Human T-cell Leukemia Virus Type 1 Infected Cells Research Articles

10221-ML-11 A CASE OF PRIMARY CENTRAL NERVOUS SYSTEM ADULT T CELL LEUKEMIA

Abstract Adult T cell leukemia (ATL) manifesting only in the central nervous system (CNS) at presentation is rare. We present a case of primary CNS ATL. 54 year old male presenting with multiple enhanced lesions in the cerebrum went under biopsy and was diagnosed as T-cell lymphoma. While systemic workup was negative, human T-cell leukemia virus type 1 (HTLV-1) antibody was found to be seropositive. Southern blotting using peripheral blood DNA revealed positivity for clonal proliferation of HTLV-1 infected cells. He was therefore clinically diagnosed as primary CNS ATL. He received chemotherapy using methotrexate, procarbazine and vincristine (MPV), and gained a complete response unconfirmed (CRu) after completion of four cycles. However, relapse occurred after the fifth cycle of MPV. Salvage treatment using etoposide, cisplatin, cytarabine and prednisone (ESHAP), whole brain radiation therapy, and lenalidomide, were delivered as second, third, and fourth line therapies, but were ineffective and disease continued to progress. He was therefore treated by best supportive care. Primary CNS ATL should be considered in the differential diagnosis of brain tumors. Southern blotting using peripheral blood might be useful for clinical diagnosis in such cases.

The unique activity of saponin: Induction of cytotoxicity in HTLV-1 infected cells

Infection with the retrovirus human T-cell leukemia virus type 1 (HTLV-1) sometimes causes diseases that are difficult to cure. To find anti-HTLV-1 natural compounds, we opted to screen using the HTLV-1-infected T-cell line, MT-2. Based on our results, an extract of the pulp/seeds of Akebia quinata Decaisne fruit killed MT-2 cells but did not affect the Jurkat cell line that was not infected with virus. To determine the active ingredients, seven saponins with one-six sugar moieties were isolated from A. quinata seeds, and their activities against the two cell lines were examined. Both cell lines were killed in a similar manner by Akebia saponins A and B. Further, Akebia saponins D, E, PK and G did not exhibit cytotoxicity. Akebia saponin C had a similar activity to the extract found in the screening. This compound was found to enhance Gag aggregation, induce the abnormal cleavage of Gag, suppress virion release, and preferentially kill HTLV-1 infected cells; however, their relationship remains elusive. Our findings may lead to the development of new therapies for infectious diseases based on the removal of whole-virus-infected cells.

Development of a Monoclonal Antibody Targeting HTLV-1 Envelope gp46 Glycoprotein and Its Application to Near-Infrared Photoimmuno-Antimicrobial Strategy.

Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, causes adult T-cell leukemia-lymphoma, HTLV-1 associated myelopathy/tropical spastic paraparesis, and HTLV-1 uveitis. Currently, no antiretroviral therapies or vaccines are available for HTLV-1 infection. This study aimed to develop an antibody against the HTLV-1 envelope protein (Env) and apply it to a near-infrared photoimmuno-antimicrobial strategy (NIR-PIAS) to eliminate HTLV-1 infected cells. We established mouse monoclonal antibodies (mAbs) against HTLV-1 Env by immunization with a complex of liposome and the recombinant protein. Detailed epitope mapping revealed that one of the mAbs bound to the proline-rich region of gp46 and exhibited no obvious neutralizing activity to inhibit viral infection. Instead, the mAb was rarely internalized intracellularly and remained on the cell surface of HTLV-1-infected cells. The antibody conjugated to the photosensitive dye IRDye700Dx recognized HTLV-1 infected cells and killed them following NIR irradiation. These results suggest that the novel mAb and NIR-PIAS could be developed as a new targeted therapeutic tool against HTLV-1 infected cells.

Role of miRNAs in Human T Cell Leukemia Virus Type 1 Induced T Cell Leukemia: A Literature Review and Bioinformatics Approach.

