Human T-cell Leukemia Virus Type 1 Infection Research Articles

The time-sequential changes of risk factors for adult T-cell leukemia development in human T-cell leukemia virus-positive patients with rheumatoid arthritis: a retrospective cohort study

Objective: This study aimed to investigate the time-sequential changes of risk factors for adult T-cell leukemia (ATL) development in human T-cell leukemia virus type 1 (HTLV-1)-positive rheumatoid arthritis (RA) patients.Methods: HTLV-1 infection was screened using particle agglutination assay and confirmed via western blotting in 365 RA patients. Twenty-three HTLV-1-positive RA patients were included in the study cohort. Blood samples were obtained from these patients at each observation time point. The values of HTLV-1 proviral load (PVL) and serum soluble IL-2 receptor (sIL2-R), which are risk factors for ATL development, were measured using real-time PCR and enzyme immunoassay, respectively.Results: The study cohort comprised 79 person-years. The median HTLV-1 PVL and sIL2-R values of the HTLV-1-positive RA patients were 0.44 copies per 100 white blood cells (WBCs) and 406 U/mL, respectively. Three HTLV-1-positive RA patients showed a high PVL value. No remarkable changes were observed in the PVL and sIL2-R values during the observation period. However, one elderly HTLV-1-positive RA patient who had a high PVL value developed ATL during treatment with methotrexate and infliximab.Conclusion: A thorough clinical assessment of the risk factors for ATL development may be necessary in daily clinical practice for RA patients in HTLV-1-endemic areas in Japan.

Dendritic Cells Promote the Spread of Human T-Cell Leukemia Virus Type 1 via Bidirectional Interactions with CD4+ T Cells

Human T-cell leukemia virus type 1 (HTLV-1) propagates within and between individuals via cell-to-cell transmission, and primary infection typically occurs across juxtaposed mucosal surfaces during breastfeeding or sexual intercourse. It is therefore likely that dendritic cells (DCs) are among the first potential targets for HTLV-1. However, it remains unclear how DCs contribute to virus transmission and dissemination in the early stages of infection. We show that an HTLV-1-infected cell line (MT-2) and naturally infected CD4+ T cells transfer p19+ viral particles to the surface of allogeneic DCs via cell-to-cell contacts. Similarly organized cell-to-cell contacts also facilitate DC-mediated transfer of HTLV-1 to autologous CD4+ T cells. These findings shed light on the cellular structures involved in anterograde and retrograde transmission and suggest a key role for DCs in the natural history and pathogenesis of HTLV-1 infection.

In vivo and in vitro immunogenicity of novel MHC class I presented epitopes to confer protective immunity against chronic HTLV-1 infection

Human T-cell leukemia virus type 1 (HTLV-1) has infected as many as 10 million people worldwide. While 90% are asymptomatic, 5% develop severe diseases including adult T-cell leukemia/lymphoka (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). No vaccine against HTLV-1 exists, and screening programs are not universal. However, patients with chronic HTLV-1 infection have high frequencies of HTLV-1-activated CD8+ T cells, and the two main HLA alleles (A2, A24) are present in 88% of infected individuals. We thus utilized an immunoproteomics approach to characterize MHC-I restricted epitopes presented by HLA-A2+, A24+ MT-2 and SLB-1 cell lines. Unlike traditional motif prediction algorithms, this approach identifies epitopes associated with cytotoxic T-cell responses in their naturally processed forms, minimizing differences in antigen processing and protein expression levels. Out of nine identified peptides, we confirmed six novel MHC-I restricted epitopes that were capable of binding HLA-A2 and HLA-A24 alleles and used in vitro and in vivo methods to generate CD8+ T cells specific for each of these peptides. MagPix MILLIPLEX data showed that in vitro generated epitope-specific CD8+ T cells secreted IFN-ɣ, granzyme B, MIP-1α, TNF-α, perforin and IL-10 when cultured in the presence of MT-2 cell line. Degranulation assay confirmed cytotoxic response through surface expression of CD107 on CD8+ T cells when cultured with MT-2 cells. A CD8+ T-cell killing assay indicated significant antiviral activity of CD8+ T cells specific against all identified peptides. In vivo generated CD8+ T cells similarly demonstrated immunogenicity on ELISpot, CD107 degranulation assay, and MagPix MILLIPLEX analysis. These epitopes are thus candidates for a therapeutic peptide-based vaccine against HTLV-1, and our results provide preclinical data for the advancement of such a vaccine.

