Human T-lymphotropic Virus Type 1 Tax Research Articles

Diversity of HLA-A2-Restricted and Immunodominant Epitope Repertoire of Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Protein: Novel Insights among N-Terminal, Central and C-Terminal Regions

The present study sought to search for the immunodominance related to the N-terminal, Central and C-terminal regions of HTLV-1 Tax using novel, cutting-edge peptide microarray analysis. In addition, in silico predictions were performed to verify the presence of nine amino acid peptides present along Tax restricted to the human leukocyte antigen (HLA)-A2.02*01 haplotype, as well as to verify the ability to induce pro-inflammatory and regulatory cytokines, such as IFN-γ and IL-4, respectively. Our results indicated abundant dose-dependent reactivity for HLA-A*02:01 in all regions (N-terminal, Central and C-terminal), but with specific hotspots. Furthermore, the results of fold-change over the Tax11-19 reactivity obtained at lower concentrations of HLA-A*02:01 reveal that peptides from the three regions contain sequences that react 100 times more than Tax11-19. On the other hand, Tax11-19 has similar or superior HLA-A*02:01 reactivity at higher concentrations of this haplotype. The in silico analysis showed a higher frequency of IFN-γ-inducing peptides in the N-terminal portion, while the C-terminal portion showed a higher frequency of IL-4 inducers. Taken together, these results shed light on the search for new Tax immunodominant epitopes, in addition to the canonic Tax11-19, for the rational design of immunomodulatory strategies for HTLV-1 chronic diseases.

Following the Clues: Usefulness of Biomarkers of Neuroinflammation and Neurodegeneration in the Investigation of HTLV-1-Associated Myelopathy Progression.

Human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurodegenerative disease due to axonal damage of the corticospinal secondary to an inflammatory response against infected T-cells. In the present work, we aimed to evaluate biomarkers of neurodegeneration and neuroinflammation in the definition of HAM/TSP prognosis. Neurofilament light (NfL) and phosphorylated heavy (pNfH) chains, total Tau protein, cellular prion protein (PrPc), inflammatory chemokines, and neopterin were quantified in paired cerebrospinal fluid (CSF) and serum samples from HAM/TSP patients (n=21), HTLV-1 asymptomatic carriers (AC) (n=13), and HTLV-1 seronegative individuals with non-inflammatory non-degenerative neurological disease (normal-pressure hydrocephalus) (n=9) as a control group. HTLV-1 proviral load in peripheral blood mononuclear cells and the expression of chemokine receptors CCR4, CCR5, and CXCR3 in infected CD4+ T-cells (HTLV-1 Tax+ cells) were also assessed. CSF levels of Tau, NfL, and pNfH were similar between groups, but PrPc and neopterin were elevated in HAM/TSP patients. Most individuals in the control group and all HTLV-1 AC had CSF/serum neopterin ratio < 1.0, and two-thirds of HAM/TSP patients had ratio values > 1.0, which positively correlated with the speed of disease progression and pNfH levels, indicating active neuroinflammation. HAM/TSP patients showed high serum levels of CXCR3-binding chemokines (CXCL9, CXCL10, and CXCL11) and elevated CSF levels of CCL2, CCL3, CCL4, CCL17, CXCL5, CXCL10, and CXCL11. Indeed, CXCL10 concentration in CSF of HAM/TSP patients was 5.8-fold and 8.7-fold higher in than in HTLV-1 AC and controls, respectively, and correlated with CSF cell counts. HAM/TSP patients with typical/rapid disease progression had CSF/serum CXCL10 ratio > 1.0 and a higher frequency of CXCR3+Tax+CD4+ T-cells in blood, which indicated a positive gradient for the migration of infected cells and infiltration into the central nervous system. In conclusion, the slow progression of HAM/TSP abrogates the usefulness of biomarkers of neuronal injury for the disease prognosis. Thus, markers of inflammation provide stronger evidence for HAM/TSP progression, particularly the CSF/serum neopterin ratio, which may contribute to overcome differences between laboratory assays.

Polymorphisms in HTLV-1 Tax-responsive elements in HTLV-1-associated myelopathy/tropical spastic paraparesis patients are associated with reduced proviral load but not with disease progression.

