HBZ Transgenic Mice Research Articles

Mouse Models for HTLV-1 Infection and Adult T Cell Leukemia.

Adult T cell leukemia (ATL) is an aggressive hematologic disease caused by human T cell leukemia virus type 1 (HTLV-1) infection. Various animal models of HTLV-1 infection/ATL have been established to elucidate the pathogenesis of ATL and develop appropriate treatments. For analyses employing murine models, transgenic and immunodeficient mice are used because of the low infectivity of HTLV-1 in mice. Each mouse model has different characteristics that must be considered before use for different HTLV-1 research purposes. HTLV-1 Tax and HBZ transgenic mice spontaneously develop tumors, and the roles of both Tax and HBZ in cell transformation and tumor growth have been established. Severely immunodeficient mice were able to be engrafted with ATL cell lines and have been used in preclinical studies of candidate molecules for the treatment of ATL. HTLV-1-infected humanized mice with an established human immune system are a suitable model to characterize cells in the early stages of HTLV-1 infection. This review outlines the characteristics of mouse models of HTLV-1 infection/ATL and describes progress made in elucidating the pathogenesis of ATL and developing related therapies using these mice.

The Road to HTLV-1-Induced Leukemia by Following the Subcellular Localization of HTLV-1-Encoded HBZ Protein.

Human T cell leukemia virus-1 (HTLV-1) is the causative agent of a severe cancer of the lymphoid lineage that develops in 3-5% of infected individuals after many years. HTLV-1 infection may also induce a serious inflammatory pathology of the nervous system designated HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two virus-encoded proteins, the viral transactivator Tax-1 and the HTLV-1 basic leucine-zipper factor HBZ, are strongly involved in the oncogenic process. Tax-1 is involved in initial phases of the oncogenic process. Conversely, HBZ seems to be involved in maintenance of the neoplastic state as witnessed by the generation of leukemic/lymphomatous phenotype in HBZ transgenic mice and the persistent expression of HBZ in all phases of the oncogenic process. Nevertheless, the intimate molecular and cellular mechanism mediated by the two viral proteins, particularly HBZ, in oncogenesis still remain elusive. An important step toward the complete comprehension of HBZ-associated oncogenicity is the clarification of the anatomical correlates of HBZ during the various phases of HTLV-1 infection to development of HTLV-1-associated inflammatory pathology and ultimately to the establishment of leukemia. In this review, I will summarize recent studies that have established for the first time a temporal and unidirectional expression of HBZ, beginning with an exclusive cytoplasmic localization in infected asymptomatic individuals and in HAM/TSP patients and ending to a progressive cytoplasmic-to-nuclear transition in leukemic cells. These results are framed within the present knowledge of HTLV-1 infection and the future lines of research that may shed new light on the complex mechanism of HTLV-1- mediated oncogenesis.

HTLV-1 induces T cell malignancy and inflammation by viral antisense factor-mediated modulation of the cytokine signaling

Human T cell leukemia virus type 1 (HTLV-1) is the etiologic agent of a T cell neoplasm and several inflammatory diseases. A viral gene, HTLV-1 bZIP factor (HBZ), induces pathogenic Foxp3-expressing T cells and triggers systemic inflammation and T cell lymphoma in transgenic mice, indicating its significance in HTLV-1-associated diseases. Here we show that, unexpectedly, a proinflammatory cytokine, IL-6, counteracts HBZ-mediated pathogenesis. Loss of IL-6 accelerates inflammation and lymphomagenesis in HBZ transgenic mice. IL-6 innately inhibits regulatory T cell differentiation, suggesting that IL-6 functions as a suppressor against HBZ-associated complications. HBZ up-regulates expression of the immunosuppressive cytokine IL-10. IL-10 promotes T cell proliferation only in the presence of HBZ. As a mechanism of growth promotion by IL-10, HBZ interacts with STAT1 and STAT3 and modulates the IL-10/JAK/STAT signaling pathway. These findings suggest that HTLV-1 promotes the proliferation of infected T cells by hijacking the machinery of regulatory T cell differentiation. IL-10 induced by HBZ likely suppresses the host immune response and concurrently promotes the proliferation of HTLV-1 infected T cells.

