ATL and its virus]

A retrovirus called Human T cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T cell leukemia (ATL). A cultured cell line called MT-2, produces constitutively HTLV-1. The characteristics of HTLV-1 produced from MT-2 has been extensively investigated. The molecular mechanism of ATL leukemogenesis by HTLV-1 is discussed.     

Human T cell leukemia virus type I and neurologic disease: events in bone marrow, peripheral blood, and central nervous system during normal immune surveillance and neuroinflammation.

Human T cell lymphotropic/leukemia virus type I (HTLV-I) has been identified as the causative agent of both adult T cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the exact sequence of events that occur during the early stages of infection are not known in detail, the initial route of infection may predetermine, along with host, environmental, and viral factors, the subset of target cells and/or the primary immune response encountered by HTLV-I, and whether an HTLV-I-infected individual will remain asymptomatic, develop ATL, or progress to the neuroinflammatory disease, HAM/TSP. Although a large number of studies have indicated that CD4(+) T cells represent an important target for HTLV-I infection in the peripheral blood (PB), additional evidence has accumulated over the past several years demonstrating that HTLV-I can infect several additional cellular compartments in vivo, including CD8(+) T lymphocytes, PB monocytes, dendritic cells, B lymphocytes, and resident central nervous system (CNS) astrocytes. More importantly, extensive latent viral infection of the bone marrow, including cells likely to be hematopoietic progenitor cells, has been observed in individuals with HAM/TSP as well as some asymptomatic carriers, but to a much lesser extent in individuals with ATL. Furthermore, HTLV-I(+) CD34(+) hematopoietic progenitor cells can maintain the intact proviral genome and initiate viral gene expression during the differentiation process. Introduction of HTLV-I-infected bone marrow progenitor cells into the PB, followed by genomic activation and low level viral gene expression may lead to an increase in proviral DNA load in the PB, resulting in a progressive state of immune dysregulation including the generation of a detrimental cytotoxic Tax-specific CD8(+) T cell population, anti-HTLV-I antibodies, and neurotoxic cytokines involved in disruption of myelin-producing cells and neuronal degradation characteristic of HAM/TSP.     

Construction of a full-length human T cell leukemia virus type I genome from MT-2 cells containing multiple defective proviruses using overlapping polymerase chain reaction

Human T cell leukemia virus type I (HTLV-I), the etiological agent of adult T cell leukemia, integrates into the host genome as a provirus. Multiple defective copies of the integrated provirus are often present in the host genome. For this reason it is difficult to clone the intact provirus from HTLV-I-infected cells using conventional techniques. Here, we used overlapping polymerase chain reaction (PCR) to construct a full-length provirus of HTLV-I directly from an HTLV-I-transformed cell line, MT-2, which contains multiple defective proviruses. First, four overlapping proviral HTLV-I fragments (1.4-3.9 kb each) were constructed from genomic MT-2 DNA using PCR. Next, the complete HTLV-I proviral DNA (9 kb) was generated from these fragments using asymmetric PCR and cloned into a plasmid vector. 293 T cells transfected with this plasmid produced virus-like particles, and we show that these particles are capable of infecting a human T cell line. We propose that this cloning technique constitutes a powerful tool for constructing infectious molecular clones from cells of patients infected with HTLV-I or other viruses.     

Anti-tumor immunity in adult T-cell leukemia

Adult T-cell leukemia (ATL) occurs in a small population of human T-cell leukemia virus type I (HTLV-I)-infected individuals. It has been noted that ATL is incidentally associated with mother-to-child infection which occurs mainly through breast-feeding, elevated levels of proviral load, and insufficiency in HTLV-I-specific cytotoxic T lymphocyte (CTL) responses. Among these, anti-tumor potentials of HTLV-I-specific CTL have been shown in ex vivo analysis of human HTLV-I-infected individuals and also in vivo experiments by using rat models of HTLV-I-infected lymphomas. In another rat model of HTLV-I-infection, orally infected rats showed significantly higher HTLV-I proviral load but lower HTLV-I-specific cellular immune responses than in intraperitoneally infected rats. As a result, persistent viral load was inversely correlated with levels of virus-specific T-cell responses. HTLV-I-specific T-cell responses in orally infected rats recovered by re-immunization. Conversion of Tax-specific T-cell responses from low to high levels was also observed in an ATL patient who obtained complete remission after hematopoietic stem cell transplantation. These findings suggest that HTLV-I-specific immune unresponsiveness associated with oral HTLV-I infection may be a potential risk factor for development of ATL, allowing expansion of the infected cell reservoir in vivo, and that immunological strategies targeting Tax may potentially reduce the risk of ATL and induce therapeutic effects on ATL.     

