- Open Access
Activation and detection of HTLV-I Tax-specific CTLs by Epitope expressing Single-Chain Trimers of MHC Class I in a rat model
© Ohashi et al; licensee BioMed Central Ltd. 2008
Received: 22 July 2008
Accepted: 08 October 2008
Published: 08 October 2008
Human T cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. Immunological studies have suggested that insufficient host T cell response to HTLV-I is a potential risk factor for ATL. To understand the relationship between host T cell response and HTLV-I pathogenesis in a rat model system, we have developed an activation and detection system of HTLV-I Tax-specific cytotoxic T lymphocytes (CTLs) by Epitope expressing Single-Chain Trimers (SCTs) of MHC Class I.
We have established expression vectors which encode SCTs of rat MHC-I (RT1.Al) with Tax180-188 peptide. Human cell lines transfected with the established expression vectors were able to induce IFN-γ and TNF-α production by a Tax180-188-specific CTL line, 4O1/C8. We have further fused the C-terminus of SCTs to EGFP and established cells expressing SCT-EGFP fusion protein on the surface. By co-cultivating the cells with 4O1/C8, we have confirmed that the epitope-specific CTLs acquired SCT-EGFP fusion proteins and that these EGFP-possessed CTLs were detectable by flow cytometric analysis.
We have generated a SCT of rat MHC-I linked to Tax epitope peptide, which can be applicable for the induction of Tax-specific CTLs in rat model systems of HTLV-I infection. We have also established a detection system of Tax-specific CTLs by using cells expressing SCTs fused with EGFP. These systems will be useful tools in understanding the role of HTLV-I specific CTLs in HTLV-I pathogenesis.
Human T-cell leukemia virus type I (HTLV-I) is etiologically linked to adult T-cell leukemia (ATL) [1, 2], a chronic progressive neurological disorder termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) [3, 4], and various other human diseases [5–8]. ATL is a malignant lymphoproliferative disease affecting a subgroup of middle-aged HTLV-I carriers characterized by the presence of mature T cell phenotype . HTLV-I genome contains a unique 3' region, designated as pX, which encodes the viral transactivator protein, Tax . Because of its broad transactivation capabilities , it is speculated that Tax plays a central role in HTLV-I associated immortalization and transformation of T cells, which may lead to the development of ATL.
Tax is also known as a major target protein recognized by cytotoxic T lymphocytes (CTL) of HTLV-I carriers . It has been reported that the levels of HTLV-I-specific CTL are quite diverse among HTLV-I carriers and that ATL patients have impaired levels of HTLV-I specific CTLs in contrast to the high levels of CTL response in HTLV-I carriers with HAM/TSP [13–15]. In addition, it has been known that HTLV-I Tax-specific CTL response was strongly activated in ATL patients who acquired complete remission after hematopoietic stem cell transplantation . Based on these observations, it is speculated that HTLV-I-specific immune response may contribute to repressing the growth of HTLV-I infected cells in the infected individuals and insufficient host T cell response against HTLV-I may be a risk factor for ATL.
To understand the mechanism of ATL development, it is very important to dissect the interplay between the virus-specific CTLs and HTLV-I infected T cells. We have previously established a rat model of ATL-like disease, which allows examination of the growth and spread of HTLV-I infected cells, as well assessment of the effects of immune T cells on the development of the disease [17, 18]. By using this model system, we also reported the therapeutic effect of Tax-coding DNA or peptide against the disease [19, 20]. For further analyzing the effects of Tax specific CTLs in the rat model, it is important to develop effective methods to activate Tax specific CTLs and to detect the virus-specific CTLs.
It has been reported that single chain trimers (SCTs) of MHC-I have the potential to efficiently stimulate and identify antigen specific T cells in both human and mouse systems [21, 22]. In this system, all three components of MHC-I complexes, such as an antigen peptide, β2-microgrobulin (β2m), and MHC-I heavy chain are covalently attached with flexible linkers. By linking together the three components into a single chain chimeric protein, a complicated cellular machinery of normal antigen processing can be bypassed, leading to stable cell surface expression of MHC-I coupled with an antigenic peptide of interest. In addition, a new system has been established to identify virus-specific T cells using the acquisition mechanism of epitope/MHC complex by CD8 T cells through MHC/TCR interaction .
