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Journal of Virology logoLink to Journal of Virology
. 1999 Aug;73(8):6436–6443. doi: 10.1128/jvi.73.8.6436-6443.1999

Establishment of a Seronegative Human T-Cell Leukemia Virus Type 1 (HTLV-1) Carrier State in Rats Inoculated with a Syngeneic HTLV-1-Immortalized T-Cell Line Preferentially Expressing Tax

Yoshihiro Koya 1, Takashi Ohashi 1, Hirotomo Kato 1, Shino Hanabuchi 1, Tomonori Tsukahara 1, Fumiyo Takemura 1,2, Ken-ichiro Etoh 3, Masao Matsuoka 3, Masahiro Fujii 1,4, Mari Kannagi 1,2,*
PMCID: PMC112724  PMID: 10400737

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) causes T-cell malignancies in a small percentage of the population infected with the virus after a long carrier state. In the present study, we established a seronegative HTLV-1 carrier state in rats inoculated with a newly established HTLV-1-infected rat T cell line, FPM1. FPM1 originated from rat thymocytes cocultured with a human HTLV-1 producer, MT-2 cells, and expressed rat CD4, CD5, CD25, and HTLV-1 Tax. However, FPM1 scarcely expressed other major HTLV-1 structural proteins and failed to induce typical antibody responses against HTLV-1 in inoculated rats. In contrast, control rats inoculated with MT-2 cells generated significant levels of anti-HTLV-1 antibodies. HTLV-1 proviruses were detected in peripheral blood cells of syngeneic rats inoculated with FPM1 for more than 1 year. Analysis of the flanking region of HTLV-1 provirus integrated into host cells suggested that FPM1 cells remained in these animals over a relatively long period of time. However, a similar seronegative HTLV-1 carrier state was induced in the rats inoculated with mitomycin C-treated FPM1 cells and also in FPM1-inoculated allogeneic rats, suggesting that FPM1 could also transmit HTLV-1 into host cells in vivo. Our findings indicated that (i) HTLV-1-immortalized T cells which preferentially express HTLV-1 Tax persisted in vivo but failed to induce any diseases in immunocompetent syngeneic rats and that (ii) suboptimal levels of HTLV-1 for antibody responses allowed the establishment of persistent HTLV-1 infection.

Human T-cell leukemia virus type 1 (HTLV-1) causes T-cell malignancies (14, 38) such as adult T-cell leukemia (ATL) (52), and chronic inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (9, 36). Although only a small population of HTLV-1-infected individuals develop malignant diseases, HTLV-1-infected cell clones in vivo possess more or less a self-proliferative characteristic, because oligoclonality of the infected cells is found not only in ATL patients but also in nonleukemic and asymptomatic HTLV-1 carriers (8, 56). This proliferative feature is thought to be due to HTLV-1 Tax, which transactivates various cellular genes that promote cell activation (7, 16, 33, 51).

Viruses use various strategies to avoid attack by the host immune system. In HTLV-1 infection, scarcity of the viral antigens in vivo may be one such strategy (20, 21), although the HTLV-1 genome is not completely silent (2, 10, 25, 55). HAM/TSP patients show relatively high viral expression associated with active immune responses (10). However, the viral expression is extremely low in ATL patients and many of the asymptomatic HTLV-1 carriers (25). Controversy exists as to whether such a low level of HTLV-1 expression in vivo is sufficient, for immortalizing infected cells, to cause infection of other cells in order to establish a variable repertoire of infected clones and for the activation of host immune mechanisms. Nevertheless, multiple HTLV-1-infected clones seem to arise in vivo, and some of them develop into more-malignant clones.

HTLV-1 carriers can be identified by serological tests that detect anti-HTLV-1 antibodies (14, 39). Serological screening of donated blood for HTLV-1-specific antibodies is now routinely performed throughout Japan. However, the seronegative HTLV-1-harboring state has been recently reported in patients with cutaneous malignancies, such as mycosis fungoides and cutaneous T-cell lymphoma, which were reported to be also associated with HTLV-1 infection (5, 11, 12, 30, 37). Most of these cases had defective HTLV-1 proviruses, which partly explained the negative host antibody responses, but some of them carried replication-competent HTLV-1 (5). It is unclear at present whether there are more seronegative HTLV-1 carriers, and what proportion of such carriers will develop T-cell malignancy. It is conceivable, however, that the host immune unresponsiveness might be advantageous for tumor development.

