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Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma, accounting for 30% of non-Hodgkin lymphomas. Although comprehensive analysis of genetic abnormalities has led to the classification of lymphomas, the exact mechanism of lymphomagenesis remains elusive. The Ets family transcription factor, PU.1, encoded by Spi1, is essential for the development of myeloid and lymphoid cells. Our previous research illustrated the tumor suppressor function of PU.1 in classical Hodgkin lymphoma and myeloma cells. In the current study, we found that patients with DLBCL exhibited notably reduced PU.1 expression in their lymphoma cells, particularly in the non-germinal center B-cell-like (GCB) subtype. This observation suggests that downregulation of PU.1 may be implicated in DLBCL tumor growth. To further assess PU.1's role in mature B cells in vivo, we generated conditional Spi1 knockout mice using Cγ1-Cre mice. Remarkably, 13 of the 23 knockout mice (56%) showed splenomegaly, lymphadenopathy, or masses, with some having histologically confirmed B-cell lymphomas. In contrast, no wild-type mice developed B-cell lymphoma. In addition, RNA-seq analysis of lymphoma cells from Cγ1-Cre Spi1 F/F mice showed high frequency of each monoclonal CDR3 sequence, indicating that these lymphoma cells were monoclonal tumor cells. When these B lymphoma cells were transplanted into immunodeficient recipient mice, all mice died within 3 weeks. Lentiviral-transduced Spi1 rescued 60% of the recipient mice, suggesting that PU.1 has a tumor suppressor function in vivo. Collectively, PU.1 is a tumor suppressor in mature B cells, and decreased PU.1 results in mature B-cell lymphoma development., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Serum immunofixation electrophoresis (IFE) is often performed for screening monoclonal proteins (M proteins) in immunoglobulin light-chain amyloidosis (AL amyloidosis). However, the performance of serum IFE for detecting M protein in AL amyloidosis patients is often insufficient. In this study, we examined the detection rate of serum M protein in newly diagnosed AL amyloidosis patients and analyzed differences in M protein detection between IFE methods. Among 60 patients newly diagnosed with AL amyloidosis, 22 had undetectable serum M protein by IFE with the Epalyzer2 system. Samples with undetectable M protein had significantly lower involved serum-free light-chain (iFLC) and a smaller difference between involved and uninvolved serum-free light-chain (dFLC) values than samples with IFE-detectable monoclonal light chains. When samples that tested negative for M protein by the Epalyzer2 system were retested by IFE with the HYDRASYS 2 system, 50% had IFE-detectable monoclonal light chains. The IFE system and reagents used may affect serum monoclonal immunoglobulin light-chain detection in AL amyloidosis patients, especially those with low iFLC or low dFLC samples. More attention should be paid to the performance of IFE systems, since it may affect the diagnostic and therapeutic evaluation of AL amyloidosis patients., (© 2024. Japanese Society of Hematology.)

Background: Although the prevalence rate of valvular heart disease (VHD) is high in patients with systemic lupus erythematosus (SLE), the predictive factors of concomitant VHD have not been fully evaluated., Methods and Results: Among 288 patients with SLE who underwent transthoracic echocardiography at Kumamoto University Hospital from 2016 to 2021, 241 patients with sufficient echocardiographic data were retrospectively analysed. Among them, 22 (9 %) had VHD (10 had mitral regurgitation, 3 had aortic regurgitation, 6 had tricuspid regurgitation, 1 had mitral regurgitation and tricuspid regurgitation, and 2 had a prosthetic cardiac valve). After excluding the two patients with a prosthetic cardiac valve, we divided the remaining patients into two groups: the VHD group and non-VHD group. Multivariate logistic regression analysis revealed that age and the platelet count were significantly and independently associated with having VHD (age: odds ratio, 1.06; 95 % confidence interval, 1.02-1.10; p < 0.01) (platelet count: odds ratio, 0.99; 95 % confidence interval, 0.98-1.00; p < 0.05). After excluding 95 patients aged < 40 years, receiver operating characteristic analysis revealed that the area under the curve of the platelet count for prediction of VHD was 0.73 with an optimal cut-off value of 166.5 × 10 3 /µL (sensitivity: 76.6 %, specificity: 60.0 %). Among patients with a low platelet count (<166.5 × 10 3 /µL), the rate of having VHD was 29 % (12/41 patients). However, among those with a high platelet count (≥166.5 × 10 3 /µL), this rate was only 8 % (8/103 patients)., Conclusion: The platelet count is useful to predict concomitant VHD in middle-aged and older patients with SLE., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Human retroviruses are derived from simian ones through cross-species transmission. These retroviruses are associated with little pathogenicity in their natural hosts, but in humans, HIV causes AIDS, and human T-cell leukemia virus type 1 (HTLV-1) induces adult T-cell leukemia-lymphoma (ATL). We analyzed the proviral sequences of HTLV-1, HTLV-2, and simian T-cell leukemia virus type 1 (STLV-1) from Japanese macaques ( Macaca fuscata ) and found that APOBEC3G (A3G) frequently generates G-to-A mutations in the HTLV-1 provirus, whereas such mutations are rare in the HTLV-2 and STLV-1 proviruses. Therefore, we investigated the mechanism of how HTLV-2 is resistant to human A3G (hA3G). HTLV-1, HTLV-2, and STLV-1 encode the so-called antisense proteins, HTLV-1 bZIP factor (HBZ), Antisense protein of HTLV-2 (APH-2), and STLV-1 bZIP factor (SBZ), respectively. APH-2 efficiently inhibits the deaminase activity of both hA3G and simian A3G (sA3G). HBZ and SBZ strongly suppress sA3G activity but only weakly inhibit hA3G, suggesting that HTLV-1 is incompletely adapted to humans. Unexpectedly, hA3G augments the activation of the transforming growth factor (TGF)-β/Smad pathway by HBZ, and this activation is associated with ATL cell proliferation by up-regulating BATF3/IRF4 and MYC . In contrast, the combination of APH-2 and hA3G, or the combination of SBZ and sA3G, does not enhance the TGF-β/Smad pathway. Thus, HTLV-1 is vulnerable to hA3G but utilizes it to promote the proliferation of infected cells via the activation of the TGF-β/Smad pathway. Antisense factors in each virus, differently adapted to control host cellular functions through A3G, seem to dictate the pathogenesis., Competing Interests: Competing interests statement:The authors declare no competing interest.