Highly enhanced expression of CD70 on human T-lymphotropic virus type 1-carrying T-cell lines and adult T-cell leukemia cells

Human T-lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL). In Japan, the number of HTLV-1 carriers is estimated to be 1.2 million and more than 700 cases of ATL have been diagnosed every year. Considering the poor prognosis and lack of curative therapy of ATL, it seems mandatory to establish an effective strategy for the treatment of ATL. In this study, we attempted to identify the cell surface molecules that will become suitable targets of antibodies for anti-ATL therapy. The expression levels of approximately 40,000 host genes of three human T-cell lines carrying HTLV-1 genomes were analyzed by oligonucleotide microarray and compared with the expression levels of the genes in an HTLV-1-negative T-cell line. The HTLV-1-carrying T-cell lines used for experiments had totally different expression patterns of viral genome. Among the genes evaluated, the expression levels of 108 genes were found to be enhanced more than 10-fold in all of the T-cell lines examined and 11 of the 108 genes were considered to generate the proteins expressed on the cell surface. In particular, the CD70 gene was upregulated more than 1,000-fold and the enhanced expression of the CD70 molecule was confirmed by laser flow cytometry for various HTLV-1-carrying T-cell lines and primary CD4(+) T cells isolated from acute-type ATL patients. Such expression was not observed for primary CD4(+) T cells isolated from healthy donors. Since CD70 expression is strictly restricted in normal tissues, such as highly activated T and B cells, CD70 appears to be a potential target for effective antibody therapy against ATL.     

Establishment of hybridomas producing cancer specific human antibodies from B cell line derived from PBL of a patient with adult T cell leukemia

Adult T cell leukemia (ATL) is a malignant disease characterized by tumorous proliferation of CD4⁺ T cells infected with retrovirus human T cell leukemia virus Type-I (HTLV-I) and concurs with an autoimmune disease and cancer due to attenuated immune response. In this study, we established ATL patient derived B-cell line TM-1 producing cancer-specific IgM antibodies, and further characterized its antigen specificity by establishing hybridomas fused with human-mouse origin hetero-myeloma cell line RF-S1. We established three hybridoma cell lines termed 2E12, 3E9, and 3E10, which continuously secreted human IgM antibodies. Immunohistochemical staining of formalin-fixed tissue section using antibodies secreted from these hybridomas showed that these antibodies specifically recognized tumor sites of human colon adenocarcinomas. Antibody produced from hybridoma 3E9 bound to some of leukemic cell lines, but not to normal human PBL, which was evidenced by the flow cytometric analysis, indicating that antibody produced from 3E9 recognizes cell surface antigen specifically expressed in the leukemic cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Interleukin 2 acts directly on a Tac-positive cloned B cell line established from a patient with adult T cell leukemia

A Tac-positive B cell line termed K3B was established from a patient with adult T cell leukemia (ATL). This cell line had EBNA antigen and human T cell leukemic virus (HTLV) provirus besides B1 antigen and surface immunoglobulin. A cloned Tac-positive B cell line termed K3B01 was obtained from K3B by the limiting dilution method. The K3B01 cells were shown to absorb IL 2 activity in a tonsillar IL 2 preparation. By using this cloned cell line and a purified recombinant IL 2 preparation, it was shown that the proliferation of K3B01 cells was enhanced by the addition of recombinant IL 2. Moreover, this response was inhibited by anti-Tac antibody. These results demonstrate definitively that IL 2 acts directly on B cells through IL 2 receptors on them.     

