Oligomannose-coated liposomes efficiently induce human T-cell leukemia virus-1-specific cytotoxic T lymphocytes without adjuvant

Human T-cell leukemia virus-1 (HTLV-1) causes adult T-cell leukemia/lymphoma, which is an aggressive peripheral T-cell neoplasm. Insufficient T-cell response to HTLV-1 is a potential risk factor in adult T-cell leukemia/lymphoma. Efficient induction of antigen-specific cytotoxic T lymphocytes is important for immunological suppression of virus-infected cell proliferation and oncogenesis, but efficient induction of antigen-specific cytotoxic T lymphocytes has evaded strategies utilizing poorly immunogenic free synthetic peptides. Here, we examined the efficient induction of an HTLV-1-specific CD8+ T-cell response by oligomannose-coated liposomes (OMLs) encapsulating the human leukocyte antigen (HLA)-A*0201-restricted HTLV-1 Tax-epitope (OML/Tax). Immunization of HLA-A*0201 transgenic mice with OML/Tax induced an HTLV-1-specific gamma-interferon reaction, whereas immunization with epitope peptide alone induced no reaction. Upon exposure of dendritic cells to OML/Tax, the levels of CD86, major histocompatibility complex class I, HLA-A02 and major histocompatibility complex class II expression were increased. In addition, our results showed that HTLV-1-specific CD8+ T cells can be efficiently induced by OML/Tax from HTLV-1 carriers compared with epitope peptide alone, and these HTLV-1-specific CD8+ T cells were able to lyse cells presenting the peptide. These results suggest that OML/Tax is capable of inducing antigen-specific cellular immune responses without adjuvants and may be useful as an effective vaccine carrier for prophylaxis in tumors and infectious diseases by substituting the epitope peptide.     

Overexpression of transforming growth factor-beta in transgenic mice carrying the human T-cell lymphotropic virus type I tax gene.

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.     

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.     

Analysis of p53 inactivation in a human T-cell leukemia virus type 1 Tax transgenic mouse model

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). The HTLV-1 Tax protein has been strongly linked to oncogenesis and is considered to be the transforming protein of this virus. A Tax transgenic mouse model was utilized to study the contribution of p53 inactivation to Tax-mediated tumorigenesis. These mice develop primary, peripheral tumors consisting of large granular lymphocytic (LGL) cells, which also infiltrate the lymph nodes, bone marrow, spleen, liver, and lungs. Primary Tax-induced tumors and tumor-derived cell lines exhibited functional inactivation of the p53 apoptotic pathway; such tumors and tumor cell lines were resistant to an apoptosis-inducing stimulus. In contrast, p53 mutations in tumors were found to be associated with secondary organ infiltration. Three of four identified mutations inhibited transactivation and apoptosis induction activities in vitro. Furthermore, experiments which involved mating Tax transgenic mice with p53-deficient mice demonstrated minimal acceleration in initial tumor formation, but significantly accelerated disease progression and death in mice heterozygous for p53. These studies suggest that functional inactivation of p53 by HTLV-1 Tax, whether by mutation or another mechanism, is not critical for initial tumor formation, but contributes to late-stage tumor progression.     

Physical interaction of human T-cell leukemia virus type 1 Tax with cyclin-dependent kinase 4 stimulates the phosphorylation of retinoblastoma protein

The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1) induces leukemia in transgenic mice and permanent T-cell growth in vitro. In transformed lymphocytes, it acts as an essential growth factor. Tax stimulates the cell cycle in the G(1) phase by activating the cyclin-dependent kinase (CDK) CDK4 and CDK6 holoenzyme complexes. Here we show that Tax directly interacts with CDK4. This binding to CDK4 was specific, since Tax did not bind to either CDK2 or CDK1. The interaction with CDK4/cyclin D complexes was observed in vitro, in transfected fibroblasts, in HTLV-1-infected T cells, and in adult T-cell leukemia-derived cultures. Binding studies with several point and deletion mutants indicated that the N terminus of Tax mediates the interaction with CDK4. The Tax/CDK complex represented an active holoenzyme which capably phosphorylates the Rb protein in vitro and is resistant to repression by the inhibitor p21(CIP). Binding-deficient Tax mutants failed to activate CDK4, indicating that direct association with Tax is required for enhanced kinase activity. Tax also increased the association of CDK4 with its positive cyclin regulatory subunit. Thus, protein-protein contact between Tax and the components of the cyclin D/CDK complexes provides a further mechanistic explanation for the mitogenic and immortalizing effects of this HTLV-1 oncoprotein.     