Human T-cell leukemia virus type 1 (HTLV-1) establishes chronic infection in humans and induces a T-cell malignancy called adult T-cell leukemia-lymphoma (ATL) and several inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Persistent HTLV-1 infection is established under the pressure of host immunity, and therefore the immune response against HTLV-1 is thought to reflect the status of the disease it causes. Indeed, it is known that cellular immunity against viral antigens is suppressed in ATL patients compared to HAM/TSP patients. In this study, we show that profiling the humoral immunity to several HTLV-1 antigens, such as Gag, Env, and Tax, and measuring proviral load are useful tools for classifying disease status and predicting disease development. Using targeted sequencing, we found that several carriers whom this profiling method predicted to be at high risk for developing ATL indeed harbored driver mutations of ATL. The clonality of HTLV-1-infected cells in those carriers was still polyclonal; it is consistent with an early stage of leukemogenesis. Furthermore, this study revealed significance of anti-Gag proteins to predict high risk group in HTLV-1 carriers. Consistent with this finding, anti-Gag cytotoxic T lymphocytes (CTLs) were increased in patients who received hematopoietic stem cell transplantation and achieved remission state, indicating the significance of anti-Gag CTLs for disease control. Our findings suggest that our strategy that combines anti-HTLV-1 antibodies and proviral load may be useful for prediction of the development of HTLV-1-associated diseases., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is a rare subtype of non-Hodgkin lymphoma (NHL) with poor prognosis, particularly in relapsed or refractory patients. Thus, timely detection of relapse and appropriate disease management are crucial. We present two patients with ENKTL, wherein positron emission tomography-computed tomography (PET-CT) with total-body coverage after induction therapy, detected newly relapsed regions in the bone marrow of the lower leg prior to progression. Case 1: A 47-year-old woman with nasal obstruction, showing 18F-fluoro-deoxyglucose (FDG) uptake in the nasal cavity (Lugano stage IE). After induction therapy (RT-2/3 DeVIC), PET-CT revealed abnormal uptake only in the right fibula. Case 2: A 68-year-old man with a skin nodule/ulcer and an enlarged right inguinal lymph node was diagnosed with advanced ENKTL. A PET-CT scan revealed abnormal uptake in the subcutaneous mass of the right medial thigh, lymph nodes, and descending colon (Lugano stage IV). After induction therapy, PET-CT revealed new abnormal uptake only in the left tibia. In both patients, CT-guided biopsy confirmed ENKTL recurrence. Moreover, PET-CT with whole-body coverage was useful for the timely assessment of relapse and detection of asymptomatic bone involvement. This approach allowed for modifications to treatment strategies in certain patients.

Acceleration of glycolysis is a common trait of cancer. A key metabolite, lactate, is typically secreted from cancer cells because its accumulation is toxic. Here, we report that a viral oncogene, HTLV-1 bZIP factor (HBZ), bimodally upregulates TAp73 to promote lactate excretion from adult T-cell leukemia-lymphoma (ATL) cells. HBZ protein binds to EZH2 and reduces its occupancy of the TAp73 promoter. Meanwhile, HBZ RNA activates TAp73 transcription via the BATF3-IRF4 machinery. TAp73 upregulates the lactate transporters MCT1 and MCT4. Inactivation of TAp73 leads to intracellular accumulation of lactate, inducing cell death in ATL cells. Furthermore, TAp73 knockout diminishes the development of inflammation in HBZ-transgenic mice. An MCT1/4 inhibitor, syrosingopine, decreases the growth of ATL cells in vitro and in vivo. MCT1/4 expression is positively correlated with TAp73 in many cancers, and MCT1/4 upregulation is associated with dismal prognosis. Activation of the TAp73-MCT1/4 pathway could be a common mechanism contributing to cancer metabolism., Significance: An antisense gene encoded in HTLV-1, HBZ, reprograms lactate metabolism and epigenetic modification by inducing TAp73 in virus-positive leukemic cells. A positive correlation between TAp73 and its target genes is also observed in many other cancer cells, suggesting that this is a common mechanism for cellular oncogenesis. This article is featured in Selected Articles from This Issue, p. 337., (©2023 The Authors; Published by the American Association for Cancer Research.)

The prognosis of aggressive adult T-cell leukemia/lymphoma (ATL) is poor, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment. In order to identify favorable prognostic patients after intensive chemotherapy, and who therefore might not require upfront allo-HSCT, we aimed to improve risk stratification of aggressive ATL patients aged <70 years. The clinical risk factors and genetic mutations were incorporated into risk modeling for overall survival (OS). We generated the m7-ATLPI, a clinicogenetic risk model for OS, that included the ATL prognostic index (PI) (ATL-PI) risk category, and non-silent mutations in seven genes, namely TP53, IRF4, RHOA, PRKCB, CARD11, CCR7, and GATA3. In the training cohort of 99 patients, the m7-ATLPI identified a low-, intermediate-, and highrisk group with 2-year OS of 100%, 43%, and 19%, respectively (hazard ratio [HR] =5.46; P<0.0001). The m7-ATLPI achieved superior risk stratification compared to the current ATL-PI (C-index 0.92 vs. 0.85, respectively). In the validation cohort of 84 patients, the m7-ATLPI defined low-, intermediate-, and high-risk groups with a 2-year OS of 81%, 30%, and 0%, respectively (HR=2.33; P=0.0094), and the model again outperformed the ATL-PI (C-index 0.72 vs. 0.70, respectively). The simplified m7-ATLPI, which is easier to use in clinical practice, achieved superior risk stratification compared to the ATLPI, as did the original m7-ATLPI; the simplified version was calculated by summing the following: high-risk ATL-PI category (+10), low-risk ATL-PI category (-4), and non-silent mutations in TP53 (+4), IRF4 (+3), RHOA (+1), PRKCB (+1), CARD11 (+0.5), CCR7 (-2), and GATA3 (-3).

Retroviruses and their host have coevolved in a delicate balance between viral replication and survival of the infected cell. In this equilibrium, restriction factors expressed by infected cells control different steps of retroviral replication such as entry, uncoating, nuclear import, expression, or budding. Here, we describe a mechanism of restriction against human T cell leukemia virus type 1 (HTLV-1) by the helicase-like transcription factor (HLTF). We show that RNA and protein levels of HLTF are reduced in primary T cells of HTLV-1-infected subjects, suggesting a clinical relevance. We further demonstrate that the viral oncogene Tax represses HLTF transcription via the Enhancer of zeste homolog 2 methyltransferase of the Polycomb repressive complex 2. The Tax protein also directly interacts with HLTF and induces its proteasomal degradation. RNA interference and gene transduction in HTLV-1-infected T cells derived from patients indicate that HLTF is a restriction factor. Restoring the normal levels of HLTF expression induces the dispersal of the Golgi apparatus and overproduction of secretory granules. By synergizing with Tax-mediated NF-κB activation, physiologically relevant levels of HLTF intensify the autophagic flux. Increased vesicular trafficking leads to an enlargement of the lysosomes and the production of large vacuoles containing viral particles. HLTF induction in HTLV-1-infected cells significantly increases the percentage of defective virions. In conclusion, HLTF-mediated activation of the autophagic flux blunts the infectious replication cycle of HTLV-1, revealing an original mode of viral restriction.