HTLV-I and leukemogenesis

Human T-cell leukemia virus type I (HTLV-I) is a causative virus of adult T-cell leukemia (ATL). ATL is a highly aggressive neoplastic disease of CD4 positive T lymphocyte, which is featured by the pleomorphic tumor cells with hypersegmented nuclei, called " flower cell". HTLV-I increases its copy number by clonal proliferation of the host cells, not by replication of the virus. Therefore, HTLV-I eventually induces ATL. Tax, encoded by HTLV-I pX region, has been recognized as a protein that plays a central role of the transformation of HTLV-I-infected cells by its pleiotropic actions. However, fresh ATL cells frequently lose Tax protein expression by several mechanisms. Recently, HBZ was identified in the complementary strand of HTLV-I and it is suggested that HBZ is a critical gene in leukemogenesis. Furthermore, there is a long latency period before onset of ATL, indicating the multistep mechanisms of leukemogenesis. Therefore, it is suggested that multiple factors, such as viral proteins, genetic and epigenetic changes of host genome, and immune status of the hosts, could be implicated in leukemogenesis of ATL.     

DNA methylation affecting the expression of murine leukemia proviruses.

The endogenous, vertically transmitted proviral DNAs of the ecotropic murine leukemia virus in AKR embryo fibroblasts were found to be hypermethylated relative to exogenous AKR murine leukemia virus proviral DNAs acquired by infection of the same cells. The hypermethylated state of the endogenous AKR murine leukemia virus proviruses in these cells correlated with the failure to express AKR murine leukemia virus and the lack of infectivity of cellular DNA. Induction of the endogenous AKR murine leukemia virus proviruses with the methylation antagonist 5-azacytidine suggested a causal connection between DNA methylation and provirus expression. Also found to be relatively hypermethylated and noninfectious were three of six Moloney murine leukemia virus proviral DNAs in an unusual clone of infected rat cells. Recombinant DNA clones which derived from a methylated, noninfectious Moloney provirus of this cell line were found to be highly active upon transfection, suggesting that a potentially active proviral genome can be rendered inactive by cellular DNA methylation. In contrast, in vitro methylation with the bacterial methylases MHpaII and MHhaI only slightly reduced the infectivity of the biologically active cloned proviral DNA. Recombinant DNA clones which derived from a second Moloney provirus of this cell line were noninfectious. An in vitro recombination method was utilized in mapping studies to show that this lack of infectivity was governed by mechanisms other than methylation.     

Detection of the integrated feline leukemia viruses in a cat lymphoid tumor cell line by fluorescence in situ hybridization

Feline leukemia virus (FeLV) is a type-C retrovirus associated with lymphoid and hematopoietic malignancies in cats. The FeLV-induced tumors are thought to be caused, at least in part, by somatically acquired insertional mutagenesis in which the integrated provirus may activate a proto-oncogene or disrupt a tumor suppressor gene. This study was undertaken to enumerate and map the acquired proviral insertions in the genome of a feline thymic lymphoma cell line (FT-1) infected with FeLV. Fluorescence in situ hybridization (FISH) combined with tyramide signal amplification was applied on the chromosome specimen of FT-1 cells and normal cat lymphocytes, with an entire FeLV-A genome used as a probe. Specific hybridization signals were detected from only the metaphases of the FT-1 cells, not from those of normal cat lymphocytes. Statistically based on the Poisson's distribution, at least six loci of chromosomal regions, A2p23-p22, B2p15-p14, B4p15-p14, D4q23-q24, E1p14-p13, and E2p13-p12, appeared to be positive for FeLV integration. Consistently, Southern blot hybridization analysis using an FeLV LTR-U3 probe specific for exogenous FeLV showed the integration of at least six FeLV proviral genomes in FT-1 cells. The cytogenetic technique employed here will provide valuable molecular tags to reveal unidentified tumor-associated genes in FeLV-associated tumor cells.     

