RESUMO
RNA interference (RNAi) has recently emerged as a specific and efficient method to silence gene expression in mammalian cells either by transfection of short interfering RNAs (siRNAs; ref. 1) or, more recently, by transcription of short hairpin RNAs (shRNAs) from expression vectors and retroviruses. But the resistance of important cell types to transduction by these approaches, both in vitro and in vivo, has limited the use of RNAi. Here we describe a lentiviral system for delivery of shRNAs into cycling and non-cycling mammalian cells, stem cells, zygotes and their differentiated progeny. We show that lentivirus-delivered shRNAs are capable of specific, highly stable and functional silencing of gene expression in a variety of cell types and also in transgenic mice. Our lentiviral vectors should permit rapid and efficient analysis of gene function in primary human and animal cells and tissues and generation of animals that show reduced expression of specific genes. They may also provide new approaches for gene therapy.
Assuntos
Lentivirus/genética , Interferência de RNA , Animais , Sequência de Bases , Antígenos CD8/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Vetores Genéticos , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/química , RNA/genética , Receptores de Interleucina-2/genética , Células-TroncoRESUMO
The effector cytokine interferon gamma (IFN-gamma) may play a role in T cell homeostasis. We have examined the requirement for IFN-gamma in one mechanism that regulates T cell expansion and survival, activation-induced cell death (AICD). CD4(+) T cells lacking IFN-gamma or the Stat1 transcription factor are resistant to AICD. IFN-gamma is required for the production of caspases, and retrovirus-mediated expression of caspase-8 restores the sensitivity of Stat1-deficient T cells to AICD. In vitro, IFN-gamma limits the expansion of T cells that are stimulated through their antigen receptors. Thus, IFN-gamma may function to control the expansion and persistence of T cells by promoting caspase-8-dependent apoptosis.
Assuntos
Interferon gama/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Interferon gama/deficiência , Interferon gama/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/deficiência , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
Signaling pathways involved in regulating T cell proliferation and survival are not well understood. Here we have investigated a possible role of the nuclear factor (NF)-kappaB pathway in regulating mature T cell function by using CD4+ T cells from p50-/- cRel-/- mice, which exhibit virtually no inducible kappaB site binding activity. Studies with these mice indicate an essential role of T cell receptor (TCR)-induced NF-kappaB in regulating interleukin (IL)-2 expression, cell cycle entry, and survival of T cells. Our results further indicate that NF-kappaB regulates TCR-induced expression of antiapoptotic Bcl-2 family members. Strikingly, retroviral transduction of CD4+ T cells with the NF-kappaB-inducing IkappaB kinase beta showed that NF-kappaB activation is not only necessary but also sufficient for T cell survival. In contrast, our results indicate a lack of involvement of NF-kappaB in both IL-2 and Akt-induced survival pathways. In vivo, p50-/- cRel-/- mice showed impaired superantigen-induced T cell responses as well as decreased numbers of effector/memory and regulatory CD4+ T cells. These findings provide the first demonstration of a role for NF-kappaB proteins in regulating T cell function in vivo and establish a critically important function of NF-kappaB in TCR-induced regulation of survival.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-rel/fisiologia , Animais , Antígenos CD28/fisiologia , Ciclo Celular , Morte Celular , Sobrevivência Celular , Quinase I-kappa B , Camundongos , Subunidade p50 de NF-kappa B , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/análiseRESUMO
Transforming growth factor B (TGF-beta) is a potent immunosuppressive cytokine that is frequently associated with mechanisms of tumor escape from immunosurveillance. We report that transplantation of murine bone marrow (BM) expressing a dominant-negative TGF-beta type II receptor (TbetaRIIDN) leads to the generation of mature leukocytes capable of a potent antitumor response in vivo. Hematopoietic precursors in murine BM from donor mice were rendered insensitive to TGF-beta via retroviral expression of the TbetaRIIDN construct and were transplanted in C57BL/6 mice before tumor challenge. After i.v. administration of 5 x 10(5) B16-F10 murine melanoma cells into TbetaRIIDN-BM transplanted recipients, survival of challenged mice at 45 days was 70% (7 of 10) versus 0% (0 of 10) for vector-control treated mice, and surviving TbetaRIIDN-BM mice showed a virtual absence of metastatic lesions in the lung. We also investigated the utility of the TGF-beta-targeted approach in a mouse metastatic model of prostate cancer, TRAMP-C2. Treatment of male C57BL/6 mice with TbetaRIIDN-BM resulted in the survival of 80% (4 of 5) of recipients versus 0% (0 of 5) in green fluorescent protein-BM recipients or wild-type controls. Cytolytic T-cell assays indicate that a specific T-cell response against B16-F10 cells was generated in the TbetaRIIDN-BM-treated mice, suggesting that a gene therapy approach to inducing TGF-beta insensitivity in transplanted BM cells may be a potent anticancer therapy.