Adult T-cell leukemia (ATL) is a peripheral T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Microsatellite instability (MSI) has been observed in ATL cells. Although MSI results from impaired mismatch repair (MMR) pathway, no null mutations in the genes encoding MMR factors are detectable in ATL cells. Thus, it is unclear whether or not impairment of MMR causes the MSI in ATL cells. HTLV-1 bZIP factor (HBZ) protein interacts with numerous host transcription factors and significantly contributes to disease pathogenesis and progression. Here we investigated the effect of HBZ on MMR in normal cells. The ectopic expression of HBZ in MMR-proficient cells induced MSI, and also suppressed the expression of several MMR factors. We then hypothesized that the HBZ compromises MMR by interfering with a transcription factor, nuclear respiratory factor 1 (NRF-1), and identified the consensus NRF-1 binding site at the promoter of the gene encoding MutS homologue 2 (MSH2), an essential MMR factor. The luciferase reporter assay revealed that NRF-1 overexpression enhanced MSH2 promoter activity, while co-expression of HBZ reversed this enhancement. These results supported the idea that HBZ suppresses the transcription of MSH2 by inhibiting NRF-1. Our data demonstrate that HBZ causes impaired MMR, and may imply a novel oncogenesis driven by HTLV-1., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Masao Matsuoka reports a relationship with Kyowa Kirin Co Ltd that includes: speaking and lecture fees. Masao Matsuoka reports a relationship with Meiji Seika Pharma Co Ltd that includes: speaking and lecture fees. Masao Matsuoka reports a relationship with Bristol Myers Squibb Co that includes: speaking and lecture fees. Masao Matsuoka reports a relationship with Chugai Pharmaceutical Co Ltd that includes: speaking and lecture fees. Akifumi Takaori-Kondo reports a relationship with Ono Pharmaceutical Co Ltd that includes: funding grants., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4 + T cells induced by human T-cell leukemia virus type I (HTLV-1). HTLV-1 maintains life-long infection in the human host by clonal proliferation of infected cells and cell-to-cell spread of the virus. Two viral genes, tax and HTLV-1 bZIP factor (HBZ), promote expansion of infected cells through the important roles they play in acceleration of cell proliferation and protection from cell death. Long-term survival of infected clones in vivo causes genetic mutations and aberrant epigenetic changes to accumulate in host genes, resulting in the emergence of an ATL clone. Recent advances in sequencing technology have revealed the broad picture of genetic and transcriptional abnormalities in ATL cells. ATL cells have hyper-proliferative and anti-apoptotic signatures like those observed in other malignancies, but also notably have traits related to immune evasion. ATL cells exhibit a regulatory T-cell-like immuno-phenotype due to both the function of HBZ and mutation of several host genes, such as CCR4 and CIC. These findings suggest that immune evasion is a critical step in the oncogenesis of ATL, and thus novel therapies that activate anti-ATL/HTLV-1 immunity may be effective in the treatment and prevention of ATL., (© 2023. Japanese Society of Hematology.)

Chemotherapy in combination with mogamulizumab (Mog) was approved in Japan in 2014 for untreated aggressive adult T-cell leukaemia-lymphoma (ATL), but the survival benefit remains unclear. Therefore, we retrospectively analysed clinical outcomes in 39 transplant-ineligible patients with untreated aggressive ATL at Kumamoto University Hospital between 2010 and 2021. The probability of four-year overall survival was 46.3% in the first-line Mog-containing treatment group compared to 20.6% in the chemotherapy-alone group (p = 0.033). Furthermore, this survival benefit was observed even in the elderly. In conclusion, first-line Mog-containing treatment can be a promising strategy for transplant-ineligible patients with ATL, especially in the elderly., (© 2022 British Society for Haematology and John Wiley & Sons Ltd.)

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm immunophenotypically resembling regulatory T cells, associated with human T-cell leukemia virus type-1. Here, we performed whole-genome sequencing (WGS) of 150 ATL cases to reveal the overarching landscape of genetic alterations in ATL. We discovered frequent (33%) loss-of-function alterations preferentially targeting the CIC long isoform, which were overlooked by previous exome-centric studies of various cancer types. Long but not short isoform-specific inactivation of Cic selectively increased CD4+CD25+Foxp3+ T cells in vivo. We also found recurrent (13%) 3'-truncations of REL, which induce transcriptional upregulation and generate gain-of-function proteins. More importantly, REL truncations are also common in diffuse large B-cell lymphoma, especially in germinal center B-cell-like subtype (12%). In the non-coding genome, we identified recurrent mutations in regulatory elements, particularly splice sites, of several driver genes. In addition, we characterized the different mutational processes operative in clustered hypermutation sites within and outside immunoglobulin/T-cell receptor genes and identified the mutational enrichment at the binding sites of host and viral transcription factors, suggesting their activities in ATL. By combining the analyses for coding and noncoding mutations, structural variations, and copy number alterations, we discovered 56 recurrently altered driver genes, including 11 novel ones. Finally, ATL cases were classified into 2 molecular groups with distinct clinical and genetic characteristics based on the driver alteration profile. Our findings not only help to improve diagnostic and therapeutic strategies in ATL, but also provide insights into T-cell biology and have implications for genome-wide cancer driver discovery., (© 2022 by The American Society of Hematology.)

There remains an unmet clinical need to identify which patients with diffuse large B-cell lymphoma (DLBCL) would benefit from central nervous system (CNS) prophylaxis, due to the low positive predictive value (PPV; 10%-15%) of the currently available predictive models. To stratify patients at high risk of developing CNS relapse, we retrospectively analyzed 182 patients with DLBCL initially treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), or a R-CHOP-like regimen. Among them, 17 patients relapsed with CNS involvement, and the 2-year rate of CNS relapse was 7.9%. Upon carrying out multivariate analysis, ≥3 extranodal sites and elevated soluble interleukin-2 receptor (sIL-2R) levels at diagnosis were identified as independent risk factors for CNS relapse. The 2-year and 3.5-year rates of CNS relapse were 57.1% and 78.6%, respectively, in patients with both elevated sIL-2R and ≥3 extranodal sites. Furthermore, combined use of these risk factors of both elevated sIL-2R and ≥3 extranodal sites resulted in a high PPV (71.4%), negative predictive value (93.1%), and overall accuracy (92.3%) for undergoing CNS relapse. In conclusion, we propose a simple and valuable tool to predict patients with DLBCL at very high risk of CNS relapse., Competing Interests: Hiro Tatetsu has received honoraria from Ono Pharmaceutical, Chugai Pharmaceutical, Eisai, and patents, and royalties from Mesoblast. Kisato Nosaka has received consultancy fee, research funding, and honoraria from Kyowa Kirin and research funding from Chugai Pharmaceutical and honoraria from Celgene, Eisai, Meiji Seika Pharma, Janssen Pharmaceutical, Abbvie Inc. and Bristol Myers Squibb. Masao Matsuoka has received research funding from Chugai Pharmaceutical and Kyowa Kirin. Takafumi Shichijo, Yusuke Higuchi, Yoshitaka Kikukawa, Yoshitaka Inoue, Kosuke Toyoda, and Jun‐ichirou Yasunaga have no conflict of interest to disclose., (© 2022 The Authors. eJHaem published by British Society for Haematology and John Wiley & Sons Ltd.)

A 62-year-old man visited the Department of Otorhinolaryngology at our hospital with a chief complaint of a pharyngeal mass. He was admitted to our department with a diagnosis of T-cell lymphoma based on a biopsy of a mesopharyngeal tumor. Although clonality analysis was not performed due to the lack of an appropriate sample, we considered the possibility of lymphoma-type (Lugano classification stage II) adult T-cell leukemia-lymphoma (ATL), as the anti-HTLV-1 antibody was positive. During the course of the disease, the peripheral blood smear revealed atypical lymphocytes with cleaved nuclei, and inverse PCR was performed with DNA extracted from those cells; however, the result showed that the pattern of HTLV-1 proviral DNA integration sites was polyclonal. Further, we performed RNA in situ hybridization targeting HTLV-1 bZIP factor (HBZ-ISH) using the formalin-fixed paraffin-embedded (FFPE) tissue samples of the mesopharyngeal tumor, and a high expression of HBZ was found in the tumor cells, leading to the diagnosis of ATL. These findings suggest the effectiveness of the novel diagnostic method using FFPE tissue samples for ATL.

