Affiliations of authors: S. Hanabuchi, T. Ohashi, Y. Koya, H. Kato, A. Hasegawa, F. Takemura, T. Masuda, M. Kannagi, Department of Immunotherapeutics, Tokyo Medical and Dental University, Medical Research Division, Tokyo, and Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama, Japan.
Correspondence to: Dr. Mari Kannagi, Department of Immunotherapeutics, Tokyo Medical and Dental University, Medical Research Division, 15-45 Yushima, Bunkyo-Ku, Tokyo 1138519, Japan (e-mail: kann.impt{at}med.tmd.ac.jp).
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ABSTRACT |
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INTRODUCTION |
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In general, CTLs may play an important role not only in viral clearance but also in tumor eradication. We and others (12,13,15,16) have demonstrated that HTLV-I-specific CD8+ CTLs in HTLV-I carriers often recognize HTLV-I Tax (13,16) and can lyse ATL cells in vitro (12,15). These observations suggest that HTLV-I-specific CTLs may be important effectors of host immunosurveillance against HTLV-I-induced tumor development. It is still unclear, however, whether these CTLs actually play a major role in controlling HTLV-I tumors in vivo or are merely a consequence of infection.
To clarify the effect of CTLs on HTLV-I leukemogenesis in vivo, we had previously developed two experimental rat model systems in which ATL-like disease was consistently reproducible (17,18). One model produced T-cell lymphomas in athymic rats following the inoculation of HTLV-I-immortalized rat cell lines (17). In this model, the adoptive transfer of immune T lymphocytes protected rats from fatal lymphomas. In the other model, development of T-cell lymphomas was induced by treatment with anti-CD80 and anti-CD86 monoclonal antibodies (mAbs) that block costimulatory signals for T-cell activation in otherwise resistant WKAH rats (18). These findings strongly intimate the contribution of host T-cell immune responses toward protection against the development of HTLV-I tumor in vivo.
Observations made in studies of CTLs in HTLV-I-infected humans and in our rat models suggest that augmentation of HTLV-I-specific CTLs in pre-ATL patients might protect them from the development of ATL. The rate of cure of ATL is extremely low among patients with lymphoproliferative disorders because of the resistance to chemotherapy. Immunologic approaches to treatment in earlier stages of disease may be worthy of consideration.
To induce effective antitumor immune responses, the precise tumor antigens must be clarified and given to the host as a strong immunogen recognized by T lymphocytes. In particular, to evoke CD8+ CTL responses, which are one of the major antigen populations directed at tumors, presentation of processed peptides by major histocompatibility complex (MHC) class I antigens as well as MHC class II antigens of appropriate antigen-presenting cells is essential (19). Because of the preference of the intrinsic pathway for antigen presentation by MHC class I antigens, a variety of viral vectors has been proposed for the delivery and expression of exogenous genes encoding target antigens (20). Direct injection of naked DNA may elicit similar effects (21). Peptide-based vaccines corresponding to CTL epitopes, which directly bind to MHC molecules, are an alternative and promising approach (22,23). This approach may also be safer than DNA-based vaccination.
In this study, we characterized the host antitumor immunity to explore the possibility of peptide-based vaccination against ATL-like disease. We investigated the specificity of effector cells against HTLV-I Tax protein by fine mapping and explored the contribution of these cells to in vivo antitumor surveillance in a rat model of HTLV-I-linked lymphoproliferative disease.
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MATERIALS AND METHODS |
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Four-week-old female F344/N Jcl-rnu/rnu (nu/nu or athymic) rats and F344/N Jcl-rnu/+ (nu/+) rats were purchased from Clea Japan, Inc. (Tokyo, Japan). These rats were maintained at the experimental animal facilities of Tokyo Medical and Dental University, and the experimental protocol was approved by the Animal Ethics Review Committee of Tokyo Medical and Dental University.
