Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia. The HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand of the provirus, is involved in both regulation of viral gene transcription and T-cell proliferation. We showed in this report that HBZ interacted with Smad2/3, and enhanced transforming growth factor-␤ (TGF-␤)/Smad transcriptional responses in a p300-dependent manner. The N-terminal LXXLL motif of HBZ was responsible for HBZ-mediated TGF-␤ signaling activation. In a serial immunoprecipitation assay, HBZ, Smad3, and p300 formed a ternary complex, and the association between Smad3 and p300 was markedly enhanced in the presence of HBZ. In addition, HBZ could overcome the repression of the TGF-␤ response by Tax. Finally, HBZ expression resulted in enhanced transcription of Pdgfb, Sox4, Ctgf, Foxp3, Runx1, and Tsc22d1 genes and suppression of the Id2 gene; such effects were similar to those by TGF-␤. In particular, HBZ induced Foxp3 expression in naive T cells through Smad3-dependent TGF-␤ signaling. Our results suggest that HBZ, by enhancing TGF-␤ signaling and Foxp3 expression, enables HTLV-1 to convert infected T cells into regulatory T cells, which is thought to be a critical strategy for virus persistence. (Blood. 2011;118(7):1865-1876) IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/ tropical spastic paraparesis (HAM/TSP). 1,2 The unique sequence of HTLV-1 between the env region and 3Ј long terminal repeat, denoted the pX region, encodes regulatory (tax and rex) and accessory (p12, p13, and p30) genes in the plus strand. 3 The pleiotropic actions of Tax are thought to play a central role in the early stage of leukemogenesis. 4 However, approximately 60% of fresh ATL cells lack Tax expression because of genetic and epigenetic changes in the HTLV-1 provirus, suggesting that Tax may not be essential for the maintenance of ATL cells in vivo. 5 The HTLV-1 bZIP factor (HBZ), which is encoded by the complementary strand of the HTLV-1 genome, is expressed in all ATL cases and supports the proliferation of ATL cells. 6,7 In addition, HBZ was found to inhibit Tax-mediated activation of viral transcription from the 5Ј-long terminal repeat by heterodimerizing with c-Jun, CREB2, and to selectively suppress the classic pathway of nuclear factor-B by 2 distinct mechanisms. [8][9][10][11] Thus, HBZ has multifunctional roles in cellular signaling and proliferation. Recently, we reported that nonsense mutations in all HTLV-1 genes except HBZ were generated by APOBEC3G before integration of the provirus in ATL cases and HTLV-1 infected persons, indicating that the HBZ gene is essential for leukemogenesis. 12 Transforming growth factor-␤ (TGF-␤) controls a variety of biologic processes, including cell growth, differentiation, apoptosis, development, and immune homeostasis. 13 The Smad proteins, which are mediators of TGF-␤ signaling, transdu...

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.

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) causes chronic infection leading to development of adult T-cell leukemia (ATL) and inflammatory diseases. Non-human primates infected with simian T-cell leukemia virus type 1 (STLV-1) are considered to constitute a suitable animal model for HTLV-1 research. However, the function of the regulatory and accessory genes of STLV-1 has not been analyzed in detail. In this study, STLV-1 in naturally infected Japanese macaques was analyzed.ResultsWe identified spliced transcripts of STLV-1 corresponding to HTLV-1 tax and HTLV-1 bZIP factor (HBZ). STLV-1 Tax activated the NFAT, AP-1 and NF-κB signaling pathways, whereas STLV-1 bZIP factor (SBZ) suppressed them. Conversely, SBZ enhanced TGF-β signaling and induced Foxp3 expression. Furthermore, STLV-1 Tax activated the canonical Wnt pathway while SBZ suppressed it. STLV-1 Tax enhanced the viral promoter activity while SBZ suppressed its activation. Then we addressed the clonal proliferation of STLV-1+ cells by massively sequencing the provirus integration sites. Some clones proliferated distinctively in monkeys with higher STLV-1 proviral loads. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain; STLV-1 provirus was integrated in the lymphoma cell genome. When anti-CCR4 antibody, mogamulizumab, was administered into STLV-1-infected monkeys, the proviral load decreased dramatically within 2 weeks. We observed that some abundant clones recovered after discontinuation of mogamulizumab administration.ConclusionsSTLV-1 Tax and SBZ have functions similar to those of their counterparts in HTLV-1. This study demonstrates that Japanese macaques naturally infected with STLV-1 resemble HTLV-1 carriers and are a suitable model for the investigation of persistent HTLV-1 infection and asymptomatic HTLV-1 carrier state. Using these animals, we verified that mogamulizumab, which is currently used as a drug for relapsed ATL, is also effective in reducing the proviral load in asymptomatic individuals.

Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T cell grafts from third-party donors will enable widespread use of this modality if allogeneic T cell responses are effectively controlled. Here, we generated HLA class I, class II, and T cell receptor triple knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA deficient T cells were targeted by natural killer cells, they persisted better than HLA sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice.When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMC compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease, but retained antitumor responses. These results demonstrated the benefit of HLA class I, class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy. SynopsisAntitumor T cell grafts concurrently ablated of HLA class I, class II, and TCR molecules evade allogeneic T cell responses and can be used as a universal T cell source for adoptive cancer immunotherapy.

Reporter assays that use luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly luciferase and Renilla luciferase are used for monitoring single gene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because one of the two reporting luciferases must be used as an internal control. We have developed a novel reporter assay system in which three luciferases that emit green, orange, and red light with a single substrate are used as reporter genes. The activities of the luciferases can be measured simultaneously and quantitatively with optical filters. This system enables us to simply and rapidly monitor multiple gene expressions in a one-step reaction.

Key Points• Vaccination with HBZ can induce cytotoxic T lymphocytes and suppress an HBZ-expressing lymphoma cell line in vivo.• Immunodominant epitopes of HBZ have been identified in mice, monkeys, and humans.

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.

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.