Human T-lymphotropic virus type 1 (HTLV-1) provirus expression is mainly directed by Tax-responsive elements (TRE) within the long terminal repeats (LTR). Mutations in TRE can reduce provirus expression and since a high proviral load (PVL) is a risk factor for the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we evaluated polymorphisms in the 5' LTR and the association with PVL and disease progression. HTLV-1 LTR and tax sequences derived from asymptomatic carriers (AC) and HAM/TSP patients followed in a longitudinal study were analysed according to PVL and clinical severity. Individuals infected with HTLV-1 presenting the canonical TRE, considering strain ATK-1 as the consensus, displayed sustained higher PVL. By contrast, an LTR A125G mutation in TRE was associated with slightly reduced PVL only in HAM/TSP patients, although it did not influence the speed of disease progression. Moreover, this polymorphism was frequent in Latin American strains of the HTLV-1 Cosmopolitan Transcontinental subtype. Therefore, polymorphisms in the 5' TRE of HTLV-1 may represent one of the factors influencing PVL in HAM/TSP patients, especially in the Latin American population. Indeed, higher PVL in the peripheral blood has been associated with an increased inflammatory activity in the spinal cord and to a poorer prognosis in HAM/TSP. However, this event was not associated with TRE polymorphisms.

In Vivo Tumorigenesis, Osteolytic Sarcomas, and Tumorigenic Cell Lines from Transgenic Mice Expressing the Human T-Lymphotropic Virus Type 1 (HTLV-1) Tax Viral Oncogene

Human T-lymphotropic virus type 1 (HTLV-1) causes adult T-cell leukemia, a disease commonly associated with hypercalcemia and osteolysis. There is no effective treatment for HTLV-1, and the osteolytic mechanisms are not fully understood. Mice expressing the HTLV-1 oncogene Tax, driven by the human granzyme B promoter (Tax+), develop osteolytic tumors. To investigate the progression of the bone-invasive malignancies, wild-type, Tax+, and Tax+/interferon-γ-/- mice were assessed using necropsy, histologic examination, IHC analysis, flow cytometry, and advanced imaging. Tax+ and Tax+/interferon-γ-/- malignancies of the ear, tail, and foot comprised poorly differentiated, round to spindle-shaped cells with prominent neutrophilic infiltrates. Tail tumors originated from muscle, nerve, and/or tendon sheaths, with frequent invasion into adjacent bone. F4/80+ and anti-mouse CD11b (Mac-1)+ histiocytic cells predominated within the tumors. Three Tax+/interferon-γ-/- cell lines were generated for invivo allografts, invitro gene expression and bone resorption assays. Two cell lines were of monocyte/macrophage origin, and tumors formed invivo in all three. Differences in Pthrp, Il6, Il1a, Il1b, and Csf3 expression invitro were correlated with differences in invivo plasma calcium levels, tumor growth, metastasis, and neutrophilic inflammation. Tax+ mouse tumors were classified as bone-invasive histiocytic sarcomas. The cell lines are ideal for further examination of the role of HTLV-1 Tax in osteolytic tumor formation and the development of hypercalcemia and tumor-associated inflammation.

First Description of Seronegative HTLV-1 Carriers in Argentina.

In some areas of Argentina endemic for human T-lymphotropic virus type 1 (HTLV-1), tropical spastic paraparesis is frequent in subjects who lack antibodies against the virus; however, the relevance of this seronegative status in the country has not been investigated. In neighboring countries, HTLV-1 seronegative status has been described in patients with different diseases; however, data regarding features of seronegative HTLV-1 carriers are scarce. We investigated the seronegative status in 124 relatives of 28 HTLV-1 infected subjects from an endemic area in Northwest Argentina. Blood samples and clinical/epidemiological data were collected. Human T-lymphotropic virus type 1 infection was diagnosed by serology and long terminal repeat (LTR) sequence, env and tax gene detection. IgG anti-Tax HTLV-1 antibody, tax gene sequence, and DNA proviral load were also evaluated. Seventy-five percent of the 124 relatives were negative for HTLV-1/2 antibodies; 35.5% were also negative by molecular assays and 64.5% were negative for HTLV-1 LTR and env sequences, but positive for two sequences of HTLV-1 tax gene. Also, 35.7% of these subjects had IgG anti-Tax antibodies. The seronegative HTLV-1 status was significantly associated with male gender, youth, and sensory symptoms/autonomic nervous system dysfunction. High rates of seronegative symptomatic and asymptomatic HTLV-1 carriers in Argentina are described. The evidence highlights that HTLV-1 prevalence may be underestimated worldwide. Larger cohort studies are required to assess disease outcome in these seronegative subjects. Also, the findings emphasize the limitations of ongoing screening assays for diagnosis and blood safety. Therefore, algorithms for HTLV-1 diagnosis should include not only serological but also molecular assays.