Human T cell leukemia virus type 1 (HTLV-1) was identified as the first pathogenic human retrovirus and is estimated to infect 5 to 10 million individuals worldwide. Unlike other retroviruses, there is no effective therapy to prevent the onset of the most alarming diseases caused by HTLV-1, and the more severe cases manifest as the malignant phenotype of adult T cell leukemia (ATL). MicroRNA (miRNA) dysfunction is a common feature of leukemogenesis, and it is no different in ATL cases. Therefore, we sought to analyze studies that reported deregulated miRNA expression in HTLV-1 infected cells and patients’ samples to understand how this deregulation could induce malignancy. Through in silico analysis, we identified 12 miRNAs that stood out in the prediction of targets, and we performed functional annotation of the genes linked to these 12 miRNAs that appeared to have a major biological interaction. A total of 90 genes were enriched in 14 KEGG pathways with significant values, including TP53, WNT, MAPK, TGF-β, and Ras signaling pathways. These miRNAs and gene interactions are discussed in further detail for elucidation of how they may act as probable drivers for ATL onset, and while our data provide solid starting points for comprehension of miRNAs’ roles in HTLV-1 infection, continuous effort in oncologic research is still needed to improve our understanding of HTLV-1 induced leukemia.

Does HTLV-1 Infection Show Phenotypes Found in Sjögren's Syndrome?

Viruses are a possible cause for Sjögren’s syndrome (SS) as an environmental factor related to SS onset, which exhibits exocrine gland dysfunction and the emergence of autoantibodies. Although retroviruses may exhibit lymphocytic infiltration into exocrine glands, human T-cell leukemia virus type 1 (HTLV-1) has been postulated to be a causative agent for SS. Transgenic mice with HTLV-1 genes showed sialadenitis resembling SS, but their phenotypic symptoms differed based on the adopted region of HTLV-1 genes. The dominance of tax gene differed in labial salivary glands (LSGs) of SS patients with HTLV 1-associated myelopathy (HAM) and adult T-cell leukemia. Although HTLV-1 was transmitted to salivary gland epithelial cells (SGECs) by a biofilm-like structure, no viral synapse formation was observed. After infection to SGECs derived from SS patients, adhesion molecules and migration factors were time-dependently released from infected SGECs. The frequency of the appearance of autoantibodies including anti-Ro/SS-A, La/SS-B antibodies in SS patients complicated with HAM is unknown; the observation of less frequent ectopic germinal center formation in HTLV-1-seropositive SS patients was a breakthrough. In addition, HTLV-1 infected cells inhibited B-lymphocyte activating factor or C-X-C motif chemokine 13 through direct contact with established follicular dendritic cell-like cells. These findings show that HTLV-1 is directly involved in the pathogenesis of SS.

Single-Cell Analysis of the Multicellular Ecosystem in Viral Carcinogenesis by HTLV-1.

Our multimodal single-cell analyses comprehensively dissect the cellular and molecular alterations of the peripheral blood in HTLV-1 infection, with and without progression to leukemia. This study not only sheds light on premalignant clonal expansion in viral carcinogenesis, but also helps to devise novel diagnostic and therapeutic strategies for HTLV-1-related disorders.

Clonality of HIV-1- and HTLV-1-Infected Cells in Naturally Coinfected Individuals.

Coinfection with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1) diminishes the value of the CD4+ T-cell count in diagnosing AIDS, and increases the rate of HTLV-1-associated myelopathy. It remains elusive how HIV-1/HTLV-1 coinfection is related to such characteristics. We investigated the mutual effect of HIV-1/HTLV-1 coinfection on their integration sites (ISs) and clonal expansion. We extracted DNA from longitudinal peripheral blood samples from 7 HIV-1/HTLV-1 coinfected, and 12 HIV-1 and 13 HTLV-1 monoinfected individuals. Proviral loads (PVL) were quantified using real-time polymerase chain reaction (PCR). Viral ISs and clonality were quantified by ligation-mediated PCR followed by high-throughput sequencing. PVL of both HIV-1 and HTLV-1 in coinfected individuals was significantly higher than that of the respective virus in monoinfected individuals. The degree of oligoclonality of both HIV-1- and HTLV-1-infected cells in coinfected individuals was also greater than in monoinfected subjects. ISs of HIV-1 in cases of coinfection were more frequently located in intergenic regions and transcriptionally silent regions, compared with HIV-1 monoinfected individuals. HIV-1/HTLV-1 coinfection makes an impact on the distribution of viral ISs and clonality of virus-infected cells and thus may alter the risks of both HTLV-1- and HIV-1-associated disease.

