Toshiyuki Hori

TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand

We recently showed that dendritic cells (DCs) activated by thymic stromal lymphopoietin (TSLP) prime naive CD4+ T cells to differentiate into T helper type 2 (Th2) cells that produced high amounts of tumor necrosis factor-α (TNF-α), but no interleukin (IL)-10. Here we report that TSLP induced human DCs to express OX40 ligand (OX40L) but not IL-12. TSLP-induced OX40L on DCs was required for triggering naive CD4+ T cells to produce IL-4, -5, and -13. We further revealed the following three novel functional properties of OX40L: (a) OX40L selectively promoted TNF-α, but inhibited IL-10 production in developing Th2 cells; (b) OX40L lost the ability to polarize Th2 cells in the presence of IL-12; and (c) OX40L exacerbated IL-12–induced Th1 cell inflammation by promoting TNF-α, while inhibiting IL-10. We conclude that OX40L on TSLP-activated DCs triggers Th2 cell polarization in the absence of IL-12, and propose that OX40L can switch IL-10–producing regulatory Th cell responses into TNF-α–producing inflammatory Th cell responses.

Plasmacytoid Dendritic Cells Regulate Th Cell Responses through OX40 Ligand and Type I IFNs

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-α in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-γ-producing Th cells depending on their capacity to residually produce IFN-α. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-α-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.

Thymic stromal lymphopoietin fosters human breast tumor growth by promoting type 2 inflammation

TSLP released from human breast cancer cells promotes OX40L expression on DCs, and these OX40L-expressing DCs drive development of inflammatory Th2 cells which promote breast tumor development.

Activation of OX40 Signal Transduction Pathways Leads to Tumor Necrosis Factor Receptor-associated Factor (TRAF) 2- and TRAF5-mediated NF-κB Activation

We investigated the intracellular signaling of OX40, a member of the tumor necrosis factor receptor family. Activation of NF-κB in OX40-transfected HSB-2 cells was detected by electrophoretic mobility shift assay within 30 min after the binding of the ligand gp34. In vitro binding experiments showed that tumor necrosis factor receptor-associated factor (TRAF) 1, TRAF2, TRAF3, and TRAF5 but not TRAF4 associated with glutathioneS-transferase-OX40 fusion protein. The cotransfection experiments using human embryo kidney cell derived HEK 293T cells showed that TRAF2, TRAF3, and TRAF5 associated with OX40 in vivo. Studies with OX40 deletion mutants demonstrated that the cytoplasmic portion consisting of amino acid sequence 256–263 (GGSFRTPI) was required for the association with TRAFs and NF-κB activation. The introduction of the dominant negative mutants of TRAF2 and TRAF5 into HSB-2-OX40 cells suppressed NF-κB activation in a dose-dependent manner. In addition, the introduction of TRAF3 together with the dominant negative mutants of TRAF2 or TRAF5 further reduced NF-κB activation. These results indicate that the NF-κB activation resulting from OX40 stimulation is mediated by both TRAF2 and TRAF5, and is likely to be negatively modulated by TRAF3.

Natural Alpha Interferon-Producing Cells Respond to Human Immunodeficiency Virus Type 1 with Alpha Interferon Production and Maturation into Dendritic Cells

ABSTRACT Natural alpha interferon (IFN-α)-producing cells (IPCs) are now recognized as identical to plasmacytoid dendritic cell (DC) precursors in human blood and are thought to play an important role in antiviral immunity. In the present study, we examined the susceptibility as well as the cellular responses of IPCs to human immunodeficiency virus type 1 (HIV-1) infection. HLA-DR+ CD11c− lineage-negative cells (IPCs) were purified from peripheral blood mononuclear cells by magnetic-bead separation and cell sorting. We substantiated that IPCs expressing the major HIV-1 coreceptors, CXCR4 and CCR5, are susceptible to infection of both T-cell-line-tropic NL4-3 and macrophage-tropic JR-CSF HIV-1 by quantification of HIV-1 p24 in the culture supernatants and by provirus integration assay using human conserved Alu-HIV-1 long terminal repeat PCR. To evaluate the cellular response of IPCs to HIV-1, we examined IFN-α production and their differentiation into DCs. After incubation with either NL4-3 or JR-CSF, IPCs produced a large amount of IFN-α and at the same time underwent morphological differentiation into DCs with upregulation of CD80 and CD86. Heat inactivation of the supernatants containing HIV-1 did not affect the IFN-α production and maturation, whereas removal of virions by ultracentrifugation completely nullified both biological effects, indicating that these cellular responses do not require actual HIV-1 infection but are elicited by interaction with HIV-1 virions or certain viral components. In conclusion, these data strongly suggest that IPC can directly recognize and respond to HIV-1 with IFN-α production, which is crucial for preventing progress of HIV-1 infection and occurrence of opportunistic infection.