Cell Lines
FPM1-V1AX is an HTLV-I-immortalized rat T cell line from nu/+ rats (17). FPM-SV is a HTLV-I-negative SV40-transformed rat kidney cell line derived from a nu/+ rat (24). TARS-1, an HTLV-I infected T cell line from WKAH rats (25), was provided by Dr. T. Yoshiki (Hokkaido University, Japan). RT1.Al /TARS-1 was newly established by transfecting RT1.Al-expressing plasmid pRep10 into TARS-1 cells followed by in vitro selection with 400 µg/mL hygromycin. RT1.Al cDNA construct in pRep10 vector was provided by Dr. S. Salgar (University of Miami, FL) (26). RT1.Al is the MHC class I antigen in F344 rats. The expression of RT1.Al in RT1.Al/TARS-1 cells was confirmed by immunofluorescence analysis. G14 is an interleukin-2-dependent HTLV-I-negative CD8+ T cell line established from the nu/+ rat. G14-Tax, a stable transfectant of G14 with Tax gene, was also used (27). All the cell lines used were maintained in RPMI-1640 medium with 10% heat-inactivated fetal calf serum (FCS) (BioWhittaker; Walkersville, MD), penicillin (100 U/mL), and streptomycin (100 µg/mL). Recombinant human interleukin-2 (rhIL-2) (Shionogi Pharmaceutical Co.; Osaka, Japan) was added to the medium at a concentration of 10 U/mL for maintaining G14 and G14-Tax.
Mouse mAbs
R110B5, a monoclonal antibody (mAb) against rat CD8 antigen, and RTH-7, a mAb against rat CD4 antigen, were purchased from Seikagaku Co. (Tokyo, Japan). B5, a mAb against rat MHC class I molecule, RT1.Al (BD PharMingen Co.; Franklin Lakes, NJ) was also used.
Recombinant Vaccinia Viruses
Recombinant vaccinia viruses (rvvs) containing HTLV-I genes were kindly provided by Dr. H. Shida (Hokkaido University, Japan) (2830). The rvvs used were WR-proenv-1 containing HTLV-I env gene, WR-gag containing HTLV-I gag gene, and WR-40X and WR-27X containing HTLV-I pX genes. WR-27X expresses p21X, p27 rex, and p40 tax proteins, whereas WR-40X encoded p21X and p40 tax proteins. WR-HA without HTLV-I gene was used as a control. Monolayers of FPM-SV cells were infected with each of the HTLV-I-rvvs at a multiplicity of infection of 10 at 37 °C for 1 hour, then cultured with RPMI-1640 medium with 10% FCS at 37 °C for 12 hours before radiolabeling for cytotoxicity assay.
Preparation of Immune T-cell Subsets
Four-week-old nu/+ rats were intraperitoneally immunized with 2 x 107 FPM1-V1AX cells twice with a 2-week interval. One week after the last immunization, splenic T cells were isolated and purified through a nylon wool column. Further purification of lymphocyte subsets was performed by using a complement lysis method. Briefly, splenic T cells were incubated with anti-rat CD8 mAb (R110B5) or anti-rat CD4 mAb (RTH-7) for 30 minutes on ice, washed, then incubated for 45 minutes at 37 °C in 2% rabbit serum as a source of complement (CEDARLANE Laboratories Ltd.; Hornby, Ontario, Canada) in RPMI-1640 medium. With this treatment, enrichment to more than 98% CD4+ or CD8+ T cells was obtained as assessed by flow cytometry analysis.
Cytotoxic Assay and Cytotoxic T-Lymphocyte Lines
Cytotoxic activities were measured by 6-hour 51Cr-release assay at various effector to target (E/T) cell ratios as described previously (31). Effector cells used were rat splenic T cells or CTL, and the target cells used were FPM1-V1AX, FPM-SV, TARS-1, RT1.Al/TARS-1, G14, and G14-Tax. Specific cytotoxicity was calculated as ([experimental 51Cr release spontaneous 51Cr release]/[maximum 51Cr release spontaneous 51Cr release]) x 100%. The [3H]thymidine ([3H]-TdR)-release assay was also used to measure cytotoxicity because some of the target cells were found to be labeled more efficiently with [3H]-TdR than with 51Cr. In this method, target cells were incubated with 3.7 MBq of [3H]-TdR per 106 cells for 12 hours at 37 °C, followed by triplicate washing. These target cells (5 x 103 cells/well) and effector cells were plated in 96-well U-bottom plates at various E/T ratios. After 6 hours of incubation at 37 °C, cells were harvested by using a Micro 96 Harvester (Skatron; Sunnyvale, CA) and [3H]-TdR remaining in target cells was measured in a microplate -counter (Micro Beta Plus, Wallac Oy; Turku, Finland). The percentage of specific cytotoxicity was calculated as ([cpm without effector cpm with effector] / cpm without effector]x 100.
