HTLV-1 Infected Cells Research Articles

Novel Treatments of Adult T Cell Leukemia Lymphoma.

Adult T cell leukemia-lymphoma (ATL) is an aggressive malignancy secondary to chronic infection with the human T cell leukemia virus type I (HTLV-I) retrovirus. ATL carries a dismal prognosis. ATL classifies into four subtypes (acute, lymphoma, chronic, and smoldering) which display different clinical features, prognosis and response to therapy, hence requiring different clinical management. Smoldering and chronic subtypes respond well to antiretroviral therapy using the combination of zidovudine (AZT) and interferon-alpha (IFN) with a significant prolongation of survival. Conversely, the watch and wait strategy or chemotherapy for these indolent subtypes allies with a poor long-term outcome. Acute ATL is associated with chemo-resistance and dismal prognosis. Lymphoma subtypes respond better to intensive chemotherapy but survival remains poor. Allogeneic hematopoietic stem cell transplantation (HSCT) results in long-term survival in roughly one third of transplanted patients but only a small percentage of patients can make it to transplant. Overall, current treatments of aggressive ATL are not satisfactory. Prognosis of refractory or relapsed patients is dismal with some encouraging results when using lenalidomide or mogamulizumab. To overcome resistance and prevent relapse, preclinical or pilot clinical studies using targeted therapies such as arsenic/IFN, monoclonal antibodies, epigenetic therapies are promising but warrant further clinical investigation. Anti-ATL vaccines including Tax peptide-pulsed dendritic cells, induced Tax-specific CTL responses in ATL patients. Finally, based on the progress in understanding the pathophysiology of ATL, and the risk-adapted treatment approaches to different ATL subtypes, treatment strategies of ATL should take into account the host immune responses and the host microenvironment including HTLV-1 infected non-malignant cells. Herein, we will provide a summary of novel treatments of ATL in vitro, in vivo, and in early clinical trials.

Inhibition of I\u03baB\u03b1 phosphorylation potentiates regulated cell death induced by azidothymidine in HTLV-1 infected cells

Adult T cell leukemia/lymphoma (ATL) can be susceptible, at least transiently, to treatments with azidothymidine (AZT) plus IFNα and/or arsenic trioxide. However, the real role of AZT in this effect is still unclear. In fact, while reverse transcriptase (RT) inhibition could explain reduction of clonal expansion and of renewal of HTLV-1 infected cells during ATL progression, this effect alone seems insufficient to justify the evident and prompt decrease of the pro-viral load in treated patients. We have previously demonstrated that AZT is endowed with an intrinsic pro-apoptotic potential towards both peripheral blood mononuclear cells from healthy donors or some tumor cell lines, but this cytotoxic potential cannot be fully achieved unless IκBα phosphorylation is inhibited. Since the constitutive activation of NF-kappa B (NF-κB) appears a common biological basis of HTLV-1-infected cells, a pharmacological inhibition of IκBα phosphorylation seems a potential strategy for treating and preventing HTLV-1 related pathologies. In this study, we have demonstrated that a combination treatment with the IκBα phosphorylation inhibitor Bay 11-7085 and AZT induced increased levels of regulated cell death (RCD) by apoptosis compared to the single treatments in HTLV-1 infected cells of different origin. Importantly, levels of RCD were considerably higher in infected cells in comparison with the uninfected ones. Inhibition of NF-κB activation following the combined treatment was confirmed by analysis of both gel-shift and functional activity of the NF-κB complex proteins, p65/p52. Moreover, a transcriptional analysis revealed that the addition of Bay 11-7085 to AZT treatment in HTLV-1-infected cells modified their transcriptional profile, by inducing the upregulation of some pro-apoptotic genes together with the downregulation of some anti-apoptotic genes. Our data suggest that addition of adequate concentrations of IκBα phosphorylation inhibitor to therapeutic regimens including AZT could be a promising strategy in ATL.

