HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP)

The HTLV-1 transactivator protein Tax is essential for malignant transformation of CD4 T cells, ultimately leading to adult T-cell leukemia/lymphoma (ATL). Malignant transformation may involve development of apoptosis resistance. In this study we investigated the molecular mechanisms by which HTLV-1 Tax confers resistance toward CD95-mediated apoptosis. We show that Tax-expressing T-cell lines derived from HTLV-1-infected patients express elevated levels of c-FLIP(L) and c-FLIP(S). The levels of c-FLIP correlated with resistance toward CD95-mediated apoptosis. Using an inducible system we demonstrated that both resistance toward CD95-mediated apoptosis and induction of c-FLIP are dependent on Tax. In addition, analysis of early cleavage of the BH3-only Bcl-2 family member Bid, a direct caspase-8 substrate, revealed that apoptosis is inhibited at a CD95 death receptor proximal level in Tax-expressing cells. Finally, using siRNA we directly showed that c-FLIP confers Tax-mediated resistance toward CD95-mediated apoptosis. In conclusion, our data suggest an important mechanism by which expression of HTLV-1 Tax may lead to immune escape of infected T cells and, thus, to persistent infection and transformation.     

High frequencies of Th1-type CD4(+) T cells specific to HTLV-1 Env and Tax proteins in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis

CD4(+) T cells are critical for inducing and maintaining efficient humoral and cellular immune responses to pathogens. The CD4(+) T-cell response in human T-lymphotropic virus 1 (HTLV-1) infection has not been studied in detail. However, CD4(+) T cells have been shown to predominate in early lesions in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). We present direct estimates of HTLV-1 Env- and Tax-specific CD4(+) T-cell frequencies in patients infected with HTLV-1. We first showed that there was a strong bias toward the Th1 phenotype in these HTLV-1-specific CD4(+) T cells in patients with HAM/TSP. We then demonstrated significantly higher frequencies of HTLV-1-specific Th1-type CD4(+) T cells in HAM/TSP patients than in asymptomatic HTLV-1 carriers. The majority of these HTLV-1-specific CD4(+) T cells did not express HTLV-1 Tax and were therefore unlikely to be infected by HTLV-1. High frequencies of activated HTLV-1-specific CD4(+) T cells of the Th1 phenotype might contribute to the initiation or pathogenesis of HAM/TSP and other HTLV-1-associated inflammatory diseases.     

Strong induction of 4-1BB, a growth and survival promoting costimulatory receptor, in HTLV-1-infected cultured and patients' T cells by the viral Tax oncoprotein

Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia, stimulates the growth of infected T cells in cultures and in nonleukemic patients. In the latter, HTLV-1 is found in long-term persisting T-cell clones. The persistence of normal T cells is controlled by the growth-stimulating and antiapoptotic functions of costimulatory receptors, while the growth-stimulating HTLV-1 functions are mediated by the viral oncoprotein Tax. Here we analyzed the impact of Tax on costimulatory receptors in T cells with repressible Tax and found that among these receptors 4-1BB (TNFRSF9/CD137/ILA) was induced most strongly. Up-regulated 4-1BB expression was a consistent feature of all HTLV-1–infected cell lines, whether patient-derived or in vitro transformed. Tax was sufficient to induce the expression of the endogenous 4-1BB gene in uninfected T cells, and it strongly activated (45-fold) the 4-1BB promoter via a single NF-B site. The ligand of 4-1BB was also found on transformed T-cell lines, opening up the possibility of autostimulation. Moreover, 4-1BB expression in patients' lymphocytes ex vivo correlated with Tax expression, strongly suggesting Tax-mediated 4-1BB activation in vivo. Thus, 4-1BB up-regulation by Tax could contribute to growth, survival, and clonal expansion of the infected cells during persistence and disease.     

Engagement of specific T-cell surface molecules regulates cytoskeletal polarization in HTLV-1-infected lymphocytes

Cell-cell contact is required for efficient transmission of human T-lymphotropic virus type 1 (HTLV-1). An HTLV-1-infected cell polarizes its microtubule-organizing center (MTOC) toward the cell-cell junction; HTLV-1 core (Gag) complexes and the HTLV-1 genome accumulate at the point of contact and are then transferred to the uninfected cell. However, the mechanisms involved in this cytoskeletal polarization and transport of HTLV-1 complexes are unknown. Here, we tested the hypothesis that engagement of a specific T-cell surface ligand is synergistic with HTLV-1 infection in causing polarization of the MTOC to the cell contact region. We show that antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54) caused MTOC polarization at a higher frequency in HTLV-1-infected cells. ICAM-1 is upregulated on HTLV-1-infected cells, and, in turn, ICAM-1 on the cell surface upregulates HTLV-1 gene expression. We propose that a positive feedback loop involving ICAM-1 and HTLV-1 Tax protein facilitates the formation of the virologic synapse and contributes to the T-cell tropism of HTLV-1. In contrast, MTOC polarization induced in T cells by antibodies to CD3 or CD28 was significantly inhibited by HTLV-1 infection.     

