Yasuko Tsunetsugu-Yokota

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4− and CD8−, but CD44+, CD25+ and cytoplasmic CD3+. This phenotype is indicative of a thymus-derived pre–T-cell phenotype, and disease development was associated with the constitutive activation of NF-κB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.

Yeast-derived human immunodeficiency virus type 1 p55(gag) virus-like particles activate dendritic cells (DCs) and induce perforin expression in Gag-specific CD8(+) T cells by cross-presentation of DCs.

ABSTRACT To evaluate the immunogenicity of human immunodeficiency virus (HIV) type 1 p55gag virus-like particles (VLPs) released by budding from yeast spheroplasts, we have analyzed the effects of yeast VLPs on monocyte-derived dendritic cells (DCs). Yeast VLPs were efficiently incorporated into DCs via both macropinocytosis and endocytosis mediated by mannose-recognizing receptors, but not the mannose receptor. The uptake of yeast VLPs induced DC maturation and enhanced cytokine production, notably, interleukin-12 p70. We showed that yeast membrane components may contribute to DC maturation partly through Toll-like receptor 2 signaling. Thus, Gag particles encapsulated by yeast membrane may have an advantage in stimulating Gag-specific immune responses. We found that yeast VLPs, but not the control yeast membrane fraction, were able to activate both CD4+ and CD8+ T cells of HIV-infected individuals. We tested the effect of cross-presentation of VLP by DCs in two subjects recruited into a long-term nonprogressor-slow progressor cohort. When yeast VLP-loaded DCs of these patients were cocultured with peripheral blood mononuclear cells for 7 days, approximately one-third of the Gag-specific CD8+ T cells were activated and became perforin positive. However, some of the Gag-specific CD8+ T cells appeared to be lost during in vitro culture, especially in a patient with a high virus load. Our results suggest that DCs loaded with yeast VLPs can activate Gag-specific memory CD8+ T cells to become effector cells in chronically HIV-infected individuals, but there still remain unresponsive Gag-specific T-cell populations in these patients.

Novel Nuclear Import of Vpr Promoted by Importin α Is Crucial for Human Immunodeficiency Virus Type 1 Replication in Macrophages

ABSTRACT Monocytes/macrophages are major targets of human immunodeficiency virus type 1 (HIV-1) infection. The viral preintegration complex (PIC) of HIV-1 enters the nuclei of monocyte-derived macrophages, but very little PIC migrates into the nuclei of immature monocytes. Vpr, one of the accessory gene products of HIV-1, is essential for the nuclear import of PIC in these cells, although the role of Vpr in the entry mechanism of PIC remains to be clarified. We have shown previously that Vpr is targeted to the nuclear envelope and then transported into the nucleus by importin α alone, in an importin β-independent manner. Here we demonstrate that the nuclear import of Vpr is strongly promoted by the addition of cytoplasmic extract from macrophages but not of that from monocytes and that the nuclear import activity is lost with immunodepletion of importin α from the cytoplasmic extract. Immunoblot analysis and real-time PCR demonstrate that immature monocytes express importin α at low levels, whereas the expression of three major importin α isoforms markedly increases upon their differentiation into macrophages, indicating that the expression of importin α is required for nuclear import of Vpr. Furthermore, interaction between importin α and the N-terminal α-helical domain of Vpr is indispensable, not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages. This study suggests the possibility that the binding of Vpr to importin α, preceding a novel nuclear import process, is a potential target for therapeutic intervention.

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

Background In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor κB (NF-κB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-κB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed. Principal Findings Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-κB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFβ-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-κB activation, were not essential for RLH-mediated NF-κB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-κB and IRF7. Conclusions/Significance Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

Abstract The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-γ and TNF-α) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.

Differentiation associated regulation of microRNA expression in vivo in human CD8+ T cell subsets.

BackgroundThe differentiation of CD8+ T lymphocytes following priming of naïve cells is central in the establishment of the adaptive immune response. Yet, the molecular events underlying this process are not fully understood. MicroRNAs have been recently shown to play a key role in the regulation of haematopoiesis in mouse, but their implication in peripheral lymphocyte differentiation in humans remains largely unknown.MethodsIn order to explore the potential implication of microRNAs in CD8+ T cell differentiation in humans, microRNA expression profiles were analysed using microarrays and quantitative PCR in several human CD8+ T cell subsets defining the major steps of the T cell differentiation pathway.ResultsWe found expression of a limited set of microRNAs, including the miR-17~92 cluster. Moreover, we reveal the existence of differentiation-associated regulation of specific microRNAs. When compared to naive cells, miR-21 and miR-155 were indeed found upregulated upon differentiation to effector cells, while expression of the miR-17~92 cluster tended to concomitantly decrease.ConclusionsThis study establishes for the first time in a large panel of individuals the existence of differentiation associated regulation of microRNA expression in human CD8+ T lymphocytes in vivo, which is likely to impact on specific cellular functions.