Follicular T-cell lymphoma (FTCL) is a rare disease, recently defined in the revised WHO classification Tumours of Haematopoietic and lymphoid tissues (4th edition). Although angioimmunoblastic T-cell lymphoma (AITL) and FTCL share similar T follicular helper (TFH) cell immunophenotypes and gene mutations, the clinical course of FTCL is not well characterized. Herein, we report the case of a 91-year-old woman with FTCL, who was successfully treated with corticosteroid. The patient, who had systemic lymphadenopathy and splenomegaly, was first diagnosed with necrotizing lymphadenitis. Re-biopsy was performed because of her persistent lymphadenopathy, which revealed FTCL. She was treated with corticosteroid because of her advanced age, poor performance, edema, and pleural effusion. After administering 100 mg prednisone, her condition improved and was discharged with prednisone tapering. Six-month positron emission tomography-computed tomography (PET-CT) scan showed complete metabolic remission. With a low dose of prednisone (6-10 mg), she remained disease-free for >3 years. Thus, these findings suggest that corticosteroid treatment is effective in some patients with peripheral T-cell lymphoma of TFH origin, including FTCL.

Human T-cell leukemia virus type 1 (HTLV-1) infects target cells primarily through cell-to-cell routes. Here, we provide evidence that cellular protein M-Sec plays a critical role in this process. When purified and briefly cultured, CD4+ T cells of HTLV-1 carriers, but not of HTLV-1- individuals, expressed M-Sec. The viral protein Tax was revealed to mediate M-Sec induction. Knockdown or pharmacological inhibition of M-Sec reduced viral infection in multiple co-culture conditions. Furthermore, M-Sec knockdown reduced the number of proviral copies in the tissues of a mouse model of HTLV-1 infection. Phenotypically, M-Sec knockdown or inhibition reduced not only plasma membrane protrusions and migratory activity of cells, but also large clusters of Gag, a viral structural protein required for the formation of viral particles. Taken together, these results suggest that M-Sec induced by Tax mediates an efficient cell-to-cell viral infection, which is likely due to enhanced membrane protrusions, cell migration, and the clustering of Gag., Competing Interests: The authors have declared that no competing interests exist.

Human retroviruses, including human T cell leukemia virus type 1 (HTLV-1) and HIV type 1 (HIV-1), encode an antisense gene in the negative strand of the provirus. Besides coding for proteins, the messenger RNAs (mRNAs) of retroviral antisense genes have also been found to regulate transcription directly. Thus, it has been proposed that retroviruses likely localize their antisense mRNAs to the nucleus in order to regulate nuclear events; however, this opposes the coding function of retroviral antisense mRNAs that requires a cytoplasmic localization for protein translation. Here, we provide direct evidence that retroviral antisense mRNAs are localized predominantly in the nuclei of infected cells. The retroviral 3' LTR induces inefficient polyadenylation and nuclear retention of antisense mRNA. We further reveal that retroviral antisense RNAs retained in the nucleus associate with chromatin and have transcriptional regulatory function. While HTLV-1 antisense mRNA is recruited to the promoter of C-C chemokine receptor type 4 ( CCR4 ) and enhances transcription from it to support the proliferation of HTLV-1-infected cells, HIV-1 antisense mRNA is recruited to the viral LTR and inhibits sense mRNA expression to maintain the latency of HIV-1 infection. In summary, retroviral antisense mRNAs are retained in nucleus, act like long noncoding RNAs instead of mRNAs, and contribute to viral persistence., Competing Interests: The authors declare no competing interest.

HTLV-1-associated myelopathy (HAM/TSP) is a chronic and progressive inflammatory disease of the central nervous system. The aim of our study was to identify genetic determinants related to the onset of HAM/TSP in the Japanese population. We conducted a genome-wide association study comprising 753 HAM/TSP patients and 899 asymptomatic HTLV-1 carriers. We also performed comprehensive genotyping of HLA-A , -B , -C , -DPB1 , -DQB1 , and -DRB1 genes using next-generation sequencing technology for 651 HAM/TSP patients and 804 carriers. A strong association was observed in HLA class I ( P = 1.54 × 10 -9 ) and class II ( P = 1.21 × 10 -8 ) loci with HAM/TSP. Association analysis using HLA genotyping results showed that HLA-C * 07:02 ( P = 2.61 × 10 -5 ), HLA-B * 07:02 ( P = 4.97 × 10 -10 ), HLA-DRB1 * 01:01 ( P = 1.15 × 10 -9 ) and HLA-DQB1 * 05:01 ( P = 2.30 × 10 -9 ) were associated with disease risk, while HLA-B * 40:06 ( P = 3.03 × 10 -5 ), HLA-DRB1 * 15:01 ( P = 1.06 × 10 -5 ) and HLA-DQB1 * 06:02 ( P = 1.78 × 10 -6 ) worked protectively. Logistic regression analysis identified amino acid position 7 in the G-BETA domain of HLA-DRB1 as strongly associated with HAM/TSP ( P = 9.52 × 10 -10 ); individuals homozygous for leucine had an associated increased risk of HAM/TSP (odds ratio, 9.57), and proline was protective (odds ratio, 0.65). Both associations were independent of the known risk associated with proviral load. DRB1-GB-7-Leu was not significantly associated with proviral load. We have identified DRB1-GB-7-Leu as a genetic risk factor for HAM/TSP development independent of proviral load. This suggests that the amino acid residue may serve as a specific marker to identify the risk of HAM/TSP even without knowledge of proviral load. In light of its allele frequency worldwide, this biomarker will likely prove useful in HTLV-1 endemic areas across the globe., Competing Interests: Competing interest statement: S.K. and F.M. are board members of GenoConcierge Kyoto Inc. S.K., M. Shimizu, and F.M. have patents pending for HLA typing software, primer sets for PCR amplification of HLA genes and its experimental protocol, and a risk marker of DRB1-GB-7 for HAM/TSP development., (Copyright © 2021 the Author(s). Published by PNAS.)

Human T-cell leukemia virus type 1 (HTLV-1) spreads through cell contact. Therefore, this virus persists and propagates within the host by two routes: clonal proliferation of infected cells and de novo infection. The proliferation is influenced by the host immune responses and expression of viral genes. However, the detailed mechanisms that control clonal expansion of infected cells remain to be elucidated. In this study, we show that newly infected clones were strongly suppressed, and then stable clones were selected, in a patient who was infected by live liver transplantation from a seropositive donor. Conversely, most HTLV-1+ clones persisted in patients who received hematopoietic stem cell transplantation from seropositive donors. To clarify the role of cell-mediated immunity in this clonal selection, we suppressed CD8+ or CD16+ cells in simian T-cell leukemia virus type 1 (STLV-1)-infected Japanese macaques. Decreasing CD8+ T cells had marginal effects on proviral load (PVL). However, the clonality of infected cells changed after depletion of CD8+ T cells. Consistent with this, PVL at 24 hours in vitro culture increased, suggesting that infected cells with higher proliferative ability increased. Analyses of provirus in a patient who received Tax-peptide pulsed dendritic cells indicate that enhanced anti-Tax immunity did not result in a decreased PVL although it inhibited recurrence of ATL. We postulate that in vivo selection, due to the immune response, cytopathic effects of HTLV-1 and intrinsic attributes of infected cells, results in the emergence of clones of HTLV-1-infected T cells that proliferate with minimized HTLV-1 antigen expression., Competing Interests: The authors have declared that no competing interests exist.