Transgenic thymocytes are refractory to transformation by the human T-cell leukemia virus type I tax gene.

Previous transgenic work demonstrated transforming activity of the human T-cell leukemia virus type I Tax protein in fibroblasts. In the present study, a Thy-1-based vector was used to express Tax in thymocytes. These mice developed no functional or neoplastic abnormalities of T cells but developed fibroblastic tumors with a longer latency than in the previous model.     

Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.     

Stable ubiquitination of human T-cell leukemia virus type 1 tax is required for proteasome binding

Human T-cell leukemia virus type 1 (HTLV-1) is the retrovirus responsible for adult T-cell leukemia and HTLV-1-associated myelopathy. Adult T-cell leukemia development is mainly due to the ability of the viral oncoprotein Tax to promote T-cell proliferation, whereas the appearance of HTLV-1-associated myelopathy involves the antigenic properties of Tax. Understanding the events regulating the intracellular level of Tax is therefore an important issue. How Tax is degraded has not been determined, but it is known that Tax binds to proteasomes, the major sites for degradation of intracellular proteins, generally tagged through polyubiquitin conjugation. In this study, we investigated the relationship between Tax, ubiquitin, and proteasomes. We report that mono- and polyubiquitinated Tax proteins can be recovered from both transfected 293T cells and T lymphocytes. We also show that lysine residues located in the carboxy-terminal domain of Tax are the principal targets of this process. Remarkably, we further demonstrate that mutation of lysine residues in the C-terminal part of Tax, which massively reduces Tax ubiquitination, impairs proteasome binding, and conversely, that a Tax mutant that binds poorly to this particle (M22) is faintly ubiquitinated, suggesting that Tax ubiquitination is required for association with cellular proteasomes. Finally, we document that comparable amounts of ubiquitinated species were found whether proteasome activities were inhibited or not, providing evidence that they are not directly addressed to proteasomes for degradation. These findings indicate that although it is ubiquitinated and binds to proteasomes, Tax is not massively degraded via the ubiquitin-proteasome pathway and therefore reveal that Tax conjugation to ubiquitin mediates a nonproteolytic function.     

Soluble HTLV-I Tax1 protein stimulates proliferation of human peripheral blood lymphocytes.

Human T-cell leukemia virus type I (HTLV-I) is associated with two human diseases, adult T-cell leukemia (ATL) and tropical spastic paraparesis/HTLV-I associated myelopathy (TSP/HAM). Lymphocytes from patients with ATL or TSP/HAM display abnormal proliferation properties in culture. Here we report that purified, soluble Tax1 protein can be taken up by, and stimulate proliferation of, uninfected human peripheral blood lymphocytes (PBLs) that have been stimulated with phytohemagglutinin (PHA). Tax1 was 40 to 70% as active as interleukin-2 (IL-2) in stimulating proliferation of PBLs. Heat inactivation, chloroform extraction, and immunoprecipitation with antisera specific for Tax1 each abolished the ability of the protein to stimulate lymphocyte proliferation. Tax1 failed to stimulate PBL proliferation in the absence of PHA. After an initial round of cell division, Tax1-treated PBLs exhibited prolonged sensitivity to IL-2-induced proliferation. These results indicate that Tax1 can stimulate lymphocyte proliferation in culture and imply that extracellular Tax1 may be involved in the spontaneous proliferation of TSP/HAM lymphocytes and the IL-2-dependent proliferation of ATL lymphocytes.     