Novel Cellular Immunotherapy Using Allogeneic Vγ9/δ2-T Cells Gene-Modified to Express HTLV-1 P40Tax-Specific TCR for the Treatment of Adult T Cell Leukemia

Background: Adult T-cell leukemia/lymphoma (ATL) is a refractory peripheral T-cell malignancy caused by human T-lymphotropic virus type 1 (HTLV-1) infection. Although only allogeneic hematopoietic stem cell transplantation (allo-HSCT) displaying the graft-vs. ATL (GvATL) can bring durable remission, allo-HSCT is largely ineligible for newly diagnosed ATL patients due to disease aggressiveness and advanced age-related conditions. Thus, a novel treatment with safety and efficacy instead of allo-HSCT still remains an unmet need, and a cellular immunotherapy using TCR or CAR gene-modified immune cells exerting GvATL could be such an option. However, to generate those effector cells from autologous T cells of heavily pre-treated ATL patients faces many obstacles. To circumvent those hurdles, an employment of unconventional allogeneic Vγ9/δ2-T cells which are potentially free from the risk of GVHD could provide greater treatment opportunity for ATL patients due to highly extended donor availability. Taking above, here, we have newly devised an adoptive immunotherapy using a novel HTLV-1 p40Tax-specific TCR gene-modified allogeneic Vγ9/δ2 T cells against ATL. Methods: After written informed conscent, we firstly established novel HLA-A24 restricted TCR-α/β genes from HTLV-1 P40Tax301-309 (SFHSLHLLF)/HLA-A24 tetramer-positive peripheral CD8+T-lymphocytes of ATL patinets in durable remission using a single cell cloning method. Then, we confirmed that T cells gene-modified with these TCR-α/β genes exerted the epitope-specific and HLA-A24-restricted responses. Next, in order to achieve highly stable expression of this TCR-α/β heterodimer on gene-modified Vγ9/δ2-T cells, we newly developed a retroviral vector co-expressing TCR-α/β and CD8 α/β genes using self-cleaving P2A and E2A peptides. Using this vector, allogeneic Vγ9/δ2-T cells from healthy donors numerously expanded with high purity in our novel culture system were subjected to gene-transfer to express relevant TCR α/β complex. Thereafter, we asssessed target-reactive cytokine production and cytocidal activity mediated by those gene-modified allogeneic Vγ9/δ2-T cells both in vitro and in vivo. Finally, we additionally assessed a potential risk of GVHD using intravenous administration of another TCR gene-modified Vγ9/δ2 T-cells in vivo. Results: To start with PBMCs from healthy donors, allogeneic Vγ9/δ2-T cells were stably multiplied greater than thousandfold with a quite high purity (≥95%) using our novel bisphosphonate derivative PTA (tetrakis-pivaloyloxymethyl2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate) combined with both 25 ng/ml of IL-7 and IL-15 in culture for 8 to 10 days. The stable expression of introduced TCR α/β heterodimer on Vγ9/δ2-T cells were successfully achieved by co-expression of CD8 α/β molecule. Those gene-modified Vγ9/δ2-T cells successfully recognized target peptide (SFHSLHLLF) in an HLA-A24 restricted fashion, and similarly demonstrated a cytocidal activity both in vitro and in vivo against HLA-A24 positive HTLV-1 infected cell lines (TL-Su and ILT#Hod), but not HLA-A24-negtive/Tax-positive cell line ILT#37 or HLA-A24-positve/Tax-negative cell line ATN-1. Furthermore, intravenously administered those TCR gene-modified Vγ9/δ2-T cells quickly and durably eradicated luciferase-gene modified TL-Su cells, but not ATN-1 cells in xenografted immunodeficient (NOG) mice, examined by in vivo imaging system. Finally, infused HLA-A2 restricted and NY-ESO-1 specific TCR (G50) gene-modified Vγ9/δ2-T cells exerted durable antitumor activity without causing GVHD using NOG mice xenografted with HLA-A2 positive melanoma cell line cells (NW-MEL-38). Conclusions: Our preclinical observations here obviously demonstrated the potential utility of TCR-α/β gene-modified allogeneic Vγ9/δ2-T cells for the treatment of ATL without causing GVHD. Further studies regarding biological behaviors of HTLV-1 Tax specific TCR-α/β gene-modified allogeneic Vγ9/δ2-T cells following target recognition in vivo are warranted, however, based on these lines of evidence and currently conducting assessments using clinical samples, we are planning to launch a novel clinical trial, particularly focusing on the applicability of HLA partially matched relative donors, as the source of gene-modified allogeneic Vγ9/δ2-T cells, which could highly extend the donor availability. Disclosures Fujiwara: BrightPath Biotherapeutics, Co.,Ltd.: Other: member of the department endowed by BrightPath Bio. Okumura:BrightPath Biotherapeutics, Co.,Ltd.: Other: member of the department endowed by BrightPth Bio.. Miyahara:BirghtPath Biotherapeutics, Co., Ltd.: Other: member of the department endowed by BrightPath Bio.. Wan:BrightPath Biotherapeutics, Co., Ltd.: Other: member of the department endowed by BrightPath Bio.. Tawara:Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding. Shiku:BrightPath Biotherapeutics, Co., Ltd.: Other: Chair of the department endowed by BrightPath Bio..

