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Background The reliable diagnosis of human T-cell leukemia virus type 1 (HTLV-1) infection is important, particularly as it can be vertically transmitted by breast feeding mothers to their infants. However, current diagnosis in Japan requires a confirmatory western blot (WB) test after screening/primary testing for HTLV-1 antibodies, but this test often gives indeterminate results. Thus, this collaborative study evaluated the reliability of diagnostic assays for HTLV-1 infection, including a WB-based one, along with line immunoassay (LIA) as an alternative to WB for confirmatory testing. Results Using peripheral blood samples from blood donors and pregnant women previously serologically screened and subjected to WB analysis, we analyzed the performances of 10 HTLV-1 antibody assay kits commercially available in Japan. No marked differences in the performances of eight of the screening kits were apparent. However, LIA determined most of the WB-indeterminate samples to be conclusively positive or negative (an 88.0% detection rate). When we also compared the sensitivity to HTLV-1 envelope gp21 with that of other antigens by LIA, the sensitivity to gp21 was the strongest. When we also compared the sensitivity to envelope gp46 by LIA with that of WB, LIA showed stronger sensitivity to gp46 than WB did. These findings indicate that LIA is an alternative confirmatory test to WB analysis without gp21. Therefore, we established a novel diagnostic test algorithm for HTLV-1 infection in Japan, including both the performance of a confirmatory test where LIA replaced WB on primary test-reactive samples and an additional decision based on a standardized nucleic acid detection step (polymerase chain reaction, PCR) on the confirmatory test-indeterminate samples. The final assessment of the clinical usefulness of this algorithm involved performing WB analysis, LIA, and/or PCR in parallel for confirmatory testing of known reactive samples serologically screened at clinical laboratories. Consequently, LIA followed by PCR (LIA/PCR), but neither WB/PCR nor PCR/LIA, was found to be the most reliable diagnostic algorithm. Conclusions Because the above results show that our novel algorithm is clinically useful, we propose that it is recommended for solving the aforementioned WB-associated reliability issues and for providing a more rapid and precise diagnosis of HTLV-1 infection.

Adult T-cell leukemia-lymphoma (ATL) is divided into two types for decision of therapeutic strategy; aggressive variant includes acute, lymphoma and chronic type with poor prognostic factor, and indolent variant includes smoldering and chronic type without any factors. Prognosis of aggressive ATL by chemotherapy alone is very poor. We have reported the possibility of improvement of prognosis in aggressive ATL by allogeneic hematopoietic stem cell transplantation (allo-HSCT) for the first time in 2001, however transplant-related mortality (TRM) was very high. In order to reduce TRM and improve outcomes of aggressive ATL, we conducted prospective clinical trials of allo-HSCT using the reduced intensity conditioning (RIC). The first clinical trial (NST-1) was performed for evaluating allo-HSCT with RIC using HLA matched sibling donor in the patients of aggressive ATL aged over 50, and subsequently NST-2 trial was applied for the same eligibility. Conditioning regimen in NST-1 included busulfan, fludarabine and antithymocyte globulin (ATG), but omitted ATG in NST-2 because of early relapse. In all 29 patients registered in NST-1 and -2, 3-year and 5-year overall survival rates (OS) were 36% and 34% respectively. Eight patients were died of TRM. Ten of the 29 patients obtained long-term survival with good performance status. Subsequently, NST-3 trial was conducted with similar eligibility with NST-2 to confirm the efficacy of the therapeutic strategy of RIC. Twenty patients were transplanted and 2-year OS was 53%. The availability of sibling donors is becoming difficult mainly due to the aging of not only patients but also donors. Therefore, NST-4 trial was performed with similar eligibility using unrelated bone marrow (UBM) as alternative stem cell source. Fifteen patients were transplanted, we have confirmed the feasibility of UBM and 2-year OS was 67%. For the further improvement of allo-HSCT for aggressive ATL, the study using cord blood (NST-5) is on the way.

An adult male patient was admitted to our hospital with systemic lymphadenopathy. He was diagnosed as having ALK-negative anaplastic large cell lymphoma (ALCL) by lymph node biopsy. Immunohistochemical staining revealed positive reaction for CD4, CD30, CD25, TIA-1, and Granzyme B, but negative for ALK and EBER. Although anti-HTLV-1 antibody in his serum was positive, monoclonal integration of HTLV-1 provirus DNA was not detected in his lymph node by Southern blot analysis. He was treated by combination chemotherapy, and complete remission was obtained by 8 courses of CHOP therapy. Subsequently he received autologous stem cell transplantation (auto-SCT). Fifteen months after auto-SCT, abnormal lymphocytes in the peripheral blood gradually increased. Southern blot analysis of abnormal lymphocytes revealed monoclonal integration of HTLV-1 provirus DNA and monoclonal rearrangement of T-cell receptor b. The patient was therefore diagnosed as chronic type of adult T-cell leukemia-lymphoma (ATL), and immediately progressed to acute type with central nervous system involvement. He died of tumor progression regardless of intensive chemotherapy. We analyzed genomic alterations of the ALCL cells and the chronic type ATL cells using high-resolution array comparative hybridization. These results revealed that the genomic alteration pattern of ALCL was different from those of the chronic type. Chronic type ATL cells had the genetic alterations such as 1q gain and CD58 loss that were frequently observed in acute type ATL than the chronic type, suggesting that these genetic events might contribute to the transformation of this case. TCR rearrangement analyses using PCR were performed for the tumor cells of ALCL and ATL to evaluate the origin of these tumor cells. Results showed that tumor cell clone of the ATL might have already proliferated in ALCL lymph node. This finding suggests that lymph nodes can serve as a niche for ATL development.

