RESUMO
A small proportion of human T-cell leukemia virus type-1 (HTLV-1)-infected individuals develop adult T-cell leukemia/lymphoma, a chemotherapy-resistant lymphoproliferative disease with a poor prognosis. HTLV-1-specific cytotoxic T lymphocytes (CTLs), potential anti-tumor/virus effectors, are impaired in adult T-cell leukemia/lymphoma patients. Here, using Japanese monkeys naturally infected with simian T-cell leukemia/T-lymphotropic virus type-1 (STLV-1) as a model, we demonstrate that short-term-cultured autologous peripheral blood mononuclear cells (PBMCs) can serve as a therapeutic vaccine to activate such CTLs. In a screening test, STLV-1-specific CTL activity was detectable in 8/10 naturally STLV-1-infected monkeys. We conducted a vaccine study in the remaining two monkeys with impaired CTL responses. The short-term-cultured PBMCs of these monkeys spontaneously expressed viral antigens, in a similar way to PBMCs from human HTLV-1 carriers. The first monkey was subcutaneously inoculated with three-day-cultured and mitomycin C (MMC)-treated autologous PBMCs, and then boosted with MMC-treated autologous STLV-1-infected cell line cells. The second monkey was inoculated with autologous PBMC-vaccine alone twice. In addition, a third monkey that originally showed a weak STLV-1-specific CTL response was inoculated with similar autologous PBMC-vaccines. In all three vaccinated monkeys, marked activation of STLV-1-specific CTLs and a mild reduction in the STLV-1 proviral load were observed. Follow-up analyses on the two monkeys vaccinated with PBMCs alone indicated that STLV-1-specific CTL responses peaked at 3-4 months after vaccination, and then diminished but remained detectable for more than one year. The significant reduction in the proviral load and the control of viral expression were associated with CTL activation but also diminished 6 and 12 months after vaccination, respectively, suggesting the requirement for a booster. The vaccine-induced CTLs in these monkeys recognized epitopes in the STLV-1 Tax and/or Envelope proteins, and efficiently killed autologous STLV-1-infected cells in vitro. These findings indicated that the autologous PBMC-based vaccine could induce functional STLV-1-specific CTLs in vivo.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T do Adulto , Vírus Linfotrópico T Tipo 1 de Símios , Linfócitos T Citotóxicos , Animais , Humanos , Leucócitos Mononucleares , Macaca fuscata , Provírus , VacinaçãoRESUMO
Activation of CD8+ Tax-specific CTL is a new therapeutic concept for adult T-cell leukemia (ATL) caused by HTLV-1. A recent clinical study of the dendritic cell vaccine pulsed with Tax peptides corresponding to CTL epitopes showed promising outcomes in ATL patients possessing limited human leukocyte antigen (HLA) alleles. In this study, we aimed to develop another immunotherapy to activate Tax-specific CTL without HLA limitation by using patients' own HTLV-1-infected cells as a vaccine. To examine the potential of HTLV-1-infected T-cells to activate CTL via antigen presenting cells, we established a unique co-culture system. We demonstrated that mitomycin C-treated HLA-A2-negative HTLV-1-infected T-cell lines or short-term cultured peripheral blood mononuclear cells (PBMC) derived from ATL patients induced cross-presentation of Tax antigen in co-cultured HLA-A2-positive antigen presenting cells, resulting in activation of HLA-A2-restricted CD8+ Tax-specific CTL. This effect was not inhibited by a reverse transcriptase inhibitor. IL-12 production and CD86 expression were also induced in antigen presenting cells co-cultured with HTLV-1-infected cells at various levels, which were improved by pre-treatment of the infected cells with histone deacetylase inhibitors. Furthermore, monocyte-derived dendritic cells induced from PBMC of a chronic ATL patient produced IL-12 and expressed enhanced levels of CD86 when co-cultured with autologous lymphocytes that had been isolated from the same PBMC and cultured for several days. These findings suggest that short-term cultured autologous PBMC from ATL patients could potentially serve as a vaccine to evoke Tax-specific CTL responses.
