Tax-specific Cytotoxic T Lymphocytes Research Articles

Development of a Cytotoxic T-Cell Assay in Rabbits to Evaluate Early Immune Response to Human T-Lymphotropic Virus Type 1 Infection

Human T-lymphotropic virus type 1 (HTLV-1) infection causes adult T-cell lymphoma/leukemia (ATL) following a prolonged clinical incubation period, despite a robust adaptive immune response against the virus. Early immune responses that allow establishment of the infection are difficult to study without effective animal models. We have developed a cytotoxic T-lymphocyte (CTL) assay to monitor the early events of HTLV-1 infection in rabbits. Rabbit skin fibroblast cell lines were established by transformation with a plasmid expressing simian virus 40 (SV40) large T antigen and used as autochthonous targets (derived from same individual animal) to measure CTL activity against HTLV-1 infection in rabbits. Recombinant vaccinia virus (rVV) constructs expressing either HTLV-1 envelope surface unit (SU) glycoprotein 46 or Tax proteins were used to infect fibroblast targets in a (51)Cr-release CTL assay. Rabbits inoculated with Jurkat T cells or ACH.2 cells (expressing ACH HTLV-1 molecule clone) were monitored at 0, 2, 4, 6, 8, 13, 21, and 34 wk post-infection. ACH.2-inoculated rabbits were monitored serologically and for viral infected cells following ex vivo culture. Proviral load analysis indicated that rabbits with higher proviral loads had significant CTL activity against HTLV-1 SU as early as 2 wk post-infection, while both low- and high-proviral-load groups had minimal Tax-specific CTL activity throughout the study. This first development of a stringent assay to measure HTLV-1 SU and Tax-specific CTL assay in the rabbit model will enhance immunopathogenesis studies of HTLV-1 infection. Our data suggest that during the early weeks following infection, HTLV-1-specific CTL responses are primarily targeted against Env-SU.

Target Epitopes of Human T Lymphotropic Virus 1 Recognized by Class I MHC-Restricted Cytotoxic T Lymphocytes in HAM/TSP Patients and Infected Patients with Autoimmune Disorders.

Abstract 2660Poster Board II-636Human T-cell leukemia virus-1 (HTLV-1) causes adult T cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) after long-term infection. A variety of collagen diseases such as Sjögren's syndrome and systemic lupus erythematosus has been reported in HTLV-1-infected individuals, although the precise relationship between these disorders and HTLV-1 infection remains unknown. We have previously reported the decreased frequency and function of HTLV-1 Tax-specific cytotoxic T lymphocytes (CTLs) in ATL patients due to insufficient cytolytic effector molecules compared with asymptomatic carriers (ACs) (ASH annual meeting 2007). However, there is no report on the repertoire of HTLV-1-specific CTLs in HAM/TSP patients or carriers with collagen diseases (CCs), both of which are characterized by abnormal immune states. In order to characterize HTLV-1-specific CTLs in ACs, HAM/TSP patients and CCs, we examined the frequency and diversity of HTLV-1-specific CTLs using 16 distinct HTLV-1 Tax and Env HLA-A*0201 and HLA-A*2402 tetramers. There was no statistically significant difference in the proportion of patients having HTLV-1 Tax specific CTLs among ACs, HAM/TSP and CC patients, but a greater proportion of patients with HAM/TSP or collagen disease had HTLV-1 Env-specific CTLs compared with ACs (Env175-183, Env239-246, Env442-450 for HLA-A*0201 and Env11-19, Env21-29, Env153-161 for HLA-A*2402) (Table 1). Within CD8+ lymphocyte subsets, the percentage of cells binding either Tax11-19/HLA-A*0201 or Tax 301-309/HLA-A*2402 tetramer (Figure 1), was significantly higher for HAM/TSP patients compared to ACs, being lowest among CCs. Additionally, the number of epitope repertoires found on HTLV-1 Env-specific CD8+ cells in ACs was considerably lower than for HAM/TSP patients and CCs (ACs, 1/6; HAM/TSP, 4/6; CCs, 6/6). This study demonstrates the relatively greater importance of CTLs recognizing HTLV-1 envelope tetramers compared to HTLV-1 Tax tetramers, thereby suggesting that the diversity, frequency and repertoire of HTLV-1-specific CTL clones, especially Env-CTLs, may be related to the hyperimmune response in HAM/TSP and carriers with collagen diseases. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Impaired Tax‐specific T‐cell responses with insufficient control of HTLV‐1 in a subgroup of individuals at asymptomatic and smoldering stages

