Human T-cell Lymphotropic Virus Type 1 Tax Research Articles

Effect of Teriflunomide on Cells From Patients With Human T-cell Lymphotropic Virus Type 1-Associated Neurologic Disease.

ObjectiveTo test the hypothesis that teriflunomide can reduce ex vivo spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) from patients with human T-cell lymphotropic virus type 1 (HTLV-1)–associated myelopathy/tropical spastic paraparesis (HAM/TSP).MethodsPBMCs from patients with HAM/TSP were cultured in the presence and absence of teriflunomide and assessed for cell viability, lymphocyte proliferation, activation markers, HTLV-1 tax and HTLV-1 hbz messenger ribonucleic acid (mRNA) expression, and HTLV-1 Tax protein expression.ResultsIn culture, teriflunomide did not affect cell viability. A concentration-dependent reduction in spontaneous proliferation of PBMCs was observed with 25 μM (38.3% inhibition), 50 μM (65.8% inhibition), and 100 μM (90.7% inhibition) teriflunomide. The inhibitory effects of teriflunomide were detected in both CD8+ and CD4+ T-cell subsets, which are involved in the immune response to HTLV-1 infection and the pathogenesis of HAM/TSP. There was no significant change in HTLV-1 proviral load (PVL) or tax mRNA/Tax protein expression in these short-term cultures, but there was a significant reduction of HTLV-1 PVL due to inhibition of proliferation of CD4+ T cells obtained from a subset of patients with HAM/TSP.ConclusionsThese results suggest that teriflunomide inhibits abnormal T-cell proliferation associated with HTLV-1 infection and may have potential as a therapeutic option in patients with HAM/TSP.

Mechanisms of Oncogenesis by HTLV-1 Tax.

The human T-cell lymphotropic virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia/lymphoma (ATLL), a neoplasm of CD4+CD25+ T cells that occurs in 2–5% of infected individuals after decades of asymptomatic latent infection. Multiple HTLV-1-encoded regulatory proteins, including Tax and HTLV-1 basic leucine zipper factor (HBZ), play key roles in viral persistence and latency. The HTLV-1 Tax oncoprotein interacts with a plethora of host cellular proteins to regulate viral gene expression and also promote the aberrant activation of signaling pathways such as NF-κB to drive clonal proliferation and survival of T cells bearing the HTLV-1 provirus. Tax undergoes various post-translational modifications such as phosphorylation and ubiquitination that regulate its function and subcellular localization. Tax shuttles in different subcellular compartments for the activation of anti-apoptotic genes and deregulates the cell cycle with the induction of DNA damage for the accumulation of genomic instability that can result in cellular immortalization and malignant transformation. However, Tax is highly immunogenic and therefore HTLV-1 has evolved numerous strategies to tightly regulate Tax expression while maintaining the pool of anti-apoptotic genes through HBZ. In this review, we summarize the key findings on the oncogenic mechanisms used by Tax that set the stage for the development of ATLL, and the strategies used by HTLV-1 to tightly regulate Tax expression for immune evasion and viral persistence.

Role of HTLV-1 Tax and HBZ in the Pathogenesis of HAM/TSP.

Human T cell lymphotropic virus type 1 (HTLV-1) infection can lead to development of adult T cell leukemia/lymphoma (ATL) or HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. Understanding the interaction between host and HTLV-1 and the molecular mechanisms associated with disease pathogenesis is critical for development efficient therapies. Two HTLV-1 genes, tax and HTLV-1 basic leucine zipper factor (HBZ), have been demonstrated to play important roles in HTLV-1 infectivity and the growth and survival of leukemic cells. Increased HTLV-1 Tax expression induces the expression of various cellular genes such as IL-2 and IL-15, which directly contributes to lymphocyte activation and immunopathogenesis in HAM/TSP patients. However, little is known about the molecular and cellular mechanism of HBZ in development of HAM/TSP. It has been reported that HBZ mRNA expression was detected in HAM/TSP patients higher than in asymptomatic carriers and correlated with proviral load and disease severity. Unlike HTLV-1 tax, HBZ escapes efficient anti-viral immune responses and therefore these reactivities are difficult to detect. Thus, it is important to focus on understanding the function and the role of HTLV-1 tax and HBZ in disease development of HAM/TSP and discuss the potential use of these HTLV-1 viral gene products as biomarkers and therapeutic targets for HAM/TSP.

Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription.