NF-κB signaling mechanisms in HTLV-1-induced adult T-cell leukemia/lymphoma.

The human T-cell leukemia virus type 1 (HTLV-1) is a complex deltaretrovirus linked to adult T-cell leukemia/lymphoma (ATLL), a fatal CD4+ malignancy in 3-5% of infected individuals. The HTLV-1 Tax regulatory protein plays indispensable roles in regulating viral gene expression and activating cellular signaling pathways that drive the proliferation and clonal expansion of T cells bearing HTLV-1 proviral integrations. Tax is a potent activator of NF-κB, a key signaling pathway that is essential for the survival and proliferation of HTLV-1-infected T cells. However, constitutive NF-κB activation by Tax also triggers a senescence response, suggesting the possibility that only T cells capable of overcoming NF-κB-induced senescence can selectively undergo clonal expansion after HTLV-1 infection. Tax expression is often silenced in the majority of ATLL due to genetic alterations in the tax gene or DNA hypermethylation of the 5'-LTR. Despite the loss of Tax, NF-κB activation remains persistently activated in ATLL due to somatic mutations in genes in the T/B-cell receptor (T/BCR) and NF-κB signaling pathways. In this review, we focus on the key events driving Tax-dependent and -independent mechanisms of NF-κB activation during the multistep process leading to ATLL.

Natural Course of Human T-Cell Leukemia Virus Type 1 Proviral DNA Levels in Carriers During Pregnancy.

The measurement of human T-cell leukemia virus type 1 (HTLV-1) proviral DNA levels by using polymerase chain reaction has been beneficial for confirming HTLV-1 infection during pregnancy. However, the influence of pregnancy on HTLV-1 infection and proviral DNA levels among pregnant women with HTLV-1 has not been clarified. We prospectively gathered blood samples from 36 pregnant women in whom HTLV-1 carriage was previously diagnosed and sequentially measured their proviral DNA levels. The HTLV-1 proviral DNA levels remained at a plateau during pregnancy but were elevated after delivery. Moreover, flow cytometry and serological analyses revealed that the regulatory T-cell population and soluble interleukin 2 receptor levels were similarly elevated after birth in comparison with those in control pregnant women. This study is the first to provide data on sequential changes in HTLV-1 proviral DNA levels during and after pregnancy. These findings will guide the establishment of a better program to prevent mother-to-child transmission of HTLV-1.

HTLV-1 HBZ Viral Protein: A Key Player in HTLV-1 Mediated Diseases.

Human T cell leukemia virus type 1 (HTLV-1) is an oncogenic human retrovirus that has infected 10–15 million people worldwide. After a long latency, 3–5% of infected individuals will develop either a severe malignancy of CD4+ T cells, known as Adult T-cell Leukemia (ATL) or a chronic and progressive inflammatory disease of the nervous system designated Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (HAM/TSP). The precise mechanism behind HTLV-1 pathogenesis still remains elusive. Two viral regulatory proteins, Tax-1 and HTLV-1 bZIP factor (HBZ) are thought to play a critical role in HTLV-1-associated diseases. Tax-1 is mainly involved in the onset of neoplastic transformation and in elicitation of the host’s inflammatory responses; its expression may be lost during cell clonal proliferation and oncogenesis. Conversely, HBZ remains constantly expressed in all patients with ATL, playing a role in the proliferation and maintenance of leukemic cells. Recent studies have shown that the subcellular distribution of HBZ protein differs in the two pathologies: it is nuclear with a speckled-like pattern in leukemic cells and is cytoplasmic in cells from HAM/TSP patients. Thus, HBZ expression and distribution could be critical in the progression of HTLV-1 infection versus the leukemic state or the inflammatory disease. Here, we reviewed recent findings on the role of HBZ in HTLV-1 related diseases, highlighting the new perspectives open by the possibility of studying the physiologic expression of endogenous protein in primary infected cells.

HTLV-1, the Other Pathogenic Yet Neglected Human Retrovirus: From Transmission to Therapeutic Treatment.