Experimental HTLV-1 infection in rats, established by inoculation of HTLV-1 producer cells, causes persistent HTLV-1 infection associated with specific antibody responses (15, 42, 48). HAM/TSP-like diseases actually occur in some strains of rats (17, 23, 26, 28). However, lymphoproliferative diseases hardly occur in these rats. This is partly explained by the time taken for clonal evolution of randomly infected cells toward a more malignant phenotype. Host immune responses established against abundant HTLV-1 antigens at primary infection could be another reason for the resistance to T-cell malignancy in these rats. In contrast, most of the human ATL patients show poor cellular immune responses against HTLV-1 accompanied by low levels of HTLV-1 expression in the tumor cells (20, 21). To mimic such a state in experimental animals, inoculation of syngeneic HTLV-1 tumor cells with low antigenicity may be preferable to HTLV-1 producer cells.

In an attempt to establish a model for the subclinical stage of HTLV-1 carriers with potential persistence of tumor cells, we describe in the present study the establishment of a rat T-cell line infected with HTLV-1 that scarcely expressed HTLV-1 structural proteins. When transferred into syngeneic rats, these cells persisted without causing overt leukemia and caused de novo infection in vivo in the absence of anti-HTLV-1 antibody responses. This model would be useful not only for understanding the mechanisms of persistence of potential tumor cells, but also for analyzing the mechanisms that allow primary infections to induce atypical seronegative HTLV-1 carriers.

MATERIALS AND METHODS

Animals and cell lines.

Inbred F344/N Jcl-rnu/+ (F344/N) and WKAH/HKm Slc (WKAH) rats were purchased from Clea Japan, Inc. (Tokyo, Japan), and Japan SLC, Inc. (Shizuoka, Japan), respectively. Established human T-cell lines included an HTLV-1 producer cell line, MT-2 (34); an HTLV-1-infected nonproducer cell line, TL-OmI (43); and an HTLV-1-negative cell line, MOLT-4 (40). HTLV-1-transformed T-cell line FPM1 was newly established in our laboratories from thymocytes of 4-week-old female F344/N Jcl-rnu/+ rats. Briefly, thymocytes were cocultured with the same number of mitomycin C (MMC; Sigma, St. Louis, Mo.)-treated MT-2 cells in RPMI 1640 medium with 10% fetal calf serum (FCS) and 20 U of recombinant human interleukin-2 (IL-2) per ml (Shionogi Co., Osaka, Japan). FPM1 required IL-2 for its growth in the initial 8 months and then gradually became IL-2 independent.

Analysis of cell surface markers.

Expression of cell surface markers was examined by flow cytometry. Briefly, 106 cells were stained with various mouse monoclonal antibodies (MAb) for 30 min on ice, washed three times with 1% FCS in phosphate-buffered saline (PBS), and then stained with fluorescein isothiocyanate-conjugated goat F(ab′)2 fragment anti-mouse IgG+IgM(H+L) (Jackson ImmunoResearch Laboratories, Inc.). After being washed, the cells were fixed with 1% formaldehyde in PBS prior to analysis on a FACSCalibur (Becton Dickinson). The MAbs utilized were anti-rat CD4 MAb RTH-7, anti-rat CD5 MAb R1-3B3, anti-rat CD8 MAb R1-10B5 (Seikagaku Co., Tokyo, Japan), anti-rat major histocompatibility complex class I (MHC-I) RT1.A MAb MRC-OX-18, anti-rat MHC-II RT1.B MAb MRC-OX-6 (Cosmo Bio, Tokyo, Japan), and anti-rat CD25 MAb OX-39 (Chemicon International).

Immunoblot analysis.