A-106 Strategy and pathogenesis of human T-cell leukemia virus type 1

Human T-cell leukemia virus type 1 (HTLV-1) causes not only adult T-cell leukemia-lymphoma (ATL) but also inflammatory diseases. The most important characteristic of HTLV-1 is that this virus can transmit only through cell-to-cell contact. Therefore, HTLV-1 increases the number of infected cells to facilitate its transmission through breast feeding and sexual intercourse. HTLV-1 promotes proliferation of infected cells, inhibits their apoptosis and facilitates escape from the host immune surveillance. The pathogenesis of these diseases is based on these characteristics of this virus. Infected cells evade the host immune responses to survive in vivo. Early studies of HTLV-1 focused on Tax that is the trans-activator of viral gene transcription. Tax can immortalize T cellsin vitroand in vivo. Tax activates NFkB pathway, and functionally inhibits p53 function. It is assumed that Tax is the viral oncogene that causes ATL. However, analyses of HTLV-1 proviruses in clinical samples revealed that Tax expression is frequently inactivated in ATL cells by 3 mechanisms: genetic changes of the taxgene (mainly non-sense mutation), deletion of 5′ long terminal repeat (LTR), the promoter/enhancer of the taxgene, and DNA methylation of 5′LTR. On the other hand, 3′ provirus is maintained in all ATL cases and HTLV-1 carriers. This region contains HTLV-1 bZIP factor (HBZ) coding region. HBZ gene is encoded in the minus strand of the provirus. These finding suggest that HBZ is critical for ATL cells and HTLV-1 infected cells. To study the function of HBZZ, we generated HBZ-transgenic mice that express HBZ in CD4+ T cells. HBZ-Tg mice developed T-cell lymphoma and inflammatory diseases, suggesting that HBZ is critical for HTLV-1 associated diseases. Thereafter, we focus on function of HBZ in biology and pathology of HTLV-1. This strategy is supported by several attributes of this virus. First, HTLV-1 infected T cells possess the specific immunophenotype; CD4, CD45RO, CCR4, CADM1, TIGIT, Foxp3 (approximately 50%) positive T cells. Effector/memory (CD45RO) T cells express higher level of LFA-1, which likely migrate into breast milk and semen. Furthermore, TIGIT suppresses the host immune responses, which facilitates infected cells to escape from immune surveillance. HBZ also induces expression of Foxp3. Foxp3 induces expression of regulatory T (Treg) cell associated molecules. On the other hand, labile Foxp3 expression is associated with inflammation by increased interferon-gproduction. Thus, HTLV-1 bZIP factor (HBZ) is mainly implicated in immunnophenotype of HTLV-1 infected cells and ATL cells. Second, HTLV-1 infected cells need persistent proliferation and survival in vivo. Both HBZ and tax are implicated in proliferation and survival. Third, HTLV-1 infected cells and ATL cells need to minimize expression of immunogenic viral proteins. Tax is a highly immunogenic protein, and a major target of cytotoxic T-lymphocytes (CTLs) in vivo. On the other hand, immunogenicity of HBZ is quite low. Therefore, HTLV-1 infected cells and ATL cells continuously express HBZ. In contrast, Tax is transiently expressed (Mahgoub et al, PNAS 2018). The intermittent expression of Tax plays an important role to maintain ATL cells, and possibly HTLV-1 infected cells. Pathogenesis of HTLV-1 is based on these characteristics of this virus; infiltration, inflammation, persistent proliferation, and anti-apoptosis. Thus, pro-inflammatory infected cells cause inflammatory diseases, and lead to development of leukemia. In this meeting, I am going to show the roles of HBZ and Tax in the pathogenesis, discuss about complex relation between virus and diseases.