The cytotoxicity of fresh splenic T cells was examined after 6 days of coculture of these cells (5 x 106 cells/well) with formalin-fixed FPM1-V1AX cells (2 x 106 cells/well) in 2 mL of RPMI-1640 medium with 10% FCS per well of 24-well plates. These splenic T cells cultured for 6 days are termed "freshly induced CTLs" in this article.
For induction of HTLV-I-specific CTL lines in long-term cultivation, 2.5 x 106 cells/well of splenic T cells were cocultured with the same number of formalin-fixed FPM1-V1AX cells in RPMI-1640 medium with 10% FCS in the presence of 20 U/mL rhIL-2 with periodic stimulation by using formalin-fixed FPM1-V1AX cells every 2 weeks.
Evaluation of the Antitumor Effect of Adoptive Transfer of Immune T Cells
A total of 2 x 107 immune T cells such as fresh splenic T cells, T cell subsets, or CTL line cells derived from immunized nu/+ rats were intraperitoneally inoculated in 4-week-old nu/nu rats, which were simultaneously inoculated subcutaneously with 2 x 107 FPM1-V1AX cells. The nu/nu rats inoculated with FPM1-V1AX alone served as controls. The size of each subcutaneous tumor was measured every other day with a caliper. In these measurements, we determined the longest surface length (in millimeters [a]) and width (in millimeters [b]) and calculated the tumor volume (V; in cubic millimeters) using the formula V = ab2/2, as described previously (17). Rats were killed if a tumor was greater than 25 mm in any dimension or if the rats appeared to be ill from the tumor burden.
Synthetic Peptides and Mapping of CTL Epitopes
Peptides were synthesized by the stepwise method of solid-phase peptide synthesis and purified as described before (32). Some of the oligopeptides were purchased from Hokudo Co. (Hokkaido, Japan).
Mapping of CTL epitopes was performed as follows. The [3H]-TdR-labeled G14 cells were washed and placed into a round-bottom 96-well plate at a concentration of 5 x 103 cells/well in RPMI-1640 medium with 10% FCS and 10 µM oligopeptides. Following 1 hour of incubation at 37 °C, 5 x 104 CTLs were added per well for a cytotoxicity assay. Oligopeptides were carried over in the cytotoxicity assay medium. The ability of the oligopeptides to sensitize target cells for CTL was evaluated by the cyotoxicity levels.
Immunization Protocols for Peptide/ImmuneStimulatory DNA SequencesOligodeoxynucleotides Vaccine In Vivo
For immunization of Tax 180188 oligopeptides in rats, we used immunestimulatory DNA sequencesoligodeoxynucleotides (ISS-ODNs) as an adjuvant (33). ISS-ODNs (5'-TGACTGTGAACGTTCGAGATGA-3') synthesized as phosphorothioate single-strand oligonucleotides were purchased from ESPEC OLIGO SERVICE (Ibaraki, Japan). Female 4-week-old nu/+ rats were injected intradermally with saline, 100 µg of Tax 180188 peptide alone, 10 nmol of ISS-ODN alone, 100 µg of Tax 180188 peptide mixed with 10 nmol of ISS-ODN (Tax 180188/ISS-ODN), or 100 µg of Influenza A matrix 5866 peptide mixed with 10 nmol of ISS-ODN (Influenza A matrix 5866/ISS-ODN). Peptide/ISS-ODN mixture was prepared by mixing 5 µL of 20 mg/mL stock solution of peptides in distilled water and 10 µL of 1 µM stock solution of ISS-ODN in phosphate-buffered saline into 200 µL of normal saline and was immediately used for inoculation. Immunization was performed twice with a 2-week interval. Two weeks after the last immunization, 107 freshly isolated T-cell enriched splenocytes from vaccinated rats were intraperitoneally inoculated into 4-week-old nu/nu rats, which were simultaneously inoculated subcutaneously with 2 x 107 FPM1-V1AX cells. The size of each subcutaneous tumor was measured every other day.