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell malignancy that is caused by human T-lymphotropic virus type 1 (HTLV-1) with poor prognosis. We have reported the feasibility and efficacy of allogeneic hematopoietic cell transplantation with reduced intensity conditioning (RIC-HCT) for elderly patients with ATL, and have also shown that in more than half of patients the HTLV-1 proviral load (HTLV-PL) decreased to an undetectable level after RIC-SCT, suggested presence of anti HTLV-1 effects. In this study, we try to compare the kinetics of HTLV-PL after RIC-SCT from HTLV-1 antibody negative sibling donor (SD) with that from unrelated donors (UD), all of whom were HTLV-1 antibody negative. Fourteen patients were included in the SD group, and fifteen patients were in UD group. The HTLV-PL decreased to an undetectable revel in 11 of 14 patients (78.6%) in the SD group and 13 of 15 (86.7%) in the UD group with in 120 days after RIC-SCT. In short term, the kinetics of the HTLV-PL after RIC-SCT from UD was almost similar to that from HTLV-1 negative SD. Looking at the long term follow-up results, the HTLV-PL remains to be an undetectable level in only 2 of 11 (18.2%) patients in the SD group (median follow up 111 months [101-122])), on the other hand, 7 of 13 (53.8%) patient in the UD group (24 month [20-36]). RIC-HCT from UD tends to have better anti-HTLV-1 effect, compared with that from SD, but not conclusive because of the short follow-up period.

Background HTLV-1 utilizes CD4 T cells as the main host cell and maintains the proviral load via clonal proliferation of infected CD4+ T cells. Infection of CD4+ T cells by HTLV-1 is therefore thought to play a pivotal role in HTLV-1-related pathogenicity, including leukemia/lymphoma of CD4+ T cells and chronic inflammatory diseases. Recently, it has been reported that a proportion of HTLV-1 infected CD4+ T cells express FoxP3, a master molecule of regulatory T cells. However, crucial questions remain unanswered on the relationship between HTLV-1 infection and FoxP3 expression. Results To investigate the effect of HTLV-1 infection on CD4+ T-cell subsets, we used flow cytometry to analyze the T-cell phenotype and HTLV-1 infection in peripheral mononuclear cells (PBMCs) of four groups of subjects, including 23 HTLV-1-infected asymptomatic carriers (AC), 10 patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), 10 patients with adult T-cell leukemia (ATL), and 10 healthy donors. The frequency of FoxP3+ cells in CD4+ T cells in AC with high proviral load and patients with HAM/TSP or ATL was higher than that in uninfected individuals. The proviral load was positively correlated with the percentage of CD4+ T cells that were FoxP3+. The CD4+FoxP3+ T cells, themselves, were frequently infected with HTLV-1. We conclude that FoxP3+ T- cells are disproportionately infected with HTLV-1 during chronic infection. We next focused on PBMCs of HAM/TSP patients. The expression levels of the Treg associated molecules CTLA-4 and GITR were decreased in CD4+FoxP3+ T cells. Further we characterized FoxP3+CD4+ T-cell subsets by staining CD45RA and FoxP3, which revealed an increase in CD45RA−FoxP3low non-suppressive T-cells. These findings can reconcile the inflammatory phenotype of HAM/TSP with the observed increase in frequency of FoxP3+ cells. Finally, we analyzed ATL cells and observed not only a high frequency of FoxP3 expression but also wide variation in FoxP3 expression level among individual cases. Conclusions HTLV-1 infection induces an abnormal frequency and phenotype of FoxP3+CD4+ T cells.

Background Human 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. Results By 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. Conclusions These 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.

The clinical course and disease activity of patients with HTLV-1 associated myelopathy / tropical spastic paraparesis (HAM/TSP) are different among patients. Therefore, the treatment plan should be designed based on these backgrounds of patients. However, there is little information about the natural history of HAM/TSP and biomarkers of disease activity that is associated with prognosis. As the candidate for biomarkers to evaluate the disease activity in HAM/TSP, HTLV-1 proviral load in PBMC, several cytokines and chemokines in serum or cerebrospinal fluid (CSF) are known to be increased in HAM/TSP patients. However, little is known which parameter of these candidates is most associated with disease severity. Therefore, we investigated the clinical course of 30 HAM/TSP patients without any history of treatment. Furthermore, we measured quantitatively the concentration of a series of cytokines and chemokines in serum and CSF, and HTLV-1 proviral DNA load in PBMC. Then, the level of these markers was evaluated for the correlation with disease severity. In HAM/TSP patients, the level of CXCL10/IP-10 and neopterin in CSF was strongly correlated with disease severity. Interestingly, the level of soluble IL-2 receptor and CXCL10/IP-10 in serum was also correlated with disease severity with statistical significance. Furthermore, based on the clinical course and laboratory findings, HAM/TSP was classified into 4 different clinical subtypes as follows; (1) Rapidly progressive (active), (2-A) Chronic progressive (active), (2-B) Chronic progressive (inactive), (3) Chronic mild (inactive). This classification might be useful to determine the therapeutic strategy for patients with HAM/TSP.