Functional Impairment of Co-Inhibitory Receptors Promotes T-Cell Proliferation in HTLV-1 Associated Adult T-Cell Leukemia Cells

IntroductionAdult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1). It has been reported that co-inhibitory receptors such as programmed cell death 1 (PD-1) and T cell immunoglobulin and ITIM domain (TIGIT) are highly expressed on ATL cells and HTLV-1 infected cells. However, ATL cells and HTLV-1 infected cells proliferate regardless of their high expression. Although it has been known that HTLV-1 bZIP factor (HBZ), which is constitutively expressed in ATL cells and HTLV-1 infected cells, promotes T cell proliferation, the detailed mechanisms remain unclear. In this study, we found that HBZ promotes T-cell proliferation by interfering the suppressive function of co-inhibitory receptors.MethodsWe analyzed T-cell proliferation of HBZ transgenic (HBZ-Tg) mice that specifically express HBZ in CD4+ T cells, and expression of co-inhibitory and co-stimulatory molecules on ATL cells and CD4+ T cells of HBZ-Tg mice. Furthermore, the function of TIGIT and PD-1 was studied using HBZ-transduced murine CD4+T cells. The co-localization of SHP-2 and PD-1 in the presence of HBZ was analyzed by immunoprecipitation and confocal microscope. The immunoprecipitation and confocal microscope were also used to analyze interaction between HBZ and THEMIS and HBZ localization in the presence of THEMIS.ResultsAlthough HBZ promotes T-cell proliferation, we found that some co-inhibitory receptors, TIGIT and PD-1, were highly expressed on CD4+ T cells of HBZ-Tg mice and ATL cells. As mechanisms, HBZ upregulated transcriptions of these genes. Based on these observations, we hypothesized that HBZ impairs suppressive functions of TIGIT and PD-1 while it increases expression of these co-inhibitory receptors. To address this question, we analyzed the suppressive activity of TIGIT and PD-1 in the presence of HBZ. We transduced HBZ by the retrovirus vector into primary murine T cells and evaluated the proliferation after stimulated TIGIT or PD-1 with anti-CD3 and its ligand. As a result, TIGIT and PD-1 did not inhibit T-cell proliferation in the presence of HBZ, indicating that HBZ impairs the suppressive function of TIGIT and PD-1. Both TIGIT and PD-1 possess SHP-2, a tyrosine phosphatase, binding domains in its cytoplasmic tail, ITIM or ITSM motif. Therefore, we next studied whether HBZ influences the interaction between PD-1 and SHP-2. Tyrosine phosphorylation of PD-1 was induced with pervanadate and then SHP-2 recruitment and PD-1/SHP-2 co-localization were investigated. HBZ inhibited recruitment of SHP-2 to the ITSM motif of PD-1. Indeed, phosphorylation of SHP-2 was decreased in CD4+T cells of HBZ-Tg mice and HBZ-transduced murine primary T cells. Furthermore, function of SHP-2 to dephosphorylate ZAP-70 and CD3-zeta was suppressed in the presence of HBZ. These data showed that HBZ inhibited recruitment of SHP-2 to ITSM motif of PD-1 and suppressed its inhibitory function. Next, we examined how HBZ inhibits the interaction between PD-1 and SHP-2. HBZ did this by interacting with THEMIS, which forms a complex with Grb2 and SHP-2. Moreover, HBZ hindered the interaction between THEMIS and Grb2. In general, THEMIS is localized in the cytoplasm, whereas it has been reported that HBZ is localized in the nucleus. When we expressed THEMIS or HBZ, THEMIS existed in the cytoplasm (50 of 50 cells: 100%) whereas HBZ was mainly localized in the nucleus (67 of 74 cells: 90.5%). Interestingly, when both proteins were expressed in the same cells, HBZ changed its localization to cytoplasm (28 of 79 cells: 35.4%) and co-localized with THEMIS. These findings suggest that THEMIS changes the localization of HBZ from nucleus to cytoplasm. Thus, HBZ functions not only in the nucleus but also in the cytoplasm by interacting with host factors. Since THEMIS is expressed only in T-lineage cells, inhibition of the suppressive effects of co-inhibitory receptors by HBZ accounts for how HTLV-1 induced proliferation only T cells in vivo.ConclusionsOur findings demonstrated that HBZ promotes T-cell proliferation upon TCR stimulation by impairing the suppressive signal of co-inhibitory receptors. This study presents the first evidence of mechanisms how HBZ attenuates the inhibitory signals and promotes T-cell proliferation. DisclosuresNo relevant conflicts of interest to declare.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