Statistical Methods
Measurement of cytotoxicities by use of either a 51Cr- release or [3H]-TdR-release assay was carried out in triplicate. Data are reported as means ± 95% confidence intervals, generated according to the Excel 97/98 Book system (Microsoft Co., Redmond, WA). Differences in the cytotoxicities or tumor size of individual groups versus controls were evaluated by using the StatView system (SAS Institute Inc. Cary, NC). P values less than .05 were considered to be statistically significant.
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RESULTS |
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We previously reported that adoptive transfer of splenic T cells from rats immunized with HTLV-I-infected cells efficiently inhibited the development of fatal lymphomas in nu/nu rats inoculated with a HTLV-I-transformed tumor line, FPM1-V1AX (17). To clarify which subsets of immune effectors are required for direct tumor regression in vivo, we isolated CD4+ or CD8+ cell populations from the nu/+ rats immunized with FPM1-V1AX and transferred each subpopulation in nu/nu rats bearing FPM1-V1AX tumor. As shown in Fig. 1, A, both CD4+ and CD8+ T cells, as well as unfractionated T cells, exhibited equivalent inhibitory effects on the growth of HTLV-I-infected tumors.
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HTLV-I Antigens Recognized by CTLs
Viral antigens recognized by freshly induced CTLs from nu/+ rats that had been inoculated with FPM1-V1AX cells were further analyzed by using rvvs (as shown in Fig. 2, A). The freshly induced CTLs exhibited significant cytotoxicity against FPM1-V1AX cells but not against FPM-SV cells. Among rvv-infected FPM-SV cells, WR-40X- and WR-27X-infected cells, which commonly express HTLV-I Tax, were most susceptible to the CTLs. The targets expressing HTLV-I envelope protein were also susceptible, but to a lesser degree. The cytotoxicity against targets expressing HTLV-I gag protein, however, was as low as that against the control rvv-infected target. Since the FPM-SV cells were positive for major histocompatibility complex (MHC) class I antigen, but negative for class II antigen as determined by immunofluorescence analysis, this cell line probably preferentially presents MHC class I-restricted antigens.
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MHC Class I Restriction of HTLV-I-Specific Cytotoxicity in Nu/+ Rats
Using the HTLV-I Tax-specific CD8+ CTL line established by long-term culture with FPM1-V1AX cell stimulation as described above, MHC class I restriction of cytotoxicity was examined next. The FPM1-V1AX cell line derived from a nu/+ rat possesses the rat MHC class I molecule, RT1.Al, whereas the TARS-1 cell line derived from a WKAH rat is negative for the RT1.Al molecule. In addition to these two cell lines, a newly established cell line, RT1.Al/TARS-1, which was stably transfected with RT1.Al cDNA in TARS-1 cells, was used. As shown in Fig. 3, RT1.Al/TARS-1 cells, but not TARS-1 cells, were substantially lysed by two CD8+ CTL lines but not by CD4+ CTL induced from FPM1-V1AX cell-immunized nu/+ rats. Thus, the cytotoxicity of the HTLV-I Tax-specific CD8+ CTL lines from nu/+ rats was restricted by the rat MHC class I RT1.Al molecule.