The viral Tax oncoprotein plays a key role in both Human T-cell lymphotropic virus type 1 (HTLV-1)-replication and HTLV-1-associated pathologies, notably adult T-cell leukemia. Tax governs the transcription from the viral 5’LTR, enhancing thereby its own expression, via the recruitment of dimers of phosphorylated CREB to cAMP-response elements located within the U3 region (vCRE). In addition to phosphorylation, CREB is also the target of O-GlcNAcylation, another reversible post-translational modification involved in a wide range of diseases, including cancers. O-GlcNAcylation consists in the addition of O-linked-N-acetylglucosamine (O-GlcNAc) on Serine or Threonine residues, a process controlled by two enzymes: O-GlcNAc transferase (OGT), which transfers O-GlcNAc on proteins, and O-GlcNAcase (OGA), which removes it. In this study, we investigated the status of O-GlcNAcylation enzymes in HTLV-1-transformed T cells. We found that OGA mRNA and protein expression levels are increased in HTLV-1-transformed T cells as compared to control T cell lines while OGT expression is unchanged. However, higher OGA production coincides with a reduction in OGA specific activity, showing that HTLV-1-transformed T cells produce high level of a less active form of OGA. Introducing Tax into HEK-293T cells or Tax-negative HTLV-1-transformed TL-om1 T cells is sufficient to inhibit OGA activity and increase total O-GlcNAcylation, without any change in OGT activity. Furthermore, Tax interacts with the OGT/OGA complex and inhibits the activity of OGT-bound OGA. Pharmacological inhibition of OGA increases CREB O-GlcNAcylation as well as HTLV-1-LTR transactivation by Tax and CREB recruitment to the LTR. Moreover, overexpression of wild-type CREB but not a CREB protein mutated on a previously described O-GlcNAcylation site enhances Tax-mediated LTR transactivation. Finally, both OGT and OGA are recruited to the LTR. These findings reveal the interplay between Tax and the O-GlcNAcylation pathway and identify new key molecular actors involved in the assembly of the Tax-dependent transactivation complex.

Genetic Sequencing of Adult T-Cell Leukemia/Lymphoma in the Caribbean Population Shows a Distinct Mutational Profile When Compared to the Japanese Cohort

IntroductionAdult T-cell leukemia/lymphoma (ATL) is a rare, aggressive disease caused by human T cell lymphotropic virus type 1 (HTLV1) that predominantly affects Japanese and Caribbean populations. (Goncalves, Proietti et al. 2010) Most studies have focused on Japanese cohorts. A recent whole genome/exome sequencing of 81 Japanese ATL cases identified alterations that overlap with the HTLV1 Tax interactome and are highly enriched for T cell receptor-NF-kB signaling, and T cell trafficking (Kataoka, Nagata et al. 2015). We recently showed that ATL in Caribbean patients has a poor prognosis and may have distinctive clinical features when compared to the Japanese cohort (Zell, Assal et al. 2016). In order to determine whether there are genomic differences between the two cohorts, we sequenced 15 ATL samples from the Caribbean cohort.MethodsIn this prospective study we perform comprehensive Next Generation Sequencing to assess the mutational spectrum of ATL in Caribbean patients. Samples were analyzed by complete genetic profiling with the Genoptix Nexcourse complete assay that contains 173 cancer related genes. Patient genomic DNA was utilized to identify relevant single nucleotide variants, insertion/deletions, copy number variations, and translocation fusion genomic alterations in a panel of up to 173 reportable genes at an average mean sequencing depth of 500X coverage.ResultsIn the 15 patients, a total of 49 genes were found to be mutated ranging from 3 to 7 mutated genes in each patient. The percent of mutated genes amongst the total analyzed ranged from 1.7-4% for each patient and the percent of mutated genes known to be pathogenic ranged from 0-2.3% for each patient. There were 18 known pathogenic mutations in a total of 70 genetic mutations identified in this cohort (25.7%). The mean number of mutations in the acute and lymphomatous subtype was approximately 0.9%.Genes most commonly mutated in out cohort were TP53 (26.7%), FAT1 (26.7%), NOTCH1 (20%) and APC (20%). Novel mutations not reported by the Japanese cohort but present in our cohort are - NRAS, KRAS, EZH2, RICTOR, XPO1, VHL (known to be pathogenic), FAT1, APC, DDX3X, KIT, NOTCH2, MYC, TSC1, PLCG2, FGFR2, FGFR4, PDGFRA, EGFR, KEAP1, MCL1, ALK, BCL6, RIPK1, IGF1R, DDR2, KDR, AKT1, ERBB2, NKX2-1, BRCA2, PBRM1, CD79A, STAG2, PTCH1, KMT2A, HIST1HE, SPEN, ASXL1 (not known to be pathogenic). Mutations common to both the Japanese and our Caribbean cohort are - CARD11, TNFAIP3, TRAF3 (TCR signaling/NFkB pathway), NOTCH1 (signaling pathway), GATA3, CEBPA, TBL1XR1 (transcriptional regulation), EP300 (epigenetic regulation), TP53, POT1 (Telomere regulation and DNA repair).Serial analysis of a patient revealed increase in the size of the pathogenic clone with XPO1 mutation. XPO1 has been involved in nuclear transport of p53 and the increase in its clone size occurred with relapse after cytotoxic chemotherapy.The overall survival (OS) was shorter for TP53 mutated patients (99.3 days) versus non TP53 mutated patients (307.8 days).DiscussionWe report on mutational profiling in the largest cohort of Caribbean ATL and an additional 8 patients will be included in the analysis to be presented at ASH. A significant number of genes found to be mutated affected known pathways in the pathogenesis of ATL. There are known genes which are common between the Japanese and Caribbean cohorts but there are some notable differences.Previously we showed that the Caribbean cohort has a poorer OS than the Japanese cohort. TP53 mutations are more frequent and associated with decreased OS in this cohort. Genes involved in Wnt pathway - FAT1, APC, NOTCH1 were more frequently mutated in our cohort. FAT1 mutations have not been described in ATL. However, they have been implicated in oncogenesis in solid tumors, chronic lymphocytic leukemia and T-cell acute lymphoblastic leukemia. FAT1 acts as a tumor suppressor gene and encodes a cadherin-like protein, which potently suppresses cancer cell growth. Inactivation of FAT1 via mutation therefore promotes Wnt signaling and tumorigenesis and affects patient survival (Morris, Kaufman et al. 2013). In in-vitro models of ATL, Wnt pathway dysregulation has been shown to promote ATL tumorigenesis (Bellon, Ko et al. 2013, Ma, Yasunaga et al. 2013). Based on this study the driver mutations of the Caribbean cohort appear to be the Wnt signaling pathway which is different from the TCR- NF-kB signaling pathway seen in the Japanese cohort. [Display omitted] DisclosuresHall:Genoptix, a Novartis Company: Employment.