HTLV-1 Viral Factor HBZ Induces CCR4 to Promote T-cell Migration and Proliferation.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and other inflammatory diseases in infected individuals. However, a complete understanding of how HTLV-1 transforms T cells is lacking. Expression of the chemokine receptor CCR4 on ATL cells and HTLV-1-infected cells suggested the hypothesis that CCR4 may mediate features of ATL and inflammatory diseases caused by HTLV-1. In this study, we show that the constitutively expressed HTLV-1 bZIP factor (HBZ) encoded by HTLV-1 is responsible for inducing CCR4 and its ability to promote T-cell proliferation and migration. Ectopic expression of HBZ was sufficient to stimulate expression of CCR4 in human and mouse T cells. Conversely, HBZ silencing in ATL cell lines was sufficient to inhibit CCR4 expression. Mechanistic investigations showed that HBZ induced GATA3 expression in CD4(+) T cells, thereby activating transcription from the CCR4 promoter. In an established air pouch model of ATL, we observed that CD4(+) T cells of HBZ transgenic mice (HBZ-Tg mice) migrated preferentially to the pouch, as compared with those in nontransgenic mice. Migration of CD4(+) T cells in HBZ-Tg mice was inhibited by treatment with a CCR4 antagonist. Proliferating (Ki67(+)) CD4(+) T cells were found to express high levels of CCR4 and CD103. Further, CD4(+) T-cell proliferation in HBZ-Tg mice was enhanced by coordinate treatment with the CCR4 ligands CCL17 and 22 and with the CD103 ligand E-cadherin. Consistent with this finding, we found that ATL cells in clinical skin lesions were frequently positive for CCR4, CD103, and Ki67. Taken together, our results show how HBZ activates CCR4 expression on T cells to augment their migration and proliferation, two phenomena linked to HTLV-1 pathogenesis. Cancer Res; 76(17); 5068-79. ©2016 AACR.

Impact of the SCF signaling pathway on leukemia stem cell-mediated ATL initiation and progression in an HBZ transgenic mouse model.

Adult T-cell leukemia (ATL) is a malignant disease caused by human T-lymphotropic virus type 1. In aggressive ATL, the response to chemotherapy is extremely poor. We hypothesized that this poor response is due to the existence of chemotherapy-resistant cells, such as leukemic stem cells. Previously, we successfully identified an ATL stem cell (ATLSC) candidate as the c-kit+/CD38−/CD71− cells in an ATL mouse model using Tax transgenic mice. Here, with a new ATL mouse model using HBZ-transgenic mice, we further discovered that the functional ATLSC candidate, which commonly expresses c-kit, is drug-resistant and has the ability to initiate tumors and reconstitute lymphomatous cells. We characterized the ATLSCs as c-kit+/CD4−/CD8− cells and found that they have a similar gene expression profile as T cell progenitors. Additionally, we found that AP-1 gene family members, including Junb, Jund, and Fosb, were up-regulated in the ATLSC fraction. The results of an in vitro assay showed that ATLSCs cultured with cytokines known to promote stem cell expansion, such as stem cell factor (SCF), showed highly proliferative activity and maintained their stem cell fraction. Inhibition of c-kit–SCF signaling with the neutralizing antibody ACK2 affected ATLSC self-renewal and proliferation. Experiments in Sl/Sld mice, which have a mutation in the membrane-bound c-kit ligand, found that ATL development was completely blocked in these mice. These results clearly suggest that the c-kit–SCF signal plays a key role in ATLSC self-renewal and in ATL initiation and disease progression.

HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule, T Cell Immunoglobulin and ITIM Domain (TIGIT).

Human T-cell leukemia virus type 1 (HTLV-1) infects CD4+ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4+ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4+ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT+CD4+ cells of HBZ-Tg mice compared with TIGIT-CD4+ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4+ T cells from HBZ-Tg mice were stimulated with TIGIT’s ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1.