Assuntos
Vacinas Anticâncer/administração & dosagem , Técnicas de Cultura de Células , Infecções por HTLV-I/terapia , Imunoterapia/métodos , Leucemia-Linfoma de Células T do Adulto/terapia , Leucócitos Mononucleares/transplante , Antígenos Virais/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Apresentação Cruzada/imunologia , Produtos do Gene tax/imunologia , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Infecções por HTLV-I/sangue , Infecções por HTLV-I/imunologia , Inibidores de Histona Desacetilases/farmacologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Leucemia-Linfoma de Células T do Adulto/sangue , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Mitomicina/farmacologia , Linfócitos T Citotóxicos/imunologia , Fatores de Tempo , Transplante AutólogoRESUMO
Immunomodulatory imide drugs (IMiDs), such as lenalidomide and pomalidomide, exert pleiotropic effects, e.g., antitumor effects in multiple myeloma, by binding the protein Cereblon and altering its substrate specificity. Lenalidomide is approved for the treatment of adult T-cell leukemia/lymphoma (ATL) caused by human T-cell leukemia virus type 1 (HTLV-1), although the precise mechanisms responsible for its effectiveness have not been fully elucidated. Here, we used HTLV-1-infected cell lines to investigate how IMiDs exert anti-ATL effects. In three of four tested HTLV-1-infected cell lines, the cells treated with lenalidomide or pomalidomide exhibited mild growth suppression without apoptosis, which was associated with decreased IRF4, c-Myc, and phosphorylated STAT3 levels as well as enhanced SOCS3 expression. Additionally, the levels of enhancer of zeste homolog 2 (EZH2) and trimethyl histone 3 Lys27 (H3K27me3) were decreased following IMiD treatment in all three susceptible cell lines. An IMiD-mediated reduction of EZH2 and H3K27me3 levels was also observed in a multiple myeloma cell line. Furthermore, treatment with an EZH2-inhibitor reproduced the IMiD-mediated effects in HTLV-1-infected cells and multiple myeloma cells. These findings strongly suggest that a reduction of EZH2 expression is involved in the mechanism underlying the antitumor effects of IMiD.
Assuntos
Antivirais/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Infecções por HTLV-I/tratamento farmacológico , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Lenalidomida/farmacologia , Talidomida/análogos & derivados , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Infecções por HTLV-I/patologia , Humanos , Testes de Sensibilidade Microbiana , Talidomida/farmacologiaRESUMO
Adult T-cell leukemia/lymphoma (ATL) is an aggressive lymphoproliferative disease caused by human T-cell leukemia virus type 1 (HTLV-1). Multi-agent chemotherapy can reduce ATL cells but frequently allows relapses within a short period of time. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) following chemotherapy is now a standard therapy for ATL in Japan as it can achieve long-term remission in approximately one-third of recipient ATL patients; however, it also has a risk of treatment-related mortality. Allo-HSCT often induces HTLV-1 Tax-specific cytotoxic T cells (CTL) as well as graft-versus-host (GVH) response in ATL patients. This observation led to development of a new therapeutic vaccine to activate Tax-specific CTL, anticipating anti-ATL effects without GVH response. The newly developed Tax-DC vaccine consists of autologous dendritic cells pulsed with Tax peptides corresponding to CTL epitopes that have been identified in post-allo-HSCT ATL patients. In a pilot study of Tax-DC therapy in three ATL patients after various initial therapies, two patients survived for more than 4 years after vaccination without severe adverse effects (UMIN000011423). The Tax-DC vaccine is currently under phase I trial, showing a promising clinical outcome so far. These findings indicate the importance of patients' own HTLV-1-specific T-cell responses in maintaining remission and provide a new approach to anti-ATL immunotherapy targeting Tax. Although Tax-targeted vaccination is ineffective against Tax-negative ATL cells, it can be a safe alternative maintenance therapy for Tax-positive ATL and may be further applicable for treatment of indolent ATL or even prophylaxis of ATL development among HTLV-1-carriers.