Assuntos
Terapia Genética/métodos , Células-Tronco Hematopoéticas/fisiologia , Melanoma Experimental/prevenção & controle , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Proteínas de Fluorescência Verde , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/virologia , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/prevenção & controle , Proteínas Serina-Treonina Quinases , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Retroviridae/genética , Linfócitos T Citotóxicos/imunologia , Transfecção , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Tumor necrosis factor-alpha (TNF-alpha) is a contributing cause of the insulin resistance seen in obesity and obesity-linked type 2 diabetes, but the mechanism(s) by which TNF-alpha induces insulin resistance is not understood. By using 3T3-L1 adipocytes and oligonucleotide microarrays, we identified 142 known genes reproducibly upregulated by at least threefold after 4 h and/or 24 h of TNF-alpha treatment, and 78 known genes downregulated by at least twofold after 24 h of TNF-alpha incubation. TNF-alpha-induced genes include transcription factors implicated in preadipocyte gene expression or NF-kappaB activation, cytokines and cytokine-induced proteins, growth factors, enzymes, and signaling molecules. Importantly, a number of adipocyte-abundant genes, including GLUT4, hormone sensitive lipase, long-chain fatty acyl-CoA synthase, adipocyte complement-related protein of 30 kDa, and transcription factors CCAAT/enhancer binding protein-alpha, receptor retinoid X receptor-alpha, and peroxisome profilerator-activated receptor gamma were significantly downregulated by TNF-alpha treatment. Correspondingly, 24-h exposure of 3T3-L1 adipocytes to TNF-alpha resulted in reduced protein levels of GLUT4 and several insulin signaling proteins, including the insulin receptor, insulin receptor substrate 1 (IRS-1), and protein kinase B (AKT). Nuclear factor-kappaB (NF-kappaB) was activated within 15 min of TNF-alpha addition. 3T3-L1 adipocytes expressing IkappaBalpha-DN, a nondegradable NF-kappaB inhibitor, exhibited normal morphology, global gene expression, and insulin responses. However, absence of NF-kappaB activation abolished suppression of >98% of the genes normally suppressed by TNF-alpha and induction of 60-70% of the genes normally induced by TNF-alpha. Moreover, extensive cell death occurred in IkappaBalpha-DN-expressing adipocytes after 2 h of TNF-alpha treatment. Thus the changes in adipocyte gene expression induced by TNF-alpha could lead to insulin resistance. Further, NF-kappaB is an obligatory mediator of most of these TNF-alpha responses.
Assuntos
Adipócitos/fisiologia , Antineoplásicos/farmacologia , Expressão Gênica/efeitos dos fármacos , Proteínas Musculares , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Núcleo Celular/metabolismo , Relação Dose-Resposta a Droga , Expressão Gênica/imunologia , Transportador de Glucose Tipo 4 , Insulina/metabolismo , Resistência à Insulina/fisiologia , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/imunologiaRESUMO
PURPOSE: Overexpression of antiapoptotic Bcl-2 family members has recently been related to resistance to chemo/radiotherapy in several human malignancies, particularly lymphomas. Hence, innovative approaches bypassing this resistance mechanism are required in the therapeutic approach. This study evaluated whether chemoresistance associated with Bcl-2 and Bcl-x(L) overexpression would be overcome by activating the death receptor pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the Jurkat cell model EXPERIMENTAL DESIGN: We made use of genetically modified Jurkat cells to evaluate the effect of Bcl-2 or Bcl-x(L) overexpression on the cytotoxic effect produced by the anticancer drugs doxorubicin, etoposide, and oxaliplatin and TRAIL. Caspase activation was detected by cleavage of caspase-8 and -3. The mitochondrial transmambrane potential was assessed by staining with DiOC(6) and flow cytometry. Caspase activity was blocked by the broad-spectrum caspase inhibitor zVAD-fmk. RESULTS: Bcl-2 and Bcl-x(L) overexpression but not lack of caspase-8 protects the Jurkat cells from the anticancer drug-induced cytolysis. However, Bcl-2/Bcl-x(L) Jurkat cells retained some susceptibility to TRAIL-induced cytolysis. A highly synergistic cytotoxic effect of the combination of TRAIL with any of the antiblastic used in this study was detected in the chemoresistant cells. This effect was associated with mitochondrial disassemblage and dependent on caspase activation CONCLUSIONS: The combination of TRAIL with conventional anticancer drugs may prove to be useful in the treatment of antiapoptotic Bcl-2 family proteins-expressing malignancies.
Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Glicoproteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Proteínas Reguladoras de Apoptose , Caspase 3 , Caspase 8 , Caspases/metabolismo , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Ativação Enzimática , Etoposídeo/farmacologia , Citometria de Fluxo , Humanos , Immunoblotting , Células Jurkat , Ligantes , Linfoma/metabolismo , Potenciais da Membrana , Compostos Organoplatínicos/farmacologia , Oxaliplatina , Ligante Indutor de Apoptose Relacionado a TNF , Fatores de Tempo , Proteína bcl-XRESUMO
RNA interference (RNAi) has emerged as a novel cellular mechanism regulating gene expression at the post-transcriptional level and as a powerful tool to control gene function experimentally. Recent advances in the biology and application of RNAi include the definition of improved criteria for selecting effective small interfering RNA (siRNA) sequences, and the generation of vectors for the delivery of siRNAs and stable silencing of genes in mammalian cells, tissues and animals. High-throughput screening projects based on RNAi have been initiated to search for genes involved in basic biological processes and in complex pathological conditions such as cancer, autoimmunity and degenerative disorders. This research is helping to identify novel therapeutic targets for a range of diseases and may translate into novel clinical applications for RNAi.
Assuntos
Regulação da Expressão Gênica , Biblioteca Gênica , Inativação Gênica , Interferência de RNA/fisiologia , RNA Interferente Pequeno/farmacologia , Animais , Marcação de Genes , Técnicas de Transferência de Genes , Humanos , RNA/metabolismo , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/uso terapêuticoRESUMO
Cancer and autoimmunity are polygenic diseases. In order to better understand the mechanisms of disease development and progression it is essential to uncover which genes are involved. Much has been learned from population studies in human patients by searching for polymorphic genetic loci associated with disease. In addition, animal models of tumor development, as well as models for various autoimmune conditions such as multiple sclerosis and Type I diabetes, have helped determine genetic loci that contribute to disease susceptibility. However, characterization of the exact genes involved is often difficult and requires lengthy and technically demanding genetic manipulations. The generation of knockout animals is the method of choice to probe single genes. However, this is not possible in all species or even in all inbred strains within a species. The recent discovery of a new post-transcriptional gene silencing pathway termed RNA interference, which is mediated by short fragments of double-stranded RNA (short-interfering RNA), has opened up new avenues for genetic manipulation of experimental animals. This review will consider how silencing genes by RNA interference within the context of experimental disease models promises to become a powerful new tool for the genetic analysis of cancer and autoimmunity. Advances in RNA interference technology now permit the relatively rapid generation of transgenic animals in a wide range of species with complex genetic backgrounds that were previously inaccessible to genetic manipulation. This novel approach should help refine the characterization of disease-associated genes, either by silencing specific candidates or even by screening a larger number of genes in vivo within a comparatively short time frame.
Assuntos
Predisposição Genética para Doença , Interferência de RNA , Animais , Doenças Autoimunes/genética , Técnicas Genéticas , Testes Genéticos , Vetores Genéticos , Lentivirus/genética , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: TGF-beta is a potent immunosuppressant. High levels of TGF-beta produced by cancer cells have a negative inhibition effect on surrounding host immune cells and leads to evasion of the host immune surveillance and tumor progression. In the present study, we report a distinct ability of tumor reactive, TGF-beta-insensitive CD8+ T cells to infiltrate into established tumors, secrete relevant cytokines, and induce apoptosis of tumor cells. METHODS: CD8+ T cells were isolated from the spleens of C57BL/6 mice, which were primed with irradiated mouse prostate cancer cells, the TRAMP-C2 cells. After ex vivo expansion, these tumor reactive CD8+ cells were rendered TGF-beta-insensitive by infection with a retroviral (MSCV)-mediated dominant negative TGF-beta type II receptor (TbetaRIIDN). Control CD8+ cells consist of those transfected with the GFP-only empty vector and naïve CD8+ T cells. Recipient mice were challenged with a single injection of TRAMP-C2 cells 21 days before adoptive transfer of CD8+ T cells was performed. Forty days after the adoptive transfer, all animals were sacrificed. The presence of pulmonary metastases was evaluated pathologically. Serial slides of malignant tissues were used for immunofluorescent staining for different kinds of immune cell infiltration, cytokines, and apoptosis analysis. RESULTS: Pulmonary metastases were either eliminated or significantly reduced in the group receiving adoptive transfer of tumor-reactive TGF-beta-insensitive CD8+ T cells (3 out of 12) when compared to GFP controls (9 out of 12), and naïve CD8+ T cells (12 out of 12). Results of immunofluorescent studies demonstrated that only tumor-reactive TGF-beta-insensitive CD8+ T cells were able to infiltrate into the tumor and mediate apoptosis when compared to CD4+ T cells, NK cells, and B cells. A large amount of cytokines such as perforin, nitric oxide, IFN-gamma, IL-2, TNF-alpha were secreted in tumor tissue treated with tumor-reactive TGF-beta-insensitive CD8+ T cells. No immune cells infiltration and cytokine secretion were detected in tumor tissues treated with naïve T cells and GFP controls. CONCLUSIONS: Our results demonstrate the mechanism of anti-tumor effect of tumor-reactive TGF-beta-insensitive CD8+ T cells that adoptive transfer of these CD8+ T cells resulted in infiltration of these immune cells into the tumor parenchyma, secretion of relevant cytokines, and induction of apoptosis in tumor cells. These results support the concept that tumor-reactive TGF-beta-insensitive CD8+ T cells may prove beneficial in the treatment of advanced cancer patients.
Assuntos
Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias da Próstata/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Linhagem Celular Tumoral , Citocinas/imunologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias da Próstata/patologia , Organismos Livres de Patógenos EspecíficosRESUMO
Over the past four years RNA interference (RNAi) has exploded onto the research scene as a new approach to manipulate gene expression in mammalian systems. More recently, RNAi has garnered much interest as a potential therapeutic strategy. In this review, we briefly summarize the current understanding of RNAi biology and examine how RNAi has been used to study the genetic basis of physiological and disease processes in mammalian systems. We also explore some of the new developments in the use of RNAi for disease therapy and highlight the key challenges that currently limit its application in the laboratory, as well as in the clinical setting.
Assuntos
Pesquisa Biomédica , Fisiologia/métodos , Interferência de RNA , Terapêutica , Animais , HumanosRESUMO
The lifespan of antigen-bearing dendritic cells (DCs) is determined by signals from pathogens and T cells. These signals regulate DC survival by modulating expression of Bcl-2 family proteins. Toll-like receptors and T cell costimulatory molecules both trigger a DC survival pathway that is dependent on Bcl-x(L). However, Toll-like receptors uniquely increase expression of Bim and trigger cell death by a pathway that is blocked by Bcl-2. This pathway serves as a molecular 'timer' that sets the lifespan of DCs and regulates the magnitude of T cell responses in vivo. Thus, signals derived from the innate and acquired immune systems control DC lifespan and immunogenicity by distinct molecular mechanisms.
Assuntos
Sobrevivência Celular/fisiologia , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Relógios Biológicos/imunologia , Relógios Biológicos/fisiologia , Sobrevivência Celular/imunologia , Homeostase/fisiologia , Camundongos , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
The regulated elimination of T cells serves to maintain normal immune function and prevents autoimmune responses. IL-2 family cytokines play an important role in controlling the survival of immature and mature T cells. These molecules activate the protein kinase, AKT/PKB. AKT has been shown to transduce an antiapoptotic signal in numerous cell types. In this study, we show that an active form of AKT can protect T cells from apoptosis following growth factor withdrawal and that IL-2 family cytokines can promote T cell survival by activating this kinase. We also provide evidence that AKT does not block death receptor-mediated killing of lymphocytes. These data suggest that AKT may serve as a common signaling element by which members of the IL-2 family of cytokines promote T cell survival.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/enzimologia , Interleucina-2/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antígenos/farmacologia , Apoptose/imunologia , Linfócitos B/citologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Citocinas/fisiologia , Ativação Enzimática/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Muramidase/imunologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Receptor fas/fisiologiaRESUMO
A population of CD25(+)CD4(+) regulatory T cells (T regs) functions to maintain immunological self tolerance by inhibiting autoreactive T cell responses. CD25(+)CD4(+) T regs are present in low, but steady, numbers in the peripheral lymphoid tissues of healthy mice. Recent studies have shown that IL-2 is an essential growth factor for these cells. How this cytokine functions to regulate CD25(+)CD4(+) T reg homeostasis and prevent autoimmune disease remains unknown. In conventional CD4(+) T cells, IL-2 triggers signaling pathways that promote proliferation and survival by activating the STAT5 transcription factor and by increasing the expression of the antiapoptotic protein, Bcl-2. We show here that bcl-2 deficiency does not affect CD25(+)CD4(+) T reg homeostasis, and that ectopic expression of this molecule fails to rescue CD25(+)CD4(+) T reg numbers or to prevent the development of autoimmunity in IL-2-deficient mice. Furthermore, transient activation of STAT5 is sufficient to increase CD25(+)CD4(+) T reg numbers in IL-2-deficient mice. Our study uncovers an essential role for STAT5 in maintaining CD25(+)CD4(+) T reg homeostasis and self-tolerance.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ligação a DNA/fisiologia , Homeostase/imunologia , Proteínas do Leite , Receptores de Interleucina-2/biossíntese , Tolerância a Antígenos Próprios/imunologia , Transdução de Sinais/imunologia , Transativadores/fisiologia , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Homeostase/genética , Interleucina-2/deficiência , Interleucina-2/genética , Interleucina-2/fisiologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Fator de Transcrição STAT5 , Tolerância a Antígenos Próprios/genética , Transdução de Sinais/genética , Transativadores/deficiência , Transativadores/genética , Transativadores/metabolismo , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
We have developed an alternative to transgenesis for producing antigen-specific T cells in vivo. In this system, clonal naive T cells with defined antigen specificity are generated by retrovirus-mediated expression of T cell antigen receptor cDNAs in RAG1-deficient murine hematopoietic precursor cells. These T cells can be stimulated to proliferate and produce cytokines by exposure to antigen in vitro, and they become activated and expand in vivo after immunization. IL-2-deficient T cells generated by this technique show decreased proliferation and cytokine production, both of which can be rescued by exogenous addition of this growth factor. Thus, retrovirus-mediated expression of T cell antigen receptor cDNAs in hematopoietic precursor cells permits the rapid and efficient analysis of the life history of antigen-specific T cells in different genetic backgrounds and may allow for the long-term production of antigen-specific T cells with different functional properties for prophylactic and therapeutic purposes.
Assuntos
DNA Complementar/genética , DNA Complementar/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Retroviridae/genética , Retroviridae/metabolismo , Animais , Células da Medula Óssea/citologia , Linfócitos T CD4-Positivos/metabolismo , Divisão Celular , Linhagem Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Técnicas de Transferência de Genes , Hibridomas , Interferon gama/biossíntese , Interleucina-2/biossíntese , Óperon Lac , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/citologia , Linfócitos T/metabolismoRESUMO
15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) is a naturally occurring cyclopentenone metabolite of PGD(2) that possesses both peroxisome proliferator-activated receptor gamma (PPAR-gamma)-dependent and PPAR-gamma-independent anti-inflammatory properties. Recent studies suggest that cyclopentenone PGs may play a role in the down-regulation of inflammation-induced immune responses. In this study, we report that 15d-PGJ(2) as well as synthetic PPAR-gamma agonists inhibit lymphocyte proliferation. However, only 15d-PGJ(2), but not the specific PPAR-gamma activators, induce lymphocyte apoptosis. We found that blocking of the death receptor pathway in Fas-associated death domain(-/-) or caspase-8(-/-) Jurkat T cells has no effect on apoptosis induction by 15d-PGJ(2). Conversely, overexpression of Bcl-2 or Bcl-x(L) completely inhibits the initiation of apoptosis, indicating that 15d-PGJ(2)-mediated apoptosis involves activation of the mitochondrial pathway. In line with these results, 15d-PGJ(2) induces mitochondria disassemblage as demonstrated by dissipation of mitochondrial transmembrane potential (Deltapsi(m)) and cytochrome c release. Both of these events are partially inhibited by the broad spectrum caspase inhibitor benzyloxycarbonil-Val-Ala-Asp-fluoromethylketone, suggesting that caspase activation may amplify the mitochondrial alterations initiated by 15d-PGJ(2). We also demonstrate that 15d-PGJ(2) potently stimulates reactive oxygen species production in Jurkat T cells, and Deltapsi(m) loss induced by 15d-PGJ(2) is prevented by the reactive oxygen species scavenger N-acetyl-L-cysteine. In conclusion, our data indicate that cyclopentenone PGs like 15d-PGJ(2) may modulate immune responses even independent of PPAR-gamma by activating the mitochondrial apoptosis pathway in lymphocytes in the absence of external death receptor signaling.
