Induction of Human T Cell Leukemia Virus Type I Receptors on Quiescent Naive T Lymphocytes by TGF-1
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免疫学杂志 2005年第7期
Abstract
The retrovirus human T cell leukemia virus (HTLV) type I (HTLV-I) is primarily transmitted by breast-feeding or sexual contact, by cell-to-cell contact between T cells. TGF-, which has been shown to enhance transmission of HTLV-I in vitro, is found at high levels in breast milk and semen. In this study, the ability of TGF- to regulate expression of molecules involved in HTLV-I binding and entry was examined. Previous studies using a soluble form of the HTLV-I envelope protein SU have shown that quiescent human T cells do not express cell surface molecules that specifically bind SU. After T cell activation, HTLV SU binding proteins are rapidly induced. In this study, we report that TGF- induces expression of proteins that bind soluble HTLV SU and HTLV virions on naive CD4+ T lymphocytes. The induction of these proteins occurred without cell cycle entry or expression of activation markers, involved TGF--induced intracellular signaling, and required de novo transcription and translation. Treatment of naive CD4+ T lymphocytes with TGF- induced expression of GLUT-1, which has recently been reported to function as a receptor for HTLV. Treatment of a TGF--sensitive human myeloid cell line increased the titer of both HTLV-I- and HTLV-II-pseudotyped viruses. Although earlier studies suggested that HTLV SU binding proteins might be an early marker of T cell activation and/or cell proliferation, we report in this study that TGF- induces binding of HTLV virions and expression of glucose transporter type 1 in primary CD4+ T lymphocytes that remain quiescent.
Introduction
The human T cell leukemia virus (HTLV)4 type I (HTLV-I) retrovirus, the first disease-causing human retrovirus isolated (1), is endemic in certain geographic areas, including southwestern Japan, the Caribbean basin, and parts of West Africa, South America, and Melanesia (2). Although the majority of 10 million infected individuals remain healthy lifelong asymptomatic carriers, HTLV-I is the etiologic agent for several diseases. The two most common are a severe lymphocyte neoplasia called adult T cell leukemia (ATL) (1, 3) and an inflammatory neurological disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (4, 5).
ATL is a malignancy of CD4+ T cells, and HTLV-I has a preferential tropism for CD4+ T cells in asymptomatic patients. Infection of both memory (CD45RO+) (6) and effector/memory (CD27–CD45RA–) (7) CD4+ T cells has been observed. Recent studies have indicated that, in HAM/TSP patients, both CD4+ and CD8+ T cells serve as viral reservoirs (8). The closely related retrovirus HTLV type II (HTLV-II), which is believed to share a common receptor with HTLV-I (9, 10), also infects both CD4+ and CD8+ T cells in vivo (11, 12). Both HTLV-I and HTLV-II can infect all human lymphocytic subsets, causing polyclonal and monoclonal T cell lymphocytoses (reviewed in Ref. 13).
Entry of retroviruses into target cells involves the interaction of two envelope (Env) glycoproteins, a surface glycoprotein (SU), and a transmembrane glycoprotein (TM), with at least one specific host cell receptor (14). In order for infection to occur, cell surface molecules must specifically bind the viral envelope, and this binding must lead to subsequent fusion of the viral and cellular membranes. For some retroviruses such as HIV, the binding and fusion molecules are separate; for others, such as ecotropic murine leukemia viruses, they appear to be a single molecule.
Because primary T cells are difficult to infect with HTLV-I in vitro, the majority of the work over the past 20 years has examined requirements for HTLV Env-mediated binding and fusion on established (often non-T) cell lines. These studies revealed that, in contrast to the limited in vivo tropism of HTLV, molecules capable of functioning as HTLV receptors are found on a wide variety of cells (15, 16, 17, 18). Cell surface molecules capable of specifically binding HTLV SU are even more widely expressed; they have been found on every vertebrate cell line tested to date (19, 20, 21).
Recently, the first identification of a specific molecule involved in HTLV binding and fusion was reported (22). The glucose transporter type 1 (GLUT-1) specifically binds a soluble form of both the HTLV-I and HTLV-II SU proteins in T cell and non-T cell lines, and was shown to be critical for efficient entry of HTLV-II-pseudotyped virions.
For some retroviruses, it is now clear that cell surface molecules other than the primary entry receptors can bind virions (recently reviewed in Ref. 23). These molecules, referred to as attachment factors, can play a critical role in the efficiency of retroviral entry. Heparan sulfates, which are widely expressed on the surface of cells, have been shown to be used by a number of viruses including the retroviruses HIV-1 and murine leukemia virus (24, 25). More recent studies have revealed that heparan sulfate proteoglycans (HSPG) can also specifically bind soluble HTLV SU and enhance entry of HTLV-I pseudotypes (26, 27). This indicates that the amount of binding of soluble SU and the titer of HTLV-pseudotyped viruses, at least on certain cell lines, reflects interactions with HSPG, as well as interactions with specific receptors. Because HSPGs are expressed at low or undetectable levels on primary CD4+ T cells (28, 29, 30), they may not play a role in the ability of HTLV-I to infect its primary target cell. Thus, it is not clear whether the requirements for HTLV-I Env-mediated fusion are identical in established cell lines and in primary T cells or how much of the binding of the soluble SU is to attachment factors rather than binding/fusion receptors.
The cell surface molecules required for efficient entry of HTLV-I virions into primary T lymphocytes have not been rigorously defined. Recent work by our group and another laboratory found that HTLV SU binding molecules are expressed on most primary cells of the immune system (21, 31), with the highest levels of binding observed on CD4+ and CD8+ T cells undergoing an immune response. In contrast to both stimulated T lymphocytes and established cell lines, quiescent T cells derived from adult and cord blood cells do not bind detectable levels of HTLV-I SU. However, SU binding proteins were rapidly induced after stimulation. Thus, it has been suggested that these HTLV SU binding proteins are an early marker of T cell activation (21, 31).
HTLV-I is primarily transmitted in vivo by breast-feeding or sexual contact. HTLV transmission appears to require passage of cells between individuals, and infection is believed to require contact between T cells. Circulating T cells are almost entirely in the G0 phase of the cell cycle; these cells do not express the HTLV SU binding protein. This suggests that viral transmission occurs in areas of extensive immune reactivity such as lymph nodes or sites of inflammation. Alternatively, humoral factors could stimulate expression of HTLV-I receptors on quiescent T cells that then could serve as targets for viral entry. For HIV-1, it has been reported that the virus can enter quiescent T cells, which can harbor the virus in an inactive state until subsequent mitogenic stimulation allows completion of the early stages of infection (32).
TGF- is a member of a superfamily of growth factors that regulate numerous physiological processes including cell cycle control, differentiation, and the immune response (33). TGF- can inhibit the growth of most epithelial and lymphoid cells by arresting cells in mid-G1 phase (34). Intracellular signaling is initiated upon the binding of TGF- to the cell surface type II TGF- receptor (TRII), which recruits type I TGF- receptor (TRI) to a multimeric complex (reviewed in Ref. 35). The propagation of the intracellular signal to the nucleus then occurs through phosphorylation of Smads, which regulate gene expression in cooperation with other transcription factors (23, 36, 37).
TGF-, which can enhance transmission of HTLV-I in vitro, is found at high levels in breast milk and semen. TGF- can induce expression of HTLV-I viral proteins in PBMCs from HAM/TSP (38) and asymptomatic carriers (39). In the latter study, it was shown that TGF- transactivates the long terminal repeat (LTR) promoter and can accelerate transmission of the virus from infected to uninfected cells. For HIV-1, TGF- has also been shown to both increase transcription from the LTR (40) and to up-regulate cell surface expression of coreceptors in target cells in the absence of T cell activation and proliferation (41, 42).
In this study, we observed that TGF- induces expression of HTLV SU binding proteins without activation of naive CD4+ T lymphocytes and can increase the titer of HTLV-I- and HTLV-II-pseudotyped viruses. Treatment of naive CD4+ T lymphocytes with TGF- induces expression of GLUT-1, a receptor for HTLV entry (22). Thus, TGF- can stimulate cell surface expression of proteins involved in HTLV binding and fusion in the absence of T cell activation and cell cycle entry.
Materials and Methods
Cell culture and isolation of lymphocytes
Cord blood samples obtained from healthy volunteer donors during vaginal births from Frederick Memorial Hospital (Frederick, MD) and leukopaks of peripheral blood from healthy donors were collected according to the National Institutes of Health approved institutional review board protocols. Mononuclear leukocytes were isolated by Ficoll-Hypaque gradient centrifugation. The light density fraction (buffy coat) was collected, washed twice with PBS, and then enriched for CD4+ T cells by removal of CD8-, CD19-, CD45RO-, CD11b-, and glycophorin-A-positive cells using MACs methods according to manufacturer’s instructions (Miltenyi Biotec). After isolation, the cells were cultured in either RPMI 1640 medium supplemented either with 20% BIT (BSA, insulin, and transferrin; StemCell Technologies) or with 10% human Ab, as indicated, along with 2 mM L-glutamine, sodium pyruvate (1 mM), and antibiotics. For TGF- treatment, cells were exposed to TGF-2 (Genzyme) or TGF-1 (BioSource) at the concentrations and times indicated. T cells were cultured in 20 U/ml IL-2 (Zeptometrix) and activated with PHA (1 μg/ml; Abbott Diagnostics) or cultured with 10 ng/ml IL-7 (PeproTech). In some studies, SB-431542 (Tocris Cookson) an inhibitor of TGF-RI signaling (43, 44) was added along with TGF-. In one study, the cells were treated with actinomycin D (Sigma-Aldrich) or cyclohexamide (Sigma-Aldrich). KPC/1 is a clone of the human myeloid cell line K562. It was generated by transfecting K562 with a plasmid encoding a neo selectable marker (pcDNA), selecting for transfected cells plated at 100 cells/well in a 96-well plate with 500 μg/ml G418, and identifying the cells most sensitive to TGF- (as judged by growth inhibition and level of Smad phosphorylation). All cells were cultured in a humidified atmosphere at 37°C in an incubator containing 5% CO2.
Expression of the recombinant HTLV-I SU envelope-Fc fusion proteins
Soluble SU proteins were generated essentially as previously described, with the following modifications. 293-T cells were transfected with either the plasmid vector encoding HTSU-IgG (HTSU-IgG/pSK100; Ref. 20) or a plasmid encoding a similar fusion protein (SUA-rIgG) containing the SU protein from the avian retrovirus ALSV-A (45). The latter was used as a negative control in all studies. Two days after transfection, the cells were washed with PBS, and then resuspended in ice-cold PBS to which proteinase inhibitor mixture (Sigma-Aldrich) had been added. The cells were then sonicated twice, and the lysates were centrifuged at 800 x g for 5 min. The clarified lysate was then aliquoted and stored at –80°C, and the concentration of immunoadhesins was determined by ELISA, as previously described (20).
Flow cytometric analysis
Specific binding of HTSU-IgG to target cells was examined essentially as previously described (20) with the following modifications. Briefly, the lysates containing immunoadhesin (either HTSU-IgG or SUA-IgG) were centrifuged at 12,000 x g for 1 min. Because HTSU-IgG can be rapidly internalized following binding to cells, and fixation does not significantly reduce the level of HTSU-IgG binding, target cells were fixed in 4% paraformaldehyde for 30 min on ice, washed with PBS, and then resuspended in PBS/2% FCS/0.02% sodium azide. The target cells (1 x 106) were then incubated on ice with lysates containing immunoadhesins to the final volume of 0.3 ml for 30 min. The cells were washed, incubated for 30 min on ice with FITC-conjugated Ab specific for rabbit Igs (BioSource International), washed again, and resuspended in PBS.
Studies for analysis of activation markers were performed as follows. The cells were washed once with PBS/2% FCS/0.02% sodium azide, and incubated with anti- CD4, CD25, CD45RA, CD69, and Ki67 Abs at 1/20 dilution in PBS/2% FCS/0.02% sodium azide to the final volume of 200 μl. For the experiments involving two-color flow cytometry, PE-labeled anti-CD25 and PerCP-labeled anti-CD69 (BD Biosciences) were used. After incubating at 4°C for 30 min, cells were washed and fixed with 4% paraformaldehyde.
