Ayako Takamori

Clinical outcomes of a novel therapeutic vaccine with Tax peptide-pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study

Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type‐I (HTLV‐I)‐infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV‐I Tax‐specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti‐ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate‐ to high‐risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax‐specific CTL responses were observed with peaks at 16–20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide‐pulsed DC vaccine is a safe and promising immunotherapy for ATL.

Interferon-α (IFN-α) suppresses HTLV-1 gene expression and cell cycling, while IFN-α combined with zidovudin induces p53 signaling and apoptosis in HTLV-1-infected cells

BackgroundHuman T-cell leukemia virus type-1 (HTLV-1) is the causative retrovirus of adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 gene expression is maintained at low levels in vivo by unknown mechanisms. A combination therapy of interferon-α (IFN-α) and zidovudin (AZT) shows therapeutic effects in ATL patients, although its mechanism is also obscure. We previously found that viral gene expression in IL-2-dependent HTLV-1-infected T-cells (ILTs) derived from ATL patients was markedly suppressed by stromal cells through a type I IFN response. Here, we investigated the effects of IFN-α with or without AZT on viral gene expression and cell growth in ILTs.ResultsILTs expressed variable but lower amounts of HTLV-1 Tax protein than HTLV-1-transformed HUT102 cells. Following the addition of IFN-α, the amounts of HTLV-1 p19 in the supernatants of these cells decreased in three days, while HTLV-1 gene expression decreased only in ILTs but not HUT102 cells. IFN-α also suppressed the spontaneous HTLV-1 induction in primary ATL cells cultured for 24 h. A time course study using ILTs revealed that the levels of intracellular Tax proteins decreased in the first 24 h after addition of IFN-α, before the reduction in HTLV-1 mRNA levels. The initial decreases of Tax protein following IFN-α treatment were observed in 6 of 7 ILT lines tested, although the reduction rates varied among ILT lines. An RNA-dependent protein kinase (PKR)-inhibitor reversed IFN-mediated suppression of Tax in ILTs. IFN-α also induced cell cycle arrest at the G0/G1 phase and suppressed NF-κB activities in these cells. AZT alone did not affect HTLV-1 gene expression, cell viability or NF-κB activities. AZT combined with IFN-α markedly induced cell apoptosis associated with phosphorylation of p53 and induction of p53-responsive genes in ILTs.ConclusionsIFN-α suppressed HTLV-1 gene expression at least through a PKR-mediated mechanism, and also induced cell cycle arrest in ILTs. In combination with AZT, IFN-α further induced p53 signaling and cell apoptosis in these cells. These findings suggest that HTLV-1-infected cells at an IL-2-dependent stage retain susceptibility to type I IFN-mediated regulation of viral expression, and partly explain how AZT/IFN-α produces therapeutic effects in ATL.

Functional impairment of Tax-specific but not cytomegalovirus-specific CD8+ T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a small percentage of infected individuals. ATL is often associated with general immune suppression and an impaired HTLV-1-specific T-cell response, an important host defense system. We previously found that a small fraction of asymptomatic HTLV-1-carriers (AC) already showed impaired T-cell responses against the major target antigen, Tax. However, it is unclear whether the impaired HTLV-1 Tax-specific T-cell response in these individuals is an HTLV-1-specific phenomenon, or merely reflects general immune suppression. In this study, in order to characterize the impaired HTLV-1-specific T-cell response, we investigated the function of Tax-specific CD8+ T-cells in various clinical status of HTLV-1 infection.ResultsBy using tetramers consisting of HLA-A*0201, -A*2402, or -A*1101, and corresponding Tax epitope peptides, we detected Tax-specific CD8+ T-cells in the peripheral blood from 87.0% of ACs (n = 20/23) and 100% of HAM/TSP patients (n = 18/18) tested. We also detected Tax-specific CD8+ T-cells in 38.1% of chronic type ATL (cATL) patients (n = 8/21), although its frequencies in peripheral blood CD8+ T cells were significantly lower than those of ACs or HAM/TSP patients. Tax-specific CD8+ T-cells detected in HAM/TSP patients proliferated well in culture and produced IFN-γ when stimulated with Tax peptides. However, such functions were severely impaired in the Tax-specific CD8+ T-cells detected in cATL patients. In ACs, the responses of Tax-specific CD8+ T-cells were retained in most cases. However, we found one AC sample whose Tax-specific CD8+ T-cells hardly produced IFN-γ, and failed to proliferate and express activation (CD69) and degranulation (CD107a) markers in response to Tax peptide. Importantly, the same AC sample contained cytomegalovirus (CMV) pp65-specific CD8+ T-cells that possessed functions upon CMV pp65 peptide stimulation. We further examined additional samples of two smoldering type ATL patients and found that they also showed dysfunctions of Tax-specific but not CMV-specific CD8+ T-cells.ConclusionsThese findings indicated that Tax-specific CD8+ T-cells were scarce and dysfunctional not only in ATL patients but also in a limited AC population, and that the dysfunction was selective for HTLV-1-specifc CD8+ T-cells in early stages.

