Kazuo Terashima

A New Humanized Mouse Model of Epstein-Barr Virus Infection That Reproduces Persistent Infection, Lymphoproliferative Disorder, and Cell-Mediated and Humoral Immune Responses

The functional human immune system, including T, B, and natural killer lymphocytes, is reconstituted in NOD/Shi-scid/IL-2Rgamma(null) (NOG) mice that receive hematopoietic stem cell transplants. Here, we show that these humanized mice can recapitulate key aspects of Epstein-Barr virus (EBV) infection in humans. Inoculation with approximately 1 x 10(3) TD(50) (50% transforming dose) of EBV caused B cell lymphoproliferative disorder, with histopathological findings and latent EBV gene expression remarkably similar to that in immunocompromised patients. Inoculation with a low dose of virus (<or=1 x 10(1) TD(50)), in contrast, resulted in apparently asymptomatic persistent infection. Levels of activated CD8(+) T cells increased dramatically in the peripheral blood of infected mice, and enzyme-linked immunospot assay and flow cytometry demonstrated an EBV-specific T cell response. Immunoglobulin M antibody specific to the EBV-encoded protein BFRF3 was detected in serum from infected mice. The NOG mouse is the most comprehensive small-animal model of EBV infection described to date and should facilitate studies of the pathogenesis, prevention, and treatment of EBV infection.

Rapid Tumor Formation of Human T-Cell Leukemia Virus Type 1-Infected Cell Lines in Novel NOD-SCID/γcnull Mice: Suppression by an Inhibitor against NF-κB

ABSTRACT We established a novel experimental model for human T-cell leukemia virus type 1 (HTLV-1)-induced tumor using NOD-SCID/γcnull (NOG) mice. This model is very useful for investigating the mechanism of tumorigenesis and malignant cell growth of adult T-cell leukemia (ATL)/lymphoma, which still remains unclear. Nine HTLV-1-infected cell lines were inoculated subcutaneously in the postauricular region of NOG mice. As early as 2 to 3 weeks after inoculation, seven cell lines produced a visible tumor while two transformed cell lines failed to do so. Five of seven lines produced a progressively growing large tumor with leukemic infiltration of the cells in various organs that eventually killed the animals. Leukemic cell lines formed soft tumors, whereas some transformed cell lines developed into hemorrhagic hard tumors in NOG mice. One of the leukemic cell lines, ED-40515(−), was unable to produce visible tumors in NOD-SCID mice with a common γ-chain after 2 weeks. In vivo NF-κB DNA binding activity of the ED-40515(−) cell line was higher and the NF-κB components were changed compared to cells in vitro. Bay 11-7082, a specific and effective NF-κB inhibitor, prevented tumor growth at the sites of the primary region and leukemic infiltration in various organs of NOG mice. This in vivo model of ATL could provide a novel system for use in clarifying the mechanism of growth of HTLV-1-infected cells as well as for the development of new drugs against ATL.

Humanized NOD/SCID/IL2Rγnull Mice Transplanted with Hematopoietic Stem Cells under Nonmyeloablative Conditions Show Prolonged Life Spans and Allow Detailed Analysis of Human Immunodeficiency Virus Type 1 Pathogenesis

ABSTRACT In a previous study, we demonstrated that humanized NOD/SCID/IL2Rγnull (hNOG) mice constructed with human hematopoietic stem cells (HSCs) allow efficient human immunodeficiency virus type 1 (HIV-1) infection. However, HIV-1 infection could be monitored for only 43 days in the animals due to their short life spans. By transplanting HSCs without any myeloablation methods, the mice successfully survived longer than 300 days with stable engraftment of human cells. The mice showed high viremia state for more than the 3 months examined, with systemic HIV-1 infection and gradual decrease of CD4+ T cells analogous to that in humans. These capacities of the hNOG mice are very attractive for modeling mechanisms of AIDS progression and therapeutic strategy.

T Cell-Mediated Control of Epstein-Barr Virus Infection in Humanized Mice

Humanized NOD/Shi-scid/interleukin-2Rgamma(null) (NOG) mice with full T cell development had significantly longer life span after Epstein-Barr virus (EBV) infection, compared with those with minimal T cell development. Removing CD3(+) or CD8(+) T cells from EBV-infected humanized mice by administration of anti-CD3 or anti-CD8 antibodies reduced their life span. CD8(+) T cells obtained from EBV-infected mice suppressed the outgrowth of autologous B cells isolated from uninfected mice and inoculated with EBV in vitro. These results indicate that humanized NOG mice are capable of T cell-mediated control of EBV infection and imply their usefulness as a tool to evaluate immunotherapeutic and prophylactic strategies for EBV infection.

