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1.
Immunity ; 49(4): 678-694.e5, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30314757

RESUMO

CD8+ T cell exhaustion impedes control of chronic viral infection; yet how new T cell responses are mounted during chronic infection is unclear. Unlike T cells primed at the onset of infection that rapidly differentiate into effectors and exhaust, we demonstrate that virus-specific CD8+ T cells primed after establishment of chronic LCMV infection preferentially generate memory-like transcription factor TCF1+ cells that were transcriptionally and proteomically distinct, less exhausted, and more responsive to immunotherapy. Mechanistically, adaptations of antigen-presenting cells and diminished T cell signaling intensity promoted differentiation of the memory-like subset at the expense of rapid effector cell differentiation, which was now highly dependent on IL-21-mediated CD4+ T cell help for its functional generation. Chronic viral infection similarly redirected de novo differentiation of tumor-specific CD8+ T cells, ultimately preventing cancer control. Thus, targeting these T cell stimulatory pathways could enable strategies to control chronic infection, tumors, and enhance immunotherapeutic efficacy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Imunidade/imunologia , Memória Imunológica/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Diferenciação Celular/genética , Doença Crônica , Perfilação da Expressão Gênica/métodos , Imunidade/genética , Memória Imunológica/genética , Imunoterapia , Coriomeningite Linfocítica/terapia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Proteômica/métodos , Fator 1 de Transcrição de Linfócitos T/genética , Fator 1 de Transcrição de Linfócitos T/imunologia , Fator 1 de Transcrição de Linfócitos T/metabolismo
2.
Microb Pathog ; 110: 325-334, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28710013

RESUMO

Rotavirus (RV) infection causes acute, watery dehydrating diarrhea and even death in infants and other young animals, resulting in a severe economic burden; however, little is known about the innate immune mechanisms associated with RV infection. Dendritic cells (DCs), which are professional antigen-presenting cells (APCs), serve as a bridge connecting the innate and adaptive immune system. In this study, the interaction between murine bone marrow-derived DCs (BMDCs) and porcine rotavirus (PRV) was investigated in vitro. Upon stimulation, the expression levels of MHC-II, CD40, CD80, CD86 and CD83 in BMDCs increased in a time-dependent manner, indicating activation and maturation by PRV. In addition, up-regulated Toll-like receptor 2 (TLR2), TLR3 and NF-κB increased the production of interleukin-12 and interferon-γ. The PRV-stimulated BMDCs also showed increased stimulatory capacity in mixed lymphocyte reactions and promoted the Th1 subtype response.


Assuntos
Medula Óssea/imunologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por Rotavirus/imunologia , Rotavirus/imunologia , Células Th1/imunologia , Imunidade Adaptativa , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Antígenos CD/metabolismo , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Antígenos CD40/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Genes MHC da Classe II , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Imunidade nas Mucosas , Imunoglobulinas/metabolismo , Interferon gama/metabolismo , Interleucina-12/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Rotavirus/patogenicidade , Suínos , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Regulação para Cima , Antígeno CD83
3.
J Virol ; 89(3): 1867-78, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25428872

RESUMO

UNLABELLED: Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease in susceptible SJL/J mice but not in resistant C57BL/6 mice. Previous studies have indicated that the major histocompatibility complex (MHC) genes play the most prominent role in the development of TMEV-induced demyelinating disease. In this study, we used C57BL/6.S (B6.S) congenic mice, which carry H-2(s) MHC genes instead of H-2(b) MHC genes in conjunction with the C57BL/6 (B6) background genes. Our data show that virus-infected B6.S mice are free from disease and have significantly lower viral loads than susceptible SJL mice, particularly in the spinal cord. A strong protective Th1-type T helper response with virtually no pathogenic Th17 response was detected in B6.S mice, in contrast to the reduced Th1- and robust Th17-type responses in SJL mice. Notably, lower levels of viral infectivity in B6.S antigen-presenting cells (APCs) correlated with the disease resistance and T-cell-type response. In vitro studies using APCs from B6.S and SJL mice show that TLR2, -3, -4, and -7, but not TLR9, signaling can replace viral infection and augment the effect of viral infection in the differentiation of the pathogenic Th17 cell type. Taken together, these results strongly suggest that the viral replication levels in APCs critically affect the induction of protective versus pathogenic Th cell types via the signaling of pattern recognition receptors for innate immune responses. Our current findings further imply that the levels of viral infectivity/replication and TLR-mediated signaling play critical roles in the pathogenesis of chronic viral diseases. IMPORTANCE: This study indicates that innate immune cytokines produced in antigen-presenting cells stimulating the T cell immune responses during early viral infection play a critical role in determining the susceptibility of mice to the development of demyelinating disease. The level of innate immune cytokines reflects the level of initial viral infection in the antigen-presenting cells, and the level determines the development of T cell types, which are either protective or pathogenic. The level of initial viral infection to the cells is controlled by a gene or genes that are not associated with the major histocompatibility antigen complex genes. This finding has an important implication in controlling not only chronic viral infections but also infection-induced autoimmune-like diseases, which are closely associated with the pathogenic type of T cell responses.