Impaired Tax‐specific T‐cell responses with insufficient control of HTLV‐1 in a subgroup of individuals at asymptomatic and smoldering stages

Human T‐cell leukemia virus type‐1 (HTLV‐1)‐specific T‐cell immunity, a potential antitumor surveillance system in vivo, is impaired in adult T‐cell leukemia (ATL). In this study, we aimed to clarify whether the T‐cell insufficiency in ATL is present before the disease onset or occurs as a consequence of the disease. We investigated T‐cell responses against Tax protein in peripheral blood mononuclear cells (PBMCs) from individuals at earlier stages of HTLV‐1‐infection, including 21 asymptomatic HTLV‐1 carriers (ACs) and four patients with smoldering‐type ATL (sATL), whose peripheral lymphocyte count was in normal range. About 30% of samples tested showed clear Tax‐specific interferon (IFN)‐γ producing responses. Proviral loads in this group were significantly lower than those in the other less‐specific response group. The latter group was further divided to two subgroups with or without emergence of Tax‐specific responses following depletion of CC chemokine receptor 4 (CCR4)+ cells that contained HTLV‐1‐infected cells. In the PBMCs with Tax‐specific responses, CD8+ cells efficiently suppressed HTLV‐1 p19 production in culture. The remaining group without the emergence of Tax‐specific response after CCR4+ cell‐depletion included at least two sATL and one AC samples, which spontaneously produced HTLV‐1 p19 in culture, where tetramer‐binding, Tax‐specific cytotoxic T‐lymphocytes were either undetectable or unresponsive. Our results indicated that HTLV‐1‐specific T‐cell responsiveness widely differed among HTLV‐1 carriers, and that impairment of HTLV‐1‐specific T‐cell responses was observed not only in advanced ATL patients but also in a subpopulation at earlier stages, which was associated with insufficient control of HTLV‐1. (Cancer Sci 2009; 100: 481–489)

Double control systems for human T-cell leukemia virus type 1 by innate and acquired immunity

Human T‐cell leukemia virus type 1 (HTLV‐1) is the causative retrovirus of adult T‐cell leukemia (ATL) and HTLV‐1‐associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV‐1‐specific T‐cell responses elicit antitumor and antiviral effects in experimental models, and are considered to be one of the most important determinants of the disease manifestation, since they are activated in HAM/TSP but not in ATL patients. The combination of low T‐cell responses and elevated HTLV‐1 proviral loads are features of ATL, and are also observed in a subpopulation of HTLV‐1 carriers at the asymptomatic stage, suggesting that these features may be underlying risk factors. These risks may potentially be reduced by vaccination to activate HTLV‐1‐specific T‐cell responses. HAM/TSP and ATL patients also differ in their levels of HTLV‐1 mRNA expression, which are generally low in vivo but slightly higher in HAM/TSP patients. Our recent study indicated that viral expression in HTLV‐1‐infected T‐cells is suppressed by stromal cells in culture through type‐I IFNs. The suppression was reversible after isolation from the stromal cells, mimicking a long‐standing puzzling phenomenon in HTLV‐1 infection where the viral expression is very low in vivo and rapidly induced in vitro. Collectively, HTLV‐1 is controlled by both acquired and innate immunity in vivo: HTLV‐1‐specific T‐cells survey infected cells, and IFNs suppress viral expression. Both effects would contribute to a reduction in viral pathogenesis, although they may potentially influence or conflict with one another. The presence of double control systems for HTLV‐1 infection provides a new concept for understanding the pathogenesis of HTLV‐1‐mediated malignant and inflammatory diseases. (Cancer Sci 2011; 102: 670–676)