Evolution of retrovirus-infected premalignant T-cell clones prior to adult T-cell leukemia/lymphoma diagnosis

AbstractAdult T-cell leukemia/lymphoma (ATL) is an aggressive hematological malignancy caused by human T-cell leukemia virus type-1 (HTLV-1). ATL is preceded by decades of chronic HTLV-1 infection, and the tumors carry both somatic mutations and proviral DNA integrated into the tumor genome. In order to gain insight into the oncogenic process, we used targeted sequencing to track the evolution of the malignant clone in 6 individuals, 2 to 10 years before the diagnosis of ATL. Clones of premalignant HTLV-1–infected cells bearing known driver mutations were detected in the blood up to 10 years before individuals developed acute and lymphoma subtype ATL. Six months before diagnosis, the total number and variant allele fraction of mutations increased in the blood. Peripheral blood mononuclear cells from premalignant cases (1 year prediagnosis) had significantly higher mutational burden in genes frequently mutated in ATL than did high-risk, age-matched HTLV-1 carriers who remained ATL-free after a median of 10 years of follow-up. These data show that HTLV-1–infected T-cell clones carrying key oncogenic driver mutations can be detected in cases of ATL years before the onset of symptoms. Early detection of such mutations may enable earlier and more effective intervention to prevent the development of ATL.

Activation of PERK-ATF4-CHOP pathway as a novel therapeutic approach for efficient elimination of HTLV-1–infected cells

AbstractPatients with adult T-cell leukemia (ATL) exhibit a poor prognosis and overall survival rate when treated with standard chemotherapy, highlighting the continued requirement for the development of novel safe and effective therapies for human T-cell leukemia virus type 1 (HTLV-1)-related diseases. In this study, we demonstrated that MK-2048, a second-generation HIV-1 integrase (IN) inhibitor, potently and selectively kills HTLV-1–infected cells. Differential transcriptome profiling revealed significantly elevated levels of gene expression of the unfolded protein response (UPR) PKR-like ER kinase (PERK) signaling pathway in ATL cell lines following MK-2048 treatment. We also identified a significant downregulation in glucose regulated protein 78 (GRP78), a master regulator of the UPR in the CD4+CADM1+ HTLV-1–infected cell population of primary HTLV-1 carrier peripheral blood mononuclear cells (PBMCs) (n = 9), suggesting that HTLV-1–infected cells are hypersensitive to endoplasmic reticulum (ER) stress-mediated apoptosis. MK-2048 efficiently reduced proviral loads in primary HTLV-1 carrier PBMCs (n = 4), but had no effect on the total numbers of these cells, indicating that MK-2048 does not affect the proliferation of HTLV-1–uninfected PBMCs. MK-2048 specifically activated the ER stress–related proapoptotic gene, DNA damage-inducible transcript 3 protein (DDIT3), also known as C/EBP homologous protein (CHOP), in HTLV-1–infected but not uninfected cells of HTLV-1–carrier PBMCs. Our findings demonstrated that MK-2048 selectively induces HTLV-1–infected cell apoptosis via the activation of the UPR. This novel regulatory mechanism of the HIV IN inhibitor MK-2048 in HTLV-1–infected cells provides a promising prophylactic and therapeutic target for HTLV-1–related diseases including ATL.

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.

Diversity of cell phenotypes among MT-2 cell lines affects the growth of U937 cells and cytokine production.