In this study, to establish an activation system of Tax-specific CTLs in our rat model system, we have generated a SCT of rat MHC-I linked to Tax epitope peptide. We have also established a detection system of Tax-specific CTLs by using cells expressing SCTs fused with EGFP. These newly established systems would be useful tools in understanding the role of HTLV-I specific CTLs in HTLV-I pathogenesis.
Production and functional capabilities of peptide-β2m-RT1.Alfusion proteins
Establishment of MOLT-4 cells stably expressing SCTs of RT1.Al
Inhibitory effects of SCTs expressing Tax180-188 on the growth of Tax-specific CTLs
Detection of Tax-specific CTLs by SCTs fused with EGFP
Detection of Tax-specific CTLs in splenocytes derived from HTLV-I infected rats
In this study, by using epitope expressing SCTs of rat MHC Class I, we have developed an activation and detection system of HTLV-I Tax-specific CTLs which can be applicable for analyzing CTL responses in a rat model system of HTLV-I infection. The SCT system has been developed in mouse and human MHC-I with its corresponding epitopes [25, 26], but not in rat MHC-I. Based on the information previously reported on the mouse system , we have designed expression vectors for SCTs of rat RT1.Al and successfully obtained the constructs which can activate epitope specific CTLs in vitro. We have further developed the CTL detection system by combining the SCT complex with EGFP, which should be transferred to epitope specific CTLs as previously reported . Because of the poor availability of MHC-I tetramers in rats, development of this system will provide various benefits in analyzing the role of CTLs in a variety of disease models in rats.
The Tax180-188 epitope used in this study was previously identified by epitope mapping analysis and was actually confirmed to be one of the major epitopes presented by RT1.Al in F344 rats infected with HTLV-I or immunized with Tax protein [20, 27]. On the other hand, NLEnv371-379 epitope was predicted by "SYFPEITHI epitope prediction algorithm"  and was given 27 points in the scoring system. Since Tax180-188 was given the same points as NLEnv371-379 scored, it would be reasonable to assume that NLEnv371-379 epitope was equivalently presented by RT1.Al in our present experiments. Nevertheless, only SCTs with Tax180, but not with NLEnv371 can recognize and activate Tax180-188 specific CTLs. Moreover, SCTs with Tax180 did not recognize another CD8+ T cell line, G14, which is not specific to Tax180-188. These results indicated that the SCTs of RT1.Al engineered in the present study appropriately presented the Tax epitope to the corresponding CTLs. However, it is still necessary to establish new CTL lines with different epitope specificities for further confirming the epitope specificity of SCTs used in this study. In addition, it is important to identify new CTL epitopes in rat model of HTLV-I infection for better understanding of the relationship between diversity of HTLV-I-specific CTLs and the virus-related diseases. Especially, recently identified HTLV-I basic leucine zipper factor (HBZ) is the most important factor to be analyzed as a CTL target because of its possible involvement in ATL development . The SCT system together with RT1.Al-EGFP complex should be applicable for the search of new epitopes in F344 rats. Indeed, Tomaru et. al successfully detected new CD8+ T cell epitope from the envelope region of HTLV-I using HLA-A2-EGFP fusion proteins .
MOLT-4/RT1AlSCTax180L cells induced the production of IFN-γ by 4O1/C8 CTLs. However, the activated 4O1/C8 cells failed to proliferate, but rather tended to decrease the number in the mixed culture (Figure 3). This is in dramatic contrast to the results observed in the mixed culture of 4O1/C8 with FPM1.BP, wherein both IFN-γ production and cell proliferation were enhanced. Although the exact mechanism of this difference is not clear, our results suggest that the failure of CTL expansion was due to the enhanced apoptosis induced by RT1AlSCTax180L. In this regard, it has been reported that the presence of CD4+ helper T cells reduced CTL susceptibility to AICD through a cell contact-dependent mechanism . Thus, it is possible that activation of 4O/1C8 by RT1AlSCTax180L may fail to induce the protective signal from AICD in the CTLs. Since a syngeneic CD4+ T cell line, FPM1.BP, was able to induce the expansion of 4O1/C8 in spite of the apparent AICD induction, it is also possible that MOLT-4/RT1AlSCTax180L cells failed to trigger the signal(s) which 4O1/C8 was able to activate for the induction of CTL proliferation. Actually, as shown in Figure 3D, we have detected the enhanced production of IL-2 in the mixed culture of 4O1/C8 with FPM1.BP, suggesting the involvement of the IL-2 signal transduction pathway in the proliferation of 4O1/C8. Further analysis is required to clarify the activation mechanisms of CTLs in the rat system for inducing better immune response by SCTs. Nevertheless, it may be still possible to apply the pEF/RT1AlSCTax180L vector for inducing Tax-specific CTL response in rats, since similar SCT complex expressing human papillomavirus-16 E6 antigen was shown to induce protective immunity against the virus in a mouse system in vivo . Thus, it will also be necessary to assess the in vivo effect of the rat SCTs for the evaluation of the system as a therapeutic tool in HTLV-I infection.