Going back to their discovery in the early 1980s, both the Human T-cell Leukemia virus type-1 (HTLV-1) and the Human Immunodeficiency Virus type-1 (HIV-1) greatly fascinated the virology scene, not only because they were the first human retroviruses discovered, but also because they were associated with fatal diseases in the human population. In almost four decades of scientific research, both viruses have had different fates, HTLV-1 being often upstaged by HIV-1. However, although being very close in terms of genome organization, cellular tropism, and viral replication, HIV-1 and HTLV-1 are not completely commutable in terms of treatment, especially because of the opposite fate of the cells they infect: death versus immortalization, respectively. Nowadays, the antiretroviral therapies developed to treat HIV-1 infected individuals and to limit HIV-1 spread among the human population have a poor or no effect on HTLV-1 infected individuals, and thus, do not prevent the development of HTLV-1-associated diseases, which still lack highly efficient treatments. The present review mainly focuses on the course of HTLV-1 infection, from the initial infection of the host to diseases development and associated treatments, but also investigates HIV-1/HTLV-1 co-infection events and their impact on diseases development.

HLA-DMB restricts human T-cell leukemia virus type-1 (HTLV-1) protein expression via regulation of ATG7 acetylation

The roles of autophagy in viral infection are complicated. While autophagy has been shown to function in host antiviral defense by eliminating intracellular viruses and regulating adaptive immunity, several viruses have evolved molecular mechanisms to get benefits from it. The deltaretrovirus human T-cell leukemia virus type-1 (HTLV-1) has been reported to profit its replication from enhancing autophagosome accumulation. Here, we reported that HLA-DMB (generally referred to here as DMB), the beta chain of the non-classical MHC-II protein HLA-DM, had strong expression in HTLV-1-transformed T-cell lines and could be induced in Hela, PMA-differentiated THP1 (PMA-THP1) or primary human monocytes by HTLV-1 infection. Immunoblot and real-time PCR assays demonstrated that overexpression of DMB decreased HTLV-1 protein expression while the knockdown of DMB increased HTLV-1 protein expression. Immunoblot and confocal microscopy assays indicated that overexpression of DMB decreased HTLV-1 induced autophagosome accumulation while the knockdown of DMB yielded the opposite effects. Coimmunoprecipitation and immunoprecipitation experiments suggested DMB interacted with autophagy-related gene (ATG) 7 and increased the acetylation of ATG7. Taken together, these results suggested DMB modulated HTLV-1 protein expression through regulation of autophagosome accumulation and our findings suggested a new mechanism by which the host cells defended against HTLV-1 infection.

Positive and Negative Regulation of Type I Interferons by the Human T Cell Leukemia Virus Antisense Protein HBZ.

The pathogenesis of human T cell leukemia virus type 1 (HTLV-1) is strongly linked to the viral regulatory proteins Tax1 and HBZ, whose opposing functions contribute to the clinical outcome of infection. Type I interferons alpha and beta (IFN-α and IFN-β) are key cytokines involved in innate immunity, and IFN-α, in combination with other antivirals, is extensively used in the treatment of HTLV-1 infection. The relationship between HTLV-1 and IFN signaling is unclear, and to date the effect of HBZ on this pathway has not been examined. Here we report that HBZ significantly enhances interferon regulatory factor 7 (IRF7)-induced IFN-α- and IFN-stimulated response element (ISRE) promoter activities and IFN-α production and can counteract the inhibitory effect of Tax1. In contrast to this, we show that HBZ and Tax1 cooperate to inhibit the induction of IFN-β and ISRE promoters by IRF3 and IFN-β production. In addition, we reveal that HBZ enhances ISRE activation by IFN-α. We further show that HBZ enhances IRF7 and suppresses IRF3 activation by TBK1 and IKKε. We demonstrate that HBZ has no effect on virus-induced nuclear accumulation of IRF3, suggesting that it may inhibit IRF3 activity at a transcriptional level. We show that HBZ physically interacts with IRF7 and IKKε but not with IRF3 or TBK1. Overall, our findings suggest that both HBZ and Tax1 are negative regulators of immediate early IFN-β innate immune responses, while HBZ but not Tax1 positively regulates the induction of IFN-α and downstream IFN-α signaling.IMPORTANCE Type I interferons are powerful antiviral cytokines and are used extensively in the treatment of HTLV-1-induced adult T cell leukemia (ATL). To date, the relationship between HTLV-1 and the IFN pathway is poorly understood, and studies so far have focused on Tax1. Our study is unique in that it examined the effect of HBZ, alone or in combination with Tax1, on type I IFN signaling. This is important because HBZ is frequently the only viral protein expressed in infected cells, particularly at later stages of infection. A better understanding of the how HBZ regulates IFN signaling may lead to the development of therapeutics that can modify such responses and improve the clinical outcome for infected individuals.

