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. (Cancer Sci 2005; 96: 249 –255)
It is estimated that approximately one million people are infected with human T-cell leukemia virus type I (HTLV-I) in Japan. Although most HTLV-I carriers are asymptomatic throughout their lives, 1–5% of infected subjects develop adult T-cell leukemia (ATL),(1–3) and another small fraction of HTLV-I carriers develop a chronic progressive neurological disorder termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and other inflammatory disorders.(4,5)
Adult T-cell leukemia is characterized by tumor cells with mostly CD4+ and CD25+ mature T-lymphocyte phenotypes, onset during middle age or later, immune suppression and poor prognosis.(6) There are four clinical subtypes of ATL: acute, lymphoma, chronic and smoldering types, based on Shimoyama's diagnostic criteria.(7) Monoclonal integration of HTLV-I provirus in ATL cells indicates that ATL arises from a single HTLV-I-infected cell that undergoes malignant phenotypic progression.(8) However, oligoclonal expansion of HTLV-I-infected cells in vivo is also observed in HAM/TSP patients and some asymptomatic HTLV-I carriers,(9) suggesting that HTLV-I-infected cells generally have proliferative potential.
The HTLV-I viral protein Tax is a multifunctional protein, interacting with many cellular proteins regulating cell growth and apoptosis resistance. Tax activates Nuclear factor κ B (NFκB), cAPM response element binding protein (CREB), serum response factor (SRF), activator protein 1 (AP-1), and represses p53 or other tumor suppressor proteins either by direct or indirect mechanisms, partly accounting for HTLV-I-induced leukemogenesis.(10)
However, the level of HTLV-I expression in freshly isolated peripheral ATL cells is extremely low. This paradoxical observation provides controversy concerning the role of Tax in HTLV-I leukemogenesis. It has been reported that fresh ATL cells exhibit constitutive activation of NFκB,(11) one of the transcription factors induced by Tax, while Tax is undetectable in these cells. This implies that either subdetectable levels of Tax or some other mechanism substituting for Tax function activates NFκB in ATL cells.
Expression of viral antigen
It has been noted that HTLV-I expression is inducible in ATL cells from some ATL patients after several hours of culture.(12) A recent study using flow cytometry indicated that similar induction of HTLV-I Tax and Gag antigens occurs in ATL cells in approximately half of the ATL cases tested (Fig. 1).(13) Earlier studies demonstrated that HTLV-I mRNA is detectable at low levels in ATL cells without culture, and is also detectable in ATL lymph nodes in situ.(14,15) Moreover inoculation of uncultured formalin-treated ATL cells into naive rats resulted in induction of a HTLV-I-specific T-cell response.(13) These observations suggest that ATL cells in approximately half of all ATL cases may express very low levels of HTLV-I antigens, which are further enhanced by in vivo culture. Similar transcriptional repression of HTLV-I expression in vitro and its induction in in vitro culture have been observed in the peripheral blood mononuclear cells (PBMC) of HAM/TSP patients and HTLV-I-carriers.(16–18)
The mechanism of this transient transcriptional repression of HTLV-I in the peripheral blood is unknown. Methylation of the CpG motif found in the 5′-long-terminal repeat (LTR) may be partly involved, but do not fully explain the phenomenon.(19–21)
Adult T-cell leukemia cells from the other half of cases fail to express HTLV-I antigens even after in vitro culture. This irreversible silencing of HTLV-I could be due to various genomic changes in the HTLV-I provirus of ATL cells, such as deletions at the 5′-LTR and gag/pol regions.(22,23)
Thus, ATL cases may be categorized into two groups in the context of their HTLV-I expression in ATL cells; HTLV-I expression is inducible in approximately half of all ATL cases, while irreversible in the other half of cases. The inducible type of viral suppression in the PBMC may be a common phenomenon in HTLV-I-infected individuals irrespective of the disease.