Previous reports suggested that insufficient T cell response against HTLV-I is a potential risk factor for ATL. Among HTLV-I infected individuals, the infrequency of HTLV-I-specific CTL induction in vitro has been reported in ATL patients [12, 13, 31]. Moreover, a recent study using various tetramers clearly demonstrated the reduction of the frequency and diversity of anti-Tax CTLs in ATL patients . The importance of HTLV-I-specific T cell immunity in anti-tumor surveillance was also supported by a previous report showing that Tax-specific CTL response was strongly activated in ATL patients who obtained complete remission after HSCT . These observations suggested the importance of Tax-specific CTLs for prevention and therapy of ATL and should be further verified using suitable animal models. Rats have been used for a number of studies on HTLV-I infection, because they are susceptible to the virus and because the virus-transformed T cell lines can be established in vitro [33, 34]. It has previously shown in a rat model that HTLV-I specific T cells were important to inhibit the growth of virus-infected cells in vivo . Moreover, the association of elevated proviral load with insufficient T cell immunity has been also observed in a rat model of oral HTLV-I infection . In this model, it has further demonstrated that re-immunization of orally HTLV-I-infected rats resulted in a reduction of the proviral load . Although these results further support the importance of Tax-specific CTLs for the prophylaxis and treatment of ATL, detailed analysis to understand the interplay between epitope-specific CTLs and HTLV-I infected cells in vivo has not been performed yet. This is mainly due to the lack of tools to identify epitope specific CTLs in rats. In this study, we have demonstrated that the SCT-EGFP system was able to detect Tax180-188 specific CTLs in splenocytes derived from an HTLV-I-infected rat and that the detection of the epitope-specific CTLs by SCT-EGFP system was comparable to the measurement of peptide-induced IFN-γ production. Thus, the activation and detection system established in this study should be useful for further verifying the strategies to fight against HTLV-I.
In this study, we have generated a SCT of rat MHC-I linked to Tax epitope peptide, which can be applicable for the induction of Tax-specific CTLs in rat model systems. We have also established a detection system of Tax-specific CTLs by using cells expressing SCTs fused with EGFP. These systems will be useful tools in understanding the role of HTLV-I specific CTLs in HTLV-I pathogenesis.
An HTLV-I-immortalized cell line, FPM1.BP, was established previously from an F344/N Jcl-rnu/+ rat . The cells were maintained in RPMI 1640 with 10% heat-inactivated FCS (Biosource, Rockville, MD), penicillin, and streptomycin. A CD8+ Tax-specific CTL line, 4O1/C8, and an IL-2-dependent HTLV-I-negative CD8+ cell line, G14, were also established previously from F344/N Jcl-rnu/+ rats . These cells were maintained in RPMI 1640 medium with 10% FCS and 20 U/ml of IL-2 (PEPROTECH, London, UK). For the maintenance of 4O1/C8 cells, periodical stimulation with formalin-fixed FPM1.BP cells is also required, because their growth is dependent on RT1.Al-restricted presentation of Tax180-188 epitope . Human 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS and MOLT-4 cells were cultured in RPMI 1640 medium with 10% FCS.
Plasmid DNA construction
Primers to construct SCTs of RT1.A1
For the generation of SCT-EGFP expression vectors, RT1AlSCTax180L or RT1AlSCNLEnv371L cDNAs were further amplified by PCR to delete a stop codon and to add KpnI and BamHI sites at the 5'- and 3'- termini, respectively. The pEF/RT1AlSCTax180L-EGFP and pEF/RT1AlSCNLEnv371L-EGFP vectors were generated by insertion of the corresponding PCR products between KpnI and BamHI sites of pEFGFP vector.
To confirm the accuracy of vectors used in this study, all established constructs were subjected to sequence analysis using ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer's instruction.