The huge amount of data acquired by high-throughput sequencing requires data reduction for effective analysis. Here we give a clustering algorithm for genome-wide open chromatin data using a new data reduction method. This method regards the genome as a string of 1s and 0s based on a set of peaks and calculates the Hamming distances between the strings. This algorithm with the systematically optimized set of peaks enables us to quantitatively evaluate differences between samples of hematopoietic cells and classify cell types, potentially leading to a better understanding of leukemia pathogenesis., Competing Interests: The authors declare that they have no conflict of interest.

Background: Simian T-cell leukemia virus type 1 (STLV-1) is disseminated among various non-human primate species and is closely related to human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Notably, the prevalence of STLV-1 infection in Japanese macaques (JMs) is estimated to be > 60%, much greater than that in other non-human primates; however, the mechanism and mode of STLV-1 transmission remain unknown. The aim of this study is to examine the epidemiological background by which STLV-1 infection is highly prevalent in JMs., Results: The prevalence of STLV-1 in the JMs rearing in our free-range facility reached up to 64% (180/280 JMs) with variation from 55 to 77% among five independent troops. Anti-STLV-1 antibody titers (ABTs) and STLV-1 proviral loads (PVLs) were normally distributed with mean values of 4076 and 0.62%, respectively, which were mostly comparable to those of HTLV-1-infected humans. Our initial hypothesis that some of the macaques might contribute to frequent horizontal STLV-1 transmission as viral super-spreaders was unlikely because of the absence of the macaques exhibiting abnormally high PVLs but poor ABTs. Rather, ABTs and PVLs were statistically correlated (p < 0.0001), indicating that the increasing PVLs led to the greater humoral immune response. Further analyses demonstrated that the STLV-1 prevalence as determined by detection of the proviral DNA was dramatically increased with age; 11%, 31%, and 58% at 0, 1, and 2 years of age, respectively, which was generally consistent with the result of seroprevalence and suggested the frequent incidence of mother-to-child transmission. Moreover, our longitudinal follow-up study indicated that 24 of 28 seronegative JMs during the periods from 2011 to 2012 converted to seropositive (86%) 4 years later; among them, the seroconversion rates of sexually matured (4 years of age and older) macaques and immature macaques (3 years of age and younger) at the beginning of study were comparably high (80% and 89%, respectively), suggesting the frequent incidence of horizontal transmission., Conclusions: Together with the fact that almost all of the full-adult JMs older than 9 years old were infected with STLV-1, our results of this study demonstrated for the first time that frequent horizontal and mother-to-child transmission may contribute to high prevalence of STLV-1 infection in JMs.

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of a T cell neoplasm and several inflammatory diseases. A viral gene, HTLV-1 bZIP factor (HBZ), induces pathogenic Foxp3-expressing T cells and triggers systemic inflammation and T cell lymphoma in transgenic mice, indicating its significance in HTLV-1-associated diseases. Here we show that, unexpectedly, a proinflammatory cytokine, IL-6, counteracts HBZ-mediated pathogenesis. Loss of IL-6 accelerates inflammation and lymphomagenesis in HBZ transgenic mice. IL-6 innately inhibits regulatory T cell differentiation, suggesting that IL-6 functions as a suppressor against HBZ-associated complications. HBZ up-regulates expression of the immunosuppressive cytokine IL-10. IL-10 promotes T cell proliferation only in the presence of HBZ. As a mechanism of growth promotion by IL-10, HBZ interacts with STAT1 and STAT3 and modulates the IL-10/JAK/STAT signaling pathway. These findings suggest that HTLV-1 promotes the proliferation of infected T cells by hijacking the machinery of regulatory T cell differentiation. IL-10 induced by HBZ likely suppresses the host immune response and concurrently promotes the proliferation of HTLV-1 infected T cells., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Human T-cell leukemia virus type 1 (HTLV-1) establishes persistent infection in vivo in two distinct ways: de novo infection and clonal proliferation of infected cells. Two viral genes, Tax and HTLV-1 bZIP factor (HBZ) play critical roles in viral transcription and promotion of T-cell proliferation, respectively. Tax is a potent transactivator not only for viral transcription but also for many cellular oncogenic pathways, such as the NF-κB pathway. HBZ is a suppressor of viral transcription and has the potential to change the immunophenotype of infected cells, conferring an effector regulatory T cell (eTreg)-like signature (CD4+ CD25+ CCR4+ TIGIT+ Foxp3+) and enhancing the proliferation of this subset. Reports that mice transgenic for either gene develop malignant tumors suggest that both Tax and HBZ are involved in leukemogenesis by HTLV-1. However, the immunogenicity of Tax is very high, and its expression is generally suppressed in vivo . Recently, it was found that Tax can be expressed transiently in a small subpopulation of adult T-cell leukemia-lymphoma (ATL) cells and plays a critical role in maintenance of the overall population. HBZ is expressed in almost all infected cells except for the rare Tax-expressing cells, and activates the pathways associated with cell proliferation. These findings indicate that HTLV-1 fine-tunes the expression of viral genes to control the mode of viral propagation. The interplay between Tax and HBZ is the basis of a sophisticated strategy to evade host immune surveillance and increase transmission - and can lead to ATL as a byproduct., (Copyright © 2020 Yasunaga.)

T cells infected with human T-cell leukemia virus type 1 (HTLV-1) transform into malignant/leukemic cells and develop adult T-cell leukemia (ATL) after a long latency period. The tax (transactivator from the X-gene region) and HBZ (HTLV-1 bZIP factor) genes of HTLV-1 play crucial roles in the development of ATL. The process and mechanism by which HTLV-1-infected T cells acquire malignancy and develop ATL remain to be elucidated. Constitutive expression of interleukin-2 (IL-2) receptor α-chain (IL-2Rα/CD25), induced by the tax and HBZ genes of HTLV-1, on ATL cells implicates the involvement of IL-2/IL-2R pathway in the growth and development of ATL cells in vivo . However, the leukemic cells in the majority of ATL patients appeared unresponsive to IL-2, raising controversies on the role of this pathway for the growth of ATL cells in vivo . Here, we report the establishment of 32 IL-2-dependent T-cell lines infected with HTLV-1 from 26 ATL patients, including eight leukemic cell lines derived from five ATL patients, while no T-cell lines were established without IL-2. We have shown that the IL-2-dependent ATL cell lines evolved into IL-2-independent/-unresponsive growth phase, resembling ATL cells in vivo . Moreover, the IL-2-dependent non-leukemic T-cell lines infected with HTLV-1 acquired IL-2-independency and turned into tumor-producing cancer cells as with the ATL cell lines. HTLV-1-infected T cells in vivo could survive and proliferate depending on IL-2 that was produced in vivo by the HTLV-1-infected T cells of ATL patients and patients with HTLV-1-associated diseases and, acts as a physiological molecule to regulate T-cell growth. These results suggest that ATL cells develop among the HTLV-1-infected T cells growing dependently on IL-2 and that most of the circulating ATL cells progressed to become less responsive to IL-2, acquiring the ability to proliferate without IL-2., (Copyright © 2020 Maeda, Tanabe-Shibuya, Miyazato, Masutani, Yasunaga, Usami, Shimizu and Matsuoka.)