Because there are no commercially available Abs that recognize external epitopes on all the glycosylated forms of GLUT-1 (22), the expression of GLUT-1 was monitored by intracellular staining. The cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences) as directed by the manufacturer. Cells were blocked with 10% human AB serum for 15 min, and then 4 μl of a 1 μg/μl stock of anti-GLUT-1 Ab (Alpha Diagnostic International) was added to a final volume of 100 μl. The cells were incubated at 4°C for 30 min, washed with the perm/wash buffer as directed, and resuspended 50 μl of perm/wash buffer/1% skim milk containing 4 μl of FITC-conjugated goat anti-rabbit Ab (BioSource International). The cells were incubated at 4°C for 30 min and washed twice in cold perm/wash buffer, and then resuspended in 400 μl of PBS. To verify the extent of permeabilization, a portion of cells (100 μl) were stained with 2 μl of FITC-labeled anti-actin Ab (Sigma-Aldrich) following fixation/permeabilization. Twenty to fifty thousand live cell events were measured on a FACScan (BD Pharmingen) and analyzed using FlowJo software (Tree Star).
Cell cycle analysis was performed by fixing cells in 75% ethanol and staining the DNA with 50 μg/ml propidium iodide (Sigma-Aldrich) for 1 h. The DNA was analyzed by flow cytometry on a FACScan. For detection of the propidium iodide, the cells were excited at 488 nm, and their emissions were collected at 600 nm.
RNA extraction and RT-PCR analysis
Total RNA was isolated from cells using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s recommendation. The concentration and purity of RNA samples were measured, and 5 μg of total RNA was used for reverse transcription using oligo(dT) as reverse primer (SuperScript First-Strand Synthesis System for RT-PCR; Invitrogen Life Technologies). Five percent of the cDNA product was used for the PCR amplification of GLUT-1 or GAPDH. The PCR conditions were as follows: incubation for 3 min at 94°C followed by 25 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, and a final extension for 7 min at 72°C in a PerkinElmer 9700 Thermal Cycler. The primers used for GLUT-1 were 5'-TCCACGAGCATCTTCGAGA-3' (sense) and 5'-AACATGTTTGCTGGTGTCTGC-3' (antisense) (46); for actin were 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (sense) and 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3' (antisense). PCR products were identified on 1.5% agarose gel stained with ethidium bromide.
Western blot analysis of GLUT-1
Exponentially growing cells were pelleted, washed twice with PBS, and lysed for 1 h on ice in Digitonin lysis buffer (PBS with 0.25% deoxycholate, 1% digitonin, and protease inhibitor mixture (Sigma-Aldrich)). Debris was removed by centrifugation at 4°C for 10 min at 16,000 x g. Protein concentration was determined (Protein Assay reagent; Bio-Rad), and equal amounts of protein (200–300 μg) were separated by SDS-PAGE electrophoresis on a 10% Tris-glycine gel (Invitrogen Life Technologies) and transferred to Immobilon-P membrane (Millipore). The membranes were blocked with 10% nonfat dry milk/1x TBST (TBS with 0.1% Triton X-100) for 30 min at room temperature, and then incubated overnight at 4°C with a polyclonal antiserum directed against GLUT-1 (Abcam) diluted 1/1000 in 5% nonfat dry milk/1x TBST. The membranes were then washed twice with 1x TBST, hybridized with HRP-conjugated anti-rabbit IgG for 1 h at room temperature, and washed three times with 1x TBST. The bands were visualized using ECL reagent (Amersham Biosciences) and exposed to film (Kodak).
Generation of pseudotyped retroviral vectors
HIV-based retroviral vectors were generated by cotransfecting 293-T cells with an HIV-based retroviral vector encoding the enhanced GFP (EGFP) (pHIV-eGFP; gift of V. KewalRamani, National Cancer Institute-Frederick) (47) and a plasmid encoding the appropriate Env protein. The plasmids encoding HTLV-I Env (CMV-ENV-LTR) and the G protein of vesicular stomatitis virus (VSV)-G have been previously described (20). The plasmid encoding HTLV-II Env (CMV-ENV-LTR-II) was constructed as follows: a full-length clone of HTLV-II (pH6neo; gift of P. Green, Ohio State University, Columbus, OH) (48, 49) was digested with SphI and ClaI (5124–7384) to generate a fragment that included the entire HTLV-II Env coding sequences. The plasmid CMV-ENV-LTR was digested with SphI and ClaI to remove the homologous fragment in the HTLV-I provirus (5145–7495 of the sequence with accession no. J02029), and the fragment replaced with the SphI-ClaI fragment from pH6neo.
To generate virus, 293-T cells were transfected with a total of 8 μg of DNA using FuGene 6 (Roche), at a 0.1:1 ratio of Env-expressing vector:pHIV-eGFP. Twenty hours after transfection, the cells were refed. Approximately 16 h later, viral supernatants were harvested, subjected to low-speed centrifugation, filtered through a 0.45-μm pore size filter, and used immediately.
Viral transduction
KPC/1 cells were resuspended in RPMI 1640 supplemented with 10% FCS at a concentration of 106 cells/ml. For each well in a six-well plate, 0.5 ml (5 x 105 cells) were added along with 1 ml of viral supernatant, and cells were transduced using a modification of the spin infection method as described previously (20). The amount of viral particles present in each sample were quantified using a gag p24 ELISA (Zeptometrix). Following the spin, the cells were incubated at 37°C for 2 h, and then washed twice in medium and incubated at 37°C. For determination of relative viral titer, target cells were incubated with 5-fold dilutions of supernatant containing the pseudotyped viruses. Negative control cultures (cells transduced with HIV vectors with no Env) were included in each experiment. Four days later, 50,000 live cells were analyzed by flow cytometry for the expression of EGFP to determine the percentage of transduced cells. Relative titers of the pseudotyped viruses were determined from the dilution with the lowest percentage that was between 5 and 40% positive for EGFP. The relative titers were determined using the following formula: Dilution factor x (% positive – % positive in negative control) x (5 x 105) (number of cells in well on at time of transduction). Titers were corrected for the amount of viral p24 protein present in the inoculum, and adjusted to 1 ml. Each sample was performed in duplicate, and the SEM was calculated.
Virion binding assay
Specific binding of HTLV virions to target cells was examined using a modification of a previously described method (50). Virus was concentrated from the supernatant of an HTLV-I-producing cell line (MT-2) by growing the cell line at a high density (3–5 x 106/ml) in the inner chamber of an Integra CL1000 flask (Integra Biosciences). After 2 days, the cells were removed by centrifugation at 800 x g for 5 min and filtering the supernatant through a 0.22-μm filter (Millipore), and the viral preparation was stored at –80°C. The amount of HTLV-1p19 in the sample was determined using an ELISA (Zeptometrix). For the viral binding assay, target cells (1 x 106) were centrifuged at 800 x g for 5 min, washed with ice-cold PBS, centrifuged again, and resuspended in 0.2 ml of PBS. Cells were then incubated with 20 μg of p19 from the viral preparation at 22°C for 30 min. As a control, cells containing no virus were incubated in parallel. After incubation, both sets of cells were washed in PBS, resuspended in 200 μl of PBS, and incubated for 30 min on ice with 1.0 μg of mAb directed against HTLV SU (anti-gp46 mAb clone 65/6C2.2.34; Zeptometrix). Cells were then washed and resuspended in 100 μl of PBS with a goat anti-mouse FITC-conjugated Ab for 30 min on ice. After the incubation, the cells were washed and immediately analyzed by flow cytometry.
Results
TGF- stimulates cell surface expression of HTLV-I Env binding proteins on CD4+ T lymphocytes
Recently, we and others have reported that naive resting CD4+ T cells do not bind detectable levels of HTLV-I SU. However, molecules that specifically bind HTLV SU proteins are rapidly induced (4–6 h) after different modes of T cell stimulation. We wanted to examine whether expression of these proteins could be induced by TGF-.
Freshly isolated lymphocytes from cord blood, enriched for CD4+ T cells, were left untreated, treated with TGF-2, or stimulated with PHA and IL-2. After 48 h of culture in serum-free medium, the cell surface expression of the HTLV-I SU binding proteins was determined from the level of soluble SU (HTSU-IgG) bound to the cells by flow cytometry analysis. As a control for nonspecific binding, the amount of binding to a similar immunoadhesin SUA-rIgG, which contains the SU protein from an unrelated avian retrovirus, was determined in parallel. A significant increase in binding with HTSU-IgG was observed when the cells were incubated with TGF-, in comparison with the cells cultured in medium alone (Fig. 1A, compare left and middle panels). As expected from previous work (21, 31), a dramatic increase in binding with HTSU-IgG was observed when the cells were stimulated following exposure to PHA and IL-2 (Fig. 1A, right panel).
FIGURE 1. TGF- induces molecules that specifically bind HTLV SU on quiescent cord blood T lymphocytes. A, Cord blood lymphocytes were enriched for naive CD4+ T cells (>95% purity) as described in Materials and Methods. Cells were then cultured in RPMI 1640 medium supplemented with BIT at a concentration at 1 x 106/ml either alone, with TGF-2 (10 ng/ml), or with PHA (1 μg/ml) and IL-2 (20 U/ml). Two days later, the cells were assayed for their ability to specifically bind HTSU-IgG performed as described in Materials and Methods. Light line, Binding to HTSU-IgG; dark line, binding to negative control SUA-IgG. Data are representative of results obtained in eight separate experiments. B, Cells were cultured as in A with an additional TGF-2 culture that contained a specific small molecule inhibitor of TGF- type I receptor signaling (SB-431542) at 20 μM added 15 min earlier. Two days later, binding assays to HTSU-IgG were performed using 500 ng/ml HTSU-IgG and SUA-IgG. Data are representative of results obtained in three separate experiments. C, CD4+ enriched naive cord blood T lymphocytes were cultured as described in A. Cells were collected at 1, 2, and 5 days after culture, HTSU-IgG binding assays were performed as above, and mean fluorescence intensity (MFI) was determined. Data are representative of results obtained in three separate experiments.
These observations indicated that treatment of naive CD4+ T lymphocytes with TGF- induced cell surface expression of molecules that specifically bound HTLV SU. TGF- signals through a receptor complex of type I (TGF-RI) and type II (TGF-RII) receptors. To verify that the effect we observed involved TGF--induced intracellular signaling via the Smad pathway, the effect of an inhibitor of Smad signaling on HTSU binding was examined. Unstimulated CD4+ T cells were treated with TGF- either alone or in the presence of a specific small molecule inhibitor of TGF- type I receptor signaling (SB-431542; Refs. 43 and 44). Two days later, the level of binding of HTSU-IgG to these cells was determined (Fig. 1B). As reported above, TGF-2 dramatically increased cell surface levels of HTLV-I SU binding proteins. In contrast, treatment of the cord blood lymphocytes with both TGF-2 and the SB-431524 inhibitor resulted in a level of HTLV SU binding proteins similar to the untreated controls. These results indicate that TGF- induces cell surface expression of molecules that specifically bind to HTLV-I SU by intracellular signaling through TGF-RI. Similar results were seen when cells were treated with the same concentrations of TGF-1 in place of TGF-2 (data not shown).
Kinetics of expression of the HTLV SU binding proteins following TGF- treatment
Previously, it has been reported that the HTLV SU binding proteins are rapidly expressed following immune activation of naive T cells (21, 31). The level of binding then diminished as the T lymphocytes returned to their resting state. We next examined the kinetics of expression of the HTLV-I SU binding protein(s) following exposure to TGF-. Unstimulated CD4+ cord blood T lymphocytes were exposed to either TGF- or PHA/IL-2. Cells bound significant levels of soluble SU 18 h after exposure to TGF- (Fig. 1C). The level of binding was increased at 48 h after exposure; by 5 days after exposure, the binding was decreased with no detectable binding at day 10 (Fig. 1C and data not shown). This pattern of expression had similar kinetics to that observed following stimulation with PHA and IL-2 (21, 31). However, the level of binding was somewhat lower for the cells exposed to TGF- than those exposed to PHA/IL-2. This was a consistent finding in >12 cord blood samples. However, the kinetics of cell surface expression of the HTLV SU binding proteins was similar in cells exposed to TGF- and PHA/IL-2. As previously described for PHA/IL-2-stimulated cells (21, 31), detectable levels of HTSU-IgG binding was observed 6 h after TGF- treatments (data not shown).