Cytokine production assay
An HTLV-I Tax-specific CTL line, 4O1/C8 (2 × 105/well), was mixed with various stimulator cells (2 × 105/well). In some experiments, stimulator cells were fixed with 1% formalin in PBS or pulsed with 10 μM of Tax180-188 or NLEnv371-379 peptide (MBL, Nagoya, Japan) before incubation with the CTL. For the stimulation of primary splenocytes with peptides, 5 × 104 of splenocytes were incubated with 10 μM of Tax180-188 or NLEnv371-379 peptides. After the indicated period of mixed culture, supernatants were harvested and were subjected to rat IFN-γ (eBioscience Inc., San Diego, CA), TNF-α ELISA (eBioscience Inc.), or IL-2 (R&D Systems Inc., Minneapolis, MN) in accordance with the manufacturer's instructions.
Flow cytometric analysis
For the assessment of SCT expression, MOLT-4 cells transfected with various SCT expression vectors were stained with an anti-rat MHC-I antibody (BD Bioscience, San Jose, CA) for 30 min on ice, washed three times with 1% FCS in PBS, and then stained with FITC-conjugated goat anti-mouse IgG+IgM. After being washed, the cells were fixed with 1% formalin in PBS prior to analysis on a FACScalibur (BD Bioscience). For the detection of Tax180-188 specific CTLs by the mixed culture with SCT-EGFP expressing cells, 4O1/C8 or splenocytes were incubated with 293T cells expressing SCT-EGFP fusion proteins for 1 hour. Cells in the mixed cultures were stained with phycoerythrin (PE)-conjugated anti-rat CD8 (clone OX-8; BD Bioscience) for 30 min on ice, washed three times with 1% FCS in PBS, fixed with 1% formalin in PBS, and then subjected to FACS analysis.
Cell growth assay
FPM1.BP or MOLT-4 cells with SCTs were fixed with 1% formalin in PBS for 20 min and then washed four times with RPMI 1640 medium. These formalin-fixed cells (1 × 105/well) were incubated with 4O1/C8 (1 × 105/well) in each well of 96-well round-bottom microtiter plates for 3 days at 37°C. The number of growing cells was determined by using a Cell Counting Kit-8 (Dojinndo Laboratories, Kumamoto, Japan) in accordance with the manufacturer's instructions.
Formalin-fixed MOLT-4 or FPM1.BP cells (1 × 105/well) were incubated with 4O1/C8 (1 × 105/well) in each well of 96-well round-bottom microtiter plates for 24 hours at 37°C. The percentage of 4O1/C8 cells undergoing apoptosis was determined by FACS analysis using the Annexin V-FITC Apoptosis Detection Kit (MBL) in combination with a PE-conjugated anti-rat CD8 antibody (BD Bioscience).
SCT-EGFP expressing 293T cells were cultured with 4O1/C8 in each well of 96-well round-bottom microtiter plates for 1 hour at 37°C. Cells in the mixed cultures were attached on slide glasses (Matsunami Glass Ind., Japan) by centrifugation, fixed with 4% paraformaldehyde in PBS and then stained with an anti-rat CD8 antibody (BD Bioscience) in combination with Cy3-conjugated goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA). Images were examined with a confocal microscope system (FluoView; Olympus, Tokyo, Japan).
Preparation of immune splenocytes
Female F344/Jcl rats were purchased from Clea Japan, Inc. (Tokyo, Japan). Four-week-old F344/Jcl rats were intraperitoneally inoculated with 1 × 107 FPM1.BP cells. The rats received two boost inoculations with the same dose at 2 and 10 weeks after initial inoculation. One week after the last inoculation, splenocytes were isolated, purified by centrifugation on a density separation medium (Lympholyte-Rat; Cedarlane, Ontario, Canada) and subjected to the analysis for the detection of Tax180-188-specific CTLs or the quantification of IFN-γ production. All rats were maintained at the P3 level animal facilities in Laboratory of Animal Experiment, Institute for Genetic Medicine, Hokkaido University. The experimental protocol was approved by the Animal Ethics Review Committee of our University.
Comparisons between individual data points were made using a Student's t-test. Two-sided P values < 0.05 were considered statistically significant.
We thank Akiko Hirano for technical assistance.
This work was supported in part by grants from the Ministry of Education, Science, Culture, and Sports of Japan.
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