We previously reported the diversity of structure and integration sites of human T-cell leukemia virus type 1 (HTLV-1) provirus among different MT-2 cell lines. This raised the question as to whether cell phenotypes also differed among MT-2 cell lines. The influence of two different MT-2 cell lines (MT-2J and MT-2B) on the growth of the promonocytic leukemia cell line, U937, was investigated. Protein levels and mRNA expression of cytokines were also investigated. In addition, Western blot analysis of HTLV-1 regulatory proteins, Tax and HBZ, was also performed. Culture supernatant from MT-2B, but not MT-2J, cells showed marked suppressive effects on U937 cell growth. MT-2B showed high tumor necrosis factor (TNF)-α, TNF-β, and interferon (IFN)-γ both in protein levels of the culture supernatant and mRNA levels of the cells. Analysis using recombinant cytokines indicated that the suppressive effects of MT-2B were due, at least in part, to high levels of TNF-β and its synergic effects with IFN-γ in the culture supernatant. Protein levels of HTLV-1 Tax and HBZ were higher in MT-2B than those in MT-2J cells. These molecules have been reported to affect the cytokine production of HTLV-1 infected cells; therefore, the difference in these molecules may have accounted for the differences in cytokine production between MT-2J and MT-2B cells. Furthermore, because MT-2 cells showed a large variation of integrated HTLV-1 proviruses as well as cell phenotypes, it is important to exercise caution in the assessment and interpretation of experimental data from MT-2 cells.

Dynamic Changes of Chromatin Structure and Transcriptome By Transient Expression of HTLV-1 Tax in ATL Cells

Adult T-cell leukemia-lymphoma (ATL) is a fatal malignancy of CD4+ T cells, which is caused by human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 encodes two oncogenic viral factors, Tax and HTLV-1 bZIP factor (HBZ) in the sense and antisense of the provirus respectively. HBZ is constitutively expressed in infected cells and plays critical roles for cell proliferation. Tax potently promotes viral replication and activates various oncogenic signaling pathways in HTLV-1 infected cells; however, Tax expression is generally suppressed in infected cells owing to its high immunogenicity. Therefore, the dynamics of Tax expression and contribution of oncogenic transformation by Tax in HTLV-1 infected cells have not been well elucidated. Recently, we have reported that Tax is transiently expressed in a small subpopulation of ATL cells, which induces drastic changes in the host transcriptional profile (Mahgoub M., et al. Proc Natl Acad Sci USA. 2018). Moreover, the single cell analysis showed that expression of anti-apoptotic and NF-kB related genes were upregulated in Tax-expressing cells compared to Tax-non-expressing cells. HBZ expression was increased in Tax-non-expressing cells than Tax-expressing cells, suggesting that HBZ may regulate cell proliferation in a different time phase from Tax. On the other hand, precise mechanisms for regulation of host gene expression by transient Tax expression are not well known. In this study, we analyzed structural change of host chromatin accompanied by transient Tax expression to clarify the transcriptional dynamics of host genes in ATL cells.ATL cell lines (MT-1 and KK-1), which express EGFP under the control of Tax, were used to sort Tax-expressing and -non-expressing cells by a cell sorter, and each population was subjected to H3K27ac ChIP-seq and ATAC-seq analyses. DNA libraries were quantified and sequenced on Illumina NextSeq 500 (single-end) for ChIP-seq, and on Illumina Hiseq 4000 (paired-end) for ATAC-seq, respectively.H3K27ac ChIP-seq data showed that the peaks correlated with Tax expression were observed in many genetic loci including NF-kB related genes. Among Tax-expressing MT-1 cells, H3K27ac were highly enriched super-enhancers, such as IL2RA, TRAF3, TNFRSF8, PHF13 loci, compared to Tax-non-expressing cells. There were more H3K27ac-marked active enhancers in Tax-expressing cells than Tax-non-expressing cells across the entire region, suggesting that global structural change may occur in Tax-expressing cells.Using the data of ATAC-seq, pathway analyses were performed by GREAT. This analysis revealed that the pathways associated with immune activation, such as Toll-like receptor signaling, TNFR2 signaling, IL-2 signaling, and Th1/Th2 differentiation pathways were significantly enriched in Tax-expressing cells. Analysis of genome-wide distribution of motifs in open chromatin region, were carried out with HOMER, and result showed that motifs of NF-kB, and AP-1 family (e.g., JunB, Fra1, Fra2) were significantly enriched in Tax-expressing cells compared to Tax-non-expressing cells, which is consistent with the results of pathway analyses of RNA-seq. The genome-wide motif footprinting analysis was performed using Protein interaction quantitation (PIQ) methods. It was found that motifs of NF-kB, and AP-1 family (e.g., JunB, Fra1, Fra2) were occupied in large number of regions and showed higher DNA accessibility in Tax-expressing cells. In contrast, motif of CTCF was likely occupied in large number of regions and exhibit higher DNA accessibility in Tax-non-expressing cells. Importantly, mRNA levels of NF-kB and AP-1 transcription factors were significantly higher in Tax-expressing than Tax-non-expressing cells.These findings suggest that structural changes of NF-kB and AP-1 family recognition sites, and activation of pathways related to these factors could trigger global transcriptional changes in host genome by transient Tax expression, which is the critical event for persistent infection of HTLV-1 and leukemogenesis of ATL. DisclosuresMatsuoka:Bristol Myers Squibb: Research Funding.

