Envelope is a major viral determinant of the distinct in vitro cellular transformation tropism of human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are related deltaretroviruses but are distinct in their disease-inducing capacity. These viruses can infect a variety of cell types, but only T lymphocytes become transformed, which is defined in vitro as showing indefinite interleukin-2-independent growth. Studies have indicated that HTLV-1 has a preferential tropism for CD4+ T cells in vivo and is associated with the development of leukemia and neurological disease. Conversely, the in vivo T-cell tropism of HTLV-2 is less clear, although it appears that CD8+ T cells preferentially harbor the provirus, with only a few cases of disease association. The difference in T-cell transformation tropism has been confirmed in vitro as shown by the preferential transformation of CD4+ T cells by HTLV-1 versus the transformation of CD8+ T cells by HTLV-2. Our previous studies showed that Tax and overlapping Rex do not confer the distinct T-cell transformation tropisms between HTLV-1 and HTLV-2. Therefore, for this study HTLV-1 and HTLV-2 recombinants were generated to assess the contribution of LTR and env sequences in T-cell transformation tropism. Both sets of proviral recombinants expressed p19 Gag following transfection into cells. Furthermore, recombinant viruses were replication competent and had the capacity to transform T lymphocytes. Our data showed that exchange of the env gene resulted in altered T-cell transformation tropism compared to wild-type virus, while exchange of long terminal repeat sequences had no significant effect. HTLV-2/Env1 preferentially transformed CD4+ T cells similarly to wild-type HTLV-1 (wtHTLV-1), whereas HTLV-1/Env2 had a transformation tropism similar to that of wtHTLV-2 (CD8+ T cells). These results indicate that env is a major viral determinant for HTLV T-cell transformation tropism in vitro and provides strong evidence implicating its contribution to the distinct pathogenesis resulting from HTLV-1 versus HTLV-2 infections.     

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 use different receptor complexes to enter T cells

Studies using adherent cell lines have shown that glucose transporter-1 (GLUT-1) can function as a receptor for human T-cell leukemia virus type 1 (HTLV). In primary CD4(+) T cells, heparan sulfate proteoglycans (HSPGs) are required for efficient entry of HTLV-1. Here, the roles of HSPGs and GLUT-1 in HTLV-1 and HTLV-2 Env-mediated binding and entry into primary T cells were studied. Examination of the cell surface of activated primary T cells revealed that CD4(+) T cells, the primary target of HTLV-1, expressed significantly higher levels of HSPGs than CD8(+) T cells. Conversely, CD8(+) T cells, the primary target of HTLV-2, expressed GLUT-1 at dramatically higher levels than CD4(+) T cells. Under these conditions, the HTLV-2 surface glycoprotein (SU) binding and viral entry were markedly higher on CD8(+) T cells while HTLV-1 SU binding and viral entry were higher on CD4(+) T cells. Binding studies with HTLV-1/HTLV-2 SU recombinants showed that preferential binding to CD4(+) T cells expressing high levels of HSPGs mapped to the C-terminal portion of SU. Transfection studies revealed that overexpression of GLUT-1 in CD4(+) T cells increased HTLV-2 entry, while expression of HSPGs on CD8(+) T cells increased entry of HTLV-1. These studies demonstrate that HTLV-1 and HTLV-2 differ in their T-cell entry requirements and suggest that the differences in the in vitro cellular tropism for transformation and in vivo pathobiology of these viruses reflect different interactions between their Env proteins and molecules on CD4(+) and CD8(+) T cells involved in entry.     

Role of human T-cell leukemia virus type 1 X region proteins in immortalization of primary human lymphocytes in culture.

Human T-cell leukemia virus type 1 (HTLV-1) immortalizes human CD4+ T lymphocytes in culture. Previous studies show that in the context of a herpesvirus saimiri vector, the sequence of the X region at the 3' end of the HTLV-1 genome is also capable of immortalizing CD4+ lymphocytes in the absence of HTLV-1 structural proteins. The X region of HTLV-1 encodes two trans-acting viral proteins, the 42-kDa Tax protein and the 27-kDa Rex protein. Infection of human cord blood cells with herpesvirus saimiri recombinants which contain HTLV-1 X region sequences defective for expression of tax, rex, or both tax and rex demonstrates that tax function is necessary and sufficient for immortalization of primary human CD4+ cord blood lymphocytes in culture in the context of the herpesvirus saimiri vector.     