Inhibition of Cell Growth by Conditional Expression of kpm, a Human Homologue of Drosophila warts/lats Tumor Suppressor

kpm is a human serine/threonine kinase that is homologous to Drosophila tumor suppressor warts/lats and its mammalian homologue LATS1. In order to define the biological function of kpm, we generated stable transfectants of wild-type kpm (kpm-wt), a kinase-dead mutant ofkpm (kpm-kd), and luciferase in HeLa Tet-Off cells under the tetracycline-responsive promoter. Western blot analysis showed that high levels of expression of kpm-wt as well as kpm-kd with an apparent mass of 150 kDa were induced after the removal of doxycycline. Induction of kpm-wt expression resulted in a marked decline in viable cell number measured by both trypan blue dye exclusion and MTT assay, whereas that of kpm-kd or luciferase had no effect. We then analyzed the cell cycle progression and apoptosis upon induction of kpm expression. 2–3 days after removal of doxycycline, cells underwent G2/M arrest, demonstrated by flow cytometric analysis of propidium iodide incorporation and MPM-2 reactivity. In vitro kinase assay showed that induction of kpm-wt led to down-regulation of kinase activity of the Cdc2-cyclin B complex, which was accompanied by an increase in the hyperphosphorylated form of Cdc2 and a change of phosphorylation status of Cdc25C. Furthermore, both DAPI staining and TUNEL assay showed that the proportion of apoptotic cells increased as kpm expression was induced. Taken together, these results indicate that kpm negatively regulates cell growth by inducing G2/M arrest and apoptotic cell death through its kinase activity.

Molecular cloning of a novel human protein kinase, kpm, that is homologous to warts/lats, a Drosophila tumor suppressor

A novel human protein kinase, designated kpm, was identified and molecularly cloned. The isolated cDNA clone had an open reading frame consisting of 1088 amino acid residues with a putative kinase domain located near the carboxy-terminus. Homology search revealed that kpm belongs to a subfamily of serine/threonine protein kinases including warts/lats, a Drosophila tumor suppressor. Among these, kpm is most homologous to, but distinct from, recently reported LATS1, a human homolog of Drosophila warts/lats. Northern blot analysis disclosed that kpm is expressed as a 6.0 kb transcript in most of the tissues examined and also as an additional shorter 4.0 kb transcript in testis. Western blotting using polyclonal rabbit anti-kpm antibody detected kpm protein as a band with an apparent Mr of 150 kD. Immune complex kinase assay of HA-tagged kpm showed that kpm had kinase activity and phosphorylated itself in vitro. Studies with synchronized HeLa cells indicated that kpm protein was expressed relatively constantly throughout the cell cycle and underwent significant phosphorylation at mitotic phase. These results suggest that kpm plays a role in cell cycle progression during mitosis and its deletion or dysfunction might be involved in certain types of human cancers.

Phenotypic and functional relationship between adult T-cell leukemia cells and regulatory T cells

Phenotypic and functional relationship between adult T-cell leukemia cells and regulatory T cells

Signaling of gp34 (OX40 ligand) induces vascular endothelial cells to produce a CC chemokine RANTES/CCL5.

We previously showed that gp34 (OX40 ligand) expressed on vascular endothelial cells is not only involved in adhesion between activated T cells and endothelial cells but also by itself able to transmit intracellular signals leading to expression of c-fos and c-jun mRNA upon OX40 binding. In the present study, we searched for genes that were induced or upregulated by gp34 signaling in human umbilical vein endothelial cells (HUVECs) to define its downstream biological events. HUVECs expressing high levels of gp34 were stimulated with recombinant soluble OX40 or mock control and subjected to analysis using cDNA expression arrays. We found that a CC chemokine RANTES (regulated upon activation, normal T cell expressed and secreted)/CCL5 is one of such inducible genes. Reverse transcriptase-PCR analysis showed that expression of RANTES mRNA was induced after incubation with soluble OX40 and this induction was inhibited by anti-gp34 mAb. We could detect expression of intracellular RANTES protein by flow cytometry in HUVECs stimulated with soluble OX40 as well as fixed OX40 transfectant cells but not those stimulated with mock supernatants or mock transfectant cells. Again, this induction of RANTES protein was inhibited by anti-gp34 mAb. These results clearly indicate that gp34 signaling induces expression of RANTES at both mRNA and protein levels in HUVECs and suggest a possible link between the OX40/gp34 system and RANTES during the process of T cell adhesion to endothelial cells and subsequent extravasation.

Atrial Natriuretic Peptide Polarizes Human Dendritic Cells Toward a Th2-Promoting Phenotype Through Its Receptor Guanylyl Cyclase-Coupled Receptor A

Atrial natriuretic peptide (ANP) is a cardiovascular hormone secreted mainly by the cardiac atria and regulates the volume-pressure homeostasis. The action of ANP is mediated by its receptor, guanylyl cyclase-coupled receptor A (GC-A). In this study, we explored the possibility that ANP and GC-A may play a role in the dendritic cell (DC)-mediated immune regulation. We first examined the expression of GC-A in human monocyte-derived DCs in comparison with monocytes and found that DCs but not monocytes express GC-A at both the mRNA and protein levels. DCs responded to ANP with an increase in intracellular cGMP in a dose-dependent manner, indicating that GC-A expressed on DCs is functional. Furthermore, treatment of DCs with ANP decreased production of IL-12 and TNF-α and conversely increased that of IL-10 upon stimulation with LPS. In accordance with this change of cytokine production, DCs treated with ANP plus LPS promoted differentiation of naive CD4+ T cells into a Th2 phenotype. Finally, we presented evidence that ANP affected cytokine production of fresh whole blood stimulated with LPS in line with the above-mentioned results. These results indicate that ANP polarizes human DCs toward a Th2-promoting phenotype through GC-A and thus can regulate immune responses.