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Journal of Virology, May 2009, p. 5101-5108, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02564-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Stromal Cell-Mediated Suppression of Human T-Cell Leukemia Virus Type 1 Expression In Vitro and In Vivo by Type I Interferon{triangledown}

Shuichi Kinpara,1 Atsuhiko Hasegawa,1 Atae Utsunomiya,2 Hironori Nishitsuji,1 Hiroyuki Furukawa,1 Takao Masuda,1 and Mari Kannagi1*

Department of Immunotherapeutics, Tokyo Medical and Dental University, Graduate School, Tokyo 113-8519,1 Department of Hematology, Imamura Bun-in Hospital, Kagoshima 890-0064, Japan2

Received 12 December 2008/ Accepted 13 February 2009


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ABSTRACT
 
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis, and other inflammatory diseases. Despite such severe outcomes of HTLV-1 infection, the level of HTLV-1 expression in vivo is very low and rapidly increases after transfer of cells to culture conditions. The mechanisms of this phenomenon have remained obscure. In the present study, we found that human and mouse stromal cells, such as epithelial cells and fibroblasts, suppressed HTLV-1 expression in ATL and non-ATL HTLV-1-infected cells. HTLV-1 mRNA and proteins in HTLV-1-infected cells markedly decreased upon coculture with human epithelial-like cells (HEK293T) or mouse embryo fibroblasts (NIH 3T3). When infected cells were reisolated from the cocultures, viral expression was restored to the original level over the following 48 h. Spontaneous induction of HTLV-1 expression in primary ATL cells in the first 24 h of culture was also inhibited by coculture with HEK293T cells. Coculture of HTLV-1-infected cells and HEK293T cells induced type I interferon responses, as detected by beta interferon (IFN-β) promoter activation and IFN-stimulated gene upregulation. HEK293T-mediated suppression of HTLV-1 expression was partly inhibited by antibodies to human IFN-{alpha}/β receptor. NIH 3T3-mediated suppression was markedly abrogated by neutralizing antibodies to mouse IFN-β. Furthermore, viral expression in HTLV-1-infected cells was significantly suppressed when the infected cells were intraperitoneally injected into wild-type mice but not IFN regulatory factor 7 knockout mice that are deficient of type I IFN responses. These findings indicate that the innate immune system suppresses HTLV-1 expression in vivo, at least through type I IFN.


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INTRODUCTION
 
Human T-cell leukemia virus type 1 (HTLV-1) is the causal agent of adult T-cell leukemia (ATL), a chronic progressive neurological disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases (5, 11, 26, 27). Although ATL patients maintain antibodies against various viral products in their serum, HTLV-1 expression in ATL cells immediately after isolation from patients is very low and becomes significantly induced after culture for several hours in vitro (10, 18, 31). This phenomenon is observed in T cells from not only ATL patients but also HAM/TSP patients and asymptomatic HTLV-1 carriers (1, 8, 17), indicating that there may be a common mechanism for transient suppression of HTLV-1 expression in vivo.

The HTLV-1 genome contains a unique 3' region, pX, that encodes the multifunctional viral protein, Tax. Tax transactivates the HTLV-1 long terminal repeat (LTR), as well as various host genes related to cell growth and apoptosis resistance, and plays a central role in the HTLV-1-associated immortalization and transformation of T cells in vitro (7, 15, 19, 35). However, the scarcity of Tax in vivo raises controversy about its contribution to ATL. Tax is also the predominant target antigen of cytotoxic T lymphocytes (CTLs) specific for HTLV-1-infected cells in HTLV-1-infected individuals (9, 14). In a rat model of HTLV-1-infected lymphomas, suppression of Tax expression in HTLV-1-infected tumor cells using shRNAs decreased their susceptibility to Tax-specific CTLs and decreased the tumorigenicity of the tumor cells in vivo (24). Reduction of viral antigens may be an important strategy for the virus to persist in the host and a reason for the long course of the disease progression.

