-) mice did not cause colitis accompanied by increasing Foxp3(+) inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4(+)CD25(-) T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4(+) T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.

Biochemical and Biophysical Research Communications


Feasibility of reduced-intensity conditioning followed by unrelated cord blood transplantation for primary hemophagocytic lymphohistiocytosis: a nationwide retrospective analysis in Japan.

A nationwide retrospective analysis was performed on patients who received allogeneic hematopoietic stem cell transplantation for primary or familial hemophagocytic lymphohistiocytosis (HLH) in Japan. The present analysis investigated whether reduced-intensity conditioning (RIC) followed by cord blood transplantation (CBT) (RIC-CBT) is feasible, compared to the outcomes of myeloablative conditioning and bone marrow transplantation. Based on the JSHCT data, 53 patients were analyzed. The overall survival rate (OS) was 65.4 ± 6.6 %. RIC-CBT (n = 13) was not inferior to other methods. Patients with a performance status of PS 4 (ECOG scale) with HLH-associated severe organ dysfunction during the initiation of conditioning had extremely poor outcomes. The OS rate in the RIC-CBT patients, excluding those with a performance status 4, was 80.0 ± 12.6 %. RIC may reduce treatment-related mortality; in addition, patients with engraftment failure, which is the main adverse event following RIC-CBT, were successfully rescued with secondary CBT. Unrelated cord blood may represent an alternative source if a patient has no related donor. As a RIC regimen for CBT, 140 mg/m(2) melphalan with fludarabine and anti-lymphocyte globulin or anti-thymocyte globulin may be feasible, but further dosage optimization should be performed in controlled clinical trials.


The ARNT-STAT3 axis regulates the differentiation of intestinal intraepithelial TCRαβ+CD8αα+cells.

Intestinal intraepithelial T cells contribute to the regulation of inflammatory responses in the intestine; however, the molecular basis for their development and maintenance is unknown. The aryl hydrocarbon receptor complexes with the aryl hydrocarbon receptor nuclear translocator (ARNT) and senses environmental factors, including gut microbiota. Here, we identify ARNT as a critical regulator of the differentiation of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. Mice deficient in either ARNT or aryl hydrocarbon receptor show a greater than- eight-fold reduction in the number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells. The number of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells is increased by treatment with an aryl hydrocarbon receptor agonist in germ-free mice and is decreased by antibiotic treatment. The Arnt-deficient precursors of TCRαβ(+)CD8αα(+) intestinal intraepithelial T cells express low amounts of STAT3 and fail to differentiate towards the TCRαβ(+)CD8αα(+) cell fate after IL-15 stimulation, a deficiency that is overcome by overexpression of Stat3. These data demonstrate that the ARNT-STAT3 axis is a critical regulator of TCRαβ(+)CD8αα(+) intestinal intraepithelial T-cell development and differentiation.


Mariner-based transposon mutagenesis for Bacteroides species.

Bacteroides is one of the most predominant groups of human gut microbiota. Recent metagenomic analyses and studies on gnotobiotic mice demonstrated the tight association of Bacteroides with epithelial function, the gut immune system and systemic metabolism in the host. The mariner family transposon shows relatively low target site specificity and has hosts ranging from prokaryotes to eukaryotes. Thereby, random mutagenesis using the mariner family transposon is expected to identify key molecules for human-Bacteroides symbiosis. In this study, we constructed the plasmid pMI07 to deliver the gene cassette (ermF/ITR), which harbors the erythromycin resistant marker (ermF) and the inverted repeat sequences (ITRs) recognized by Himar1 transposase, to Bacteroides via electrotransformation. pMI07 successfully delivered ermF/ITR to the Bacteroides genomes and generated thousands of insertion mutants/g of pMI07 in B. thetaiotaomicron, B. fragilis, B. ovatus, and also, although to a lesser extent, B. vulgatus. Analyses of the ermF/ITR insertion sites in B. thetaiotaomicron and B. vulgatus revealed that the cassette targeted the dinucleotide TA and integrated into the genomes in an unbiased manner. The data reported here will provide useful information for transposon mutagenesis in Bacteroides species, which will enable identification of the genes responsible for their unique phenotypes.


PhoB regulates the survival of Bacteroides fragilis in peritoneal abscesses.

In response to phosphate limitation, bacteria employ the Pho regulon, a specific regulatory network for phosphate acquisition. The two-component signal transduction system of PhoRB plays a crucial role in the induction of Pho regulon genes, leading to the adaptation to phosphate starvation. Herein, we identified the PhoRB system in Bacteroides fragilis, a commensal gut bacterium, and evaluated its role in gut colonization and survival in peritoneal abscesses. BF1575 and BF1576 encoded PhoR (sensor histidine kinase) and PhoB (response regulator) in the sequenced B. fragilis strain YCH46, respectively. Transcriptome analysis revealed that deletion of phoB affected the expression of 585 genes (more than 4-fold change) in B. fragilis, which included genes for stress response (chaperons and heat shock proteins), virulence (capsular polysaccharide biosynthesis) and phosphate metabolism. Deletion of phoB reduced the ability of the bacterium to persist in peritoneal abscesses induced by an intra-abdominal challenge of B. fragilis. Furthermore, PhoB was necessary for survival of this anaerobe in peritoneal abscesses but not for in vitro growth in rich media or in intestinal colonization. These results indicate that PhoB plays an important role in the survival of B. fragilis under stressful extraintestinal conditions.


Dll4-Fc, an inhibitor of Dll4-Notch signaling, suppresses liver metastasis of small cell lung cancer cells through down-regulation of NF-kappa-B activity

Notch signaling regulates cell-fate decisions during development and postnatal life. Little is known, however, about the role of Delta-like-4 (Dll4)-Notch signaling between cancer cells, or how this signaling affects cancer metastasis. We, therefore, assessed the role of Dll4-Notch signaling in cancer metastasis. We generated a soluble Dll4 fused to the IgG1 constant region (Dll4-Fc) that acts as a blocker of Dll4-Notch signaling and introduced it into human small cell lung cancer (SCLC) cell lines expressing either high levels (SBC-3 and H1048) or low levels (SBC-5) of Dll4. The effects of Dll4-Fc on metastasis of SCLC were evaluated using a mouse model. Although Dll4-Fc had no effect on the liver metastasis of SBC-5, the number of liver metastasis inoculated with SBC-3 and H1048 cells expressing Dll4-Fc was significantly lower than that injected with control cells. To study the molecular mechanisms of the effects of Dll4-Fc on liver metastasis, a PCR array analysis was conducted. Because the expression of NF-κB target genes was affected by Dll4-Fc, we conducted an electrophoretic mobility shift assay and observed that NF-κB activities, both with and without stimulation by TNF-α, were downregulated in Dll4-Fc-overexpressing SBC-3 and H1048 cells compared with control cells. Moreover, Dll4-Fc attenuates, at least in part, the classical and alternative NF-κB activation pathway by reducing Notch1 signaling. These results suggest that Dll4-Notch signaling in cancer cells plays a critical role in liver metastasis of SCLC by regulating NF-κB signaling.