Identification of TIGIT as an HBZ-induced gene by genome-wide analyses: its association with evasion of host defense

Human T-cell leukemia virus type 1 (HTLV-1) is a causative virus of adult T-cell leukemia (ATL), and inflammatory diseases. HTLV-1 bZIP factor (HBZ) is encoded on the minus strand of HTLV-1, and expressed in all ATL cases. We performed RNA-seq and ChIP-seq using HBZ transduced T cells, and found that expressions of several Treg-related genes, including Foxp3, CD25, CCR4, CCR5, PD-1, NRP1, IKZF family genes, and T-cell immunoglobulin and ITIM domain receptor (Tigit), were upregulated by HBZ. We focused on TIGIT in this study. TIGIT expression was also upregulated in HTLV-1 infected cells in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients and CD4+ T cells of ATL patients. HBZ induced the histone modification of the promoter of TIGIT and enhanced its expression. HBZ also enhanced acetylation of H3K18, which is the specific target of p3. HBZ augments transcription of Tigit gene in the presence of PMA/Ionomycin stimulation. Although the detailed mechanism of HBZ mediated activation of Tigit transcription is still unclear, p3 and activated pathway by PMA/Ionomycin is likely involved in its regulation. Furthermore, we analyzed the function of Tigit in HBZ-expressing T cells and CD4+ T cells from HBZ-transgenic mice (HBZ-Tg), and found that stimulation of Tigit with its ligand, PVR, enhanced expression of inhibitory cytokines, such as IL-10, in HBZ-Tg. These results suggest that the enhanced Tigit expression by HBZ may play a key role to modulate the microenvironment where anti-tumor immune response is attenuated by increased expression of IL-10. This environment seems to be associated with the evasion of HTLV-1 infected cells from anti-tumor host immune response and the pathogenesis of HTLV-1 associated inflammatory diseases and ATL.

Interferon-γ Promotes Inflammation and Development of T-Cell Lymphoma in HTLV-1 bZIP Factor Transgenic Mice.

Human T-cell leukemia virus type 1 (HTLV-1) is an etiological agent of several inflammatory diseases and a T-cell malignancy, adult T-cell leukemia (ATL). HTLV-1 bZIP factor (HBZ) is the only viral gene that is constitutively expressed in HTLV-1-infected cells, and it has multiple functions on T-cell signaling pathways. HBZ has important roles in HTLV-1-mediated pathogenesis, since HBZ transgenic (HBZ-Tg) mice develop systemic inflammation and T-cell lymphomas, which are similar phenotypes to HTLV-1-associated diseases. We showed previously that in HBZ-Tg mice, HBZ causes unstable Foxp3 expression, leading to an increase in regulatory T cells (Tregs) and the consequent induction of IFN-γ-producing cells, which in turn leads to the development of inflammation in the mice. In this study, we show that the severity of inflammation is correlated with the development of lymphomas in HBZ-Tg mice, suggesting that HBZ-mediated inflammation is closely linked to oncogenesis in CD4+ T cells. In addition, we found that IFN-γ-producing cells enhance HBZ-mediated inflammation, since knocking out IFN-γ significantly reduced the incidence of dermatitis as well as lymphoma. Recent studies show the critical roles of the intestinal microbiota in the development of Tregs in vivo. We found that even germ-free HBZ-Tg mice still had an increased number of Tregs and IFN-γ-producing cells, and developed dermatitis, indicating that an intrinsic activity of HBZ evokes aberrant T-cell differentiation and consequently causes inflammation. These results show that immunomodulation by HBZ is implicated in both inflammation and oncogenesis, and suggest a causal connection between HTLV-1-associated inflammation and ATL.

HTLV-1 bZIP Factor Induces Inflammation through Labile Foxp3 Expression

Human T-cell leukemia virus type 1 (HTLV-1) causes both a neoplastic disease and inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the proviral DNA and is constitutively expressed in infected cells and ATL cells. HBZ increases the number of regulatory T (Treg) cells by inducing the Foxp3 gene transcription. Recent studies have revealed that some CD4+Foxp3+ T cells are not terminally differentiated but have a plasticity to convert to other T-cell subsets. Induced Treg (iTreg) cells tend to lose Foxp3 expression, and may acquire an effector phenotype accompanied by the production of inflammatory cytokines, such as interferon-γ (IFN-γ). In this study, we analyzed a pathogenic mechanism of chronic inflammation related with HTLV-1 infection via focusing on HBZ and Foxp3. Infiltration of lymphocytes was observed in the skin, lung and intestine of HBZ-Tg mice. As mechanisms, adhesion and migration of HBZ-expressing CD4+ T cells were enhanced in these mice. Foxp3−CD4+ T cells produced higher amounts of IFN-γ compared to those from non-Tg mice. Expression of Helios was reduced in Treg cells from HBZ-Tg mice and HAM/TSP patients, indicating that iTreg cells are predominant. Consistent with this finding, the conserved non-coding sequence 2 region of the Foxp3 gene was hypermethylated in Treg cells of HBZ-Tg mice, which is a characteristic of iTreg cells. Furthermore, Treg cells in the spleen of HBZ-transgenic mice tended to lose Foxp3 expression and produced an excessive amount of IFN-γ, while Foxp3 expression was stable in natural Treg cells of the thymus. HBZ enhances the generation of iTreg cells, which likely convert to Foxp3−T cells producing IFN-γ. The HBZ-mediated proinflammatory phenotype of CD4+ T cells is implicated in the pathogenesis of HTLV-1-associated inflammation.