Assuntos
Células Apresentadoras de Antígenos/virologia , Infecções por Cardiovirus/patologia , Infecções por Cardiovirus/virologia , Doenças Desmielinizantes/patologia , Doenças Desmielinizantes/virologia , Theilovirus/isolamento & purificação , Carga Viral , Animais , Células Apresentadoras de Antígenos/imunologia , Infecções por Cardiovirus/imunologia , Doenças Desmielinizantes/imunologia , Feminino , Genes MHC Classe I , Camundongos Endogâmicos C57BL , Células Th1/imunologia , Células Th17/imunologia , Theilovirus/imunologia , Receptores Toll-Like/imunologia
4.
J Immunol ; 193(3): 1223-32, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24951814

RESUMO

Adenoviral vectors have long been forerunners in the development of effective CD8 T cell-based vaccines; therefore, it is imperative that we understand the factors controlling the induction of robust and long-lasting transgene-specific immune responses by these vectors. In this study, we investigated the organ sites, molecules, and cell subsets that play a critical role in the priming of transgene-specific CD8 T cells after vaccination with a replication-deficient adenoviral vector. Using a human adenovirus serotype 5 (Ad5) vector and genetically engineered mice, we found that CD8(+) and/or CD103(+) dendritic cells in the draining lymph node played a critical role in the priming of Ad5-induced CD8 T cell responses. Moreover, we found that CD80/86, but not CD28, was essential for efficient generation of both primary effectors and memory CD8 T cells. Interestingly, the lack of CD28 expression resulted in a delayed primary response, whereas memory CD8 T cells generated in CD28-deficient mice appeared almost normal in terms of both phenotype and effector cytokine profile, but they exhibited a significantly reduced proliferative capacity upon secondary challenge while retaining immediate in vivo effector capabilities: in vivo cytotoxicity and short-term in vivo protective capacity. Overall, our data point to an absolute requirement for professional APCs and the expression of the costimulatory molecules CD80/86 for efficient CD8 T cell priming by adenoviral vectors. Additionally, our results suggest the existence of an alternative receptor for CD80/86, which may substitute, in part, for CD28.


Assuntos
Infecções por Adenovirus Humanos/prevenção & controle , Adenovírus Humanos/imunologia , Antígeno B7-1/deficiência , Antígeno B7-2/deficiência , Antígenos CD28/deficiência , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Engenharia Genética , Infecções por Adenovirus Humanos/genética , Adenovírus Humanos/genética , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/virologia , Antígeno B7-1/genética , Antígeno B7-2/genética , Antígenos CD28/genética , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Ligantes , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vacinação
5.
Immunology ; 146(2): 312-26, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26173587

RESUMO

Self-amplifying mRNAs (SAM(®) ) are a novel class of nucleic acid vaccines, delivered by a non-viral delivery system. They are effective at eliciting potent and protective immune responses and are being developed as a platform technology with potential to be used for a broad range of targets. However, their mechanism of action has not been fully elucidated. To date, no evidence of in vivo transduction of professional antigen-presenting cells (APCs) by SAM vector has been reported, while the antigen expression has been shown to occur mostly in the muscle fibres. Here we show that bone-marrow-derived APCs rather than muscle cells are responsible for induction of MHC class-I restricted CD8 T cells in vivo, but direct transfection of APCs by SAM vectors is not required. Based on all our in vivo and in vitro data we propose that upon SAM vaccination the antigen is expressed within muscle cells and then transferred to APCs, suggesting cross-priming as the prevalent mechanism for priming the CD8 T-cell response by SAM vaccines.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Células da Medula Óssea/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Fibras Musculares Esqueléticas/imunologia , RNA Mensageiro/imunologia , RNA Viral/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas do Core Viral/imunologia , Animais , Células Apresentadoras de Antígenos/virologia , Células da Medula Óssea/virologia , Transplante de Medula Óssea , Linfócitos T CD8-Positivos/virologia , Comunicação Celular , Linhagem Celular , Cricetinae , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/genética , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Musculares Esqueléticas/virologia , Proteínas do Nucleocapsídeo , RNA Mensageiro/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Transfecção , Quimeras de Transplante , Proteínas do Core Viral/genética
6.
J Virol ; 88(5): 2508-18, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24352453