A Transgenic Drosophila melanogaster Model To Study Human T-Lymphotropic Virus Oncoprotein Tax-1-Driven Transformation In Vivo.

Human T-cell lymphotropic virus type 1 (HTLV-1)-induced adult T-cell leukemia/lymphoma is an aggressive malignancy. HTLV-2 is genetically related to HTLV-1 but does not cause any malignant disease. HTLV-1 Tax transactivator (Tax-1) contributes to leukemogenesis via NF-κB. We describe transgenic Drosophila models expressing Tax in the compound eye and plasmatocytes. We demonstrate that Tax-1 but not Tax-2 induces ommatidial perturbation and increased plasmatocyte proliferation and that the eye phenotype is dependent on Kenny (IKKγ/NEMO), thus validating this new in vivo model.

High IFN-γ/IL-10 Expression Ratio and Increased Frequency of Persistent Human T-Cell Lymphotropic Virus Type 1-Infected Clones Are Associated with Human T-Cell Lymphotropic Virus Type 1-Associated Myelopathy/Tropical Spastic Paraparesis Development

Background/Aims: Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes a persistent infection, and only 0.5-5% of infected individuals will develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Therefore, we investigated parameters to discriminate HTLV-1 asymptomatic carriers (ACs) with an increased chance to develop HAM/TSP. Methods: We evaluated integration patterns of HTLV-1 provirus, the relative expression of HTLV-1 tax and HBZ mRNAs and of IFN-γ and IL-10 mRNAs, in addition to proviral load (PVL) levels. Results: HAM/TSP patients presented a higher number of large persistent HTLV-1-carrying clones compared to ACs, and the expression of the HTLV-1 tax and HBZ genes by infected cells was detected at low levels and correlated positively with PVL. In addition, HAM/TSP patients and ACs with high PVL expressed higher levels of IFN-γ mRNA in comparison to IL-10, while ACs with low PVL presented an equilibrate IFN-γ/IL-10 ratio. Conclusions: The presence of large persistent HTLV-1-infected clones in association with viral gene expression, even at small levels, could stimulate the intense inflammatory response in HTLV-1-infected individuals. This was supported by a high ratio of IFN-γ/IL-10 relative expression in HAM/TSP patients and ACs with high PVL, indicating that these parameters could aid the identification of ACs with a high risk to develop HAM/TSP.

Universal cytotoxic activity of a HTLV-1 Tax-specific T cell clone from an HLA-A*24:02+ patient with adult T-cell leukemia against a variety of HTLV-I-infected T-cells

Adult T cell leukemia/lymphoma (ATL) is an aggressive mature T cell malignancy that is causally associated with human T cell lymphotropic virus type 1 (HTLV-1) infection. The HTLV-1 regulatory protein Tax aggressively accelerates the proliferation of host cells and is also an important target antigen for CD8+ cytotoxic T cells (CTLs). We previously reported that several predominant HLA-A*24:02-restricted HTLV-1 Tax301–309-specific CTL clones commonly expressed a particular amino acid sequence motif (P-D-R) in complementarity-determining region 3 of T-cell receptor (TCR)-β chain among unrelated ATL patients who underwent allogeneic stem cell transplantation (allo-HSCT). Furthermore, a PDR-motif+ CTL clone persistently existed in a long-term survivor as a central CTL clone with strong CTL activities after HSCT. Although a larger analysis of the relationship between PDR-motif+ CTLs and the clinical course is required, the expression of PDR-motif+ TCR on CD8+ T cells may play a critical role in the management of anti-HTLV-1 activities for HLA-A24:02+ ATL patients. Therefore, in this study, we prepared an HTLV-1 Tax301–309 peptide-specific CTL clone (HT-9) expressing PDR-motif+ TCR isolated from a long-term survivor after HSCT, and evaluated its CTL activity against a variety of HTLV-1-infected T-cells from HLA-A*24:02+ ATL patients. Before the assay of CTL function, we confirmed that HT-9 expressed less-differentiated effector-memory phenotypes (CD45RA−CCR7−CD27+CD28+/−CD57+/−) and T-cell exhaustion marker PD-1+. In assays of CTL function, HT-9 recognized HTLV-1 Tax in an HLA-restricted fashion and demonstrated strong CTL activities against a variety of HTLV-1-infected T-cells from HLA-A*24:02+ ATL patients regardless of whether the sources were autologous or allogeneic, but not normal cells. These data indicate that PDR-motif+ TCR could be an important TCR candidate for TCR-gene immunotherapy for HLA-A24:02+ ATL patients, provided that the CTL activities against HTLV-1 are reproduced in in vivo experiments using mouse models.