HTLV type 1 infection in squirrel monkeys (Saimiri sciureus): a promising animal model for HTLV type 1 human infection

We show that the squirrel monkey Saimiri sciureus is susceptible to experimental infection with either syngeneic or allogeneic HTLV-1-immortalized cells. As in humans, such experimental inoculation leads to chronic infection, and HTLV-1 provirus was detected in PBMCs by PCR. Chronically infected monkeys developed high titers of antibodies against the structural proteins of the virus, as do HTLV-1-infected humans. Furthermore, in serially sacrificed squirrel monkeys infected with HTLV-1, proviral DNA was detected at primary phases of infection in PBMCs, spleens, and lymph nodes. Tax/rex mRNA was also detected by RT-PCR in the PBMCs of two monkeys at 12 days after inoculation and in the spleen and lymph nodes of the monkey sacrificed on Day 12. In this animal, scattered HTLV-1-tax/rex mRNA-positive lymphocytes were detected by in situ hybridization in frozen sections of the spleen. These results indicate that PBMCs, spleen, and lymph nodes serve as major reservoirs for HTLV-1 during the early phase of infection. To evaluate the relationship between viral expression and the immune response during infection, humoral and cytotoxic T cell responses (CTL) were studied at various times after inoculation. Antibodies to HTLV-1 were detected 3 weeks after infection and anti-p40Tax and anti-Env CTL activity was detected 2 months after infection and remained detectable thereafter. Our results indicate that the squirrel monkey provides a useful animal model for studying the pathogenesis of HTLV-1 and for evaluating new candidate vaccines.     

Majority of interferon-gamma-producing CD4+ cells in patients infected with human T cell lymphotrophic virus do not express tax protein

The present study was conducted to determine whether interferon (IFN)-gamma production by CD4(+) cells in patients infected with human T cell lymphotropic virus (HTLV) is associated with expression of Tax, an HTLV type 1 (HTLV-1) transactivator. The frequency of IFN-gamma production from CD4(+) cells was greater in HTLV-1-infected patients (n=21) than in uninfected (n=3) and Strongyloides stercoralis-infected patients (n=4), and greater in patients with HTLV-1 with detectable Tax than in patients with HTLV-1 with undetectable Tax. In the patients with HTLV-1 with detectable Tax, the majority of CD4(+) cells making IFN-gamma did not express Tax.     

Anti-Apoptotic Effect of Tax: An NF-κB Path or a CREB Way?

The NF-κB pathway is intimately linked to the survival of mammalian cells, and its activation by Tax has consequently been considered important for human T-cell leukemia/lymphoma virus type 1 (HTLV-1)-infected cell resistance to death. Very little emphasis has been given to other mechanisms, although Tax regulates the expression and activity of several cellular genes. The finding that CREB protein is activated in HTLV-1 infected cells underlines the possibility that other mechanisms of survival may be implicated in HTLV-1 infection. Indeed, CREB activation or overexpression plays a role in normal hematopoiesis, as well as in leukemia development, and CREB is considered as a survival factor in various cell systems. A better understanding of the different molecular mechanisms used by Tax to counteract cell death will also help in the development of new therapeutic strategies for HTLV-1 associated diseases.     

Short communication an interferon-γ ELISPOT assay with two cytotoxic T cell epitopes derived from HTLV-1 tax region 161-233 discriminates HTLV-1-associated myelopathy/tropical spastic paraparesis patients from asymptomatic HTLV-1 carriers in a Peruvian population

HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic and progressive disorder caused by the human T-lymphotropic virus type 1 (HTLV-1). In HTLV-1 infection, a strong cytotoxic T cell (CTL) response is mounted against the immunodominant protein Tax. Previous studies carried out by our group reported that increased IFN-γ enzyme-linked immunospot (ELISPOT) responses against the region spanning amino acids 161 to 233 of the Tax protein were associated with HAM/TSP and increased HTLV-1 proviral load (PVL). An exploratory study was conducted on 16 subjects with HAM/TSP, 13 asymptomatic carriers (AC), and 10 HTLV-1-seronegative controls (SC) to map the HAM/TSP-associated CTL epitopes within Tax region 161-233. The PVL of the infected subjects was determined and the specific CTL response was evaluated with a 6-h incubation IFN-γ ELISPOT assay using peripheral blood mononuclear cells (PBMCs) stimulated with 16 individual overlapping peptides covering the Tax region 161-233. Other proinflammatory and Th1/Th2 cytokines were also quantified in the supernatants by a flow cytometry multiplex assay. In addition, a set of human leukocyte antigen (HLA) class I alleles that bind with high affinity to the CTL epitopes of interest was determined using computational tools. Univariate analyses identified an association between ELISPOT responses to two new CTL epitopes, Tax 173-185 and Tax 181-193, and the presence of HAM/TSP as well as an increased PVL. The HLA-A*6801 allele, which is predicted to bind to the Tax 181-193 peptide, was overpresented in the HAM/TSP patients tested.