Reduced-intensity conditioning in unrelated donor cord blood transplantation for familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a disorder of immune homeostasis characterized by fever, cytopenias, hepatosplenomegaly, and coagulopathy. We studied the outcomes of 13 FHL patients who underwent the first unrelated cord blood transplantation (UCBT) after non-myeloablative conditionings. The major regimen consisted of fludarabine (FLU; n = 12)+melphalan (MEL; n = 11)± low-dose total body irradiation (TBI 2-4 Gy; n = 6). The median age at presentation and period to UCBT were 6 and 5 months, respectively. Central nervous system (CNS) disease developed in one infant at diagnosis, and in two others until UCBT. HLH activity was controlled in all but one at the time of UCBT. Ten patients had early engraftment on median day 21 with no grade >2 treatment-related toxicity and two controllable grade >2 acute GVHD. Two patients with early rejection successfully underwent subsequent UCBT after myeloablative conditioning. Two others had late graft failure following mixed donor chimerism. Two deaths occurred from HLH; early liver failure and late CNS disease. Of 11 FLU+MEL-conditioned patients, the frequency of disease-free complete engraftment was higher for MEL (120 mg/m(2) )+TBI, or high-dose MEL (180 mg/m(2) ) than for others (83% vs. 25%, p = 0.036). The FLU+MEL-based non-myeloablative regimen was acceptable for FHL infants undergoing UCBT, although further studies will be needed for confirmation.


Manipulation of CD98 resolves type 1 diabetes in nonobese diabetic mice.

The interplay of CD4(+) and CD8(+) T cells targeting autoantigens is responsible for the progression of a number of autoimmune diseases, including type 1 diabetes mellitus (T1D). Understanding the molecular mechanisms that regulate T cell activation is crucial for designing effective therapies for autoimmune diseases. We probed a panel of Abs with T cell-modulating activity and identified a mAb specific for the H chain of CD98 (CD98hc) that was able to suppress T cell proliferation. The anti-CD98hc mAb also inhibited Ag-specific proliferation and the acquisition of effector function by CD4(+) and CD8(+) T cells in vitro and in vivo. Injection of the anti-CD98hc mAb completely prevented the onset of cyclophosphamide-induced diabetes in NOD mice. Treatment of diabetic NOD mice with anti-CD98hc reversed the diabetic state to normal levels, coincident with decreased proliferation of CD4(+) T cells. Furthermore, treatment of diabetic NOD mice with CD98hc small interfering RNA resolved T1D. These data indicate that strategies targeting CD98hc might have clinical application for treating T1D and other T cell-mediated autoimmune diseases.


Notch2 regulates the development of marginal zone B cells through Fos.

B cells are classified into several subsets depending on their functions, marker expression pattern and localization. Marginal zone B (MZB) cells are a distinct lineage from follicular B cells, and regulate host defenses against blood-borne pathogens. Notch2/RBP-J signaling regulates the development of MZB cells by interacting with delta-like 1 ligand, although the target genes for Notch2 signaling remain unclear. We identified Fos as an upregulated gene in LPS-stimulated B cells that received Notch2 signaling. Fos is expressed in CD21(high)CD23(low) MZB cells at a higher level compared to CD21(Int)CD23(high) follicular B cells. Deleting the Notch2 gene in CD19(+) B cells decreased Fos expression in B cells. Overexpression of Fos in Notch2-deficient B cells or bone marrow cells partially restored MZB development. Fos promoter activity was upregulated by Notch2 signaling, indicating that Notch2 directly controls Fos transcription associated with MZB development. These data identify Fos as one of the target genes for Notch2 signaling that is crucial for MZB development.

Biochemical and Biophysical Research Communications


Low-dose lipopolysaccharide affects lung allergic responses by regulating Jagged1 expression on antigen-pulsed dendritic cells.

These data demonstrate that LPS regulates the expression of Jagged1 on BMDCs, which is essential for the full development of lung allergic responses.

International Archives of Allergy and Immunology


A mutation in the immunoproteasome subunit PSMB8 causes autoinflammation and lipodystrophy in humans.

Proteasomes are multisubunit proteases that play a critical role in maintaining cellular function through the selective degradation of ubiquitinated proteins. When 3 additional subunits, expression of which is induced by IFN-, are substituted for their constitutively expressed counterparts, the structure is converted to an immunoproteasome. However, the underlying roles of immunoproteasomes in human diseases are poorly understood. Using exome analysis, we found a homozygous missense mutation (G197V) in immunoproteasome subunit, type 8 (PSMB8), which encodes one of the subunits induced by IFN- in patients from 2 consanguineous families. Patients bearing this mutation suffered from autoinflammatory responses that included recurrent fever and nodular erythema together with lipodystrophy. This mutation increased assembly intermediates of immunoproteasomes, resulting in decreased proteasome function and ubiquitin-coupled protein accumulation in the patient's tissues. In the patient's skin and B cells, IL-6 was highly expressed, and there was reduced expression of PSMB8. Downregulation of PSMB8 inhibited the differentiation of murine and human adipocytes in vitro, and injection of siRNA against Psmb8 in mouse skin reduced adipocyte tissue volume. These findings identify PSMB8 as an essential component and regulator not only of inflammation, but also of adipocyte differentiation, and indicate that immunoproteasomes have pleiotropic functions in maintaining the homeostasis of a variety of cell types.


High phosphate diet reduces atherosclerosis formation in apolipoprotein E-deficient mice.

Although higher serum phosphate level is a risk factor for cardiovascular diseases in general population as well as chronic kidney disease patients, it has not been clarified whether higher phosphate can affect atherosclerotic plaque formation. In this study, we investigated the effect of prolonged-intake of different concentrations of phosphate on atherosclerosis formation using apolipoprotein E-deficient mice. Apolipoprotein E-deficient mice were fed with high fat diet including 0.6%, 1.2% or 1.8% phosphate. After 20-week treatment, atherosclerotic plaque formation in aorta in 1.8% phosphate diet group was unexpectedly less than that in the other groups. To elucidate mechanisms of suppression of plaque formation by high phosphate diet, we hypothesized that high phosphate diet may modify a profile of monocytes/macrophages suppressing plaque formation. We confirmed that elevated peripheral monocytes (CD11b+, F4/80+ cell numbers) in apolipoprotein E-deficient mice were decreased by feeding with 1.8% P diet. In addition, ex vivo study indicated that high dose of phosphate induced macrophage apoptosis. These observations suggest that excess phosphate intake decreased atherosclerosis formation, at least in part, by changing the profile of peripheral monocytes or inducing apoptosis of macrophages in apolipoprotein E-deficient mice.