Degradation of p47 by autophagy contributes to CADM1 overexpression in ATLL cells through the activation of NF-\u03baB

Cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is identified as a novel cell surface marker for human T-cell leukemia virus (HTLV-1)-infected T cells. Adult T-cell leukemia/lymphoma (ATLL) is developed in HTLV-1-infected T-cells after a long infection period. To examine the mechanism of CADM1 overexpression in ATLL, we first identified that CADM1 is transcriptionally up-regulated by a transcriptional enhancer element through NF-κB signaling pathway. In HTLV-1-infected T-cells, CADM1 expression is dependent on HTLV-1/Tax through activation of canonical and non-canonical NF-κB; however, in ATLL cells with frequent loss of Tax expression, the activation of canonical NF-κB only enhances the CADM1 expression. Along with active mutations in signaling molecules under T-cell recepor (TCR) signaling, degradation of p47, a negative regulator of NF-κB, was essential for activation of canonical NF-κB through stabilization of NEMO (NF-κB essential modulator). The mechanism of p47 degradation is primarily dependent on activation of lysosomal-autophagy and the autophagy is activated in most of the HTLV-infected and ATLL cells, suggesting that the p47 degradation may be a first key molecular event during HTLV-1 infection to T-cells as a connector of two important signaling pathways, NF-κB and autophagy.

Sensing of cell-associated HTLV by plasmacytoid dendritic cells is regulated by dense β-galactoside glycosylation.

Human T Lymphotropic virus (HTLV) infection can persist in individuals resulting, at least in part, from viral escape of the innate immunity, including inhibition of type I interferon response in infected T-cells. Plasmacytoid dendritic cells (pDCs) are known to bypass viral escape by their robust type I interferon production. Here, we demonstrated that pDCs produce type I interferons upon physical cell contact with HTLV-infected cells, yet pDC activation inversely correlates with the ability of the HTLV-producing cells to transmit infection. We show that pDCs sense surface associated-HTLV present with glycan-rich structure referred to as biofilm-like structure, which thus represents a newly described viral structure triggering the antiviral response by pDCs. Consistently, heparan sulfate proteoglycans and especially the cell surface pattern of terminal β-galactoside glycosylation, modulate the transmission of the immunostimulatory RNA to pDCs. Altogether, our results uncover a function of virus-containing cell surface-associated glycosylated structures in the activation of innate immunity.

P-G2 Development of engineered T-cell immmunotherapy for treating Adult-T cell leukemia caused by HTLV-1

HTLV-1 is perhaps the most oncogenic virus in humans. Five percent to ten percent of individuals infected by HTLV-1 develop a fatal T-cell leukemia (ATL) after 2-3 decades of latency. Conventional chemotherapy only creates escape mutants in ATL. Novel treatments including retroviral drugs, Arsenic Trioxide/IFNa combination, or Allogeneic stem cell transplant have increased the 5-year survival, but there is still need to establish a cure. We chose an immunotherapy approach because all leukemic cells express some HTLV-1 viral proteins allowing to distinguish normal and HTLV-1 infected cells by the host immune system. In addition, anti-HTLV-1 immunity is suppressed in ATL patients. We aim to target HBZ (an antisense protein of HTLV-1) for this purpose because; 1) the frequency of T cells recognizing HBZ correlates well with low HTLV-1 proviral loads in contrast to T cells against Tax-1 or Env. 2) Unlike Tax-1, HBZ is constantly expressed by all stages of ATL cells. The challenge is that HBZ is an extremely weak immunogen. We identified a few epitopes of HBZ that stably find to class I HLA molecules. We tested them using an in vivo model of ATL in humanized mice. Immunization by HBZ showed little therapeutic effects in these mice. DC immunization showed prolonged survival, but did not protect mice from ATL. Thus, we grew anti-HBZ CD8 T cells by immunizing normal T cells ex vivo, enriched them by HBZ-tetramer sorting. T-cell therapy involving anti-HBZ CD8 T cells successfully protected host mice, prompting us to rely on an “engineered T cell” strategy as novel treatment of acute/lymphomatous ATL cases.