Although crucial roles of microRNA have begun to emerge, detailed studies with ATL patients have not been achieved. Using 40 primary ATL samples and 22 samples of normal CD4+ T-cells, we determined the microRNA signatures of ATL and revealed loss of miR-31, which has recently been reported as a metastasis-associated miRNA. All ATL cases invariably showed undetectable or very low levels of miR-31, clearly implying that miR-31 loss is involved in ATL development. As a novel miR-31 target gene, we identified NF-κB inducing kinase (NIK) that plays central roles in noncanonical signaling and constitutive activation of NF-κB in various cancers, including ATL. Restoration of miR-31 downregulated the levels of NIK and NF-κB activity, resulting in reduction of malignant phenotypes, containing proliferative index, anti-apoptosis, and chemotaxis in ATL cells. Furthermore, lentivirus-introduced miR-31 could induce strong apoptosis in primary tumor cells freshly isolated from ATL patients, indicating pivotal functions of miR-31 as a tumor suppressor. Global copy number profiling demonstrated that 21 cases out of 168 (12.5%) have genomic loss of 9p21 containing miR-31 region. Furthermore, expression profiling and ChIP assay showed requirement of overexpression of histone methyltransferase in epigenetic suppression of miR-31 and aberrant NF-κB activation in primary ATL cells. Knockdown of methyltransferase complex restored the miR-31 expression and consequently inhibited NIK-dependent NF-κB cascade. These findings illustrated that genetic and epigenetic abnormalities link to NF-κB activation through the loss of miR-31. Considering aberrant epigenomics associated with cancers, the emerging relationship provides us a conceptual advance in understanding the broad-acting oncogenic signaling.

The level of HTLV-I expression is low in vivo. We found that recombinant interferon (IFN)-α and β suppressed viral expression in HTLV-I-infected cells. Non-lymphoid stromal cells also suppressed HTLV-I expression through type-I IFNs. The suppression was reversible after isolation of infected cells from the source of IFNs, mimicking the status of viral expression in freshly isolated ATL cells which is rapidly induced in culture. Nevertheless, HTLV-I-infected individuals maintain acquired immune responses against HTLV-I such as antibodies and cytotoxic T lymphocytes (CTLs), indicating the presence of HTLV-I proteins in vivo. Analysis on Tax-specific CTLs revealed that they were activated in HAM/TSP but unresponsive in ATL patients. We found that a subpopulation of HTLV-I carriers at asymptomatic stage exhibited impaired Tax-specific T-cell response and elevated HTLV-I proviral load. This combination is a feature of ATL and likely to be an underlying risk of ATL. Collectively, HTLV-I is doubly controlled by acquired and innate immunity; HTLV-I-specific CTLs eliminate infected cells, and IFNs suppress viral expression. Both would contribute the reduction of viral pathogenesis, while the efficiency of CTLs could be partial because of limited viral expression. An increase in viral expression would activate CTLs but also accelerate inflammation. When the viral expression is well-controlled, viral pathogenesis may not be apparent until infected cell clones with a malignant phenotype finally emerge, which may occur earlier without proper CTL responses. Diversity in innate and acquired immune responses among individuals might be important determinants of disease manifestation in HTLV-I infection.

Cell adhesion molecule 1 (CADM1/TSLC1/IgSF4) was recently identified as a novel cell surface maker for adult T-cell leukemia/lymphoma (ATLL). In this manuscript, we developed several kinds of antibodies for diagnostic tools identifying CADM1-positive ATLL cells. 107 kDa of CADM1 protein was detected in the ATLL cell lines and a 72 kDa of soluble CADM1 protein was detected in serum from ATLL patients. In analysis by flow cytometry, percentages of CADM1+/CD4+ positive (DP) cells in the peripheral blood are well correlated with the percentages of CD4+/CD25+ DP cells in the peripheral bloods from various types of ATLL patients and HTLV-1 carriers. Percentages of CADM1+/CD4+ DP cell fraction were well correlated with the percentages of abnormal lymphocytes and DNA copy numbers of HTLV-1 infected or ATLL cells in the peripheral blood, particularly, with high DNA copy numbers of HTLV-1-infected lymphocytes from HTLV-1 carriers. Expression of soluble-form CADM1 is also detected in the peripheral blood from acute-type of ATLL patients with correlation of the level of soluble IL2Ra. Moreover, lymphomas derived from ATLL in paraffin-embedded tissue sections were strongly and specifically stained by CADM1 antibody, suggesting that CADM1 is one of very useful markers for identifying various types of ATLL cells.