Journal of Clinical Biochemistry and Nutrition


The lifestyle of the segmented filamentous bacterium: a non-culturable gut-associated immunostimulating microbe inferred by whole-genome sequencing.

Numerous microbes inhabit the mammalian intestinal track and strongly impact host physiology; however, our understanding of this ecosystem remains limited owing to the high complexity of the microbial community and the presence of numerous non-culturable microbes. Segmented filamentous bacteria (SFBs), which are clostridia-related Gram-positive bacteria, are among such non-culturable populations and are well known for their unique morphology and tight attachment to intestinal epithelial cells. Recent studies have revealed that SFBs play crucial roles in the post-natal maturation of gut immune function, especially the induction of Th17 lymphocytes. Here, we report the complete genome sequence of mouse SFBs. The genome, which comprises a single circular chromosome of 1 620 005 bp, lacks genes for the biosynthesis of almost all amino acids, vitamins/cofactors and nucleotides, but contains a full set of genes for sporulation/germination and, unexpectedly, for chemotaxis/flagella-based motility. These findings suggest a triphasic lifestyle of the SFB, which comprises two types of vegetative (swimming and epicellular parasitic) phases and a dormant (spore) phase. Furthermore, SFBs encode four types of flagellin, three of which are recognized by Toll-like receptor 5 and could elicit the innate immune response. Our results reveal the non-culturability, lifestyle and immunostimulation mechanisms of SFBs and provide a genetic basis for the future development of the SFB cultivation and gene-manipulation techniques.


Regulation of the development of acute hepatitis by IL-23 through IL-22 and IL-17 production.

IL-23 plays a critical role in the expansion of highly proinflammatory Th17 cells secreting IL-17 and IL-22. Recently, we demonstrated that Notch signaling drives IL-22 secretion through the aryl hydrocarbon receptor (AHR) and plays a protective role in Con A-induced hepatitis. In this study, we investigated the role of IL-23 in hepatitis using IL-23p19- and IL-17-deficient mice. In WT mice, the injection of Con A induced the upregulation of various cytokines which included IL-23, IL-22, IL-17, IFN- and TNF-. In IL-23p19-deficient mice, exacerbated hepatitis was observed and serum IL-22 and IL-17 levels were greatly reduced, whereas in IL-17-deficient mice, ameliorated hepatitis was observed. The injection of exogenous IL-22 protected p19-deficient mice from hepatitis, whereas the injection of exogenous IL-23 significantly increased the serum levels of not only IL-22 but also IL-17, and less effectively protected against hepatitis in IL-17-dependent and -independent manners. Finally, it was revealed that STAT3, STAT4 and Notch contributed to the production of both cytokines, and that the AHR was important only for IL-22 production in response to Con A and IL-23 in liver mononuclear cells. These results suggest that IL-23 plays a protective role in hepatitis through IL-22 production and also a pathological role via IL-17-dependent and -independent mechanisms.


Notch signaling regulates the development of a novel type of Thy1-expressing dendritic cell in the thymus.

Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs.


Notch2 signaling is required for proper mast cell distribution and mucosal immunity in the intestine.

Notch receptor-mediated signaling is involved in the developmental process and functional modulation of lymphocytes, as well as in mast cell differentiation. Here, we investigated whether Notch signaling is required for antipathogen host defense regulated by mast cells. Mast cells were rarely found in the small intestine of wild-type C57BL/6 mice but accumulated abnormally in the lamina propria of the small-intestinal mucosa of the Notch2-conditional knockout mice in naive status. When transplanted into mast cell-deficient W(sh)/W(sh) mice, Notch2-null bone marrow-derived mast cells were rarely found within the epithelial layer but abnormally localized to the lamina propria, whereas control bone marrow-derived mast cells were mainly found within the epithelial layer. After the infection of Notch2 knockout and control mice with L3 larvae of Strongyloides venezuelensis, the abundant number of mast cells was rapidly mobilized to the epithelial layer in the control mice. In contrast, mast cells were massively accumulated in the lamina propria of the small intestinal mucosa in Notch2-conditional knockout mice, accompanied by impaired eradication of Strongyloides venezuelensis. These findings indicate that cell-autonomous Notch2 signaling in mast cells is required for proper localization of intestinal mast cells and further imply a critical role of Notch signaling in the host-pathogen interface in the small intestine.


Notch signaling drives IL-22 secretion in CD4+ T cells by stimulating the aryl hydrocarbon receptor.

CD4(+) helper T (Th) cells differentiate toward distinct effector cell lineages characterized by their distinct cytokine expression patterns and functions. Multiple Th cell populations secrete IL-22 that contributes to both protective and pathological inflammatory responses. Although the differentiation of IL-22-producing Th cells is controlled by the aryl hydrocarbon receptor (AhR), little is known about the regulatory mechanisms inducing physiological stimulators for AhR. Here, we show that Notch signaling enhances IL-22 production by CD4(+) T cells by a mechanism involving AhR stimulation. Notch-mediated stimulation of CD4(+) T cells increased the production of IL-22 even in the absence of STAT3. CD4(+) T cells from RBP-J-deficient mice had little ability to produce IL-22 through T cell receptor-mediated stimulation. RBP-J-deficient mice were highly susceptible to the detrimental immunopathology associated with ConA-induced hepatitis with little IL-22 production by CD4(+) T cells. Exogenous IL-22 protected RBP-J-deficient mice from ConA-induced hepatitis. Notch signaling promoted production of endogenous stimulators for AhR, which further augmented IL-22 secretion. Our studies identify a Notch-AhR axis that regulates IL-22 expression and fine-tunes immune system control of inflammatory responses.

Proceedings of the National Academy of Sciences of the United States of America


Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading.

Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine(608)-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.


Jagged1 on dendritic cells and Notch on CD4+ T cells initiate lung allergic responsiveness by inducing IL-4 production.