Immunoblot analysis was performed for the detection of HTLV-1 antigens in the cell line. Cells were lysed in a lysis buffer (20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 10% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.1% aprotinin, and 0.1% leupeptin) on ice for 30 min. Cell lysates were centrifuged at 12,000 × g for 5 min at 4°C and mixed with an equal volume of a twofold concentration sample buffer containing 125 mM Tris-HCl (pH 8.0), 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.004% bromophenol blue, and 2% 2-mercaptoethanol. These samples were boiled for 5 min and then subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on 12.5% separation gels; they were then blotted onto Clear Blot Membrane-p (Atto Co., Tokyo, Japan). The sheets were treated overnight with Block Ace (Dainippon Pharmaceutical Co., Osaka, Japan) at 4°C, washed twice with PBS, and incubated overnight with various antibodies diluted with 10% Block Ace and 0.5% Tween 20 in PBS at 4°C. The membranes were washed three times with 0.5% Tween 20 in PBS and incubated with horseradish peroxidase-labeled second antibodies (Amersham International plc., Buckinghamshire, United Kingdom) for 1 h. After a thorough washing, the bound antibodies were visualized with the ECL detection reagent (Amersham) and developed on Hyperfilm-ECL (Amersham). The anti-HTLV-1 MAbs utilized were anti-Tax1 mouse MAb Lt-4 (47), anti-p19 mouse MAb GIN14 (46), and anti-gp46 rat MAb REY30 (49), all of which were kindly provided by Y. Tanaka (Kitasato University). Human sera that were positive or negative for HTLV-1 antibodies were also used. A particular HTLV-1-infected human serum containing antibodies to HTLV-1 Tax was kindly provided by K. Matsumoto (Osaka Red Cross Blood Center, Osaka, Japan) (32).

Southern blot hybridization.

Genomic DNA prepared by using DNA ZOL reagent (GIBCO BRL) was digested with EcoRI, and the DNA fragments were separated by electrophoresis in 0.8% agarose gel and then denatured and transferred to a Biodyne B membrane (Pall Biosupport); they were then fixed by baking them at 80°C. The filter was prehybridized for 3 h at 65°C in a hybridization solution containing 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 50 mM Tris-HCl (pH 7.5), 1% SDS, 5× Denhardt solution, and 100 μg of denatured salmon sperm DNA per ml. (Stratagene, La Jolla, Calif.) and then hybridized with a 32P-labeled HTLV-1 long terminal repeat (LTR) probe overnight at 65°C in a hybridization solution. The filter was washed three times with 0.5× SSC containing 0.1% SDS at 65°C for 1 h. An autoradiogram was constructed by using a BAS 1500 imaging analyzer (Fuji Photofilm). The HTLV-1 LTR probe used in these assays was a 755-bp DNA fragment, which was originally amplified from DNA templates of F344-S1 cells (17) with the primers LTR-S (5′-TGACAATGACCATGAGCCCC-3′) and LTR-R (5′-TGTGTACTAAATTTCTCTCC-3′) by PCR and then cloned into pCR 2.1 (TA Cloning Kit, Invitrogen, Calif.).

Long PCR.

The long PCR method with the Expand Long Template PCR system (Boehringer Mannheim, Mannheim, Germany) was also used to detect HTLV-1 proviruses. The primers of HTLV-1 LTR used were primer 1 (5′-GTTCCACCCCTTTCCCTTTCATTCACGACTGACTGC-3′) and primer 2 (5′-GGCTCTAAGCCCCCGGGGGAT-3′) (45). The expected size of the amplified fragments with these primers from a full-length HTLV-1 provirus template was 7.7 kbp.

RT-PCR.