However, the essential mechanisms involved in the suppression of HTLV-1 expression in vivo have remained obscure. Early studies indicated that HTLV-1 expression in vivo may be suppressed at the transcriptional level (31). The transcription of HTLV-1 is mainly regulated by CRE-like repeats in the HTLV-1 LTR, called Tax-responsive elements, where Tax transactivates HTLV-1 transcription by by-passing the association between CREB and CBP/p300 (3, 28, 35). Inducible cyclic AMP early repressor inhibits Tax-mediated transactivation and potentially suppresses HTLV-1 expression in vitro (2) (23).

Interestingly, a reporter system for HTLV-1 Tax-mediated transcription is suppressed even in mice (4). A recent report about a rat model of HAM/TSP-like disease indicated that WKAH strain rats susceptible to this disease exhibited malfunction of an interleukin-12 (IL-12) receptor and impaired gamma interferon (IFN-{gamma}) production in the spinal cord (20). Increased Tax expression in the spinal cord has also been reported in this rat strain after HTLV-1 infection (30). These findings suggest that innate immunity is involved in disease development and viral expression after HTLV-1 infection.

In the present study, we investigated whether innate immunity is involved in the inhibition of HTLV-1 expression. Transcriptional activation of cytokines, such as type I IFNs (IFN-{alpha} and IFN-β), is an important part of the antiviral innate immune response. We demonstrate that stromal cells, such as fibroblasts and epithelial cells, can inhibit HTLV-1 expression in HTLV-1-infected T cells via type I IFN responses. The levels of HTLV-1 mRNA recovered following separation from the stromal cells. We further demonstrate that HTLV-1 expression in infected cells is suppressed after injection into wild-type (WT) mice but not after injection into IRF-7 knockout (–/–) mice. These observations strongly suggest that innate immune responses contribute to repression of HTLV-1 expression in vivo.


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MATERIALS AND METHODS
 
Cells. Peripheral blood mononuclear cells (PBMC) were isolated from blood samples donated from a patient with chronic ATL, after written informed consent was provided, by using a Ficoll-Paque PLUS (GE Healthcare UK, Ltd., Buckinghamshire, United Kingdom) density gradient centrifugation and then stored frozen in liquid nitrogen until use. Among the IL-2-dependent HTLV-1-infected human T-cell lines used, ILT-Hod (33) and ILT-#29 were derived from chronic ATL patients, and ILT-Myj was derived from a HAM/TSP patient (16). These cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 10 U of recombinant human IL-2 (rhIL-2; Shionogi, Osaka, Japan)/ml. MT-2 cells derived from healthy cord blood T cells exogenously infected with HTLV-1 (21) were maintained without IL-2. NIH 3T3, a mouse embryo fibroblast (MEF) line derived from a Swiss mouse; HEK293T, a human embryo kidney epitheliumlike cell line; and HeLa, a human cervical carcinoma cell line were maintained in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics.

Mice. IRF-7 knockout (–/–) mice with a C57BL/6J background (13) were obtained from RIKEN BioResource Center (Ibaraki, Japan) under the permission of Tadatsugu Taniguchi (University of Tokyo, Tokyo, Japan). Wild-type C57BL/6J mice were purchased from Clea Japan, Inc. (Tokyo, Japan). MEFs were isolated from mice that had been pregnant for 13 days. All mice were maintained at the experimental animal facilities of Tokyo Medical and Dental University. The Animal Ethics Review Committee of our university approved the experimental protocol.

Cocultures of HTLV-1-infected cells with nonlymphoid cells. HTLV-1-infected cells (106) were cocultured with HEK 293T cells, NIH 3T3 cells, or MEFs growing as monolayers in 12-well plates in RPMI 1640 containing 10% FBS and 10 U of rhIL-2/ml. Nonadherent cells after coculture for various periods were harvested for evaluation of HTLV-1 expression. In some experiments, HTLV-1-infected cells were cocultured with HEK 293T or NIH 3T3 cells for 24 h, and then the floating cells were isolated, washed, and cultured in new wells with fresh medium containing rhIL-2 for several days in order to observe the recovery of viral expression.