The novel defucosylated humanized monoclonal antibody against CC chemokine receptor 4 (CCR4), mogamulizumab, exhibited strong effects on adult T-cell leukemia-lymphoma (ATL) in a phase II clinical study. It has been also reported that mogamulizumab frequently developed cutaneous adverse reactions (CAR) and a close association between CAR and the prognosis. [1] We studied the effects of mogamulizmab on ATL and CAR in 32 patients with ATL. Informed consent was obtained prior to study. Nineteen patients received more than 4 cycles of mogamulizumab therapy. Twenty patients developed an infusion reaction, and eight patients suffered from CAR. These eight patients were administered mogamulizumab more than 4 cycles (median: 7 cycles). The severity of CAR according to CTCAE v3.0 was grade 1 in one patient, 2 in two, 3 in three and 4 in two patients respectively. In six of 8 patients, CARs appeared or most worsened in more than 4 weeks after the last administration. All CARs fortunately subsided later. One patient, however, suffered from toxic epidermal necrolysis and other CARs (erythroderma and generalized exudative erythema in two each patients) prolonged for several months. As for anti-tumor effects with more than 4 cycles of treatments, overall response rate (complete remission (CR) and partial remission (PR)) was 58% (CR: 6, progressive disease (PD): 2) with CAR, and 36% (CR:3, PR:1, stable disease (SD):4, PD:3) without CAR. These results suggest that 1) the incidence of CAR were associated with the frequency of mogamulizumab administration, 2) CARs might be favorable signs of the effect, and 3) follow-up with careful attention to CAR was recommended for at least several months after the finish of treatments.

Human T-lymphotropic virus type I (HTLV-1) can cause HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma (ATL). Since the prognosis for HAM/TSP patients is extremely poor, there is a strong demand for a novel therapeutic strategy, especially one which would effectively reduce HTLV-1 proviral load, which is well-correlated with disease prognosis. CD4+CCR4+ T cells are the main HTLV-1 reservoir, and the defucosylated humanized anti-CCR4 antibody KW0761 has been approved in Japan as a treatment for ATL. KW0761 strongly binds to Fcγ receptor IIIa (FcγRIIIa) on natural killer cells and elicits powerful antibody-dependent cellular cytotoxicity (ADCC) against the CCR4+ cells. In this study, we evaluated KW0761 as a treatment for HAM/TSP using primarily ex vivo cell cultures. In addition, given that KW0761 would also target CD8+CCR4+ T cells, we sought to determine how this would likely affect HAM/TSP patients by elucidating the role of CD8+CCR4+ T cells in HAM/TSP pathogenesis. When applied to cultures of PBMCs from HAM/TSP patients (n=11), KW0761 effectively reduced HTLV-1 proviral load (56.4% mean reduction at 0.01μg/mL), spontaneous proliferation, and production of pro-inflammatory cytokines including IFN-γ. Like CD4+CCR4+ T cells, CD8+CCR4+ T cells from HAM/TSP patients exhibited high proviral loads and spontaneous IFN-γ production, unlike their CCR4-counterparts. CD8+CCR4+ T cells from HAM/TSP patients contained more IFN-γ+ cells and less IL-4+ cells than those from healthy donors. Notably, Tax-specific cytotoxic T lymphocytes that may help control the HTLV-1 infection were overwhelmingly CCR4-. In conclusion, we determined that CD8+CCR4+ T cells as well as CD4+CCR4+ T cells are prime therapeutic targets for treating HAM/TSP, and that KW0761 shows promise as a new treatment. Based on the results of this study, we have begun conducting an investigator -led Phase I/IIa clinical trial to test the safety and efficacy of KW0761 on HAM/TSP patients (UMIN000012655).

The plasticity inherent to the CD4+ T cell differentiation program especially as it pertains to regulatory T (Treg) cells has been implicated in the pathogeneses of multiple inflammatory diseases. Human T-lymphotropic virus type 1 (HTLV-1) is thought to effect transcriptional changes in infected T cells via HTLV-1 Tax that can cause once suppressive CD4+CD25+CCR4+ Treg cells to lose FOXP3 expression and produce IFN-γ. We hypothesized that spawning of such inflammatory Th1-like cells from infected CCR4+ T cells plays a key role in the pathogenesis of the neurodegenerative inflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). In this study, we demonstrated that Tax in cooperation with specificity protein 1 (Sp1) boosts the expression of the Th1 master regulator T box transcription factor (T-bet/Tbx21) and consequently IFN-γ. We established the presence of abundant CD4+CCR4+ T cells co-expressing the Th1 marker CXCR3 and producing T-bet/Tbx21 and IFN-γ in the CSF and spinal cord lesions of HAM/TSP patients. Finally, we tested treatments on ex vivo cell cultures from patients and found evidence that a therapy targeting CCR4+ T cells via antibody-dependent cellular cytotoxicity may represent a viable treatment option for HAM/TSP.

