Adult T-cell Leukemia Patients Research Articles

Cellular Factors Involved in HTLV-1 Entry and Pathogenicit

Human T cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia (ATL) and HTLV-1 – associated myelopathy and tropical spastic paraparesis (HAM/TSP). HTLV-1 has a preferential tropism for CD4 T cells in healthy carriers and ATL patients, while both CD4 and CD8 T cells serve as viral reservoirs in HAM/TSP patients. HTLV-1 has also been detected other cell types, including monocytes, endothelial cells, and dendritic cells. In contrast to the limited cell tropism of HTLV-1 in vivo, the HTLV receptor appears to be expressed in almost all human or animal cell lines. It remains to be examined whether this cell tropism is determined by host factors or by HTLV-1 heterogeneity. Unlike most retroviruses, cell-free virions of HTLV-1 are very poorly infectious. The lack of completely HTLV-1-resistant cells and the low infectivity of HTLV-1 have hampered research on the HTLV entry receptor. Entry of HTLV-1 into target cells is thought to involve interactions between the env (Env) glycoproteins, a surface glycoprotein (surface unit), and a transmembrane glycoprotein. Recent studies have shown that glucose transporter GLUT1, heparan sulfate proteoglycans (HSPGs), and neuropilin-1 (NRP-1) are the three proteins important for the entry of HTLV-1. Studies using adherent cell lines have shown that GLUT1 can function as a receptor for HTLV. HSPGs are required for efficient entry of HTLV-1 into primary CD4 T cells. NRP-1 is expressed in most established cell lines. Further studies have shown that these three molecules work together to promote HTLV-1 binding to cells and fusion of viral and cell membranes. The virus could first contact with HSPGs and then form complexes with NRP-1, followed by association with GLUT1. It remains to be determined whether these three molecules can explain HTLV-1 cell tropism. It also remains to be more definitively proven that these molecules are sufficient to permit HTLV-1 entry into completely HTLV-1-resistant cells.

The roles of acquired and innate immunity in human T-cell leukemia virus type 1-mediated diseases

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis in small subsets of HTLV-1 carriers. HTLV-1-specific T-cell responses play critical roles in anti-viral and anti-tumor host defense during HTLV-1 infections. Some HTLV-1 carriers exhibit selective loss or anergy of HTLV-1-specific T-cells at an asymptomatic stage. This is also observed in ATL patients and may therefore be an underlying risk factor of ATL in combination with elevated proviral loads. HTLV-1-specific T-cells often recognize the viral oncoprotein Tax, indicating expression of Tax protein in vivo, although levels of HTLV-1 gene expression are known to be very low. A type-I interferon (IFN) response can be induced by HTLV-1-infected cells and suppresses HTLV-1 expression in vitro, suggesting a role of type-I IFN response in viral suppression and pathogenesis in vivo. Both acquired and innate immune responses control the status of HTLV-1-infected cells and could be the important determinants in the development of HTLV-1-mediated malignant and inflammatory diseases.

HTLV-1感染症とmiRNA

Although human T cell leukemia virus type I (HTLV-I) is undoubtedly involved in the immortalization and leukemogenesis of infected cells, mechanistic underpinnings of its molecular pathophysiology in long latent period of Adult T-cell leukemia (ATL) remain to be elucidated. One of the most significant recent advances in biomedical research has been the discovery of small noncoding RNAs designated microRNA (miRNA), which affect the field of virology including HTLV-1 research. Mounting evidence indicates that viruses use these miRNAs to manipulate both cellular and viral gene expression. Viral infection also can exert a profound impact on the cellular miRNA expression profile. Some studies have demonstrated that some deregulations of miRNA are involved in the pathogenesis of HTLV-1. Furthermore, global analyses of ATL patient samples have provided a conceptual progress that Polycomb family induces miR-31 silencing, resulting in overexpression of NF- kappaB inducing kinase (NIK) following NF-kappaB activation. Given that miRNAs act as pleiotropic molecules essential in all cellular events, deregulation of miRNA signature caused by HTLV-1 infection strongly involves the imbalance of molecular network of lymphocytes. Recognition and understanding of the widespread molecular applicability of miRNAs will increasingly have much effect on the development of novel strategies to treat the HTLV-1-associated diseases. Here we discuss our current knowledge of viral miRNAs and virally influenced cellular miRNAs and their relationship to ATL.