Interferon Lambda Family along with HTLV-1 Proviral Load, Tax, and HBZ Implicated in the Pathogenesis of Myelopathy/Tropical Spastic Paraparesis

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic neuroinflammatory disease related to human T lymphotropic virus type 1 (HTLV-1) infection. Interferon type III (IFN-λ), which includes IL28, IL29, and IL28R, and affects the outcome of viral infections, might be complicated in the progression of HAM/TSP. Here, we investigated the host-virus interactions in the manifestation of HAM/TSP, using IL28B, IL29, IL28R, HTLV-1 Tax, HTLV-1 basic leucine zipper factor (HBZ), and proviral load (PVL). The study groups consisted of 20 patients with HAM/TSP, 20 asymptomatic HTLV-1 carriers (ACs), and 20 healthy controls (HCs). The means of PVL, Tax, and HBZ gene expressions in the HAM/TSP group (p = 0.004, 0.006, and < 0.0001, respectively) were significantly higher than in the AC group. The comparison of IL28B, IL29, and IL28R expression in the HAM/TSP, AC, and HC groups revealed no significant difference between the first 2, but lower concentrations in the HCs (IL28B: p = 0.03, 0.01; IL29: p = 0.07, 0.01; and IL28R: p < 0.0001, respectively). In the HAM/TSP group, correlations were seen between Tax and HBZ (R = 0.61, p = 0.004) and between Tax and IL29 (R = 0.45, p = 0.04). Negative correlations were observed between Tax and IL28B (R = –0.49, p = 0.02) and between HBZ and IL28R (R = –0.43, p = 0.06). In the ACs, an inverse correlation was found between Tax and IL28B (R = –0.42, p = 0.06). These findings suggest that IL29, IL28B, and IL28R interfere in the infection of HAM/TSP, mainly via Tax activation.

A novel medium-throughput biological assay system for HTLV-1 infectivity and drug discovery.