Assuntos
Vacinas Anticâncer/imunologia , Produtos do Gene tax/imunologia , Leucemia-Linfoma de Células T do Adulto/imunologia , Animais , Infecções por HTLV-I/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Projetos Piloto , Linfócitos T Citotóxicos/imunologiaRESUMO
Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and other inflammatory diseases. There is no disease-specific difference in viral strains, and it is unclear how HTLV-1 causes such different diseases manifesting as lymphoproliferation or inflammation. Although some progress has been made in therapies for these diseases, the prognosis for ATL is still dismal and HAM/TSP remains an intractable disease. So far, two regulatory proteins of HTLV-1, Tax and HBZ, have been well studied and shown to have pleiotropic functions implicated in viral pathogenesis. Tax in particular can strongly activate NFκB, which is constitutively activated in HTLV-1-infected cells and considered to contribute to both oncogenesis and inflammation. However, the expression level of Tax is very low in vivo, leading to confusion in understanding its role in viral pathogenesis. A series of studies using IL-2-dependent HTLV-1-infected cells indicated that IL-10, an anti-inflammatory/immune suppressive cytokine, could induce a proliferative phenotype in HTLV-1-infected cells. In addition, type I interferon (IFN) suppresses HTLV-1 expression in a reversible manner. These findings suggest involvement of host innate immunity in the switch between lymphoproliferative and inflammatory diseases as well as the regulation of HTLV-1 expression. Innate immune responses also affect another important host determinant, Tax-specific cytotoxic T lymphocytes (CTLs), which are impaired in ATL patients, while activated in HAM/TSP patients. Activation of Tax-specific CTLs in ATL patients after hematopoietic stem cell transplantation indicates Tax expression and its fluctuation in vivo. A recently developed anti-ATL therapeutic vaccine, consisting of Tax peptide-pulsed dendritic cells, induced Tax-specific CTL responses in ATL patients and exhibited favorable clinical outcomes, unless Tax-defective ATL clones emerged. These findings support the significance of Tax in HTLV-1 pathogenesis, at least in part, and encourage Tax-targeted immunotherapy in ATL. Host innate and acquired immune responses induce host microenvironments that modify HTLV-1-encoded pathogenesis and establish a complicated network for development of diseases in HTLV-1 infection. Both host and viral factors should be taken into consideration in development of therapeutic and prophylactic strategies in HTLV-1 infection.
Assuntos
Genes pX , Infecções por HTLV-I/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Imunoterapia , Leucemia-Linfoma de Células T do Adulto/terapia , Animais , Infecções por HTLV-I/terapia , Humanos , Leucemia-Linfoma de Células T do Adulto/imunologia , Paraparesia Espástica Tropical/imunologia , Paraparesia Espástica Tropical/terapiaRESUMO
Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical role of guanosine (G) number at 5'-terminal of HIV-1 RNA for successful strand-transfer of -sscDNA. In this study, role(s) of the cap consisting of 7-methyl guanosine (7mG), a hallmark of transcripts generated by RNA polymerase II, at the 5'-end G nucleotide (5'-G) of HIV-1 RNA were examined. In parallel, contribution of highly conserved GGG tract located at the U3/R boundary in 3' terminal region of viral RNA (3'-GGG tract) was also addressed. The in vitro reverse transcription analysis using synthetic HIV-1 RNAs possessing the 5'-G with cap or triphosphate form demonstrated that the 5'-cap significantly increased strand-transfer efficiency of -sscDNA. Meanwhile, effect of the 5'-cap on the strand-transfer was retained in the reaction using mutant HIV-1 RNAs in which two Gs were deleted from the 3'-GGG tract. Lack of apparent contribution of the 3'-GGG tract during strand-transfer events in vitro was reproduced in the context of HIV-1 replication within cells. Instead, we noticed that the 3'-GGG tract might be required for efficient gene expression from proviral DNA. These results indicated that 7mG of the cap on HIV-1 RNA might not be reverse-transcribed and a possible role of the 3'-GGG tract to accept the non-template nucleotide addition during -sscDNA synthesis might be less likely. The 5'-G modifications of HIV-1 RNAs by the cap- or phosphate-removal enzyme revealed that the cap or monophosphate form of the 5'-G was preferred for the 1st strand-transfer compared to the triphosphate or non-phosphate form. Taken together, a status of the 5'-G determined strand-transfer efficiency of -sscDNA without affecting the non-template nucleotide addition, probably by affecting association of the 5'-G with 3'-end region of viral RNA.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , Capuzes de RNA/genética , RNA Viral/genética , Transcrição Reversa , Sequência de Bases , Linhagem Celular , Sequência Conservada , DNA Complementar/química , DNA Complementar/genética , Guanosina/química , Guanosina/genética , HIV-1/química , Humanos , Capuzes de RNA/química , RNA Viral/químicaRESUMO