We studied the effects of Mogamulizmab on ATL and its adverse reactions in 10 patients with ATL, among them 6 patients had skin lesions. Informed consent was obtained prior to study. Four out of 6 patients with cutaneous involvement showed complete response (CR), with 4 to 8 cycles of treatments. Of interest were two cases which appeared to have worsened in the early phase of treatment because of enlargement of cutaneous nodules or tumors. It, however, was found to be actually improving determined from histopathological examinations, i.e., more inflammatory cell infiltration and edema with less number of lymphoma cells in the skin. Eventually, these two patients showed CR. Furthermore, 2 each patients with 4 CR patients showed no recurrence with ASR, i.e., erythema and plaque, and showed reccurence without ASR. ASR were observed in 4 out of 10 patients. All these ASR fortunately subsided later. Immunohistopathological examinations revealed the infiltration of cytotoxic T-lymphocytes in the dermis. These results suggest that 1) Mogamulizmab had excellent effects (4/6) to suppress the growth of cutaneous lesions in ATL, 2) ASR might be favorable signs of the effects (2/4), and 3) ASR (4/10) were not serious. Studies of plasma and tissue levels of Mogamulizmab and T-reg cells may reveal the action mechanism of this novel anti-CCR4 agent in detail.

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a debilitating neurologic disorder characterized by chronic inflammation in the spinal cord. However, the precise mechanism by which chronic inflammatory lesions in HAM/TSP are formed and maintained has not been discovered. Since it is believed that chemokines play a central role in lymphocyte recruitment to sites of inflammation, we hypothesized that a positive feedback loop driven by chemokines may be responsible for the chronic inflammation in HAM/TSP. We aimed to determine the identity of these chemokines, where they are produced, and how they drive chronic inflammation in HAM/TSP. We found that HAM/TSP patients have extraordinarily high levels of the chemokine CXCL10 and an abundance of cells expressing the CXCL10-binding receptor CXCR3 in the cerebrospinal fluid. Histological analysis revealed that astrocytes are the main producers of CXCL10 in the spinal cords of HAM/TSP patients. Co-culture of human astrocytoma cells with CD4+ T-cells from HAM/TSP patients revealed that astrocytes produce CXCL10 in response to IFN-γ secreted by CD4+ T-cells. Chemotaxis assays results suggest that CXCL10 induces migration of peripheral blood mononuclear cells to the central nervous system (CNS) and anti-CXCL10 neutralizing antibody can disrupt this migration. In short, HTLV-1-infected cells in the CNS produce IFN-γ that induces astrocytes to secrete CXCL10 that recruits more infected cells to the area via CXCR3, constituting a Th1-centric positive feedback loop that results in chronic inflammation.

In the last decade, various kinds of clinical trials of hematopoietic stem cell transplantation (HSCT) have been performed for ATL patients, and showed therapeutic effects with long-term remission in a part of recipients. Our previous finding of activation of CD8+ Tax-specific cytotoxic T-lymphocytes (CTL) in post-HSCT ATL patients indicated the presence of Tax expression in vivo and potential contributions of CTL to GVL effects. These findings let us attempt developing an anti-ATL therapeutic vaccine consisting of autologous dendritic cells (DC) pulsed with Tax peptides corresponding to the major epitopes of Tax-specific CTL previously identified from HLA-A2, A24 or A11-possessing post-HSCT ATL patients. In preliminary experiments, the DC induced with a conventional method showed matured phenotype and produced IL-12 in 2 of 3 ATL patients tested. Under the official approval by the institutional ethical committees, we conducted the phase-I clinical study of anti-ATL immunotherapy for ATL patients at stable conditions after other therapy. The peptide-pulsed DC was subcutaneously injected for three times with 2 weeks intervals. The first patient showed a significant reduction in the proviral loads in 1 week after administration of DC, which gradually increase during the treatment, then decreased again at 8 weeks after the initiation of the therapy. Reduction in the size of surface lymph nodes by CT confirmed the therapeutic effects. So far two patients completed the course of the study without severe side effects except for fever and skin rash, and their clinical outcomes were partial remission and stable disease respectively.

Background Human T-cell leukemia virus type-1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 gene expression is maintained at low levels in vivo by unknown mechanisms. A combination therapy of interferon-α (IFN-α) and zidovudin (AZT) shows therapeutic effects in ATL patients, although its mechanism is also obscure. We previously found that viral gene expression in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients was markedly suppressed by stromal cells through a type I IFN response. Here, we investigated the effects of IFN-α with or without AZT on viral gene expression and cell growth in ILTs. Results ILTs expressed variable but lower amounts of HTLV-1 Tax protein than HTLV-1-transformed HUT102 cells. Following the addition of IFN-α, the amounts of HTLV-1 p19 in the supernatants of these cells decreased in three days, while HTLV-1 gene expression decreased only in ILTs but not HUT102 cells. IFN-α also suppressed the spontaneous HTLV-1 induction in primary ATL cells cultured for 24 h. A time course study using ILTs revealed that the levels of intracellular Tax proteins decreased in the first 24 h after addition of IFN-α, before the reduction in HTLV-1 mRNA levels. The initial decreases of Tax protein following IFN-α treatment were observed in 6 of 7 ILT lines tested, although the reduction rates varied among ILT lines. An RNA-dependent protein kinase (PKR)-inhibitor reversed IFN-mediated suppression of Tax in ILTs. IFN-α also induced cell cycle arrest at the G0/G1 phase and suppressed NF-κB activities in these cells. AZT alone did not affect HTLV-1 gene expression, cell viability or NF-κB activities. AZT combined with IFN-α markedly induced cell apoptosis associated with phosphorylation of p53 and induction of p53-responsive genes in ILTs. Conclusions IFN-α suppressed HTLV-1 gene expression at least through a PKR-mediated mechanism, and also induced cell cycle arrest in ILTs. In combination with AZT, IFN-α further induced p53 signaling and cell apoptosis in these cells. These findings suggest that HTLV-1-infected cells at an IL-2-dependent stage retain susceptibility to type I IFN-mediated regulation of viral expression, and partly explain how AZT/IFN-α produces therapeutic effects in ATL.