TGF--induced expression of HTLV SU binding proteins on quiescent T cells requires de novo protein synthesis
It has been reported previously that the activation of HTLV SU binding molecules on CD4+ lymphocytes required de novo protein synthesis (21). We wanted to determine whether induction of HTLV binding proteins by TGF- also required de novo protein synthesis. Naive cord blood lymphocytes, enriched for CD4+ cells, were exposed to TGF- or PHA and IL-2, either alone or in the presence of cycloheximide. Three days later, binding of HTSU-IgG to these cells was determined. When protein synthesis was blocked, neither TGF- nor PHA/IL-2 induced the expression of HTLV SU binding proteins (Fig. 2, right panels). Treatment with actinomycin D, an inhibitor of transcription, also blocked expression of these proteins, suggesting that de novo transcription as well as translation was required (Fig. 2, middle panels).
FIGURE 2. TGF--induced expression of HTLV SU binding proteins requires de novo transcription and translation. Naive CD4+ enriched cord blood lymphocytes were resuspended at 1 x 106 in RPMI 1640/10% human AB sera. The cells were then either left untreated, or treated with 10 μg/ml actinomycin D, or 5 μg/ml cyclohexamide. For each condition, the cells were divided into two flasks and additionally treated with either TGF-2 (10 ng/ml), or with PHA and IL-2. Three days later, the cells were harvested, and the amount of binding of HTSU-IgG was determined. Light line, Binding to HTSU-IgG; dark line, binding to negative control SUA-IgG. Data are representative of results obtained in two separate experiments.
Specific binding of HTSU-IgG following TGF- treatment occurs without activation of quiescent CD4+ T lymphocytes
Previously, it has been reported that several different modes of T cell activation all lead to the rapid induction of HTLV-I-SU binding on naive CD4+ T lymphocytes (21, 31). At early times after activation, T cells that were still phenotypically naive could bind soluble HTLV SU. This led to the suggestion that this protein is a very early marker of T cell activation.
We next examined whether induction of HTLV-I-SU binding proteins by TGF- required T cell activation. The percentage of cells positive for cell surface expression of two early activation markers, CD25 (IL-2R -chain) and CD69, was determined, along with percentage of cells binding HTSU-IgG (Fig. 3A). As expected, naive cord blood lymphocytes treated with PHA and IL-2 expressed high levels of CD25 and CD69, as well as HTLV SU binding proteins, on their cell surface. In contrast, TGF- induced cell surface expression of the HTLV SU binding proteins but not the activation markers. Further evidence that TGF- induced HTSU-IgG binding in the absence of activation came from the observations that there was no expression of Ki-67, a marker of proliferation, the cells remained CD45RA positive, and no increases in forward- and side-angle light scattering of the TGF--treated T cells was observed (data not shown). Furthermore, cell cycle analysis revealed that 40 h after treatment, the percentage of the T cells in S/G2M in the TGF--treated cells and the untreated cells was similar. In contrast, for the same cells treated in parallel with PHA and IL-2, the percentage of cells in S/G2M had increased to 27% over the control. Thus, HTLV SU binding proteins can be induced in the absence of T cell activation by TGF-.
FIGURE 3. Specific binding of soluble SU and HTLV virions following TGF- treatment occurs without activation of quiescent cord blood T lymphocytes. A, Naive cord blood CD4+ T cells were either left untreated in RPMI 1640/BIT medium, or treated with TGF-2 (10 ng/ml), or with PHA and IL-2. After 2 days in culture, the cells were harvested, and the amount of binding of HTSU-IgG, anti-CD25, and anti-CD69 Abs were determined. The percent positive for HTLV SU binding was calculated from the following formula: (% positive for HTSU-IgG) – (% positive for SUA-IgG); for the Abs, the formula used was as follows: (% positive for Ab) – (% positive for isotype control). Data are representative of results obtained in three separate experiments. B, Naive CD4+ cord blood T lymphocytes were treated as described above. Two days later, with no detectable cell activation, a virion binding assay was performed as described in Materials and Methods. Detection of the amount of virions bound was determined with an Ab directed against the HTLV SU. Black, Binding of HTLV-I virions; white, negative control (binding to cells incubated with medium without virus).
It has been reported that IL-7 differentially regulates cell cycle progression in neonatal and adult CD4+ T cells (51) and that IL-7-treated quiescent adult T cells, unlike quiescent neonatal T cells, do not enter S phase and do not express molecules that bind HTLV-I SU (21). We also examined the effect of TGF- on adult quiescent CD4+ T cells to determine whether it differed from that observed in neonatal cells. We observed that TGF-, unlike IL-7, induced the expression of HTLV-I SU binding proteins on both quiescent adult and cord blood T lymphocytes (data not shown).
Treatment of quiescent T cells with TGF- increases binding of HTLV-I virions
To complement the studies of the binding of soluble HTLV SU protein, we also examined the effect of TGF- on the binding of HTLV-I virions. CD4+ naive T lymphocytes were cultured for 3 days in either medium alone, or with medium containing either TGF- or PHA/IL-2. The cells were then incubated with concentrated cell-free HTLV-I virions, and the relative amount of virion binding was determined using a recently described flow-based assay (50). The binding of HTLV-I virions was similar to that observed for binding of the soluble HTLV SU alone. Treatment with TGF- induced the binding of HTLV-I virions to these cells. Activation of the cells by PHA and IL-2 also induced virion binding, at a higher level than that observed for TGF- (Fig. 3B). Thus, TGF- treatment of quiescent T lymphocytes induces cell surface expression of molecules that specifically bind both HTLV-I virions and soluble HTLV SU, in the absence of markers of activation.
TGF- induces expression of GLUT-1 in primary T lymphocytes
While this study was in progress, it was reported that the glucose transporter protein GLUT-1 could specifically bind soluble HTLV SU and was critical for efficient HTLV-II Env-mediated entry (22). Because those studies were performed in established cell lines, we examined the level of GLUT-1 in primary CD4+ T cells after exposure to TGF-.
As a first step, GLUT-1 expression was examined on treated and untreated cord blood lymphocytes. The level of GLUT-1 protein in the cells was determined by flow cytometry, as described in Materials and Methods. Cells cultured without any additional supplements except BIT medium did not express significant levels of GLUT-1 (Fig. 4A, left panel). Similar results were obtained with freshly isolated CD4+ cord blood lymphocytes (data not shown). Two days after activation with PHA/IL-2, the lymphocytes expressed high levels of GLUT-1 (Fig. 4A, right panel). TGF- also induced expression of GLUT-1, but at a lower level than PHA/IL-2-activated T cells (Fig. 4A, middle panel). Levels of HTLV SU binding, determined in parallel, were also higher for the lymphocytes activated by PHA and IL-2 (data not shown).
FIGURE 4. TGF- and PHA/IL-2 induce GLUT-1 expression in naive cord blood T lymphocytes. A, Naive CD4+ cord blood T lymphocytes were cultured with TGF-2, PHA and IL-2, or BIT medium alone. Three days later, the cells were examined for the level of expression of GLUT-1. B, Naive cord blood CD4+ T lymphocytes were isolated and treated with TGF-2 (10 ng/ml) alone or in the presence of 20 μM inhibitor of TGF- type I receptor signaling activity as described in Fig. 1B. Two days later, expression level of GLUT-1 was determined. For both A and B, GLUT-1 expression levels were determined by intracellular staining with an anti-GLUT-1 Ab as described in Materials and Methods. Light line, Binding of anti-GLUT-1 Ab; dark line, binding of isotype control. Data are representative of results obtained in four (A) and two (B) separate experiments. C, RT-PCR analysis of GLUT-1 mRNA expression. Naive cord blood CD4+ T lymphocytes were isolated and total RNA was extracted as described in Materials and Methods. The remaining cells were cultured as described above. Three days later, total RNA was extracted and reverse transcribed, and relative GLUT-1 mRNA expression was determined by nonsaturating PCR as described in Materials and Methods. Data are representative of results obtained from two separate experiments.
To verify that GLUT-1 expression was induced by intracellular signaling following TGF- stimulation, we examined the effect of a TGF- inhibitor. GLUT-1 expression was determined 4 days after exposure to TGF-, either with or without the inhibitor of TGF- type I receptor kinase described above. As shown in Fig. 4B, the induction of GLUT-1 by TGF- was completely blocked by the inhibitor.
TGF- and PHA/IL-2 treatment of naive CD4+ T cells also induced GLUT-1 mRNA expression (Fig. 4C). GLUT-1 mRNA was not detected in freshly isolated CD4+ T cells (Fig. 4C, lane 1). Following 2 days of treatment with either TGF- (Fig. 4C, lane 3) or PHA/IL-2 (lane 4), a dramatic increase in the level of GLUT-1 mRNA was observed. In the cells cultured in medium alone, a low but significant level of GLUT-1 mRNA was present (lane 2). This is consistent with the low levels of HTSU-IgG binding observed following several days of culture in medium (see Figs. 1, B and C, and 3), and may reflect the activation of GLUT-1 transcription by the insulin present in the BIT supplement.
Treatment of target cells with TGF- increases the titer of HTLV Env-pseudotyped viruses
Because requirements for retroviral Env-mediated fusion can differ from those of binding alone, we wanted to determine the effect of TGF- on the titer of HTLV-pseudotyped viruses. HTLV virions are poorly infectious (52, 53), and HTLV-pseudotyped viruses do not transduce primary T cells at detectable levels (our unpublished data). We therefore determined the effect of TGF- treatment on a TGF--sensitive cell line on the titer of HTLV-pseudotyped viruses. Numerous cell lines have lost their sensitivity to TGF-. Recently, we have generated a variant of K562 that was sensitive to signaling by TGF- as measured by growth inhibition and Smad2 phosphorylation.
Initially, we wanted to determine whether treatment with TGF- increased HTLV-I SU binding to KPC/1 cells. K562 have previously been shown to express molecules capable of binding HTLV-I Env (54). Similarly, KPC/1, like all other vertebrate cell lines tested to date, binds HTSU-IgG. To examine whether binding was increased by TGF-, cells were treated with different doses of the cytokine for 3 days. TGF- increased cell surface expression of HTLV SU binding proteins in a dose-dependent manner with 5 ng/ml being the optimal dose (Fig. 5A; data not shown).
FIGURE 5. TGF- increases binding of HTLV SU to KPC/1 and expression of GLUT-1 mRNA and protein in a dose-dependent manner. A, The human myeloid cell line KPC/1 was suspended at 0.5 x 106/ml in RPMI 1640 with either 0, 1, 2, or 5 ng/ml TGF-2. Two days later, the cells were refed with TGF--containing medium. Three days after culture initiation, the cells were harvested and tested for their ability to bind of HTSU-IgG. Data are the average of results obtained in two separate experiments. B, KPC/1 was treated with different doses of TGF-. After 2 days, RNA was extracted, and relative GLUT-1 mRNA expression was determined by nonsaturating PCR. C, KPC/1 was treated with different doses of TGF-1 as indicated. After 2 days, the cells were lysed, and immunoblot analysis of GLUT-1 was performed as described in Materials and Methods. Data are representative of results obtained in two separate experiments.
The induction of GLUT-1 in KPC/1 cells following exposure to TGF- was also examined. Treatment of these cells with different concentrations of TGF- increased the level of GLUT-1 mRNA cells (Fig. 5B). The level of GLUT-1 protein was also increased following exposure of KPC/1 to different concentrations of TGF- (Fig. 5C). Thus, treatment of KPC/1 cells with TGF- increased binding of the soluble HTLV SU, as well as GLUT-1 mRNA and protein levels.
We next tested the effect of TGF- on entry into KPC/1 cells. HIV-based indicator proviruses, pseudotyped with either HTLV-I Env, HTLV-II Env, or VSV-G proteins, were generated and used to transduce either treated or untreated KPC/1 cells as described in Materials and Methods. Three days later, the titers were determined. Treatment of KPC/1 with TGF-2 increased the titer of HTLV-I and HTLV-II (3.5- and 2-fold, respectively) (Fig. 6). Treatment with TGF-1 gave similar results (data not shown). This appeared to be a specific effect on HTLV Env-mediated entry, because the titer of VSV-G-pseudotyped virus in KPC/1 was not significantly affected by TGF- (Fig. 6, right panel). Thus, TGF- is capable of up-regulating HTLV Env-mediated virus/cell fusion and entry.