Tax-inducible production of CC chemokine ligand 22 by human T cell leukemia virus type 1 (HTLV-1)-infected T cells promotes preferential transmission of HTLV-1 to CCR4-expressing CD4+ T cells

Adult T cell leukemia is a mature CD4+ T cell malignancy which predominantly expresses CCR4 and is etiologically associated with human T cell leukemia virus type 1 (HTLV-1). Because HTLV-1 transmission depends on close cell-cell contacts, HTLV-1-infected T cells may preferentially interact with CCR4+CD4+ T cells for efficient viral transmission. In terms of gene expression and protein secretion, we found a strong correlation between HTLV-1 Tax oncoprotein and CCL22, a CCR4 ligand, in HTLV-1-infected T cells. Transient Tax expression in an HTLV-1-negative T cell line activated the CCL22 promoter and induced CCL22. Additionally, tax gene knockdown by small interference RNA reduced CCL22 expression in the infected T cells. These findings indicate that CCL22 is a cellular target gene of Tax. In chemotaxis assays, the culture supernatants of HTLV-1-infected T cells selectively attracted CCR4+CD4+ T cells in PBMCs. This was blocked by pretreating the supernatants with anti-CCL22 Ab or PBMCs with a synthetic CCR4 antagonist. In coculture experiments, primary CCR4+CD4+ T cells significantly adhered to Tax-expressing cells. This adhesion was blocked by the CCR4 antagonist or pertussis toxin. Interestingly, CCR4 was redistributed to the contact region, and in some cases, this was accompanied by a polarized microtubule-organizing center, which is an indicator of virological synapse formation, in the infected T cells. Finally, anti-CCL22 Ab treatment also blocked HTLV-1 transmission to primary CD4+ T cells in coculture experiments with HTLV-1 producer cells. Thus, HTLV-1-infected T cells produce CCL22 through Tax and selectively interact with CCR4+CD4+ T cells, resulting in preferential transmission of HTLV-1 to CCR4+CD4+ T cells.     

Reduced frequency, diversity, and function of human T cell leukemia virus type 1-specific CD8+ T cell 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-gamma, 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 Tax11-19 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 and Tax301-309/HLA-A*2402 tetramers between AC and ATL. Anti-HTLV-1 Tax CD8+ T cells in AC and ATL produced IFN-gamma in response to Tax. In contrast, perforin and granzyme B expression in anti-HTLV-1 CD8+ T cells of ATL was significant lower than that of AC. Frequency of Tax-specific CD8+ T cells in AC was related to proviral load in HLA-A*0201. These results suggest that decreased frequency, diversity, and function of anti-HTLV-1 Tax CD8+ T cell clones may be one of the risks of ATL development.     

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.     

Human T-cell leukemia virus type 1 tax oncoprotein suppression of multilineage hematopoiesis of CD34+ cells in vitro

Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis, bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2, respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors, and transduced cells were cultured in a semisolid medium permissive for the development of erythroid, myeloid, and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro, in contrast to Tax2-transduced cells, which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected, suggesting that Tax1 inhibited the maturation of relatively early, uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis, lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells, an activity that is not displayed by Tax2, and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since hematopoietic progenitor cells may serve as a latently infected reservoir for HTLV infection in vivo, the different abilities of HTLV-1 and -2 Tax to suppress hematopoiesis may play a role in the respective clinical outcomes after infection with HTLV-1 or -2.     

Human T Lymphotropic Virus Type 1 SU Residue 195 Plays a Role in Determining the Preferential CD4+ T Cell Immortalization/Transformation Tropism