The roles of acquired and innate immunity in human T-cell leukemia virus type 1-mediated diseases.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis in small subsets of HTLV-1 carriers. HTLV-1-specific T-cell responses play critical roles in anti-viral and anti-tumor host defense during HTLV-1 infections. Some HTLV-1 carriers exhibit selective loss or anergy of HTLV-1-specific T-cells at an asymptomatic stage. This is also observed in ATL patients and may therefore be an underlying risk factor of ATL in combination with elevated proviral loads. HTLV-1-specific T-cells often recognize the viral oncoprotein Tax, indicating expression of Tax protein in vivo, although levels of HTLV-1 gene expression are known to be very low. A type-I interferon (IFN) response can be induced by HTLV-1-infected cells and suppresses HTLV-1 expression in vitro, suggesting a role of type-I IFN response in viral suppression and pathogenesis in vivo. Both acquired and innate immune responses control the status of HTLV-1-infected cells and could be the important determinants in the development of HTLV-1-mediated malignant and inflammatory diseases.

Potential Contribution of a Novel Tax Epitope–Specific CD4+ T Cells to Graft-versus-Tax Effect in Adult T Cell Leukemia Patients after Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for adult T cell leukemia/lymphoma (ATL) caused by human T cell leukemia virus type 1 (HTLV-1). We previously reported that Tax-specific CD8+ cytotoxic T lymphocyte (CTL) contributed to graft-versus-ATL effects in ATL patients after allo-HSCT. However, the role of HTLV-1–specific CD4+ T cells in the effects remains unclear. In this study, we showed that Tax-specific CD4+ as well as CD8+ T cell responses were induced in some ATL patients following allo-HSCT. To further analyze HTLV-1–specific CD4+ T cell responses, we identified a novel HLA-DRB1*0101–restricted epitope, Tax155–167, recognized by HTLV-1–specific CD4+ Th1-like cells, a major population of HTLV-1–specific CD4+ T cell line, which was established from an ATL patient at 180 d after allo-HSCT from an unrelated seronegative donor by in vitro stimulation with HTLV-1–infected cells from the same patient. Costimulation of PBMCs with both the identified epitope (Tax155–167) and known CTL epitope peptides markedly enhanced the expansion of Tax-specific CD8+ T cells in PBMCs compared with stimulation with CTL epitope peptide alone in all three HLA-DRB1*0101+ patients post–allo-HSCT tested. In addition, direct detection using newly generated HLA-DRB1*0101/Tax155–167 tetramers revealed that Tax155–167-specific CD4+ T cells were present in all HTLV-1–infected individuals tested, regardless of HSCT. These results suggest that Tax155–167 may be the dominant epitope recognized by HTLV-1–specific CD4+ T cells in HLA-DRB1*0101+–infected individuals and that Tax-specific CD4+ T cells may augment the graft-versus-Tax effects via efficient induction of Tax-specific CD8+ T cell responses.

DNA-dependent activator of IFN-regulatory factors enhances the transcription of HIV-1 through NF-κB.

Pattern recognition receptors (PRRs) play a pivotal role in host innate immune responses against microbial infection. Viruses are primarily recognized by PRRs such as Toll-like receptor 3, 7, 8 and 9, and RIG-I-like receptors. Recent studies have demonstrated that DNA-dependent activator of IFN-regulatory factors (DAI) is a cytosolic sensor molecule for dsDNA, and is implicated in antiviral responses to some DNA viruses. Soon after infection, human immunodeficiency virus type-1 (HIV-1) synthesizes viral dsDNA in the cytoplasm by reverse transcriptase. In addition, an immune compromised state due to chronic HIV-1 infection results in opportunistic infection with some microbes that potentially activate DAI. However, it has not been elucidated whether DAI affects HIV-1 replication, or its possible mechanisms. Here, we showed that forced expression of DAI markedly enhanced HIV-1 replication, which was largely impaired by mutations at κB sites in HIV-1 LTR or by suppressing activation of NF-κB. Moreover, intact structure around the D3 region (174-232 aa) and two RIP homotypic interaction motifs (198-214 aa and 256-272 aa) within DAI were critical for its activity. These results suggest that activation of DAI might contribute to augment HIV-1 replication through DAI- NF-κB pathway.