RESUMO

UNLABELLED: Antigen persistence in chronic infections and cancer upregulates inhibitory networks, such as the PD-1 and interleukin-10 (IL-10) pathways, that impair immunity and lead to disease progression. These pathways are attractive targets for immunotherapy, as demonstrated by recent clinical trials of PD-1/PD-L1 blockade in cancer patients. However, in HIV-1 infection not all subjects respond to inhibition of either pathway and the mechanistic interactions between these two networks remain to be better defined. Here we demonstrate that in vitro blockade of PD-L1 and/or IL-10Rα results in markedly different profiles of HIV-1-specific CD4 T cell restoration. Whereas PD-L1 blockade leads to balanced increase in gamma interferon (IFN-γ), IL-2, and IL-13 secretion, IL-10Rα blockade preferentially restores IFN-γ production. In viremic subjects, combined PD-L1/IL-10Rα blockade results in a striking 10-fold increase in IFN-γ secretion by HIV-1-specific CD4 T cells that is not observed in subjects with spontaneous (elite controllers) or therapy-induced control of viral replication. In contrast to the dramatic increase in IFN-γ production, concurrent blockade has a marginal additive effect on IL-2 production, IL-13 secretion, and HIV-1-specific CD4 T cell proliferation. IFN-γ produced by Thelper cells upregulates PD-L1, HLA I/II, and IL-12 expression by monocytes. The effect of combined blockade on IFN-γ was dependent on reciprocal reinforcement through IL-12. These studies provide crucial information on the different immunoregulatory qualities of PD-1 and IL-10 in progressive disease and link exhausted virus-specific CD4 T cells and monocytes in the regulation of IFN-γ and IL-12 secretion. IMPORTANCE: Infection with HIV results in most people in uncontrolled viral replication and progressive weakening of the body defenses. In the absence of antiviral therapy, this process results in clinical disease, or AIDS. An important reason why HIV continues to multiply is that a population of white blood cells called CD4 T cells that targets the virus fails to work properly. At least part of this impairment is under the control of inhibitory mechanisms that can be blocked to improve the function of these CD4 T cells. In this report, we show that blocking one or two of the molecules involved, called PD-1 and IL-10, has different effects on the individual functions of these cells and that one is strongly improved. We investigate how these effects are caused by interactions between CD4 T cells and antigen-presenting cells. These observations can have implications for new therapeutic approaches in HIV infection.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , HIV-1/imunologia , Interleucina-10/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Anticorpos Monoclonais/farmacologia , Células Apresentadoras de Antígenos/virologia , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Citocinas/biossíntese , Epitopos de Linfócito T/imunologia , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Humanos , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-10/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
J Immunol ; 190(6): 2767-77, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23390296

RESUMO

CMV can infect dendritic cells (DCs), and direct Ag presentation could, therefore, lead to the priming of CMV-specific CD8(+) T cells. However, CMV-encoded immune evasins severely impair Ag presentation in the MHC class I pathway; thus, it is widely assumed that cross-presentation drives the priming of antiviral T cells. We assessed the contribution of direct versus cross priming in mouse CMV (MCMV) infection using recombinant viruses. DCs infected with an MCMV strain encoding the gB498 epitope from HSV-1 were unable to stimulate in vitro naive gB498-specific CD8(+) T cells from TCR transgenic mice. Infection of C57BL/6 mice with this recombinant virus led, however, to the generation of abundant numbers of gB498-specific T cells in vivo. Of the DC subsets isolated from infected mice, only CD8α(+) DCs were able to stimulate naive T cells, suggesting that this DC subset cross-presents MCMV-encoded Ag in vivo. Upon infection of mice with MCMV mutants encoding Ag that can either be well or hardly cross-presented, mainly CD8(+) T cells specific for cross-presented epitopes were generated. Moreover, even in the absence of immune evasion genes interfering with MHC class I-mediated Ag presentation, priming of T cells to Ag that can only be presented directly was not observed. We conclude that the host uses mainly DCs capable of cross-presentation to induce the CMV-specific CD8(+) T cell response during primary, acute infection and discuss the implications for the development of a CMV vaccine.


Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Apresentação Cruzada/imunologia , Infecções por Herpesviridae/imunologia , Ativação Linfocitária/imunologia , Muromegalovirus/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Linfócitos T CD8-Positivos/patologia , Células Clonais , Epitopos de Linfócito T/imunologia , Feminino , Infecções por Herpesviridae/patologia , Infecções por Herpesviridae/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muromegalovirus/genética
8.
Nature ; 461(7265): 788-92, 2009 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-19776740

RESUMO

The innate immune system is critical for the early detection of invading pathogens and for initiating cellular host defence countermeasures, which include the production of type I interferon (IFN). However, little is known about how the innate immune system is galvanized to respond to DNA-based microbes. Here we show that STING (stimulator of interferon genes) is critical for the induction of IFN by non-CpG intracellular DNA species produced by various DNA pathogens after infection. Murine embryonic fibroblasts, as well as antigen presenting cells such as macrophages and dendritic cells (exposed to intracellular B-form DNA, the DNA virus herpes simplex virus 1 (HSV-1) or bacteria Listeria monocytogenes), were found to require STING to initiate effective IFN production. Accordingly, Sting-knockout mice were susceptible to lethal infection after exposure to HSV-1. The importance of STING in facilitating DNA-mediated innate immune responses was further evident because cytotoxic T-cell responses induced by plasmid DNA vaccination were reduced in Sting-deficient animals. In the presence of intracellular DNA, STING relocalized with TANK-binding kinase 1 (TBK1) from the endoplasmic reticulum to perinuclear vesicles containing the exocyst component Sec5 (also known as EXOC2). Collectively, our studies indicate that STING is essential for host defence against DNA pathogens such as HSV-1 and facilitates the adjuvant activity of DNA-based vaccines.


Assuntos
DNA Bacteriano/imunologia , DNA Viral/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Espaço Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/microbiologia , Células Apresentadoras de Antígenos/virologia , Vesículas Citoplasmáticas/metabolismo , DNA Bacteriano/metabolismo , DNA Viral/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Fibroblastos/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Interferon Tipo I/biossíntese , Espaço Intracelular/imunologia , Listeria monocytogenes/genética , Listeria monocytogenes/imunologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Plasmídeos/genética , Plasmídeos/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/imunologia , Proteínas de Transporte Vesicular/metabolismo
9.
Proc Natl Acad Sci U S A ; 109(30): 12117-22, 2012 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-22778433

RESUMO

A coordinated innate and adaptive immune response, orchestrated by antigen presenting cells (APCs), is required for effective clearance of influenza A virus (IAV). Although IAV primarily infects epithelial cells of the upper respiratory tract, APCs are also susceptible. To determine if virus transcription in these cells is required to generate protective innate and adaptive immune responses, we engineered IAV to be selectively attenuated in cells of hematopoietic origin. Incorporation of hematopoietic-specific miR-142 target sites into the nucleoprotein of IAV effectively silenced virus transcription in APCs, but had no significant impact in lung epithelial cells. Here we demonstrate that inhibiting IAV replication in APCs in vivo did not alter clearance, or the generation of IAV-specific CD8 T cells, suggesting that cross-presentation is sufficient for cytotoxic T lymphocyte activation. In contrast, loss of in vivo virus infection, selectively in APCs, resulted in a significant reduction of retinoic acid-inducible gene I-dependent type I IFN (IFN-I). These data implicate the formation of virus replication intermediates in APCs as the predominant trigger of IFN-I in vivo. Taking these data together, this research describes a unique platform to study the host response to IAV and provides insights into the mechanism of antigen presentation and the induction of IFN-I.