We used the reverse transcription-PCR (RT-PCR) method to detect HTLV-1 mRNA. Total RNA was extracted from the cells with Isogen (Nippon Gene Co., Toyama, Japan). It was then treated with DNase I (GIBCO BRL) and subjected to RT-PCR. The one-step RT-PCR method with dual functional EZ rTth RNA PCR kit (Perkin-Elmer) was used when the primers were designed to detect spliced RNA. To detect unspliced RNA, the two-step RT-PCR method was used with an “RT-PCR High” kit (Toyobo Co., Osaka, Japan). The primers used were gag 4 (5′-CCCCACTGCCAAAGACCTCCAAGA-3′; gag 5 (5′-TCTTTAGCACTCCCCGGCAGG-3′), RENV1 (5′-ACGCCGGTTGAGTCGCGTTCT-3′), RENV4 (5′-CACCGAAGATGAGGGGGCAGA-3′), RPX3 (5′-ATCCCGTGGA-GACTCCTCAA-3′), and RPX4 (5′-AACACGTAGACTGGGTATCC-3′). The primer sets gag 4 and gag 5 amplify 242-bp fragments from unspliced HTLV-1 mRNA. RENV1 and RENV4 are located upstream and downstream, respectively, of the first splice junction site of env mRNA, which amplify 316-bp fragments. RPX3 and RPX4 are located upstream and downstream, respectively, of the second splice junction site of tax/rex mRNA, which amplify 145-bp fragments. As an internal control, rat glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was also detected by the primer set of RT-G3PDH5′ (5′-CATTGACCTCAACTACATGG-3′) and RT-G3PDH3′ (5′-AGTGATGGCATGGACTGTGG-3′), which amplifies 435-bp cDNA fragments of G3PDH mRNA spliced over five intron regions. One-step RT-PCR was performed at 60°C for 60 min for RT followed by a single step of 94°C for 2 min and 30 cycles of a three-temperature PCR (94°C for 30 s, 55°C for 30 s, and 72°C for 30 s). For the two-step RT-PCR, RT at 42°C for 30 min was terminated at 99°C for 5 min and cooled off at 4°C for 5 min, and then 35 cycles of a three-temperature PCR (94°C for 30 s, 60°C for 30 s, and 72°C for 30 s) were performed. A thermal cycler (Touch Down, Hybaid, Middlesex, United Kingdom) was used for all PCR amplifications.

Inoculation of HTLV-1 infected cells.

A total of 107 FPM1 or MT-2 cells were intravenously inoculated into each of four female F344/N rats at 4 weeks of age. The presence of HTLV-1 provirus and antibodies to HTLV-1 in the peripheral blood was monitored every other week. HTLV-1 provirus in 3 μl of the whole peripheral blood was detected by a nested PCR method by using the Single-Tube PCR Kit (Takara, Kyoto, Japan) with HTLV-1 pX-specific primers. The outer primer sets were pX1 (5′-CCCACTTCCCAGGGTTTGGACAGAGTCTTC-3′) and pX4 (5′-GGGGAAGGAGGGGAGTCGAGGGATAAGGAA-3′), and the inner primer sets were pX2 (5′-CGGATACCCAGTCTACGTGTTTGGAGACTGT-3′) and pX3 (5′-GAGCCGATAACGCGTCCATCGATGGGGTCC-3′) (1). As an internal control, primer sets G3PDH5′ (5′-ACCACAGTCCATGCCATCAC-3′) and G3PDH3′ (5′-TCCACCACCCTGTTGCTGTA-3′) were used to amplify 555-bp fragments of the G3PDH gene. Amplification with each primer set was performed by subjection to 30 cycles of a three-temperature PCR (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min). Blood samples obtained from naive animals never showed any positive results by nested PCR with the HTLV-1 pX primers, thus supporting the reliability of this method. The titer of the antibody against HTLV-1 antigens in the rat plasma was determined by the particle agglutination method by using Serodia HTLV-1 (Fuji Rebio, Tokyo, Japan). The specificity of the antibodies against HTLV-1 antigens in the rat plasma was confirmed by using immunoblot strips which contain known HTLV-1 antigens (Problot HTLV-1; Fuji Rebio).

In some experiments, 4-week-old female F344/N rats were intraperitoneally inoculated with 2 × 107 MMC-treated FPM1 cells or the same number of MMC-treated MT-2 cells. To confirm the absence of surviving cells in the inoculum, a small fraction of the MMC-treated cells used for inoculation was simultaneously cultured in vitro for at least 2 weeks. In other experiments, 4-week-old female WKAH rats were intraperitoneally inoculated with 2 × 107 live FPM1 or MT-2 cells. The presence of HTLV-1 proviruses and antibodies in these rats was monitored as described above.

Analysis of HTLV-1 flanking region.