Mogamulizumab (Anti-CCR4) in HTLV-1–Associated Myelopathy

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) causes the debilitating neuroinflammatory disease HTLV-1–associated myelopathy–tropical spastic paraparesis (HAM–TSP) as well as adult T-cell leukemia–lymphoma (ATLL). In patients with HAM–TSP, HTLV-1 infects mainly CCR4+ T cells and induces functional changes, ultimately causing chronic spinal cord inflammation. We evaluated mogamulizumab, a humanized anti-CCR4 monoclonal antibody that targets infected cells, in patients with HAM–TSP.MethodsIn this uncontrolled, phase 1–2a study, we assessed the safety, pharmacokinetics, and efficacy of mogamulizumab in patients with glucocorticoid-refractory HAM–TSP. In the phase 1 dose-escalation study, 21 patients received a single infusion of mogamulizumab (at doses of 0.003 mg per kilogram of body weight, 0.01 mg per kilogram, 0.03 mg per kilogram, 0.1 mg per kilogram, or 0.3 mg per kilogram) and were observed for 85 days. Of those patients, 19 continued on to the phase 2a study and received infusions, over a period of 24 weeks, of 0.003 mg per kilogram, 0.01 mg per kilogram, or 0.03 mg per kilogram at 8-week intervals or infusions of 0.1 mg per kilogram or 0.3 mg per kilogram at 12-week intervals.ResultsThe side effects of mogamulizumab did not limit administration up to the maximum dose (0.3 mg per kilogram). The most frequent side effects were grade 1 or 2 rash (in 48% of the patients) and lymphopenia and leukopenia (each in 33%). The dose-dependent reduction in the proviral load in peripheral-blood mononuclear cells (decrease by day 15 of 64.9%; 95% confidence interval [CI], 51.7 to 78.1) and inflammatory markers in cerebrospinal fluid (decrease by day 29 of 37.3% [95% CI, 24.8 to 49.8] in the CXCL10 level and of 21.0% [95% CI, 10.7 to 31.4] in the neopterin level) was maintained with additional infusions throughout the phase 2a study. A reduction in spasticity was noted in 79% of the patients and a decrease in motor disability in 32%.ConclusionsMogamulizumab decreased the number of HTLV-1–infected cells and the levels of inflammatory markers. Rash was the chief side effect. The effect of mogamulizumab on clinical HAM–TSP needs to be clarified in future studies. (Funded by the Japan Agency for Medical Research and Development and the Ministry of Health, Labor, and Welfare; UMIN trial number, UMIN000012655.)

Assessment of HTLV-1 proviral load, LAT, BIM, c-FOS and RAD51 gene expression in adult T cell leukemia/lymphoma.

Adult T cell leukemia/lymphoma (ATLL) is a life-threatening malignancy of HTLV-1 infected Th lymphocytes. In the present study host-virus interactions were investigated by assessment of HTLV-1 proviral load (PVL) and host gene expression. A cross-sectional study was carried out on 18 ATLL, 10 HAM/TSP patients and 18 HTLV-1 asymptomatic carriers (ACs). DNA and mRNA of the peripheral blood mononuclear cells were extracted for PVL and LAT, BIM, c-FOS and RAD51 gene expression measurement using qRT-PCR. The mean PVL in ATLL patients was 11,430±3770copies/104 which was statistically higher than ACs, 530±119copies/104, (p<0.001). The expression of BIM, and c-FOS in ATLL patients were higher than HTLV-1 ACs; however, there were no statistically significant differences. The expression of RAD51 as an essential player on DNA repair showed around 160 times increase in ATLL group (166±95) compared to ACs (1.04±0.34) which is statistically significant (p<0.001). Interestingly, there was a positive correlation between RAD51 expression and HTLV-PVL. The expression of LAT as a central adaptor in TCR signaling interestingly was around 36 times higher in ATLL group than ACs (ATLL; 41.33±19.91 vs. ACs; 1.15±0.22, p<0.001). This finding showed that TCR signaling pathway mainly provides the growth factors for transformed cells. Furthermore, the overexpression of RAD51 which has been induced in HTLV-1 infected cells as a consequence of virus replication is not able to overcome the DNA damage toward cell transformation.

Isolation of Exosomes from HTLV-Infected Cells.