Assuntos
Imunidade Adaptativa/imunologia , Células Apresentadoras de Antígenos/virologia , Regulação Viral da Expressão Gênica/imunologia , Imunidade Inata/imunologia , Vírus da Influenza A/imunologia , Replicação Viral/imunologia , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular , Clonagem Molecular , Primers do DNA/genética , Cães , Fibroblastos , Citometria de Fluxo , Regulação Viral da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interferon Tipo I/metabolismo , Macrófagos , Camundongos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Nucleoproteínas/genética , Nucleoproteínas/metabolismo
10.
Immunology ; 141(4): 531-9, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24205828

RESUMO

Vaccinia virus (VV) has been used globally as a vaccine to eradicate smallpox. Widespread use of this viral vaccine has been tempered in recent years because of its immuno-evasive properties, with restrictions prohibiting VV inoculation of individuals with immune deficiencies or atopic skin diseases. VV infection is known to perturb several pathways for immune recognition including MHC class II (MHCII) and CD1d-restricted antigen presentation. MHCII and CD1d molecules associate with a conserved intracellular chaperone, CD74, also known as invariant chain. Upon VV infection, cellular CD74 levels are significantly reduced in antigen-presenting cells, consistent with the observed destabilization of MHCII molecules. In the current study, the ability of sustained CD74 expression to overcome VV-induced suppression of antigen presentation was investigated. Viral inhibition of MHCII antigen presentation could be partially ameliorated by ectopic expression of CD74 or by infection of cells with a recombinant VV encoding murine CD74 (mCD74-VV). In contrast, virus-induced disruptions in CD1d-mediated antigen presentation persisted even with sustained CD74 expression. Mice immunized with the recombinant mCD74-VV displayed greater protection during VV challenge and more robust anti-VV antibody responses. Together, these observations suggest that recombinant VV vaccines encoding CD74 may be useful tools to improve CD4⁺ T-cell responses to viral and tumour antigens.


Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vacina Antivariólica/imunologia , Vaccinia virus/imunologia , Vacínia/prevenção & controle , Proteínas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/virologia , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Vacina Antivariólica/administração & dosagem , Vacina Antivariólica/genética , Vacina Antivariólica/metabolismo , Fatores de Tempo , Transfecção , Vacinação , Vacinas Sintéticas/imunologia , Vacínia/imunologia , Vacínia/metabolismo , Vacínia/virologia , Vaccinia virus/genética , Vaccinia virus/metabolismo , Proteínas Virais/genética
11.
Biochem Biophys Res Commun ; 450(1): 61-6, 2014 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-24866241

RESUMO

Chronic hepatitis B virus (HBV) infection is the result of an inadequate antiviral immune response to the virus. In this study, we aimed to investigate whether the soluble CD40 ligand-activated B (CD40-B) cells could present antigen and induce specific cytotoxic T lymphocytes (CTLs) in patients with chronic HBV infection. We observed that after activated by sCD40L, the expression of CD80, CD86, major histocompatibility complex (MHC) I and II molecules on the CD40-B cells was significantly increased. Cytometry and fluorescence microscopy showed that more than 41.34% CD40-B cells were loaded by the HBcAg peptide. Furthermore, after been activated and HBcAg18-27 antigen peptide pulsed, B cells obtained from patients with chronic HBV infection could induce HBcAg18-27 specific CTLs in vitro. Taken together, our results show that B cells from patients with chronic HBV infection can be activated by sCD40L and may function as antigen presenting cells and induce HBV-specific CTLs.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Antígenos CD40/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Hepatite B Crônica/virologia , Linfócitos T Citotóxicos/imunologia , Células Apresentadoras de Antígenos/virologia , Linfócitos B/virologia , Células Cultivadas , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Humanos , Solubilidade , Linfócitos T Citotóxicos/virologia
12.
PLoS Pathog ; 8(8): e1002885, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22952446

RESUMO

Measles remains a significant childhood disease, and is associated with a transient immune suppression. Paradoxically, measles virus (MV) infection also induces robust MV-specific immune responses. Current hypotheses for the mechanism underlying measles immune suppression focus on functional impairment of lymphocytes or antigen-presenting cells, caused by infection with or exposure to MV. We have generated stable recombinant MVs that express enhanced green fluorescent protein, and remain virulent in non-human primates. By performing a comprehensive study of virological, immunological, hematological and histopathological observations made in animals euthanized at different time points after MV infection, we developed a model explaining measles immune suppression which fits with the "measles paradox". Here we show that MV preferentially infects CD45RA(-) memory T-lymphocytes and follicular B-lymphocytes, resulting in high infection levels in these populations. After the peak of viremia MV-infected lymphocytes were cleared within days, followed by immune activation and lymph node enlargement. During this period tuberculin-specific T-lymphocyte responses disappeared, whilst strong MV-specific T-lymphocyte responses emerged. Histopathological analysis of lymphoid tissues showed lymphocyte depletion in the B- and T-cell areas in the absence of apoptotic cells, paralleled by infiltration of T-lymphocytes into B-cell follicles and reappearance of proliferating cells. Our findings indicate an immune-mediated clearance of MV-infected CD45RA(-) memory T-lymphocytes and follicular B-lymphocytes, which causes temporary immunological amnesia. The rapid oligoclonal expansion of MV-specific lymphocytes and bystander cells masks this depletion, explaining the short duration of measles lymphopenia yet long duration of immune suppression.