Anti-tumor immunity in HTLV-I infection
Cytotoxic T-lymphocyte response in human HTLV-I-infected individuals. Although no consistent differences have been observed among HTLV-I strains isolated from ATL and HAM/TSP patients,(24,25) immunological studies have found a clear difference in HTLV-I-specific T-cell responses between these two diseases. HTLV-I-specific cytotoxic T-lymphocytes (CTL) are highly activated in HAM/TSP patients and are sometimes readily detectable in PBMC without any stimulation in vitro. Similar CTL can be induced in PBMC culture from many asymptomatic HTLV-I carriers when stimulated with autologous HTLV-I-infected cells in vitro. However, HTLV-I-specific CTL are only rarely induced in ATL patients.(26–29) A recent report demonstrated that HTLV-I-specific CTL are present in ATL patients but expand insufficiently,(30) suggesting involvement of some immune suppression or tolerance.
Human T-cell leukemia virus type I core, envelope, polymerase and Tax proteins are recognized by HTLV-I-specific CTL.(28,31,32) In addition, oligopeptides of Tof and Rof were shown to induce CTL from HTLV-I-infected individuals.(33) Among these antigens, HTLV-I Tax, a critical viral protein for T-cell immortalization, is the most popular target for HTLV-I-specific CTL found in HTLV-I-infected individuals.(28,31) HTLV-I Tax-specific CTL are capable of killing short-term cultured ATL cells expressing viral antigens in vitro.(34)
Experimental tumor vaccine. To understand the influence of host immunity to HTLV-I leukemogenesis in vivo, a series of experiments using rat models for HTLV-I-infected T-cell lymphomas were carried out. In these models, a syngeneic HTLV-I-transformed T-cell line underwent phenotypic evolution to cause fatal lymphomas in immune-suppressed rats.(35) However, this cell line did not cause tumors in immune-competent rats. Immunological analysis revealed that the antitumor effects in immune-competent rats were mediated by CTL predominantly directed to HTLV-I Tax.(36) It is intriguing that the major target of HTLV-I-specific CTL is Tax both in rats and humans.
Antitumor effects of Tax-specific CTL were further confirmed by vaccine experiments, in which T-cells from immune-competent rats vaccinated with Tax-encoded DNA could eradicate fatal T-cell lymphomas in athymic rats when transferred.(37) Similar results were obtained by vaccination with oligopeptides corresponding to the major CTL epitope.(36)
Immune response in post-hematopoietic stem cell transplantation ATL patients
Although the rat models described above may mimic some aspects of HTLV-I leukemogenesis, they differ from full-blown ATL in humans as ATL develops in immune-competent individuals following over 40 years of incubation, whereas rat lymphoma consists of HTLV-I-transformed cells and develops only in immune-suppressed hosts.
Allogeneic hematopoietic stem cell transplantation (HSCT) has been used to treat ATL and achieved long-lasting complete remission in some ATL patients.(38) Graft-versus-host (GVH) or graft-versus-leukemia (GVL) responses are presumed to contribute to antitumor effects in these patients. Because the GVH/GVL response is mediated primarily by T cells, we investigated T-cell responses in ATL patients who obtained complete remission following non-myeloablative allogeneic peripheral blood HSCT from human leukocyte antigen (HLA)-identical sibling donors (Fig. 2).(39) In that study, as the target of the GVH response, mitogen-stimulated IL-2-dependent T-cell lines (ILT) were established from ATL patients before HSCT, which express antigens originating from the recipient. These cells were also infected spontaneously with HTLV-I. When the PBMC from the same patients after HSCT were stimulated in culture with formalin-treated ILT cells in vitro, CD8+ CTL capable of killing ILT cells proliferated vigorously. Further analysis revealed that most of these CTL predominantly recognized a limited number of Tax epitopes; Tax 11-19 restricted by HLA-A2 in one patient and Tax 301-309 restricted by HLA-A24 in another. However, PBMC from these ATL patients before HSCT did not show such CTL responses.
Similar oligoclonal expansion of Tax 11-19-specific CTL was reported previously in HLA-A2 + HAM/TSP patients.(40) This phenomenon is explained by a highly activated host CTL response against abundant HTLV-I antigens in HAM/TSP patients. Thus, the Tax-specific CTL expansion observed in the post-HSCT ATL patients implies that these patients may be in a similar status to HAM/TSP patients in the context of their activated levels of T-cell response and/or Tax antigen presentation in vivo. Significant reduction in the proviral load in these patients following HSCT might be partly due to such a strong anti-Tax CTL response.