CD25+CCR4+ cells as a marker of HTLV-1-infected cells in peripheral blood

The diagnosis of Human T cell Lymphotropic Virus type 1 (HTLV-1) infection is based on serology and PCR. The burden of HTLV-1 viral infection is monitored by measuring the proviral load [PVL] in peripheral blood. This assay is not useful for isolation of HTLV-1-infected cells. HTLV-1 Tax upregulates CD25 expression on infected CD4+ T cells. However, CD4+CD25+ is also the phenotype of activated T cells. CCR4 is a chemokine receptor expressed on subsets of activated T cells. HTLV-1-infected cells secrete the CCR4 ligand CCL22, and preferentially infect CCR4+ Cells. We studied the expression of CD25 and CCR4 in lymphocytes and its relation to PVL. We performed 11-colour immunophenotyping and HTLV-1 PVL quantification on 53 samples obtained from 36 HTLV-1 infected patients, [10 asymptomatic carriers (AC); 11 patients with HTLV-1-associated myelopathy (HAM); 4 co-infected with HIV; 11 with chronic/smouldering adult T-cell leukaemia/lymphoma (ATL)] and 3 uninfected individuals. Increased frequency of CD25+CCR4+ T cells [median: 14.7%, range: 1.95-91.3%] was observed in all HTLV-1 infected patients; the frequency correlated with PVL [Spearman r=0.89, P <0.001, Linear R2 = 0.61]. CD4+ and CD8+ cells from 12 patients [6ACs and 6 HAMs] were separated for PVL estimation. CD25+ CCR4+ cell counts correlated closely with PVL in CD4+ cells [Spearman r=0.816, P <0.001, linear R2=0.886] but not in CD8+ cells. Frequency of CD25+CCR4+ T cells correlated with PVL change in treated ATL patients. We conclude that CD25+CCR4+ cells can be used as a specific marker for monitoring of HTLV-1 infection and isolation of HTLV-1 infected cells.

Profound Immune Exhaustion and Downregulation Of Interleukin-7 Alpha Receptor (CD127) In CD8 and CD4 T-Cell Subsets In Jamaican Patients Co-Infected With Human T-Cell Lymphotropic Virus Type-1 and Human Immunodeficiency Virus Type-1 Despite Normal Or Above Normal CD4 Counts

BackgroundNext to Sub-Saharan Africa, the Caribbean has the second highest prevalence of human immunodeficiency virus type 1 (HIV-1) infection; and human T-cell lymphotropic virus type 1 (HTLV-1), another human retrovirus, is also endemic. Fewer than five percent of persons with HTLV-1, predominantly transmitted through breast feeding, progress to any of the myriad associated diseases, including adult T-cell leukemia, HTLV-1 associated myelopathy/Tropical Spastic Paraparesis (HAM/TSP), or polymyositis. HTLV-1, through its oncoprotein-like transactivating (Tax) protein, results in aberrant proliferation of CD4 cells through interaction with cell cycle regulators, and activation of nuclear factor kappa B and the interleukin-2 pathway. Elevated CD4 counts, often above normal range, observed in patients with HTLV-1/HIV-1 co-infection, may be spurious, posing a challenge for laboratory monitoring of lymphoproliferative disorders or HIV-1 progression. MethodsWe compared CD4 counts, HIV-1 viral load, and clinical parameters in a cohort of patients co-infected with HTLV-1 and HIV-1 (syphilis, hepatitis B and C non-reactive), with controls with HIV-1 infection only matched for age, sex, and duration of antiretroviral therapy, as well as donors without HTLV-1 or HIV-1 infection. We used flow cytometry to characterize CD4 and CD8 naïve and memory T-cell subsets, expression of T-cell survival and homeostatic cytokine interleukin-7 alpha receptor (CD127), and examined the role of co-inhibitory programmed death-1 in HTLV-1 infected (intracellular HTLV-1 Tax protein-expressing) and HIV-1 infected (intracellular HIV-1 Gag protein-expressing) CD4 cells in immune evasion. Additionally, we assessed the effects of exogenous interleukin-7 (IL-7) and programmed death-1 pathway blockade on the function of responding CD8 cytotoxic T-lymphocyte (CTL) subsets ex vivo. ResultsAll patients with HTLV-1 and HIV-1 co-infection (n = 5) were female and of median age 42 years (range, 22 to 49 years), with normal or above normal CD4 counts. Four of five patients with HTLV-1/HIV-1 co-infection were WHO Stage 4 HIV/AIDS at diagnosis (e.g., oesophageal candidiasis, HIV nephropathy). Median duration since diagnosis with HIV-1 infection was six years (range, 1 to 18 years), and unexpectedly high CD4 count was the reason for HTLV-1/2 testing in all cases. Nadir CD4 count among patients with HTLV-1/HIV-1 co-infection was significantly higher than controls with HIV-1 infection only (n = 12) matched for age, sex, and duration of antiretroviral therapy (median 684, range 467 to 1474; and median 36, range, 8 to 352 cells per microliter, respectively; p = 0.007, Mann-Whitney U test). Flow cytometric analyses of CD8 and CD4 T-cell memory subsets revealed more profound loss of CD127 in the CD8 compared to the CD4 compartment; and CD8(+) effector memory T-cells showed the most significant downregulation of CD127 when patients with HTLV-1/HIV-1 co-infection were compared with seronegative donors (40.3±17.0%, n =5; and 73.1±7.48%, n = 10, respectively; p = 0.032, Mann-Whitney U). In ex vivo experiments in the context of peripheral blood mononuclear cells, preliminary data suggest that patients with HTLV-1/HIV-1 co-infection have impaired CD8(+) CTL function in response to HLA-restricted HTLV-1 Tax11-19 and cytomegalovirus (CMV) pp65 peptide stimulation. Further, we sought to describe expression of co-inhibitory marker programmed death-1 ligand (PDL1) on intracellular HTLV-1 Tax protein- as well as intracellular HIV-1 Gag protein-expressing CD4 cells. Blockade of PDL1 resulted in partial recovery of CTL function in patients with HTLV-1/HIV co-infection, and enhanced killing of target cells infected with either HTLV-1 or HIV-1. ConclusionsPatients with HTLV-1/HIV-1 co-infection show profound CTL exhaustion; and CD4 cells, although within or above normal CD4 count range, show aberrant naïve and memory T-cell distribution and possible immune evasion by upregulation of the programmed death-1 pathway, blockade of which showed a tendency towards enhanced virus-specific CTL function and killing of target cells infected with HTLV-1 or HIV-1. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

