RESUMO
Persistent human papillomavirus type 16 (HPV16) infection is associated with the development of more than 50% of cervical cancers. The HPV16 E6 and E7 oncoproteins are constitutively expressed in cervical carcinomas and are attractive targets for cytotoxic T lymphocyte (CTL)-based immunotherapy. However, cervical carcinomas may possess multiple evasion mechanisms for HPV16 E6/E7-specific CTL. In this study, we investigated whether HPV16(+) cervical carcinoma cell lines (CaCxCL) could evade all effector functions of HPV16 E6(29-38)-specific T cells. Such CD8(+) T cells were detected in the blood (4/10) or invaded lymph node (1/1) of cervical cancer patients using HLA-A*0201/HPV16 E6(29-38) tetramers after in vitro stimulation. T cells cultured from 3 different donors killed HPV16 E6(29-38) peptide-pulsed target cells but not HPV16(+) CaCxCL in (51)Cr release assays. The absence of killing correlated with limited T-cell degranulation against CaCxCL, but this was not due to antigen processing defects per se; CaCxCL could induce specific T-cell release of IFN-gamma and TNF-alpha, and CaCxCL could be killed in longer cytotoxicity assays (>20 hr). Interestingly, the 'slow' killing of CaCxCL could be partially inhibited by concanamycin A, a known perforin inhibitor. The results suggest that CaCxCL was only partially activating T cells, but this was still sufficient for slow killing. Overall, our results highlight the need to examine multiple T-cell effector functions in the context of endogenous antigen presentation by tumour cells. In this study, testing for cytotoxicity using short-term assays only would have ruled out a candidate epitope for immunotherapy.
Assuntos
Proteínas Oncogênicas Virais/imunologia , Proteínas Repressoras/imunologia , Linfócitos T/imunologia , Neoplasias do Colo do Útero/imunologia , Estudos de Casos e Controles , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , MasculinoAssuntos
Pesquisa Biomédica/tendências , Diabetes Mellitus/prevenção & controle , Cardiopatias/prevenção & controle , Acidente Vascular Cerebral/prevenção & controle , Doença Crônica/economia , Doença Crônica/epidemiologia , Doença Crônica/prevenção & controle , Atenção à Saúde/tendências , Diabetes Mellitus/economia , Diabetes Mellitus/epidemiologia , Educação em Saúde/tendências , Cardiopatias/economia , Cardiopatias/epidemiologia , Humanos , Pobreza , Fatores de Risco , Acidente Vascular Cerebral/economia , Acidente Vascular Cerebral/epidemiologia , UrbanizaçãoAssuntos
Difusão de Inovações , Aceitação pelo Paciente de Cuidados de Saúde , Prevenção Primária/métodos , Vacinas/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Ensaios Clínicos como Assunto , Países em Desenvolvimento , Feminino , Saúde Global , Política de Saúde , História do Século XVIII , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Masculino , Vacina contra Sarampo-Caxumba-Rubéola/administração & dosagem , Vacina contra Sarampo-Caxumba-Rubéola/efeitos adversos , Prevenção Primária/história , Varíola/prevenção & controle , Vacina Antivariólica/administração & dosagem , Vacina Antivariólica/história , Fatores Socioeconômicos , Tuberculose/epidemiologia , Tuberculose/história , Tuberculose/prevenção & controle , Tuberculose/terapia , Neoplasias do Colo do Útero/prevenção & controle , Vacinas/históriaRESUMO
The inhibitory leukocyte Ig-like receptor 1 (LIR-1, also known as ILT2, CD85j, or LILRB1) was identified by its high affinity for the human CMV (HCMV) MHC class I homolog gpUL18. The role of this LIR-1-gpUL18 interaction in modulating NK recognition during HCMV infection has previously not been clearly defined. In this study, LIR-1(+) NKL cell-mediated cytotoxicity was shown to be inhibited by transduction of targets with a replication-deficient adenovirus vector encoding UL18 (RAd-UL18). Fibroblasts infected with an HCMV UL18 mutant (DeltaUL18) also exhibited enhanced susceptibility to NKL killing relative to cells infected with the parental virus. In additional cytolysis assays, UL18-mediated protection was also evident in the context of adenovirus vector transduction and HCMV infection of autologous fibroblast targets using IFN-alpha-activated NK bulk cultures derived from a donor with a high frequency of LIR-1(+) NK cells. A single LIR-1(high) NK clone derived from this donor was inhibited by UL18, while 3 of 24 clones were activated. CD107 mobilization assays revealed that LIR-1(+) NK cells were consistently inhibited by UL18 in all tested donors, but this effect was often masked in the global response by UL18-mediated activation of a subset of LIR-1(-) NK cells. Although Ab-blocking experiments support UL18 inhibition being induced by a direct interaction with LIR-1, the UL18-mediated activation is LIR-1 independent.