Exosomes are small vesicles, approximately 30-100 nm in diameter, that transport various cargos, such as proteins and nucleic acids, between cells. It has been previously shown that exosomes can also transport viral proteins, such as the HTLV protein Tax, and viral RNAs, potentially contributing to disease pathogenesis. Therefore, it is important to understand their impact on recipient cells. Here, we describe methods of isolating and purifying exosomes from cell culture or tissue through ultracentrifugation, characterizing exosomes by surface biomarkers, and assays that evaluate the effect of exosomes on cells.

CADM1 Overexpression and CD7 Downregulation in Strongyloides stercoralis and HTLV-1 Coinfection as Possible Markers for Adult T-cell Leukemia/Lymphoma progression

Background Human T-cell lymphotropic virus (HTLV-1) is a retrovirus endemic in Latin America, Africa, and Asia. Increasing numbers are being reported in the United States. HTLV-1 causes Adult T-cell Leukemia/Lymphoma (ATL) in 3–5% of HTLV-1 carriers, usually only after a prolonged latent period. Co-infection with the nematode Strongyloides stercoralis (SS) is associated with early onset of ATL. The exact mechanism by which SS accelerates ATL development in HTLV-1 subjects is not understood. CADM1 has been recently identified as a surface marker of HTLV-1 infected cells; CD7 is a probable marker of early cell transformation. We hypothesize that previous SS infection will increase the proportion of infected T cells (CADM1+) and lead to transformation of infected cells (CADM1+CD7low) in HTLV-1 subjects.Methods In this pilot study, we tested seven subjects that were diagnosed with HTLV-1 between 2006 and 2008 and actively followed up at the HTLV-1 Cohort Clinic at the Tropical Medicine Institute in Lima, Peru. Five had previous SS infection. We performed surface flow cytometry staining of peripheral blood mononuclear cells with monoclonal antibodies against CADM1, CD7, CD4, and CD3. Current SS infection was ruled out by the Baermann technique in a stool sample.ResultsAverage age was 53 years (range 30–79). Average time since diagnosis of HTLV-1 was 10 years (range 9–11). One 30-year-old patient with prior SS infection was diagnosed with ATL and hospitalized in the ICU at the time of follow-up and excluded from group comparison. There was a trend for higher proportions of CADM1+ T-cells in subjects with previous SS co-infection compared with those without SS (median 16.20% vs. 9.55%, P = 0.13, Mann–Whitney). The proportion of PBMCs that were CADM1+CD7− was also increased in some of those with previous SS (Figure 1).Conclusion Our pilot data suggest that SS co-infection is associated with prolonged effects on HTLV-1 infection. We noted trends towards increased numbers of HTLV-1 infected cells (CADM1+) and a predisposition towards malignant transformation (CADM1+CD7low). Further studies are needed to confirm this observation and to determine whether CADM1 and CD7 expression may be useful as a screening tool to monitor HTLV-1 subjects at high risk of developing ATL.Figure 1.Representative flow cytometry.Disclosures All authors: No reported disclosures.

HTLV-1 Viral Factor HBZ Induces CCR4 to Promote T-cell Migration and Proliferation.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and other inflammatory diseases in infected individuals. However, a complete understanding of how HTLV-1 transforms T cells is lacking. Expression of the chemokine receptor CCR4 on ATL cells and HTLV-1-infected cells suggested the hypothesis that CCR4 may mediate features of ATL and inflammatory diseases caused by HTLV-1. In this study, we show that the constitutively expressed HTLV-1 bZIP factor (HBZ) encoded by HTLV-1 is responsible for inducing CCR4 and its ability to promote T-cell proliferation and migration. Ectopic expression of HBZ was sufficient to stimulate expression of CCR4 in human and mouse T cells. Conversely, HBZ silencing in ATL cell lines was sufficient to inhibit CCR4 expression. Mechanistic investigations showed that HBZ induced GATA3 expression in CD4(+) T cells, thereby activating transcription from the CCR4 promoter. In an established air pouch model of ATL, we observed that CD4(+) T cells of HBZ transgenic mice (HBZ-Tg mice) migrated preferentially to the pouch, as compared with those in nontransgenic mice. Migration of CD4(+) T cells in HBZ-Tg mice was inhibited by treatment with a CCR4 antagonist. Proliferating (Ki67(+)) CD4(+) T cells were found to express high levels of CCR4 and CD103. Further, CD4(+) T-cell proliferation in HBZ-Tg mice was enhanced by coordinate treatment with the CCR4 ligands CCL17 and 22 and with the CD103 ligand E-cadherin. Consistent with this finding, we found that ATL cells in clinical skin lesions were frequently positive for CCR4, CD103, and Ki67. Taken together, our results show how HBZ activates CCR4 expression on T cells to augment their migration and proliferation, two phenomena linked to HTLV-1 pathogenesis. Cancer Res; 76(17); 5068-79. ©2016 AACR.