Human T-cell leukemia virus type-1 (HTLV-1) causes two distinct diseases, adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since there are no disease-specific differences among HTLV-1 strains, the etiological mechanisms separating these respective lymphoproliferative and inflammatory diseases are not well understood. In this study, by using IL-2-dependent HTLV-1-infected T-cell lines (ILTs) established from patients with ATL and HAM/TSP, we demonstrate that the anti-inflammatory cytokine IL-10 and its downstream signals potentially act as a switch for proliferation in HTLV-1-infected cells. Among six ILTs used, ILTs derived from all three ATL patients grew much faster than those from three HAM/TSP patients. Although most of the ILTs tested produced IFN-γ and IL-6, the production of IL-10 was preferentially observed in the rapid-growing ILTs. Interestingly, treatment with exogenous IL-10 markedly enhanced proliferation of the slow-growing HAM/TSP-derived ILTs. The IL-10-mediated proliferation of these ILTs was associated with phosphorylation of STAT3 and induction of survivin and IRF4, all of which are characteristics of ATL cells. Knockdown of STAT3 reduced expression of IL-10, implying a positive-feedback regulation between STAT3 and IL-10. STAT3 knockdown also reduced survivin and IRF4 in the IL-10- producing or IL-10- treated ILTs. IRF4 knockdown further suppressed survivin expression and the cell growth in these ILTs. These findings indicate that the IL-10-mediated signals promote cell proliferation in HTLV-1-infected cells through the STAT3 and IRF4 pathways. Our results imply that, although HTLV-1 infection alone may not be sufficient for cell proliferation, IL-10 and its signaling pathways within the infected cell itself and/or its surrounding microenvironment may play a critical role in pushing HTLV-1-infected cells towards proliferation at the early stages of HTLV-1 leukemogenesis. This study provides useful information for understanding of disease mechanisms and disease-prophylactic strategies in HTLV-1 infection.
Assuntos
Proliferação de Células/fisiologia , Vírus Linfotrópico T Tipo 1 Humano , Interleucina-10/metabolismo , Leucemia-Linfoma de Células T do Adulto/imunologia , Transdução de Sinais , Citocinas/metabolismo , Humanos , Fatores Reguladores de Interferon/metabolismo , Paraparesia Espástica Tropical/imunologia , Fator de Transcrição STAT3/metabolismoRESUMO
Adult T cell leukemia/lymphoma (ATL), a CD4+ T cell malignancy with a poor prognosis, is caused by human T cell leukemia virus type 1 (HTLV-1) infection. High proviral load (PVL) is a risk factor for the progression to ATL. We previously reported that some asymptomatic carriers had severely reduced functions of CTLs against HTLV-1 Tax, the major target Ag. Furthermore, the CTL responses tended to be inversely correlated with PVL, suggesting that weak HTLV-1-specific CTL responses may be involved in the elevation of PVL. Our previous animal studies indicated that oral HTLV-1 infection, the major route of infection, caused persistent infection with higher PVL in rats compared with other routes. In this study, we found that Tax-specific CD8+ T cells were present, but not functional, in orally infected rats as observed in some human asymptomatic carriers. Even in the infected rats with immune unresponsiveness against Tax, Tax-specific CTL epitope-pulsed dendritic cell (DC) therapy reduced the PVL and induced Tax-specific CD8+ T cells capable of proliferating and producing IFN-γ. Furthermore, we found that monocyte-derived DCs from most infected individuals still had the capacity to stimulate CMV-specific autologous CTLs in vitro, indicating that DC therapy may be applicable to most infected individuals. These data suggest that peptide-pulsed DC immunotherapy will be useful to induce functional HTLV-1-specific CTLs and decrease PVL in infected individuals with high PVL and impaired HTLV-1-specific CTL responses, thereby reducing the risk of the development of ATL.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Produtos do Gene tax/imunologia , Infecções por HTLV-I/terapia , Tolerância Imunológica , Vacinação , Animais , Linhagem Celular , Feminino , Infecções por HTLV-I/imunologia , Infecções por HTLV-I/virologia , Humanos , Interferon gama/biossíntese , Provírus/isolamento & purificação , Ratos , Ratos Endogâmicos F344 , Carga ViralRESUMO
Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. IMPORTANCE: Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.