In Memoriam – Hugh A. Smythe, 1927–2012

<h3>Objective</h3> To test the hypothesis that teriflunomide can reduce ex vivo spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) from patients with human T-cell lymphotropic virus type 1 (HTLV-1)–associated myelopathy/tropical spastic paraparesis (HAM/TSP). <h3>Methods</h3> PBMCs from patients with HAM/TSP were cultured in the presence and absence of teriflunomide and assessed for cell viability, lymphocyte proliferation, activation markers, HTLV-1 <i>tax</i> and HTLV-1 <i>hbz</i> messenger ribonucleic acid (mRNA) expression, and HTLV-1 Tax protein expression. <h3>Results</h3> In culture, teriflunomide did not affect cell viability. A concentration-dependent reduction in spontaneous proliferation of PBMCs was observed with 25 μM (38.3% inhibition), 50 μM (65.8% inhibition), and 100 μM (90.7% inhibition) teriflunomide. The inhibitory effects of teriflunomide were detected in both CD8⁺ and CD4⁺ T-cell subsets, which are involved in the immune response to HTLV-1 infection and the pathogenesis of HAM/TSP. There was no significant change in HTLV-1 proviral load (PVL) or <i>tax</i> mRNA/Tax protein expression in these short-term cultures, but there was a significant reduction of HTLV-1 PVL due to inhibition of proliferation of CD4⁺ T cells obtained from a subset of patients with HAM/TSP. <h3>Conclusions</h3> These results suggest that teriflunomide inhibits abnormal T-cell proliferation associated with HTLV-1 infection and may have potential as a therapeutic option in patients with HAM/TSP.

ROLE OF CD48 IN REGULATION OF T-CELL MEDIATED IMMUNITY IN HTLV-1 INFECTION

Event Abstract Back to Event ROLE OF CD48 IN REGULATION OF T-CELL MEDIATED IMMUNITY IN HTLV-1 INFECTION Chioma E. Chibueze-Nnorom1, Yohann White2, Makoto Yoshimitsu1* and Naomichi Arima1* 1 Center for Chronic Viral Diseases Graduate School of Medical and Dental Sciences Kagoshima University, Hematology and Immunology, Japan 2 University of West Indies, Mona Kingston, Jamaica HTLV-1 (Human T cell lymphotropic virus type 1) infection is associated with an aggressive T cell malignancy ATLL (Adult T-cell leukemia/lymphoma), the virus also establishes lifelong persistent infection in humans resulting in a carrier state (clinically asymptomatic). CD48 is a GPI anchored cell surface molecule ubiquitously expressed on various hematopoietic cells, its expression has been shown to be up-regulated in viral infections as a sign of ongoing breach of immunity and therefore facilitates immune response. In HTLV-1 infection, one of the factors suggested to play a role in its persistence is the increased expression of the trans-activating viral gene encoding HTLV-1 tax among others; tax protein is the dominant target antigen of the T cell response to HTLV-1 in infected individuals. In this study, we investigate the role of CD48 in HTLV-infection, we show that it is down regulated in HTLV-1 infection; the presence of HTLV-1 tax protein also suppresses CD48 expression. On blockade using anti-CD48 AB (antibody), the effector function of T cells in terms of cytokine production improved. Thus, we propose a possible means of immune evasion by HTLV-1 virus by Cd48 down-regulation and a possible inhibitory role in HTLV-1 infection.