Human T-cell leukemia virus type-1 (HTLV-1)-specific T-cell immunity, a potential antitumor surveillance system in vivo, is impaired in adult T-cell leukemia (ATL). In this study, we aimed to clarify whether the T-cell insufficiency in ATL is present before the disease onset or occurs as a consequence of the disease. We investigated T-cell responses against Tax protein in peripheral blood mononuclear cells (PBMCs) from individuals at earlier stages of HTLV-1-infection, including 21 asymptomatic HTLV-1 carriers (ACs) and four patients with smoldering-type ATL (sATL), whose peripheral lymphocyte count was in normal range. About 30% of samples tested showed clear Tax-specific interferon (IFN)-gamma producing responses. Proviral loads in this group were significantly lower than those in the other less-specific response group. The latter group was further divided to two subgroups with or without emergence of Tax-specific responses following depletion of CC chemokine receptor 4 (CCR4)(+) cells that contained HTLV-1-infected cells. In the PBMCs with Tax-specific responses, CD8(+) cells efficiently suppressed HTLV-1 p19 production in culture. The remaining group without the emergence of Tax-specific response after CCR4(+) cell-depletion included at least two sATL and one AC samples, which spontaneously produced HTLV-1 p19 in culture, where tetramer-binding, Tax-specific cytotoxic T-lymphocytes were either undetectable or unresponsive. Our results indicated that HTLV-1-specific T-cell responsiveness widely differed among HTLV-1 carriers, and that impairment of HTLV-1-specific T-cell responses was observed not only in advanced ATL patients but also in a subpopulation at earlier stages, which was associated with insufficient control of HTLV-1.

Discrepant Expression Between mRNA and Protein of HTLV-I Basic Leucine Zipper Factor (HBZ) Makes HTLV-I Infected CD4 Lymphocytes and Adult T Cell Leukemia Cells Less Susceptible to HBZ-Specific Cytotoxic T Lymphocytes