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We then performed peptide mapping analysis to identify target epitopes recognized by HTLV-I Tax-specific CD8+ CTLs from nu/+ rats immunized with FPM1-V1AX cells. Initially, we examined the cytotoxic response of the freshly induced CTLs against syngeneic G14 cells in the presence of synthetic peptides corresponding to the amino acid sequence of HTLV-I Tax. Among a series of 29 partially overlapping synthetic peptides (13 to 24 amino acids long), freshly induced CTLs lysed the target cells effectively in the presence of peptides Tax 177200 and Tax 181195. The CTLs also exhibited low levels of cytotoxicity for the target cells in the presence of peptides Tax 1740, Tax 3356, Tax 81104, and Tax 97120 (Table 1). Considerable cytolysis in the presence of Tax 177200 and Tax 181195 was also observed when long-cultured CTL lines were used (Table 1
, experiment 3). Neither Tax 177200 nor Tax 181195 alone exhibited notable toxicity for target cells.
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To determine the central epitope of Tax-specific CTLs among Tax 180188, Tax 181189, and Tax 182190, the corresponding oligopeptides were serially diluted and evaluated for their relative ability to sensitize G14 cells to CTL (Fig. 4, A). Only 103 pM of peptide Tax 180188 was sufficient to sensitize target cells, whereas 1 µM of Tax 181189 and 10 µM of Tax 182190 were required to obtain a similar effect. Target cells treated with the control peptide Tax 1119 were not lysed by the CTLs at any concentrations tested. None of these peptides exhibited direct toxicity when added alone to the target cells (data not shown). Thus, the amino acid sequence GAFLTNVPY (Tax 180188) was identified as the central sequence of the CTL epitope.
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Adoptive Transfer of CTL Recognizing Tax 180188 Induces HTLV-I Tumor Regression In Vivo
We next investigated whether Tax 180188-specific CTLs protect against the growth of HTLV-I tumor cells in vivo. A long-cultured CTL line shown above to recognize Tax 180188 was used for passive immunization. Nu/nu rats were subcutaneously inoculated either with FPM1-V1AX cells alone or simultaneously with intraperitoneal inoculation of the CTL line cells. The kinetics of tumor growth in these rats is shown in Fig. 5. Substantial suppression of tumor growth was observed in the rats treated with the CTLs compared with the control rats without CTLs. These results indicated that the HTLV-I Tax-specific CTLs recognizing Tax 180188 contributed to the regression of HTLV-I-infected tumor growth in vivo.
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Finally, we examined whether immunization with Tax 180188 peptides elicits host immune responses, which protect against the growth of HTLV-I-infected tumors in vivo. ISS-ODN was used as an adjuvant for the peptide-based vaccine (33). Nu/+ rats were immunized with Tax 180188 peptide mixed with ISS-ODN (Tax 180188/ISS-ODN) twice, with a 2-week interval. Freshly isolated splenic T cells from these rats were intraperitoneally transferred into nu/nu rats that were subcutaneously inoculated with FPM1-V1AX cells. As shown in Fig. 6, substantial suppression of tumor growth was observed in the first week of FPM1-V1AX cell inoculation (two rats). None of the other groups (two rats in each group) of FPM1-V1AX-inoculated nu/nu rats that were either untreated or treated with Tax 180188 alone, ISS-ODN alone, or Influenza A matrix 5866/ISS-ODN-immunized T cells exhibited tumor regression during the observation period. Thus, vaccination with a combination of Tax 180188 peptide and ISS-ODN effectively elicited T cell immunity that protected against HTLV-I-infected tumor in vivo in both rats tested.
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DISCUSSION |
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Analogous to human HTLV-I carriers, HTLV-I Tax is one of the major target antigens recognized by HTLV-I-specific CTLs (13,16) in this rat experimental model. Besides Tax, HTLV-I env was also recognized by the rat CTLs, whereas in humans, CTLs against HTLV-I Env, Gag, Pol, and p12 proteins have been found (13,14,16,34,35). The difference in variety of CTL target molecules between rats and humans may be caused in part by the difference in the pattern of HTLV-I expression between them. The HTLV-I-infected FPM1-V1AX cells used in this study express Tax but almost no other HTLV-I structural proteins such as Env or Gag (24). In our previous study, immunocompetent rats inoculated with FPM1-V1AX cells did not exhibit antibody responses against HTLV-I structural proteins (24). It is intriguing that FPM1-V1AX cells could induce Env-specific CTLs in immunized rats. The amount of Env protein required for priming and maintaining CTLs in vivo may be much lower than the levels that are detectable by serologic means. The fact that HTLV-I Tax is a common target for both human and rat CTLs suggests the presence of sufficient amounts of this antigen for CTL responses in vivo.