Functional impairment of Tax-specific but not cytomegalovirus-specific CD8+ T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8+ T-cells in various clinical status of HTLV-1 infection.ResultsBy using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8+ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8+ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8+ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8+ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8+ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8+ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8+ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8+ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8+ T-cells.ConclusionsThese findings indicated that Tax-specific CD8+ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8+ T-cells in early stages.

Pathogenesis of Metastatic Calcification and Acute Pancreatitis in Adult T-Cell Leukemia under Hypercalcemic State

Human T-cell leukemia virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL). Hypercalcemia is common in patients with ATL. These patients rarely develop metastatic calcification and acute pancreatitis. The underlying pathogenesis of this condition is osteoclast hyperactivity with associated overproduction of parathyroid hormone-related protein, which results in hypercalcemia in association with bone demineralization. The discovery of the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL), its receptor RANK, and its decoy receptor osteoprotegerin (OPG), enhanced our understanding of the mechanisms of ATL-associated hypercalcemia. Macrophage inflammatory protein-1-α, tumor necrosis factor-α, interleukin-1, and interleukin-6 are important molecules that enhance the migration and differentiation of osteoclasts and the associated enhanced production of RANKL for osteoblast formation. In this paper, we focus on metastatic calcification and acute pancreatitis in ATL, highlighting recent advances in the understanding of the molecular role of the RANKL/RANK/OPG system including its interaction with various cytokines and calciotropic hormones in the regulation of osteoclastogenesis for bone resorption in hypercalcemic ATL patients.

Human Telomerase Reverse Transcriptase-Specific T-Cell Receptor Gene Transfer Redirects T-Lymphocytes to Exert Effective Antitumor Reactivity Against Adult T-Cell Leukemia,

Abstract 4169 Background and PurposeCurrently adult T-cell leukemia (ATL) is one of the most chemotherapy-resistant T cell malignancies and the prognosis of ATL patients still remains very poor. Although several novel treatment options, for example anti-chemokine receptor 4 (CCR4) monoclonal antibody, look promise, development of other novel treatment options still remains warranted. Currently activated status of human telomerase reverse transcriptase (hTERT) in ATL cells has been underscored. Thus, in this study, in order to develop a novel T cell-based immunotherapy for the treatment of ATL, we investigated the feasibility of redirected T-cell using hTERT-specific T-cell receptor (TCR) gene transfer. MethodsApproval for this study was obtained from the Institutional Review Board of Ehime University Hospital. HLA-A*24:02-restricted and hTERT461–469epitope (residues: VYGFVRACL)-specific TCR -a/b genes (Tajima K et al. Int J Cancer 2004) cloned from our established hTERT-specific CTL clone were inserted into a novel GaLV-pseudotyped retroviral vector encoding built-in siRNAs for constant regions of endogenous TCR a /b genes (hTERT-siTCR vector). hTERT-siTCR vector was transfected into normal CD8+ T cells in RetroNectin (Takara Bio Inc.) coated-plates and these redirected CD8+ T cells (hTERT-siTCR CD8) were used as effector cells. HLA-A*24:02+ or A*24:02− ATL cell lines, HTLV-1 infected T-cell lines and freshly isolated ATL cells from patients were examined as target cells. All patients gave written informed consents in accordance with the declaration of Helsinki. Normal HLA-A*24:02+CD4+ T cells andHLA-A*24:02+cord blood CD34+ mononuclear cells (CB-CD34+) were similarly obtained from healthy volunteers. Expression of hTERT mRNA and hTERT protein in ATL cell lines, freshly isolated ATL cells, and HTLV-1 infected T cells were evaluated by quantitative real time PCR (QRT-PCR) using DCt method and Western blotting. Antileukemia reactivity mediated by hTERT-siTCR CD8 against ATL cells were examined by standard 51chromium release assay and flow-based CD107a assay. For the safety assessment, effector cells-mediated cytotoxicity against CB-CD34+ cells as normal hematopoietic progenitors was similarly examined. Peripheral blood mononuclear cells (PBMCs) from ATL patients were obtained on admission and in stable disease or remission after receiving treatments, and hTERT461–469 epitope responsive CD8+ T cells were detected using hTERT461–469peptide/ HLA-A*2402 tetramer or ELISPOT assay. ResultsQRT-PCR revealed that both freshly isolated ATL cells and ATL cell lines markedly overexpressed hTERT mRNA, while that in normal CD4+ cells and CB-CD34+ cells was less than undetectable. hTERT-siTCR CD8 cells were more than 40% positive for hTERT/HLA-A*2402 tetramer and successfully displayed HLA-A*2402-restricted and hTERT461–469-specific cytocidal effect against target-loaded C1R-A24 cells. Additionally, hTERT-siTCR CD8 cells successfully discriminated A*2402+ ATL cell lines (ATN-1, TL-Su) from A*2402− ones (TLO-m1, HUT-102); those all cell lines similarly overexpressed hTERT. Furthermore, those effector cells successfully killed freshly isolated HLA-A*2402+ ATL cells, but not A*2402− ones. Finally, hTERT461–469-specific cytotoxic T-lymphocyte precursors were detected at variable levels in PBMCs obtained from HLA-A*2402+ ATL patients, which shows hTERT overexpressed by ATL cells may be naturally processed in vivo. ConclusionAlthough much further invetigations are warranted, in our preliminary study, we have shown for the first time that hTERT could be a potential therapeutic target of redirected T cell-based immunotherapy for the treatment of ATL. We are now in detail examining efficacy and safety of those redirected T cells using xenograft mouse model. Disclosures:No relevant conflicts of interest to declare.