Assuntos
Terapia de Imunossupressão , Vírus do Sarampo/imunologia , Sarampo/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Linfócitos B/imunologia , Linfócitos B/virologia , Modelos Animais de Doenças , Feminino , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Humanos , Memória Imunológica , Antígenos Comuns de Leucócito/imunologia , Leucopenia/imunologia , Leucopenia/virologia , Depleção Linfocítica , Tecido Linfoide/imunologia , Tecido Linfoide/virologia , Macaca , Masculino , Sarampo/virologia , Vírus do Sarampo/fisiologia , Linfócitos T/virologia , Viremia/imunologia , Viremia/virologia
13.
BMC Microbiol ; 14: 126, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24886142

RESUMO

BACKGROUND: Previous findings suggested that Lactobacillus rhamnosus CRL1505 is able to increase resistance of children to intestinal viral infections. However, the intestinal cells, cytokines and receptors involved in the immunoregulatory effect of this probiotic strain have not been fully characterized. RESULTS: We aimed to gain insight into the mechanisms involved in the immunomodulatory effect of the CRL1505 strain and therefore evaluated in vitro the crosstalk between L. rhamnosus CRL1505, porcine intestinal epithelial cells (IECs) and antigen presenting cells (APCs) from swine Peyer's patches in order to deepen our knowledge about the mechanisms, through which this strain may help preventing viral diarrhoea episodes. L. rhamnosus CRL1505 was able to induce IFN-α and -ß in IECs and improve the production of type I IFNs in response to poly(I:C) challenge independently of Toll-like receptor (TLR)-2 or TLR9 signalling. In addition, the CRL1505 strain induced mRNA expression of IL-6 and TNF-α via TLR2 in IECs. Furthermore, the strain significantly increased surface molecules expression and cytokine production in intestinal APCs. The improved Th1 response induced by L. rhamnosus CRL1505 was triggered by TLR2 signalling and included augmented expression of MHC-II and co-stimulatory molecules and expression of IL-1ß, IL-6, and IFN-γ in APCs. IL-10 was also significantly up-regulated by CRL1505 in APCs. CONCLUSIONS: It was recently reviewed the emergence of TLR agonists as new ways to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than IFN therapies. The use of L. rhamnosus CRL1505 as modulator of innate immunity and inductor of antiviral type I IFNs, IFN-γ, and regulatory IL-10 clearly offers the potential to overcome this challenge.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Células Epiteliais/imunologia , Células Epiteliais/virologia , Fatores Imunológicos/farmacologia , Lacticaseibacillus rhamnosus/imunologia , Probióticos/farmacologia , Animais , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Suínos
14.
J Immunol ; 189(2): 638-45, 2012 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-22706075

RESUMO

By the peak of the CD8(+) T cell response, the effector cell pool consists of a heterogeneous population of cells that includes both those with an increased propensity to become long-lived memory cells (memory precursor effector cells; MPEC) and those that are terminally differentiated cells (short-lived effector cells; SLEC). Numerous studies have established the critical role that functional avidity plays in determining the in vivo efficacy of CD8(+) effector cells. Currently, how functional avidity differs in MPEC versus SLEC and the evolution of this property within these two populations during the expansion and contraction of the response are unknown. The data presented in this study show that at the peak of the effector response generated after poxvirus infection, SLEC were of higher functional avidity than their MPEC counterpart. Over time, however, SLEC exhibited a decrease in peptide sensitivity. This is in contrast to MPEC, which showed a modest increase in peptide sensitivity as the response reached equilibrium. The decrease in functional avidity in SLEC was independent of CD8 modulation or the amount of Ag receptor expressed by the T cell. Instead, the loss in sensitivity was correlated with decreased expression and activation of ZAP70 and Lck, critical components of TCR membrane proximal signaling. These results highlight the potential contribution of avidity in the differentiation and evolution of the T cell effector response after viral infection.