Interleukin-13 overexpression by tax transactivation: a potential autocrine stimulus in human T-cell leukemia virus-infected lymphocytes

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein induces growth transformation and is critical for the pathogenesis of the HTLV-1-induced adult T-cell leukemia (ATL). It stimulates the cell cycle and transactivates cellular genes. Here we show that the expression of interleukin-13 (IL-13) is upregulated as a consequence of Tax in HTLV-1-transformed T cells and ATL-derived cultures. IL-13 exerts proliferative and antiapoptotic functions and is linked to leukemogenesis, since it stimulates Hodgkin lymphoma cells by an autocrine mechanism. Overexpression of IL-13 RNA and protein was confirmed in HTLV-1-positive and Tax-transformed cells. Induction of endogenous IL-13 levels in tax-transfected Jurkat cells and in conditional Tax-expressing transformed T lymphocytes suggested that Tax can replace signals required for IL-13 synthesis. For functional analysis, the IL-13 promoter and deletion variants were cloned into luciferase reporter plasmids. Experiments with transfected human T lymphocytes revealed a 16-fold stimulation of the IL-13 promoter by Tax. Experiments with Tax mutants indicated that none of the classical transactivation pathways (SRF, CREB, and NF-kappaB) is sufficient for the transactivation; at least two different Tax functions are required for full transactivation. The IL-13 promoter is stimulated via two elements; one is a NF-AT binding P element, and the other is a putative AP-1 site. The following observations suggest that IL-13 may stimulate HTLV-1-transformed cells by an autocrine mechanism: (i) the HTLV-1-transformed cells express the IL-13 receptor on their surface, and (ii) STAT6, a downstream effector of IL-13 signaling, is constitutively activated. Thus, in summary, Tax, by transactivating the promoter, induces IL-13 overexpression that possibly leads to an autocrine stimulation of HTLV-1-infected cells.     

Apoptosis induced by the histone deacetylase inhibitor FR901228 in human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells

Inhibition of histone deacetylase (HDAC) activity induces growth arrest, differentiation, and, in certain cell types, apoptosis. FR901228, FK228, or depsipeptide, is an HDAC inhibitor effective in T-cell lymphomas. Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. We examined whether FR901228 is effective for treatment of ATL by assessing its ability to induce apoptosis of HTLV-1-infected T-cell lines and primary leukemic cells from ATL patients. FR901228 induced apoptosis of Tax-expressing and -unexpressing HTLV-1-infected T-cell lines and selective apoptosis of primary ATL cells, especially those of patients with acute ATL. FR901228 also efficiently reduced the DNA binding of NF-kappaB and AP-1 in HTLV-1-infected T-cell lines and primary ATL cells and down-regulated the expression of Bcl-x(L) and cyclin D2, regulated by NF-kappaB. Although the viral protein Tax is an activator of NF-kappaB and AP-1, FR901228-induced apoptosis was not associated with reduced expression of Tax. In vivo use of FR901228 partly inhibited the growth of tumors of HTLV-1-infected T cells transplanted subcutaneously in SCID mice. Our results indicated that FR901228 could induce apoptosis of these cells and suppress the expression of NF-kappaB and AP-1 and suggest that FR901228 could be therapeutically effective in ATL.     

Human T-cell leukemia virus type 1 Tax transactivates the matrix metalloproteinase 7 gene via JunD/AP-1 signaling

Adult T-cell leukemia (ATL) is a T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1) and characterized by visceral invasion. Degradation of the extracellular matrix by matrix metalloproteinases (MMPs) is a crucial process in invasion of tumors and metastasis. MMP-7 (or matrilysin), is a “minimal domain MMP” with proteolytic activity against components of the extracellular matrix. To determine the involvement of MMP-7 in visceral spread in ATL, this study investigated MMP-7 expression in ATL. MMP-7 expression was identified in HTLV-1-infected T-cell lines, peripheral blood ATL cells and ATL cells in lymph nodes, but not in uninfected T-cell lines or normal peripheral blood mononuclear cells. MMP-7 expression was induced following infection of a human T-cell line with HTLV-1, and specifically by the viral protein Tax. Functionally, MMP-7 promoted cell migration of HTLV-1-infected T cells. The MMP-7 promoter activity was increased by Tax and reduced by deletion of the activator protein-1 (AP-1) binding site. Electrophoretic mobility shift assay showed high levels of AP-1 binding proteins, including JunD, in HTLV-1-infected T-cell lines and ATL cells, and Tax elicited JunD binding to the MMP-7 AP-1 element. Tax-induced MMP-7 activation was inhibited by dominant negative JunD and augmented by JunD/JunD homodimers. Short interfering RNA against JunD inhibited MMP-7 mRNA expression in HTLV-1-infected T-cell lines. These results suggest that the induction of MMP-7 by Tax is regulated by JunD and that MMP-7 could facilitate visceral invasion in ATL.