Regression of Adult T-Cell Leukemia with Anti-CD25 Recombinant Immunotoxin LMB-2 Preceded by Chemotherapy

Abstract 2575 Background:Adult T-cell leukemia (ATL) is a highly aggressive CD25+ HTLV-1-associated malignancy with median survivals of 4 and 9 months for leukemic and lymphomatous forms, respectively. There is no standard therapy capable of altering median survival by more than a few months. The anti-CD25 recombinant immunotoxin LMB-2 as a single agent was previously reported in phase I testing to have activity, but limited by rapid tumor growth between cycles and immunogenicity. Human CD25+ xenografts in mice contained soluble CD25 (sCD25) levels >100-fold higher than in serum, which were rapidly depleted by chemotherapy. Treatment with fludarabine was previously reported to be associated with lower immunogenicity to murine antibodies (HAMA), and the fludarabine-cyclophosphamide (FC) combination was reported associated with reductions in normal T- and B-cells. Methods:To prevent the formation of neutralizing antibodies and enhance anti-tumor activity, leukemic or lymphomatous ATL patients received FC chemotherapy with fludarabine 25 mg/m2 and cyclophosphamide 250 mg/m2 on days 1, 2, and 3, and 2 weeks later began cycles every 3 weeks in which FC was administered on days 1, 2 and 3, followed by LMB-2 30–40 ug/Kg on days 3, 5 and 7. Formation of neutralizing antibodies and clinical response were determined with each cycle. Results:Interim results with 8 evaluable patients receiving 28 cycles (2–5 each) include an ORR of 50% with 2 CRs and 2 PRs. In 6 patients with circulating ATL cells detectable in the blood, reductions of ATL cells varied from 90.8 to 100% compared to baseline. CRs were associated with complete resolution of chest wall and pelvic masses, circulating ATL cells, and bone marrow involvement as assessed by immunohistochemistry and flow cytometry. One of 2 PRs is ongoing for nearly 1 year after complete resolution of skin involvement and partial decrease in circulating ATL cells. One of 2 CRs recurred after 6 months only in a sanctuary site (testis). The toxicity of LMB-2 was not increased by FC and 40 ug/Kg of LMB-2 days 3, 5 and 7 was not associated with dose-limiting toxicity (DLT). While the original doses of FC used were also without DLT, escalation to 30 and 300 mg/m2/day x3 resulted in severe thrombocytopenia in 1 of 2 patients. The most common toxicities, based on percent of cycles observed, were neutropenia (69%), lymphopenia (52%), leukopenia and nausea (48%), and anemia, thrombocytopenia and fever (41%). With FC, normal T-cells were reduced 0–95% (median 70%), but normal B-cells were reduced 91–100% (median 96%). Two (25%) patients made high levels of neutralizing antibodies after 3–4 cycles of LMB-2. Conclusions:LMB-2 is effective in ATL when preceded by FC, and can achieve major responses including CR. Additional patients will be needed to determine if FC can delay immunogenicity significantly, and allow enough cycles to result in long-term remission from this disease (Supported in part by NCI, intramural research program, NIH). Disclosures:Kreitman:NIH: Patents & Royalties. Off Label Use: Use of investigational agent and FC chemotherapy for treatment of ATL. Pastan:NIH: Patents & Royalties.