ELISA. The HTLV-1 p19 concentrations in supernatants from ILT-Hod cells were measured using a Retro-Tek HTLV-I/II p19 antigen enzyme-linked immunosorbent assay (ELISA; ZeptoMetrix Corp., Buffalo, NY) according to the manufacturer's instructions.

Quantitative RT-PCR. Aliquots (0.5 µg) of total RNA extracted from cells using Isogen (Nippon Gene, Tokyo, Japan) were treated with DNase (Ambion, Austin, TX) and subjected to reverse transcription-PCR (RT-PCR) with HTLV-1 gag-specific primers (forward, 5'-CCTTACCACGCCTTCGTAGAACGCCTCAACATAGC-3'; reverse, 5'-TTTGTCTTTGGGGGTCCAGGTCTGACAAGCCCGCA-3') and human GAPDH-specific primers (forward, 5'-ACCAGGGCTGCTTTTAACTC-3'; reverse, 5'-TTGATTTTGGAGGGATCTCG-3') using LightCycler Fast Start DNA Master SYBR green I (Roche Diagnostics, Mannheim, Germany) after RT with oligo(dT)20 primers. The PCR amplifications consisted of an initial denaturation step at 95°C for 5 min and 40 cycles of denaturation at 95°C for 15 s, annealing at 62°C for 10 s, extension at 72°C for 10 s, and denaturation at 85°C for 2 s. The RT-PCR products were quantified and standardized by simultaneously amplifying GAPDH mRNA and quantifying the GAPDH copy number. Amplification of myxomavirus resistance protein A (MxA) mRNA was similarly performed using MxA-specific primers (forward, 5'-AGTATGGTGTCGACATACCGGA-3'; reverse, 5'-GAGTCTGGTAAACAGCCGAATG-3') (32), and the levels were standardized by the corresponding GAPDH mRNA levels.

Flow cytometry. To detect intracellular HTLV-1 antigens, cells were fixed and permeabilized as described previously (33) and serially stained with anti-HTLV-1 p19 mouse monoclonal antibody GIN-7 (29) or control ascites and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) plus IgM (IgG+IgM) antibodies. The stained cells were analyzed by using a flow cytometer (FACSCalibur; Becton Dickinson, San Jose, CA).

Antibodies. Anti-HTLV-1 p19 mouse monoclonal antibody GIN-7 (29) was kindly provided by Yuetsu Tanaka (University of the Ryukyus, Okinawa, Japan). Polyclonal rabbit antibodies against mouse IFN-{alpha} and IFN-β (PBL Interferon Source, Piscataway, NJ) were added to cocultures of ILT-Hod and NIH 3T3 cells. Mouse monoclonal antibodies against human IFN-{alpha}/β receptor (PBL Biomedical Laboratories, New Brunswick, NJ) and control mouse IgG2a,k (BioLegend) were added to cocultures of ILT-Hod and HEK293T cells. A polyclonal rabbit antibody against mouse/rat asialo-GM1 (Acris Antibodies GmbH, Hiddenhausen, Germany) was used to eliminate natural killer cells in vivo.

Cytokines. In some experiments, rhIFN-{alpha} (Sigma-Aldrich), rhIFN-β (Sigma-Aldrich), and rhIL-10 (R&D Systems, Inc.) were added to the culture.

Cell growth and viability. Cells were cultured in the presence or absence of 10 µM bromodeoxyuridine (BrdU) for 24 h and stained first with phycoerythrin (PE)-conjugated anti-CD25 monoclonal antibody or isotype control antibody (R&D Systems, Inc.). Cells were then permeabilized and stained with FITC-BrdU flow kits (BD Pharmingen), and then the proportions of CD25+ BrdU+ cells were measured by two-color flow cytometry.