FIGURE 6. TGF- increases titer of HTLV-I- and HTLV-II-pseudotyped viruses in KPC/1 cells. KPC/1 cells were suspended at 1 x 106/ml in RPMI 1640, and either left untreated or treated with 5 ng/ml TGF-2. Two days later, the cells were transduced with HTLV-I-, HTLV-II-, or VSV-G-pseudotyped virus particles, generated as described in Materials and Methods. The relative amount of viral particles present in the supernatants, quantified using a p24 ELISA, were determined to be as follows: HTLV-I, 24 ng/ml; HTLV-II, 33 ng/ml; VSV-G, 29 ng/ml. Four days later, the cells were harvested, and the titer was determined as described in Materials and Methods, and normalized for the amount of p24 present in the inoculum. Each sample was performed in duplicate, and the SEM was determined. White, No treatment; black, treated with TGF-. Data are representative of results obtained in three separate experiments.
Discussion
For retroviral infection to occur, cell surface molecules must bind the viral envelope, leading to the subsequent fusion of the viral and cellular membranes. For some retroviruses such as HIV, more than one molecule is involved, whereas for ecotropic murine leukemia virus, a single molecule confers entry. Recently, the ubiquitous GLUT-1 was shown to specifically bind a soluble form of both the HTLV-I and HTLV-II SU proteins in T cell and non-T cell lines, and was shown to be critical for efficient entry of HTLV-II-pseudotyped virions into epithelial cell lines.
Recent studies have shown that initial binding of a retrovirus to the surface of target cells can involve molecules other than the binding/fusion receptors. For HIV-1, a number of molecules that promote initial attachment of virions to the cell surface have been identified; these include HSPGs, LFA-1, and nucleolin (23, 55). Recently, it has been reported that HSPGs can specifically bind soluble HTLV SU and enhance entry of HTLV-I pseudotypes (26, 27). Although the role of HSPGs in the transmission of HTLV in vivo is not yet clear, these observations indicate that HSPG, as well as HTLV binding/fusion receptors, contribute to the level of binding of soluble SU and the titer of HTLV-pseudotyped viruses, at least on certain cell lines.
The cell surface molecules required for efficient entry of HTLV-I virions into primary T lymphocytes have not been rigorously defined. The highest levels of HTLV SU binding on primary cells are observed on CD4+ and CD8+ T cells undergoing an immune response (21, 31). In contrast, quiescent naive and memory T cells derived from adult and cord blood cells do not bind detectable levels of HTLV-I SU. In this study, we show that TGF- can induce expression of the HTLV SU binding proteins on quiescent T cells.
HTLV-I is primarily transmitted vertically from mother to infant, primarily by breast milk, and horizontally by sexual contact or by blood products. The virus is believed to spread primarily by T cell-to-T cell contact. Contact between T cells has been shown to result in polarization of the cytoskeleton, leading to transfer of viral cores to the uninfected cell (56). Circulating T cells are almost entirely in the G0 phase of the cell cycle and are not expressing the HTLV SU binding proteins. The observations that HTLV binding proteins are induced by immune activation, and that virus can be transferred during interactions between T cells, suggests that viral transmission may occur primarily in areas of extensive immune reactivity such as lymph nodes or sites of inflammation. This study suggests that, alternatively, TGF--induced expression of HTLV receptors could allow binding of HTLV-I virions to T cells in the absence of immune activation. For HIV-1, it has been previously shown that the virus could interact with quiescent T cells, which led to productive infection when those cells were subsequently stimulated (32).
TGF-, which has been shown to enhance transmission of HTLV-I in vitro (39), is found at high levels in breast milk and semen. TGF- has previously been shown to increase virus production in PMBCs isolated from infected individuals and transcription from the HTLV-I LTR (38, 39). In the current study, we observed that treatment of these cells with TGF- induces expression of molecules which specifically bind both a soluble form of HTLV SU (Fig. 1A) and HTLV-I virions (Fig. 3B). The effect of TGF- on these cells involves TGF--induced intracellular signaling via the Smad pathway, because the effect was blocked by an inhibitor of TGF- type I receptor signaling (43, 44) (Fig. 1B). The cell surface expression of HTLV-I SU binding proteins required de novo transcription and translation (Fig. 2).
We also examined the level of GLUT-1 proteins in treated and untreated naive CD4+ T lymphocytes. As previously reported (57), GLUT-1 was not expressed at detectable levels in unstimulated CD4+ T lymphocytes (Fig. 4). Following exposure of the cells to TGF-, expression of both GLUT-1 protein (Fig. 4A) and mRNA (C) was induced. The GLUT-1 expression was blocked by a specific inhibitor of TGF- type I receptor signaling (Fig. 4B). This observation is consistent with previous reports that TGF- up-regulates GLUT-1 in mesenchymal cells (58, 59) and in mouse fibroblast cells (60).
GLUT-1 expression was also observed following activation of the cells by PHA and IL-2. The kinetics of the expression of GLUT-1 (Fig. 4) was similar to what has been previously reported for expression of HTLV SU binding proteins following treatment of naive T cells by either PHA and IL-2 or TGF- (21, 31). Although indirect, these observations in primary CD4+ T cells are consistent with the results from established cell lines showing that GLUT-1 can directly bind HTLV SU proteins.
Previous observations that HTLV-I SU binding proteins are rapidly induced on quiescent CD4+ T lymphocytes following different modes of activation led to the suggestion that this protein is a very early marker of T cell activation (21, 31). Furthermore, IL-7, which had been shown to induce proliferation in neonatal but not adult T cells (51), was later shown to induce expression of HTLV-I SU binding proteins on neonatal but not adult cells. These observations led to the suggestion that the SU binding proteins are distinct activation markers in neonatal and adult CD4+ lymphocytes (21).
In the current study, we found that TGF- can induce binding of soluble HTLV SU and expression of GLUT-1 in these cells without activation or proliferation of the T lymphocytes. Induction of HTLV SU binding was observed in the absence of expression of two early markers of T cell activation, CD25 and CD69 (Fig. 3A). The cells also retained their CD45RAhigh, Ki67– phenotype, and did not form blasts. Cell cycle analysis confirmed that, as expected from previous work (34), the TGF--treated cells and the untreated controls had little or no cells in S-G2M. Previously, it has been reported that IL-7 (51) and TGF- (34) treatments of adult cells result in a block in the G1b stage of the cell cycle. In contrast to IL-7, which does not induce HTLV-I SU binding on quiescent adult T cells (21, 51), TGF- can stimulate HTLV-I SU binding on the same quiescent cells.
It has been reported recently that stimulation of the TCR on naive CD4+CD25– T cells in the presence of TGF- can result in differentiation of the cells to CD4+CD25+ regulatory T (Treg) cells (61). Although induction of CD25 was not observed following exposure to TGF- alone, we further examined whether differentiation toward Treg cells occurred in these cultures by looking at expression of the FoxP3-specific marker for Treg cells. As previously reported (61), real-time RT-PCR analysis revealed that exposure of naive CD4+ T cells to TGF- and anti-CD3/CD28 induced FoxP3 mRNA expression, whereas exposure to TGF- alone did not induce FoxP3 expression (data not shown). Thus, it appears unlikely that the binding of HTLV SU observed following exposure of naive CD4+ T cells to TGF- reflects differentiation toward Treg cells.
The observation that TGF- can induce expression of HTLV receptors without activating T cells parallels what has previously been reported for the coreceptors of HIV-1. TGF- increases expression of CXCR4 but not CD4 on resting T cells without inducing expression of CD25, CD69, or Ki67 (62), even after 10 days in culture. The induction of GLUT-1 expression in the absence of proliferation is consistent with another study demonstrating that TGF- induces GLUT-1 expression without stimulating DNA synthesis in quiescent murine fibroblasts (60). Because quiescent T cells have a very low metabolic rate, it is not surprising that GLUT-1 is induced very rapidly. In contrast, because TGF- is known to be a potent immunosuppressive molecule (33), we are investigating what other rapidly induced molecules might be involved in this immunosuppression.
The effect of TGF- on HTLV Env-mediated entry was also examined. TGF- treatment increased the titer of both HTLV-I- and HTLV-II-pseudotyped viruses, but not the titer of VSV-G pseudotypes, in a TGF--sensitive K562 cell line (Fig. 6). One drawback of these studies, as discussed previously for the studies identifying GLUT-1 as an HTLV receptor (63), is that the effect of TGF- on the titer and the level of HTLV SU binding is being measured on cells that can be transduced by HTLV pseudotypes and bind HTLV SU before treatment. Indeed, recent work showing that HTLV-I SU binds HSPG and that blocking HSPG/SU interactions by dextran sulfate dramatically reduces the titer of HTLV-I Env-pseudotyped viral particles (27) indicates that more than one molecule can be required for efficient HTLV Env-mediated entry. Thus, it is unclear whether treatment with TGF- is up-regulating molecules (GLUT-1 and/or other) that are necessary, or only enhance, HTLV Env-mediated entry. Studies to directly examine whether GLUT-1 mediates all of the effects of TGF- on HTLV SU binding to quiescent T cells are beyond the scope of this report. Studies to define conditions for small interfering RNA entry into primary quiescent T cells without activation are in progress.
The observation that TGF- up-regulates the expression of binding receptors on naive T lymphocytes and increases the titer of HTLV-I- and HTLV-II-pseudotyped virions suggests that TGF- plays a role in transmission of HTLV-I virus in vivo. In vitro studies have shown that TGF- increases production of virus in cultures of HTLV-I-infected cells (38, 39, 64). This reflects at least in part activation of the HTLV-I LTR by TGF- (39). In that study, it appeared that TGF- enhanced transmission as well as virus production, because TGF- increased virus production more dramatically in cocultures of infected and uninfected nonactivated T cells. Our observations suggest that TGF- can induce HTLV-I receptors on the target cells and are consistent with a previous report that TGF- cannot prevent HTLV-I-mediated T cell activation (65).
Although HTLV-I infection leads to T cell activation, it, paradoxically, increases production of TGF- (66, 67). Studies with TGF-1-null mice have previously shown that TGF- is the prime in vivo block of T cell activation (68, 69). Both ATL and HAM/TSP cells are resistant to the growth-inhibitory effects of TGF- (38, 64, 65). However, when spontaneously growing HTLV-I-infected T clones were further stimulated by cross-linking of the CD3/TCR complex, the superimposed proliferation was inhibited by TGF-. This indicated that the TGF- signaling pathway in these cells is intact but is bypassed by HTLV-I-induced T cell activation (65).
It has recently been reported that independent expression of the HTLV-I transactivator protein Tax blocks growth suppression by TGF- by perturbing Smad-dependent TGF- signaling (64, 70, 71). Observations from this study that TGF- can regulate the expression of proteins important for HTLV-I entry suggest another function for the blocking of TGF- signaling by Tax. Many enveloped viruses, including retroviruses, down-regulate cell surface expression of their receptor on infected cells to prevent superinfection (14). One way viruses accomplish this is by Env-receptor interactions, either on the cell surface or in the endoplasmic reticulum. Alternatively, other virally encoded proteins can down-regulate the receptor. In cells infected with HIV-1, the viral regulatory Vpu and Nef proteins participate along with Env in CD4 down-regulation (recently reviewed in Ref. 72). In the current study, we observed that TGF- induces cell surface expression of proteins that bind HTLV Env proteins. These observations, taken together with previous reports that Tax can block TGF- signaling, raise the possibility that Tax could down-regulate expression of the binding receptor during HTLV infection. Studies to directly examine whether Tax can down-regulate the amount of binding of HTLV SU and/or the level of GLUT-1 expression by interfering with TGF- signaling are in progress.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Pat Green and Vineet KewalRamani for providing reagents, helpful suggestions, and encouragement, and Chris Grant and Steve Jacobson for their help with PCR.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This publication has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400.
2 The content of this publication does not reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
3 Address correspondence and reprint requests to Dr. Kathryn S. Jones, Building 567, Room 253, Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD 21702-1201. E-mail address: jonesk@ncifcrf.gov
4 Abbreviations used in this paper: HTLV, human T cell leukemia virus; ATL, adult T cell leukemia; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; GLUT-1, glucose transporter type 1; HSPG, heparan sulfate proteoglycan; LTR, long terminal repeat; EGFP, enhanced GFP; VSV, vesicular stomatitis virus; Treg, regulatory T.
Received for publication August 6, 2004. Accepted for publication January 11, 2005.
