Norihiro Takenouchi

An animal model of adult T-cell leukemia: humanized mice with HTLV-1–specific immunity

Human T-cell leukemia virus type 1 (HTLV-1) is causally associated with adult T-cell leukemia (ATL), an aggressive T-cell malignancy with a poor prognosis. To elucidate ATL pathogenesis in vivo, a variety of animal models have been established; however, the mechanisms driving this disorder remain poorly understood due to deficiencies in each of these animal models. Here, we report a novel HTLV-1-infected humanized mouse model generated by intra-bone marrow injection of human CD133(+) stem cells into NOD/Shi-scid/IL-2Rγc null (NOG) mice (IBMI-huNOG mice). Upon infection, the number of CD4(+) human T cells in the periphery increased rapidly, and atypical lymphocytes with lobulated nuclei resembling ATL-specific flower cells were observed 4 to 5 months after infection. Proliferation was seen in both CD25(-) and CD25(+) CD4 T cells with identical proviral integration sites; however, a limited number of CD25(+)-infected T-cell clones eventually dominated, indicating an association between clonal selection of infected T cells and expression of CD25. Additionally, HTLV-1-specific adaptive immune responses were induced in infected mice and might be involved in the control of HTLV-1-infected cells. Thus, the HTLV-1-infected IBMI-huNOG mouse model successfully recapitulated the development of ATL and may serve as an important tool for investigating in vivo mechanisms of ATL leukemogenesis and evaluating anti-ATL drug and vaccine candidates.

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.

Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo

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.

HLA-A24-restricted HTLV-I-specific CTL response reduces the HTLV-I proviral load but the HLA increases the risk of HAM/TSP

It is controversial whether HTLV-I-specific CTLs are beneficial or harmful to the host in the development of HAM/TSP. HLA-A2 reduces the risk of HAM/TSP and HLA-A2-restricted HTLV-I Tax11-19-specific CTL response reduces HTLV-I proviral load in asymptomatic HTLV-I carriers (ACs), suggesting that HLA-A2-restricted CTLs are beneficial to the host. Recently, HTLV-I Tax301-309 is newly identified as an immunodominant epitope restricted to HLA-A24 and frequency of Tax301-309-specific CTLs is high in HTLV-I-infected individuals. We investigated whether HLA-A24 also reduces the risk of HAM/TSP and compared the differences between HLA-A2- and HLA-A24-restricted Tax-specific CTL responses. We found that the allele frequency of HLA-A24 was significantly increased in HAM/TSP patients compared to ACs. The frequency of HTLV-I Tax301-309-specific CTLs was higher in HAM/TSP patients than that in ACs and negatively correlated with the HTLV-I proviral load in both HAM/TSP patients and ACs. In the comparison between HLA-A2/Tax11-19-specific CTLs and HLA-A24/Tax301-309-specific CTLs, the maximum responses by antigen stimulation were not different in IFN-gamma and MIP-1beta productions and CD107a expression, however, the functional avidity of the CTLs was 50-fold stronger in Tax301-309-specific CTLs than in Tax11-19-specific CTLs. This suggests that Tax301-309-specific CTLs more efficiently recognize HTLV-I-infected cells when the cells express low levels of viral proteins. Our data suggest that HLA-A24 increases the risk of HAM/TSP and that Tax301-309-specific CTLs may play a role in the pathogenesis of HAM/TSP even though they reduce the proviral load, or other factors related to HLA-A24 may affect the risk.

Inverse correlation between Tax and CD25 expressions in HTLV-1 infected CD4 T-cells in vivo

Tax has pleiotropic actions that induce proliferation and inhibit apoptosis of T-cells and thus is considered to play a critical role in leukemogenesis. However, Tax expression is frequently lost in fresh adult T-cell leukemia (ATL) cells by genetic changes or epigenetic modifications of proviral genome. To clarify the significance of tax gene in leukemogenesis, we analyzed the expression of tax gene in the HTLV-1 infected human CD4+ T-lymphocytes in humanized mouse system. NOG-SCID mouse were transplanted with human hematopoietic stem cells and infected with HTLV-1 in vivo by peritoneal injection of MT-2 cells after 4 months of transplantation. HTLV-1 infection induced the rapid proliferation of CD4+ T-cells, irrespective of the expression of CD25, and resulted in the severe splenomegaly with CD25+ CD4+ T-cells. Tax gene expression was low in the isolated splenocytes but greatly increased by the ex vivo culture for 24 hours as seen in infected peripheral blood T-cells from HTLV-1 carrier. As the gene expression of CD25 was also activated in proportion to the tax induction, function of Tax was indicated to be responsible for the CD25 expression. When CD4+ T-cells from HTLV-1 infected splenocytes were separated into CD25+ and CD25- cells, however, tax expression was observed mainly in CD25- CD4+ T-cells. These results indicate that the expression of CD25 may not necessarily involve the Tax function in vivo. We will discuss the function of Tax in CD25- CD4+ T-cells and a significance of CD25 expression without the action of Tax in vivo.