[Background]Adult T cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and still remains one of the chemotherapy-resistant leukemias. Accumulating the successful evidence of allogeneic hematopoietic stem cell transplantation (allo-HSCT), the immunotherapeutic strategy for ATL has become to look promising. The fact that more than half of primary ATL cells lack the immunogenic oncoprotien HTLV-1 Tax has been promoting researchers to find an alternative target antigen to develop the cellular immunotherapy for ATL. Recently, HTLV-1 basic leucine zipper factor (HBZ), which is encoded by the minus strand of HTLV-1 proviral genome and is transcribed from 3'-LTR, has been highlighted as the important molecule in ATL leukemogenesis. Since almost all ATL cells express HBZ mRNA, we attempted to verify whether HBZ can be a target of cellular immunotherapy for ATL.[Methods] At first, we synthesized a variety of HBZ-derived 9 amino acid peptides (9 mer) that are predicted to have high binding affinity to HLA-A*0201 molecule. CD8+ T lymphocytes from an HLA-A*0201+ healthy donor were stimulated with peptide-loaded autologous monocyte-derived mature dendritic cells repetitively. Thereafter, epitope specificity, HLA-restriction, and cytotoxic activity of induced cytotoxic T lymphocytes (CTLs) were determined by standard 51Cr-release assays. HBZ and Tax mRNA expression levels of target cells, including primary ATL cells, HTLV-1-infected cell lines, peripheral blood lymphocytes isolated from HTLV-1 carriers and HTLV-1-uninfected individuals, and K562-A*0201 and C1R-A*0201 cells transfected with HBZ gene, were simultaneously measured by real-time quantitative PCR (RQ-PCR). The relative expression levels of HBZ and Tax mRNA were determined by comparative Ct method, and the results were shown as the relative values to those of HTLV-1-infected cell line MT4. HBZ protein expression was determined by Western blotting using anti-HBZ serum which was produced by immunizing rabbits with purified six-His-tagged HBZ polypeptide corresponding to the bZIP domain of HBZ. The detection of HBZ-specific and Tax-specific CTLs was performed by tetramer assays.[Results] We identified an HBZ-derived 9-mer epitope, HBZ26–34 (GLLSLEEEL), with high binding affinity to HLA-A*0201 measured by using HLA-A*0201 gene-transfected T2 cell. We successfully established an HBZ26–34 peptide-specific CTL clone designated as HBZ-1. HBZ-1 exerted cytotoxicity against HBZ gene-transfected K562-A*0201 and C1R-A*0201 cells, but not against HLA-A*0201-positive HTLV-1-infected cells or ATL cells. HTLV-1 infection and HBZ gene transfection did not alter the HLA class I expression of target cells. Expression levels of HBZ mRNA appeared to be higher in ATL cells, HTLV-1-infected cell lines, and HBZ gene-transfected cells than those in HTLV-1 carrier cells. Abundant HBZ protein expression was detected in only HBZ gene-transfected K562-A*0201 and C1R-A*0201 cells, but HBZ protein expression levels in HTLV-1-infected cell lines and ATL cells appeared to be very low. We further examined Tax-specific and HBZ-specific CTLs in an HLA-A*0201-positive ATL patient who received allo-HSCT from the HLA-identical sibling donor. In this patient, Tax mRNA and HBZ mRNA were similarly expressed in ATL cells; however, only Tax-specific CTLs but not HBZ-specific CTLs were detected in both before and after allo-HSCT at full donor chimerism.[Conclusions] Although HBZ mRNA was apparently detected in all HTLV-1-infected cell lines and ATL cells, HBZ protein in these cells was insufficiently expressed to be recognized by HBZ-specific CTLs. Our observations in a transplanted patient with ATL also suggest that HBZ protein may be less immunogenic due to discrepant expression level between mRNA and protein.

Reduced Foxp3 expression with increased cytomegalovirus-specific CTL in HTLV-I-associated myelopathy

Human T-lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients show high immune responses to HTLV-I. However, it is unclear whether the cytotoxic T lymphocyte (CTL) responses to other chronic viruses also increase. We investigated the responses in the peripheral blood by using HLA-A*0201/peptide pentamers. The frequency of cytomegalovirus (CMV)-specific CTL tended to be higher in HAM/TSP patients than in healthy controls (HCs). The frequency of CMV-specific CTL positively correlated with that of HTLV-I Tax-specific CTL. The frequency of Foxp3+ cells in CD4+ lymphocytes tended to be higher in HAM/TSP patients than in ACs and HCs. The expression level of Foxp3 was lower in HAM/TSP patients than in HCs and was inversely correlated with the CMV-specific CTL frequency. A percentage of Foxp3+ cells showed a positive correlation with the HTLV-I proviral load. These results suggest that a decrease in the Foxp3 expression may contribute to the high immune response to CMV and that the Foxp3+ regulatory T cells may play a role in the immune surveillance of HTLV-I.

Activation and detection of HTLV-I Tax-specific CTLs by Epitope expressing Single-Chain Trimers of MHC Class I in a rat model

BackgroundHuman 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.ResultsWe 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.ConclusionWe 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.