Augmentation of donor-derived Tax-specific CTL responses by a novel Tax epitope-specific CD4+ helper T-cells in ATL patients after allogeneic hematopoietic stem cell transplantation

Adult T-cell leukemia/ lymphoma (ATL) is an aggressive T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1) and characterized by extremely poor prognosis because of intrinsic drug resistance to cytotoxic agents. Recently, allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been shown to be an effective treatment for ATL due to graft-versus-leukemia effects. However, donor-derived HTLV-1-specific T-cell responses in ATL patients after allo-HSCT are not well understood. In this study, blood samples from 15 ATL patients at 180 days following allo-HSCT from seronegative donors were screened for IFN-g production of donor-derived T-cells against Tax protein. Among 15 patients, Tax-specific T-cell responses were observed in 11 patients. Direct detection with Tax/MHC class I tetramers revealed that donor-derived Tax-specific CD8+ cytotoxic T-lymphocytes (CTLs) were present in 12 of 15 patients. We then identified a novel Tax epitope (Tax155-167) presented by HLA-DR1 (HLA-DRB1*0101) and examined the presence of Tax-specific CD4+ T-cells in 3 HLA-DR1+ patients after allo-HSCT using a newly generated Tax155-167/HLA-DR1 tetramer. Tax155-167-specific CD4+ T-cells were found to be present in all HLA-DR1+ patients tested. Furthermore, in vitro stimulation of PBMCs from HLA-DR1+ HLA-A*2402+ patients with both Tax155-167 and HLA-A*2402-restricted CTL epitope (Tax301-309) led to robust expansion of Tax301-309-specific CTLs. Our results suggest that donor-derived HTLV-1-specific both CD4+ and CD8+ T-cells could be induced in ATL patients after allo-HSCT from seronegative donors, probably due to exposure to HTLV-1 antigens from recipient-derived ATL cells, and the HTLV-1-specific CD4+ T-cells may contribute to efficient induction of donor-derived HTLV-1-specific CTL responses.

Induction of IL-10- and IFN-γ-producing T-cell responses by autoreactive T-cells expressing human T-cell leukemia virus type I Tax

Human T-cell leukemia virus type I (HTLV-I) is associated with adult T-cell leukemia, HTLV-I-associated myelopathy/tropical spastic paraparesis and various autoimmune-like disorders. T-cell immune suppression is also associated with HTLV-I infection. Mechanisms of diverse immune dysregulation in HTLV-I infection are obscure. Here, we investigated a potential link between autoimmunity and immune suppression in HTLV-I infection. G14, an IL-2-dependent HTLV-I-negative CD4(+)CD8(+) T-cell line previously established from an HTLV-I-infected rat, constantly proliferated and produced IFN-gamma. IFN-gamma production by G14 cells was dependent on interactions between CD4 and MHC-II, suggesting that G14 cells recognized self-antigens presented by MHC-II on themselves. To examine immune response to G14 cells, we inoculated G14 cells into syngeneic naive rats. Interestingly, T-cells isolated from these rats vigorously proliferated when stimulated with G14-Tax cells that stably expressed HTLV-I Tax, but not with G14 cells. G14-Tax-mediated T-cell proliferation was abrogated by antibodies to CD80 and CD86 that were up-regulated in G14-Tax cells. T-cells propagated by repetitive G14-Tax cell stimulations in culture with IL-2 expressed CD4, CD25 and cytolytic T lymphocyte-associated antigen 4 (CTLA-4), produced abundant amounts of IL-10 and IFN-gamma in response to G14 cells and suppressed growth of G14 cells mainly through supernatant-mediated mechanisms. Similar IL-10- and IFN-gamma-producing CD4(+)CD25(+)CTLA-4(+) T-cells were predominantly induced in culture of splenocytes from HTLV-I-infected rats following stimulation with G14-Tax cells. These results implied that expression of Tax in the otherwise low immunogenic autoreactive T-cells induced IL-10- and IFN-gamma-producing T-cell responses with regulatory effects against the autoreactive cells. Our findings provide new insights into the complex immune conditions underlying HTLV-I-associated diseases.