HTLV-1 bZIP Factor Enhances T-Cell Proliferation by Impeding the Suppressive Signaling of Co-inhibitory Receptors.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases. To enhance cell-to-cell transmission of HTLV-1, the virus increases the number of infected cells in vivo. HTLV-1 bZIP factor (HBZ) is constitutively expressed in HTLV-1 infected cells and ATL cells and promotes T-cell proliferation. However, the detailed mechanism by which it does so remains unknown. Here, we show that HBZ enhances the proliferation of expressing T cells after stimulation via the T-cell receptor. HBZ promotes this proliferation by influencing the expression and function of multiple co-inhibitory receptors. HBZ suppresses the expression of BTLA and LAIR-1 in HBZ expressing T cells and ATL cells. Expression of T cell immunoglobulin and ITIM domain (TIGIT) and Programmed cell death 1 (PD-1) was enhanced, but their suppressive effect on T-cell proliferation was functionally impaired. HBZ inhibits the co-localization of SHP-2 and PD-1 in T cells, thereby leading to impaired inhibition of T-cell proliferation and suppressed dephosphorylation of ZAP-70 and CD3ζ. HBZ does this by interacting with THEMIS, which associates with Grb2 and SHP-2. Thus, HBZ interacts with the SHP containing complex, impedes the suppressive signal from PD-1 and TIGIT, and enhances the proliferation of T cells. Although HBZ was present in both the nucleus and the cytoplasm of T cells, HBZ was localized largely in the nucleus by suppressed expression of THEMIS by shRNA. This indicates that THEMIS is responsible for cytoplasmic localization of HBZ in T cells. Since THEMIS is expressed only in T-lineage cells, HBZ mediated inhibition of the suppressive effects of co-inhibitory receptors accounts for how HTLV-1 induces proliferation only of T cells in vivo. This study reveals that HBZ targets co-inhibitory receptors to cause the proliferation of infected cells.

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.

How does HTLV-1 induce ATL?

Human T-cell leukemia virus type 1 (HTLV-1) spreads through cell-to-cell transmission in vivo. HTLV-1 mainly infects CD4+ T cells in vivo. Viral genes enable infected cells to proliferate and escape detection by the host immune system. HTLV-1 infected cells tend to infiltrate tissues, and enter breast milk and semen, thereby infecting a new host. HTLV-1 bZIP factor (HBZ) causes overproduction of interferon gamma, which is associated with inflammation and lymphomagenesis. Thus, the strategies by which HTLV-1 proliferates and survives in infected cells are promoted, which is thought to induce ATL and inflammatory diseases in infected individuals.