Favorable Outcome of Reduced-Intensity Stem Cell Transplantation for Adult T-Cell Leukemia

Abstract 4480 Introduction:Adult T-cell leukemia (ATL) endemic in southwestern Japan is an HTLV-I-induced mature T-cell neoplasm. It has a dismal prognosis when treated with conventional chemotherapy, mainly because ATL cells overexpress multidrug-resistance genes. Allogeneic hematopoietic stem cell transplantation (allo-SCT) has been reported to improve the prognosis. However, it is also known that allo-SCT shows high treatment-related mortality (TRM) because of disease characteristics of severely deteriorated cellular immunity. Therefore, we evaluated the outcome of reduced-intensity stem cell transplantation (RIST), compared to conventional stem cell transplantation (CST) for ATL patients (pts). Patients and methods:We retrospectively analyzed consecutive 20 pts with aggressive ATL (acute type, 16; lymphoma type, 4) who underwent allo-SCT between 2000 and 2010 in our institute. The age of pts who received RIST was significantly older than that of CST (P = <.001). The other parameters were balanced between the two groups (Table 1). [Display omitted] All pts received induction chemotherapy with Japan Clinical Oncology Group (VCAP-AMP-VECP) regimen or biweekly CHOP prior to SCT. Nine pts received a conventional myeloablative conditioning with CY/TBI-based (n=8) or BU/CY-based (n=1) regimens and 11 pts received a reduced-intensity regimen with fludarabine(Flu)/melphalan(Mel)±TBI (Flu 25 mg/m2 on days -6 to -2 + Mel 40 mg/m2 on days -4 and -3 ± TBI 4 Gy on day -1). Graft-versus-host-disease (GVHD) prophylaxis consisted of cyclosporine plus short-term methotrexate (sMTX) for HLA-matched sibling donor recipients and tacrolimus plus sMTX for the others. Cyclosporine or tacrolimus was promptly tapered when complete chimerism was achieved. Results and discussion:The estimated 3-year overall survival (OS) and 3-year progression-free survival (PFS) rates for all pts were 59% and 55%, respectively (Fig. 1). At the median follow-up duration of 25 months (1–67), 12 pts were alive and 8 pts dead. Causes of death were disease progression (n=4) and treatment-related complications including acute GVHD and infection (n=4). The cumulative incidences of disease-associated mortality and TRM at 3 years were 21% and 20%, respectively. Importantly, after 1 year post-transplantation, the curve of PFS reached plateau phase. Therefore, allo-SCT could be a curable therapeutic option for aggressive ATL. [Display omitted] It is notable that RIST showed excellent 3-year OS. Although not significant, 3-year OS for RIST and CST were 70% and 44% (P =.25), respectively (Fig. 2). TRM of RIST trended to be low compared with that of CST (18% vs 22%, P =.87). In addition, chronic GVHD was observed in 91% of the evaluable 11 pts (limited type, 6; extensive type, 5). It is suggested that both improvement of TRM and the induction of graft-versus-leukemia effects may contribute to the favorable outcome in RIST for ATL. Furthermore, it should be stressed that older pts who preferentially received RIST rather than CST achieved favorable survival comparable to younger pts (≥ 50 years: 58% vs < 50 years: 60%, P =.94). We would suggest that RIST should be applied to all ATL pts eligible for allo-SCT, regardless of the age. [Display omitted] Univariate analysis for all pts revealed that only male sexuality was a significant poor prognostic factor for OS (male: 25% vs female: 82%, P =.006). Regarding disease status at SCT, CR or non-CR did not significantly affect OS (CR: 67% vs non-CR: 58%, P =.98). However, PD was an adverse impact on 3-year OS (PD: 33% vs non-PD: 64%, P =.11). It is recommended that ATL pts receive allo-SCT in early phase before the disease becomes chemo-resistant, even if they do not achieve CR. Conclusion:Allo-SCT for ATL remarkably improved the poor prognosis. Especially, RIST is a promising procedure for excellent outcome. Disclosures:No relevant conflicts of interest to declare.