Assuntos
Apoptose/efeitos dos fármacos , Ciclopentanos/farmacologia , Mitocôndrias/fisiologia , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacologia , Receptores do Fator de Necrose Tumoral/fisiologia , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Apoptose/imunologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Inibidores do Crescimento/farmacologia , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/imunologia , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/imunologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Permeabilidade/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Citoplasmáticos e Nucleares/fisiologia , Rosiglitazona , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Tiazolidinedionas/metabolismo , Tiazolidinedionas/farmacologia , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
TGF-beta regulation of immune homeostasis has been investigated in the context of cytokine knockout (TGF-beta null) mice, in which particular TGF-beta isoforms are disrupted throughout the entire organism, as well as in B and T cell-specific transgenic models, but to date the immunoregulatory effects of TGF-beta have not been addressed in the context of an in vivo mouse model in which multi-isoform TGF-beta signaling is abrogated in multiple leukocyte lineages while leaving nonhemopoietic tissue unaffected. Here we report the development of a murine model of TGF-beta insensitivity limited to the hemopoietic tissue of adult wild-type C57BL/6 mice based on retroviral-mediated gene transfer of a dominant negative TGF-beta type II receptor targeting murine bone marrow. Unlike the lymphoproliferative syndrome observed in TGF-beta1-deficient mice, the disruption of TGF-beta signaling in bone marrow-derived cells leads to dramatic expansion of myeloid cells, primarily monocytes/macrophages, and is associated with cachexia and mortality in lethally irradiated mice reconstituted with dominant negative receptor-transduced bone marrow. Surprisingly, there was a notable absence of T cell expansion in affected animals despite the observed differentiation of most cells in the T cell compartment to a memory phenotype. These results indicate not only that TGF-beta acts as a negative regulator of immune function, but that lack of functional TGF-beta signaling in the myeloid compartment of adult mice may trigger suppression of lymphocytes, which would otherwise proliferate when rendered insensitive to TGF-beta.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células Progenitoras Mieloides/patologia , Quimera por Radiação/imunologia , Receptores de Fatores de Crescimento Transformadores beta/administração & dosagem , Receptores de Fatores de Crescimento Transformadores beta/genética , Transdução Genética , Células 3T3 , Animais , Caquexia/genética , Caquexia/imunologia , Caquexia/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Regulação Viral da Expressão Gênica/imunologia , Técnicas de Transferência de Genes , Vetores Genéticos/análise , Vetores Genéticos/síntese química , Vetores Genéticos/fisiologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/virologia , Receptores de Hialuronatos/biossíntese , Memória Imunológica/genética , Imunofenotipagem , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Selectina L/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Células Progenitoras Mieloides/imunologia , Proteínas Serina-Treonina Quinases , Quimera por Radiação/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Interleucina-2/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Retroviridae/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Transgenes/imunologiaRESUMO
A brief antigenic stimulus can promote T cell proliferation, but the duration and nature of intracellular signals required for survival are unclear. Here we show that in the absence of OX40 costimulation, antigen-activated CD4+ cells are short-lived because the activity of protein kinase B (PKB; also known as Akt) is not maintained over time. Activated T cells that express a dominant-negative variant of PKB also undergo apoptosis, reproducing the OX40-deficient phenotype. In contrast, an active form of PKB prevents downregulation of antiapoptotic proteins in OX40-deficient T cells, rescues antigen-induced cell survival in vivo, and controls inflammation in recall responses. Thus, sustained and periodic PKB signaling has an integral role in regulating T cell longevity.