Various minor histocompatibility antigens (mHA) have been postulated to act as the target of the GVH/GVL response in HSCT.(41) In cultures of post-HSCT ATL patients, a minor population of CTL induced against ILT cells was directed to an unknown antigen other than Tax, probably related to the GVH response. The role of the anti-Tax CTL response in relation GVL effects remains to be clarified. However, the strong HTLV-I-specific response observed in the patients after complete remission suggests that HTLV-I-specific CTL as well as the GVH effectors might participate in the maintenance of remission.
Immunological risk factors for ATL development
The insufficient HTLV-I-specific T-cell response observed generally in ATL patients could be either a consequence of ATL or a risk associated with ATL development. If this were a risk associated with ATL, a wide survey of HTLV-I-specific T-cell responses among HTLV-I carriers would be useful to identify a high-risk group, to whom prophylactic strategies should be applied.
Epidemiological risk factors for ATL. In cohort studies of HTLV-I carriers, it appears that the risk factors for ATL might include vertical HTLV-I infection, and increasing numbers of abnormal lymphocytes.(3,42,43) HTLV-I transmits from mother to child mainly through breast milk, and from male to female by sexual contact. Blood transfusion or intravenous drug use also causes HTLV-I transmission.(44–46) Among these, mother-to-child transmissions are the major natural route in Japan. The higher incidence of ATL in males is attributed to the relatively higher ratio of vertical infection in males. The presence of typical HLA haplotypes for ATL in an endemic area(47) also implies that vertical infection might transmit some determinants of HTLV-I leukemogenesis.
Oral infection as a determinant of insufficient T-cell response in rats. In a rat model, the routes of infection strongly affect HTLV-I-specific immunity (Fig. 3).(48) Among immune-competent adult rats infected with HTLV-I through various routes, rats inoculated orally showed very low levels of HTLV-I-specific T-cell response, whereas significant responses were detected in rats infected through other routes.(49) In contrast, HTLV-I proviral load in the spleen cells, examined several months after infection, was significantly higher in orally infected rats. Because HTLV-I proviruses are associated with infected cells, the increase in proviral load indicates the increase in infected cell number.
Together with the fact that oral infection through mothers’ milk is a major route of vertical HTLV-I infection in humans,(44) the results of the rat experiments of oral HTLV-I infection strongly suggest that the epidemiological risks of ATL (i.e. vertical HTLV-I infection and high viral load) link to the immunological risk (i.e. low T-cell responses to HTLV-I).
Balance between host immunity and HTLV-I in natural HTLV-I infection
Positive or negative correlation between host immunity and the virus. In the rat experiment described above, there was an inverse correlation between HTLV-I proviral load and HTLV-I-specific T-cell proliferation,(49) indicating that HTLV-I-specific T-cell responses might contribute to limiting expansion the of HTLV-I-infected cells in vivo, and that oral infection may be a reason for insufficient T-cell immunity to HTLV-I.
Infants born to HTLV-I-carrying mothers are fed approximately 1 × 108 HTLV-I-infected cells before weaning,(45) and a number of infantile carriers stay seronegative for HTLV-I for a certain period of time,(50) probably due to some immunological tolerance during this period. Most of these children show seroconversion by the age of 3 years.(51) Although T-cell immune responses to HTLV-I in children are not known, many adult HTLV-I carriers show HTLV-I-specific CTL responses, suggesting that the T-cell response might recover spontaneously later in life, just as happens in vertical hepatitis B virus infection.
In contrast to the results of rat experiments, HTLV-I proviral load in human adult HTLV-I carriers correlate positively with the HTLV-I-specific T-cell response.(52,53) This discrepancy between rats and humans may be partly explained by the difference in the period of HTLV-I infection; several months in the rat experiments but many years in human HTLV-I carriers. If the T-cell response recovered after a long period of insufficient response to HTLV-I, the magnitude of the recovered response would correlate positively with the elevated levels of pre-existing proviral load in vivo.