Molecular cloning and characterization of a novel human papillomavirus, HPV 126, isolated from a flat wart-like lesion with intracytoplasmic inclusion bodies and a peculiar distribution of Ki-67 and p53

Infection with certain human papillomavirus types induces warts with specific macroscopic and microscopic features. We observed multiple flat wart-like lesions on the chest, neck and extremities of an adult T-cell leukemia patient. Histologically, atypical intracytoplasmic inclusion bodies currently known to be pathognomonic for genus gamma or mu papillomaviruses were disclosed in some cells of the epidermis showing histological features compatible with flat warts. In the present study, a novel human papillomavirus was identified and its whole genome, 7326bp in length, was cloned and characterized. Phylogenetic analysis showed the virus designated as HPV126 to be a novel type of genus gamma papillomavirus. Strikingly, Ki-67 and p53 expression was found to be increased in all layers of the epidermis except for horny layer, contrasting to expression restricted to the basal and lower spinous layers in ordinary flat warts.

Guest editorial: a new era of ATL and HTLV-1 research

In the mid 1970s, Dr. Uchiyama, Dr. Yodoi and Dr. Takatsuki noticed that the frequency of T-cell leukemia in Japan was higher than that in Western countries. A novel finding was that patients with T-cell leukemia had similar clinical features: frequent skin lesions, hypercalcemia, the presence of characteristic leukemic cells in the peripheral blood, and opportunistic infections. Based on these clinical observations, they proposed this disease as a distinct clinical entity, adult T-cell leukemia (ATL). Surprisingly, the birthplaces of ATL patients were concentrated in Kyushu, suggesting that this disease was caused by a specific pathogen. In 1980, Dr. Gallo’s group at NIH found the first human retrovirus, now called human T-cell leukemia virus, from a patient with Mycosis fungoides. In 1981, Dr. Hinuma’s group reported that ATL patients had antibodies to an ATL cell line, indicating that ATL was caused by a pathogen. Thereafter, Dr. Yoshida revealed the sequence of the virus in ATL cells, finally proving that HTLV is a causative agent of ATL. HTLV is the first human retrovirus discovered to cause a human disease. Later, it was reported that HTLV-1 induced inflammatory diseases in addition to ATL. It goes without saying that the discovery of HTLV-1 has had tremendous impacts on medicine, virology and human society. Thereafter, Dr. Uchiyama moved to NIH as a postdoctoral fellow in Dr. Waldmann’s laboratory, and started to generate monoclonal antibodies to T cells. Quite by accident, he used cultured T cells from the same patient from whom Dr. Gallo’s group established a cell line and identified HTLV. He succeeded in generating a monoclonal antibody to activated T cells, anti-Tac. Later, this antibody was found to react to the IL-2 receptor alpha chain, which is abundantly expressed on HTLV-1 infected cells. This was a great serendipity. Thus, research on HTLV-1 and ATL has made a great contribution to not only virology, but also immunology and hematology. However, many problems remain. First, the frequency of HTLV-1 infection does not appear to be decreasing as rapidly as expected. Dr. Watanabe reports on the recent status of HTLV-1 infection in Japan and the world. It was speculated that the number of HTLV-1 infected individuals would decrease rapidly. However, recent studies show that the number of HTLV-1 carriers remains unchanged in Japan, and rather has increased in the Tokyo and Kansai areas. These findings require us to pay attention to HTLV-1 infection in the non-endemic areas of Japan. Second, the pathogenesis of ATL is still not fully understood. Previous studies suggested the significance of Tax in the leukemogenesis of ATL. However, Tax expression is frequently disrupted in ATL cases. The HTLV-1 bZIP factor (HBZ) gene is expressed in all ATL cases, and promotes the proliferation of ATL cells. HBZ expression converts cells to regulatory T cells. Dr. Yasunaga and I summarize recent progress on the understanding of HTLV-1, especially HBZ. Finally and most importantly, new therapies are needed for ATL, a fatal disease. Stem cell transplantation can cure some ATL patients. However, many ATL patients cannot receive this treatment since the disease is not controllable or the donors are not found. Dr. Ishida and Dr. Ueda reported the development of an anti-CCR4 monoclonal antibody as a novel therapeutic agent for ATL. This new agent will be a lamp of hope for ATL patients. Thirty-five years after the discovery of ATL, a new era of research on HTLV-1 and ATL has finally arrived. M. Matsuoka (&) Institute for Virus Research, Kyoto University, 53 Shogoin Kawara-cho, Kyoto 606-8507, Japan e-mail: mmatsuok@virus.kyoto-u.ac.jp

The evaluation of FOXP3 and GITR gene expression in ATL patients before and after treatment with triple arsenic regimen using real time PCR TaqMan method