Assuntos
Integrase de HIV/química , Transcriptase Reversa do HIV/química , HIV-1/genética , Subunidades Proteicas/química , Tirosina/química , Montagem de Vírus , Sequência de Aminoácidos , Capsídeo/química , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Expressão Gênica , Genes Reporter , Células HEK293 , Integrase de HIV/genética , Integrase de HIV/metabolismo , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/metabolismo , HIV-1/ultraestrutura , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Modelos Moleculares , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Tirosina/metabolismo , Replicação ViralRESUMO
Human T-cell leukaemia virus type 1 (HTLV-1) is a human retrovirus that is a causative agent of adult T-cell leukaemia/lymphoma (ATL) and is mainly transmitted from an infected mother to her child via breastfeeding. Such an HTLV-1 infection during childhood is believed to be a risk factor for ATL development. Although it has been suggested that an increased proviral load (PVL), a higher titre of antibody (Ab) in the infected mother and prolonged breastfeeding are associated with an increased risk of mother-to-child transmission (MTCT), the mechanisms underlying MTCT of HTLV-1 remain largely unknown. In this study, we developed an MTCT model using orally HTLV-1-infected rats that have no Ab responses against viral antigens, such as Gag and Env. In this model, HTLV-1 could be transmitted from the infected mother rats to their offspring at a high rate (50-100â%), and the rate of MTCT tended to be correlated with the PVL of the infected mother rats. Furthermore, passive immunization of uninfected adult rats and an infected mother rat with a rat anti-HTLV-1 Env gp46-neutralizing mAb was unable to suppress primary oral HTLV-1 infection to the adult rats and vertical HTLV-1 transmission to the offspring, respectively. Our findings indicate that this MTCT model would be useful to investigate not only the mechanisms of MTCT but also the role of anti-HTLV-1 Ab in MTCT of HTLV-1. They also provide some information on the role of maternal Abs in MTCT, which should be considered when designing a strategy for prevention of MTCT of HTLV-1.
Assuntos
Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Infecções por HTLV-I/transmissão , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Transmissão Vertical de Doenças Infecciosas , Animais , RatosRESUMO
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.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Produtos do Gene tax/imunologia , Leucemia-Linfoma de Células T do Adulto/imunologia , Leucemia-Linfoma de Células T do Adulto/terapia , Fragmentos de Peptídeos/imunologia , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/efeitos adversos , Citocinas/metabolismo , Feminino , Produtos do Gene tax/química , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Imunoterapia , Leucemia-Linfoma de Células T do Adulto/diagnóstico , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Epitopos de Linfócito T/imunologia , Produtos do Gene tax/imunologia , Efeito Enxerto vs Leucemia/imunologia , Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma de Células T do Adulto/imunologia , Idoso , Linfócitos T CD4-Positivos/metabolismo , Feminino , Produtos do Gene tax/administração & dosagem , Transplante de Células-Tronco Hematopoéticas/métodos , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Leucemia-Linfoma de Células T do Adulto/cirurgia , Masculino , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
BACKGROUND: Human 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. RESULTS: ILTs 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. CONCLUSIONS: IFN-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.
Assuntos
Antivirais/farmacologia , Apoptose , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Interferon-alfa/imunologia , Linfócitos T/virologia , Proteína Supressora de Tumor p53/metabolismo , Zidovudina/farmacologia , Pontos de Checagem do Ciclo Celular , Humanos , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , eIF-2 Quinase/metabolismoRESUMO
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.