Jagged1, a Notch ligand, and Notch have been implicated in Th2 differentiation, but their role in initiating IL-4 production and Th2 differentiation in vivo and the development of allergic airway responses has not been defined. In this study, we show that Jagged1 is up-regulated on bone marrow-derived dendritic cells (BMDCs) pulsed with allergen and that the transfer of these BMDCs before allergen challenge induces airway hyperresponsiveness (AHR) and eosinophilic airway inflammation. Treatment of CD4(+) T cells with a gamma-secretase inhibitor (GSI), which inhibits Notch signaling, resulted in decreased cytokine production when the cells were cocultured with allergen-pulsed, Jagged1-expressing BMDCs and, after the transfer of allergen-pulsed BMDCs, IL-4-deficient (IL-4(-/-)) recipients of GSI-treated naive CD4(+) T cells developed lower levels of AHR, reduced numbers of eosinophils, and lower Th2 cytokine levels when challenged with allergen. In vivo treatment of wild-type mice with Jagged1-Fc enhanced AHR and airway inflammation, whereas the transfer of BMDC transfected with Jagged1 small interfering RNA (siRNA) cells into WT or IL-4(-/-) mice before transfer of CD4(+) T cells resulted in decreased AHR, inflammation, and Th2 cytokines, indicating the critical role for Jagged1 expression on APCs. These data identify the essential role of the interactions between Notch on CD4(+) T cells and Jagged1 on APCs in the initiation of IL-4 production and Th2 differentiation for the development of AHR and allergic airway inflammation.


Notch activation induces the generation of functional NK cells from human cord blood CD34-positive cells devoid of IL-15.

The development of NK cells from hematopoietic stem cells is thought to be dependent on IL-15. In this study, we demonstrate that stimulation of human cord blood CD34(+) cells by a Notch ligand, Delta4, along with IL-7, stem cell factor, and Fms-like tyrosine kinase 3 ligand, but no IL-15, in a stroma-free culture induced the generation of cells with characteristics of functional NK cells, including CD56 and CD161 Ag expression, IFN-gamma secretion, and cytotoxic activity against K562 and Jurkat cells. Addition of gamma-secretase inhibitor and anti-human Notch1 Ab to the culture medium almost completely blocked NK cell emergence. Addition of anti-human IL-15-neutralizing Ab did not affect NK cell development in these culture conditions. The presence of IL-15, however, augmented cytotoxicity and was required for a more mature NK cell phenotype. CD56(+) cells generated by culture with IL-15, but without Notch stimulation, were negative for CD7 and cytoplasmic CD3, whereas CD56(+) cells generated by culture with both Delta4 and IL-15 were CD7(+) and cytoplasmic CD3(+) from the beginning and therefore more similar to in vivo human NK cell progenitors. Together, these results suggest that Notch signaling is important for the physiologic development of NK cells at differentiation stages beyond those previously postulated.


Association study of TRAF1-C5 polymorphisms with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in Japanese.

The primary aim of this study was to investigate the association of polymorphisms of TRAF1-C5, a newly identified rheumatoid arthritis (RA) risk locus in Caucasians, with susceptibility to RA and systemic lupus erythematosus (SLE) in Japanese populations. Gene expression levels of TRAF1 and C5 to assess the functional significance of genotypes were also analysed. A multicentre association study consisting of 4 RA case-control series (4397 cases and 2857 controls) and 3 SLE case-control series (591 cases and 2199 shared controls) was conducted. Genotyping was performed using TaqMan genotyping assay for two single nucleotide polymorphisms (SNPs) that showed the best evidence of association in the previous Caucasian studies. Quantifications of TRAF1 and C5 expression were performed with TaqMan expression assay. Significant differences in allele frequency for both SNPs were observed between RA and control subjects (combined odds ratio = 1.09), while no significant difference was detected between patients with SLE and controls. Interestingly, alleles rs3761847 A and rs10818488 G had increased the risk for RA in the present study, while they decreased the risk in the original studies. A significant difference was found between risk allele carriers and non-carriers of rs10818488 for the expression level of TRAF1 in phorbol myristate acetate-stimulated lymphoblastoid cell lines (p = 0.04). Association of TRAF1-C5 locus with RA susceptibility was detected in the Japanese populations with modest magnitude, while no significant association was observed for SLE. Significant positive effect of genotype on the expression of TRAF1 might support the genetic association between TRAF1 and RA.


Notch2 integrates signaling by the transcription factors RBP-J and CREB1 to promote T cell cytotoxicity.

The acquisition of cytotoxic effector function by CD8(+) T cells is crucial for the control of intracellular infection and tumor invasion. However, it remains unclear which signaling pathways are required for the differentiation of CD8(+) cytotoxic T lymphocytes. We show here that Notch2-deficient T cells had impaired differentiation into cytotoxic T lymphocytes. In addition, dendritic cells with lower expression of the Notch ligand Delta-like 1 induced the differentiation of cytotoxic T lymphocytes less efficiently. We found that the intracellular domain of Notch2 interacted with a phosphorylated form of the transcription factor CREB1, and together these proteins bound the transcriptional coactivator p300 to form a complex on the promoter of the gene encoding granzyme B. Our results suggest that the highly regulated, dynamic control of T cell cytotoxicity depends on the integration of Notch2 and CREB1 signals.


Association of STAT4 with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in the Japanese population.

OBJECTIVE: STAT4 encodes a transcriptional factor that transmits signals induced by several key cytokines, and it might be a key molecule in the development of autoimmune diseases. Recently, a STAT4 haplotype was reported to be associated with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in Caucasian populations. This was replicated in a Korean RA population. Interestingly, the degree of risk of RA susceptibility with the STAT4 haplotype was similar in the Caucasian and Korean populations. The present study was undertaken to investigate the effect of STAT4 on susceptibility to RA and SLE in the Japanese. METHODS: We performed an association study using 3 independent Japanese RA case-control populations (total 3,567 cases and 2,199 controls) and 3 independent Japanese SLE populations (total 591 cases). All samples were genotyped using the TaqMan fluorogenic 5' nuclease assay for single-nucleotide polymorphism (SNP) rs7574865, which tags the susceptibility haplotype. The association of the SNP with disease susceptibility in each case-control study was calculated using Fisher's exact test, and the results were combined, using the Mantel-Haenszel method, to obtain combined odds ratios (ORs). RESULTS: We observed a significant association of the STAT4 polymorphism with susceptibility to both RA and SLE. The combined ORs for RA and SLE, respectively, were 1.27 (P = 8.4 x 10(-9)) and 1.61 (P = 2.1 x 10(-11)) for allele frequency distribution; these ORs were quite similar to those previously observed in the Caucasian population. CONCLUSION: We conclude that STAT4 is associated with RA and SLE in the Japanese. Our results indicate that STAT4 is a common genetic risk factor for autoimmune diseases, with similar strength across major racial groups.


Dendritic cell-mediated NK cell activation is controlled by Jagged2-Notch interaction.