Cell viabilities were evaluated by using a Cell Counting Kit-8 (Dojindo), which measures formazan color in the viable cells. Cell Counting Kit-8 solution (10 µl) was added into 100-µl portions of cell cultures in a 96-well microplate. After incubation in a CO2 incubator for 3 h, the color changes (i.e., the optical density at 450 nm) in culture supernatants were measured by a microplate reader (Bio-Rad). The negative control value of the well containing medium alone was subtracted from the sample values.

Reporter assays. A reporter cell line, 293T/IFN-β-luc, was established by transfecting p55-luc plasmids containing the IFN-β promoter (kindly provided by Takashi Fujita, Institute for Virus Research, Kyoto University, Kyoto, Japan) (34). The luciferase activities in cell lysates were measured by using a luciferase assay system (Promega, Madison, WI).

Injection of HTLV-1-infected cells into mice. Mice were intraperitoneally (i.p.) injected with an anti-asialo-GM1 antibody (Acris Antibodies) at 0.3 mg/head on the day before an ILT-Hod cell injection and then i.p. injected with 2 x 107 ILT-Hod cells. The cells in the peritoneal cavities of the mice were harvested at 16 h after the ILT-Hod cell injection by washing the peritoneal cavities with 10 ml of phosphate-buffered saline.


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RESULTS
 
Suppression of HTLV-1 expression by coculture with human and mouse stromal cells. First, we examined whether nonlymphoid stromal cells suppressed HTLV-1 expression in HTLV-1-infected cells during coculture. When ILT-Hod cells derived from a chronic ATL patient were cocultured with a monolayer of human embryonic kidney-derived HEK293T cells for 3 days, the amount of HTLV-1 p19 in the supernatant decreased (Fig. 1A, left). In primary PBMC from a patient with chronic ATL, the amount of HTLV-1 p19 produced during the initial 24-h culture was also decreased by coculture with HEK293T cells (Fig. 1A, right). Suppression of HTLV-1 gag mRNA expression in HTLV-1-infected cells after a 24-h coculture with HEK293T cells was confirmed by a quantitative RT-PCR analysis in the floating cell fractions (Fig. 1B). HEK293T cell-mediated suppression of HTLV-1 expression was also observed in ILT-Myj cells derived from a HAM/TSP patient and in HTLV-1-transformed MT-2 cells in a lesser degree (Fig. 1B). Similar suppression of HTLV-1 transcription was observed when ILT-Hod cells were cocultured with HeLa cells derived from human cervical cancer or NIH 3T3 cells derived from MEFs (Fig. 1C). Although we normalized the HTLV-1 mRNA levels by the GAPDH mRNA levels in the same sample, the reduction in HTLV-1 mRNA was not a relative artifact due to contamination by uninfected NIH 3T3 cells because the RT-PCR primers used for GAPDH amplification were specific for human GAPDH mRNA.


Figure 1
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  		FIG. 1. Suppression of HTLV-1 expression by coculture with stromal cells. (A) ILT-Hod cells (left panel) or primary PBMC from a chronic ATL patient (right panel) were cultured with or without HEK293T cells for 72 h or 24 h, respectively, and the HTLV-1 p19 concentrations in the supernatants were measured by ELISA. The data are the means and standard deviations (SD) of duplicate wells. (B) ILT-Hod, ILT-Myj, or MT-2 cells were cultured with or without HEK293T for 24 h, and the HTLV-1 gag mRNA levels in floating cells were measured by quantitative RT-PCR. The HTLV-1 gag mRNA copy numbers were standardized by the human GAPDH mRNA copy numbers and are presented as the fold changes compared to each HTLV-1-infected cell line cultured in medium alone. (C) ILT-Hod cells were cultured with or without HEK293T, HeLa, or NIH 3T3 cells for 24 h, and the HTLV-1 gag mRNA levels in floating cells were measured by quantitative RT-PCR as described above. The results shown are representatives of several independent experiments for each cell line. (D) Intracellular HTLV-1 Gag proteins in floating ILT-Hod cells were stained before coculture (green line) and after 24 h (blue line) and 48 h (red line) of coculture with NIH 3T3 cells and analyzed by using a flow cytometer. The closed histogram indicates control staining with ascites and a FITC-labeled anti-mouse IgG+IgM antibody. (E) ILT-Hod cells were cocultured with (right panel) or without (left panel) HEK293T cells in the presence of 10 µM BrdU for 24 h, and the floating cells were stained for surface CD25 (y axis) and intracellular BrdU (x axis) and then analyzed by a flow cytometer. The proportions of CD25+ BrdU+ cells are indicated. All culture media used contained 10 U of rhIL-2/ml.