Monocyte-derived dendritic cells represent a transient stage of differentiation in the myeloid lineage

Cultivation of human peripheral blood monocytes with granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-4 facilitates generation of strongly antigen-presenting dendritic cells (DC). These monocyte-derived DC (mdDC) were used here to further delineate differentiation pathways in the myeloid lineage. Incubation of mdDC with TNF or soluble CD40L led to enhanced MHC and accessory surface antigen expression with significantly elevated T cell stimulatory activity, indicative of DC maturation. In contrast, after cytokine withdrawal or incubation with M-CSF, mdDC differentiated to macrophages. Cells became adherent, monocyte/macrophage surface markers were upregulated, and MHC and accessory surface proteins were downregulated. Furthermore, the multilaminar MHC class II compartments (MIIC) were lost and the T cell stimulating capacity largely diminished. Thus, mdDC show a high developmental plasticity by retaining their ability to become macrophages or to continue their differentiation towards mature DC.

Selective transmission of R5 HIV-1 over X4 HIV-1 at the dendritic cell-T cell infectious synapse is determined by the T cell activation state.

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of T cell immunity against HIV. On the other hand, due to the susceptibility of DCs to HIV infection, virus replication is strongly enhanced in DC–T cell interaction via an immunological synapse formed during the antigen presentation process. When HIV-1 is isolated from individuals newly infected with the mixture of R5 and X4 variants, R5 is predominant, irrespective of the route of infection. Because the early massive HIV-1 replication occurs in activated T cells and such T-cell activation is induced by antigen presentation, we postulated that the selective expansion of R5 may largely occur at the level of DC–T cell interaction. Thus, the immunological synapse serves as an infectious synapse through which the virus can be disseminated in vivo. We used fluorescent recombinant X4 and R5 HIV-1 consisting of a common HIV-1 genome structure with distinct envelopes, which allowed us to discriminate the HIV-1 transmitted from DCs infected with the two virus mixtures to antigen-specific CD4+ T cells by flow cytometry. We clearly show that the selective expansion of R5 over X4 HIV-1 did occur, which was determined at an early entry step by the activation status of the CD4+ T cells receiving virus from DCs, but not by virus entry efficiency or productivity in DCs. Our results imply a promising strategy for the efficient control of HIV infection.

Polyfunctional CD4+ T-Cell Induction in Neutralizing Antibody-Triggered Control of Simian Immunodeficiency Virus Infection

ABSTRACT Rapid depletion of memory CD4+ T cells and delayed induction of neutralizing antibody (NAb) responses are characteristics of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. Although it was speculated that postinfection NAb induction could have only a limited suppressive effect on primary HIV replication, a recent study has shown that a single passive NAb immunization of rhesus macaques 1 week after SIV challenge can result in reduction of viral loads at the set point, indicating a possible contribution of postinfection NAb responses to virus control. However, the mechanism accounting for this NAb-triggered SIV control has remained unclear. Here, we report rapid induction of virus-specific polyfunctional T-cell responses after the passive NAb immunization postinfection. Analysis of SIV Gag-specific responses of gamma interferon, tumor necrosis factor alpha, interleukin-2, macrophage inflammatory protein 1β, and CD107a revealed that the polyfunctionality of Gag-specific CD4+ T cells, as defined by the multiplicity of these responses, was markedly elevated in the acute phase in NAb-immunized animals. In the chronic phase, despite the absence of detectable NAbs, virus control was maintained, accompanied by polyfunctional Gag-specific T-cell responses. These results implicate virus-specific polyfunctional CD4+ T-cell responses in this NAb-triggered virus control, suggesting possible synergism between NAbs and T cells for control of HIV/SIV replication.

Distinct fucosylation of M cells and epithelial cells by Fut1 and Fut2, respectively, in response to intestinal environmental stress

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyers patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells.