RESUMO
More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II-dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta-restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis.
Assuntos
Antígenos Virais/análise , Herpesvirus Humano 4/imunologia , Mononucleose Infecciosa/imunologia , Superantígenos/análise , Adulto , Animais , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Linfócitos B/imunologia , Linfócitos B/virologia , Transformação Celular Viral , Antígenos HLA-DR/análise , Humanos , Hibridomas/química , Lectinas Tipo C , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Linfócitos T/imunologia , Linfócitos T/virologiaRESUMO
The expression of exogenous genes in long-lived primary T cells is potentially beneficial for the treatment of various diseases including cancer, AIDS, genetic defects of the lymphoid compartment, and systemic enzyme deficiencies such as hemophilia. One approach for genetic modification of T cells is to introduce therapeutic genes into hematopoietic stem cells that would give rise to cells of the lymphoid lineage. Efficient gene transfer and expression in stem cells is often problematic, however. A more direct approach is to introduce the genes into mature primary T lymphocytes since the transferred genes can be maintained and expressed for long periods by long-lived peripheral T cells. In this report, we describe the adoptive transfer into SCID mice of both murine and human primary T cells that have been efficiently transduced with exogenous genes. Primary murine T cells transduced with a retroviral vector containing the human adenosine deaminase (ADA) gene persisted for at least 5 months in lymphoid organs of SCID mice, continuously expressing the exogenous gene. Primary human T cells were also used as target cells for transfer of the beta-galactosidase (lacZ) gene. Expression of the lacZ gene could be detected in over 20% of the transduced primary T cells before adoptive transfer into SCID mice. Transduced human T cells were injected into SCID mice intraperitoneally (ip), and the beta-galactosidase activity could be detected in cells collected from peritoneal exudate washes of recipient mice 6 weeks post-injection. These results demonstrate the availability of a murine model in which the long-term effects of expression of exogenous genes in both murine and human T cells can be tested.
Assuntos
Camundongos SCID/genética , Linfócitos T/citologia , Adenosina Desaminase/deficiência , Adenosina Desaminase/genética , Transferência Adotiva , Animais , Sequência de Bases , Compartimento Celular/genética , Exsudatos e Transudatos/citologia , Exsudatos e Transudatos/enzimologia , Técnicas de Transferência de Genes , Humanos , Óperon Lac/genética , Camundongos , Camundongos Endogâmicos BALB C , Peritônio/citologia , Provírus/química , Linfócitos T/metabolismo , Fatores de Tempo , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Superantigens are microbial agents that have a strong effect on the immune response of the host. Their initial target is the T lymphocyte, but a whole cascade of immunological reactions ensues. It is thought that the microbe engages the immune system of the host to its own advantage, to facilitate persistent infection and/or transmission. In this review, we discuss in detail the structure and function of the superantigen encoded by the murine mammary tumor virus, a B-type retrovirus which is the causative agent of mammary carcinoma. We will also outline what has more recently become known about superantigen activity associated with two human herpesviruses, cytomegalovirus and Epstein-Barr virus. It is likely that we have only uncovered the tip of the iceberg in our discovery of microbial superantigens, and we predict a flood of new information on this topic shortly.
Assuntos
Antígenos Virais/imunologia , Superantígenos/imunologia , Animais , Antígenos Virais/genética , Citomegalovirus/genética , Citomegalovirus/imunologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Vírus do Tumor Mamário do Camundongo/genética , Vírus do Tumor Mamário do Camundongo/imunologia , Camundongos , Vírus da Raiva/genética , Vírus da Raiva/imunologia , Superantígenos/genéticaRESUMO
The efficient introduction of exogenous genes into primary lymphocytes is potentially important both for somatic cell gene therapy and for studying lymphocyte biology. We describe the use of retroviral vectors to efficiently introduce exogenous genes into primary, mature murine lymph node T and B cells, and primary, immature murine CD4- CD8- double-negative (DN) thymocytes. Efficient infection of primary cells was achieved by cocultivation of target cells with lethally irradiated helper cells that produce high titers of retroviral vectors containing either the neomycin phosphotransferase II (neo) gene, or both the neo and the human adenosine deaminase (ADA) genes, in the presence of lymphokines and/or mitogens. Two days postinfection, without neomycin selection, one to five copies of the exogenous genes per cell were detected by Southern blot analysis. Expression of the exogenous human ADA protein was detected at levels comparable to the endogenous murine ADA protein in the mature T and B lymphocytes, and was somewhat lower for the immature DN thymocytes.
Assuntos
Subpopulações de Linfócitos/fisiologia , Transfecção/métodos , Adenosina Desaminase/genética , Animais , Feminino , Expressão Gênica , Genes , Vetores Genéticos , Canamicina Quinase , Linfonodos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosfotransferases/genética , RNA Mensageiro/genética , Retroviridae/genética , Timo/citologiaRESUMO
Superantigens (SAgs) are proteins produced by pathogenic microbes to elicit potent, antigen-independent T cell responses that are believed to enhance the microbes' pathogenicity. Here we show that the human lymphotropic herpesvirus Epstein-Barr virus (EBV) transcriptionally activates the env gene of an endogenous retrovirus, HERV-K18, that possesses SAg activity. SAg activity was demonstrated by MHC class II dependent preferential activation of TCRVB13 T cells in response to murine B cells transfected with the HERV-K18 env gene. This is a unique demonstration of a pathogen inducing a host-encoded Sag and accounts for the previously described EBV associated Sag activity. The T cell activation elicited by the Sag could play a central role in EBV infection and associated diseases.
Assuntos
Retrovirus Endógenos/genética , Retrovirus Endógenos/imunologia , Infecções por Vírus Epstein-Barr/genética , Produtos do Gene env/imunologia , Herpesvirus Humano 4/patogenicidade , Superantígenos/genética , Alelos , Animais , Anticorpos Antivirais/imunologia , Linfoma de Burkitt/genética , Linfoma de Burkitt/imunologia , Linhagem Celular Transformada , Células Cultivadas , Retrovirus Endógenos/metabolismo , Infecções por Vírus Epstein-Barr/imunologia , Regulação Viral da Expressão Gênica , Produtos do Gene env/biossíntese , Produtos do Gene env/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Ativação Linfocitária , Camundongos , RNA Viral/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Superantígenos/biossíntese , Superantígenos/imunologia , Linfócitos T/imunologia , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Mature primary B lymphocytes represent a potentially important cellular target for somatic cell gene therapy, which could prove advantageous for the treatment of certain metabolic and immunologic disorders. Their capacity to serve as antigen-presenting cells could be utilized for triggering and/or potentiating immune responses to tumors and viruses. Alternatively, B cells expressing an autoantigen could be manipulated to induce antigen-specific unresponsiveness for treatment of autoimmune diseases. Efficient expression of an exogenous gene product in long-lived B lymphocytes could be particularly useful for providing a corrected gene product in the bloodstream. Despite these advantages, efficient gene transfer into mature primary B cells has not been reported. One reason for this is that current protocols for retroviral vector-mediated gene transfer into lymphocytes rely on in vitro expansion and/or drug selection. This precludes the use of mature primary B cells as targets, since they cannot be readily cultured for long periods of time. In this report, we describe an efficient and rapid protocol for the introduction of exogenous genes into primary B cells without the need for drug selection. We have used retroviral vectors containing the human adenosine deaminase gene as a marker gene, since the biological activity of this enzyme is easy to measure and is readily distinguishable from that of the endogenous mouse adenosine deaminase. Upon adoptive transfer into SCID mice, infected B cells continuously expressing one to three copies of the human adenosine deaminase gene could be found in the spleens of recipient animals for at least 3 months.
Assuntos
Adenosina Desaminase/genética , Linfócitos B , Terapia Genética/métodos , Animais , Sequência de Bases , Primers do DNA/química , Feminino , Humanos , Linfonodos/citologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Dados de Sequência Molecular , Baço/citologia , Fatores de TempoRESUMO
The current paradigm concerning the kinetics of hematopoiesis is that only the most primitive pluripotential bone marrow stem cells can support prolonged hematopoiesis whereas more differentiated, lineage-committed stem cells can only contribute to a particular lineage for a limited period of time. In this study, we present evidence that in mice, the spleen contains a long-lived myeloid-committed stem cell population(s) that continuously replenishes the mature myeloid lineage for at least 9 months. After myeloid-specific retroviral-mediated gene transfer, the exogenous gene could be detected in thioglycollate-induced macrophages and granulocytes by Southern blot analysis and by in situ polymerase chain reaction on an individual cell basis. The targeted stem cell population does not repopulate the bone marrow in secondary recipients and did not give rise to cells other than cells of the myeloid lineage. It therefore represents the first nonpluripotential stem cell population capable of replenishing a hemopoietic lineage for a long period of time. The ability to target a myeloid-specific stem cell could facilitate gene therapy of congenital disorders of the myeloid system such as lysosomal storage diseases. It also offers a unique opportunity to assess the immunologic consequences of expressing an exogenous gene of choice exclusively in the myeloid lineage.
Assuntos
Linfócitos B/transplante , Células da Medula Óssea/citologia , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Baço/citologia , Animais , Linfócitos B/citologia , Linhagem da Célula , Feminino , Genes Reporter , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hibridização In Situ , Canamicina Quinase/análise , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Macrófagos Peritoneais/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vírus da Leucemia Murina de Moloney/genética , Reação em Cadeia da Polimerase , Quimera por Radiação , Proteínas Recombinantes de Fusão/análise , TransfecçãoRESUMO
An in vitro culture system has been developed for the long-term maintenance of primary, human peripheral blood and umbilical cord blood T lymphocytes, which does not rely on the use of stimulatory cytokines, antigen, or mitogens. In these cultures, a monolayer of adherent cells, some spindle-shaped and some resembling macrophages, developed within a week. All adherent cells were positive for the extracellular matrix proteins laminin and fibronectin, the intermediate filament vimentin, and for the surface markers major histocompatibility complex class II, platelet-endothelial cell adhesion molecule l (CD31), and E-Selectin (ELAM-1; CD62E). They were negative for the leukocyte common antigen (CD45), the macrophage marker MO-2 (CD14), muscle-specific actin, and Factor VIII-related antigen. These monolayers supported the maintenance of nonadherent, resting, mature T cells for up to 3 months, and these cells retained their ability to respond to mitogens and allogeneic cells. Both CD4+ and CD8+ cells were supported. The proportion of CD4+ and CD8+ cells remained unchanged after 3 months in culture. We have also used T cells from 2-month-old cultures as target cells for retroviral vector-mediated gene transfer. Up to 30% of the long-term T cells expressed the transferred lacZ gene after infection with a retroviral vector. The infection efficiency was similar to that obtained for fresh peripheral blood T cells, indicating that the long-term-cultured cells might be suitable for certain gene therapy applications.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Técnicas de Cultura/métodos , Endotélio Vascular/citologia , Adolescente , Adulto , Biomarcadores/análise , Relação CD4-CD8 , Comunicação Celular , Células Cultivadas , Criança , Pré-Escolar , Replicação do DNA , Endotélio Vascular/metabolismo , Proteínas da Matriz Extracelular/análise , Feminino , Sangue Fetal/citologia , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos , Substâncias de Crescimento/metabolismo , Humanos , Lactente , Recém-Nascido , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/biossíntese , Sarcoma de Kaposi/patologia , Fatores de Tempo , TransfecçãoRESUMO
Mouse mammary tumor viruses encode superantigens that interact with MHC class II proteins and stimulate T cells. We show here that presentation of mouse mammary tumor virus superantigen does not require DM. Furthermore, we have identified a strong class II peptide binding motif in the Mtv-7 superantigen, and we show that this motif is necessary for association with class II molecules in in vitro translation and in vivo functional assays. Our results suggest that endogenously synthesized viral superantigen can bind to MHC class II heterodimers during biosynthesis in the endoplasmic reticulum in a manner analogous to that used by the class II-associated invariant chain.
Assuntos
Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Vírus do Tumor Mamário do Camundongo/imunologia , Superantígenos/imunologia , Superantígenos/metabolismo , Motivos de Aminoácidos , Animais , Apresentação de Antígeno/genética , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos Virais , Sistema Livre de Células/imunologia , Dimerização , Cães , Antígeno HLA-DR1/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Camundongos , Modelos Imunológicos , Mutação , Oligopeptídeos/imunologia , Oligopeptídeos/metabolismo , Ligação Proteica/imunologia , Biossíntese de Proteínas/imunologia , Coelhos , Superantígenos/biossíntese , Superantígenos/genéticaRESUMO
We earlier proposed that a human endogenous retroviral (HERV) superantigen (SAg) IDDMK(1,2)22 may cause type I diabetes by activating autoreactive T cells. Viral infections and induction of interferon-alpha (IFN-alpha) are tightly associated with the onset of autoimmunity. Here we establish a link between viral infections and IFN-alpha-regulated SAg expression of the polymorphic and defective HERV-K18 provirus. HERV-K18 has three alleles, IDDMK(1,2)22 and two full-length envelope genes, that all encode SAgs. Expression of HERV-K18 SAgs is inducible by IFN-alpha and this is sufficient to stimulate V beta 7 T cells to levels comparable to transfectants constitutively expressing HERV-K18 SAgs. Endogenous SAgs induced via IFN-alpha by viral infections is a novel mechanism through which environmental factors may cause disease in genetically susceptible individuals.