IL-10-mediated signals act as a switch for lymphoproliferation in Human T-cell leukemia virus type-1 infection by activating the STAT3 and IRF4 pathways.

Human T-cell leukemia virus type-1 (HTLV-1) causes two distinct diseases, adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since there are no disease-specific differences among HTLV-1 strains, the etiological mechanisms separating these respective lymphoproliferative and inflammatory diseases are not well understood. In this study, by using IL-2-dependent HTLV-1-infected T-cell lines (ILTs) established from patients with ATL and HAM/TSP, we demonstrate that the anti-inflammatory cytokine IL-10 and its downstream signals potentially act as a switch for proliferation in HTLV-1-infected cells. Among six ILTs used, ILTs derived from all three ATL patients grew much faster than those from three HAM/TSP patients. Although most of the ILTs tested produced IFN-γ and IL-6, the production of IL-10 was preferentially observed in the rapid-growing ILTs. Interestingly, treatment with exogenous IL-10 markedly enhanced proliferation of the slow-growing HAM/TSP-derived ILTs. The IL-10-mediated proliferation of these ILTs was associated with phosphorylation of STAT3 and induction of survivin and IRF4, all of which are characteristics of ATL cells. Knockdown of STAT3 reduced expression of IL-10, implying a positive-feedback regulation between STAT3 and IL-10. STAT3 knockdown also reduced survivin and IRF4 in the IL-10- producing or IL-10- treated ILTs. IRF4 knockdown further suppressed survivin expression and the cell growth in these ILTs. These findings indicate that the IL-10-mediated signals promote cell proliferation in HTLV-1-infected cells through the STAT3 and IRF4 pathways. Our results imply that, although HTLV-1 infection alone may not be sufficient for cell proliferation, IL-10 and its signaling pathways within the infected cell itself and/or its surrounding microenvironment may play a critical role in pushing HTLV-1-infected cells towards proliferation at the early stages of HTLV-1 leukemogenesis. This study provides useful information for understanding of disease mechanisms and disease-prophylactic strategies in HTLV-1 infection.

Early growth response gene-1 regulates host cell autophagy in HTLV-1 infection

Objective To investigate the influence of early growth response gene-1 (EGR1) on the autophagy of host cells following infection with human T cell leukemia virus type 1 (HTLV-1). Methods A HTLV-1-positive cell line MT2 was co-cultured with HeLa cells for 24 h to construct the virus early infection model. Immunoblotting assay was used to detect the expression of HTLV-1 core protein p19 and EGR1. Luciferase reporter gene analysis was used to detect the transcriptional activity of 5′-regulatory sequence of EGR1 at different time points after co-culturing. An effective small interfering RNA (siRNA) targeting EGR1 was screened out and transfected into HeLa cells by Lipofectamine 2000. Then the transfected HeLa cells were co-cultured with the HTLV-1-positive cell line MT2 for 24 h. Immunoblotting assay was used to detect HTLV-1 core protein p19, EGR1 and autophagy-related protein LC3. Real-time PCR was performed to detect viral load. Autophagosome was analyzed by immunofluorescence after co-culturing. Results The expression of EGR1 and the transcriptional activity of pEGR1-luc gradually increased after co-culturing HeLa cells with MT2 cells for 8 h (P<0.01). The expression of EGR1 was positively correlated with host cell autophagy following HTLV-1 infection. The effective siRNA for silencing the expression of EGR1 was obtained and named as siE2. The viral load, the expression of HTLV-1 core protein p19 and the proportion of LC3B/LC3A in the co-culture model were markedly down-regulated by RNA interference with siE2, which was concomitant with a persistent decrease of intracellular autophagosome (P<0.01). Conclusion EGR1 is associated with host cell autophagy and viral replication in HTLV-1 infection. Key words: Early growth response gene-1; Human T-cell leukemia virus type 1; Autophagy; Virus replication

Genetic alterations in adult T-cell leukemia/lymphoma.