HTLV-I is the etiologic agent of adult T-cell leukemia (ATL), a fatal T-cell malignancy that is associated with profound immunosuppression. In this study, comprehensive gene expression profiling was performed using massively parallel signature sequencing (MPSS) to investigate virus–host interactions in acutely HTLV-1 transformed cells. The analysis revealed the modulation of numerous genes across different functional classes, many of which have not been previously implicated in HTLV-1 transformation or ATL. Differences in the transcriptomes of transformed cell lines were observed that have provided clues on how different clonal populations of cells respond to virus transformation. Quantitation of HTLV-1 transcription was possible, thus making MPSS a useful tool to study emerging pathogens and unknown microbial causes of human diseases. Importantly, overexpression of GITR, an activation marker that has not been previously reported to be upregulated by HTLV-1-infection or in transformed/leukemic cells and that is associated with the suppressor phenotype of CD4+CD25+ regulatory T-cells (Tregs), was also observed. The deep and quantitative gene expression profile generated by MPSS should provide additional leads for discovery research that can be applied to better understand the pathobiology of HTLV-1 transformation and ATL as well as to developing new therapies.

HBZ impedes the Menin function and up-regulates the transcription of the hTERT gene in leukemic cells

Leukemic cells from Adult T-cell leukemia (ATL) patients display elevated telomerase activity, resulting mainly from transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). We have previously shown that HBZ (HTLV-1 bZip) protein cooperates with JunD transcription factor to enhance hTERT expression after JunD anchoring to Sp1 bound to Sp1 sites within the hTERT proximal promoter. In normal somatic cells, telomerase expression is negatively regulated by tumor suppressor gene products, such as Menin, encoded by the multiple endocrine neoplasia type 1 (MEN-1) gene. Interestingly, the interaction of Menin with JunD has been shown to repress its transcriptional activity. We report here that, in HBZ-expressing cells, Menin and HBZ exert opposite effects on JunD-mediated regulation of hTERT transcription. Chromatin immuno-precipitation as well as functional assays demonstrate that this antagonism is linked to the recruitment of p300 by HBZ and HDACs by Menin. Furthermore, knock-down of Menin in the ATL Tl-Om1 (only expressing HBZ) cells results in an increase of hTERT expression and of telomerase activity, whereas a knock-down of HBZ exerts opposite effects. Interestingly, primary leukemic cells isolated from ATL patients that express high amounts of hTERT transcripts, are also characterized by an elevated expression of HBZ and MEN-1 genes. Thus, in leukemic cells, HBZ behaves as a key factor impeding the Menin function and sustaining hTERT transcription. These findings underline the critical role of HBZ as a tumor-promoting protein during the development of the HTLV-1-induced leukemogenic process.

Involvement of osteopontin and its signaling molecule CD44 in clinicopathological features of adult T cell leukemia

We previously reported that the osteopontin (OPN) gene as well as CD44 is trans-activated by the Tax protein of HTLV-1, however the synthesis of both in adult T cell leukemia (ATL) has not been described yet. Here we showed the expression of these molecules in plasma and tissue of ATL. Significant differences were found among the normal and four subtypes of 27 ATL patients in plasma levels of OPN (p=3.6×10−6) and soluble CD44 (p<0.001) and they were significantly related to each other (p<0.002). Also they were significantly associated with the performance status, total number of involved lesions, and lactic dehydrogenase, and inversely with lymphocyte count (p<0.01). Immunohistochemical staining of lymph-nodes and skin from 7 ATL patients using anti-OPN and anti-CD44 antibodies demonstrated that both expressions were weak/moderate in ATL cells but moderate/strong in infiltrated macrophages in 6 patients. These results demonstrate that OPN and CD44 play important roles in tumor formations and their products in plasma could be markers of the severity in ATL.

Proteomic profiling of HTLV-1 infected T-cells for the identification of potential biomarkers and therapeutic targets for HTLV-1 associated myelopathy/ tropical spastic paraparesis and adult T-cell leukemia

