Naoto Ishii

Positional identification of TNFSF4, encoding OX40 ligand, as a gene that influences atherosclerosis susceptibility

Ath1 is a quantitative trait locus on mouse chromosome 1 that renders C57BL/6 mice susceptible and C3H/He mice resistant to diet-induced atherosclerosis. The quantitative trait locus region encompasses 11 known genes, including Tnfsf4 (also called Ox40l or Cd134l), which encodes OX40 ligand. Here we report that mice with targeted mutations of Tnfsf4 had significantly (P ≤ 0.05) smaller atherosclerotic lesions than did control mice. In addition, mice overexpressing Tnfsf4 had significantly (P ≤ 0.05) larger atherosclerotic lesions than did control mice. In two independent human populations, the less common allele of SNP rs3850641 in TNFSF4 was significantly more frequent (P ≤ 0.05) in individuals with myocardial infarction than in controls. We therefore conclude that Tnfsf4 underlies Ath1 in mice and that polymorphisms in its human homolog TNFSF4 increase the risk of myocardial infarction in humans.

Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40.

An emerging immunotherapeutic strategy for T-cell-mediated diseases is to directly target antigen-specific T cells that are responsible for the clinical effects, without causing general widespread immunosuppression. A T-cell co-stimulatory molecule, OX40, which is transiently expressed after antigen recognition, fits these criteria in several immune-mediated diseases. In vivo blockade of OX40 signalling specifically suppresses the function of recently activated autoantigen-specific T cells, leading to inhibition of autoimmune disease without severe immunosuppression. In addition, deliberate ligation of OX40 in tumour-bearing hosts enhances anticancer immunity. We discuss how targeting OX40 is potentially an ideal strategy for immune-based therapies in several human diseases.

Critical role for OX40 ligand in the development of pathogenic Th2 cells in a murine model of asthma

Bronchial asthma is characterized by massive infiltration of eosinophils and airway hyperreactivity (AHR), which are caused by overproduction of Th2 cytokines (IL‐4, IL‐5, and IL‐13) by allergen‐specific Tu2004cells. We recently demonstrated a critical contribution of OX40 ligand (OX40L) to the development of Th2‐mediated experimental leishmaniasis. In this study, we have examined the role of OX40L in the development of Th2‐mediated pulmonary inflammation by utilizing OX40L‐deficient mice and a neutralizing anti‐OX40L mAb in a murine model of asthma. Sensitization and airway challenge with ovalbumin in wild‐type BALB/c mice induced a typical allergic asthma characterized by AHR, accumulation of eosinophils, increased mucus production, and high levels of Th2 cytokines in the lung.All these asthmatic responses were not induced in OX40L‐deficient BALB/c mice. Administration of neutralizing anti‐OX40L mAb in wild‐type BALB/c mice during the sensitization period also abolished the induction of asthmatic responses. In contrast, administration of anti‐OX40L mAb during the challenge period did not inhibit the asthmatic responses. These results indicate a critical role for OX40L in the induction phase, which leads to the development of pathogenic Th2 cells, but not in the effector phase, which includes migration and activation of pathogenic Th2 cells in the lung.

Critical Involvement of OX40 Ligand Signals in the T Cell Priming Events During Experimental Autoimmune Encephalomyelitis

OX40 ligand (OX40L) expressed on APCs, and its receptor, OX40 present on activated T cells, are members of the TNF/TNFR family, respectively, and have been located at the sites of inflammatory conditions. We have observed in OX40L-deficient mice (OX40L−/−) an impaired APC capacity and in our recently constructed transgenic mice expressing OX40L (OX40L-Tg), a markedly enhanced T cell response to protein Ags. Using these mice, we demonstrate here the critical involvement of the OX40L-OX40 interaction during the T cell priming events in the occurrence of experimental autoimmune encephalomyelitis (EAE). In OX40L−/− mice, abortive T cell priming greatly reduced the clinical manifestations of actively induced EAE, coupled with a reduction in IFN-γ, IL-2, and IL-6 production in vitro. Adoptive transfer experiments however revealed an efficient transfer of disease to OX40L−/− mice using wild-type donor T cells, indicating an intact capacity of OX40L−/− mice to initiate effector responses. On the other hand, OX40L−/− donor T cells failed to transfer disease to wild-type recipient mice. Furthermore, OX40L-Tg mice developed a greater severity of EAE despite a delayed onset, while both OX40L-Tg/CD28−/− and OX40L-Tg/CD40−/− mice failed to develop EAE demonstrating a requisite for these molecules. These findings indicate a pivotal role played by OX40L in the pathogenesis of EAE.