Immunologic Control of Human T-Cell Leukemia Virus Type I and Adult T-Cell Leukemia

Host T-cell responses to human T-cell leukemia virus type I (HTLV-I) control the expansion of HTLV-I-infected cells and are determinants of the equilibrium proviral load in vivo. Insufficient T-cell responses are regarded as an immunologic risk factor for adult T-cell leukemia (ATL) because they allow increased proviral loads, which represent an epidemiologic risk factor for ATL. ATL cells from approximately half of ATL cases retain the ability to express HTLV-I Tax, a major target antigen of HTLV-I-specific cytotoxic T-lymphocytes (CTL), whereas Tax-specific CTL in ATL patients are inactive. Tax-specific CTL responses are strongly activated after hematopoietic stem cell transplantation in some ATL patients in long-term remission, indicating that HTLV-I Tax is expressed in vivo rather than being silent, and that the donor-derived T-cell system can recognize it. These findings strongly suggest that reactivation of Tax-specific CTL by vaccines may be promising for prophylaxis of ATL in the high-risk group of HTLV-I carriers and for therapy of ATL in patients whose tumor cells are capable of expressing Tax.

Anti‐HTLV‐1 tax antibody and tax‐specific cytotoxic T lymphocyte are associated with a reduction in HTLV‐1 proviral load in asymptomatic carriers

Previous studies have suggested that higher anti-human T-lymphotropic virus 1 (HTLV-1) antibody titer and lower anti-HTLV-1 Tax antibody reactivity are risk factors for adult T-cell leukemia/lymphoma. In the present study, we analyzed the relationships between these factors and clarified their significance. Forty-five carriers were examined for anti-HTLV-1 and anti-Tax antibody by ELISA. In addition, 43 of the 45 carriers with HLA-A*0201 and/or A*2402 were examined for frequency of Tax-specific cytotoxic T lymphocytes (CTLs) using HTLV-1/HLA tetramers, and 44 were examined for proviral load by real-time PCR. The relationships between these factors were analyzed statistically. The frequencies of Tax11-19 and Tax301-309-specific CTLs were significantly higher in the anti-Tax antibody-positive group as compared with the antibody-negative group (P = 0.002 and 0.033, respectively). Anti-HTLV-1 antibody titer had a positive correlation with proviral load (P = 0.019), whereas anti-Tax antibody did not show a significant correlation. Higher frequencies of both Tax11-19 and Tax301-309-specific CTLs are related to a reduction in proviral load (P = 0.017 and 0.015, respectively). Synergistic interactions of humoral and cellular immunity against Tax protein were demonstrated in HTLV-1 carriers. Tax-specific CTL may reduce HTLV-1 proviral load to prevent asymptomatic carriers from developing adult T-cell leukemia/lymphoma.

Use of human antigen presenting cell gene array profiling to examine the effect of human T-cell leukemia virus type 1 Tax on primary human dendritic cells

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.

Identification of Two New HLA-A*1101-Restricted Tax Epitopes Recognized by Cytotoxic T Lymphocytes in an Adult T-Cell Leukemia Patient after Hematopoietic Stem Cell Transplantation

We previously reported that Tax-specific CD8(+) cytotoxic T lymphocytes (CTLs), directed to single epitopes restricted by HLA-A2 or A24, expanded in vitro and in vivo in peripheral blood mononuclear cells (PBMC) from some adult T-cell leukemia (ATL) patients after but not before allogeneic hematopoietic stem cell transplantation (HSCT). Here, we demonstrated similar Tax-specific CTL expansion in PBMC from another post-HSCT ATL patient without HLA-A2 or A24, whose CTLs equally recognized two newly identified epitopes, Tax88-96 and Tax272-280, restricted by HLA-A11, suggesting that these immunodominant Tax epitopes are present in the ATL patient in vivo.

Tumor immunity against adult T‐cell leukemia

Human T-cell leukemia virus type-I (HTLV-I) causes adult T-cell leukemia (ATL) in a small population of infected individuals after a long incubation period. Although the process of clonal evolution of ATL cells may involve multiple steps, ATL cells from half of the ATL cases still retain the ability to express HTLV-I Tax, a key molecule of HTLV-I leukemogenesis. A recent finding of reactivation of Tax-specific cytotoxic T lymphocytes (CTL) in ATL patients after hematopoietic stem cell transplantation suggests the presence of Tax expression in vivo and potential contribution of the CTL to antitumor immunity. This is consistent with the results of a series of animal experiments indicating that Tax-specific CTL limit the growth of HTLV-I-infected cells in vivo, although the animal model mimics only an early phase of HTLV-I infection and leukemogenesis. Establishment of an insufficient HTLV-I-specific T-cell response and an increased viral load in orally HTLV-I-infected rats suggests that host HTLV-I-specific T-cell response at a primary HTLV-I infection can be a critical determinant of persistent HTLV-I levels thereafter. These findings indicate that Tax-targeted vaccines may be effective for prophylaxis of ATL in a high-risk group, and also for therapy of ATL in at least half the cases.