Downregulation of proinflammatory cytokines in HTLV-1-infected T cells by Resveratrol

BackgroundHuman T-cell leukemia virus (HTLV-1) is a lymphotropic retrovirus associated to adult T cell leukemia (ATL) and to non-neoplastic inflammatory conditions affecting the central nervous system, lung or skin. The inflammatory disorders associated to HTLV-1 are mediated by different proinflammatory cytokines as IL-1α, IL-6, TNF-α. The release and the role of IL-17 is still debated. Aims of this study were to analyze IL-17 induction by HTLV-1 infection and to determine whether resveratrol (RES) is able to down regulate the pathway of cytokines production either in HTLV-1 chronically infected MT-2 cell line or in human CD4+ cells infected in vitro with HTLV-1.MethodsMT-2 and HTLV-1 infected CD4+ cells were analyzed for proinflammatory cytokine production before or after RES treatment. The concentrations of IL-17, IL-1α, IL-6, and TNF-α were measured in cell culture supernatants by ELISA and SearchLight™ technology. The IL-17 mRNA expression was evaluated by RT-PCR. NF-kB activation was detected by non-radioactive, Electro Mobility Shift Assay (EMSA). HTLV-1 RNA expression was detected by Real-time-PCR (RQ-PCR).ResultsWe found that RES is capable of inducing a dose-dependent inhibition of IL-1α, IL-6 and TNF-α production in vitro and can down regulate the expression of IL-17 at both mRNA and protein levels in HTLV-1 infected cells. This effect was associated with a dose-dependent inhibition of the of the nuclear factor kappa-B (NF-kB) activity. Conversely, RES did not apparently affect HTLV-1 proliferation.ConclusionsThese results support the anti-inflammatory properties of RES, suggesting that it might be a useful therapeutic agent for the treatment of HTLV-1 related inflammatory diseases.

Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells.

Polycomb-dependent epigenetic landscape in adult T-cell leukemia

AbstractAdult T-cell leukemia-lymphoma (ATL) shows global gene expression alterations that confer cellular characteristics and unfavorable prognosis. However, molecular mechanisms of the sustained expression changes are largely unknown, because there is no study addressing the relationship between landscapes of the gene expression and epigenetic modifications. Here, we analyzed ATL epigenome and integrated it with transcriptome from primary ATL cells and those from corresponding normal CD4+ T cells to decipher ATL-specific “epigenetic code” that was critical for cell identity. We found that polycomb-repressive complex 2 (PRC2)-mediated trimethylation at histone H3Lys27 (H3K27me3) was significantly and frequently reprogrammed at half of genes in ATL cells. A large proportion of the abnormal gene downregulation was detected at the early stage of disease progression and was explained by H3K27me3 accumulation. The global H3K27me3 alterations involved ATL-specific gene expression changes that included several tumor suppressors, transcription factors, epigenetic modifiers, miRNAs, and developmental genes, suggesting diverse outcomes by the PRC2-dependent hierarchical regulation. Interestingly, a key enzyme, EZH2, was sensitive to promiscuous signaling network including the NF-κB pathway and was functionally affected by human T-cell leukemia virus type I (HTLV-1) Tax. The Tax-dependent immortalized cells showed H3K27me3 reprogramming that was significantly similar to that of ATL cells. Of note, a majority of the epigenetic silencing has occurred in leukemic cells from indolent ATL and also in HTLV-1–infected T cells from asymptomatic HTLV-1 carriers. Because pharmacologic inhibition of EZH2 reversed epigenetic disruption and selectively eliminated leukemic and HTLV-1–infected cells, targeting the epigenetic elements will hold great promise in treatment and prevention of the onset of ATL and HTLV-1–related diseases.