Constitutive OX40/OX40 Ligand Interaction Induces Autoimmune-Like Diseases

The interaction between OX40 and OX40 ligand (OX40L) is suggested to provide T cells with an effective costimulatory signals during T cell-APC interaction. To examine the in vivo effect of constitutive OX40/OX40L interaction during immune regulation, we report the establishment of OX40L-transgenic (OX40L-Tg) mice that constitutively express OX40L on T cells. Markedly elevated numbers of effector memory CD4+ T cells, but not CD8+ T cells, were observed in the secondary lymphoid organs of OX40L-Tg mice. Upon immunization with keyhole limpet hemocyanin in the absence of adjuvant, profound T cell proliferative responses and cytokine productions were seen in the OX40L-Tg mice as compared with wild-type mice. Furthermore, in OX40L-Tg mice administrated with superantigen, this constitutive OX40/OX40L interaction on CD4+ T cells completely prevented normal in vivo clonal T cell deletion. Interestingly, OX40L-Tg mice on the C57BL/6 background spontaneously developed interstitial pneumonia and inflammatory bowel disease that was accompanied with a significant production of anti-DNA Ab in the sera. Surprisingly, these diseases were not evident on the OX40L-Tg mice on the BALB/c strain. However, such inflammatory diseases were successfully reproducible in recombination-activating gene (RAG)2-deficient mice upon transfer of OX40L-Tg CD4+ T cells. Blockade of OX40/OX40L interaction in the recipient RAG2-deficient mice completely prevented disease development. The present results orchestrated in this study indicate that OX40/OX40L interaction may be a vital link in our understanding of T cell-mediated organ-specific autoimmunity.

OX40 (CD134) and OX40 ligand interaction plays an adjuvant role during in vivo Th2 responses

The role of OX40‐OX40 ligand (OX40L) interaction in Th cell differentiation remains contentious. In vitro studies have revealed a Th2‐biased effect by OX40 signals in T cells. However, in vivo studies demonstrated that OX40‐OX40L interaction is involved in responses either Th1 or Th2, or both, which appears to be dependent on the experimental conditions used. We document in our report Th cell differentiation in OX40L‐deficient and OX40L‐transgenic (Tg) mice in response to protein antigens (Ag) and to Leishmania major (L. major) infection. Upon immunization with protein Ag, we demonstrate the adjuvant effect of OX40 signals during in vivo Th2 responses. However, adjuvant treatment to mice ameliorates the Th2‐specific effect of OX40‐OX40L interaction and rather induces concurrent promotion of both Th1 and Th2 responses via OX40 signals. Thus, previous reports showing promotion of Th1 response by OX40‐OX40L interaction may in actual fact be affected by the adjuvant effects mediated by the various experimental conditions. Indeed, constitutive OX40–OX40L interactions in OX40L‐Tg mice converted the normally resistant C57BL/6 strain, into a susceptible status following L. major infection due to an extraordinary elevated Th2 response. These results provide convincing evidence demonstrating that the OX40‐OX40L interaction is paramount in the development of Th2 responses in vivo.

Ibudilast, a nonselective phosphodiesterase inhibitor, regulates Th1/Th2 balance and NKT cell subset in multiple sclerosis

We investigated the immunoregulatory effects of ibudilast, a nonselective phosphodiesterase inhibitor, at a clinically applicable dose (60 mg/day p.o. for four weeks) in multiple sclerosis (MS) patients. Sensitive real-time PCR for quantifying cytokine mRNA in the blood CD4- cells revealed that the ibudilast monotherapy significantly reduced tumour necrosis factor-a and interferon (IFN)-g mRNA and the IFN-g/interleukin-4 mRNA ratio, suggesting a shift in the cytokine profile from Th1 toward Th2 dominancy. In a flow cytometric analysis, natural killer T cells, which have been reported to relate to Th2 responses in MS and its animal model (experimental autoimmune encephalomyelitis), increased significantly after the therapy. None of the significant immunological changes were seen in healthy subjects or untreated MS patients. Ibudilast may be a promising therapy for MS and its clinical effects warrant further study.