Objective(s):Here, a reporter cell line containing two reporter vectors were developed, in order to monitor the Human T-Lymphotropic Virus type1(HTLV-1) infectivity and the cell viability simultaneously.Materials and Methods:The reporter cell line was constructed by stably transfected baby hamster’s kidney cell line (BHK-21), with the genomes expressing two different reporters in separate plasmids. The first reporter gene is transactivated by the HTLV-1 tax protein, while the second reporter is continuously expressed when introduced into a mammalian cell. In order to show its functionality, the effect of the drug mix on HTLV-1 was assayed by this system and was compared to the results obtained by other methods.Results:HTLV-1 reporter cell line was found to produce high level of luciferase when co-cultured with MT-2 and Hut-102 cells but not with Jurkat cell. Moreover, the combination therapy against HTLV-1 can reduce luciferase expression of the cell when co-cultured with MT-2 and Hut-102 comparable to the ELISA (R=0.932, P-value =0.002). In addition, the results revealed the superiority of the present system over the molecular methods.Conclusion:The results demonstrated that the biological assay system is a beneficial tool for the medium-throughput anti-HTLV-1 drug screening and inhibitory effect.

HTLV-1 Tax impairs K63-linked ubiquitination of STING to evade host innate immunity

The cellular antiviral innate immune system is essential for host defense and viruses have evolved a variety of strategies to evade the innate immunity. Human T lymphotropic virus type 1 (HTLV-1) belongs to the deltaretrovirus family and it can establish persistent infection in human beings for many years. However, how this virus evades the host innate immune responses remains unclear. Here we report a new strategy used by HTLV-1 to block innate immune responses. We observed that stimulator of interferon genes (STING) limited HTLV-1 protein expression and was critical to HTLV-1 reverse transcription intermediate (RTI) ssDNA90 triggered interferon (IFN)-β production in phorbol12-myristate13-acetate (PMA)-differentiated THP1 (PMA-THP1) cells. The HTLV-1 protein Tax inhibited STING overexpression induced transcriptional activation of IFN-β. Tax also impaired poly(dA:dT), interferon stimulatory DNA (ISD) or cyclic GMP-AMP (cGAMP) −stimulated IFN-β production, which was dependent on STING activation. Coimmunoprecipitation assays and confocal microscopy indicated that Tax was associated with STING in the same complex. Mechanistic studies suggested that Tax decreased the K63-linked ubiquitination of STING and disrupted the interactions between STING and TANK-binding kinase 1 (TBK1). These findings may shed more light on the molecular mechanisms underlying HTLV-1 infection.

T Cell Receptor Vβ Staining Identifies the Malignant Clone in Adult T cell Leukemia and Reveals Killing of Leukemia Cells by Autologous CD8+ T cells.

There is growing evidence that CD8+ cytotoxic T lymphocyte (CTL) responses can contribute to long-term remission of many malignancies. The etiological agent of adult T-cell leukemia/lymphoma (ATL), human T lymphotropic virus type-1 (HTLV-1), contains highly immunogenic CTL epitopes, but ATL patients typically have low frequencies of cytokine-producing HTLV-1-specific CD8+ cells in the circulation. It remains unclear whether patients with ATL possess CTLs that can kill the malignant HTLV-1 infected clone. Here we used flow cytometric staining of TCRVβ and cell adhesion molecule-1 (CADM1) to identify monoclonal populations of HTLV-1-infected T cells in the peripheral blood of patients with ATL. Thus, we quantified the rate of CD8+-mediated killing of the putative malignant clone in ex vivo blood samples. We observed that CD8+ cells from ATL patients were unable to lyse autologous ATL clones when tested directly ex vivo. However, short in vitro culture restored the ability of CD8+ cells to kill ex vivo ATL clones in some donors. The capacity of CD8+ cells to lyse HTLV-1 infected cells which expressed the viral sense strand gene products was significantly enhanced after in vitro culture, and donors with an ATL clone that expressed the HTLV-1 Tax gene were most likely to make a detectable lytic CD8+ response to the ATL cells. We conclude that some patients with ATL possess functional tumour-specific CTLs which could be exploited to contribute to control of the disease.

Molecular and clinical analyses of HTLV-1 subgroups associated with the risk of developing HAM/TSP.