Exosomes derived from HTLV-1 infected cells contain viral proteins and mRNA

The HTLV-1 transactivator protein Tax controls many critical cellular pathways including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Recently, exosomes have been shown to play critical roles during pathogenic viral infections as delivery vehicles for host and viral components including proteins, mRNA and miRNA. We hypothesized that exosomes derived from HTLV-1 infected cells contain unique host and viral proteins that may contribute to pathogenesis. We have characterized exosomes released from uninfected and HTLV-1 infected cell lines, as well as ATL and HAM/TSP material. The functional impact of exosomes derived from HTLV-1 infected cells on naive recipient cells was evaluated by utilizing transcription and reactive oxygen species (ROS) assays. Exosomes from HTLV-1 infected cells displayed unique proteomic profiles distinct from exosomes derived from uninfected cells. For instance, proinflammatory mediators are contained within the exosomes, as well as viral mRNA transcripts including Tax, HBZ, and Env. We found that exosomes from infected cells deliver functional Tax to naA¯ve recipient cells as well as cytokines. The release of factors was through Calcium and Calcium channels. We observed that exosomes released from HTLV-1 infected, Tax-expressing cells contributed to enhanced survival of target cells treated with α-FAS. Two other critical proteins were found in these exosomes that could contribute to overall activation of neighboring cells including Tax1BP1 and PrP. Both proteins are critical for activation of NFkB patway in recipient cells. Collectively, our results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. Furthermore, exosomes derived from infected cells are capable of inducing an ROS response in naA¯ve cells and contribute to an anti-apoptotic phenotype in cells treated with α-FAS.

HTLV-1 infected CD4+T cells of HAM/TSP patients are susceptible to tunicamycin-induced endoplasmic reticulum stress

Lectin array analysis for membrane proteins from CD4+Tcells of four respective cases of HAM, healthy carriers (HC), and negative controls (NC) showed significant high signal for Urtica dioica agglutinin (UDA) and Solanum tuberosum (Potato) (STL) lectins, that specifically recognize N-glycan, N-acetyllactosamine (LacNAc) which consist of repeats of the disaccharide βGal(1-4)βGlcNAc(1-3). We verified that LacNAc is significantly expressed in CD4+T cells in HAM by lectin ELISA for each ten cases of HAM, HC, and NC, followed by qRT-PCR of β1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) gene which was reported as main responsible enzyme in LacNAc biosynthesis. Using tunicamycin(TM), known as a N-glycan inhibitor and an apoptosis inducing agent through endoplasmic reticulum (ER) stress, we investigated the effect of LacNAc suppression on CD4+T cells. In NC, both of LacNAc+ and LacNAc-cells showed similar survival regardless of treatment with TM of 2μM for 24h or without TM, however, both cells in HAM showed worse survival than in NC and LacNAc+cells showed better survival when treated with TM. Moreover in HAM, Tax-cells showed better survival regardless of treatment with TM and LacNAc+cells when with treatment with TM survived significantly better than when without TM. Tax+cells showed worse survival than Tax-cells and tended to decrease when treated with TM. Collectively, we hypothesized that LacNAc is a resistant factor to TM-induced apoptosis in HTLV-1 uninfected cells but HTLV-1 infected cells may be susceptible to TM even if LacNAc are expressed. To confirm this hypothesis, we performed qRT-PCR of main effector genes in ER stress pathway: x-box binding protein 1, spliced variant (XBP1s), heat shock 70kDa protein 5(GRP78), and heat shock protein 90kDa beta, member 1(GRP94). Only GRP94 was significantly suppressed in HAM and HC. We conclude that HTLV-1 infected cells may be under ER stress and be susceptible to TM, and that LacNAc positive cells are resultant survivors.