Human T-cell leukemia virus type I Tax induces the expression of dendritic cell markers associated with maturation and activation

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of both adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the genesis of HAM/TSP likely involves several steps, the generation of a highly specific and effective population of Tax-specific CD8+ cytotoxic T lymphocytes (CTLs) that migrate to the central nervous system (CNS) is of pivotal importance in this neuropathologic process. Presentation of Tax peptides by activated dendritic cells (DCs) to naive CD8+ T cells likely plays an important role in the induction of a Tax-specific CTL response and the eventual neurologic dysfunction observed in HAM/TSP. The immune response mounted during HTLV-I infection is primarily targeted against Tax with both Tax-specific antibodies and CTLs found in HTLV-I-infected individuals, indicating that Tax is available for immune recognition. Studies have suggested that Tax may be secreted from HTLV-I-infected cells and act as an extracellular cytokine, be internalized and processed for presentation, or be transported to the nucleus where it may act as a transcriptional activator. The authors report in this article that purified Tax induces DC activation involving an increase in the production of CD80 and CD86 mRNA in the absence of corresponding protein synthesis. Furthermore, intracellular Tax down-regulates the protein expression of molecules involved in antigen presentation. This implies a difference in the mechanism of Tax activity depending upon its location. Additionally, treatment of JAWS II DCs with extracellular Tax decreases the ability of DCs to present a major histocompatibility complex (MHC) class I-restricted peptide, indicating that Tax likely matures the DCs to the point where presentation of a secondary antigen is restricted. The implication of the experimental results with respect to the generation of a Tax-specific CTL compartment that participates in the genesis of HAM/TSP is discussed.

Mediators of central nervous system damage during the progression of human T-cell leukemia type I-associated myelopathy/tropical spastic paraparesis.

Human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) represents one of the most devastating diseases associated with HTLV-I infection. Despite the delineation of clinical features associated with this neurologic disease, more progress needs to be made with respect to understanding the molecular mechanisms relating to the genesis of HAM/TSP. Several factors have been hypothesized to contribute to whether an HTLV-I-infected individual remains asymptomatic, develops adult T-cell leukemia (ATL), or progresses to HAM/TSP. Among the most intriguing of these factors is the immune response mounted by the host against HTLV-I. Several cell populations are crucial with respect to generating an efficient immune response against the virus. This includes CD4(+) T cells, CD8(+) T cells, dendritic cells (DCs), monocytes/macrophages, and HTLV-I-infected cells that interact with immune cells to stimulate their effector functions. Although all of these cell types likely play important roles in the etiology of HAM/TSP, this review focuses specifically on the potential function of the CD8(+) T-cell population during the progression of HTLV-I-induced neurologic disease. The immune response in HAM/TSP patients may transition from a beneficial response aimed at controlling the viral infection, to a detrimental response that ultimately participates in mediating the pathology observed in HAM/TSP. In this respect, the generation of a hyperactive CD8(+) cytotoxic T lymphocyte (CTL) response primarily targeting the HTLV-I Tax protein likely plays a key role in the genesis of pathologic abnormalities associated with HAM/TSP. The efficiency and activity of Tax-specific CD8(+) CTLs may be regulated at a number of levels, and deregulation of Tax-specific CTL activation may contribute to HAM/TSP. This review focuses on potential mechanisms of central nervous system (CNS) damage associated with the genesis of HAM/TSP following HTLV-I infection, focusing on the role of the Tax-specific CTL compartment.

Presentation of a new H-2D(k)-restricted epitope in the Tax protein of human T-lymphotropic virus type I is enhanced by the proteasome inhibitor lactacystin.