Among human T-lymphotropic virus type 1 (HTLV-1)-infected individuals, the lifetime risk of developing HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) differs between ethnic groups, also, there is an association between HTLV-1 tax gene subgroups (i.e., tax-A or -B) and the risk of HAM/TSP. In an attempt to understand the molecular mechanism responsible for this association, we studied the full-length proviral genome sequences of various HTLV-1-infected cell lines and patient samples, the functional difference in the viral transcriptional regulator Tax and HBZ between each subgroup, and the relationship between tax subgroups and the clinical and laboratory characteristics of HAM/TSP patients. The results indicated that (1) the distinct nucleotide substitutions corresponding to each tax subgroup are also associated with the nucleotide substitutions of viral structural, regulatory, and accessory genes; (2) mRNA expression of HBZ in HTLV-1-infected cells was significantly higher in HAM/TSP patients with tax-B than those with tax-A; (3) there was a positive correlation between HBZ and the expression of its target FoxP3 in HAM/TSP patients with tax-B but not in patients with tax-A; (4) transcriptional changes in inducible cells that express each subgroup of Tax or HBZ protein under the control of an inducible promoter showed different target gene profiles; however, (5) there were no clear differences in clinical and laboratory characteristics between HAM/TSP patients with tax-A and tax-B; and (6) there were no functional differences in Tax and HBZ between each subgroup evaluated by reporter gene assays. Our results indicate that although different HTLV-1 tax subgroups cause different patterns of viral and host gene expression in HAM/TSP patients via independent mechanisms of direct transcriptional regulation, these differences do not significantly affect the clinical and laboratory characteristics of HAM/TSP patients. Further studies on other viral genes, as well as clinical samples of asymptomatic carriers and adult T-cell leukemia (ATL) patients, are in progress to clarify the role of HTLV-1 tax subgroups.

HTLV-1 Tax induces Th1 master regulator T-bet and thus IFN-γ in CD4+CCR4+ T-cells of virus-associated myelopathy patients

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

The structure and genomic integration site of the HTLV-1 provirus determine selective clonal expansion and transformation to adult T cell leukaemia/lymphoma

Following infection and integration into the genome of host CD4+ T-lymphocytes, human T-lymphotropic virus type-1 (HTLV-1) persists by driving proliferation of infected T-cell clones which are counter-selected by HTLV-1-specific cytotoxic T-lymphocytes (CTLs). HTLV-1 Tax protein is the dominant target antigen recognized by these CTLs, and Tax is thought to cause the infected T-cell proliferation. In 40% of cases of adult T-cell leukaemia/lymphoma (ATLL) Tax protein is not expressed, suggesting that the provirus has down-regulated Tax to allow immune escape and viral persistence. Here, we tested the hypothesis that the genomic site of proviral integration determines the degree of clonal expansion and therefore determines the risk of ATLL. Proviral integration sites in 242 ATLL cases and 96 asymptomatic carriers were mapped and quantified using our recently described high-throughput technique (Gillet et al 2011, Blood, Cook et al 2012, Blood). In the ATLL cases, we identified promoter deletions predicted to cause tax gene silencing, we sequenced the tax gene and quantified promoter methylation. The results showed that 65% cases of ATLL could silence Tax by promoter deletion, tax gene nonsense mutations or tax promoter hypermethylation. High-throughput sequencing data showed that ATLL cases were not invariably monoclonal and that the provirus is typically integrated into a transcriptionally active region of the host genome. The results also suggest that transcriptional activity of the flanking host genome contributes to tax gene silencing, allowing CTL escape and clonal expansion.

Nonsense-mediated mRNA decay inhibition by HTLV-1 Tax protein

We have previously observed that degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway is indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed to a limited extent by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E, which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). Tax expression also alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments is increased by Tax, whereas that of INT6 is decreased. In line with these effects, the level of the phosphorylated form of UPF1 is increased in the presence of Tax. To further investigate how Tax can act on the stability of some viral and cellular transcripts which are prone to NMD, we examined whether Tax can directly bind such RNAs. By performing RNA immunoprecipitation analyses we indeed observed that Tax associates specifically with NMD-sensitive RNAs and the factors determining this binding are currently under investigation. These data further strengthen the notion that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control, but also involve posttranscriptional activities.