Flow cytometric analysis demonstrated that intracellular HTLV-1 Gag proteins were still detected at 24 h and decreased to undetectable levels at 48 h after initiation of the coculture with NIH 3T3 cells (Fig. 1D). This confirmed the suppression of HTLV-1 protein expression following coculture with NIH 3T3 cells at the single cell level.

We further examined whether coculture with stromal cells affects the growth of ILT-Hod cells by measuring BrdU uptakes in floating CD25+ cells in coculture. As shown in Fig. 1E, the proportion of CD25+BrdU+ ILT-Hod cells did not decrease but even increased after coculture with HEK293T cells.

Recovery of HTLV-1 expression in infected cells after separation from stromal cells. Next, we examined whether the suppressed viral expression in HTLV-1-infected cells after coculture with NIH 3T3 or HEK293T cells could recover after separation from these adherent cells. ILT-Hod cells were isolated after a 24-h coculture with NIH 3T3 cells and subsequently cultured alone. HTLV-1 mRNA expression in ILT-Hod cells remained at low levels for 24 h after separation from NIH 3T3 cells but then recovered by 72 h after the separation. Similar kinetics were observed for ILT-#29, another IL-2-dependent HTLV-1-infected cell line (Fig. 2A).


Figure 2
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  		FIG. 2. Recovery of HTLV-1 expression in infected cells after separation from stromal cells. (A) ILT-Hod ({circ}, •) or ILT-#29 ({triangleup}, {blacktriangleup}) cells were cultured with (•, {blacktriangleup}) or without ({circ}, {triangleup}) NIH 3T3 cells for 24 h, and then nonadherent cells were isolated and cultured in new wells with fresh medium. The HTLV-1 gag mRNA levels in floating cells were measured before coculture (control), at 24 h after coculture (day 0), and at 1 and 3 days after separation. The HTLV-1 gag mRNA copy numbers were standardized by the human GAPDH mRNA copy numbers and are presented as the fold changes compared to ILT-Hod cells before coculture. The results represent the means and SD of duplicate wells. (B and C) ILT-Hod cells cocultured with NIH 3T3 (B) or 293T (C) cells for 24 h were isolated and cultured in new wells with fresh medium. Flow cytometric analyses for intracellular HTLV-1 Gag proteins were performed at the indicated time points after separation. The green histogram indicates ILT-Hod cells without coculture, and the closed histogram indicates control staining with ascites and an FITC-labeled anti-mouse IgG+IgM antibody. All culture media used contained 10 U of rhIL-2/ml.

The kinetics of intracellular HTLV-1 Gag protein expression in ILT-Hod cells reisolated after a 24-h coculture with NIH 3T3 cells were assessed by flow cytometry. As shown in Fig. 2B, Gag protein expression remained suppressed for 24 h after separation from NIH 3T3 cells but then recovered at 48 to 72 h after the separation, similar to the what was found for HTLV-1 mRNA. When ILT-Hod cells were cocultured with HEK293T cells, intracellular HTLV-1 Gag proteins in ILT-Hod cells became undetectable 24 h after coculture, which was earlier than for coculture with NIH 3T3. After isolation from HEK293T cells, viral expression in ILT-Hod cells gradually recovered to the initial level by 48 h after the separation (Fig. 2C).