Prompt tumor formation and maintenance of constitutive NF-κB activity of multiple myeloma cells in NOD/SCID/γCnull mice

Clinically and biologically relevant animal models are indispensable to evaluate both the pathophysiology and strategies for diagnosis and treatment of multiple myeloma (MM). We examined the tumorigenicity of MM cell lines KMM‐1 and U‐266 in an in vivo cell proliferation model using NOD/SCID/γCnull (NOG) mice. Two cell lines were inoculated either subcutaneously (s.c.) in the post‐auricular region or intravenously (i.v.) in the tail of NOG mice. The KMM‐1 cell line produced a progressively growing large tumor with infiltration of the cells expressing human λ‐chain in various organs of all NOG mice, while the U‐266 cell line failed to do so. Tumor cells grown in NOG mice maintained the original histomorphology, as well as expression patterns of tumor markers human λ, Ig light chain and VEGF. Tumor progression in mice also correlated with elevation of serum human soluble IL‐6R and gp130. Tumor cells sustained a strong NF‐κB activity in vivo and induced NF‐κB components were indistinguishable from those in cells cultured in vitro. The rapid and efficient engraftment of the MM cell line in NOG mice suggests that this is a very useful animal model which could provide a novel system in which to clarify the mechanism of growth of cancer cells, as well as to develop new therapeutic regimens against MM.

A Monoclonal Antibody to the α2 Domain of Murine Major Histocompatibility Complex Class I that Specifically Kills Activated Lymphocytes and Blocks Liver Damage in the Concanavalin A Hepatitis Model

We earlier found that a rat monoclonal antibody (mAb) RE2 can induce rapid death of murine activated, but not resting, lymphocytes and lymphocyte cell lines, in a complement-independent manner, a cell death differing from typical apoptosis or necrosis. We here found that this cell death is independent of pathways involving Fas, caspase, and phosphoinositide-3 kinase. With the advantage of producing human B cell line transfectants with stable expression of human/mouse xeno-chimeric MHC class I genes, we found that RE2 epitope resides on the murine class I α2 domain. However, the α3 domain plays a key role in transducing the death signal, which mediates extensive aggregation of the MHC class I-integrin-actin filament system, giving rise to membrane blebs and pores. In mouse models with T/NKT cell activation-associated fulminant hepatitis, administration of mAb RE2 almost completely inhibited the development of liver cell injuries. Taken collectively, this form of cell death may be involved in homeostatic immune regulation, and induction of this form of cell death using the mAbs may be potentially therapeutic for subjects with immunological diseases mediated by activated lymphocytes.

Hodgkin's lymphoma cells are efficiently engrafted and tumor marker CD30 is expressed with constitutive nuclear factor-κB activity in unconditioned NOD/SCID/γcnull mice

As there are very few reproducible animal models without conditioning available for the study of human B‐cell‐type Hodgkins lymphoma (HL), we investigated the ability of HL cells to induce tumors using novel NOD/SCID/γcnull (NOG) mice. Four human Epstein–Barr virus‐negative cell lines (KM‐H2 and L428 originated from B cells, L540 and HDLM2 originated from T cells) were inoculated either subcutaneously in the postauricular region or intravenously in the tail of unmanipulated NOG mice. All cell lines successfully engrafted and produced tumors with infiltration of cells in various organs of all mice. Tumor cells had classical histomorphology as well as expression patterns of the tumor marker CD30, which is a cell surface antigen expressed on HL. Tumor progression in mice inoculated with B‐cell‐type, but not T‐cell‐type, HL cells correlated with an elevation in serum human interleukin‐6 levels. Tumor cells from the mice also retained strong nuclear factor (NF)‐κB DNA binding activity, and the induced NF‐κB components were indistinguishable from those cultured in vitro. The reproducible growth behavior and preservation of characteristic features of both B‐cell‐type and T‐cell‐type HL in the mice suggest that this new xenotransplant model can provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth, and to develop novel anticancer therapies. (Cancer Sci 2005; 96: 466–473)