Tax, the trans-activator of human T-lymphotropic virus type I (HTLV-I), is the dominant target antigen for cytotoxic T lymphocytes (CTLs) in the majority of infected individuals, although the reason for this immunodominance is not clear. Tax has been shown to associate physically with the proteasome, a protease that is responsible for the generation of the majority of major histocompatibility complex (MHC) class I ligands recognized by CTLs. This association could lead to the preferential targeting of Tax to the MHC class I pathway and account for its high immunogenicity. Here, the CTL response to Tax was investigated in mice by priming with a Tax expression vector and boosting with a Tax recombinant vaccinia virus (modified vaccinia virus Ankara strain). This approach led to the identification of a new H-2D(k)-restricted epitope in Tax, amino acid residues 38-46, sequence ARLHRHALL. Surprisingly, presentation of this epitope was found to be enhanced by the proteasome inhibitor lactacystin, although Tax was shown to associate with proteasomes in murine cells. The difficulties encountered in generating Tax-specific CTL responses and the results of enzyme-linked immunospot (ELISpot) analysis suggested that Tax is only poorly immunogenic for CTLs in mice. Therefore, the immunodominance of Tax in human CTL responses to HTLV-I is probably not due to an intrinsic property of the protein itself, such as an association with the proteasome, but instead may result from the fact that Tax is the predominant protein synthesized early after infection.

Prevention of adult T-cell leukemia-like lymphoproliferative disease in rats by adoptively transferred T cells from a donor immunized with human T-cell leukemia virus type 1 Tax-coding DNA vaccine.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) in infected individuals after a long incubation period. To dissect the mechanisms of the development of the disease, we have previously established a rat model of ATL-like disease which allows examination of the growth and spread of HTLV-1 infected tumor cells, as well assessment of the effects of immune T cells on the development of the disease. In the present study, we induced HTLV-1 Tax-specific cytotoxic T lymphocyte (CTL) immunity by vaccination with Tax-coding DNA and examined the effects of the DNA vaccine in our rat ATL-like disease model. Our results demonstrated that DNA vaccine with Tax effectively induced Tax-specific CTL activity in F344/N Jcl-rnu/+ (nu/+) rats and that these CTLs were able to lyse HTLV-1 infected syngeneic T cells in vitro. Adoptive transfer of these immune T cells effectively inhibited the in vivo growth of HTLV-1-transformed tumor in F344/N Jcl-rnu/rnu (nu/nu) rats inoculated with a rat HTLV-1 infected T cell line. Vaccination with mutant Tax DNA lacking transforming ability also induced efficient anti-tumor immunity in this model. Our results indicated a promising effect for DNA vaccine with HTLV-1 Tax against HTLV-1 tumor development in vivo.

High Activated and Memory Cytotoxic T-cell Responses to HTLV-1 in Healthy Carriers and Patients with Tropical Spastic Paraparesis

The cytotoxic T-lymphocyte (CTL) response to HTLV-1 is directed mainly against the Tax protein. Circulating, activated Tax-specific CTL can be found in a majority of healthy carriers and patients with the HTLV-1-associated disease tropical spastic paraparesis (HAM/TSP). In this study we present data on the Tax-specific CTL response of 26 HTLV-1 carriers, including 10 newly recruited subjects. Rex-specific CTL were not found in any subjects investigated. Activated and memory CTL responses were determined separately in 4 healthy carriers, 3 HAM/TSP patients, and 1 “seronegative HAM/TSP.” In all subjects, the mean frequency of peptide-specific memory cells per epitope (1/1307) was high. There was no significant difference in mean memory CTL frequency per epitope or in the proportion of subjects with activated CTL between healthy carriers and HAM/TSP patients. One individual with HAM/TSP had an unusually high frequency response to two peptides, suggesting immunodominance of epitope recognition in this individual. We conclude that the magnitude and components of the HTLV-1-specific CTL response do not differ between healthy carriers and HAM/TSP patients. These data do not support a specific CTL-mediated component in the pathogenesis of HAM/TSP.