Human T lymphotropic virus type 1 (HTLV-1) mainly causes adult T cell leukemia and predominantly immortalizes/transforms CD4⁺ T cells in culture. HTLV-2 is aleukemic and predominantly immortalizes/transforms CD8⁺ T cells in culture. We have shown previously that the viral envelope is the genetic determinant of the differential T cell tropism in culture. The surface component (SU) of the HTLV-1 envelope is responsible for binding to the cellular receptors for entry. Here, we dissect the HTLV-1 SU further to identify key domains that are involved in determining the immortalization tropism. We generated HTLV-1 envelope recombinant virus containing the HTLV-2 SU domain. HTLV-1/SU2 was capable of infecting and immortalizing freshly isolated peripheral blood mononuclear cells in culture. HTLV-1/SU2 shifted the CD4⁺ T cell immortalization tropism of wild-type HTLV-1 (wtHTLV-1) to a CD8⁺ T cell preference. Furthermore, a single amino acid substitution, N195D, in HTLV-1 SU (Ach.195) resulted in a shift to a CD8⁺ T cell immortalization tropism preference. Longitudinal phenotyping analyses of the in vitro transformation process revealed that CD4⁺ T cells emerged as the predominant population by week 5 in wtHTLV-1 cultures, while CD8⁺ T cells emerged as the predominant population by weeks 4 and 7 in wtHTLV-2 and Ach.195 cultures, respectively. Our results indicate that SU domain independently influences the preferential T cell immortalization tropism irrespective of the envelope counterpart transmembrane (TM) domain. We further showed that asparagine at position 195 in HTLV-1 SU is involved in determining this CD4⁺ T cell immortalization tropism. The slower emergence of the CD8⁺ T cell predominance in Ach.195-infected cultures suggests that other residues/domains contribute to this tropism preference.     

Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax.

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.     

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.     

Direct evidence for a chronic CD8+-T-cell-mediated immune reaction to tax within the muscle of a human T-cell leukemia/lymphoma virus type 1-infected patient with sporadic inclusion body myositis

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) infection can lead to the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), concomitantly with or without other inflammatory disorders such as myositis. These pathologies are considered immune-mediated diseases, and it is assumed that migration within tissues of both HTLV-1-infected CD4(+) T cells and anti-HTLV-1 cytotoxic T cells represents a pivotal event. However, although HTLV-1-infected T cells were found in inflamed lesions, the antigenic specificity of coinfiltrated CD8(+) T cells remains to be determined. In this study, we performed both ex vivo and in situ analyses using muscle biopsies obtained from an HTLV-1-infected patient with HAM/TSP and sporadic inclusion body myositis. We found that both HTLV-1-infected CD4(+) T cells and CD8(+) T cells directed to the dominant Tax antigen can be amplified from muscle cell cultures. Moreover, we were able to detect in two successive muscle biopsies both tax mRNA-positive mononuclear cells and T cells recognized by the Tax11-19/HLA-A*02 tetramer and positive for perforin. These findings provide the first direct demonstration that anti-Tax cytotoxic T cells are chronically recruited within inflamed tissues of an HTLV-1 infected patient, which validates the cytotoxic immune reaction model for the pathogenesis of HTLV-1-associated inflammatory disease.     

Conserved CDR3 regions in T-cell receptor (TCR) CD8(+) T cells that recognize the Tax11-19/HLA-A*0201 complex in a subject infected with human T-cell leukemia virus type 1: relationship of T-cell fine specificity and major histocompatibility complex/peptide/TCR crystal structure

We investigated the T-cell receptor (TCR) repertoire of CD8(+) T cells that recognize the Tax11-19 immunodominant epitope of Tax protein expressed by human T-cell leukemia virus (HTLV-1) that is implicated in the disease HTLV-1-associated myelopathy (HAM/TSP). A panel of Tax11-19-reactive CD8(+) T-cell clones was generated by single-cell cloning of Tax11-19/HLA-A*0201 tetramer-positive peripheral blood lymphocytes from an HTLV-1-infected individual. The analyses of TCR usage revealed that the combination of diverse TCR alpha and beta chains could be used for the recognition of Tax11-19 but the major population of T-cell clones (15 of 24 clones) expressed the TCR V beta 13S1 and V alpha 17 chain. We found striking similarities in CDR3 regions of TCR alpha and beta chains between our major group of CD8(+) T-cell clones and those originating from different subjects as previously reported, including TCRs with resolved crystal structures. A 3-amino-acid sequence (PG-G) in the CDR3 region of the V beta chain was conserved among all the Tax11-19-reactive T-cell clones expressing V beta 13S1 and V alpha 17 chains. Conserved amino acids in the CDR3 region do not directly contact the Tax11-19 peptide, as corroborated by the crystal structure of B7-TCR, a TCR that is almost identical to VB13S1 clones isolated in this study. Analysis of fine peptide specificity using altered peptide ligands (APL) of Tax11-19 revealed a similar recognition pattern among this panel of T-cell clones. These data suggest that the PG-G amino acids in the CDR3 beta loop provide a structural framework necessary for the maintenance of the tertiary TCR structure.