Adult T‐cell leukemia/lymphoma (ATL) is a peripheral T‐cell neoplasm with a dismal prognosis. It is caused by human T‐cell leukemia virus type‐1 (HTLV‐1) retrovirus. A long latency period from HTLV‐1 infection to ATL onset suggests that not only HTLV‐1 proteins, such as Tax and HBZ, but also additional genetic and/or epigenetic events are required for ATL development. Although many studies have demonstrated the biological functions of viral genes, alterations of cellular genes associated with ATL have not been fully investigated. Recently, a large‐scale integrated genetic analysis revealed the entire landscape of somatic aberrations in ATL. This neoplasm is characterized by frequent gain‐of‐function alterations in components of the T‐cell receptor/NF‐κB signaling pathway, including activating mutations in the PLCG1,PRKCB,CARD11 and VAV1 genes, and CTLA4‐CD28 and ICOS‐CD28 fusions. Importantly, molecules associated with immune surveillance, such as HLA‐A/B,CD58 and FAS, are affected recurrently. Among them, one notable lesion occurs as frequent structural variations that truncate the PD‐L1 3′‐untranslated region, leading to its overexpression. Other genetic targets include transcription factors (IRF4,IKZF2, and GATA3) and chemokine receptors (CCR4,CCR7 and GPR183), which are functionally relevant in normal T cells. A substantial proportion of ATL cases show widespread accumulation of repressive epigenetic changes, such as trimethylation of histone H3 lysine 27 and DNA hypermethylation of CpG islands, which coordinately modulate multiple pathways, including Cys2‐His2 zinc finger genes involved in silencing retroelements. Here we review the current understanding of the genetic/epigenetic aberrations in ATL, focusing on their relevance in its molecular pathogenesis.

HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling

Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target.

A novel mother-to-child human T-cell leukaemia virus type 1 (HTLV-1) transmission model for investigating the role of maternal anti-HTLV-1 antibodies using orally infected mother rats.

Human T-cell leukaemia virus type 1 (HTLV-1) is a human retrovirus that is a causative agent of adult T-cell leukaemia/lymphoma (ATL) and is mainly transmitted from an infected mother to her child via breastfeeding. Such an HTLV-1 infection during childhood is believed to be a risk factor for ATL development. Although it has been suggested that an increased proviral load (PVL), a higher titre of antibody (Ab) in the infected mother and prolonged breastfeeding are associated with an increased risk of mother-to-child transmission (MTCT), the mechanisms underlying MTCT of HTLV-1 remain largely unknown. In this study, we developed an MTCT model using orally HTLV-1-infected rats that have no Ab responses against viral antigens, such as Gag and Env. In this model, HTLV-1 could be transmitted from the infected mother rats to their offspring at a high rate (50-100 %), and the rate of MTCT tended to be correlated with the PVL of the infected mother rats. Furthermore, passive immunization of uninfected adult rats and an infected mother rat with a rat anti-HTLV-1 Env gp46-neutralizing mAb was unable to suppress primary oral HTLV-1 infection to the adult rats and vertical HTLV-1 transmission to the offspring, respectively. Our findings indicate that this MTCT model would be useful to investigate not only the mechanisms of MTCT but also the role of anti-HTLV-1 Ab in MTCT of HTLV-1. They also provide some information on the role of maternal Abs in MTCT, which should be considered when designing a strategy for prevention of MTCT of HTLV-1.

Influence of cellular autophagy on replication dynamics of human T cell leukemia virus typeI(HTLV-1) in HeLa cells

Objective To investigate the replication dynamics and cellular states of host cells with human T cell leukemia virus type 1 (HTLV-1) infection. Methods HTLV-1 positive cell line MT2 was co-cultured with HeLa cells for 4 to 36 hours to acquire the virus early infection model. Immunoblotting was used to detect HTLV-1 core protein p19, apoptosis-related proteins caspase-3/9 and autophagy-related protein LC3 at different time points. Real-time PCR was used to measure viral loads. Luciferase reporter gene assay was used to detect the enzymatic activities of caspase-3/7/9 and the transcriptional activities of 3′-LTR at different time points after co-culturing. Autophagosomes were analyzed by immunofluorescence after co-culturing. The HTLV-1 long terminal repeat reporter gene (pHTLV-1-LTR-luc) was transfected into HeLa cells by lipidosome 2000-mediated transfection and the luciferase activities at different time points were detected. Results The HTLV-1 infection model was successfully constructed by co-culturing MT2 cells with HeLa cells in vitro. Viral load and transcriptional activity of the 3′-LTR gradually increased at 4 to 16 h after virus infection (P 0.05), but the proportion of LC3B/LC3A showed a gradual increasing trend with the persistent increase of autophagosomes (P<0.01). Conclusion Viral replication dynamics of HTLV-1 is closely related to autophagy in host cells. Key words: Human T cell leukemia virus type 1; Replication dynamics; Apoptosis; Autophagy

Analysis of HTLV-1 proviral load (PVL) and antibody detected with various kinds of tests in Japanese blood donors to understand the relationship between PVL and antibody level and to gain insights toward better antibody testing.