Natural killer (NK) cells regulate various immune responses by exerting cytotoxic activity or secreting cytokines. The interaction of NK cells with dendritic cells (DC) contributes to NK cell-mediated antitumor or antimicrobial responses. However, the cellular and molecular mechanisms for controlling this interaction are largely unknown. Here, we show an involvement of Jagged2-Notch interaction in augmenting NK cell cytotoxicity mediated by DC. Enforced expression of Jagged2 on A20 cells (Jag2-A20 cells) suppressed their growth in vivo, which was abrogated by depleting NK cells. Moreover, Jag2-A20 cells exerted a suppression on the growth of nonmanipulated A20 cells in SCID mice in an NK-dependent manner. Consistently, coinoculation of A20 cells with DC overexpressing Jagged2 (Jag2-DC) suppressed the growth of A20 cells in mice. Stimulation of NK cells with Jagged2 directly enhanced their cytotoxicity, IFN-gamma production, and proliferation. Ligation of Notch2 on NK cells enhanced their cytotoxic activity, and Jag2-DC or CpG-treated DC-mediated NK cell cytotoxicity was suppressed by a gamma-secretase inhibitor. These results indicate that the Jagged2-Notch axis plays a crucial role in DC-mediated NK cell cytotoxicity. Furthermore, manipulation of this interaction may provide an approach to induce potent tumor immunity or to inhibit certain autoimmune diseases caused by NK cell activation.


Maturational arrest of thymocyte development is caused by a deletion in the receptor-like protein tyrosine phosphatase κ gene in LEC rats

The Long-Evans Cinnamon (LEC) rat has a spontaneous mutation, T helper immunodeficiency (thid), which causes a markedly reduced CD4(+) thymocyte population. Here we positionally clone the locus and identify a deletion in the gene encoding a receptor-like protein tyrosine phosphatase kappa (Ptprk) that led to complete loss of the transcript. The rat Ptprk gene exhibits 98% identity with the human and mouse counterparts and is expressed most abundantly in the CD4(-)CD8(-) double-negative stage. The downregulation of Ptprk in mouse immature thymocytes by RNA interference mimicked the thid phenotype. These results indicate that thid maps to the Ptprk locus and that functional Ptprk is crucial for lineage commitment or progression of CD4(+) T cells. We also found that Ptprk appears to function in parallel with or downstream of Th-POK/cKrox (also known as ZBTB7B), a master regulator of T cell lineage decision.


Water extract of Cordyceps sinensis (WECS) inhibits the RANKL-induced osteoclast differentiation.

It has been reported that Cordyceps sinensis, a traditional Chinese medicine, has various pharmacological effects. The aim of this study was to clarify the effect of water extract of Cordyceps sinensis (WECS) on osteoclast differentiation in vitro. In mouse bone marrow cells and monocyte/macrophage cell line RAW264.7, WECS dose-dependently inhibited the receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL)-induced osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) staining. In fact, cytotoxic effect was not observed in the RAW264.7 cells treated with WECS. Moreover, the mRNA expression of osteoclast related genes (calcitonin receptor, cathepsin K, matrix metalloprotease 9 and nuclear factor of activated T cells c1) was also inhibited by WECS. Investigation of inhibitory mechanism by using electrophoretic mobility shift assay (EMSA) and Western blot analysis revealed that WECS inhibited the activation of NF-kappaB through the prevention of IkappaBalpha phosphorylation. In conclusion, the present results demonstrate for the first time that WECS is a potent inhibitor of the RANKL-induced osteoclast differentiation through a mechanism involving the NF-kappaB pathway.


Lunatic fringe controls T cell differentiation through modulating Notch signaling.

T cells differentiate from bone marrow-derived stem cells by expressing developmental stage-specific genes. We here searched arrays of genes that are highly expressed in mature CD4-CD8+ (CD8 single-positive (SP)) T cells but little in CD4+CD8+ (double-positive (DP)) cells by cDNA subtraction. Lunatic fringe (Lfng), a modulator of Notch signaling, was identified to be little expressed in DP cells and highly expressed in CD8SP T cell as well as in CD4-CD8- (double-negative (DN)) and mature CD4+CD8- (CD4SP) T cells. Thus, we examined whether such change of expression of Lfng plays a role in T cell development. We found that overexpression of Lfng in Jurkat T cells strengthened Notch signaling by reporter gene assay, indicating that Lfng is a positive regulator for Notch signaling in T cells. The enforced expression of Lfng in thymocytes enhanced the development of immature CD8SP cells but decreased mature CD4SP and CD8SP cells. In contrast, the down-regulation of Lfng in thymocytes suppressed DP cells development due to the defective transition from CD44+CD25- stage to subsequent stage in DN cells. The overexpression of Lfng in fetal liver-derived hemopoietic stem cells enhanced T cell development, whereas its down-regulation suppressed it. These results suggested that the physiological high expression of Lfng in DN cells contributes to enhance T cell differentiation through strengthening Notch signaling. Shutting down the expression of Lfng in DP cells may have a physiological role in promoting DP cells differentiation toward mature SP cells.


Clinical roles of increased populations of Foxp3+CD4+ T cells in peripheral blood from advanced pancreatic cancer patients.

Further metastasis should be avoided in pancreatic cancer (PC) patients for effective surgical treatment. Regulatory T cells (Foxp3CD4 T cells including CD4CD25 T cells and CD4CD25 T cells) play important roles in tumor immunity. This study aimed to investigate whether regulatory T cells participate in metastasis. Peripheral blood was withdrawn from PC patients, as well as healthy volunteer donors as controls. The peripheral blood mononuclear cells (PBMCs) were subjected to FACScan analysis after labeling with anti-CD4, anti-CD25, and anti-Foxp3 antibodies. Tumor markers, including DUPAN2 and CA19-9, surface markers, such as the CD4/CD8 ratio, and the CD57 cell population were assessed. Clinical stages were classified according to the TNM classification. The Foxp3CD4 T-cell population among the PBMCs was significantly increased in PC patients (8.10% +/- 4.65%) compared with healthy donors (2.47 +/- 0.78%) (P < 0.001). No significant relationships existed for the tumor markers, CD4/CD8 ratio, and CD57 cells. However, a significant correlation was found between Foxp3CD4 T cells among the PBMCs and the TNM stage (P < 0.05). Foxp3CD4 T cells are good markers for metastasis detection in PC patients and more accurate than other conventional tumor markers, especially at advanced stages of the disease.


Resistance to GITR signaling in antigen specific CD4+CD25+ regulatory T cells during plasmodium yoelii infection.