Background Prognosis of indolent types of adult T-cell leukemialymphoma (ATL) including the smoldering type was recently reported to be poorer than that shown in previous studies. Prognostic factors of smoldering-type ATL have not been defined. Cutaneous-type, which is defined as cases of smoldering type predominantly involve skin with or without peripheral blood involvement has been proposed as a distinct clinical subtype. Prognosis of cutaneous-type ATL was reported to be poorer than that of smoldering-type without cutaneous involvement. Aim To determine prognostic factors for survival and disease progression of smoldering-type ATL. Patients Thirty-one patients with smoldering-type ATL including 21 with cutaneous lesion. Methods Multivariate Cox proportional hazards model was used to identify variables associated with survival and disease progression. Peripheral blood abnormal lymphocytes (

Background ATL is an aggressive T-cell malignancy caused by the virus HTLV-1 with a very poor outcome. ATL, and is characterized by its cell surface expression of CC chemokine receptor 4 (CCR4), to which KW-0761, a defucosylated, humanized antibody with enhanced antibodydependent cellular cytotoxicity (ADCC), binds. In a phase I study of KW-0761 in 13 patients (pts) with CCR4-positive relapsed ATL, encouraging efficacy of KW-0761 was observed (ORR of 31%; 2CRs and 2PRs, ref. 3). Here, we report the result of a pivotal phase II study of KW-0761 in pts with CCR4-positive relapsed ATL.

HTLV-1 causes ATLL in 3-5% of infected individuals after a long latent period of 40-60 years. ATLL is divided into four stages: namely, smoldering, chronic, lymphoma and acute types. The smoldering and chronic types are indolent, but the acute and lymphoma types are aggressive ATLL characterized by resistance to chemotherapy and a poor prognosis. Such a long latent period suggests that a multi-step leukemogenic/lymphomagenic mechanism is involved in the development of ATLL, although the critical events in the progression have not been characterized. To determine whether epigenetic abnormalities are playing important roles in the progression of ATLL, we analyzed the methylation profiles, showing that number of CpG island methylated genes increased with disease progression and aberrant hyper-methylation in specific genes was detected even in HTLV-1 carriers and correlated with progression to ATLL. The CpG island methylator phenotype (CIMP) was observed most frequently in lymphoma type ATLL and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes and increase of CIMP incidence were shown to be unfavorable prognostic factors and correlated with a shorter overall survival with the Kaplan-Meyer analysis. Increase of aberrant DNA methylation density was observed during the progression of an ATLL patient. The present findings strongly suggest that the multi-step accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the initiation and progression of ATLL not only epidemiologically but also in the clinical course of a specific ATLL patient.

Adult T-cell leukemia-lymphoma (ATL) is a rare and aggressive T-cell malignancy associated with human T-cell lymphotropic virus type 1 (HTLV-1). ATL remains an extremely difficult disease to treat, and therefore, the development of novel therapies is highly needed. Because CC chemokine receptor 4 (CCR4) is frequently overexpressed on ATL cells from patients (Clin Cancer Res. 2003;9:3625), mogamulizumab, a defucosylated humanized anti-CCR4 antibody, has been developed for the treatment of ATL in Japan. In a phase I study in patients with relapsed CCR4-positive T-cell malignancies, mogamulizumab was well tolerated up to 1 mg/kg and encouraging efficacy was observed (JCO 2010;28:1591). In a subsequent pivotal phase II study in CCR4-positive relapsed ATL patients, mogamulizumab exhibited an overall response rate (ORR) of 50% including 8 complete responses (CR) (JCO 2012;30:837), leading to its approval in Japan in 2012 for relapsed/refractory ATL. Furthermore, with the aim of establishing a new standard therapy for untreated ATL, we conducted a randomized phase II study of VCAP-AMP-VECP, a dose-intensified multi-agent chemotherapy, with or without mogamulizumab (arm-A or arm-B). The CR rate (primary endpoint) and ORR (secondary endpoint) were higher in arm-A than in arm-B (52% vs. 33% and 86% vs. 75%, respectively). Median progression-free survival was also longer in arm-A (259 days vs. 192 days). The most common treatment-related AEs were hematological toxicity in both arms. These results suggest that mogamulizumab could provide a new effective treatment option for both relapsed/refractory and untreated ATL. Trial registration http://ClinicalTrials.gov NCT01173887