Immune Exhaustion and 2B4 Expression in Human T-Cell Lymphotropic Virus Type 1(HTLV-1) Infection

AbstractAbstract 4807 Background:The role of the 2B4/CD48 pathway in chronic viral infections has been controversial, with some studies suggesting an enhancing and others an inhibitory effect on virus specific CD8 (+) cytotoxic T-lymphocytes. Human T-cell lymphotropic virus type 1 (HTLV-1) trans-activating (Tax) protein is crucial for viral replication and activates HTLV-1 gene transcription. Methods:Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll centrifugation and cryopreserved until use. For staining, cells were incubated with fluorochrome-conjugated antibodies against 2B4, CD48, CD4, CD8. CD8 (+) lymphocytes were identified using phycoerythrin (PE) labeled tetramers HLA*0201 or 2402 Tax, CMV and EBV immune-dominant epitopes. In some, cells were fixed and permeabilized prior to staining with FITC-conjugated anti-Tax protein (clone Lt-4) antibodies or a control IgG. To examine the effect of CD48 blockade on Cd8+ T lymphocyte function, PBMCs (1 × 106) were cultured in complete medium (RPMI-1640 supplemented with 100U/ml penicillin, 0.1mg/ml streptomycin, 0.05mM 2-mercaptoethanol, 50U/ml recombinant interleukin-2 and 10% inactivated FCS) with/without Tax peptide and/or anti-CD48 blocking antibody (5μg/ml) in the presence of brefeldin or monensin for intracellular perforin or surface degranulation of CD107a, respectively. Acquisition was done using FACS Calibur or FACS Scan flow-cytometer and analyzed with Flowjo software. Differences were analyzed by Mann-Whitney U test; changes pre- and post- ex-vivo culture were analyzed by Wilcoxon matched pairs test using Stata software version 9 (StataCorp LP). P-values <0.05 were considered significant. Results:The expression of 2B4 in total CD8 (+) T-cells was significantly greater in HTLV-1 infected individuals compared to healthy controls(67.2±17.6 and 51.6±21.4, respectively; p 0.03; n = 11 HCs, 44 infected; Figure 1b), there was no difference in 2B4 expression in HTLV-1 tetramer specific CD8 (+) CTLs between asymptomatic HTLV-1 infected (ACs) and ATLL (81.0±18.5 and 73.2±24.8, respectively; n = 23 ACs, 19 ATLLs; Figure 1d) individuals.In CMV specific CTLs, 2B4 expression was significantly higher in HTLV-1 infected individuals compared with uninfected healthy controls (77.4±21.8 and 46.7±29.5, respectively; p = 0.01, n = 28 infected and 7 HCs; Figure 1D); and 2B4 expression was also greater in EBV-specific CTLs in HTLV-1 infected individuals compared with uninfected healthy controls (72.7±22.1 and 46.5±26.3, respectively; p = 0.02; n = 27 infected and 6 HCs; Figure 1d).HTLV-1 infected cells express intracellular HTLV-1 Tax protein in ex vivo cultures, peaking at 16 to 18 hours followed by a marked fall to undetectable levels. In our study, we noticed a trend whereby CD48 expression was lower in intracellular Tax-protein expressing (infected) CD4 (+) cells compared to Tax protein-negative (uninfected cells) in short term ex vivo cultures (50.6±7.3% versus 90.1±5.6%, respectively); and CD48 expression in total CD4 (+) cells was downregulated as Tax protein became expressed in cultures (93.3±3.3 at start versus 86.7±6.1 after culture; 3 independent experiments; Figure 2). Programmed death ligand 1 (PDL1) is known to be upregulated in Tax-protein expressing compared with Tax-negative cells, which is believed to permit immune evasion by HTLV-1 infected cells, but the proportion of HTLV-1 infected cells that are CD48(+) appears to be down regulated in this study. In vitro infection of target cells with EBV has been shown to upregulate CD48 expression at the mRNA and protein levels; thus, indicating that downregulation of CD48 in HTLV-1 infected cells is unique, possibly related to interactions of the trans-activating viral protein with host gene regulatory mechanisms at molecular level.Further, perforin production by CD8(+) lymphocytes is known to mediate lysis of HTLV-1 infected cells, in our study, the CD48 antibody resulted in increased perforin production by CD8 (+) CTLs, suggesting an inhibitory role for the 2B4/CD48 pathway in HTLV-1 infection(Figure 3c).Similarly, expression of the degranulation marker, CD107a in CD8 (+) lymphocytes was augmented with CD48 blocking antibody (Figure 3a, b). Conclusion:The pattern of 2B4 expression in HTLV-1 infection and enhanced CD8 (+) lymphocyte function with 2B4/CD48 blockade suggest involvement of this pathway in immune exhaustion, and a possible immunotherapeutic target. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Detection of human T-cell lymphotropic virus Type-1 among patients with malignant hematological diseases in Capital of Iran, Tehran

Human T-cell lymphotropic virus type-1 (HTLV-1) is a deltaretrovirus linked causally to adult T-cell leukemia or lymphoma (ATL), and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The aim of this study was to detect HTLV-1 infection in patients with malignant hematological diseases and also determining the prevalence of HTLV-1 in these patient groups. Sixty patients with malignant hematological diseases were included in the study and tested by enzyme-linked immunosorbent assay (ELISA) for anti-HTLV-1, and Real time-PCR for the sequences from HTLV-1 tax gene. The mean age of patients was 33.9 ± 18.3 years. 18 of the subjects were found HTLV-1 seropositive using ELISA and the viral prevalence by Real time-PCR was 12%. HTLV-1 was found in 25% of patients with acute myelogenous leukemia (AML), 58.3% of patients with chronic myelogenous leukemia (CML), 16.7% of patients with acute lymphoblastic leukemia (ALL), and no detected in patients with lymphoma. The present study revealed that HTLV-1 is prevalent in patients with malignant hematological diseases and in our study. The major HTLV-1 associated syndromes were chronic myelogenous leukemia and acute lymphoblastic leukemia. Key words: Human T-cell lymphotropic virus type-1, malignant hematological diseases, prevalence, Iran

Increased Frequency of HTLV-1 Tax Specific CD8+ Regulatory T-Cells in HTLV-1 Infected Individuals.