How human T-cell leukemia virus type 1 induces diseases

Human T-cell leukemia virus type1 (HTLV-1) is the causal agent of adult T-cell leukemia (ATL), and inflammatory diseases, such as HTLV-1 associated myelopathy (HAM), uveitis, alveolitis and infective dermatitis. Approximately, 5% of carrier will develop ATL after a long latent period. A characteristic of this virus is that HTLV-1 can transmit only by cell-to-cell contact. Therefore, HTLV-1 increases infected cells in vivo to facilitate its transmission. Previous studies suggest that Tax plays central roles in leukemogenesis by HTLV-1. However, Tax expression is frequently impaired in ATL cells. The HTLV-1 bZIP factor (HBZ) gene is transcribed from minus strand of provirus using 3’ long terminal repeat (LTR) as the promoter/ enhancer. Among ATL cases, only the HBZ gene is expressed in all ATL cases, suggesting an importance of the HBZ gene in ATL cells. Indeed, transgenic expression of the HBZ gene induced T-cell lymphoma and inflammatory diseases. Furthermore, regulatory T (Treg) cells increased in HBZ-transgenic (HBZ-Tg) mice. As a mechanism, HBZ induces transcription of the Foxp3 gene through enhanced Smad/ TGF-β pathway and converts infected cells to Treg cells. Concurrently, HBZ impairs function of Treg via interaction of HBZ with Foxp3 and associated factors. Thus, HBZ increases functionally impaired Treg cells in vivo, which resembles to phenotype of HTLV-1 infected cells and ATL cells. It is speculated that HTLV-1 infected Treg cells escape the host immune surveillance by acquired phenotype of Treg cells. Treg cells derived from HBZ-Tg mice progressively lost Foxp3 expression in vitro and produced IFN-γ, indicating that HBZ induces inflammation through unstable Foxp3 expression. Thus, HBZ is responsible for both T-cell lymphoma and inflammatory diseases. HBZ has pleiotropic functions as well as Tax. HBZ enhances TGF-β/Smad pathway while Tax inhibits it. Likewise, HBZ suppresses canonical NF-κB whereas Tax activates canonical and noncanonical NF-κB pathways. Thus, HBZ has opposite effects to Tax in many pathways. Interplay between HBZ and Tax likely coordinates to promote viral replication and proliferation of infected cells, leading to important roles in the pathogenesis.

HTLV-1 bZIP factor遺伝子によるHTLV-1病原性発現機構の解析

HTLV-1 is a retrovirus associated with human diseases, such as ATL or HAM/TSP. More than thirty years have passed since HTLV-1 was discovered, but the precise mechanism of HTLV-1 pathogenesis still remains elusive. HTLV-1 bZIP factor (HBZ) was reported ten years ago as a viral gene encoded in the minus strand of HTLV-1. We have elucidated that HBZ is constitutively detectable in all ATL cells examined whereas tax expression is frequently lost. Furthermore, we and other researchers have reported that HBZ expression contributes to the proliferation of infected cells. We have shown that HBZ has the potential to transform T cells in vivo by analyzing HBZ-transgenic mice. Further investigations will uncover a more detailed role of HBZ in HTLV-1 pathogenesis. This paradigm shift of HTLV-1 research should provide novel target in prevention or treatment of HTLV-1-related human diseases.