HTLV-1 flanking regions of FPM1 cells were obtained by using an inverse PCR method (44). Briefly, Sau3AI-digested genome DNA of FPM1 cells was self-ligated and amplified with HTLV-1 LTR-specific primers. The amplified fragments were inserted into pCR2.1 (TA Cloning Kit), and the DNA sequence of one of these clones was determined by the dideoxy method by using the DNA Sequence Kit (Applied Biosystems). To amplify the HTLV-1 flanking region of FPM1, a nested PCR was performed with the outer primer set, FPM1-GEN1 and U5-4, and the inner primer set, FPM1-GEN2 and U5-5. FPM1-GEN1 (5′-TGCCCTGGTCATGGTGTCTC-3′) and FPM1-GEN2 (5′-CAGCCAGTGAACAAGGTACC-3′) are the primers for the host side of the HTLV-1 flanking region. U5-4 (5′-CCAGCGACAGCCCATTCTAT-3′) and U5-5 (5′-TCCAGGAGAGAAATTTAGTACACA-3′) are HTLV-1 LTR-specific primers. Amplification with each primer set was performed by 30 cycles of a three-temperature PCR (94°C for 1 min, 55°C for 1 min, and 72°C for 1 min).

RESULTS

Phenotype of FPM1 cell line.

In the first step, an HTLV-1-infected cell line, designated FPM1, was established from F344/N Jcl-rnu/+ rat thymocytes by coculture with MMC-treated human HTLV-1 producer MT-2 cells. FPM1 expressed rat CD4, CD5, CD25, MHC-I, and MHC-II but not CD8 (Fig. 1). These results indicated that FPM1 originated from rat T cells but not MT-2. The phenotype of FPM-1 was compatible with that of human ATL cells, which are typically CD4+ T cells that express CD25 (13).

FIG. 1.

FIG. 1

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Expression of cell surface makers on FPM1 cells was analyzed by using a flow cytometer. Solid histograms indicate cells stained with MAbs to rat CD4 (a), CD5 (b), CD8 (c), CD25 (d), MHC-I (e), or MHC-II (f). Open histograms represent cells stained with control mouse immunoglobulin. The mean fluorescence in the stained cells in panels a to f were 6.6, 11.4, 3.8, 13.8, 51.9, and 15.5, respectively, while that of the control staining was 3.5.

Absence of HTLV-1 antigens apart from HTLV-1 Tax in FPM1 cells.

FPM1 did not react with a standard human serum positive for HTLV-1 antibodies when examined by immunofluorescence staining methods (data not shown). Further analysis of the expression of HTLV-1 antigens was performed by immunoblot assay by using MAbs (Fig. 2). HTLV-1 structural antigens, such as gp46 and p19, could not be detected in FPM1 cells. However, significant amounts of HTLV-1 p40 Tax were detected in these cells. Selective expression of HTLV-1 Tax was confirmed by using a particular human serum containing high titers of antibodies to HTLV-1 antigens including Tax. On the immunoblot of FPM1 cell lysates, this serum detected only HTLV-1 Tax but not other HTLV-1 antigens, whereas the same serum detected multiple HTLV-1 antigens in MT-2 cell lysates (Fig. 2d). Therefore, only HTLV-1 Tax among HTLV-1 antigens was detected in FPM1 by serological tests.

FIG. 2.

FIG. 2

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Expression of HTLV-1 antigens in cell lines was analyzed by immunoblot assay. First, 75 μg of whole lysates of MOLT-4 (lane 1) and FPM1 (lane 2) and 25 μg of MT-2 (lane 3) were separated by SDS-PAGE; these were then transferred to blotting sheets and reacted with MAbs REY-30 (a), GIN14 (b), and Lt-4 (c), which detect HTLV-1 gp46, p19, and p40 Tax, respectively. Immunoblot assay was also performed with a human serum positive for antibodies to HTLV-1 antigens including HTLV-1 Tax (d).

HTLV-1 provirus integration and gene expression in FPM1.

HTLV-1-infected cells that lack HTLV-1 expression sometimes have defective HTLV-1 proviruses (3, 27, 31). To test such a possibility in FPM1 cells, we conducted a Southern blot analysis with FPM1 DNA digested with EcoRI, which did not cut the HTLV-1 genome internally (41). As shown in Fig. 3a, FPM1 contained two EcoRI-fragments of 13 and 8.3 kbp hybridized with HTLV-1-specific probe. To assess whether these fragments include full-length of HTLV-1 proviruses, a long-PCR analysis with HTLV-1 LTR primers was performed (Fig. 3b). These primers amplified two types of PCR products with FPM1 template DNA, and the size of the longer product was compatible with the expected size from the full-length of HTLV-1 (7.7 kbp). These results indicated that at least one copy of full-length HTLV-1 provirus and another copy of defective HTLV-1 provirus were integrated into the FPM1 genome.