Assuntos
Antígenos CD8/biossíntese , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Diferenciação Celular/imunologia , Senescência Celular/imunologia , Memória Imunológica , Vírus da Coriomeningite Linfocítica/imunologia , Vaccinia virus/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/virologia , Antígenos CD8/fisiologia , Linfócitos T CD8-Positivos/metabolismo , Adesão Celular/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Fatores de Tempo
15.
Proc Natl Acad Sci U S A ; 108(9): 3743-8, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21321191

RESUMO

Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, Bay11, which blocks both the canonical and the alternative NF-κB pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-κB pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-κB pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-κB pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-κB pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-κB inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.


Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Imunidade/genética , NF-kappa B/metabolismo , Transdução de Sinais , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/virologia , Sítios de Ligação , Citocinas/metabolismo , Dependovirus/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Imunidade/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Camundongos , NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Infecções por Parvoviridae/genética , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/virologia , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Sequências Repetidas Terminais/genética , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transgenes
16.
Bull Exp Biol Med ; 157(1): 56-61, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24915947

RESUMO

We analyzed local reactions of immune homeostasis in the human skin, in particular, effector immune cells CD68 responsible for antigen presentation, during human papillomavirus infection. Under conditions of long-term papillomavirus infection, CD68 markers were identifi ed only in the connective tissue of the skin (derma) and were completely absent in the epidermis, where they were found during physiological and reparative regeneration after thermal injury. We concluded that hypertrophy of the epidermis and connective tissue of the dermal papillary layer in human papillomavirus infection is related to the absence of CD68 immune cells in the epithelial plate and their accumulation in the connective tissue adjacent to the basement membrane of the epidermis. The possibility of epithelium contamination with the virus depends on local immune homeostasis. Therefore, induction of proper CD68 distribution in appropriate structures can contribute to normalization of epithelial-connective tissue interactions.


Assuntos
Células Apresentadoras de Antígenos/patologia , Membrana Basal/patologia , Tecido Conjuntivo/patologia , Derme/patologia , Epiderme/patologia , Infecções por Papillomavirus/patologia , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Membrana Basal/imunologia , Membrana Basal/virologia , Biomarcadores/metabolismo , Diferenciação Celular , Tecido Conjuntivo/imunologia , Tecido Conjuntivo/virologia , Derme/imunologia , Derme/virologia , Epiderme/imunologia , Epiderme/virologia , Humanos , Imunofenotipagem , Papillomaviridae/fisiologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Técnicas de Cultura de Tecidos
17.
Biochem Biophys Res Commun ; 437(3): 446-51, 2013 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-23831627

RESUMO

Allergen exposure and rhinovirus infections that propagate from the upper to the lower airways are the most frequent causes of asthma exacerbation. In patients at increased risk of disease exacerbations, chronic airway inflammation is associated with the airway recruitment of circulating fibrocytes, bone marrow-derived CD34(+)CD45RO(+)CD11b(+)CD13(+)HLA-DR(+) progenitors that have antigen-presenting function and fibroblast-like properties. This study demonstrates that allergen-pulsed circulating fibrocytes from patients with allergic asthma are potent inducer of the predominant release of the T helper type (Th)2 cytokines IL-4 and IL-5 from autologous naïve and memory CD4(+) T cells. This study also provides evidence that circulating fibrocytes from allergic asthmatics are susceptible to rhinovirus infection. Infected cells release high amounts of pro-inflammatory cytokines with minimal production of IFN-α/ß. Moreover, allergen-pulsed fibrocytes support prolonged rhinovirus replication and release larger quantities of pro-inflammatory cytokines upon rhinovirus infection than unpulsed fibrocytes. Thus, fibrocytes may amplify allergen-induced, Th2 cell-driven inflammatory responses and promote further inflammation by functioning as a reservoir for rhinovirus replication in asthmatic airways. Through these mechanisms, fibrocytes may play an important role in the provocation of disease exacerbations.