Role of Follicular Dendritic Cells in the Early HIV-1 Infection: In vitro Model without Specific Antibody

About 90% of HIV‐1 RNA in the lymph nodes is reported to localize in follicular dendritic cells‐network (FDC‐NW) as early as several days after infection and as much as that in the late stage. But the mechanism remains to be fully understood. To elucidate the role of follicular dendritic cells (FDC) in the early stage of HIV‐1 infection, FDC‐like cell strains (FDCLC) were established and they were characterized in the co‐culture system with T cells for their effect on HIV‐1 trapping and replication in p24 immunoassay, immunohistochemistry as well as confocal and electronmicroscopy. Established FDCLC were positive for CNA‐42, S‐100α and intercellular desmosome‐like junctions. L‐SIGN and DC‐SIGN were also detected in FDCLC. Alu‐HIV‐1 PCR analysis showed no HIV‐1 integration in FDCLC. FDCLC trapped HIV‐1 and transferred them to uninfected MOLT‐4 T cells (MOLT‐4) efficiently in the absence of specific antibody. FDCLC also accelerated HIV‐1 replication in HIV‐1‐pre‐exposed MOLT‐4. These unique FDCLC effects were explained, at least partly, by the fact that FDCLC up‐regulated CD4 expression in MOLT‐4 and helped T cells escape from apoptosis in the co‐culture. These data suggest that FDC/FDCLC engage not only in trapping but also in active expansion of HIV‐1 in the absence of specific antibody.

Follicular Dendritic Cells Activate HIV-1 Replication in Monocytes/Macrophages through a Juxtacrine Mechanism Mediated by P-Selectin Glycoprotein Ligand 1

Follicular dendritic cells (FDCs) are located in the lymphoid follicles of secondary lymphoid tissues and play a pivotal role in the selection of memory B lymphocytes within the germinal center, a major site for HIV-1 infection. Germinal centers are composed of highly activated B cells, macrophages, CD4+T cells, and FDCs. However, the physiological role of FDCs in HIV-1 replication remains largely unknown. We demonstrate in our current study that FDCs can efficiently activate HIV-1 replication in latently infected monocytic cells via an intercellular communication network mediated by the P-selectin/P-selectin glycoprotein ligand 1 (PSGL-1) interaction. Upon coculture with FDCs, HIV-1 replication was significantly induced in infected monocytic cell lines, primary monocytes, or macrophages. These cocultures were found to synergistically induce the expression of P-selectin in FDCs via NF-κB activation and its cognate receptor PSGL-1 in HIV-1-infected cells. Consistent with this observation, we find that this response is significantly blocked by antagonistic Abs against PSGL-1 and almost completely inhibited by PSGL-1 small interfering RNA. Moreover, a selective inhibitor for Syk, which is a downstream effector of PSGL-1, blocked HIV-1 replication in our cultures. We have thus elucidated a novel regulatory mechanism in which FDCs are a potent positive bystander that facilitates HIV-1 replication in adjacent infected monocytic cells via a juxtacrine signaling mechanism.

Follicular dendritic cell sarcoma with microtubuloreticular structure and virus-like particle production in vitro.

Neoplasm of follicular dendritic cells (FDC), follicular dendritic cell sarcoma (FDCS), is a rare tumor of intermediate to high‐grade malignancy in lymph nodes and visceral organs. Reported herein is a case of FDCS arising from cervical lymph nodes in a 16‐year‐old Japanese boy, who died of the disease 3 years after diagnosis. The tumor cells were pale eosinophilic and elongated with euchromatic nuclei and were positive for CD21, clusterin, and CNA‐42 on immunohistochemistry, as well as desmosome‐like junctions on electron microscopy. The presence of microtubuloreticular structures (MTRS) in the tumor cells and associated lymphocytes characterized this case, suggesting some viral infection, although qualitative polymerase chain reaction of genomic and complementary DNA obtained from the tumor failed to demonstrate any viral infection at the laboratory level. The stimulation of dispersed tumor cells and peripheral blood mononuclear cells with mAb to CD3 and interleukin‐2 was attempted; and the cell line established by the authors (FDCS‐Sa) was stimulated with iododeoxyuridine. Virus‐like particles (VLP) were successfully induced from each cellular source. The VLP, 100 nm in diameter, showed an electron‐dense thorny envelope and granular core. This is the first case of FDCS with MTRS accompanying VLP production in vitro.