These observations indicate that viral expression in HTLV-1-infected cells is reversible, even after suppression by contact with stromal cells.

Involvement of type I IFNs in suppression of HTLV-1 expression by stromal cells. Since type I IFNs are produced by many kinds of cells, including epithelial cells and fibroblasts, we assessed the involvement of type I IFNs in the stromal cell-mediated suppression of HTLV-1 (Fig. 3). When rhIFN-{alpha} or rhIFN-β was directly added to the culture of ILT-Hod cells, the amounts of HTLV-1 p19 produced in the supernatants decreased in a dose-dependent manner (Fig. 3A). This was not associated with alteration of the viable cell number (Fig. 3B). These type I IFNs also efficiently suppressed the spontaneous induction of HTLV-1 expression in primary ATL cells in the initial 24 h of culture (Table 1).


Figure 3
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  		FIG. 3. Involvement of type I IFNs in suppression of HTLV-1 expression by stromal cells. (A and B) ILT-Hod cells were cultured in the presence of rhIFN-{alpha}, rhIFN-β, or rhIL-10 at the indicated concentrations for 72 h, and the concentrations of HTLV-1 p19 in the supernatants (A) and viable cell numbers (B) were evaluated by ELISA or using Cell Counting Kit-8, respectively. The results indicate means and SD of duplicate wells. (C) The HTLV-1 gag mRNA expression levels in ILT-Hod cells were measured after a 24-h coculture without (open bars) or with (closed bars) NIH 3T3 cells in the presence of anti-mouse IFN-{alpha} or IFN-β neutralizing antibodies (4 x 103 neutralizing units/ml). (D) ILT-Hod cells were pretreated with or without 1 µg of anti-human IFN {alpha}/β receptor monoclonal antibody or control mouse IgG2a/ml for 1 h prior to coculture with HEK293T cells for 2 h, and then the HTLV-1 gag mRNA expression levels in ILT-Hod cells were evaluated. The data in panels C and D are the fold changes compared to control cells cultured in medium alone and represent the means and SD of duplicate wells. (E) Reporter 293T cells containing an IFN-β reporter plasmid (293T/IFN-β-luc) were cultured with or without ILT-Hod cells or ILT-Hod cells fixed with 1% formaldehyde, and the luciferase activities in the whole-cell lysates were measured after coculture for 24 h. The results are means and SD of duplicate samples. (F) The MxA ({circ}) and HTLV-1 gag (•) mRNA levels in ILT-Hod cells were measured after coculture with HEK293T cells for the indicated periods. The data represent the fold changes compared to control ILT-Hod cells cultured alone for 1 h.


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  		TABLE 1. Suppressive effects of type I IFNs on spontaneous HTLV-1 induction from primary ATL cellsa

Addition of polyclonal neutralizing antibodies against mouse IFN-β but not IFN-{alpha} efficiently abrogated the suppression of HTLV-1 expression in ILT-Hod cells cocultured with NIH 3T3 cells (Fig. 3C), indicating that IFN-β produced by NIH 3T3 cells contributed to the repression of HTLV-1 expression. Antibodies against human IFN-{alpha}/β receptor partly, but not completely, abrogated the effects of coculture with HEK293T cells (Fig. 3D).

Induction of IFN-β in cocultures with ILT-Hod cells was further examined using the reporter cell line 293T/IFN-β-luc, comprising a stable transfectant with a luciferase reporter plasmid driven by an IFN-β promoter. Coculture of ILT-Hod cells with 293T/IFN-β-luc cells markedly induced reporter luciferase activities, whereas formalin-fixed ILT-Hod cells had no effect (Fig. 3E). A rapid increase in the mRNA of MxA, an IFN-stimulated gene, was also detected in HTLV-1-infected cells after coculture with HEK293T cells (Fig. 3F).

These findings indicate that IFN-β contributes to viral suppression almost exclusively in cocultures with NIH 3T3 cells and partially in cocultures with HEK293T cells.