HTLV-1 bZIP factor induces systemic inflammations in vivo

Human T-cell leukemia virus type 1 (HTLV-1) causes both neoplastic and inflammatory diseases, which include HTLV-1 associated myelopathy/tropic spastic paraparesis and uveitis. Recently, we have reported that transgenic expression of HTLV-1 bZIP factor (HBZ) in CD4+ T cells caused dermatitis and alveolitis in mice. In this study, we investigated the production of cytokines in HBZ transgenic (HBZ-Tg) mice to elucidate the mechanism of its pro-inflammatory phenotype. IFN-γ production in CD4+ T cells was remarkably increased in splenocytes, lungs and PBMCs from HBZ-Tg compared with non-transgenic littermates, whereas there was no difference in levels of IL-4 and IL-17. We also found that production of IFN-γ was remarkably enhanced in CD4+Foxp3- fraction. Recent studies have reported that CD4+Foxp3+ T cells are not terminally differentiated but have a plasticity to convert to other T cell subsets. Induced regulatory T cells (iTreg) tend to lose Foxp3 expression, and may acquire an effector phenotype accompanied by the production of inflammatory cytokines, such as IFN-γ. We observed that the percentage of naturally occurring Treg cells was lower in HBZ-Tg mice than non-Tg mice, although total number of Treg was increased in HBZ-Tg mice. It is suggested that the enhanced generation of iTreg cells and instability of Foxp3 expression in HBZ-induced iTreg is a possible mechanism for increased number IFN-γ producing cells in HBZ-Tg mice, leading to systemic inflammation.

HTLV-1 bZIP factor perturbs immune response to the pathogens in vivo by inhibiting IFN-gamma production

HTLV-1 carriers and the patients with adult T-cell leukemia (ATL) frequently suffer from the opportunistic infections. Although it has been known that HTLV-1 infection evokes cell-mediated immune deficiency, its mechanism remains unknown. HTLV-1 bZIP factor (HBZ) is encoded in the minus strand of HTLV-1, which is constitutively expressed and involved in the proliferation of HTLV-1-infected cells. In this study, we investigated the role of HBZ in immunodeficiency in a mice model. HBZ transgenic (HBZ-Tg) mice were infected with HSV-2 and Listeria monocytogenes and evaluated for cellular immunity to those pathogens. The clearance of both pathogens was impaired in HBZ-Tg mice compared with the non-transgenic littermate. In the both infection, production of IFN-gamma in CD4+ T cells was significantly reduced in HBZ-Tg mice. In addition, ectopic expression of HBZ in primary human CD4+ T cells impaired IFN-gamma production in vitro. As the molecular mechanisms, we found that HBZ suppressed transcription of IFNG promoter. Our results suggest that HBZ plays a crucial role for cellular immunodeficiency in HTLV-1-infected subjects.

HTLV-1 bZIP factor induces T-cell lymphoma and systemic inflammation in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) is the causal agent of a neoplastic disease of CD4+ T cells, adult T-cell leukemia (ATL), and inflammatory diseases including HTLV-1 associated myelopathy/tropical spastic paraparesis, dermatitis, and inflammatory lung diseases. ATL cells, which constitutively express CD25, resemble CD25+CD4+ regulatory T cells (Treg). Approximately 60% of ATL cases indeed harbor leukemic cells that express FoxP3, a key transcription factor for Treg cells. HTLV-1 encodes an antisense transcript, HTLV-1 bZIP factor (HBZ), which is expressed in all ATL cases. In this study, we show that transgenic expression of HBZ in CD4+ T cells induced T-cell lymphomas and systemic inflammation in mice, resembling diseases observed in HTLV-1 infected individuals. In HBZ-transgenic mice, CD4+Foxp3+ Treg cells and effector/memory CD4+ T cells increased in vivo. As a mechanism of increased Treg cells, HBZ expression directly induced Foxp3 gene transcription in T cells. The increased CD4+Foxp3+ Treg cells in HBZ transgenic mice were functionally impaired while their proliferation was enhanced. HBZ could physically interact with Foxp3 and NFAT, thereby impairing the suppressive function of Treg cells. Thus, the expression of HBZ in CD4+ T cells is a key mechanism of HTLV-1-induced neoplastic and inflammatory diseases.