Assuntos
Proteínas do Capsídeo/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/antagonistas & inibidores , Adenoviridae/genética , Antígenos CD/análise , Antígenos CD/imunologia , Proteínas do Capsídeo/genética , Células Cultivadas , Citotoxicidade Imunológica , Vetores Genéticos/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Células Matadoras Naturais/química , Receptor B1 de Leucócitos Semelhante a Imunoglobulina , Ativação Linfocitária , Receptores Imunológicos/análise , Receptores Imunológicos/imunologia , Deleção de SequênciaRESUMO
Dendritic cells (DCs) play a pivotal role in the generation of virus-specific cytotoxic T-cell responses, but some viruses can render DCs inefficient in stimulating T cells. We studied whether infection of DCs with human cytomegalovirus (HCMV) results in a suppression of DC function which may assist HCMV in establishing persistence. The effect of HCMV infection on the phenotype and function of monocyte-derived DCs and on their ability to mature following infection with an endothelial cell-adapted clinical HCMV isolate were studied. HCMV infection induced no maturation of DCs; instead, it efficiently down-regulated the expression of surface major histocompatibility complex (MHC) class I, CD40, and CD80 molecules. Slight down-regulation of MHC class II and CD86 molecules was also observed. Lipopolysaccharide (LPS)-induced maturation of infected DCs was strongly inhibited, as indicated by lower levels of surface expression of MHC class I, class II, costimulatory, and CD83 molecules. The down-regulation or inhibition of these surface markers occurred only in HCMV antigen-positive DCs. DCs produced no interleukin 12 (IL-12) and only low levels of tumor necrosis factor alpha (TNF-alpha) upon HCMV infection. Furthermore, cytokine production upon stimulation with LPS or CD40L was significantly impaired. Inhibition of cytokine production did not depend on viral gene expression as UV-irradiated HCMV resulted in the same effect. Proliferation and cytotoxicity of T cells specific to a recall antigen presented by DCs were also reduced when DCs were HCMV infected. This study shows that HCMV inhibits DC function, revealing a powerful viral strategy to delay or prevent the generation of virus-specific cytotoxic T cells.
Assuntos
Citomegalovirus/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Antígenos CD/metabolismo , Diferenciação Celular , Citocinas/metabolismo , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Células Dendríticas/citologia , Regulação para Baixo , Antígenos HLA/metabolismo , Humanos , Terapia de Imunossupressão , Interleucina-12/metabolismo , Ativação Linfocitária/imunologia , Monócitos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Human cytomegalovirus (HCMV) exploits a range of strategies to evade and modulate the immune response. Its capacity to down-regulate MHC I expression was anticipated to render infected cells vulnerable to natural killer (NK) attack. Kinetic analysis revealed that during productive infection, HCMV strain AD169 first enhanced and then inhibited lysis of primary skin fibroblasts by a CD94/NKG2A(+)NKG2D(+)ILT2(+) NK line. The inhibition of cytotoxicity against strain AD169-infected fibroblasts was abolished by prior treatment of targets or effectors with anti-MHC I and anti-CD94 monoclonal antibodies, respectively, implying a CD94/HLA-E-dependent mechanism. An HCMV strain AD169, UL40 deletion mutant could not inhibit CD94/NKG2A(+) NK killing against skin fibroblasts. The contribution of UL40 to evasion of primary NK cells then was tested in a system where targets and effectors were MHC-matched. Primary NK cells activated with IFNalpha as well as cultured primary NK cell lines showed increased killing against DeltaUL40-infected fibroblasts compared with AD169-infected targets. This effect was abrogated by depletion of CD94(+) cells. These findings demonstrate that HCMV encodes a mechanism of evasion specifically targeted against a proportion of CD94(+) NK cells and show that this system functions during a productive infection.