Suppression of HTLV-1 expression by MEFs from WT but not IRF-7–/– mice. We further examined whether primary MEFs can suppress HTLV-1 expression in a type I IFN-dependent manner. For this purpose, we used IRF-7–/– mice (13). MEFs of IRF7–/– mice are impaired in their ability to induce IFN-{alpha}/β in response to viruses and multiple pathogen-associated molecular patterns, including poly(I:C), lipopolysaccharide, poly(U), CpG-A, and CpG-B. As shown in Fig. 4A, HTLV-1 mRNA expression in ILT-Hod cells was markedly suppressed after a 24-h coculture with MEFs freshly isolated from WT C57BL/6J mice. In contrast, HTLV-1 expression was not repressed, but rather enhanced, in MEFs from IRF-7–/– C57BL/6J mice (Fig. 4A).


Figure 4
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  		FIG. 4. Suppression of HTLV-1 expression in wild-type but not IRF-7–/– mice. (A) ILT-Hod cells were cocultured with or without MEFs isolated from WT or IRF-7–/– C57BL/6J mice as indicated for 24 h, and the HTLV-1 gag mRNA levels in floating ILT-Hod cells were measured. The data are the fold changes compared to control wells cultured in medium alone, and represent the means and SD of duplicate wells. Similar results were obtained in two other independent experiments. (B) WT and IRF-7–/– C57BL/6J mice were i.p. injected with an anti-asialo-GM1 antibody on the day before an ILT-Hod injection and then i.p. injected with ILT-Hod cells (2 x 107 cells/head). The cells in the peritoneal cavity were harvested with 10 ml of PBS at 16 h after the ILT-Hod injection and analyzed by quantitative RT-PCR. The HTLV-1 gag mRNA levels standardized by the human GAPDH mRNA levels are indicated as the fold changes compared to the control value of ILT-Hod cells cultured in IL-2-containing medium. The HTLV-1 gag mRNA expression level in ILT-Hod cells cultured without IL-2 in vitro is also indicated. The data represent the means and SD of duplicate wells. A representative result of at least three independent experiments is shown.

Suppression of HTLV-1 expression in WT but not IRF-7–/– mice in vivo. Finally, we investigated whether type I IFN responses occur and repress HTLV-1 expression in vivo. We injected ILT-Hod cells into the peritoneal cavity of WT or IRF-7–/– C57BL/6J mice. To avoid the possible clearance of ILT-Hod cells by murine NK cells, we treated the mice with anti-mouse/rat asialo-GM1 antibody (0.3 mg/mouse) on the day before the ILT-Hod cell injection. The cells in the peritoneal cavity were harvested 16 h after the injection. The HTLV-1 mRNA levels were very low in ILT-Hod cells harvested from WT mice (Fig. 4B).

We prepared two control ILT-Hod cultures in medium with or without IL-2, because human IL-2 does not exist in mice. The HTLV-1 mRNA level in ILT-Hod cells cultured in the absence of IL-2 was significantly higher than that in the cells cultured with IL-2, in agreement with our previous report (33). Nevertheless, the HTLV-1 expression in ILT-Hod cells injected into WT mice was significantly lower than that in control ILT-Hod cells. In contrast, HTLV-1 expression in ILT-Hod cells harvested from IRF-7–/– mice was comparable to or greater than that in the control ILT-Hod cells (Fig. 4B). These results indicate that the suppression of HTLV-1 expression in ILT-Hod cells in vivo occurs via an IRF-7-dependent pathway in mice.


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DISCUSSION
 
In the present study, we demonstrated suppression of HTLV-1 expression after contact with stromal cells and restoration of viral expression following separation from the stromal cells. There is a striking similarity to the phenomena observed in PBMC from HTLV-1-infected individuals, which exhibit very low levels of HTLV-1 mRNA and undetectable levels of viral protein when isolated from peripheral blood but increasingly express these molecules after a short time in culture. In fact, the spontaneous induction of HTLV-1 expression in primary ATL cells in the first 24 h of culture was efficiently inhibited by coculture with HEK293T cells (Fig. 1A).