In this study, both CD4+ and CD8+ T cell subsets of Tax-specific CTLs were inducible in the rats immunized with HTLV-I-infected tumor cells. The HTLV-I Tax molecule is therefore the source of naturally processed epitopes presented by both MHC class I and class II antigens, and these epitopes are capable of stimulating both CD4+ and CD8+ T cell responses directed against tumors in vivo. The importance of CD4+ T cells in the induction and maintenance of anti-tumor responses is generally accepted (36,37). The presence of CD4+ CTL in human melanoma (3840) and HAM/TSP patients has also been reported (41). Our results suggest a potential anti-tumor effector function for CD4+ as well as CD8+ CTLs in our rat model of HTLV-I tumor. The contribution of CD4+ CTLs to antitumor surveillance in human HTLV-I carriers remains to be clarified.
The doseresponse analysis and cold inhibition assay indicate that peptide Tax 180188 is the dominant epitope recognized by the HTLV-I-specific CTLs used. Similar results were reproducibly obtained with the use of several long-cultured CTL lines independently established from nu/+ rats immunized with FPM1-V1AX. The freshly induced CTLs also exhibited dominant cytotoxic activity specific for this region, suggesting that the predominance of the epitope Tax 180188 was not merely a result of in vitro selection.
It is striking that peptide-based vaccination with a single epitope for CD8+ CTL elicited the immunity protective against HTLV-I-infected tumor in rats (Fig. 6). This indicates that the presence of Tax 180188-specific CTL alone is sufficient for tumor eradication, although an appropriate adjuvant such as ISS-ODN was required for the induction of these CTLs by peptide vaccination. In humans, some of the tumor antigen epitopes of several tumors are in clinical trials for use in cancer vaccines (42). In human HTLV-I carriers, multiple CTL epitopes of HTLV-I antigens have been identified (14,32,34,41,43,44). These epitopes are possible candidates for peptide-based vaccine trials in ATL patients.
The therapeutic potential of vaccine inducing Tax-specific CTLs in HTLV-I-induced tumor was suggested by this study and our recent study of Tax-DNA vaccination in rats (27). The contribution of Tax-specific CTL to antitumor surveillance in human HTLV-I carriers has been suggested because of both the scarcity of these CTLs in ATL patients and the susceptibility of ATL cells to these CTLs in vitro (12). However, in the rat model system, the responder animal for the vaccine was immunocompetent. For clinical application of vaccination against ATL, a strong adjuvant would be required, since ATL patients may be in a state of immunosuppression (1,45). Further studies are required to establish effective strategies for induction of sufficient immunity in immunodeficient patients.
In conclusion, the newly identified T-cell epitope Tax 180188 restricted by rat MHC-I, RT1.Al is one of the major epitopes of the HTLV-I Tax-specific CTLs contributing to regression of HTLV-I-infected tumor growth in vivo. This study provides a useful model system for the development of peptide-based immunotherapeutic strategies against HTLV-I-infected lymphoproliferative disease.
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NOTES |
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We thank Dr. Shashi K. Salgar (Liver/GI Transplant, Department of Surgery, University of Miami, FL) for providing the RT1.Al cDNA clone. We also thank Dr. Hisatoshi Shida (Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan) and Dr. Takashi Yoshiki (Department of Pathology/Pathophysiology, Hokkaido University Graduate School of Medicine, Sapporo, Japan) for recombinant vaccinia viruses expressing human T-cell leukemia virus type I (HTLV-I) antigens and TARS-1, an HTLV-I-infected T-cell line from WKAH rats, respectively.
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Manuscript received March 5, 2001; revised June 12, 2001; accepted September 20, 2001.
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