Adult T-cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T-cell leukemia virus type 1 (HTLV-1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti-HTLV-1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real-time PCR and other tests in 600 HTLV-1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV-1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV-1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV-1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus-positive samples, probably suggesting that antibody-based screening tests should incorporate multiple HTLV-1 antigens, such as Gag and Env antigens.

HTLV-1 Tax-Specific CTL Epitope-Pulsed Dendritic Cell Therapy Reduces Proviral Load in Infected Rats with Immune Tolerance against Tax.

Adult T cell leukemia/lymphoma (ATL), a CD4+ T cell malignancy with a poor prognosis, is caused by human T cell leukemia virus type 1 (HTLV-1) infection. High proviral load (PVL) is a risk factor for the progression to ATL. We previously reported that some asymptomatic carriers had severely reduced functions of CTLs against HTLV-1 Tax, the major target Ag. Furthermore, the CTL responses tended to be inversely correlated with PVL, suggesting that weak HTLV-1-specific CTL responses may be involved in the elevation of PVL. Our previous animal studies indicated that oral HTLV-1 infection, the major route of infection, caused persistent infection with higher PVL in rats compared with other routes. In this study, we found that Tax-specific CD8+ T cells were present, but not functional, in orally infected rats as observed in some human asymptomatic carriers. Even in the infected rats with immune unresponsiveness against Tax, Tax-specific CTL epitope-pulsed dendritic cell (DC) therapy reduced the PVL and induced Tax-specific CD8+ T cells capable of proliferating and producing IFN-γ. Furthermore, we found that monocyte-derived DCs from most infected individuals still had the capacity to stimulate CMV-specific autologous CTLs in vitro, indicating that DC therapy may be applicable to most infected individuals. These data suggest that peptide-pulsed DC immunotherapy will be useful to induce functional HTLV-1-specific CTLs and decrease PVL in infected individuals with high PVL and impaired HTLV-1-specific CTL responses, thereby reducing the risk of the development of ATL.

Hematopoietic Stem Cell Infected with HTLV-1 Functions As a Viral Reservoir In Vivo

▪Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of a malignant disease of peripheral CD4+ T cells called adult T-cell leukemia-lymphoma (ATL) and several inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although major target of HTLV-1 is CD4+ T cells, other hematopoietic cells such as CD8+ T cells and monocytes are also infected with HTLV-1. Since the receptors of HTLV-1 are glucose transporter 1 and neuropilin 1, which are found on various cell surfaces, it is possible that HTLV-1 infects various hematopoietic cells and hematopoietic stem cells (HSCs). However, the previous studies could not detect HTLV-1 in HSCs. To assess the distribution of infected cells and expression of viral genes in various tissues, a nonhuman primate model, Japanese macaques (JMs) infected with simian T-cell leukemia virus type 1 (STLV-1) was utilized in this study. STLV-1 is a close relative of HTLV-1, and the dynamics of viral replication and proliferation of infected cells are very similar to each other. Indeed, STLV-1 caused malignant lymphomas in STLV-1 infected monkeys. Therefore, STLV-1 infected JMs are good models of HTLV-1 carriers. Using this model, we first analyzed transcription level of two viral genes, tax and STLV-1 bZIP factor, in multiple tissues, and found that tax was highly expressed in bone marrow compared to other tissues. Since Tax is a potent activator of viral transcription, this result suggested that viral replication occurred in bone marrow. To evaluate which cells express Tax in bone marrow, we performed flow cytometric analysis of bone marrow mononuclear cells from STLV-1 infected monkeys, and found that not only CD4+ T cells but also non T cells (CD3 negative cells) expressed Tax. To determine whether hematopoietic stem cells are infected with HTLV-1, we next performed highthroughput sequencing of HTLV-1 integration sites in multiple cell lineages using a next generation sequencer. Since HTLV-1 is randomly integrated into genome of infected cells, and each infected cell can proliferate clonally, we can interpret that cells sharing same integration sites are derived from the same precursor cell. Blood samples from patients with HAM/TSP were separated into five cell types: CD4+ T cells, CD8+ T cells, B cells, monocytes, and neutrophils. Integration sites of HTLV-1 provirus were analyzed by next generation sequencing using different chips for each cell type in order to avoid cross contamination between the samples. The results showed that some of these cells have same integration sites between different cell types. More than 40 percent of HTLV-1 infected B cells, monocytes and neutrophils had shared the same integration sites, suggesting that HSCs in the HAM/TSP patients were infected with HTLV-1. Importantly, most HTLV-1 infected CD4+ T cells and CD8+ T cells had independent integration sites, while some of them shared the same integration sites with non T cell subsets. These results implied that most T-cell clones were generated by de novo infection in the periphery, but a part of infected T cells were derived from infected HSCs. To assess whether HTLV-1 infected HSCs persist in vivo, we again analyzed integration sites in neutrophils from the same HAM/TSP patients after 1 year. About 50 percent of integration sites in HTLV-1 infected neutrophils were detected after 1 year. Considering short lifespan of neutrophils, this result suggests that HTLV-1 infected HSCs could be maintained for at least 1 year in vivo. To visualize HTLV-1 infection in myeloid cells, we performed immunofluorescence staining of neutrophils from HAM/TSP patients. Tax and myeloperoxidase were detected in neutrophils. Finally, we evaluated the significance of infected monocytes in viral transmission. We isolated monocytes from HAM/TSP patients and co-cultured with JET WT35 cell, which is a subline of Jurkat containing a fluorescent reporter of viral infection. The result demonstrated that monocyte from HAM/TSP patients could be a source of infective HTLV-1. In conclusion, our findings suggest that HSCs infected with HTLV-1 survive for long time in vivo, and could be reservoirs of the virus. Since ATL is a difficult disease to cure, further studies are required to understand the nature of HTLV-1 infection. DisclosuresNo relevant conflicts of interest to declare.