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The retrovirus human T cell leukemia virus (HTLV) type I (HTLV-I) is primarily transmitted by breast-feeding or sexual contact, by cell-to-cell contact between T cells. TGF-, which has been shown to enhance transmission of HTLV-I in vitro, is found at high levels in breast milk and semen. In this study, the ability of TGF- to regulate expression of molecules involved in HTLV-I binding and entry was examined. Previous studies using a soluble form of the HTLV-I envelope protein SU have shown that quiescent human T cells do not express cell surface molecules that specifically bind SU. After T cell activation, HTLV SU binding proteins are rapidly induced. In this study, we report that TGF- induces expression of proteins that bind soluble HTLV SU and HTLV virions on naive CD4+ T lymphocytes. The induction of these proteins occurred without cell cycle entry or expression of activation markers, involved TGF--induced intracellular signaling, and required de novo transcription and translation. Treatment of naive CD4+ T lymphocytes with TGF- induced expression of GLUT-1, which has recently been reported to function as a receptor for HTLV. Treatment of a TGF--sensitive human myeloid cell line increased the titer of both HTLV-I- and HTLV-II-pseudotyped viruses. Although earlier studies suggested that HTLV SU binding proteins might be an early marker of T cell activation and/or cell proliferation, we report in this study that TGF- induces binding of HTLV virions and expression of glucose transporter type 1 in primary CD4+ T lymphocytes that remain quiescent.
Introduction
The human T cell leukemia virus (HTLV)4 type I (HTLV-I) retrovirus, the first disease-causing human retrovirus isolated (1), is endemic in certain geographic areas, including southwestern Japan, the Caribbean basin, and parts of West Africa, South America, and Melanesia (2). Although the majority of 10 million infected individuals remain healthy lifelong asymptomatic carriers, HTLV-I is the etiologic agent for several diseases. The two most common are a severe lymphocyte neoplasia called adult T cell leukemia (ATL) (1, 3) and an inflammatory neurological disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (4, 5).
ATL is a malignancy of CD4+ T cells, and HTLV-I has a preferential tropism for CD4+ T cells in asymptomatic patients. Infection of both memory (CD45RO+) (6) and effector/memory (CD27–CD45RA–) (7) CD4+ T cells has been observed. Recent studies have indicated that, in HAM/TSP patients, both CD4+ and CD8+ T cells serve as viral reservoirs (8). The closely related retrovirus HTLV type II (HTLV-II), which is believed to share a common receptor with HTLV-I (9, 10), also infects both CD4+ and CD8+ T cells in vivo (11, 12). Both HTLV-I and HTLV-II can infect all human lymphocytic subsets, causing polyclonal and monoclonal T cell lymphocytoses (reviewed in Ref. 13).
Entry of retroviruses into target cells involves the interaction of two envelope (Env) glycoproteins, a surface glycoprotein (SU), and a transmembrane glycoprotein (TM), with at least one specific host cell receptor (14). In order for infection to occur, cell surface molecules must specifically bind the viral envelope, and this binding must lead to subsequent fusion of the viral and cellular membranes. For some retroviruses such as HIV, the binding and fusion molecules are separate; for others, such as ecotropic murine leukemia viruses, they appear to be a single molecule.
Because primary T cells are difficult to infect with HTLV-I in vitro, the majority of the work over the past 20 years has examined requirements for HTLV Env-mediated binding and fusion on established (often non-T) cell lines. These studies revealed that, in contrast to the limited in vivo tropism of HTLV, molecules capable of functioning as HTLV receptors are found on a wide variety of cells (15, 16, 17, 18). Cell surface molecules capable of specifically binding HTLV SU are even more widely expressed; they have been found on every vertebrate cell line tested to date (19, 20, 21).
Recently, the first identification of a specific molecule involved in HTLV binding and fusion was reported (22). The glucose transporter type 1 (GLUT-1) specifically binds a soluble form of both the HTLV-I and HTLV-II SU proteins in T cell and non-T cell lines, and was shown to be critical for efficient entry of HTLV-II-pseudotyped virions.
For some retroviruses, it is now clear that cell surface molecules other than the primary entry receptors can bind virions (recently reviewed in Ref. 23). These molecules, referred to as attachment factors, can play a critical role in the efficiency of retroviral entry. Heparan sulfates, which are widely expressed on the surface of cells, have been shown to be used by a number of viruses including the retroviruses HIV-1 and murine leukemia virus (24, 25). More recent studies have revealed that heparan sulfate proteoglycans (HSPG) can also specifically bind soluble HTLV SU and enhance entry of HTLV-I pseudotypes (26, 27). This indicates that the amount of binding of soluble SU and the titer of HTLV-pseudotyped viruses, at least on certain cell lines, reflects interactions with HSPG, as well as interactions with specific receptors. Because HSPGs are expressed at low or undetectable levels on primary CD4+ T cells (28, 29, 30), they may not play a role in the ability of HTLV-I to infect its primary target cell. Thus, it is not clear whether the requirements for HTLV-I Env-mediated fusion are identical in established cell lines and in primary T cells or how much of the binding of the soluble SU is to attachment factors rather than binding/fusion receptors.
The cell surface molecules required for efficient entry of HTLV-I virions into primary T lymphocytes have not been rigorously defined. Recent work by our group and another laboratory found that HTLV SU binding molecules are expressed on most primary cells of the immune system (21, 31), with the highest levels of binding observed on CD4+ and CD8+ T cells undergoing an immune response. In contrast to both stimulated T lymphocytes and established cell lines, quiescent T cells derived from adult and cord blood cells do not bind detectable levels of HTLV-I SU. However, SU binding proteins were rapidly induced after stimulation. Thus, it has been suggested that these HTLV SU binding proteins are an early marker of T cell activation (21, 31).
HTLV-I is primarily transmitted in vivo by breast-feeding or sexual contact. HTLV transmission appears to require passage of cells between individuals, and infection is believed to require contact between T cells. Circulating T cells are almost entirely in the G0 phase of the cell cycle; these cells do not express the HTLV SU binding protein. This suggests that viral transmission occurs in areas of extensive immune reactivity such as lymph nodes or sites of inflammation. Alternatively, humoral factors could stimulate expression of HTLV-I receptors on quiescent T cells that then could serve as targets for viral entry. For HIV-1, it has been reported that the virus can enter quiescent T cells, which can harbor the virus in an inactive state until subsequent mitogenic stimulation allows completion of the early stages of infection (32).
TGF- is a member of a superfamily of growth factors that regulate numerous physiological processes including cell cycle control, differentiation, and the immune response (33). TGF- can inhibit the growth of most epithelial and lymphoid cells by arresting cells in mid-G1 phase (34). Intracellular signaling is initiated upon the binding of TGF- to the cell surface type II TGF- receptor (TRII), which recruits type I TGF- receptor (TRI) to a multimeric complex (reviewed in Ref. 35). The propagation of the intracellular signal to the nucleus then occurs through phosphorylation of Smads, which regulate gene expression in cooperation with other transcription factors (23, 36, 37).
TGF-, which can enhance transmission of HTLV-I in vitro, is found at high levels in breast milk and semen. TGF- can induce expression of HTLV-I viral proteins in PBMCs from HAM/TSP (38) and asymptomatic carriers (39). In the latter study, it was shown that TGF- transactivates the long terminal repeat (LTR) promoter and can accelerate transmission of the virus from infected to uninfected cells. For HIV-1, TGF- has also been shown to both increase transcription from the LTR (40) and to up-regulate cell surface expression of coreceptors in target cells in the absence of T cell activation and proliferation (41, 42).
In this study, we observed that TGF- induces expression of HTLV SU binding proteins without activation of naive CD4+ T lymphocytes and can increase the titer of HTLV-I- and HTLV-II-pseudotyped viruses. Treatment of naive CD4+ T lymphocytes with TGF- induces expression of GLUT-1, a receptor for HTLV entry (22). Thus, TGF- can stimulate cell surface expression of proteins involved in HTLV binding and fusion in the absence of T cell activation and cell cycle entry.
Materials and Methods
Cell culture and isolation of lymphocytes
Cord blood samples obtained from healthy volunteer donors during vaginal births from Frederick Memorial Hospital (Frederick, MD) and leukopaks of peripheral blood from healthy donors were collected according to the National Institutes of Health approved institutional review board protocols. Mononuclear leukocytes were isolated by Ficoll-Hypaque gradient centrifugation. The light density fraction (buffy coat) was collected, washed twice with PBS, and then enriched for CD4+ T cells by removal of CD8-, CD19-, CD45RO-, CD11b-, and glycophorin-A-positive cells using MACs methods according to manufacturer’s instructions (Miltenyi Biotec). After isolation, the cells were cultured in either RPMI 1640 medium supplemented either with 20% BIT (BSA, insulin, and transferrin; StemCell Technologies) or with 10% human Ab, as indicated, along with 2 mM L-glutamine, sodium pyruvate (1 mM), and antibiotics. For TGF- treatment, cells were exposed to TGF-2 (Genzyme) or TGF-1 (BioSource) at the concentrations and times indicated. T cells were cultured in 20 U/ml IL-2 (Zeptometrix) and activated with PHA (1 μg/ml; Abbott Diagnostics) or cultured with 10 ng/ml IL-7 (PeproTech). In some studies, SB-431542 (Tocris Cookson) an inhibitor of TGF-RI signaling (43, 44) was added along with TGF-. In one study, the cells were treated with actinomycin D (Sigma-Aldrich) or cyclohexamide (Sigma-Aldrich). KPC/1 is a clone of the human myeloid cell line K562. It was generated by transfecting K562 with a plasmid encoding a neo selectable marker (pcDNA), selecting for transfected cells plated at 100 cells/well in a 96-well plate with 500 μg/ml G418, and identifying the cells most sensitive to TGF- (as judged by growth inhibition and level of Smad phosphorylation). All cells were cultured in a humidified atmosphere at 37°C in an incubator containing 5% CO2.
Expression of the recombinant HTLV-I SU envelope-Fc fusion proteins
Soluble SU proteins were generated essentially as previously described, with the following modifications. 293-T cells were transfected with either the plasmid vector encoding HTSU-IgG (HTSU-IgG/pSK100; Ref. 20) or a plasmid encoding a similar fusion protein (SUA-rIgG) containing the SU protein from the avian retrovirus ALSV-A (45). The latter was used as a negative control in all studies. Two days after transfection, the cells were washed with PBS, and then resuspended in ice-cold PBS to which proteinase inhibitor mixture (Sigma-Aldrich) had been added. The cells were then sonicated twice, and the lysates were centrifuged at 800 x g for 5 min. The clarified lysate was then aliquoted and stored at –80°C, and the concentration of immunoadhesins was determined by ELISA, as previously described (20).
Flow cytometric analysis
Specific binding of HTSU-IgG to target cells was examined essentially as previously described (20) with the following modifications. Briefly, the lysates containing immunoadhesin (either HTSU-IgG or SUA-IgG) were centrifuged at 12,000 x g for 1 min. Because HTSU-IgG can be rapidly internalized following binding to cells, and fixation does not significantly reduce the level of HTSU-IgG binding, target cells were fixed in 4% paraformaldehyde for 30 min on ice, washed with PBS, and then resuspended in PBS/2% FCS/0.02% sodium azide. The target cells (1 x 106) were then incubated on ice with lysates containing immunoadhesins to the final volume of 0.3 ml for 30 min. The cells were washed, incubated for 30 min on ice with FITC-conjugated Ab specific for rabbit Igs (BioSource International), washed again, and resuspended in PBS.
Studies for analysis of activation markers were performed as follows. The cells were washed once with PBS/2% FCS/0.02% sodium azide, and incubated with anti- CD4, CD25, CD45RA, CD69, and Ki67 Abs at 1/20 dilution in PBS/2% FCS/0.02% sodium azide to the final volume of 200 μl. For the experiments involving two-color flow cytometry, PE-labeled anti-CD25 and PerCP-labeled anti-CD69 (BD Biosciences) were used. After incubating at 4°C for 30 min, cells were washed and fixed with 4% paraformaldehyde.