CD4+ T cells are the major effector T cells against blood-stage Plasmodium yoelii infection. On the other hand, the lethal strain of P. yoelii (PyL) has acquired an escape mechanism from host T cell immunity by activating CD4+CD25+ regulatory T cells (Treg). Although the activation of Treg during PyL infection precludes the clearance of PyL from mice, it remains unclear whether activation of Treg is attributable to a specific response against PyL infection. Thus, we examined here whether Treg proliferate in an antigen-dependent manner during PyL infection. We also investigated the effector and regulatory mechanisms of Treg. Infection with PyL increased the number of CD4+CD25+ T cells, in which expression of Foxp3 mRNA is up-regulated. The Treg that were transferred into mice infected with PyL, but not with a non-lethal strain of P. yoelii (PyNL), proliferated during the initial 5 days following infection. The Treg from PyL-infected mice showed strong suppression compared with those from naive or PyNL-infected mice, and could suppress T cell activation by recognizing PyL- but not PyNL-derived antigens. Furthermore, the suppressive function of Treg activated in PyL-infected but not in naive mice could not be inhibited by treatment with an anti-glucocorticoid-induced TNFR family-related protein (GITR) mAb. These findings indicate that PyL infection specifically activates Treg that are specific for PyL-derived antigens. The infection also induces resistance for Treg to GITR signaling, and this eventually contributes to the escape of parasites from host T cell immunity.


Effective Treatment of a Mouse Model of Sjogren's Syndrome With Eyedrop Administrasiton of Anti-CD4 Monoclonal Antibody

To determine whether eyedrop administration of an anti-CD4 monoclonal antibody (mAb) is effective in the treatment of Sjögren's syndrome (SS) using a mouse model of the disease. The anti-CD4 mAb was administered daily into the eyes of mice with SS from ages 4 to 8 weeks or ages 10 to 12 weeks. During treatment, tear volume was monitored and after final treatment, histologic features of the lacrimal and salivary glands, the phenotypes and function of T cells, and serum titers of anti-alpha-fodrin antibody were examined. Eyedrop administration of anti-CD4 mAb before the onset of SS prevented the autoimmune pathology seen in the lacrimal glands but not that in the salivary glands. Furthermore, eyedrop administration of anti-CD4 mAb after the development of SS inhibited mononuclear cell infiltration and the destruction of parenchyma only in the lacrimal glands. Eyedrop administration of anti-CD4 mAb suppressed the local activation of CD4+ T cells rather than deleting CD4+ T cells, which reduced the expansion of pathologic CD4+ T cells against alpha-fodrin. These results demonstrate the remarkable efficacy of anti-CD4 mAb eyedrops in the treatment of SS eye symptoms, which illustrates a new antibody-based therapeutic strategy for patients with eye problems caused by SS as well as other diseases.


Transforming growth factor-β in renal disease with glycogen storage disease I

We report a 14-year-old patient with Japanese glycogen storage disease I (GSD-I) who was found to have proteinuria. Renal biopsy revealed massive tubular atrophy and interstitial fibrosis with mononuclear cell infiltration, but the glomeruli were almost normal. The epithelial cells of tubules contained periodic acid-Schiff-positive glycogen deposits digested by diastase. In an immunohistological study, transforming growth factor (TGF)-beta expression was increased in tubular epithelial cells compared with a normal control kidney specimen. These data suggest that increased TGF-beta expression is involved in the pathophysiology of renal interstitial fibrosis in a patient with GSD-I.


Antigen-Specific T Cell Repertoire Modification of CD4+CD25+ Regulatory T Cells

T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4(+) T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4(+)CD25(+) regulatory T cells (T(reg)) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T(reg). In this study, we demonstrate that after a primary Ag challenge, T(reg) proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T(reg) after primary Ag challenge augmented the total suppressive function of T(reg) against TCR repertoire modification but not against the proliferation of memory CD4(+) T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T(reg), which would be crucial for limiting excess primary and memory CD4(+) T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T(reg) is an ideal strategy for the clinical use of T(reg).


Sisters with α-mannosidosis and systemic lupus erythematosus

Alpha-mannosidosis is an autosomal recessive disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). Here, we report two sisters with alpha-mannosidosis who developed systemic lupus erythematosus (SLE). The sisters were both homozygous for a one bp deletion within the LAMAN gene resulting in a truncated gene product. The coincidence of alpha-mannosidosis and SLE are discussed with regard to both clinical and molecular findings. CONCLUSION: alpha-mannnosidosis may contribute to the onset of systemic lupus erythematosus in predisposed patients.


Escape of malaria parasites from host immunity requires CD4+CD25+ regulatory T-cells

Infection with malaria parasites frequently induces total immune suppression, which makes it difficult for the host to maintain long-lasting immunity. Here we show that depletion of CD4(+)CD25(+) regulatory T cells (T(reg)) protects mice from death when infected with a lethal strain of Plasmodium yoelii, and that this protection is associated with an increased T-cell responsiveness against parasite-derived antigens. These results suggest that activation of T(reg) cells contributes to immune suppression during malaria infection, and helps malaria parasites to escape from host immune responses.


Deltal-Notch3 interactious bias the functional differentiation of activated CD4+ T-Cells

Following activation by antigen, naive CD4+ T helper precursor cells execute distinct genetic programs that result in their differentiation toward the type 1 or type 2 helper T cell (Th1 or Th2) phenotype. Although the differentiation and function of these Th subsets has been well studied, little is known about the contribution to these differentiation events of cell surface receptors other than those for soluble cytokines, such as IL-12 or IL-4. Here, we provide direct evidence that the Delta1 interaction with Notch3 on CD4+ T cells transduces signals, promoting development toward the Th1 phenotype. The positive role of Notch signaling in effector cell differentiation was dose dependent, with high levels of stimulation resulting in reduced T cell activation. Our data revealed a clear contribution of Notch pathways to Th1 versus Th2 fate decisions, while also providing insight into another mechanism for inhibition of CD4+ T cell activation.


Breakdown of peripheral T-cell tolerance by chronic IL-15 elevation

Thymic deletion purges the repertoire of most developing T cells with the potential for overt self-reactivity, but some self-specific cells do emerge into the peripheral pool. Under most conditions, these potentially autoaggressive cells remain in a quiescent state. However, in some circumstances, they become activated and acquire effector function, leading to immune disease. It is thus important to clarify the mechanism(s) responsible for determining the balance between such inappropriate T-cell activation and the normal state of peripheral tolerance. In this article, we show that chronic elevation of interleukin-15 levels interferes with the tolerant state of CD8+ T cells through a process that involves activation of nonlymphoid antigen-presenting cells by CD4+asialo-GM1+ (ASGM1) or both CD4+ASGM1- and CD4-ASGM1+ cells. These findings suggest a potential role for dysregulated interleukin-15 production in promoting tolerance breakdown. This new information may be of potential use in improving tumor vaccines to self-antigens and in ameliorating autoimmune or graft-versus-host disease.