Assuntos
Imunidade Adaptativa , Infecções por HTLV-I/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Linfoma de Células T/imunologia , Adulto , Infecções por HTLV-I/virologia , Humanos , Linfoma de Células T/virologiaRESUMO
BACKGROUND: Human 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. RESULTS: By 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. CONCLUSIONS: These 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.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/imunologia , Produtos do Gene tax/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Infecções Assintomáticas , Linfócitos T CD8-Positivos/virologia , Proliferação de Células , Células Cultivadas , Infecções por HTLV-I/imunologia , Humanos , Interferon gama/metabolismo , Lectinas Tipo C/metabolismo , Leucemia-Linfoma de Células T do Adulto/imunologia , Ativação Linfocitária , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Paraparesia Espástica Tropical/imunologia , Paraparesia Espástica Tropical/virologia , Fosfoproteínas/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas da Matriz Viral/imunologiaRESUMO
Infection of erythroid cells by Friend spleen focus-forming virus (SFFV) leads to acute erythroid hyperplasia in mice due to expression of its unique envelope glycoprotein, gp55. Erythroid cells expressing SFFV gp55 proliferate in the absence of their normal regulator, erythropoietin (Epo), because of interaction of the viral envelope protein with the erythropoietin receptor and a short form of the receptor tyrosine kinase Stk (sf-Stk), leading to constitutive activation of several signal transduction pathways. Our previous in vitro studies showed that phosphatidylinositol 3-kinase (PI3-kinase) is activated in SFFV-infected cells and is important in mediating the biological effects of the virus. To determine the role of PI3-kinase in SFFV-induced disease, mice deficient in the p85alpha regulatory subunit of class IA PI3-kinase were inoculated with different strains of SFFV. We observed that p85alpha status determined the extent of erythroid hyperplasia induced by the sf-Stk-dependent viruses SFFV-P (polycythemia-inducing strain of SFFV) and SFFV-A (anemia-inducing strain of SFFV) but not by the sf-Stk-independent SFFV variant BB6. Our data also indicate that p85alpha status determines the response of mice to stress erythropoiesis, consistent with a previous report showing that SFFV uses a stress erythropoiesis pathway to induce erythroleukemia. We further showed that sf-Stk interacts with p85alpha and that this interaction depends upon sf-Stk kinase activity and tyrosine 436 in the multifunctional docking site. Pharmacological inhibition of PI3-kinase blocked proliferation of primary erythroleukemia cells from SFFV-infected mice and the erythroleukemia cell lines derived from them. These results indicate that p85alpha may regulate sf-Stk-dependent erythroid proliferation induced by SFFV as well as stress-induced erythroid hyperplasia.
Assuntos
Leucemia Eritroblástica Aguda/virologia , Fosfatidilinositol 3-Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Vírus Formadores de Foco no Baço/patogenicidade , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilinositol 3-Quinases/deficiênciaRESUMO
Protection from primary human immunodeficiency virus type 1 (HIV-1) infection has not yet been accomplished by vaccines inducing HIV-1-specific acquired immunity. Nevertheless, it has been reported that a small subgroup of women remain resistant to HIV-1 infection under natural conditions. If similar conditions can be induced in uninfected individuals, it will contribute the first line of protection against HIV-1 infection, and also improve the effects of anti-HIV-1 vaccines. We reasoned that innate immunity may be involved in the resistance to HIV-1 infection, and investigated the effects of various Toll-like receptor (TLR) ligands and commensal bacteria on HIV-1 replication in macrophages, one of the initial targets of HIV-1 infection and also the main mediators of innate immunity. We established the HIV-1 reporter monocytic cell line, THP-1/NL4-3luc, which could be differentiated into macrophage-like cells in vitro. In these cells, stimulation of TLR3 and TLR4 by their ligands suppressed HIV-1 expression partly through type I interferon (IFN). Among the commensal bacteria tested, Escherichia coli, Veillonella parvula and Neisseria mucosa suppressed HIV-1 expression, whereas Lactobacillus acidophilus, Prevotella melaninogenica, P. bivia and Mycobacterium smegmatis enhanced it. The bacteria with suppressive effects preferentially stimulated TLR4, whereas the ones with enhancing effects stimulated TLR2. Neutralizing antibodies against TLR4 and IFN-α/ß receptor abrogated bacterially mediated HIV-1 suppression. Suppressive effects of E. coli, V. parvula and N. mucosa on HIV-1 replication were reproducible in primary monocyte-derived macrophages following acute HIV-1 infection. These findings suggest that certain commensal bacteria preferentially stimulating TLR4 potentially produce local environments resistant to HIV-1 infection.