EZH2 dependent epigenetic landscape in adult T cell leukemia and Tax immortalized cells

Epigenetic regulations globally determine gene transcription. Recent studies have revealed that expression changes and genetic mutations of epigenetic factors cause epigenetic imbalance in cancers. We previously reported that aberrant expression of Polycomb repressive complex 2 (PRC2) components causes constitutive NF-κB activation through silencing of miR-31 in ATL cells. However, the underlying mechanisms by which the epigenetic imbalance is induced and maintained remain to be elucidated. Here, we conducted ChIP-on-chip and transcriptome analyses of ATL and normal CD4+ T cells and found that the epigenetic reprogramming closely associates with ATL specific gene expression signature. Leukemic cell-specific silencing of cell cycle regulator, CDNK1A, was correlated with H3K27me3 level. In addition, orchestrated loss of microRNAs and multiple transcription factors appears to be mediated by the epigenetic mechanism. EZH2 upregulation and H3K27me3 accumulation were found in HTLV-1-infected populations derived from ATL patients as well as asymptomatic carriers. Furthermore, EZH2 inhibition blocked Tax-dependent cell growth and its immortalization in vitro. Intriguingly, the Tax-triggered immortalizing cells partially mimicked the methylation pattern observed in ATL cells, suggesting that the epigenetic alterations are closely involved in immortalization of infected cells and disease progression. In parallel, we found that over expression of PRC2 core components was induced by active signaling cascades including NF-κB in ATL cells. Collectively, our results suggest a coherent positive feedback mechanism comprised of PRC2 and NF-κB signaling, which stabilizes the epigenetic rearrangement and phenotypic outcomes in HTLV-1 infected cells. Since pharmacological inhibition of EZH2 selectively killed ATL and HTLV-1 infected cells in ex vivo culture, targeting the epigenetic elements will hold great promise in treatment and prevention of ATL and HTLV-1-related diseases.

Anti-CCR4 antibody activates virus specific immune response in STLV-1 infected Japanese monkey

Transgenic mouse model is useful to study pathogenesis of viral genes like tax and HBZ. However, it is impossible to study the host immune responses by these transgenic mice. We used simian T-cell leukemia virus type 1 (STLV-1) infected Japanese monkeys (JMs) to analyze host immune responses after treatment by anti-CCR4 monoclonal antibody (mAb). In the previous study, we reported that anti-CCR4 mAb treatment in STLV-1 infected JMs significantly reduced CCR4 positive cells and STLV-1 proviral load (PVL). In this study, we investigated the long-term effect (48 weeks) on PVL and immune responses to STLV-1 Tax (sTax) and bZIP factor (SBZ) after the anti-CCR4 mAb treatment. STLV-1 PVL in the JMs was kept at the lower level than 0 week even 48 weeks later. To further investigate the antiCCR4 mAb induced antiviral effect, we analyzed regulatory T (Treg) cells and sTax and SBZ specific T-cell responses. Treg cells, which were CCR4 positive, were rapidly reduced after the treatment. The number of activated Treg cells (most functional population) was severely suppressed. Furthermore, anti-CCR4 mAb treatment of infected CD4 T cells from the JMs enhanced engulfment by phagocytes, which likely enhances antigen-presentation. Treated JMs showed spontaneous activation of cytotoxic T-lymphocytes (CTLs) to sTax and SBZ. However, the immune response to other antigen was not enhanced. In consistent with these data, the enhanced T-cell responses (anti-Tax and HBZ) were also observed in anti-CCR4 mAb treated ATL patients. Taken together, anti-CCR4 mAb induces CTLs to sTax/Tax and HBZ/SBZ by two different mechanisms: reduced Treg cells and enhanced antigen presentation by antibodydependent cell-mediated phagocytosis. These effects of anti-CCR4 mAb induce prolonged suppression of PVL, which might enable long-term control of ATL and HTLV-1 infected cells.