FIG. 3.

FIG. 3

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Detection of HTLV-1 proviruses integrated in FPM1 cells by Southern blot hybridization (a) and long-PCR (b) methods. In panel a, 20 μg of DNA extracted from MT-2 (lane 1) and FPM1 cells (lane 2) was digested with EcoRI and hybridized with radiolabeled HTLV-1-specific probe. In panel b, 500 ng of DNA from MOLT-4 (lane 1), TL-OmI (lane 2), FPM1 (lane 3), and MT-2 (lane 4) were subjected to long PCR with primers for 5′ and 3′ HTLV-1 LTR. The expected size of the PCR products from a full-length HTLV-1 provirus is 7.7 kbp.

In the next step, we examined the expression of HTLV-1 mRNA in FPM1 cells. For this purpose, RT-PCR was used for the detection of HTLV-1 mRNA by using primers that selectively amplified doubly spliced HTLV-1 pX, single spliced env, or unspliced gag mRNA. The results are shown in Fig. 4 and 5. Significant levels of RT-PCR products were amplified with pX primers and, to a lesser degree, with gag and env primers. Analysis with serially diluted template RNA showed that the level of pX mRNA expression in FPM1 was almost equivalent to that of MT-2, but the level of expression of env and gag mRNAs was 10- to 100-fold less than in MT-2, respectively (Fig. 5).

FIG. 4.

FIG. 4

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RT-PCR analysis of HTLV-1 mRNA. A total of 300 ng of DNase-treated total RNA from MT-2 (a) or FPM1 (b) was subjected to 30 cycles of one-step RT-PCR with primers for G3PDH (lane 1), gag (lane 2), env (lane 3), and pX (lane 4). The expected size of the amplified products was 435 bp for G3PDH, 242 bp for HTLV-1 gag, 316 bp for env, and 145 bp for pX.

FIG. 5.

FIG. 5

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Relative quantitation of HTLV-1 mRNA expressed in FPM1 (top) and MT-2 (bottom) cells by RT-PCR with the HTLV-1 pX, env, and gag primers listed in the legend to Fig. 4. A 300-ng RNA sample of each cell line was 10-fold diluted serially with MOLT-4 RNA and subjected to 30 cycles of one-step RT-PCR for pX and env regions and 35 cycles of two-step RT-PCR for gag regions. The dilutions are indicated as log10 values.

Lack of antibody response and persistence of HTLV-1 provirus in FPM1-inoculated rats.

In the next series of experiments, FPM1 cells were injected intravenously into four 4-week-old syngeneic F344/N rats. As a positive control, four animals were inoculated with MT-2 cells. Figure 6 shows serial changes that occurred in HTLV-1 proviruses and titers of anti-HTLV-1 antibodies detected in the peripheral blood. Antibodies against HTLV-1 antigens were detected in the sera of MT-2-inoculated rats as early as 2 weeks after injection and gradually increased thereafter (Fig. 6A). The antibody titers ranged between 1:16 and 1:8,192 in these animals. The specificity of the antibodies to HTLV-1 antigens was confirmed by immunoblot analysis (data not shown). In contrast, none of the rats inoculated with FPM1 cells produced antibodies against HTLV-1 antigens after injection (Fig. 6B). HTLV-1 proviruses were intermittently detected by PCR in peripheral blood samples of all rats inoculated with FPM1 or MT-2 cells. Two animals from each group were further followed up for at most 53 weeks after inoculation, and HTLV-1 proviruses were present in both groups of animals.

FIG. 6.

FIG. 6

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Detection of HTLV-1 provirus and antibodies to HTLV-1 antigens in the peripheral blood of F344/N rats injected with MT-2 (A) or FPM1 (B) cells. Four animals of each group were designated as animals 1 (□), 2 (◊), 3 (○), and 4 (▵). An asterisk indicates the time when the animal was sacrificed.

Persistence of FPM1 in vivo.