Genome-wide Determinants of Proviral Targeting, Clonal Abundance and Expression in Natural HTLV-1 Infection

The regulation of proviral latency is a central problem in retrovirology. We postulate that the genomic integration site of human T lymphotropic virus type 1 (HTLV-1) determines the pattern of expression of the provirus, which in turn determines the abundance and pathogenic potential of infected T cell clones in vivo. We recently developed a high-throughput method for the genome-wide amplification, identification and quantification of proviral integration sites. Here, we used this protocol to test two hypotheses. First, that binding sites for transcription factors and chromatin remodelling factors in the genome flanking the proviral integration site of HTLV-1 are associated with integration targeting, spontaneous proviral expression, and in vivo clonal abundance. Second, that the transcriptional orientation of the HTLV-1 provirus relative to that of the nearest host gene determines spontaneous proviral expression and in vivo clonal abundance. Integration targeting was strongly associated with the presence of a binding site for specific host transcription factors, especially STAT1 and p53. The presence of the chromatin remodelling factors BRG1 and INI1 and certain host transcription factors either upstream or downstream of the provirus was associated respectively with silencing or spontaneous expression of the provirus. Cells expressing HTLV-1 Tax protein were significantly more frequent in clones of low abundance in vivo. We conclude that transcriptional interference and chromatin remodelling are critical determinants of proviral latency in natural HTLV-1 infection.

WIP1 deficiency inhibits HTLV-1 Tax oncogenesis: novel therapeutic prospects for treatment of ATL?

Attenuation of p53 activity appears to be a major step in Human T-lymphotropic virus type 1 (HTLV-1) Tax transformation. However, p53 genomic mutations are late and rather infrequent events in HTLV-1 induced Adult T cell leukemia (ATL). The paper by Zane et al. shows that a mediator of p53 activity, Wild-type p53-induced phosphatase 1 (Wip1), contributes to Tax-induced oncogenesis in a mouse model. Wip1 may therefore be a novel target for therapeutic approaches.

Human retrovirus promotes the plasticity of regulatory T cells into T helper type 1-like cells through the T-bet transcriptional activation in neuroinflammatory disease

Recently, it has become increasingly clear that some committed effecter and regulatory T (Treg) cells are not stable, and the plasticity of these T-cells may be related to the pathogenesis of autoimmunity and inflammatory diseases [1]. However, the precise mechanisms that allow for T cell plasticity have not yet been clearly understood. Human T-lymphotropic virus type 1 (HTLV-1) is a retrovirus that is associated with multiorgan inflammatorydisorders such as HTLV-1- associated myelopathy (HAM/TSP), HTLV-1- associated arthropathy (HAAP), uveitis, Sjogren syndrome, and polymyositis [2-5]. HTLV-1-infected T cells may contribute to development of these disorders, since the number of HTLV-1-infected T cells circulating in the peripheral blood is higher in patients [6]. HTLV-1 mainly infects CD4+ T helper (Th) cells that play central roles in adaptive immune responses. Based on their functions, patterns of cytokine secretion, and expression of specific transcription factors and chemokine receptors, Th cells differentiated from naive CD4+ T-cells are classified into 4 major lineages: Th1, Th2, Th17, and T regulatory (Treg) cells. We recently demonstrated that CD4+CD25+CCR4+ T cells, which mainly include suppressive T-cell subsets such as Treg and Th2 under healthy conditions, are the predominant viral reservoir of HTLV-1 in both adult T-cell leukemia/lymphoma (ATL) and HAM/TSP [7]. Interestingly, T-cells of this subset become Th1-like cells with overproduction of IFN-γ in HAM/TSP, suggesting that HTLV-1 may intracellularly induce Tcell plasticity from Treg to IFN-γ+ T cells [7].In this study, using human T-cell line and HTLV-1 infected CD4+CD25+CCR4+ T-cells of HAM/TSP patients, the virus-encoded transactivating HTLV-1 Tax protein was demonstrated to induce the IFN-γ production through the expression of T-box 21(Tbx21)/T-bet, a transcription factor that is known to direct the differentiation of naive CD4+ cells into IFN-γ-expressing Th1 cell. HTLV-1 Tax was also demonstrated to enhance promoter activity of Tbx21/T-bet cooperatively with transcription factor Specificity Protein 1 (Sp1). Furthermore, transfer of HTLV-1 tax gene in CD4+CD25+CCR4+ T-cells using a lentiviral vector resulted in the loss of regulatory function of these T cells. This is the first report to our knowledge demonstrating the role of a specific viral product (HTLV-1 Tax) on the expression of genes associated with T-cell differentiation resulting in plasticity of Treg cells into Th1-like cells. These results suggest that HTLV-1 infection-induced immune dysregulation may play an important role in the development and pathogenesis of HTLV-associated immunological diseasesthrough its interference in the equilibrium maintained among host immune responses.