Loss of Hippocampal CA3 Pyramidal Neurons in Mice Lacking STAM1

ABSTRACT STAM1, a member of the STAM (signal transducing adapter molecule) family, has a unique structure containing a Src homology 3 domain and ITAM (immunoreceptor tyrosine-based activation motif). STAM1 was previously shown to be associated with the Jak2 and Jak3 tyrosine kinases and to be involved in the regulation of intracellular signal transduction mediated by interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. Here we generated mice lacking STAM1 by using homologous recombination with embryonic stem cells. STAM1−/− mice were morphologically indistinguishable from their littermates at birth. However, growth retardation in the third week after birth was observed for the STAM1−/− mice. Unexpectedly, despite the absence of STAM1, hematopoietic cells, including T- and B-lymphocyte and other hematopoietic cell populations, developed normally and responded well to several cytokines, including IL-2 and GM-CSF. However, histological analyses revealed the disappearance of hippocampal CA3 pyramidal neurons in STAM1−/− mice. Furthermore, we observed that primary hippocampal neurons derived from STAM1−/− mice are vulnerable to cell death induced by excitotoxic amino acids or an NO donor. These data suggest that STAM1 is dispensable for cytokine-mediated signaling in lymphocytes but may be involved in the survival of hippocampal CA3 pyramidal neurons.

Expression of gp34(OX40 Ligand)and OX40 on Human T Cell Clones

gp34, which we previously cloned, is a ligand of OX40 (CD 134), a costimulatory molecule involved in T cell activation. To elucidate the role of human OX40/OX40L interaction, we examined the expression of gp34 (OX40L) and OX40 in normal human hematopoietic cells by using flow cytometry. OX40 expression is observed on activated T cells, while OX40L is expressed in antigen‐presenting cells. However, cytotoxic T lymphocyte (CTL) clones specific for Epstein‐Barr virus (EBV)‐transformed autologous lymphoblastic cell lines (LCLs) induced both OX40 and OX40L expression after antigen or T cell receptor (TCR) stimulation. This study suggests a possible function of OX40L/OX40, through T cell‐T cell interaction, in the reactivation of memory T cells in an auto‐crine manner, with implications for the pathogenesis of viral infections and neoplasms.

Consequences of OX40-OX40 ligand interactions in Langerhans cell function: enhanced contact hypersensitivity responses in OX40L-transgenic mice

Langerhans cells (LC) represent the dominant antigen‐presenting cells (APC) in the epidermis and thus play an important role in cutaneous immune responses to approaching pathogens. These responses are mediated by several costimulatory molecules after antigenic challenge. OX40 ligand (OX40L), a member of TNF superfamily, is expressed on several APC such as splenic dendritic cells (DC) andactivated B cells. This molecule has been reported to provide potent costimulation in APC‐T cell interactions upon binding to its cognate receptor, OX40, on activated T cells. Little is known, however, regarding OX40L expression and function on LC. In the present study, we report the expression of both OX40L and OX40 on differentiated LC derived from draining lymph nodes in the FITC‐sensitized mice. During contact hypersensitivity responses, OX40L‐deficient mice demonstrated a significant reduction in both hapten‐induced ear swelling and hapten‐specific T cell responses despite intact migratory responses. Conversely, these responses were markedly increased in two different OX40L‐transgenic strains with variations in OX40L overexpression. In the LC‐induced MLR, OX40L‐deficient and OX40L‐overexpressing LC were capable of reducing and elevating the responses of allogeneic CD4+ T cells, respectively. Thus the requirement of OX40L during the antigen presentation function of LC inT cell priming is here demonstrated.