Hypothetical relationship among host immunity, disease development and the route of infection. Figure 4 gives a schematic demonstration of our current hypothesis on the immunological risks of ATL in the natural course of HTLV-I infection. Vertically infected HTLV-I carriers harbor risks of ATL (i.e. insufficient HTLV-I-specific T-cell response and expansion of infected cells). However, such risks may be reduced in many HTLV-I carriers by spontaneous recovery of the HTLV-I-specific T-cell response. If there is a small group of adult HTLV-I carriers still showing insufficient T-cell responses to HTLV-I despite an abundant viral load, this might be a high-risk group for ATL, to whom prophylactic vaccines targeting Tax may be beneficial.
Favorable levels of T-cell response and a lower risk of ATL are expected in individuals infected through horizontal routes, although a small fraction of this group might develop HAM/TSP. The genetic determinants of HAM/TSP are not known. In Japan, many HAM/TSP cases arise from the vertically infected population, suggesting that T-cell immune conversion has occurred at some stage. The oligoclonal expansion of HTLV-I-infected cell clones often seen in HAM/TSP patients indicates that these clones might have been in the process of leukemogenesis.(9) Nevertheless, the incidence of ATL among HAM/TSP patients is limited, probably due to the activated host HTLV-I-specific T-cell immunity. In this respect, administration of immunosuppressive reagents to HAM/TSP patients might increase the risk of ATL development. In addition, the post-HSCT patients with T-cell immune conversion should be followed up carefully, although development of HAM/TSP in post-HSCT ATL patients has not been reported so far.
Prophylaxis and therapy for ATL
The findings of the immunological studies described above suggest that Tax-targeted vaccines may be beneficial for prophylactic use against the high-risk group. For this purpose, a handy method to detect HTLV-I-specific T-cell immune response would be required for a wide survey among HTLV-I carriers.
A number of combination chemotherapy protocols have been applied, and the median survival time of a recent protocol (JCOG9303) was 13 months.(54) In addition, several kinds of experimental therapies have also been applied to ATL. A small number of ATL patients respond to intravenous administration of anti-CD25 monoclonal antibody.(55) The combination therapy of azidothymidine (AZT) and interferon α achieved a high response rate but did not prevent relapse of ATL.(56,57) The mechanisms for the antitumor effects of these antiretroviral drugs are not clear, as HTLV-I proliferation occurs mainly by proliferation of infected cells, not by viral replication. A recent study indicated that AZT and interferon α suppress NFκB activity and induce TRAIL expression, respectively.(58) A combination of arsenic and interferon α(59) and some other NFκB-targeted therapies have been proposed.
Recently, allogeneic but not autologous HSCT achieved long-lasting complete remission in some ATL patients.(38,60) However, there is also a risk of GVH disease, which is sometimes lethal. If Tax-specific CTL induced in post-HSCT ATL patients makes any contribution to GVL effects, Tax-targeted immunotherapy might be worth trying either with or without HSCT, and selective GVL effects would be expected. Indication of Tax-targeted immunotherapy, however, should be limited to those cases whose ATL cells retain the ability to express Tax.
Although the peripheral HTLV-I-infected cells do not express detectable levels of HTLV-I antigens, they retain the ability to express Tax in most HTLV-I-infected individuals, including asymptomatic HTLV-I carriers, HAM/TSP patients and approximately half of all ATL cases. Immunological findings support the contribution of Tax-specific CTL to antitumor immunity in these hosts, encouraging immunological prophylaxis and therapy for ATL.
We thank many young clinical collaborators for supplying important blood samples. We also thank the former research staff in our laboratory: Drs Shino Hanabuchi (University of Texas), Atsuhiko Hasegawa (Tulane National Primate Research Center), Hirotomo Kato (Yamaguchi University), and Yoshihiro Koya (National Institute of Infectious Diseases) for their excellent achievements on the series of studies using animal models. This work was supported by grants from the Ministry of Education, Science, Culture and Sports of Japan, and the Ministry of Health, Welfare, and Labor of Japan.