Since FPM1 did not express detectable amounts of HTLV-1 structural proteins, we wondered whether the persistence of HTLV-1 proviruses in inoculated animals was due to the survival of inoculated FPM1 cells. To assess this possibility, the HTLV-1 flanking region of FPM1 was sequenced, and the presence of this region in the peripheral blood of inoculated animals was analyzed by PCR. The results are shown in Fig. 7. Both DNA fragments specific for pX and HTLV-1 flanking region of FPM1 were amplified 37 weeks after inoculation in blood samples of FPM1-inoculated rats. In contrast, simultaneous samples of an MT-2-inoculated rat failed to generate PCR products specific for HTLV-1-flanking region of FPM1, although pX fragments were amplified in this animal. FPM1-specific region was detected in peripheral blood samples from both of the two FPM1-inoculated rats, also at 32 weeks after inoculation. However, a similar analysis of several tissues, including the spleen, liver, lymph nodes, Peyer’s patches, and submandibular gland of one of these rats at autopsy, 53 weeks after inoculation, failed to amplify the FPM1 flanking region, while all of these samples were positive for pX regions.

FIG. 7.

FIG. 7

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Identification of HTLV-1 flanking region of FPM1. A 1-μg portion of DNA was extracted from the peripheral blood cells of a F344/N rat inoculated with MT-2 cells (lane 1) and two rats inoculated with FPM1 cells (lanes 2 and 3) 37 weeks after inoculation. The DNA was subjected to PCR with primers specific for G3PDH (top), HTLV-1 pX (middle), and HTLV-1 flanking region of FPM1 (bottom).

In vivo infectivity of FPM1.

We then assessed whether FPM1 can potentially cause de novo infection in vivo, by two kinds of experiments. In the first set of experiments, MMC-treated FPM1 cells were inoculated into syngeneic F344/N rats, and in the second set of experiments, live FPM1 cells were inoculated into allogeneic WKAH rats. As shown in Fig. 8A, the peripheral blood samples of these rats at 5 or 6 weeks after inoculation were positive for HTLV-1 proviruses but negative for anti-HTLV-1 antibodies. On the other hand, the plasma of the control rats inoculated with MMC-treated or live MT-2 cells contained high titers of antibodies specific for HTLV-1 antigens p19, p24, and p53 (Fig. 8A). Two each of the syngeneic rats inoculated with MMC-treated FPM1 cells and the allogeneic rats inoculated with live FPM1 cells were monitored for a longer period. None of these rats generated detectable levels of HTLV-1-specific antibodies for at least 15 weeks after inoculation, whereas all of them remained positive for HTLV-1 proviruses as detected by a nested-PCR method. FPM1 flanking region was not detectable in the same samples (Fig. 8B). These findings suggest that de novo HTLV-1 infection of the host cells occurred in FPM1-inoculated rats, although FPM1 expressed suboptimal levels of viral antigens for the host antibody responses or for serological detection in vitro.

FIG. 8.

FIG. 8

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In vivo infectivity of FPM1 cells. (A) Presence of HTLV-1-specific antibodies and HTLV-1 proviruses in F344/N rats inoculated with 2 × 107 MMC-treated MT-2 (lane 1) or MMC-treated FPM1 (lanes 2 to 4) cells and in WKAH rats inoculated with 2 × 107 live MT-2 (lane 5) and FPM1 (lanes 6 to 8) cells. Each lane represents the peripheral blood sample collected from individual rats six (lanes 1 to 4) or five (lanes 5 to 8) weeks after inoculation. The top panel showed the immunoblots (Problot HTLV-1) stained with each rat plasma diluted at 1:50. The molecular weights of the HTLV-1 p19, p24, and p53 were indicated at the left. The titers of HTLV-1-specific antibodies measured by Serodia HTLV-1 of the rat plasma used in lanes 1 to 8 were 4,096, <16, <16, <16, >8,192, <16, <16, and <16, respectively. The bottom panel showed the presence of HTLV-1 proviruses in 3 μl of blood samples detected by nested PCR amplifying HTLV-1 pX region. (B) Nested-PCR analysis of 2-μg DNA samples extracted from the peripheral blood of two F344/N rats inoculated with MMC-treated FPM1 cells (lanes 1 and 2) and two WKAH rats inoculated with live FPM1 cells (lanes 3 and 4) 15 weeks after inoculation, with the primers specific for HTLV-1-pX (top) and the HTLV-1 flanking region of FPM1 (bottom). A 1-μg portion of DNA template from FPM1 cells was used as a positive control (lane 5).