IL8 Gene Polymorphism SNP rs4073 analysis between HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis and HTLV-1 Carriers

Human T-cell lymphotropic Virus Type I (HTLV-1) is a retrovirus that generates an inflammatory response that can trigger a neurological disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) which appears in 5-10% of infected people around the world. This inflammatory process is due the action of anti-Tax CD8+ lymphocytes that employ cytokines in order to stop the predominant infiltration of infected CD4+ T lymphocytes. IL8 is a pro-inflammatory cytokine that induces the activation and migration of neutrophils towards the sites of infection and it is continuously expressed in HTLV-1 infected cells. The SNP rs4073, located in the promoter of IL-8 gene, has been found associated to some inflammatory diseases and we hypothesized that it could be also a risk factor for the HAM/TSP development. The aim of our study was to evaluate the association of IL8 gene polymorphism SNP rs4073 and the presence of HAM/TSP. The study includes 243 infected subjects between asymptomatic (AC) and HAM/TSP patients. The genotyping of the polymorphisms was performed using the tetra-primer ARMS-PCR (Amplification Refractory Mutation System) method, 37 ancestry informative markers (AIMs) were used to correct by ethnicity and to avoid false positive results due to population stratification effect. The statistical analysis was conducted using the chi-square and Kruskal-Wallis test for possible differences among the groups. Linear and logistic regression analysis was performed using age, gender and ethnicity. The results show that none of the SNP rs4073 genotypes achieved a significant difference between AC and HAM/TSP, neither significant difference was found with proviral load (p>0.05). Our results suggest that the increase of the inflammatory response in HAM/TSP patients may not be caused by the SNP rs4073 in the promoter site of IL8, but the over expression of these gen could be possibly manage by another SNP or any other mutation in the enhancer region.

Anti-CCR4 monoclonal antibody KW-0761 (mogamulizumab) or investigator's choice in subjects with relapsed or refractory adult T-cell leukemia-lymphoma (ATL)

The receptor for macrophage derived chemokine (MDC) and thymus- and activation-regulated chemokine (TARC) CC chemokine receptor 4 (CCR4) is over expressed in several T-cell malignancies including HTLV-1 related ATL where approximately 90% of malignant cells have been shown to over express this chemokine receptor. The HTLV-1 transactivator gene (Tax) does not directly induce CCR4 expression. Rather, expression of CCR4 is controlled by the constitutive activation of several transcription factors in HTLV-1 infected cells. Inhibiting the expression of these transcription factors with small-interfering RNAs has been shown to block CCR4 expression and also reduce proliferation of the affected cells. Patients with CCR4 positive ATL are more likely to have skin involvement and shorter overall survival (OS; median 9.5 months) compared with CCR4-negative (20.6 months) patients. Mogamulizumab is a defucosylated, humanized, monoclonal antibody with enhanced antibody-dependent cellular cytotoxicity (Potelligent®) against primary ATL cells that bind to CCR4.

Tax peptide vaccine suppressed the leukemia in humanized mouse model of ATL

Cytotoxic T-lymphocyte (CTL) against HTLV-1 Tax has been demonstrated to play a vital role in controlling HTLV-1 infected cells in the infected carrier people. It was also shown that DNA immunization of mice with Tax expression vector suppressed the tumor growth of EL-4 lymphoma cells expressing Tax (EL4/Gax) through the activation of anti-Tax CTL [1]. Recently we established an HTLV-1-infected humanized mouse model (hu-NOG) to recapitulate ATL development and HTLV-1-specific immunity [2]. Here the efficacy of Tax-peptide vaccination was examined in the hu-NOG mouse model. Mixture of twelve overlapping peptides of 40-42 amino acids long encompassing whole Tax protein was inoculated subcutaneously three times weekly to hu-NOG mouse and then -irradiated HTLV-1 producing Jurkat cells were intraperitoneally injected to infect HTLV-1. While the control mice without immunization died of leukemia within two months of infection, Tax-immunization retarded the out growth of human lymphocytes after HTLV-1 infection and two out offive mice survived with alimited number of infected T-cells. IL-12 induction and the enhanced expression of Tax-specific CD8 T-cell were observed in the peripheral blood of immunized mice before infection. Furthermore, it was shown that the intranasal inoculation of Tax peptides has comparable effect on the onset of leukemia in the hu-NOG systems. These results suggest that the Tax peptide vaccination can elicit protective immunity against HTLV-1 infection and/or ATL developments.