Abstract 3676Poster Board III-612Regulatory T-cells (Tregs) mediate self-tolerance and moderate the immune response to various antigens. Their frequency and suppressive function are reduced in various autoimmune diseases like type 1 diabetes mellitus, systemic lupus, and rheumatoid arthritis, but may be excessive in certain malignancies. The human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus with myriad clinical manifestations, most notably adult T-cell leukemia/lymphoma (ATLL) and HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP). We have previously described the decreased frequency and function of HTLV-1 specific CD8+ T-cells in ATLL patients compared to asymptomatic carriers (ACs) and healthy controls (HCs). We have also reported the relative exhaustion of HTLV-1 specific cytotoxic T-lymphocytes (CTLs) compared to cytomegalovirus (CMV)-specific CTLs in ACs, as demonstrated by upregulation of the co-inhibitory molecule, programmed death 1 (PD-1). Recent evidence supports the existence of CD8+ Tregs in humans, defined primarily by forkhead box P3 (FOXP3) transcription factor expression. Although a classic Treg phenotypic marker, FOXP3's aberrant expression makes it a less reliable marker in the setting of ATLL. Low expression or absence of interleukin 7 receptor (IL-7R) (CD127lo/-) on CD25+ T-cells has provided a useful alternative Treg phenotype, allowing preservation of cell viability for functional assays. Existing studies have focused mostly on CD4+ Tregs and a few on CD8+ Tregs in malignancy, but little is known about their involvement in the persistence of chronic viral infections like HTLV-1. In chronic HTLV-1 infection, FOXP3+CD25+ and CD127lo/-CD25+ Tregs comprise 8.3±4.6% and 8.1±4.3% of overall CD8+ T-cells, respectively (n=14). Expression of the FOXP3+CD25+ phenotype in CD8+ T-cells had a tendency towards correlation with the CD127lo/-CD25+ phenotype (r=0.561, p=0.073, n=11). Among ACs, we observed the CD127lo/-CD25+ Treg phenotype in HTLV-1 Tax-specific CD8+ T-cells (45±16.5%, n=8), which was significantly greater than CMV-specific CD8+ Tregs (25±15.8%, n=6) (p<0.05, Figure 1), as characterized by phycoerythrin-labelled HLA-A*2402 Tax301-309 or HLA-A*0201 Tax11-19 and HLA-A*24/ HLA-A*02 CMV tetramers respectively. We have demonstrated that the CD8+CD127lo/-CD25+ phenotype could be an alternative marker of CD8+ Tregs. These observations suggest that HTLV-1 specific CD8+ Tregs could be implicated in the impaired clearance of HTLV-1 infected and transformed cells. This surrogate marker may facilitate further exploration of the immunological significance of antigen specific CD8+ Tregs in chronic viral infection. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Mechanisms of SHP-1 P2 promoter regulation in hematopoietic cells and its silencing in HTLV-1-transformed T cells

The Src homology-2-containing protein-tyrosine phosphatase 1 (SHP-1), is a negative regulator of cell signaling. It is also considered a tumor suppressor gene because of its ability to antagonize the action of tyrosine kinases. Although SHP-1 is expressed strongly in hematopoietic cells, decreased expression has been observed in various hematological malignancies, which suggests a central involvement of SHP-1 in leukemogenesis. We have shown previously that human T cell lymphotropic virus type-1 (HTLV-1) Tax-induced promoter silencing (TIPS) is an early event causing down-regulation of SHP-1 expression, which is dependent on NF-kappaB. In this study, DNase I footprinting and EMSA also revealed binding of transcription factors, specificity protein 1 (Sp1) and octamer-binding transcription factor 1 (Oct-1) to the P2 promoter, and site-directed mutagenesis confirmed that these factors contribute to the basal P2 promoter activity. Chromatin immunoprecipitation (CHIP) assays showed that Sp1, Oct-1, NF-kappaB, CREB-1, and RNA polymerase II interacted with the core SHP-1 P2 promoter in CD4+ T cells and Jurkat cells but not in HTLV-1-transformed MT-2 and HUT102 cells when HTLV-1 Tax is present. Furthermore, bisulfite sequencing of the SHP-1 P2 core region revealed heavy CpG methylation in HTLV-1-transformed cells compared with freshly isolated CD4+ T cells and HTLV-1-noninfected T cell lines. A significant inverse correlation between degree of CpG methylation and expression of SHP-1 mRNA or protein was observed. Taken together, our data support the notion that in HTLV-1-transformed CD4+ T cells, TIPS causes dissociation of transcription factors from the core SHP-1 P2 promoter, which in turn leads to subsequent DNA methylation, an important early step for leukemogenesis.