Because there are no commercially available Abs that recognize external epitopes on all the glycosylated forms of GLUT-1 (22), the expression of GLUT-1 was monitored by intracellular staining. The cells were fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences) as directed by the manufacturer. Cells were blocked with 10% human AB serum for 15 min, and then 4 μl of a 1 μg/μl stock of anti-GLUT-1 Ab (Alpha Diagnostic International) was added to a final volume of 100 μl. The cells were incubated at 4°C for 30 min, washed with the perm/wash buffer as directed, and resuspended 50 μl of perm/wash buffer/1% skim milk containing 4 μl of FITC-conjugated goat anti-rabbit Ab (BioSource International). The cells were incubated at 4°C for 30 min and washed twice in cold perm/wash buffer, and then resuspended in 400 μl of PBS. To verify the extent of permeabilization, a portion of cells (100 μl) were stained with 2 μl of FITC-labeled anti-actin Ab (Sigma-Aldrich) following fixation/permeabilization. Twenty to fifty thousand live cell events were measured on a FACScan (BD Pharmingen) and analyzed using FlowJo software (Tree Star).
Cell cycle analysis was performed by fixing cells in 75% ethanol and staining the DNA with 50 μg/ml propidium iodide (Sigma-Aldrich) for 1 h. The DNA was analyzed by flow cytometry on a FACScan. For detection of the propidium iodide, the cells were excited at 488 nm, and their emissions were collected at 600 nm.
RNA extraction and RT-PCR analysis
Total RNA was isolated from cells using TRIzol reagent (Invitrogen Life Technologies), according to the manufacturer’s recommendation. The concentration and purity of RNA samples were measured, and 5 μg of total RNA was used for reverse transcription using oligo(dT) as reverse primer (SuperScript First-Strand Synthesis System for RT-PCR; Invitrogen Life Technologies). Five percent of the cDNA product was used for the PCR amplification of GLUT-1 or GAPDH. The PCR conditions were as follows: incubation for 3 min at 94°C followed by 25 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C, and a final extension for 7 min at 72°C in a PerkinElmer 9700 Thermal Cycler. The primers used for GLUT-1 were 5'-TCCACGAGCATCTTCGAGA-3' (sense) and 5'-AACATGTTTGCTGGTGTCTGC-3' (antisense) (46); for actin were 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' (sense) and 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3' (antisense). PCR products were identified on 1.5% agarose gel stained with ethidium bromide.
Western blot analysis of GLUT-1
Exponentially growing cells were pelleted, washed twice with PBS, and lysed for 1 h on ice in Digitonin lysis buffer (PBS with 0.25% deoxycholate, 1% digitonin, and protease inhibitor mixture (Sigma-Aldrich)). Debris was removed by centrifugation at 4°C for 10 min at 16,000 x g. Protein concentration was determined (Protein Assay reagent; Bio-Rad), and equal amounts of protein (200–300 μg) were separated by SDS-PAGE electrophoresis on a 10% Tris-glycine gel (Invitrogen Life Technologies) and transferred to Immobilon-P membrane (Millipore). The membranes were blocked with 10% nonfat dry milk/1x TBST (TBS with 0.1% Triton X-100) for 30 min at room temperature, and then incubated overnight at 4°C with a polyclonal antiserum directed against GLUT-1 (Abcam) diluted 1/1000 in 5% nonfat dry milk/1x TBST. The membranes were then washed twice with 1x TBST, hybridized with HRP-conjugated anti-rabbit IgG for 1 h at room temperature, and washed three times with 1x TBST. The bands were visualized using ECL reagent (Amersham Biosciences) and exposed to film (Kodak).
Generation of pseudotyped retroviral vectors
HIV-based retroviral vectors were generated by cotransfecting 293-T cells with an HIV-based retroviral vector encoding the enhanced GFP (EGFP) (pHIV-eGFP; gift of V. KewalRamani, National Cancer Institute-Frederick) (47) and a plasmid encoding the appropriate Env protein. The plasmids encoding HTLV-I Env (CMV-ENV-LTR) and the G protein of vesicular stomatitis virus (VSV)-G have been previously described (20). The plasmid encoding HTLV-II Env (CMV-ENV-LTR-II) was constructed as follows: a full-length clone of HTLV-II (pH6neo; gift of P. Green, Ohio State University, Columbus, OH) (48, 49) was digested with SphI and ClaI (5124–7384) to generate a fragment that included the entire HTLV-II Env coding sequences. The plasmid CMV-ENV-LTR was digested with SphI and ClaI to remove the homologous fragment in the HTLV-I provirus (5145–7495 of the sequence with accession no. J02029), and the fragment replaced with the SphI-ClaI fragment from pH6neo.
To generate virus, 293-T cells were transfected with a total of 8 μg of DNA using FuGene 6 (Roche), at a 0.1:1 ratio of Env-expressing vector:pHIV-eGFP. Twenty hours after transfection, the cells were refed. Approximately 16 h later, viral supernatants were harvested, subjected to low-speed centrifugation, filtered through a 0.45-μm pore size filter, and used immediately.
Viral transduction
KPC/1 cells were resuspended in RPMI 1640 supplemented with 10% FCS at a concentration of 106 cells/ml. For each well in a six-well plate, 0.5 ml (5 x 105 cells) were added along with 1 ml of viral supernatant, and cells were transduced using a modification of the spin infection method as described previously (20). The amount of viral particles present in each sample were quantified using a gag p24 ELISA (Zeptometrix). Following the spin, the cells were incubated at 37°C for 2 h, and then washed twice in medium and incubated at 37°C. For determination of relative viral titer, target cells were incubated with 5-fold dilutions of supernatant containing the pseudotyped viruses. Negative control cultures (cells transduced with HIV vectors with no Env) were included in each experiment. Four days later, 50,000 live cells were analyzed by flow cytometry for the expression of EGFP to determine the percentage of transduced cells. Relative titers of the pseudotyped viruses were determined from the dilution with the lowest percentage that was between 5 and 40% positive for EGFP. The relative titers were determined using the following formula: Dilution factor x (% positive – % positive in negative control) x (5 x 105) (number of cells in well on at time of transduction). Titers were corrected for the amount of viral p24 protein present in the inoculum, and adjusted to 1 ml. Each sample was performed in duplicate, and the SEM was calculated.
Virion binding assay
Specific binding of HTLV virions to target cells was examined using a modification of a previously described method (50). Virus was concentrated from the supernatant of an HTLV-I-producing cell line (MT-2) by growing the cell line at a high density (3–5 x 106/ml) in the inner chamber of an Integra CL1000 flask (Integra Biosciences). After 2 days, the cells were removed by centrifugation at 800 x g for 5 min and filtering the supernatant through a 0.22-μm filter (Millipore), and the viral preparation was stored at –80°C. The amount of HTLV-1p19 in the sample was determined using an ELISA (Zeptometrix). For the viral binding assay, target cells (1 x 106) were centrifuged at 800 x g for 5 min, washed with ice-cold PBS, centrifuged again, and resuspended in 0.2 ml of PBS. Cells were then incubated with 20 μg of p19 from the viral preparation at 22°C for 30 min. As a control, cells containing no virus were incubated in parallel. After incubation, both sets of cells were washed in PBS, resuspended in 200 μl of PBS, and incubated for 30 min on ice with 1.0 μg of mAb directed against HTLV SU (anti-gp46 mAb clone 65/6C2.2.34; Zeptometrix). Cells were then washed and resuspended in 100 μl of PBS with a goat anti-mouse FITC-conjugated Ab for 30 min on ice. After the incubation, the cells were washed and immediately analyzed by flow cytometry.
Results
TGF- stimulates cell surface expression of HTLV-I Env binding proteins on CD4+ T lymphocytes
Recently, we and others have reported that naive resting CD4+ T cells do not bind detectable levels of HTLV-I SU. However, molecules that specifically bind HTLV SU proteins are rapidly induced (4–6 h) after different modes of T cell stimulation. We wanted to examine whether expression of these proteins could be induced by TGF-.
Freshly isolated lymphocytes from cord blood, enriched for CD4+ T cells, were left untreated, treated with TGF-2, or stimulated with PHA and IL-2. After 48 h of culture in serum-free medium, the cell surface expression of the HTLV-I SU binding proteins was determined from the level of soluble SU (HTSU-IgG) bound to the cells by flow cytometry analysis. As a control for nonspecific binding, the amount of binding to a similar immunoadhesin SUA-rIgG, which contains the SU protein from an unrelated avian retrovirus, was determined in parallel. A significant increase in binding with HTSU-IgG was observed when the cells were incubated with TGF-, in comparison with the cells cultured in medium alone (Fig. 1A, compare left and middle panels). As expected from previous work (21, 31), a dramatic increase in binding with HTSU-IgG was observed when the cells were stimulated following exposure to PHA and IL-2 (Fig. 1A, right panel).
FIGURE 1. TGF- induces molecules that specifically bind HTLV SU on quiescent cord blood T lymphocytes. A, Cord blood lymphocytes were enriched for naive CD4+ T cells (>95% purity) as described in Materials and Methods. Cells were then cultured in RPMI 1640 medium supplemented with BIT at a concentration at 1 x 106/ml either alone, with TGF-2 (10 ng/ml), or with PHA (1 μg/ml) and IL-2 (20 U/ml). Two days later, the cells were assayed for their ability to specifically bind HTSU-IgG performed as described in Materials and Methods. Light line, Binding to HTSU-IgG; dark line, binding to negative control SUA-IgG. Data are representative of results obtained in eight separate experiments. B, Cells were cultured as in A with an additional TGF-2 culture that contained a specific small molecule inhibitor of TGF- type I receptor signaling (SB-431542) at 20 μM added 15 min earlier. Two days later, binding assays to HTSU-IgG were performed using 500 ng/ml HTSU-IgG and SUA-IgG. Data are representative of results obtained in three separate experiments. C, CD4+ enriched naive cord blood T lymphocytes were cultured as described in A. Cells were collected at 1, 2, and 5 days after culture, HTSU-IgG binding assays were performed as above, and mean fluorescence intensity (MFI) was determined. Data are representative of results obtained in three separate experiments.
These observations indicated that treatment of naive CD4+ T lymphocytes with TGF- induced cell surface expression of molecules that specifically bound HTLV SU. TGF- signals through a receptor complex of type I (TGF-RI) and type II (TGF-RII) receptors. To verify that the effect we observed involved TGF--induced intracellular signaling via the Smad pathway, the effect of an inhibitor of Smad signaling on HTSU binding was examined. Unstimulated CD4+ T cells were treated with TGF- either alone or in the presence of a specific small molecule inhibitor of TGF- type I receptor signaling (SB-431542; Refs. 43 and 44). Two days later, the level of binding of HTSU-IgG to these cells was determined (Fig. 1B). As reported above, TGF-2 dramatically increased cell surface levels of HTLV-I SU binding proteins. In contrast, treatment of the cord blood lymphocytes with both TGF-2 and the SB-431524 inhibitor resulted in a level of HTLV SU binding proteins similar to the untreated controls. These results indicate that TGF- induces cell surface expression of molecules that specifically bind to HTLV-I SU by intracellular signaling through TGF-RI. Similar results were seen when cells were treated with the same concentrations of TGF-1 in place of TGF-2 (data not shown).
Kinetics of expression of the HTLV SU binding proteins following TGF- treatment
Previously, it has been reported that the HTLV SU binding proteins are rapidly expressed following immune activation of naive T cells (21, 31). The level of binding then diminished as the T lymphocytes returned to their resting state. We next examined the kinetics of expression of the HTLV-I SU binding protein(s) following exposure to TGF-. Unstimulated CD4+ cord blood T lymphocytes were exposed to either TGF- or PHA/IL-2. Cells bound significant levels of soluble SU 18 h after exposure to TGF- (Fig. 1C). The level of binding was increased at 48 h after exposure; by 5 days after exposure, the binding was decreased with no detectable binding at day 10 (Fig. 1C and data not shown). This pattern of expression had similar kinetics to that observed following stimulation with PHA and IL-2 (21, 31). However, the level of binding was somewhat lower for the cells exposed to TGF- than those exposed to PHA/IL-2. This was a consistent finding in >12 cord blood samples. However, the kinetics of cell surface expression of the HTLV SU binding proteins was similar in cells exposed to TGF- and PHA/IL-2. As previously described for PHA/IL-2-stimulated cells (21, 31), detectable levels of HTSU-IgG binding was observed 6 h after TGF- treatments (data not shown).