Specific cytolysis of fresh tumor cells by an autologous killer T cell line derived from an adult T cell leukemia/lymphoma patient

Cytotoxic T cells (Tc) derived from one patient with adult T cell leukemia/lymphoma (ATL) killed fresh autologous lymphoma cells in vitro. The Tc were induced from peripheral blood leukocytes (PBL) of this patient during remission by multiple in vitro stimulations with an autologous ATLV-bearing cell line (ILT) that was previously established by cloning of PBL in the presence of interleukin 2 (IL 2). PBL from eight other ATL patients were stimulated in the same manner, and responder cells from a patient in remission also showed cytotoxicity specific for ATL virus (ATLV)-bearing cells. Fresh lymphoma cells were obtained in relapse and were used as target cells for the autologous Tc induced. They became susceptible to the Tc within 4 hr of in vitro incubation, and their susceptibility increased with incubation time for at least 12 hr. ATLV antigens on the cell surface of these lymphoma cells, however, were not detected by radioimmunoassay during these incubation periods, but were detectable after 16 hr of incubation. In addition, cytotoxicity against lymphoma cells was completely inhibited by autologous ILT cells used as "cold" target competitor cells. These findings indicate that the target antigen of the Tc was expressed on both autologous ILT cells and lymphoma cells, and it may be different from ATLV antigens detected by serologic methods. In addition, the data suggested allogeneic restriction of the Tc in that the preferentially killed allogeneic ATLV-bearing cells share several HLA antigens.     

Efficient propagation of human herpesvirus 6B in a T-cell line derived from a patient with adult T-cell leukemia/lymphoma.

The efficient propagation of the OK strain of the B variant of human herpesvirus 6 (HHV-6B) was demonstrated in a line of T cells, TaY, established from the peripheral blood lymphocytes of a patient with adult T-cell leukemia/lymphoma (ATL). Growth of TaY cells depends on the presence of IL-2 and the cells harbor HTLV-I genomes. A severe cytopathic effect (CPE) was observed in many HHV-6B(OK)-infected TaY cells one week after infection. The release of virus from HHV-6B(OK)-infected TaY cells [TaY(OK)] was first detected after three days and increased rapidly for up to seven days after infection, as demonstrated by PCR. The titer of HHV-6B(OK) in the supernatant was comparable to the value of 10(3.5) TCID50/ml obtained with PHA-activated cord blood lymphocytes (CBL) that had been infected with HHV-6B(OK). The replication of the virus was shown to depend to a considerable extent on cell viability. Electron microscopy revealed many herpesvirus-type capsid- and enveloped-viruses in the nuclei and cytoplasm of degenerated cells in TaY(OK) cultures. The U1102 strain of HHV-6A and the Z29 strain of HHV-6B also infected TaY cells productively, as detected by PCR and an immunofluorescence test. These results suggest that the activation of CD4+ T lymphocytes with mitogens such as PHA or IL-2 and the expression of some cellular gene or the HTLV-I gene might be essential for efficient propagation of HHV-6B. TaY cells should play an important role in future investigations of cell-virus interactions and genetic variations or cell tropism of HHV-6 isolates since no cell line that shows propagation of both HHV-6A and HHV-6B has been reported to date.     

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.     

Human leukemia-associated antigens: detection on cells of established lymphoblastoid lines.

Primate and rabbit antisera to different morphologic classes of human leukemia cells, after appropriate absorptions, detected leukemia-associated antigens present on cultured lymphoblastoid cell lines derived from leukemia patients. The primate antisera distinguished antigens on cells derived from myeloid leukemia patients from those on cells derived from lymphocytic leukemia patients. Of particular interest was the fact that antigens of myeloid leukemia, but not of lymphatic leukemia, were detected on lymphoid cell lines established from blood of patients with myeloid leukemia. One of four lymphoblastoid cell lines derived from normal donors expressed antigens of lymphatic leukemia. Leukemia-associated antigens were not found on the HRIK lymphoblastoid line derived from a Burkitt's lymphoma patient on skin fibroblasts or HeLa cells. Expression of these antigens on cultured cells derived from leukemia patients could not be related to the presence of the EB virus or the EB virus genome. Rabbit antisera detected antigens common to cells from patients with myeloid and lymphocytic leukemia. Absorption experiments demonstrated that the antigens detected on cell lines derived from leukemia patients are similar to those detected by the primate and rabbit antisera on fresh peripheral blood leukemic cells. The serologic detection of leukemia-associated antigens on lymphoblastoid cell lines indicates that some of these cultures contain cells with antigenic properties similar to those of human peripheral blood leukemic cells.     