T cell development and homeostasis requires IL-7R α-chain (IL-7Rα) signaling. Tyrosine Y449 of the IL-7Rα is essential to activate STAT5 and PI3K, whereas PI3K recruitment requires IL-7Rα methionine M452. How IL-7Rα activates and regulates both signaling pathways differentially remains unclear. To characterize differential signaling, we established two lines of IL-7Rα mutant mice: IL-7R-Y449F mice and IL-7R-M452L mice. IL-7R-Y449F mice showed decreased PI3K and STAT5 signals, whereas IL-7R-M452L mice showed decreased PI3K but significantly increased STAT5 signaling, owing to a competition between PI3K and STAT5 signaling through Y449 of IL-7Rα. The number of T, B, and mature innate lymphoid cells were markedly reduced in IL-7R-Y449F mice, whereas IL-7R-M452L mice showed impaired early T cell development and memory precursor effector T cell maintenance with the downregulation of transcription factor T cell factor-1. Peripheral T cell numbers increased in IL-7R-M452L mice with enhanced survival and homeostatic proliferation. Furthermore, although wild type and IL-7R-Y449F mice showed comparable Th1/Th2 differentiation, IL-7R-M452L mice exhibited impaired Th17 differentiation. We conclude that PI3K competes with STAT5 under IL-7Rα and maintains an appropriate signal balance for modulating T cell development and homeostasis. To our knowledge, this study provides a new insight into complex regulation of IL-7Rα signaling, which supports immune development and responses., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Human T-cell leukemia virus type 1 (HTLV-1) infection causes T-cell leukemia and inflammatory diseases, most notably including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The underlying mechanism for the pathogenesis of HAM/TSP remains unclear. According to a recent clinical trial, a humanized antibody that targets CCR4 + cells ameliorates inflammation by reducing the number of infected cells in the central nervous system; this result suggests that the transmigration of HTLV-1-infected cells plays a crucial role in HAM/TSP. Partly due to the blood-brain barrier, current treatments for HAM/TSP are mostly palliative. Pentosan polysulfate (PPS), a semisynthetic glycosaminoglycan, has recently been used to treat HAM/TSP and was found to alleviate the symptoms. In this study, we investigated the effect of PPS on HTLV-1-infected cells and provide evidence for its efficacy in HAM/TSP. PPS was cytotoxic to certain HTLV-1-infected cells and significantly suppressed HTLV-1 virion production. PPS also efficiently inhibited HTLV-1 cell-cell transmission in T cells. In addition, PPS blocked HTLV-1 infection of primary endothelial cells (human umbilical vascular endothelial cells) and suppressed the subsequent induction of proinflammatory cytokine expression. Furthermore, PPS was found to inhibit the adhesion and transmigration of HTLV-1-infected cells. We also confirmed the anti-HTLV-1 effect of PPS in vivo using two mouse models. PPS blocked HTLV-1 infection in a mouse model with peripheral blood mononuclear cell (PBMC)-humanized NOD-scid IL2Rgamma null (huPBMC NSG) mice. PPS was also found to suppress the development of dermatitis and lung damage in HTLV-1 bZIP factor (HBZ)-transgenic (HBZ-Tg) mice, an HTLV-1 transgenic mouse model in which the mice develop systemic inflammation. IMPORTANCE HTLV-1 is the first human retrovirus to have been identified and is endemic in certain areas worldwide. HTLV-1 infection leads to the development of an inflammatory disease called HAM/TSP, a myelopathy characterized by slowly progressive spastic paraparesis. There have been no effective therapeutics available for HAM/TSP, but recently, a semisynthetic glycosaminoglycan, named pentosan polysulfate (PPS), has been found to alleviate the symptoms of HAM/TSP. Here we conducted a comprehensive study on the effect of PPS both in vitro and in vivo PPS demonstrated anti-HTLV-1 potential in infected cell lines, as shown by its suppressive effects on HTLV-1 replication and transmission and on the transmigration of infected T cells. Moreover, results obtained from two HTLV-1 mouse models demonstrate that PPS inhibits HTLV-1 infection and inflammation development in vivo Our work offers insights into the treatment of HAM/TSP by PPS and also suggests its possible use for treating other HTLV-1-induced inflammatory diseases., (Copyright © 2019 American Society for Microbiology.)

Human T-cell leukemia virus type 1 (HTLV-1) is the world's first retrovirus with pathogenicity to cause adult T-cell leukemia-lymphoma (ATL) and chronic inflammatory diseases,such as HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and HTLV-1 uveitis. As the virological characteristic, HTLV-1 can transmit efficiently only through cell-to-cell contact. Spread of infection and viral persistence is ingeniously driven by several viral genes as exemplified by HTLV-1 bZIP factor (HBZ) and tax. After the infection, the virus promotes proliferation and immortalization of the infected cells with acculturating immunophenotype into effector/memory T cells. In addition, HBZ enhances expression of co-inhibitory receptors on the surface of infected cells, potentially leading to suppression of host immune responses. These viral strategies can also result in unforeseen by-product, the pathogenicity of HTLV-1-associated diseases. In this review, with recent progress of HTLV-1 researches, we focus on astute regulation systems of the viral genes developed by HTLV-1.

Adult T-cell leukemia (ATL) is a highly aggressive T-cell malignancy induced by human T-cell leukemia virus type 1 (HTLV-1) infection. Long noncoding RNA (lncRNA) plays a critical role in the development and progression of multiple human cancers. However, the function of lncRNA in HTLV-1-induced oncogenesis has not been elucidated. In the present study, we show that the expression level of the lncRNA ANRIL was elevated in HTLV-1-infected cell lines and clinical ATL samples. E2F1 induced ANRIL transcription by enhancing its promoter activity. Knockdown of ANRIL in ATL cells repressed cellular proliferation and increased apoptosis in vitro and in vivo As a mechanism for these actions, we found that ANRIL targeted EZH2 and activated the NF-κB pathway in ATL cells. This activation was independent of the histone methyltransferase (HMT) activity of EZH2 but required the formation of an ANRIL/EZH2/p65 ternary complex. A chromatin immunoprecipitation assay revealed that ANRIL/EZH2 enhanced p65 DNA binding capability. In addition, we observed that the ANRIL/EZH2 complex repressed p21/CDKN1A transcription through H3K27 trimethylation of the p21/CDKN1A promoter. Taken together, our results implicate that the lncRNA ANRIL, by cooperating with EZH2, supports the proliferation of HTLV-1-infected cells, which is thought to be critical for oncogenesis. IMPORTANCE Human T-cell leukemia virus type 1 (HTLV-1) is the pathogen that causes adult T-cell leukemia (ATL), which is a unique malignancy of CD4 + T cells. A role for long noncoding RNA (lncRNA) in HTLV-1-mediated cellular transformation has not been described. In this study, we demonstrated that the lncRNA ANRIL was important for maintaining the proliferation of ATL cells in vitro and in vivo ANRIL was shown to activate NF-κB signaling through forming a ternary complex with EZH2 and p65. Furthermore, epigenetic inactivation of p21/CDKN1A was involved in the oncogenic function of ANRIL. To the best of our knowledge, this is the first study to address the regulatory role of the lncRNA ANRIL in ATL and provides an important clue to prevent or treat HTLV-1-associated human diseases., (Copyright © 2018 American Society for Microbiology.)