γδ T cells, a small subset of T lymphocytes, are involved in innate immunity. It has been demonstrated that γδ T cells have cytotoxic activities against cells infected with a variety of viruses. However, there is little evidence suggesting a cytotoxic activity of γδ T cells against HTLV-1-infected cells. Therefore, we investigated whether γδ T cells play a protective role in the defense against HTLV-1. Using PBMCs from asymptomatic carriers (ACs) and HAM/TSP patients, we assayed the frequency of CD3+Vγ9+ (γδ T) cells and the correlation between its frequency and the HTLV-1 load. The frequency of γδ T cells was significantly decreased in HAM/TSP patients compared with that in ACs. The frequency of γδ T cells was inversely correlated with the proviral load. These results suggest that γδ T cells have a protective effect on HTLV-1-infected individuals. Next, CD3+Vγ9+ cells and CD3+Vγ9- cells were separated from PBMCs of HTLV-1-infected persons by FACS and the proviral load of each population was quantified by real-time PCR. The proviral load in γδ T cells was markedly lower than that in CD3+ lacking γδ T cells. Furthermore, we cultured PBMCs from HTLV-1-infected individuals in the presence of IL-2 and zoledronate. In some cases, the majority of cells contained in these cultures became γδ T cells and the proviral load was markedly decreased. The cultured PBMCs showed strong cytotoxic activities against a HTLV-1-infected cell line as well as an ATL cell line. These results raise the possibility of γδ T cell immunotherapy in HTLV-1-infected individuals.

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.

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.

Objective We evaluated the safety and feasibility of allogeneic hematopoietic stem cell transplantation with RIC from matched sibling donors (MSD) for ATLL pts using a conditioning regimen consisting of fludarabine and busulfan. Low-dose antithymocyte globulin was added in the 1st study (NST-1), while it was omitted for the 2nd study (NST-2). We present the results of long-term follow-up of the two trials as well as the longitudinal patterns of changes in HTLV-1 proviral load in survivors.

HTLV-1 expression is maintained at low levels in vivo through unknown mechanisms. Interferon-a (IFN-a) has been used in combination with zidovudin (AZT) to treat adult T-cell leukemia/lymphoma (ATL) patients with favorable effects, although the mechanism is also unclear. We previously reported that HTLV-1 gene expression could be markedly suppressed by stromal cells through a type-I IFN response in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients. Here, we found that treatment with IFN-a alone produced suppressive effects on HTLV-1 gene expression in ILTs and also on the spontaneous HTLV-1 induction in short-term cultured primary ATL cells. Following IFN-a treatment, the levels of intracellular Tax protein decreased earlier than those of HTLV-1 mRNA in ILTs. An RNA-dependent protein kinase (PKR) inhibitor reversed IFN-a-mediated suppression of Tax in ILTs, suggesting the involvement of a post-transcriptional mechanism mediated by PKR in the suppression. IFN-a also induced cell cycle arrest at the G0/G1 phase in ILTs. We further found that AZT combined with IFN-a induced cell apoptosis in ILTs, associated with phosphorylation of p53 and enhanced expression of genes responsive to p53, whereas AZT alone did not affect cell viability or viral expression. Our results suggest that ILTs and ATL cells retain susceptibility to type-I IFNs, which suppress HTLV-1 gene expression primarily at a posttranscriptional level and potentially allow for signaling through the p53 pathway in combination with AZT. These findings would partly explain how HTLV-1 gene expression is regulated in vivo, and how AZT/IFN-a produces therapeutic effects in ATL.

Adult T cell leukemia/lymphoma (ATL) is an aggressive malignant disease of CD4 positive T lymphocytes caused by infection with human T cell leukemia virus type I (HTLV-I). HTLV-1 causes ATL in 3-5% of infected individuals after a long latent period of 40 to 60 years. The acute and lymphoma types are aggressive ATL characterized by resistance to chemotherapy and a poor prognosis. Leukemia/lymphoma cells and rapidly proliferating cells preferentially accumulate endogenous photosensitizer protoporphyrin IX (PpIX) when supplemented with 5-aminolevulinic acid (ALA). Treatment with 1mM ALA for 48h induced 10 to 100 times accumulation of PpIX in ATL leukemic cell lines and HTLV-I (+) T cell lines than that in healthy PBMCs. Specific induction of apoptosis was observed after 10 min light exposure (28 mW/cm 2 ) using Na-Li lamp in ATL leukemic cell lines and HTLV-I (+) T cell lines. ATL patient PBMC specimen showed strong accumulation of PpIX with the treatment of ALA compared to the healthy donor and HTLV-I carrier PBMCs, which could be useful for the diagnostic purposes and monitoring the patient status with high sensitivity. Photodynamic therapy is potentially hopeful treatment especially for lymphoma type ATL as a relatively selective, minimal or no scarring, non-invasive, safe, simultaneous and repeatable multiple lesions treatable modality.