Human T-lymphotropic virus type-1 (HTLV-1) can cause the development of HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia (ATL). These nervous system inflammation and cancer occur only in a very small proportion of infected individuals, indicating that several host and viral factors might associate with the risk of HAM/TSP or ATL. The aim of this study is to identify a subset of proteins which would be applicable as the therapeutic targets or biomarkers for HAM/TSP or ATL. For this purpose, we employed comprehensive quantitative proteomics technologies. Peripheral blood mononuclear cells (PBMC) were collected from 6 uninfected volunteers, 4 asymptomatic carriers (AC), 10 HAM/TSP patients, and 9 ATL patients, followed by the specific selection of CD4+CD25+CCR4+ T cells, since this cell population was known as the predominant viral reservoir. The sorted T cells from 29 individuals were lysed, digested, and separately subjected to LC/MS/MS analyses. We integrated the 29 LC/MS/MS data into the Expressionist proteome database server platform and conducted label-free quantification analysis. The raw mass spectrometric data sets were processed and quantified on the RefinerMS module and statistical analysis was performed on the Analyst module. Eventually the results of ANOVA and the leave-one-out cross validation test revealed that the 100 peptide-panel allowed the clear classification of four pathological groups. In conclusion, our proteome profiling of CD4+CD25+CCR4+ T cells enabled us to identify the potential therapeutic or biomarker targets for HAM/TSP and/or ATL, while further validation studies with larger number of samples should be necessary.

Pattern mRNA expression similarities between ATL patients and patients coinfected with HTLV-I-Strongyolides stercoralis

HTLV-I is associated to Adult T-cell leukemia (ATL) characterized by genetic disorders and an altered mRNA expression. Individuals who have a coinfection between HTLV-I and Strongyolides stercoralis (SS) might have an increased risk to develop ATL. The mechanism for the synergism between these two infectious agents is unclear. In this study we evaluate the mRNA expression in Peripheral Blood Mononuclear Cells (PBMC) from three different groups, to determine mRNA profiles similarities or divergences among patients with a)ATL, b)HTLV-I-SS and c)Asymptomatic carriers (AC).

Functional impairment of Tax-specific but not CMV-specific CTLs in a minor population of asymptomatic HTLV-1-carriers

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T cell response. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) also showed impaired T cell responses against the major target antigen Tax. However, it is unclear whether the impaired HTLV-1-specific T cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, we investigated the function of Tax-specific cytotoxic T lymphocytes (CTLs) using tetramers consisting of major CTL epitopes and HLA-A0201, A2402, or A1101 in various HTLV-1-infected individuals possessing these HLAs. Tax-specific CTLs were detected in 33.3% (n=7/21), 100% (n=18/18), and 86.4% (n=19/22) of chronic ATL (cATL) patients, HAM/TSP patients, and AC, respectively. Tax-specific CTLs of all HAM/TSP patients tested proliferated well in culture and produced IFN-γ when stimulated with Tax peptides, while Tax-specific CTLs detected in cATL patients did not. In ACs, the Tax-specific CTL responses were retained in most cases. However, Tax-specific CTLs in one AC hardly produced IFN-γ and failed to proliferate or express a degranulation (CD107a) marker upon Tax peptide stimulation. In contrast, cytomegalovirus (CMV) pp65-specific CTLs in the same donor could be fully activated by pp65 peptide stimulation. These findings indicated that HTLV-1-specific T cell function is impaired in a limited population in an HTLV-1-specific manner during an asymptomatic stage.

JunD/HBZ enhances HBZ enhances HTLV-1 antisense transcription

Infection with the human T-cell leukemia virus type 1 (HTLV-1) results in a variety of diseases including adult T-cell leukemia (ATL), a fatal malignancy characterized by the uncontrolled proliferation of virally infected CD4+ T cells. The HTLV-1 basic leucine zipper factor (HBZ) is believed to contribute to development and maintenance of ATL. Unlike the other HTLV-1 genes, the hbz gene is encoded on the complementary strand of the provirus and therefore is not under direct control of the promoter within the 5' long terminal repeat (LTR) of the provirus. This promoter can undergo inactivating genetic or epigenetic changes during the course of ATL that eliminates expression of all viral genes except that of hbz. In contrast, repressive modifications are not known to occur on the hbz promoter located in the 3' LTR, and hbz expression has been consistently detected in all ATL patient samples. Although Sp1 regulates basal transcription from the HBZ promoter, other factors that activate transcription remain undefined. In this study, we used a proviral reporter construct deleted of the 5' LTR to show that HBZ upregulates its own expression through cooperation with JunD. Activation of antisense transcription was apparent in serum-deprived cells in which the level of JunD was elevated, and elimination of JunD expression by gene knockout or shRNA-mediated knockdown abrogated this effect. Activation through HBZ and JunD additionally required Sp1 binding at the hbz promoter. These data favor a model in which JunD is recruited to the promoter through Sp1, where it heterodimerizes with HBZ thereby enhancing its activity. Separately, hbz gene expression led to an increase in JunD abundance, and this effect correlated with emergence of features of transformed cells in immortalized fibroblasts. Overall, our results suggest that JunD represents a novel therapeutic target for the treatment of ATL.