The Frequency, Diversity, and Function of Human T-Cell Leukemia Virus Type 1-Specific CD8+ T-Cell Is Reduced in Adult T-Cell Leukemia Patients.

Human T-cell lymphotropic virus type 1 (HTLV-1)-specific CTL are thought to be immune effectors that reduce the risk of adult T-cell leukemia (ATL). However, in vivo conditions of anti-HTLV-1 CTL before and after ATL development have yet to be determined. To characterize anti-HTLV-1 CTL in asymptomatic HTLV-1 carriers (AC) and ATL patients, we analyzed the frequency and diversity of HTLV-1-specific CD8+ T cells in PBMC of 35 AC and 32 ATL patients using 16 distinct epitopes of HTLV-1 Tax or Env/HLA tetramers along with intracellular cytolytic effector molecules (IFN-γ , perforin, and granzyme B). Overall frequency of subjects possessing Tax-specific CD8+ T cells was significantly lower in ATL than AC (53% vs. 90%, p < 0.001) whereas the difference in Env-specific CD8+ T cells was not statistically significant. AC possessed Tax11-19/HLA-A*0201-specific tetramer+ cells by 90% and Tax301-309/HLA-A*2402-specific tetramer+ cells by 92%. Some AC recognized more than one epitope. In contrast, ATL recognized only Tax 11-19 with HLA-A*0201 and Tax301-309 with HLA-A*2402 at frequencies of 30% and 55%. There were also significant differences in percentage of cells binding Tax11-19/HLA-A*0201, Tax301-309/HLA-A*2402, and CMV/HLA tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-γ in response to Tax. Contrarily, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC, but no differences in anti-CMV CD8+ T cells. In addition, expression of perforin and granzyme B in HTLV-1 Tax tetramer+/CD8+ T lymphocytes were diminished in comparison to those in CMV tetramer+/CD8+ T lymphocytes in both AC and ATL. Frequency of Tax-specific CD8+ T cells in AC were related with proviral load in HLA-A*0201. These results suggest that our HTLV-1/HLA tetramer assay enabled analysis of anti-HTLV-1 CD8+ T cells in PBMC of AC and ATL patients and demonstrated deletion of anti-Tax CD8+ T cells in ATL patients. Intracellular cytokine expression in anti-HTLV-1 CD8+ T cells had significant difference between AC and ATL, but not in anti-CMV CD8+ T cells. The reduced frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be related to the development of ATL. This HLA tetramer assay can be used for monitoring the in vivo status of CTL and it may be possible to identify the high risk group in AC of developing to ATL.Summary of HTLV-1-specific tetramer and HTLV-1 proviral load in AC and ATL patientsACATLSubjects positive for Tax tetramer†28 (n = 31)15 (n = 28) *Subjects positive for Env tetramer†4 (n = 31)0 (n = 28)Tax11-19 tetramer+ CD8+ T cells†9 (n = 10)3 (n = 10) **Tax301-309 tetramer+ CD8+ T cells†22 (n = 24)12 (n = 22) *HTLV-1 proviral load††65.4 ± 7.4 (n = 35)1095.4 ± 194.1 (n = 29) *†The number of subject positive for tetramers; the percentage of HTLV-1/HLA tetramer+/CD8+ T cells in the CD8+/CD45+ T lymphocytes > 0.1% are counted as subject positives for tetramer. ††The HTLV-1 proviral load is the ± S.E., copies/1000 PBMC.*p < 0.001; **p < 0.05, Significant differences between AC and ATL.

Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity ofthe Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interactionwith Tax

In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).

Histone deacetylase 3 represses HTLV-1 tax transcription

We examined the epigenetic mechanisms involved in human T-cell lymphotropic virus type 1 (HTLV-1) Tax expression. Blockade of histone deacetylation with trichostatin A (TSA) resulted in Tax upregulation. Using a chromatin immunoprecipitation (ChIP) assay, we verified local histone hyperacetylation at the HTLV-1 LTR in response to TSA. In agreement, HDAC3 transfection led to reductions in both Tax expression and histone acetylation. HDAC3 mutations and deletions spanning the catalytic site had variable ability to repress Tax, but HDAC activity was not essential for repression. Immunoprecipitation studies revealed that Tax co-exists in a complex containing both histone deacetylase 1 (HDAC1) and 3 (HDAC3). Our results suggest that HDACs may actively participate in the repression of HTLV-1 Tax transcription.

Advances in HTLV-1 Peptide Vaccines and Therapeutics

In the past two decades a large initiative has been put forth to understand the biological and pathogenic properties of the human T-cell lymphotropic virus type 1 (HTLV-1); this has ultimately led to the development of various experimental vaccination and therapeutic strategies to combat HTLV-1 infection. The focus of this work is to outline key targets for the design of therapeutics for HTLV-1, such as fusion mediated by the envelope glycoprotein, and to discuss reports of novel vaccines or therapeutics. These strategies include peptide, recombinant protein, DNA, and viral vectors. The final focus of this review is to acquaint the reader with vaccine approaches developed in our laboratory over the last decade. These strategies include the development of envelope glycoprotein derived B-cell epitopes for the induction of neutralizing antibodies, as well as a strategy to generate a multivalent cytotoxic T-lymphocyte (CTL) response against the HTLV-1 Tax antigen.