ATL-like overgrowth and clonal expansion of HTLV-1 infected CD25+ CD4+ T- lymphocyte in humanized-NOG mouse model

Humanized mice (huNOG) established by the intrabone marrow transplantation of NOG-SCID mouse with CD133+ hematopoietic stem cells purified from human cord blood were infected with HTLV-1 in vivo by peritonial injection of g-ray-irradiated MT-2 cells in 3 to 4 months after transplantation. While normal differentiation of human T lymphocytes was observed in the spleen of uninfected huNOG mouse, HTLV-1 infection increased the number of CD25+ CD4+ Tlymphocytes and resulted in the splenomegaly within several months. In the late period of infection, where almost all of the blood cells in the mouse were composed of infected human T-lymphocytes, cells with highly lobulated or flower-shaped nuclei appeared in the peripheral blood. Inverse PCR analysis of provirus integration sites revealed the polyclonal infection in the early phase and the oligoclonal expansion of infected T cells mostly in the population of CD25+ CD4+ Tcells in the late phase. Since substantial amount of anti-Gag antibodies and Tax-specific CTLs were detected in the serum and the spleen of infected mice, respectively, the involvement of immune system against HTLV-1 was suggested in the clonal selection of HTLV-1 infected T-cells in this system. Thus, the HTLV-1 infected huNOG mouse model should provide a valuable system for the analysis of ATL pathogenesis and the development of treatments against various HTLV-1 associated diseases. Results from the analysis of gene expression in HTLV-1 infected T-cells during the course of infection and the effects of in vivo administration of various anti-tumor or anti-viral drugs on the overgrowth of infected T-cells will be discussed.

Acute myelitis resembling human T-lymphotropic virus type 1-associated myelopathy presenting as high-signal-intensity long cord lesions on T2-weighted magnetic resonance images combined with lymphoma-type adult T-cell leukemia/lymphoma

• We describe an autopsy case of acute myelitis resembling human T-lymphotropic virus type 1-associated myelopathy (HAM) combined with adult T-cell leukemia/lymphoma.

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.

Induction of APOBEC3B cytidine deaminase gene in HTLV-1 infected T-cells of ATL model mouse

A variety of mutations are accumulated in the genome of HTLV-1 infected T-cells during ATL development. To elucidate the mechanism of ATL development a mouse model of ATL was established by infecting HTLV-1 to humanized NOG mice and the infected mice recapitulate the ATL-like symptoms and die of leukemia within several months of infection. Analysis of gene expressions in the humanized mouse model of ATL demonstrated the induction of APOBEC3B (A3B) gene in the HTLV-1 infected human T-cells. A3B is a member of the APOBEC family of cellular cytidine deaminase and was recently identified as the mutational source in multiple human cancers. We have previously shown that HTLV-1 infected CD25 (-) CD4 T-cells but not CD25 (+) CD4 T-cells in ATL model mouse express a small amount of Tax mRNA even though both cell populations consist of identical infected-cell clones. As the A3B expression in HTLV-1 infected CD25 (+) T-cells was similar to, or rather higher than that in CD25 (-)T-cells, Tax appears not to be involved in the induction A3B. In contrast, ex vivo culture of infected T-cells, in which Tax expression was highly enhanced in both CD25 (+) and CD25 (-)T-cells, resulted in the suppression of A3B expression. However, ectopic expression of HTLV-1 in Jurkat cell by the transfection of HTLV-1 molecular clone plasmid enhanced the expression of A3B gene. It is thus suggested that HTLV-1 infection but not Tax induces the A3B expression. Interplay between HTLV-1 infection and A3B and the possible involvement of A3B in the mutagenesis of host cell genome are currently investigated.