Suppression of HTLV-1 expression by mouse NIH 3T3 cells took a longer time than human HEK293T cells (Fig. 1D and 2), presumably owing to differences in the species. Induction of type I IFN responses in the coculture of HTLV-1-infected cells and HEK293T cells was evidenced by IFN-β promoter activation in HEK293T cells and MxA expression in HTLV-1-infected cells. However, neutralizing antibodies against human IFN-{alpha}/β receptor only partly abrogated HEK293T cell-mediated suppression of HTLV-1 expression, whereas neutralization of mouse IFN-β almost completely blocked NIH 3T3 cell-mediated suppression. These results suggest that multiple factors, including type I IFNs, may contribute to stromal cell-mediated HTLV-1 suppression in humans.

Although IFN-β seemed to predominantly contribute to stromal cell-mediated viral suppression in the present system, both rhIFN-{alpha} and -β were capable of directly suppressing HTLV-1 expression in HTLV-1-infected cell line and primary ATL cells (Fig. 3 and Table 1). A recent report indicated that plasmacytoid dendritic cells, the major source of IFN-{alpha}, are functionally impaired in HTLV-1-infected individuals (12). Earlier studies indicated suppression of HTLV-1 expression by type I IFNs in vitro (25), and favorable clinical effects of a combination therapy of IFN-{alpha} and zidovudine for ATL (6). A recent report indicated that overexpression of HTLV-1 Tax inhibited the effects of type I IFN signals by competing with CBP/p300 (36). This may partly explain why the suppressive effect of stromal cells was weak on MT-2 cells (Fig. 1B) that express abundant amounts of HTLV-1 Tax, although such a state of infected cells differs from that in vivo.

Lymphoid and other tissues contain numerous stromal cells, which can contact with circulating lymphocytes and produce innate immune responses. HTLV-1 expression in infected cells was suppressed in WT mice but not IRF7–/– mice in vivo (Fig. 4B), suggesting that IRF7-dependent innate immune responses in mice could mimic the phenomenon occurring in humans. Since efficient HTLV-1 expression is dependent on transactivation of its own LTR by Tax protein (3, 28, 35), limitation of this protein below a certain level by stromal cells may contribute to the maintenance of HTLV-1 expression at low levels even in the presence of small amounts of viral mRNA in vivo.

Around the time of the submission of the present study, Nagai et al. reported that primary ATL cells grew by coculture with mouse bone marrow-derived stromal cells with reduced levels of viral expression in a long-term culture (22). This is consistent with our data that BrdU uptake in HTLV-1-infected cells was enhanced by coculture with stromal cells (Fig. 1E), although its mechanism remains to be clarified.

In conclusion, the present findings strongly indicate that the innate immune system suppresses HTLV-1 expression in vitro and in vivo, at least through type-I IFN.


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ACKNOWLEDGMENTS
 
We thank Tadatsugu Taniguchi (University of Tokyo, Tokyo, Japan) for permission to use the IRF-7–/– mice. We also thank Takashi Fujita (Institute for Virus Research, Kyoto University) and Yuetsu Tanaka (University of the Ryukyus) for providing the p55-luc plasmids and anti-HTLV-1 Gag monoclonal antibody, respectively.

This study was supported by grant 17013028 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and a grant for the anticancer project from the Ministry of Health, Welfare, and Labor of Japan.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Immunotherapeutics, Tokyo Medical and Dental University, Graduate School, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Phone: 81-3-5803-5798. Fax: 81-3-5803-0235. E-mail: kann.impt{at}tmd.ac.jp Back

{triangledown} Published ahead of print on 4 March 2009. Back


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Journal of Virology, May 2009, p. 5101-5108, Vol. 83, No. 10
0022-538X/09/$08.00+0     doi:10.1128/JVI.02564-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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