Plasmatic proinflammatory chemokines levels are tricky markers to monitoring HTLV-1 carriers

The human T-cell leukemia virus type 1 (HTLV-1) is present throughout the world and is associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and other inflammatory conditions. The pathogenesis of HAM/TSP involves a chronic inflammatory response in central nervous system (CNS), with the presence of HTLV-1 infected cells and HTLV-1-specific CD8+ lymphocytes. Chemokines may have a role in the infiltration of these cells into the CNS. In this context, the present study analyzed the level of plasmatic chemokines CCL2 (MCP-1), CCL5 (RANTES), IL8 (CXCL8), CXCL9 (MIG), and CXCL10 (IP-10) and HTLV-1 proviral load from peripheral blood in 162 asymptomatic carriers and 136 HAM/TSP patients to determine the differences that be associated with the clinical status of the HTLV-1 infection. The results showed that patients with HAM/TSP have significantly higher levels of IL8 and CXCL9, and that the level of IL8, CXCL9 and CXCL10 was significantly greater in HTLV-1 infected individuals with high (>1%) than those with low proviral load (<1%). However, the levels of the chemokines tested have not showed high sensitivity to discriminate HAM/TSP patients from asymptomatic carriers. In addition, chemokine profiles in asymptomatic carriers and HAM/TSP groups were similar, with no significant increased frequency of higher producers of chemokines in HAM/TSP individuals. Results indicate that the heterogeneity of the individuals in the groups regarding time of infection, duration of disease, proviral load level and other possible confound factors may impair the use of chemokines levels to monitor HTLV-1 carriers in clinical practice. J. Med. Virol. 88:1438-1447, 2016. © 2016 Wiley Periodicals, Inc.

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.

T memory stem cells are the hierarchical apex of adult T-cell leukemia

AbstractAdult T-cell leukemia (ATL) is a peripheral CD4+ T-cell neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). Despite several investigations using human specimens and mice models, the exact origin of ATL cells remains unclear. Here we provide a new insight into the hierarchical architecture of ATL cells. HTLV-1–infected cells and dominant ATL clones are successfully traced back to CD45RA+ T memory stem (TSCM) cells, which were recently identified as a unique population with stemlike properties, despite the fact that the majority of ATL cells are CD45RA–CD45RO+ conventional memory T cells. TSCM cells from ATL patients are capable of both sustaining themselves in less proliferative mode and differentiating into other memory T-cell populations in the rapidly propagating phase. In a xenograft model, a low number of TSCM cells efficiently repopulate identical ATL clones and replenish downstream CD45RO+ memory T cells, whereas other populations have no such capacities. Taken together, these findings demonstrate the phenotypic and functional heterogeneity and the hierarchy of ATL cells. TSCM cells are identified as the hierarchical apex capable of reconstituting identical ATL clones. Thus, this is the first report to demonstrate the association of a T-cell malignancy with TSCM cells.

Human T-cell leukemia virus type 1 (HTLV-1) tax requires CADM1/TSLC1 for inactivation of the NF-κB inhibitor A20 and constitutive NF-κB signaling.

Persistent activation of NF-κB by the Human T-cell leukemia virus type 1 (HTLV-1) oncoprotein, Tax, is vital for the development and pathogenesis of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). K63-linked polyubiquitinated Tax activates the IKK complex in the plasma membrane-associated lipid raft microdomain. Tax also interacts with TAX1BP1 to inactivate the NF-κB negative regulatory ubiquitin-editing A20 enzyme complex. However, the molecular mechanisms of Tax-mediated IKK activation and A20 protein complex inactivation are poorly understood. Here, we demonstrated that membrane associated CADM1 (Cell adhesion molecule1) recruits Ubc13 to Tax, causing K63-linked polyubiquitination of Tax, and IKK complex activation in the membrane lipid raft. The c-terminal cytoplasmic tail containing PDZ binding motif of CADM1 is critical for Tax to maintain persistent NF-κB activation. Finally, Tax failed to inactivate the NF-κB negative regulator ubiquitin-editing enzyme A20 complex, and activate the IKK complex in the lipid raft in absence of CADM1. Our results thus indicate that CADM1 functions as a critical scaffold molecule for Tax and Ubc13 to form a cellular complex with NEMO, TAX1BP1 and NRP, to activate the IKK complex in the plasma membrane-associated lipid rafts, to inactivate NF-κB negative regulators, and maintain persistent NF-κB activation in HTLV-1 infected cells.