Background: Among human T cell leukemia virus type 1 (HTLV-1)-infected individuals, there is an association between HTLV-1 tax subgroups (subgroup-A or subgroup-B) and the risk of HAM/TSP in the Japanese population. To investigate the role of HTLV-1 subgroups in viral pathogenesis, we studied the functional difference in the subgroup-specific viral transcriptional regulators Tax and HBZ using microarray analysis, reporter gene assays, and evaluation of viral-host protein-protein interaction., Results: (1) Transcriptional changes in Jurkat Tet-On human T-cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter revealed different target gene profiles; (2) the number of differentially regulated genes induced by HBZ was 2-3 times higher than that induced by Tax; (3) Tax and HBZ induced the expression of different classes of non-coding RNAs (ncRNAs); (4) the chemokine CXCL10, which has been proposed as a prognostic biomarker for HAM/TSP, was more efficiently induced by subgroup-A Tax (Tax-A) than subgroup-B Tax (Tax-B), in vitro as well as in unmanipulated (ex vivo) PBMCs obtained from HAM/TSP patients; (5) reporter gene assays indicated that although transient Tax expression in an HTLV-1-negative human T-cell line activated the CXCL10 gene promoter through the NF-κB pathway, there was no difference in the ability of each subgroup of Tax to activate the CXCL10 promoter; however, (6) chromatin immunoprecipitation assays showed that the ternary complex containing Tax-A is more efficiently recruited onto the promoter region of CXCL10, which contains two NF-κB binding sites, than that containing Tax-B., Conclusions: Our results indicate that different HTLV-1 subgroups are characterized by different patterns of host gene expression. Differential expression of pathogenesis-related genes by subgroup-specific Tax or HBZ may be associated with the onset of HAM/TSP.

Viruses causing chronic infection artfully manipulate infected cells to enable viral persistence in vivo under the pressure of immunity. Human T-cell leukemia virus type 1 (HTLV-1) establishes persistent infection mainly in CD4+ T cells in vivo and induces leukemia in this subset. HTLV-1-encoded Tax is a critical transactivator of viral replication and a potent oncoprotein, but its significance in pathogenesis remains obscure due to its very low level of expression in vivo. Here, we show that Tax is expressed in a minor fraction of leukemic cells at any given time, and importantly, its expression spontaneously switches between on and off states. Live cell imaging revealed that the average duration of one episode of Tax expression is ∼19 hours. Knockdown of Tax rapidly induced apoptosis in most cells, indicating that Tax is critical for maintaining the population, even if its short-term expression is limited to a small subpopulation. Single-cell analysis and computational simulation suggest that transient Tax expression triggers antiapoptotic machinery, and this effect continues even after Tax expression is diminished; this activation of the antiapoptotic machinery is the critical event for maintaining the population. In addition, Tax is induced by various cytotoxic stresses and also promotes HTLV-1 replication. Thus, it seems that Tax protects infected cells from apoptosis and increases the chance of viral transmission at a critical moment. Keeping the expression of Tax minimal but inducible on demand is, therefore, a fundamental strategy of HTLV-1 to promote persistent infection and leukemogenesis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Adult T-cell leukemia/lymphoma (ATL) is a heterogeneous group of peripheral T-cell malignancies characterized by human T-cell leukemia virus type-1 infection, whose genetic profile has recently been fully investigated. However, it is still poorly understood how these alterations affect clinical features and prognosis. We investigated the effects of genetic alterations commonly found in ATL on disease phenotypes and clinical outcomes, based on genotyping data obtained from 414 and 463 ATL patients using targeted-capture sequencing and single nucleotide polymorphism array karyotyping, respectively. Aggressive (acute/lymphoma) subtypes were associated with an increased burden of genetic and epigenetic alterations, higher frequencies of TP53 and IRF4 mutations, and many copy number alterations (CNAs), including PD-L1 amplifications and CDKN2A deletions, compared with indolent (chronic/smoldering) subtypes. By contrast, STAT3 mutations were more characteristic of indolent ATL. Higher numbers of somatic mutations and CNAs significantly correlated with worse survival. In a multivariate analysis incorporating both clinical factors and genetic alterations, the Japan Clinical Oncology Group prognostic index high-risk, older age, PRKCB mutations, and PD-L1 amplifications were independent poor prognostic factors in aggressive ATL. In indolent ATL, IRF4 mutations, PD-L1 amplifications, and CDKN2A deletions were significantly associated with shorter survival, although the chronic subtype with unfavorable clinical factors was only marginally significant. Thus, somatic alterations characterizing aggressive diseases predict worse prognosis in indolent ATL, among which PD-L1 amplifications are a strong genetic predictor in both aggressive and indolent ATL. ATL subtypes are further classified into molecularly distinct subsets with different prognosis. Genetic profiling might contribute to improved prognostication and management of ATL patients., (© 2018 by The American Society of Hematology.)

Chronic infection is one of the major causes of cancer, and there are several mechanisms for infection-mediated oncogenesis. Some pathogens encode gene products that behave like oncogenic factors, hijacking cellular pathways to promote the survival and proliferation of infected cells in vivo. Some of these viral oncoproteins trigger a cellular damage defense response leading to senescence; however, other viral factors hinder this suppressive effect, suggesting that cooperation of those viral factors is important for malignant transformation. Coinfection with multiple agents is known to accelerate cancer development in certain cases. For example, parasitic or bacterial infection is a risk factor for adult T-cell leukemia-lymphoma induced by human T-cell leukemia virus type 1, and Epstein-Barr virus and malaria are closely associated with endemic Burkitt lymphoma. Human immunodeficiency virus type 1 infection is accompanied by various types of infection-related cancer. These findings indicate that these oncogenic pathogens can cooperate to overcome host barriers against cancer development. In this review, the authors focus on the collaborative strategies of pathogens for oncogenesis from two different points of view: (i) the cooperation of two or more different factors encoded by a single pathogen; and (ii) the acceleration of oncogenesis by coinfection with multiple agents., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Liver metabolism undergoes robust circadian oscillations in gene expression and enzymatic activity essential for liver homeostasis, but whether the circadian clock controls homeostatic self-renewal of hepatocytes is unknown. Here we show that hepatocyte polyploidization is markedly accelerated around the central vein, the site of permanent cell self-renewal, in mice deficient in circadian Period genes. In these mice, a massive accumulation of hyperpolyploid mononuclear and binuclear hepatocytes occurs due to impaired mitogen-activated protein kinase phosphatase 1 (Mkp1)-mediated circadian modulation of the extracellular signal-regulated kinase (Erk1/2) activity. Time-lapse imaging of hepatocytes suggests that the reduced activity of Erk1/2 in the midbody during cytokinesis results in abscission failure, leading to polyploidization. Manipulation of Mkp1 phosphatase activity is sufficient to change the ploidy level of hepatocytes. These data provide clear evidence that the Period genes not only orchestrate dynamic changes in metabolic activity, but also regulate homeostatic self-renewal of hepatocytes through Mkp1-Erk1/2 signaling pathway.

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.