DISCUSSION

The major finding of the present study was that the persistent presence of HTLV-1 without antibody responses was successfully established experimentally in syngeneic rats inoculated with an HTLV-1-infected cell line scarcely expressing major HTLV-1 structural proteins. Persistent HTLV-1 infection in the FPM1-inoculated rats was induced by both the persistence of FPM1 cells themselves and the transmission of HTLV-1 to the host cells. This finding is in contrast to a number of previous studies showing that persistent HTLV-1 infection can be established in rats by inoculating MT-2 or other HTLV-1 producer cells, accompanied by anti-HTLV-1 antibody responses (15, 17, 42, 48, 50).

The main reason for the negative antibody responses in FPM1-inoculated rats would be the extremely low amounts of HTLV-1 antigens in these cells. The production of various cytokines by HTLV-1-infected cells could also affect host immunity (24, 35, 53, 54). On the other hand, host-related problems can be excluded, as the rats produced high amounts of HTLV-1-specific antibody when inoculated with MT-2. The use of syngeneic rats for FPM1 is also excluded, since a similar seronegative state was induced in allogeneic WKAH rats inoculated with FPM1 cells. Previous studies demonstrated that a similar seronegative HTLV-1 carrier state can be induced in newborn rats inoculated with HTLV-1 producer cells (17) and in adult rats orally inoculated with MT-2 (22), but these cases are presumed to be due to host immunological immaturation or tolerance. Failure of host serologic responses in some rabbits inoculated with HTLV-1-infected cells was also reported (4, 29). This was associated with failure in the establishment of persistent infection and presumed to be due to the poor infectious ability of the virus strain utilized.

HTLV-1 Tax but not other structural proteins tested were detectable in FPM1 cells. However, even in a small amount, detection of all of the three major HTLV-1 mRNAs support the potential presence of a very small amount of viral antigens in FPM1 cells. The unproportional expression of multiple spliced HTLV-1 mRNAs implied that the expression of HTLV-1 proteins in this cell line might be regulated at the splicing level. The precise mechanisms of the poor antigen expression in FPM1 have yet to be clarified.

It is surprising that such low levels of viral antigens were sufficient for in vivo infection. Since HTLV-1 is known to cause infection in a cell-to-cell manner, only a small amount of the HTLV-1 envelope may be sufficient for infection. It is likely that the absence of neutralizing antibodies to HTLV-1 may further enhance the development of in vivo infection. Alternatively, there could be an envelope-independent infection mechanism in the cell-cell infection of HTLV-1.

Detection of the FPM1-specific cellular flanking region in inoculated animals suggested that FPM1 persists in vivo for some time. This is in agreement with the clinical findings in humans, where HTLV-1-infected clones identified in the peripheral blood can be detected over several years in the same HTLV-1 carrier (6). However, PCR failed to amplify FPM1-specific regions in the FPM1-inoculated rat at autopsy, despite the positive results for pX regions. This is partially explained by the lower efficiency of FPM1-specific primers than of pX-specific ones, but it more likely results from selection of more proliferative clones raised among secondary HTLV-1 infected cells during long-term HTLV-1 infection.

HTLV-1 Tax can be a strong target for cellular immunity (18, 19) but to a lesser degree for humoral immunity. In fact, FPM1-inoculated animals showed strong T-cell proliferative responses to FPM1, but antibodies to HTLV-1 Tax were not detected in the sera of these rats (data not shown). We recently found that FPM1 and its subclones exhibit tumorigenic activity when inoculated into syngeneic athymic rats. The present system, therefore, would also be suitable as a model for investigating anti-HTLV-1 tumor immunity and might potentially be modified to allow study of the development of leukemia.

In conclusion, we established a novel model of a seronegative HTLV-1 carrier state by using FPM1 cells. Dissociation in the ability of this cell line to induce in vivo infection and antibody responses highlighted another aspect of HTLV-1 infection.

ACKNOWLEDGMENTS

We thank Y. Tanaka (Kitasato University, Kanagawa, Japan) and K. Matsumoto (Osaka Red Cross Center, Osaka, Japan) for providing anti-HTLV-1 MAbs and a rare human serum that reacts with HTLV-1 antigens, including Tax, respectively. We also thank F. G. Issa, Word-Medex, Sydney, Australia, for careful reading and editing of the manuscript.

This work was supported in part by grants from the Agency of Science and Technology of Japan and the Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation.

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