視力低下を契機に発見された小児褐色細胞腫の1例

視力低下を契機に発見された小児褐色細胞腫の1例

Japanese Journal of Clinical Ophthalmology


Mutation in DNASE I in people with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a highly prevalent human autoimmune diseases that causes progressive glomerulonephritis, arthritis and an erythematoid rash. Mice deficient in deoxyribonuclease I (Dnase1) develop an SLE-like syndrome. Here we describe two patients with a heterozygous nonsense mutation in exon 2 of DNASE1, decreased DNASE1 activity and an extremely high immunoglobulin G titer against nucleosomal antigens. These data are consistent with the hypothesis that a direct connection exists between low activity of DNASE1 and progression of human SLE.


CD4+ T-cell survival is not directly linked to self-MHC induced TCR signaling

T cell receptor (TCR) signaling triggered by recognition of self-major histocompatibility complex (MHC) ligands has been proposed to maintain the viability of naïve T cells and to provoke their proliferation in T cell-deficient hosts. Consistent with this, the partially phosphorylated state of TCR zeta chains in naïve CD4+ and CD8+ T cells in vivo was found to be actively maintained by TCR interactions with specific peptide-containing MHC molecules. TCR ligand-dependent phosphorylation of TCR zeta was lost within one day of cell transfer into MHC-deficient hosts, yet the survival of transferred CD4+ lymphocytes was the same in recipients with or without MHC class II expression for one month. Thus, despite clear evidence for TCR signaling in nonactivated naïve T cells, these data argue against the concept that such signaling plays a predominant role in determining lymphocyte lifespan.


Pepstatin A sensitive aspartic proteases in lysosome are involved in degradation of the invariant chain and antigen-processing in antigen presenting cells of mice infected with leishmania major

We previously reported that CA074, a specific inhibitor of cathepsin B, significantly deviated immune responses from the disease-promoting Th2 type to the protective Th1 type in BALB/c mice infected with Leishmania major. Herein, we found that pepstatin A-sensitive aspartic proteases (PSAP) in lysosomes seem to play a different role from that of cathepsin B in antigen-processing and Ii-degradation. That is, cathepsin B appears to digest 16-, 28-, and 31-kDa peptides of soluble leishmania antigen (SLA), whereas PSAP seems to process mainly 28-kDa peptides. Furthermore, the latter protease contributed to the degradation of Ii but cathepsin B did not. Following treatment with pepstatin A, both Th1 and Th2 responses were profoundly suppressed in resistant DBA/2 mice (H-2(d)) and in susceptible BALB/c mice (H-2(d)), and both strains of mice became markedly susceptible compared with the untreated groups, probably owing to failure in degradation of Ii and partly to failure in digestion of 28-kDa peptide.

Biochemical and Biophysical Research Communications


TCR signaling for initiation and completion of thymocyte positive selection has distinct requirements for ligand quality and presenting cell type

Thymocyte selection involves signaling by TCR engaging diverse self-peptide:MHC molecule ligands on various cell types in the cortex and medulla. Here we separately analyze early and late stages of selection to better understand how presenting cell type, ligand quality, and the timing of TCR signaling contribute to intrathymic differentiation. TCR transgenic CD4+CD8+ thymocytes (double positive (DP)) from MHC-deficient mice were stimulated using various presenting cells and ligands. The resulting CD69high cells were isolated and evaluated for maturation in reaggregate cultures with wild-type or MHC molecule-deficient thymic stroma with or without added hemopoietic dendritic cells (DC). Production of CD4+ T cells required TCR signaling in the reaggregates, indicating that transient recognition of self-ligands by DP is inadequate for full differentiation. DC bearing a potent agonist ligand could initiate positive selection, producing activated thymocytes that matured into agonist-responsive T cells in reaggregates lacking the same ligand. DC could also support the TCR signaling necessary for late maturation. These results argue that despite the negative role assigned to DC in past studies, neither the peptide:MHC molecule complexes present on DC nor any other signals provided by these cells stimulate only thymocyte death. These findings also indicate that unique epithelial ligands are not necessary for positive selection. They provide additional insight into the role of ligand quality in selection events and support the concept that following initiation of maturation from the DP state, persistent TCR signaling is characteristic of and perhaps required by T cells.


The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate

Signals elicited by binding of the T-cell antigen receptor and the CD4/CD8 co-receptor to major histocompatibility complex (MHC) molecules control the generation of CD4+ (helper) or CD8+ (cytotoxic) T cells from thymic precursors that initially express both co-receptor proteins. These precursors have unique, clonally distributed T-cell receptors with unpredictable specificity for the self-MHC molecules involved in this differentiation process. However, the mature T cells that emerge express only the CD4 (MHC class II-binding) or CD8 (MHC class I-binding) co-receptor that complements the MHC class-specificity of the T-cell receptor. How this matching of co-receptor-defined lineage and T-cell-receptor specificity is achieved remains unknown, as does whether signalling by the T-cell receptors, co-receptors and/or general cell-fate regulators such as Notch-1 contributes to initial lineage choice, to subsequent differentiation processes or to both. Here we show that the CD4 versus CD8 lineage fate of immature thymocytes is controlled by the co-receptor-influenced duration of initial T-cell receptor-dependent signalling. Notch-1 does not appear to be essential for this fate determination, but it is selectively required for CD8+ T-cell maturation after commitment directed by T-cell receptors. This indicates that the signals constraining CD4 versus CD8 lineage decisions are distinct from those that support subsequent differentiation events such as silencing of co-receptor loci.


Thymocyte resistance to glucocorticoids leads to antigen-specific unresponsiveness due to "holes" in the T cell repertoire

We have proposed that glucocorticoids antagonize TCR-mediated activation and influence which TCR avidities result in positive or negative selection. We now analyze the immune response of mice whose thymocytes express antisense transcripts to the glucocorticoid receptor (TKO mice). TKO mice responded normally to the complex antigen PPD but were proliferative nonresponders to pigeon cytochrome c 81-104 (PCC), having a large decrease in the frequency of PCC-responsive CD4+ T cells. Unlike wild-type T cells, few TKO T cells in PCC-specific cell lines expressed V alpha11+Vbeta3+. Furthermore, for naive CD4+ T cells from unimmunized TKO mice, the frequencies of many of the molecular features common to the CDR3 regions of PCC-responsive V alpha11+Vbeta3+ cells were substantially decreased. Thus, thymocyte glucocorticoid hyporesponsiveness resulted in loss of cells capable of responding to PCC, corresponding to an antigen-specific "hole" in the T cell repertoire.