The molecular hallmarks of ATL comprise outstanding deregulations of signaling pathways that control cell cycle, apoptosis resistance, and proliferation of leukemic cells. Using integrative analyses of primary ATL cells, we discovered unique molecular characteristics of ATL (Yamagishi et al., Cancer Cell, 2012). Genetic and epigenetic disruption leads to numerous gene expression alterations that dominate disorders of homeostasis and characteristics of ATL. In particular, a novel tumor suppressor miR-31 is completely lost in all ATL cases, leading to constitutive NF-kB activation via NIK overexpression. Polycomb family directly involves in the silencing of miR-31, providing a novel interconnection between epigenetic reprogramming and NF-kB pathway. In addition, we recently unveiled molecular mechanisms how Polycomb-dependent epigenetic perturbation is abnormally sustained in HTLV-1 infected and leukemic cells. We discuss the recent discovery of molecular hallmarks of potential generality, an abnormal miRNA pattern and epigenetic reprogramming, which strongly involve the imbalance of the molecular network of lymphocytes. Because epigenetic marks are potentially reversible, development of genuine epigenetic-targeted therapy drugs holds great promise in HTLV-1-related diseases. Furthermore, we also introduce additional signaling pathways affecting leukemic cell fate. Pathway analysis based on our comprehensive dataset and biological studies suggest breaking down of the essential signaling pathways such as Hedgehog and p38 pathways, which support the biological properties of ATL. Because these organized principles may be directly associated with the clinical traits of ATL, targeting the one outstanding hallmark or co-targeting of multiple molecular hallmarks in mechanism-guided combinations will result in more effective and durable therapies for aggressive ATL.

Accumulation of genetic disorders in HTLV-1 infected cells underlies ATL leukemogenesis, yet the actual genetic events responsible for cellular transformation have not been fully elucidated. Based on gene expression profiling in 52 ATL patients, 40 HTLV-1 carriers, and 21 healthy volunteers, we determined several potential risk-indicator genes of ATL, including cMyb. cMyb is the proto-oncogene of vMyb, the oncoprotein of avian myeloblastosis virus, governing hematopoietic cell differentiation. Required for differentiation of DN3, survival of DP, and generation of CD4+-SP cells, cMyb is not expressed at a detectable level in mature T-cells. Among well-known 7 isoforms, cMyb-9A and -10A, lacking the cis-acting negative regulatory domain (NRD) same as vMyb oncoprotein, are known to be molecules of “gain of oncogenic function”. We demonstrated that the mRNA levels of cmyb-9a and -10a were drastically elevated in ATL cells. Moreover, cMyb-9A protein was overexpressed in PBMC of HTLV-1 carriers and ATL patients. cMyb-9A showed the highest transactivation of HTLV-1 LTR, which is one of the cMyb targets, among 7 isoforms. The level of cMyb is known to be regulated by SUMOylation through the NRD. As expected, SUMOylation assay showed that cMyb-9A was not effectively SUMOylated, and its activity was not suppressed. Finally, cMyb-9A exhibited a significantly higher transforming activity than WT-cMyb. Upon confirming that cMyb-9A is released from the negative-regulatory circuit of cMyb, we speculate that overexpression of cMyb-9A in HTLV-1 infected cells has a strong link to disorders in cellular homeostasis by overruling its target gene expression, thus accelerating transformation process to ATL.

Adult T-cell leukemia/ lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) and characterized by extremely poor prognosis because of intrinsic drug resistance to cytotoxic agents. Recently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been shown to be an effective treatment for ATL due to graft-versus-leukemia effects. However, donor-derived HTLV-1-specific T-cell responses in ATL patients after allo-HSCT are not well understood. In this study, blood samples from 15 ATL patients at 180 days following allo-HSCT from seronegative donors were screened for IFN-g production of donor-derived T-cells against Tax protein. Among 15 patients, Tax-specific T-cell responses were observed in 11 patients. Direct detection with Tax/MHC class I tetramers revealed that donor-derived Tax-specific CD8+ cytotoxic T-lymphocytes (CTLs) were present in 12 of 15 patients. We then identified a novel Tax epitope (Tax155-167) presented by HLA-DR1 (HLA-DRB1*0101) and examined the presence of Tax-specific CD4+ T-cells in 3 HLA-DR1+ patients after allo-HSCT using a newly generated Tax155-167/HLA-DR1 tetramer. Tax155-167-specific CD4+ T-cells were found to be present in all HLA-DR1+ patients tested. Furthermore, in vitro stimulation of PBMCs from HLA-DR1+ HLA-A*2402+ patients with both Tax155-167 and HLA-A*2402-restricted CTL epitope (Tax301-309) led to robust expansion of Tax301-309-specific CTLs. Our results suggest that donor-derived HTLV-1-specific both CD4+ and CD8+ T-cells could be induced in ATL patients after allo-HSCT from seronegative donors, probably due to exposure to HTLV-1 antigens from recipient-derived ATL cells, and the HTLV-1-specific CD4+ T-cells may contribute to efficient induction of donor-derived HTLV-1-specific CTL responses.