Dual effects of HTLV-1 bZIP factor in suppression of interferon regulatory factor 1

Human T-cell leukemia virus type-1 (HTLV-1) causes ATL in 2.5% of carriers after a long period of latent infection. Moreover, half of adult T-cell leukemia (ATL) patients succumb to this disease within 1 year of onset. HTLV-1 bZIP factor (HBZ) is constitutively expressed in all the ATL cells. Thus, suggesting that HBZ may play a key role in cellular leukemogenesis. Herein we present evidence that interferon regulatory factor IRF-1, which is a member of IRF transcription family, interacts with HBZ. The N-terminal of HBZ interacted with IRF-1. HBZ reduced both IRF-1 DNA-binding activity and stability via a proteasome-dependent pathway. In addition, IRF-1-mediated apoptosis is significantly reduced by ectopic production of the HBZ. These results suggested that HBZ has dual suppressive effects on IRF-1 function, which may contribute to HTLV-1 related pathogenesis.

Double control systems for human T‐cell leukemia virus type 1 by innate and acquired immunity

Human T-cell leukemia virus type 1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1-specific T-cell responses elicit antitumor and antiviral effects in experimental models, and are considered to be one of the most important determinants of the disease manifestation, since they are activated in HAM/TSP but not in ATL patients. The combination of low T-cell responses and elevated HTLV-1 proviral loads are features of ATL, and are also observed in a subpopulation of HTLV-1 carriers at the asymptomatic stage, suggesting that these features may be underlying risk factors. These risks may potentially be reduced by vaccination to activate HTLV-1-specific T-cell responses. HAM/TSP and ATL patients also differ in their levels of HTLV-1 mRNA expression, which are generally low in vivo but slightly higher in HAM/TSP patients. Our recent study indicated that viral expression in HTLV-1-infected T-cells is suppressed by stromal cells in culture through type-I IFNs. The suppression was reversible after isolation from the stromal cells, mimicking a long-standing puzzling phenomenon in HTLV-1 infection where the viral expression is very low in vivo and rapidly induced in vitro. Collectively, HTLV-1 is controlled by both acquired and innate immunity in vivo: HTLV-1-specific T-cells survey infected cells, and IFNs suppress viral expression. Both effects would contribute to a reduction in viral pathogenesis, although they may potentially influence or conflict with one another. The presence of double control systems for HTLV-1 infection provides a new concept for understanding the pathogenesis of HTLV-1-mediated malignant and inflammatory diseases.

High incidence of cytomegalovirus, human herpesvirus-6, and Epstein-Barr virus reactivation in patients receiving cytotoxic chemotherapy for Adult T cell leukemia

The etiology of cytomegalovirus (CMV), human herpesvirus-6 (HHV-6), and Epstein-Barr virus (EBV) reactivation and the potential for complications following cytotoxic chemotherapy in the absence of allogeneic transplantation are not clearly understood. Patients with adult T cell leukemia (ATL) are susceptible to opportunistic infections. In this study, the incidence, kinetics and clinical significance of reactivation of CMV, HHV-6, and EBV in ATL patients were investigated. Viral DNA in a total of 468 plasma samples from 34 patients was quantified using real-time PCR. The probability of CMV, HHV-6, and EBV reactivation by 100 days after the start of chemotherapy was 50.6%, 52.3%, and 21.6%, respectively. Although most CMV reactivations were self-limited, plasma CMV DNA tended to persist or increase if the CMV DNA levels in plasma reached ≥ 10(4) copies/ml. CMV reactivation was negatively associated with survival, but the P-value for this association was near the borderline of statistical significance (P=0.052). One patient developed fatal interstitial pneumonia concomitant with peak CMV DNA accumulation (1.6 × 10(6) copies/ml plasma). Most HHV-6 and EBV reactivations were self-limited, and no disease resulting from HHV-6 or EBV was confirmed. HHV-6 and EBV reactivation were not associated with reduced survival (P=0.35 and 0.11, respectively). These findings demonstrated that subclinical reactivation of CMV, HHV-6, and EBV were common in ATL patients receiving chemotherapy. There were differences in the viral reactivation patterns among the three viruses. A CMV load ≥ 10(4) copies/ml plasma was indicative of subsequent exacerbation of CMV reactivation and developing serious clinical course.