Kazuko Murata

Distinct Roles for the OX40-OX40 Ligand Interaction in Regulatory and Nonregulatory T Cells

The OX40 (CD134) molecule is induced primarily during T cell activation and, as we show in this study, is also expressed on CD25+CD4+ regulatory T (Treg) cells. A necessary role for OX40 in the development and homeostasis of Treg cells can be inferred from the reduced numbers of the cells present in the spleens of OX40-deficient mice, and their elevated numbers in the spleens of mice that overexpress the OX40 ligand (OX40L). The homeostatic proliferation of Treg cells following transfer into lymphopenic mice was also found to be potentiated by the OX40-OX40L interaction. Suppression of T cell responses by Treg cells was significantly impaired in the absence of OX40, indicating that, in addition to its homeostatic functions, OX40 contributes to efficient Treg-mediated suppression. However, despite this, we found that CD25−CD4+ T cells became insensitive to Treg-mediated suppression when they were exposed to OX40L-expressing cells, or when they were treated with an agonistic OX40-specific mAb. OX40 signaling could also abrogate the disease-preventing activity of Treg cells in an experimental model of inflammatory bowel disease. Thus, although the data reveal important roles for OX40 signaling in Treg cell development, homeostasis, and suppressive activity, they also show that OX40 signals can oppose Treg-mediated suppression when they are delivered directly to Ag-engaged naive T cells.

Hgs (Hrs), a FYVE Domain Protein, Is Involved in Smad Signaling through Cooperation with SARA

ABSTRACT Smad proteins are effector molecules that transmit signals from the receptors for the transforming growth factor β (TGF-β) superfamily to the nucleus; of the Smad proteins, Smad2 and Smad4 are essential components for mouse early embryogenesis. We demonstrated that Hgs, a FYVE domain protein, binds to Smad2 in its C-terminal half and cooperates with another FYVE domain protein, the Smad anchor for receptor activation (SARA), to stimulate activin receptor-mediated signaling through efficient recruitment of Smad2 to the receptor. Furthermore, a LacZ knock-in allele of the C-terminal half-deletion mutant of mouse Hgs was created by gene targeting. The introduced mutation causes an embryonic lethality between embryonic days 8.5 and 10.5. Mutant cells showed significantly decreased responses to stimulation with activin and TGF-β. These findings suggest that the two FYVE domain proteins, Hgs and SARA, are prerequisites for receptor-mediated activation of Smad2.

STAM, Signal Transducing Adaptor Molecule, Is Associated with Janus Kinases and Involved in Signaling for Cell Growth and c-myc Induction

We previously identified a putative signal transducing adaptor molecule, named STAM, that contains an Src homology 3 (SH3) domain and immunoreceptor tyrosine-based activation motif (ITAM). In this report, we demonstrate the functional significance of STAM in cytokine-mediated signal transduction. STAM is associated with Jak3 and Jak2 tyrosine kinases via its ITAM region and phosphorylated by Jak3 and Jak2 upon stimulation with IL-2 and GM-CSF, respectively. An SH3 deletion mutant of STAM confers a dominant-negative effect on DNA synthesis mediated by IL-2 and GM-CSF. Furthermore, the wild-type STAM, but not STAM mutants deleted of SH3 and ITAM, significantly enhances c-myc induction mediated by IL-2 and GM-CSF. These results strongly implicate STAM in the signaling pathways for cell growth and c-myc induction immediately downstream of the Jaks associated with the cytokine receptors.

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.

Inhibition of Tumor Growth and Metastasis by Depletion of Vesicular Sorting Protein Hrs: Its Regulatory Role on E-Cadherin and β-Catenin

Abnormally high signals from receptor tyrosine kinases (RTK) are associated with carcinogenesis, and impaired deactivation of RTKs may also be a mechanism in cancer. Hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs) is one of the master regulators that sort activated receptors toward lysosomes and shut down their signals. Hrs contains a ubiquitin-interacting motif and is involved in the endosomal sorting of monoubiquitinated membrane proteins, such as growth factor receptor and E-cadherin. Here, we investigated the role of Hrs in determining the malignancy of cancer cells and discovered that the targeted disruption of Hrs by small interfering RNA effectively attenuated the proliferation, anchorage-independent growth, tumorigenesis, and metastatic potential of HeLa cells in vitro and in vivo . The restoration of Hrs expression increased cell proliferation and anchorage-independent growth in a mouse embryonic fibroblast line established from a Hrs knockout mouse. Further analysis revealed that Hrs depletion was associated with the up-regulation of E-cadherin and reduced β-catenin signaling. The aberrant accumulation of E-cadherin most likely resulted from impaired E-cadherin degradation in lysosomes. These results suggest that Hrs may play a critical role in determining the malignancy of cancer cells by regulating the degradation of E-cadherin. [Cancer Res 2007;67(11):5162–71]

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.

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.

Signal-Transducing Adaptor Molecules STAM1 and STAM2 Are Required for T-Cell Development and Survival

ABSTRACT We previously reported that the STAM family members STAM1 and STAM2 are phosphorylated on tyrosine upon stimulation with cytokines through the γc-Jak3 signaling pathway, which is essential for T-cell development. Mice with targeted mutations in either STAM1 or STAM2 show no abnormality in T-cell development, and mice with double mutations for STAM1 and STAM2 are embryonically lethal; therefore, here we generated mice with T-cell-specific double mutations for STAM1 and STAM2 using the Cre/loxP system. These STAM1−/− STAM2−/− mice showed a significant reduction in thymocytes and a profound reduction in peripheral mature T cells. In proliferation assays, thymocytes derived from the double mutant mice showed a defective response to T-cell-receptor (TCR) stimulation by antibodies and/or cytokines, interleukin-2 (IL-2) and IL-7. However, signaling events downstream of receptors for IL-2 and IL-7, such as activations of STAT5, extracellular signal-regulated kinase (ERK), and protein kinase B (PKB)/Akt, and c-myc induction, were normal in the double mutant thymocytes. Upon TCR-mediated stimulation, prolonged activations of p38 mitogen-activated protein kinase and Jun N-terminal protein kinase were seen, but activations of ERK, PKB/Akt, and intracellular calcium flux were normal in the double mutant thymocytes. When the cell viability of cultured thymocytes was assessed, the double mutant thymocytes died more quickly than controls. These results demonstrate that the STAMs are indispensably involved in T-cell development and survival in the thymus through the prevention of apoptosis but are dispensable for the proximal signaling of TCR and cytokine receptors.