TGF--induced expression of HTLV SU binding proteins on quiescent T cells requires de novo protein synthesis
It has been reported previously that the activation of HTLV SU binding molecules on CD4+ lymphocytes required de novo protein synthesis (21). We wanted to determine whether induction of HTLV binding proteins by TGF- also required de novo protein synthesis. Naive cord blood lymphocytes, enriched for CD4+ cells, were exposed to TGF- or PHA and IL-2, either alone or in the presence of cycloheximide. Three days later, binding of HTSU-IgG to these cells was determined. When protein synthesis was blocked, neither TGF- nor PHA/IL-2 induced the expression of HTLV SU binding proteins (Fig. 2, right panels). Treatment with actinomycin D, an inhibitor of transcription, also blocked expression of these proteins, suggesting that de novo transcription as well as translation was required (Fig. 2, middle panels).
FIGURE 2. TGF--induced expression of HTLV SU binding proteins requires de novo transcription and translation. Naive CD4+ enriched cord blood lymphocytes were resuspended at 1 x 106 in RPMI 1640/10% human AB sera. The cells were then either left untreated, or treated with 10 μg/ml actinomycin D, or 5 μg/ml cyclohexamide. For each condition, the cells were divided into two flasks and additionally treated with either TGF-2 (10 ng/ml), or with PHA and IL-2. Three days later, the cells were harvested, and the amount of binding of HTSU-IgG was determined. Light line, Binding to HTSU-IgG; dark line, binding to negative control SUA-IgG. Data are representative of results obtained in two separate experiments.
Specific binding of HTSU-IgG following TGF- treatment occurs without activation of quiescent CD4+ T lymphocytes
Previously, it has been reported that several different modes of T cell activation all lead to the rapid induction of HTLV-I-SU binding on naive CD4+ T lymphocytes (21, 31). At early times after activation, T cells that were still phenotypically naive could bind soluble HTLV SU. This led to the suggestion that this protein is a very early marker of T cell activation.
We next examined whether induction of HTLV-I-SU binding proteins by TGF- required T cell activation. The percentage of cells positive for cell surface expression of two early activation markers, CD25 (IL-2R -chain) and CD69, was determined, along with percentage of cells binding HTSU-IgG (Fig. 3A). As expected, naive cord blood lymphocytes treated with PHA and IL-2 expressed high levels of CD25 and CD69, as well as HTLV SU binding proteins, on their cell surface. In contrast, TGF- induced cell surface expression of the HTLV SU binding proteins but not the activation markers. Further evidence that TGF- induced HTSU-IgG binding in the absence of activation came from the observations that there was no expression of Ki-67, a marker of proliferation, the cells remained CD45RA positive, and no increases in forward- and side-angle light scattering of the TGF--treated T cells was observed (data not shown). Furthermore, cell cycle analysis revealed that 40 h after treatment, the percentage of the T cells in S/G2M in the TGF--treated cells and the untreated cells was similar. In contrast, for the same cells treated in parallel with PHA and IL-2, the percentage of cells in S/G2M had increased to 27% over the control. Thus, HTLV SU binding proteins can be induced in the absence of T cell activation by TGF-.
FIGURE 3. Specific binding of soluble SU and HTLV virions following TGF- treatment occurs without activation of quiescent cord blood T lymphocytes. A, Naive cord blood CD4+ T cells were either left untreated in RPMI 1640/BIT medium, or treated with TGF-2 (10 ng/ml), or with PHA and IL-2. After 2 days in culture, the cells were harvested, and the amount of binding of HTSU-IgG, anti-CD25, and anti-CD69 Abs were determined. The percent positive for HTLV SU binding was calculated from the following formula: (% positive for HTSU-IgG) – (% positive for SUA-IgG); for the Abs, the formula used was as follows: (% positive for Ab) – (% positive for isotype control). Data are representative of results obtained in three separate experiments. B, Naive CD4+ cord blood T lymphocytes were treated as described above. Two days later, with no detectable cell activation, a virion binding assay was performed as described in Materials and Methods. Detection of the amount of virions bound was determined with an Ab directed against the HTLV SU. Black, Binding of HTLV-I virions; white, negative control (binding to cells incubated with medium without virus).
It has been reported that IL-7 differentially regulates cell cycle progression in neonatal and adult CD4+ T cells (51) and that IL-7-treated quiescent adult T cells, unlike quiescent neonatal T cells, do not enter S phase and do not express molecules that bind HTLV-I SU (21). We also examined the effect of TGF- on adult quiescent CD4+ T cells to determine whether it differed from that observed in neonatal cells. We observed that TGF-, unlike IL-7, induced the expression of HTLV-I SU binding proteins on both quiescent adult and cord blood T lymphocytes (data not shown).
Treatment of quiescent T cells with TGF- increases binding of HTLV-I virions
To complement the studies of the binding of soluble HTLV SU protein, we also examined the effect of TGF- on the binding of HTLV-I virions. CD4+ naive T lymphocytes were cultured for 3 days in either medium alone, or with medium containing either TGF- or PHA/IL-2. The cells were then incubated with concentrated cell-free HTLV-I virions, and the relative amount of virion binding was determined using a recently described flow-based assay (50). The binding of HTLV-I virions was similar to that observed for binding of the soluble HTLV SU alone. Treatment with TGF- induced the binding of HTLV-I virions to these cells. Activation of the cells by PHA and IL-2 also induced virion binding, at a higher level than that observed for TGF- (Fig. 3B). Thus, TGF- treatment of quiescent T lymphocytes induces cell surface expression of molecules that specifically bind both HTLV-I virions and soluble HTLV SU, in the absence of markers of activation.
TGF- induces expression of GLUT-1 in primary T lymphocytes
While this study was in progress, it was reported that the glucose transporter protein GLUT-1 could specifically bind soluble HTLV SU and was critical for efficient HTLV-II Env-mediated entry (22). Because those studies were performed in established cell lines, we examined the level of GLUT-1 in primary CD4+ T cells after exposure to TGF-.
As a first step, GLUT-1 expression was examined on treated and untreated cord blood lymphocytes. The level of GLUT-1 protein in the cells was determined by flow cytometry, as described in Materials and Methods. Cells cultured without any additional supplements except BIT medium did not express significant levels of GLUT-1 (Fig. 4A, left panel). Similar results were obtained with freshly isolated CD4+ cord blood lymphocytes (data not shown). Two days after activation with PHA/IL-2, the lymphocytes expressed high levels of GLUT-1 (Fig. 4A, right panel). TGF- also induced expression of GLUT-1, but at a lower level than PHA/IL-2-activated T cells (Fig. 4A, middle panel). Levels of HTLV SU binding, determined in parallel, were also higher for the lymphocytes activated by PHA and IL-2 (data not shown).
FIGURE 4. TGF- and PHA/IL-2 induce GLUT-1 expression in naive cord blood T lymphocytes. A, Naive CD4+ cord blood T lymphocytes were cultured with TGF-2, PHA and IL-2, or BIT medium alone. Three days later, the cells were examined for the level of expression of GLUT-1. B, Naive cord blood CD4+ T lymphocytes were isolated and treated with TGF-2 (10 ng/ml) alone or in the presence of 20 μM inhibitor of TGF- type I receptor signaling activity as described in Fig. 1B. Two days later, expression level of GLUT-1 was determined. For both A and B, GLUT-1 expression levels were determined by intracellular staining with an anti-GLUT-1 Ab as described in Materials and Methods. Light line, Binding of anti-GLUT-1 Ab; dark line, binding of isotype control. Data are representative of results obtained in four (A) and two (B) separate experiments. C, RT-PCR analysis of GLUT-1 mRNA expression. Naive cord blood CD4+ T lymphocytes were isolated and total RNA was extracted as described in Materials and Methods. The remaining cells were cultured as described above. Three days later, total RNA was extracted and reverse transcribed, and relative GLUT-1 mRNA expression was determined by nonsaturating PCR as described in Materials and Methods. Data are representative of results obtained from two separate experiments.
To verify that GLUT-1 expression was induced by intracellular signaling following TGF- stimulation, we examined the effect of a TGF- inhibitor. GLUT-1 expression was determined 4 days after exposure to TGF-, either with or without the inhibitor of TGF- type I receptor kinase described above. As shown in Fig. 4B, the induction of GLUT-1 by TGF- was completely blocked by the inhibitor.
TGF- and PHA/IL-2 treatment of naive CD4+ T cells also induced GLUT-1 mRNA expression (Fig. 4C). GLUT-1 mRNA was not detected in freshly isolated CD4+ T cells (Fig. 4C, lane 1). Following 2 days of treatment with either TGF- (Fig. 4C, lane 3) or PHA/IL-2 (lane 4), a dramatic increase in the level of GLUT-1 mRNA was observed. In the cells cultured in medium alone, a low but significant level of GLUT-1 mRNA was present (lane 2). This is consistent with the low levels of HTSU-IgG binding observed following several days of culture in medium (see Figs. 1, B and C, and 3), and may reflect the activation of GLUT-1 transcription by the insulin present in the BIT supplement.
Treatment of target cells with TGF- increases the titer of HTLV Env-pseudotyped viruses
Because requirements for retroviral Env-mediated fusion can differ from those of binding alone, we wanted to determine the effect of TGF- on the titer of HTLV-pseudotyped viruses. HTLV virions are poorly infectious (52, 53), and HTLV-pseudotyped viruses do not transduce primary T cells at detectable levels (our unpublished data). We therefore determined the effect of TGF- treatment on a TGF--sensitive cell line on the titer of HTLV-pseudotyped viruses. Numerous cell lines have lost their sensitivity to TGF-. Recently, we have generated a variant of K562 that was sensitive to signaling by TGF- as measured by growth inhibition and Smad2 phosphorylation.
Initially, we wanted to determine whether treatment with TGF- increased HTLV-I SU binding to KPC/1 cells. K562 have previously been shown to express molecules capable of binding HTLV-I Env (54). Similarly, KPC/1, like all other vertebrate cell lines tested to date, binds HTSU-IgG. To examine whether binding was increased by TGF-, cells were treated with different doses of the cytokine for 3 days. TGF- increased cell surface expression of HTLV SU binding proteins in a dose-dependent manner with 5 ng/ml being the optimal dose (Fig. 5A; data not shown).
FIGURE 5. TGF- increases binding of HTLV SU to KPC/1 and expression of GLUT-1 mRNA and protein in a dose-dependent manner. A, The human myeloid cell line KPC/1 was suspended at 0.5 x 106/ml in RPMI 1640 with either 0, 1, 2, or 5 ng/ml TGF-2. Two days later, the cells were refed with TGF--containing medium. Three days after culture initiation, the cells were harvested and tested for their ability to bind of HTSU-IgG. Data are the average of results obtained in two separate experiments. B, KPC/1 was treated with different doses of TGF-. After 2 days, RNA was extracted, and relative GLUT-1 mRNA expression was determined by nonsaturating PCR. C, KPC/1 was treated with different doses of TGF-1 as indicated. After 2 days, the cells were lysed, and immunoblot analysis of GLUT-1 was performed as described in Materials and Methods. Data are representative of results obtained in two separate experiments.
The induction of GLUT-1 in KPC/1 cells following exposure to TGF- was also examined. Treatment of these cells with different concentrations of TGF- increased the level of GLUT-1 mRNA cells (Fig. 5B). The level of GLUT-1 protein was also increased following exposure of KPC/1 to different concentrations of TGF- (Fig. 5C). Thus, treatment of KPC/1 cells with TGF- increased binding of the soluble HTLV SU, as well as GLUT-1 mRNA and protein levels.
We next tested the effect of TGF- on entry into KPC/1 cells. HIV-based indicator proviruses, pseudotyped with either HTLV-I Env, HTLV-II Env, or VSV-G proteins, were generated and used to transduce either treated or untreated KPC/1 cells as described in Materials and Methods. Three days later, the titers were determined. Treatment of KPC/1 with TGF-2 increased the titer of HTLV-I and HTLV-II (3.5- and 2-fold, respectively) (Fig. 6). Treatment with TGF-1 gave similar results (data not shown). This appeared to be a specific effect on HTLV Env-mediated entry, because the titer of VSV-G-pseudotyped virus in KPC/1 was not significantly affected by TGF- (Fig. 6, right panel). Thus, TGF- is capable of up-regulating HTLV Env-mediated virus/cell fusion and entry.