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/imunologia , HIV-1/crescimento & desenvolvimento , Macrófagos/microbiologia , Macrófagos/virologia , Receptor 4 Toll-Like/imunologia , Replicação Viral , Linhagem Celular , Sobrevivência Celular , Genes Reporter , Humanos , Interferon Tipo I/imunologia , Luciferases/genética , Luciferases/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 3 Toll-Like/imunologiaRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL), HTLV-1-associated myelopathy/tropical spastic paraparesis, and other inflammatory diseases. Despite such severe outcomes of HTLV-1 infection, the level of HTLV-1 expression in vivo is very low and rapidly increases after transfer of cells to culture conditions. The mechanisms of this phenomenon have remained obscure. In the present study, we found that human and mouse stromal cells, such as epithelial cells and fibroblasts, suppressed HTLV-1 expression in ATL and non-ATL HTLV-1-infected cells. HTLV-1 mRNA and proteins in HTLV-1-infected cells markedly decreased upon coculture with human epithelial-like cells (HEK293T) or mouse embryo fibroblasts (NIH 3T3). When infected cells were reisolated from the cocultures, viral expression was restored to the original level over the following 48 h. Spontaneous induction of HTLV-1 expression in primary ATL cells in the first 24 h of culture was also inhibited by coculture with HEK293T cells. Coculture of HTLV-1-infected cells and HEK293T cells induced type I interferon responses, as detected by beta interferon (IFN-beta) promoter activation and IFN-stimulated gene upregulation. HEK293T-mediated suppression of HTLV-1 expression was partly inhibited by antibodies to human IFN-alpha/beta receptor. NIH 3T3-mediated suppression was markedly abrogated by neutralizing antibodies to mouse IFN-beta. Furthermore, viral expression in HTLV-1-infected cells was significantly suppressed when the infected cells were intraperitoneally injected into wild-type mice but not IFN regulatory factor 7 knockout mice that are deficient of type I IFN responses. These findings indicate that the innate immune system suppresses HTLV-1 expression in vivo, at least through type I IFN.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/imunologia , Interferon beta/imunologia , Leucemia-Linfoma de Células T do Adulto/imunologia , Células Estromais/imunologia , Animais , Técnicas de Cocultura , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Imunidade Inata , Leucemia-Linfoma de Células T do Adulto/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células NIH 3T3 , Receptor de Interferon alfa e beta/imunologia , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
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.
Assuntos
Produtos do Gene tax/imunologia , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Leucemia-Linfoma de Células T do Adulto/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos CD/biossíntese , Autoimunidade , Antígenos CD4/biossíntese , Antígeno CTLA-4 , Linhagem Celular , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Ratos , Ratos Endogâmicos F344 , Linfócitos T Reguladores/imunologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by selective degeneration of motor neurons and glial activation. Cell-specific transcriptional regulation induced by oxidative stress may contribute to the survival and activation of astrocytes in the face of motor neuron death. In the present study, we demonstrate an age-dependent increase in Bcl-xL and Ets-2 immunoreactivity that correlates with an increase of glial fibrillary acidic protein (GFAP)-positive cells in the ventral horn of the spinal cord in both ALS transgenic mice [mutant SOD1 (G93A)] and affected humans. Chromatin immunoprecipitation (ChIP) analysis verified that Ets-2 preferentially occupies the Ets-2 binding element in the promoter of Bcl-xL in primary astrocytes under oxidative stress conditions as well as in G93A spinal cords. Ets-2 small-interfering RNA down-regulated the transcriptional activity of Bcl-xL. In primary glial cultures, Bcl-xL overexpression and mutant SOD1 (G93A) both conferred resistance to oxidative stress-induced cell death. Our findings suggest that Ets-2 transcription factor activation of Bcl-xL gene may protect glia from constitutive oxidative stress that is thought to be a key mechanism contributing to the pathogenesis of ALS. This survival pathway may contribute to the glial survival and activation seen in the spinal cord of ALS patients.