Absolute quantification of HTLV-1 basic leucine zipper factor (HBZ) protein and its plasma antibody in HTLV-1 infected individuals with different clinical status.

BackgroundHuman T cell leukemia virus type 1 (HTLV-1) basic leucine zipper factor (HBZ), which is encoded by a minus strand mRNA, is thought to play important roles in the development of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). However, a comprehensive analysis of HBZ, including mRNA and protein expression, humoral immunoreactivity against HBZ, and HTLV-1 proviral load (PVL), in HTLV-1-infected individuals with different clinical status has not been reported previously.ResultsIn this study, using novel monoclonal antibody-based in-house enzyme-linked immunosorbent assay systems, we report the absolute quantification of HBZ protein and its plasma antibody in clinical samples from HTLV-1-infected individuals with different clinical status. The data were compared to both HBZ mRNA levels and PVL. The results showed that plasma anti-HBZ antibody was detectable only in 10.4 % (5/48) of asymptomatic carriers (ACs), 10.8 % (13/120) of HAM/TSP patients, and 16.7 % (7/42) of ATL patients. HBZ protein was detected in three out of five patients with acute ATL, but was not detected in patients with HAM/TSP (0/10) or ACs (0/4). Thus, an antibody response to HBZ was not associated with the PVL or the expression of HBZ (both at the mRNA and protein levels) or the clinical status of the infection.ConclusionsThe present results emphasize the extremely low expression and immunogenicity of HBZ in natural HTLV-1 infection. However, there is a possibility that the low but distinct expression of HBZ protein in PBMCs is associated with the survival of HTLV-1-infected cells and the development of ATL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0263-z) contains supplementary material, which is available to authorized users.

Genetic Markers of the Host in Persons Living with HTLV-1, HIV and HCV Infections.

Human T-cell leukemia virus type 1 (HTLV-1), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) are prevalent worldwide, and share similar means of transmission. These infections may influence each other in evolution and outcome, including cancer or immunodeficiency. Many studies have reported the influence of genetic markers on the host immune response against different persistent viral infections, such as HTLV-1 infection, pointing to the importance of the individual genetic background on their outcomes. However, despite recent advances on the knowledge of the pathogenesis of HTLV-1 infection, gaps in the understanding of the role of the individual genetic background on the progress to disease clinically manifested still remain. In this scenario, much less is known regarding the influence of genetic factors in the context of dual or triple infections or their influence on the underlying mechanisms that lead to outcomes that differ from those observed in monoinfection. This review describes the main factors involved in the virus–host balance, especially for some particular human leukocyte antigen (HLA) haplotypes, and other important genetic markers in the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and other persistent viruses, such as HIV and HCV.