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.