FIGURE 6. TGF- increases titer of HTLV-I- and HTLV-II-pseudotyped viruses in KPC/1 cells. KPC/1 cells were suspended at 1 x 106/ml in RPMI 1640, and either left untreated or treated with 5 ng/ml TGF-2. Two days later, the cells were transduced with HTLV-I-, HTLV-II-, or VSV-G-pseudotyped virus particles, generated as described in Materials and Methods. The relative amount of viral particles present in the supernatants, quantified using a p24 ELISA, were determined to be as follows: HTLV-I, 24 ng/ml; HTLV-II, 33 ng/ml; VSV-G, 29 ng/ml. Four days later, the cells were harvested, and the titer was determined as described in Materials and Methods, and normalized for the amount of p24 present in the inoculum. Each sample was performed in duplicate, and the SEM was determined. White, No treatment; black, treated with TGF-. Data are representative of results obtained in three separate experiments.
Discussion
For retroviral infection to occur, cell surface molecules must bind the viral envelope, leading to the subsequent fusion of the viral and cellular membranes. For some retroviruses such as HIV, more than one molecule is involved, whereas for ecotropic murine leukemia virus, a single molecule confers entry. Recently, the ubiquitous GLUT-1 was shown to specifically bind a soluble form of both the HTLV-I and HTLV-II SU proteins in T cell and non-T cell lines, and was shown to be critical for efficient entry of HTLV-II-pseudotyped virions into epithelial cell lines.
Recent studies have shown that initial binding of a retrovirus to the surface of target cells can involve molecules other than the binding/fusion receptors. For HIV-1, a number of molecules that promote initial attachment of virions to the cell surface have been identified; these include HSPGs, LFA-1, and nucleolin (23, 55). Recently, it has been reported that HSPGs can specifically bind soluble HTLV SU and enhance entry of HTLV-I pseudotypes (26, 27). Although the role of HSPGs in the transmission of HTLV in vivo is not yet clear, these observations indicate that HSPG, as well as HTLV binding/fusion receptors, contribute to the level of binding of soluble SU and the titer of HTLV-pseudotyped viruses, at least on certain cell lines.
The cell surface molecules required for efficient entry of HTLV-I virions into primary T lymphocytes have not been rigorously defined. The highest levels of HTLV SU binding on primary cells are observed on CD4+ and CD8+ T cells undergoing an immune response (21, 31). In contrast, quiescent naive and memory T cells derived from adult and cord blood cells do not bind detectable levels of HTLV-I SU. In this study, we show that TGF- can induce expression of the HTLV SU binding proteins on quiescent T cells.
HTLV-I is primarily transmitted vertically from mother to infant, primarily by breast milk, and horizontally by sexual contact or by blood products. The virus is believed to spread primarily by T cell-to-T cell contact. Contact between T cells has been shown to result in polarization of the cytoskeleton, leading to transfer of viral cores to the uninfected cell (56). Circulating T cells are almost entirely in the G0 phase of the cell cycle and are not expressing the HTLV SU binding proteins. The observations that HTLV binding proteins are induced by immune activation, and that virus can be transferred during interactions between T cells, suggests that viral transmission may occur primarily in areas of extensive immune reactivity such as lymph nodes or sites of inflammation. This study suggests that, alternatively, TGF--induced expression of HTLV receptors could allow binding of HTLV-I virions to T cells in the absence of immune activation. For HIV-1, it has been previously shown that the virus could interact with quiescent T cells, which led to productive infection when those cells were subsequently stimulated (32).
TGF-, which has been shown to enhance transmission of HTLV-I in vitro (39), is found at high levels in breast milk and semen. TGF- has previously been shown to increase virus production in PMBCs isolated from infected individuals and transcription from the HTLV-I LTR (38, 39). In the current study, we observed that treatment of these cells with TGF- induces expression of molecules which specifically bind both a soluble form of HTLV SU (Fig. 1A) and HTLV-I virions (Fig. 3B). The effect of TGF- on these cells involves TGF--induced intracellular signaling via the Smad pathway, because the effect was blocked by an inhibitor of TGF- type I receptor signaling (43, 44) (Fig. 1B). The cell surface expression of HTLV-I SU binding proteins required de novo transcription and translation (Fig. 2).
We also examined the level of GLUT-1 proteins in treated and untreated naive CD4+ T lymphocytes. As previously reported (57), GLUT-1 was not expressed at detectable levels in unstimulated CD4+ T lymphocytes (Fig. 4). Following exposure of the cells to TGF-, expression of both GLUT-1 protein (Fig. 4A) and mRNA (C) was induced. The GLUT-1 expression was blocked by a specific inhibitor of TGF- type I receptor signaling (Fig. 4B). This observation is consistent with previous reports that TGF- up-regulates GLUT-1 in mesenchymal cells (58, 59) and in mouse fibroblast cells (60).
GLUT-1 expression was also observed following activation of the cells by PHA and IL-2. The kinetics of the expression of GLUT-1 (Fig. 4) was similar to what has been previously reported for expression of HTLV SU binding proteins following treatment of naive T cells by either PHA and IL-2 or TGF- (21, 31). Although indirect, these observations in primary CD4+ T cells are consistent with the results from established cell lines showing that GLUT-1 can directly bind HTLV SU proteins.
Previous observations that HTLV-I SU binding proteins are rapidly induced on quiescent CD4+ T lymphocytes following different modes of activation led to the suggestion that this protein is a very early marker of T cell activation (21, 31). Furthermore, IL-7, which had been shown to induce proliferation in neonatal but not adult T cells (51), was later shown to induce expression of HTLV-I SU binding proteins on neonatal but not adult cells. These observations led to the suggestion that the SU binding proteins are distinct activation markers in neonatal and adult CD4+ lymphocytes (21).
In the current study, we found that TGF- can induce binding of soluble HTLV SU and expression of GLUT-1 in these cells without activation or proliferation of the T lymphocytes. Induction of HTLV SU binding was observed in the absence of expression of two early markers of T cell activation, CD25 and CD69 (Fig. 3A). The cells also retained their CD45RAhigh, Ki67– phenotype, and did not form blasts. Cell cycle analysis confirmed that, as expected from previous work (34), the TGF--treated cells and the untreated controls had little or no cells in S-G2M. Previously, it has been reported that IL-7 (51) and TGF- (34) treatments of adult cells result in a block in the G1b stage of the cell cycle. In contrast to IL-7, which does not induce HTLV-I SU binding on quiescent adult T cells (21, 51), TGF- can stimulate HTLV-I SU binding on the same quiescent cells.
It has been reported recently that stimulation of the TCR on naive CD4+CD25– T cells in the presence of TGF- can result in differentiation of the cells to CD4+CD25+ regulatory T (Treg) cells (61). Although induction of CD25 was not observed following exposure to TGF- alone, we further examined whether differentiation toward Treg cells occurred in these cultures by looking at expression of the FoxP3-specific marker for Treg cells. As previously reported (61), real-time RT-PCR analysis revealed that exposure of naive CD4+ T cells to TGF- and anti-CD3/CD28 induced FoxP3 mRNA expression, whereas exposure to TGF- alone did not induce FoxP3 expression (data not shown). Thus, it appears unlikely that the binding of HTLV SU observed following exposure of naive CD4+ T cells to TGF- reflects differentiation toward Treg cells.
The observation that TGF- can induce expression of HTLV receptors without activating T cells parallels what has previously been reported for the coreceptors of HIV-1. TGF- increases expression of CXCR4 but not CD4 on resting T cells without inducing expression of CD25, CD69, or Ki67 (62), even after 10 days in culture. The induction of GLUT-1 expression in the absence of proliferation is consistent with another study demonstrating that TGF- induces GLUT-1 expression without stimulating DNA synthesis in quiescent murine fibroblasts (60). Because quiescent T cells have a very low metabolic rate, it is not surprising that GLUT-1 is induced very rapidly. In contrast, because TGF- is known to be a potent immunosuppressive molecule (33), we are investigating what other rapidly induced molecules might be involved in this immunosuppression.
The effect of TGF- on HTLV Env-mediated entry was also examined. TGF- treatment increased the titer of both HTLV-I- and HTLV-II-pseudotyped viruses, but not the titer of VSV-G pseudotypes, in a TGF--sensitive K562 cell line (Fig. 6). One drawback of these studies, as discussed previously for the studies identifying GLUT-1 as an HTLV receptor (63), is that the effect of TGF- on the titer and the level of HTLV SU binding is being measured on cells that can be transduced by HTLV pseudotypes and bind HTLV SU before treatment. Indeed, recent work showing that HTLV-I SU binds HSPG and that blocking HSPG/SU interactions by dextran sulfate dramatically reduces the titer of HTLV-I Env-pseudotyped viral particles (27) indicates that more than one molecule can be required for efficient HTLV Env-mediated entry. Thus, it is unclear whether treatment with TGF- is up-regulating molecules (GLUT-1 and/or other) that are necessary, or only enhance, HTLV Env-mediated entry. Studies to directly examine whether GLUT-1 mediates all of the effects of TGF- on HTLV SU binding to quiescent T cells are beyond the scope of this report. Studies to define conditions for small interfering RNA entry into primary quiescent T cells without activation are in progress.
The observation that TGF- up-regulates the expression of binding receptors on naive T lymphocytes and increases the titer of HTLV-I- and HTLV-II-pseudotyped virions suggests that TGF- plays a role in transmission of HTLV-I virus in vivo. In vitro studies have shown that TGF- increases production of virus in cultures of HTLV-I-infected cells (38, 39, 64). This reflects at least in part activation of the HTLV-I LTR by TGF- (39). In that study, it appeared that TGF- enhanced transmission as well as virus production, because TGF- increased virus production more dramatically in cocultures of infected and uninfected nonactivated T cells. Our observations suggest that TGF- can induce HTLV-I receptors on the target cells and are consistent with a previous report that TGF- cannot prevent HTLV-I-mediated T cell activation (65).
Although HTLV-I infection leads to T cell activation, it, paradoxically, increases production of TGF- (66, 67). Studies with TGF-1-null mice have previously shown that TGF- is the prime in vivo block of T cell activation (68, 69). Both ATL and HAM/TSP cells are resistant to the growth-inhibitory effects of TGF- (38, 64, 65). However, when spontaneously growing HTLV-I-infected T clones were further stimulated by cross-linking of the CD3/TCR complex, the superimposed proliferation was inhibited by TGF-. This indicated that the TGF- signaling pathway in these cells is intact but is bypassed by HTLV-I-induced T cell activation (65).
It has recently been reported that independent expression of the HTLV-I transactivator protein Tax blocks growth suppression by TGF- by perturbing Smad-dependent TGF- signaling (64, 70, 71). Observations from this study that TGF- can regulate the expression of proteins important for HTLV-I entry suggest another function for the blocking of TGF- signaling by Tax. Many enveloped viruses, including retroviruses, down-regulate cell surface expression of their receptor on infected cells to prevent superinfection (14). One way viruses accomplish this is by Env-receptor interactions, either on the cell surface or in the endoplasmic reticulum. Alternatively, other virally encoded proteins can down-regulate the receptor. In cells infected with HIV-1, the viral regulatory Vpu and Nef proteins participate along with Env in CD4 down-regulation (recently reviewed in Ref. 72). In the current study, we observed that TGF- induces cell surface expression of proteins that bind HTLV Env proteins. These observations, taken together with previous reports that Tax can block TGF- signaling, raise the possibility that Tax could down-regulate expression of the binding receptor during HTLV infection. Studies to directly examine whether Tax can down-regulate the amount of binding of HTLV SU and/or the level of GLUT-1 expression by interfering with TGF- signaling are in progress.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Pat Green and Vineet KewalRamani for providing reagents, helpful suggestions, and encouragement, and Chris Grant and Steve Jacobson for their help with PCR.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This publication has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400.
2 The content of this publication does not reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
3 Address correspondence and reprint requests to Dr. Kathryn S. Jones, Building 567, Room 253, Basic Research Program, Science Applications International Corporation-Frederick, Inc., National Cancer Institute-Frederick, Frederick, MD 21702-1201. E-mail address: jonesk@ncifcrf.gov
4 Abbreviations used in this paper: HTLV, human T cell leukemia virus; ATL, adult T cell leukemia; HAM/TSP, HTLV-I-associated myelopathy/tropical spastic paraparesis; GLUT-1, glucose transporter type 1; HSPG, heparan sulfate proteoglycan; LTR, long terminal repeat; EGFP, enhanced GFP; VSV, vesicular stomatitis virus; Treg, regulatory T.
Received for publication August 6, 2004. Accepted for publication January 11, 2005.
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