Alpha1beta1 integrin-mediated collagen matrix remodeling by rat mesangial cells is differentially regulated by transforming growth factor-beta and platelet-derived growth factor-BB

Pathologic remodeling of mesangial matrix after glomerular injury is the central biologic feature of glomerular scarring (sclerosis). Transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF)-BB have been implicated in the development of glomerular scarring in rat and human glomerulonephritis. To clarify molecular and cellular mechanisms involved in abnormal mesangial remodeling, this study focused on the role of alpha1beta1 integrin, a collagen/laminin receptor, in rat mesangial cells, using collagen gel contraction as an experimental model of in vivo collagen matrix remodeling and scar formation. In addition, the influence of TGF-beta and PDGF-BB on mesangial cell (MC)-mediated collagen gel contraction in association with the alpha1beta1 integrin expression was evaluated. Integrin function blocking studies using anti-alpha1, beta1 subunit antibodies indicated that MC-alpha1beta1 integrin is essentially required not only for collagen-dependent adhesion/migration, but also for gel contraction. Protein synthesis and mRNA analysis experiments demonstrated that TGF-beta, but not PDGF-BB, increases the expression of alpha1beta1 integrin in mesangial cells cultured on plastic surface and in collagen gels. The upregulation of alpha1beta1 integrin expression by TGF-beta correlated with increases in gel contraction and collagen-dependent adhesion but not migration of mesangial cells. On the other hand, PDGF-BB enhanced MC-mediated gel contraction and migration without affecting cell adhesion to collagen I. Growth factor-induced collagen-dependent adhesion, migration, and gel contraction were significantly attenuated by incubation with anti-alpha1, beta1 subunit antibodies. Thus, these data indicate that alpha1beta1 integrin-mediated collagen matrix remodeling can be modulated by TGF-beta and PDGF-BB via different mechanisms. Alpha1 integrin-mediated mesangial matrix remodeling induced by TGF-beta or PDGF-BB may be a pathogenic mechanism leading to glomerular scarring.

Journal of the American Society of Nephrology


Divergent changes in the sensitivity of maturing T cell to structurally related ligands underlies formation of a useful T cell repertoire


Heat shock protein 65 induced by gammadelta T cells prevents apoptosis of macrophages and contributes to host defense in mice infected with Toxoplasma gondii


The Fas-deficient SCID mouse exhibits the development of T cells in the thymus


Transduction of retrovirus-mediated NeoR gene into CD34+ cells purified from granulocyte colony-stimulating factor(G-CSF) mobilized infant and cord blood


Antigen-specific B cells are required for the secondary response of T cells but not for their priming

We studied the potential role of B cells in T cell responses using severe-combined immunodeficient (SCID) mice grafted with the thymus from fetal C.B-17 mice (TG mice). These mice developed both CD4+ and CD8+ T cells, but not B cells within 2 months after transplantation. TG mice showed normal delayed-type hypersensitivity responses against the immunizing antigen ovalbumin (OVA). Lymph node (LN) cells of TG mice proliferated well in response to concanavalin A (Con A). Further, Con A stimulation induced the production of interleukin (IL)-2, IL-6 and interferon (IFN)-gamma and the expression of IL-4 mRNA. Thus, TG mice were reconstituted without remarkable immunodeficiency. However, these T cells failed to proliferate to OVA stimulation. Response to OVA was also inhibited in SCID mice grafted with fetal C.B-17 liver cells when B cells were depleted in the proliferation assay. Unresponsiveness against immunizing antigen was restored by the addition of antigen-primed B cells, but not by naive B cells, lipopolysaccharide-activated B cells or B cells primed with sheep red blood cells. Next, we examined whether antigen-primed B cells could induce T cell responses without professional antigen-presenting cells (APC). T and B cells were purified from OVA-immunized mice by cell sorter. These T cells proliferated in response to OVA and produced IFN-gamma in the absence of non-B APC. When anti-CD80 or anti-CD86 was added in the assay, proliferation and IFN-gamma production was inhibited. These results indicate that B cells activated specifically with antigen are required for the secondary response of T cells, but not for their priming.


Establishment of a xenogeneic chimera without GVHD in NK cell-depleted SCID mice by grafting rat fetal liver cells

Rat lymphopoiesis did not develop when naive SCID mice were transplanted with rat fetal liver cells. However, when SCID mice were depleted of NK cells by administration of anti-murine IL-2R beta mAb before transplantation, remarkable reconstitution of both rat T and B cells was observed in these mice without any evidence of graft-versus-host disease macroscopically or histologically. T cells in these reconstituted mice proliferated well in response to Con A and third-party rat and mouse antigens, whereas no response was seen to the stimulation with either donor rat- or host mouse-type cells. When these xenogeneic chimera mice had been immunized with SRBC, these mice exhibited DTH reaction and antibody production against the homologous antigen. These results indicate that rat fetal liver cells can differentiate to functional T and B cells in the xenogeneic microenvironment of SCID mice, if host NK cells are depleted beforehand. These rat-type T cells develop within SCID thymuses and acquire tolerance to either donor F344 rat or host SCID mouse antigens.


Defective T cells from gld mice play a pivotal role in development of Thy-1.2+B220+ cells and autoimmunity

The gld mouse represents a fascinating animal model of autoimmune disease, which is characterized by massive development of Thy-1.2+B220+ CD4-CD8- cells. These cells thus have double positive markers for T and B cells, but are double negative for CD4 and CD8 markers and are thus designated DN cells in the present context. An additional important feature in gld mice is a defect in expression of Fas ligand. To investigate the regulatory role of bone marrow-derived cells for the development of these DN cells and of gld autoimmunity, we constructed chimeric mice transplanted with fetal liver cells or fetal thymus from gld mice into nonirradiated severe combined immunodeficient (SCID) mice. These chimeric mice regenerated, developed both these DN cells and the gld autoimmune syndrome and also generalized lymphoproliferative disorders. However, when fetal liver cells from both gld and non-gld mice (C57BL/10 Thy-1.1 mice) were co-transplanted into SCID mice, the development of DN cells was apparently inhibited. Further, this inhibition was also seen in SCID mice that had been grafted with both gld and non-gld fetal thymus revealing the pivotal role played by T cells in development of DN cells. When B cells purified from non-gld (C3H+/+) mice were transplanted into SCID mice grafted with gld fetal thymus, the development of DN cells was not inhibited. Taken together, these findings indicate that T cells from non-gld mice inhibit the expression of gld features, e.g., lymphoproliferation, immune-based nephritic disease, and autoantibody production. These findings also suggest that the Fas ligand is selectively expressed on T cells.