Design and development of a quantitative real time PCR assay for monitoring of HTLV-1 provirus in whole blood

BackgroundProviral load quantification of human T-lymphotropic virus type 1 (HTLV-1) is an essential marker for disease progression. Therefore, accurate and precise quantification of the virus is important. However, many articles published about detection and quantification of HTLV-1 virus neither reported any databank for the pre-validation of their primer and probe sequences nor stressed on its importance. Consequently, this failure may cause proviral load measurement variations of different HTLV-1 strains. ObjectiveThe aim of this study was to develop a TaqMan assay for HTLV-1 proviral load quantification which is based on a conserved region of tax gene with minimal sequence variability. Study designFor the purpose of finding the most conserved region of tax gene, all the HTLV-1 Gene Bank records including tax gene sequence (524 records by December 2009) were aligned in order to design on the most conserved region of this gene. The specificity, sensitivity, inter and intra assay and the dynamic range of the assay were experimentally determined by their respective methodology. ResultThe assay has a dynamic range of 10–107 HTLV-1 plasmid DNA/rxn (reaction) and the limit of detection (LOD) less than 10 copies/rxn. The assay gave coefficient of variation (CV) for the Ct values of less than 1% and 4.8% for intra and inter assay, respectively. Clinical sensitivity and specificity were determined to be 97.8% and 100%, respectively. ConclusionThis TaqMan assay is able to reliably quantify proviral load due to the fact that it has been designed on a conserved region of HTLV-1 tax gene with minimal sequence variability.

Can thymic epithelial cells be infected by human T-lymphotropic virus type 1?

The human T-lymphotropic virus type-1 (HTLV-1) is the cause of adult T cell leukaemias/lymphoma. Because thymic epithelial cells (TEC) express recently defined receptors for the virus, it seemed conceivable that these cells might be a target for HTLV-1 infection. We developed an in vitro co-culture system comprising HTLV-1+-infected T cells and human TECs. Infected T cells did adhere to TECs and, after 24 h, the viral proteins gp46 and p19 were observed in TECs. After incubating TECs with culture supernatants from HTLV-1+-infected T cells, we detected gp46 on TEC membranes and the HTLV-1 tax gene integrated in the TEC genome. In conclusion, the human thymic epithelium can be infected in vitro by HTLV-1, not only via cell-cell contact, but also via exposure to virus-containing medium.

The plasticity of HTLV-1 infected CD4+CD25+CCR4+ T-cells through HTLV-1 tax in HAM/TSP

Recently, it has become increasingly clear that some committed effecter and regulatory T (Treg) cells are not stable, and the plasticity of committed T-cells may be related to autoimmunity and inflammatory disease. However, the environmental (extrinsic) molecules that allow for plasticity have not yet been clearly understood. In Human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy /tropical spastic paraparesis (HAM/TSP), the pathogenesis is known as HTLV-1 infected CD4+ T-cells triggered hyper immune response, which leads to chronic inflammation of the central nervous system. In our previous study, we demonstrated that the majority of CD4+CD25+CCR4+ T-cells were infected with HTLV-1 and that this T-cell subset was increased in HAM/TSP. Although CD4+CD25+CCR4+ T-cells of healthy condition include suppressive T cell subsets such as Treg and Th2, this T-cell subset becomes Th1-like cells with overproduction of IFN-γ in HAM/TSP patients (PLoS ONE 2009). Since HTLV-1 tax is known to up-regulate the expression of several proinflammatory cytokines, and importantly, the level of HTLV-1 tax mRNA expression is reported to correlate with disease severity in HAM/TSP patients, we hypothesize that HTLV-1 tax may convert HTLV-1 infected T-cells into abnormal IFN-γ producing Th1-like T-cells in HAM/TSP. In this study, we present the molecular mechanisms underlying the plasticity of HTLV-1 infected CD4+CD25+CCR4+ T-cells through HTLV-1 tax.