Adult T-cell leukemia (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). We recently reported that abacavir, an anti-HIV-1 drug, potently and selectively kills ATL cells. This effect was attributed to the reduced expression of tyrosyl-DNA-phosphodiesterase 1 (TDP1), a DNA repair enzyme, in ATL cells. However, the molecular mechanism underlying the downregulation of TDP1 in ATL cells remains elusive. Here we identified the core promoter of the TDP1 gene, which contains a conserved nuclear respiratory factor 1 (NRF-1) binding site. Overexpression of NRF-1 increased TDP1-promoter activity, whereas the introduction of dominant-negative NRF-1 repressed such activity. Overexpression of NRF-1 also upregulated endogenous TDP-1 expression, while introduction of shNRF-1 suppressed TDP1 in Jurkat T cells, making them susceptible to abacavir. These results indicate that NRF-1 is a positive transcriptional regulator of TDP1-gene expression. Importantly, we revealed that HTLV-1 bZIP factor (HBZ) protein which is expressed in all ATL cases physically interacts with NRF-1 and inhibits the DNA-binding ability of NRF-1. Taken together, HBZ suppresses TDP1 expression by inhibiting NRF-1 function in ATL cells. The HBZ/NRF-1/TDP1 axis provides new therapeutic targets against ATL and might explain genomic instability leading to the pathogenesis of ATL.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. To enhance cell-to-cell transmission of HTLV-1, the virus increases the number of infected cells in vivo. HTLV-1 bZIP factor (HBZ) is constitutively expressed in HTLV-1 infected cells and ATL cells and promotes T-cell proliferation. However, the detailed mechanism by which it does so remains unknown. Here, we show that HBZ enhances the proliferation of expressing T cells after stimulation via the T-cell receptor. HBZ promotes this proliferation by influencing the expression and function of multiple co-inhibitory receptors. HBZ suppresses the expression of BTLA and LAIR-1 in HBZ expressing T cells and ATL cells. Expression of T cell immunoglobulin and ITIM domain (TIGIT) and Programmed cell death 1 (PD-1) was enhanced, but their suppressive effect on T-cell proliferation was functionally impaired. HBZ inhibits the co-localization of SHP-2 and PD-1 in T cells, thereby leading to impaired inhibition of T-cell proliferation and suppressed dephosphorylation of ZAP-70 and CD3ζ. HBZ does this by interacting with THEMIS, which associates with Grb2 and SHP-2. Thus, HBZ interacts with the SHP containing complex, impedes the suppressive signal from PD-1 and TIGIT, and enhances the proliferation of T cells. Although HBZ was present in both the nucleus and the cytoplasm of T cells, HBZ was localized largely in the nucleus by suppressed expression of THEMIS by shRNA. This indicates that THEMIS is responsible for cytoplasmic localization of HBZ in T cells. Since THEMIS is expressed only in T-lineage cells, HBZ mediated inhibition of the suppressive effects of co-inhibitory receptors accounts for how HTLV-1 induces proliferation only of T cells in vivo. This study reveals that HBZ targets co-inhibitory receptors to cause the proliferation of infected cells., Competing Interests: The authors have declared that no competing interests exist.

Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that induces a fatal T-cell malignancy, adult T-cell leukemia (ATL). Among several regulatory/accessory genes in HTLV-1, HTLV-1 bZIP factor (HBZ) is the only viral gene constitutively expressed in infected cells. Our previous study showed that HBZ functions in two different molecular forms, HBZ protein and HBZ RNA. In this study, we show that HBZ protein targets retinoblastoma protein (Rb), which is a critical tumor suppressor in many types of cancers. HBZ protein interacts with the Rb/E2F-1 complex and activates the transcription of E2F-target genes associated with cell cycle progression and apoptosis. Mouse primary CD4(+) T cells transduced with HBZ show accelerated G1/S transition and apoptosis, and importantly, T cells from HBZ transgenic (HBZ-Tg) mice also demonstrate enhanced cell proliferation and apoptosis. To evaluate the functions of HBZ protein alone in vivo, we generated a new transgenic mouse strain that expresses HBZ mRNA altered by silent mutations but encoding intact protein. In these mice, the numbers of effector/memory and Foxp3(+) T cells were increased, and genes associated with proliferation and apoptosis were upregulated. This study shows that HBZ protein promotes cell proliferation and apoptosis in primary CD4(+) T cells through activation of the Rb/E2F pathway, and that HBZ protein also confers onto CD4(+) T-cell immunophenotype similar to those of ATL cells, suggesting that HBZ protein has important roles in dysregulation of CD4(+) T cells infected with HTLV-1., Competing Interests: The authors declare no conflict of interest.

Adult T-cell leukemia (ATL) is a fatal neoplasm derived from CD4-positive T-lymphocytes, and regardless of intensive chemotherapy, its mean survival time is less than 1 year. Nuclear factor-kappaB (NF-kappaB) activation was reported in HTLV-I associated cells, and has been implicated in oncogenesis and resistance to anticancer agents and apoptosis. We studied the effect of a proteasome inhibitor, bortezomib (formerly known as PS-341), on ATL cells in vitro and in vivo. Bortezomib could inhibit the degradation of IkappaBalpha in ATL cells, resulting in suppression of NF-kappaB and induction of cell death in ATL cells in vitro. Susceptibilities to bortezomib were well correlated with NF-kappaB activation, suggesting that suppression of the NF-kappaB pathway was implicated in the cell death induced by bortezomib. Although the majority of the cell death was apoptosis, necrotic cell death was observed in the presence of a caspase inhibitor, z-VAD-fmk. When bortezomib was administered into SCID mice bearing tumors, it suppressed tumor growth in vivo, showing that bortezomib was effective against ATL cells in vivo. These studies revealed that bortezomib is highly effective against ATL cells in vitro and in vivo by induction of apoptosis, and its clinical application might improve the prognosis of patients with this fatal disease.

Opportunistic infections frequently occur in patients with adult T-cell leukemia (ATL) and human T-cell leukemia virus type I (HTLV-I) carriers. However, the underlying mechanisms of such infections remain unknown. To clarify the mechanism of immunodeficiency in those infected with HTLV-I, this study analyzed the T-cell subsets in HTLV-I carriers and patients with HTLV-I-associated myelopathy/tropical spastic paraparesis and ATL using 3-color fluorescence with CD62L and CD45RA coexpression either with CD4(+) or CD8(+) T cells. The number of naive T lymphocytes was markedly suppressed in patients with ATL, particularly in those with acute form, compared with uninfected control individuals. The number of naive T cells was low in HTLV-I-infected individuals under 50 years old compared with uninfected individuals, whereas the number of memory T lymphocytes was greater in HTLV-I-infected individuals. Although the increase of memory T lymphocytes correlated with HTLV-I provirus loads, no relationship was found between naive T-cell counts and provirus loads. T-cell receptor rearrangement excision circles (TRECs), which are generated by DNA recombination during early T lymphopoiesis, were quantified to evaluate thymic function in HTLV-I-infected individuals. TREC levels were lower in HTLV-I-infected individuals than in uninfected individuals. In HTLV-I carriers less than 70 years old, an increase of Epstein-Barr virus DNA in peripheral blood mononuclear cells was observed in 6 of 16 (38%) examined, whereas it was detectable in only 1 of 11 uninfected controls. These results suggested that the low number of naive T lymphocytes was due to suppressed production of T lymphocytes in the thymus, which might account for immunodeficiency observed in HTLV-I-infected individuals.