Assuntos
Asma/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Infecções por Picornaviridae/imunologia , Rhinovirus/imunologia , Células Th2/imunologia , Adulto , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/patologia , Células Apresentadoras de Antígenos/virologia , Antígenos de Dermatophagoides/fisiologia , Asma/patologia , Asma/virologia , Células da Medula Óssea/virologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Doença Crônica , Citocinas/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Células HeLa , Humanos , Memória Imunológica , Inflamação/imunologia , Inflamação/patologia , Inflamação/virologia , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Macrófagos/patologia , Macrófagos/virologia , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/virologia , Células-Tronco/imunologia , Células-Tronco/patologia , Células-Tronco/virologia , Células Th2/patologia , Células Th2/virologia
18.
J Virol ; 86(1): 527-41, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22031944

RESUMO

The transporter associated with antigen processing (TAP) delivers the viral proteolytic products generated by the proteasome in the cytosol to the endoplasmic reticulum lumen that are subsequently recognized by cytotoxic T lymphocytes (CTLs). However, several viral epitopes have been identified in TAP-deficient models. Using mass spectrometry to analyze complex human leukocyte antigen (HLA)-bound peptide pools isolated from large numbers of TAP-deficient vaccinia virus-infected cells, we identified 11 ligands naturally presented by four different HLA-A, HLA-B, and HLA-C class I molecules. Two of these ligands were presented by two different HLA class I alleles, and, as a result, 13 different HLA-peptide complexes were formed simultaneously in the same vaccinia virus-infected cells. In addition to the high-affinity ligands, one low-affinity peptide restricted by each of the HLA-A, HLA-B, and HLA-C class I molecules was identified. Both high- and low-affinity ligands generated long-term memory CTL responses to vaccinia virus in an HLA-A2-transgenic mouse model. The processing and presentation of two vaccinia virus-encoded HLA-A2-restricted antigens took place via proteasomal and nonproteasomal pathways, which were blocked in infected cells with chemical inhibitors specific for different subsets of metalloproteinases. These data have implications for the study of the effectiveness of early empirical vaccination with cowpox virus against smallpox disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Vaccinia virus/imunologia , Vacínia/imunologia , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Ligantes , Camundongos , Camundongos Knockout , Vacínia/genética , Vacínia/virologia , Vaccinia virus/genética
19.
J Virol ; 86(11): 6246-57, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22491458

RESUMO

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200.


Assuntos
Antígenos CD/metabolismo , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Tolerância Imunológica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Linfócitos T/imunologia , Proteínas Virais/metabolismo , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Antígenos CD/imunologia , Humanos , Interferon gama/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Receptores de Orexina , Receptores Acoplados a Proteínas G/imunologia , Receptores de Neuropeptídeos/imunologia , Proteínas Virais/imunologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo
20.
Clin Exp Immunol ; 174(1): 172-8, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23773130

RESUMO

There is increasing interest in the role of T cell exhaustion and it is well known that the natural history of chronic hepatitis C virus infection (HCV) is modulated by CD8(+) T cell immunobiology. There are many pathways that alter the presence of exhaustive T cells and, in particular, they are functionally impaired by inhibitory receptors, such as programmed death-1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3). We obtained spleen, liver and peripheral blood (before and after splenectomy) lymphoid cells from 25 patients with HCV-related cirrhosis undergoing liver transplantation for end-stage disease or splenectomy for portal hypertension. In all samples we performed an extensive phenotypic study of exhaustion markers [PD-1, Tim-3, interferon (IFN)-γ) and their ligands (PD-L1, PD-L2, galectin-9] in CD8(+) T cell subpopulations (both total and HCV-specific) and in antigen-presenting cells (APC; monocytes and dendritic cells). In the spleen, total and HCV-specific CD8(+) T cells demonstrated enhanced markers of exhaustion, predominantly in the effector memory subpopulation. Similarly, splenic APC over-expressed inhibitory receptor ligands when compared to peripheral blood. Finally, when peripheral blood CD8(+) T cells were compared before and after splenectomy, markers of exhaustion were reduced in splenic CD8(+) T cells and APC. Our data in HCV-related cirrhosis suggest that CD8(+) T cells in the spleen manifest a significantly higher exhaustion compared to peripheral blood and may thus contribute to the failure to control HCV. Counteracting this process may contribute to inducing an effective immune response to HCV.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/patologia , Idoso , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/patologia , Células Apresentadoras de Antígenos/virologia , Biomarcadores/sangue , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/virologia , Feminino , Receptor Celular 2 do Vírus da Hepatite A , Hepatite C Crônica/sangue , Humanos , Cirrose Hepática/sangue , Cirrose Hepática/imunologia , Cirrose Hepática/patologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/sangue , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/biossíntese , Receptor de Morte Celular Programada 1/sangue , Baço/imunologia , Baço/patologia , Baço/virologia , Esplenectomia , Trombocitopenia/complicações , Trombocitopenia/imunologia , Trombocitopenia/patologia
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