Distinct IkappaB kinase regulation in adult T cell leukemia and HTLV-I-transformed cells

We have recently shown constitutive IkappaB kinase (IKK) activation and aberrant p52 expression in adult T cell leukemia (ATL) cells that do not express human T cell leukemia virus type I (HTLV-I) Tax, but the mechanism of IKK activation in these cells has remained unknown. Here, we demonstrate distinct regulation of IKK activity in ATL and HTLV-I-transformed T cells in response to protein synthesis inhibition or arsenite treatment. Protein synthesis inhibition for 4 h by cycloheximide (CHX) barely affects IKK activity in Tax-positive HTLV-I-transformed cells, while it diminishes IKK activity in Tax-negative ATL cells. Treatment of ATL cells with a proteasome inhibitor MG132 prior to protein synthesis inhibition reverses the inhibitory effect of CHX, and MG132 alone greatly enhances IKK activity. In addition, treatment of HTLV-I-transformed cells with arsenite for 1 h results in down-regulation of IKK activity without affecting Tax expression, while 8 h of arsenite treatment does not impair IKK activity in ATL cells. These results indicate that a labile protein sensitive to proteasome-dependent degradation governs IKK activation in ATL cells, and suggest a molecular mechanism of IKK activation in ATL cells distinct from that in HTLV-I-transformed T cells.     

Viral RNA content of bovine leukemia virus-infected cells

A bovine leukemia virus (BLV)-producing cell line, fetal lamb kidney cells infected with BLV (FLK) contains one or a few copies of BLV proviral DNA in its genome. These cells contain 0.002% of viral RNA which sediments, in a sucrose gradient, at about 35S and between 18S and 28S.In cattle affected by enzootic bovine leukosis, tumor cells and circulating lymphocytes also contain one or a few copies of BLV proviral DNA integrated in their genome. However, in all cases tested (except one), no viral RNA was detected in these cells in conditions where one or two copies of viral genomic RNA per cell would have been easily detected.     

HTLV-1 evades type I interferon antiviral signaling by inducing the suppressor of cytokine signaling 1 (SOCS1)

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of Adult T cell Leukemia (ATL) and the neurological disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the majority of HTLV-1-infected individuals remain asymptomatic carriers (AC) during their lifetime, 2-5% will develop either ATL or HAM/TSP, but never both. To better understand the gene expression changes in HTLV-1-associated diseases, we examined the mRNA profiles of CD4+ T cells isolated from 7 ATL, 12 HAM/TSP, 11 AC and 8 non-infected controls. Using genomic approaches followed by bioinformatic analysis, we identified gene expression pattern characteristic of HTLV-1 infected individuals and particular disease states. Of particular interest, the suppressor of cytokine signaling 1--SOCS1--was upregulated in HAM/TSP and AC patients but not in ATL. Moreover, SOCS1 was positively correlated with the expression of HTLV-1 mRNA in HAM/TSP patient samples. In primary PBMCs transfected with a HTLV-1 proviral clone and in HTLV-1-transformed MT-2 cells, HTLV-1 replication correlated with induction of SOCS1 and inhibition of IFN-α/β and IFN-stimulated gene expression. Targeting SOCS1 with siRNA restored type I IFN production and reduced HTLV-1 replication in MT-2 cells. Conversely, exogenous expression of SOCS1 resulted in enhanced HTLV-1 mRNA synthesis. In addition to inhibiting signaling downstream of the IFN receptor, SOCS1 inhibited IFN-β production by targeting IRF3 for ubiquitination and proteasomal degradation. These observations identify a novel SOCS1 driven mechanism of evasion of the type I IFN antiviral response against HTLV-1.