RESUMO
The magnitude and complexity of Ag-specific CD8(+) T cell responses is determined by intrinsic properties of the immune system and extrinsic factors, such as vaccination. We evaluated mechanisms that regulate the CD8(+) T cell response to two distinct determinants derived from the same protein Ag, SV40 T Ag (T Ag), following immunization of C57BL/6 mice with T Ag-transformed cells. The results show that direct presentation of T cell determinants by T Ag-transformed cells regulates the magnitude of the CD8(+) T cell response in vivo but not the immunodominance hierarchy. The immunodominance hierarchy was reversed in a dose-dependent manner by addition of excess naive T cells targeting the subdominant determinant. However, T cell competition played only a minor role in limiting T cell accumulation under physiological conditions. We found that the magnitude of the T cell response was regulated by the ability of T Ag-transformed cells to directly present the T Ag determinants. The hierarchy of the CD8(+) T cell response was maintained when Ag presentation in vivo was restricted to cross-presentation, but the presence of T Ag-transformed cells capable of direct presentation dramatically enhanced T cell accumulation at the peak of the response. This enhancement was due to a prolonged period of T cell proliferation, resulting in a delay in T cell contraction. Our findings reveal that direct presentation by nonprofessional APCs can dramatically enhance accumulation of CD8(+) T cells during the primary response, revealing a potential strategy to enhance vaccination approaches.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos Transformantes de Poliomavirus/fisiologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Epitopos de Linfócito T/imunologia , Transferência Adotiva/métodos , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Antígenos Transformantes de Poliomavirus/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/transplante , Linhagem Celular , Transformação Celular Viral/imunologia , Células Cultivadas , Testes Imunológicos de Citotoxicidade/métodos , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/metabolismo , Feminino , Esquemas de Imunização , Epitopos Imunodominantes/administração & dosagem , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/metabolismo , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fatores de TempoRESUMO
The required activities of CD4(+) T cells and antibody against the virally encoded oncoprotein simian virus 40 (SV40) Tag have previously been demonstrated by our laboratory to be mediators in achieving antitumor responses and tumor protection through antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we further characterize the necessary immune cell components that lead to systemic tumor immunity within an experimental pulmonary metastatic model as the result of SV40 Tag immunization and antibody production. Immunized animals depleted of CD8(+) T cells at the onset of experimental tumor cell challenge developed lung tumor foci and had an overall decreased survival due to lung tumor burden, suggesting a role for CD8(+) T cells in the effector phase of the immune response. Lymphocytes and splenocytes harvested from SV40 Tag-immunized mice experimentally inoculated with tumor cells synthesized increased in vitro levels of the Th1 cytokine gamma interferon (IFN-gamma), as assessed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry assays. CD8(+) T-cell activity was also heightened in SV40 Tag-immunized and tumor cell-challenged mice, based upon intracellular production of perforin, confirming the cytolytic properties of CD8(+) T cells against tumor cell challenge. Altogether, these data point to the role of recombinant SV40 Tag protein immunization in initiating a cytotoxic T-lymphocyte (CTL) response during tumor cell dissemination and growth. The downstream activity of CD8(+) T cells within this model is likely initiated from SV40 Tag-specific antibody mediating ADCC tumor cell destruction.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Pulmonares/imunologia , Vírus 40 dos Símios/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Anticorpos Antivirais/sangue , Antígenos Transformantes de Poliomavirus/administração & dosagem , Linhagem Celular Transformada , Imunidade Humoral , Imunização , Rim/citologia , Rim/virologia , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/prevenção & controle , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Linfócitos T Citotóxicos , Células Th1/imunologia , Infecções Tumorais por Vírus/mortalidade , Infecções Tumorais por Vírus/prevenção & controleRESUMO
Immunodominance limits the TCR diversity of specific antiviral CD8 T cell responses elicited by vaccination or infection. To prime multispecific T cell responses, we constructed DNA vaccines that coexpress chimeric, multidomain Ags (with CD8 T cell-defined epitopes of the hepatitis B virus (HBV) surface (S), core (C), and polymerase (Pol) proteins and/or the OVA Ag as stress protein-capturing fusion proteins. Priming of mono- or multispecific, HLA-A*0201- or K(b)-restricted CD8 T cell responses by these DNA vaccines differed. K(b)/OVA(257-264)- and K(b)/S(190-197)-specific CD8 T cell responses did not allow priming of a K(b)/C(93-100)-specific CD8 T cell response in mice immunized with multidomain vaccines. Tolerance to the S- Ag in transgenic Alb/HBs mice (that express large amounts of transgene-encoded S- Ag in the liver) facilitated priming of subdominant, K(b)/C(93-100)-specific CD8 T cell immunity by multidomain Ags. The "weak" (i.e., easily suppressed) K(b)/C(93-100)-specific CD8 T cell response was efficiently elicited by a HBV core Ag-encoding vector in 1.4HBV-S(mut) tg mice (that harbor a replicating HBV genome that produces HBV surface, core, and precore Ag in the liver). K(b)/C(93-100)-specific CD8 T cells accumulated in the liver of vaccinated 1.4HBV-S(mut) transgenic mice where they suppressed HBV replication. Subdominant epitopes in vaccines can hence prime specific CD8 T cell immunity in a tolerogenic milieu that delivers specific antiviral effects to HBV-expressing hepatocytes.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/genética , Vacinas contra Hepatite B/genética , Vacinas contra Hepatite B/imunologia , Epitopos Imunodominantes/genética , Ativação Linfocitária/imunologia , Vacinas de DNA/imunologia , Replicação Viral/imunologia , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/metabolismo , Feminino , Antígenos H-2/administração & dosagem , Antígenos H-2/genética , Antígenos H-2/metabolismo , Proteínas de Choque Térmico HSP70/administração & dosagem , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/genética , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vacinas contra Hepatite B/administração & dosagem , Antígenos E da Hepatite B/genética , Antígenos E da Hepatite B/imunologia , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Humanos , Epitopos Imunodominantes/administração & dosagem , Epitopos Imunodominantes/metabolismo , Hepatopatias/imunologia , Hepatopatias/prevenção & controle , Hepatopatias/virologia , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/genética , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Replicação Viral/genéticaRESUMO
PURPOSE: To recapitulate the generation of cancer stem cells in the context of an intact animal using a retroviral vector capable of in vivo delivery of oncogenes to primitive endothelial and hematopoietic stem cells. EXPERIMENTAL DESIGN: Targeting of these progenitors was achieved using transgenic mice in which the avian TVA retroviral receptor was placed under the control of the stem cell leukemia (scl/tal-1) gene promoter and SCL +19 enhancer. RESULTS: Injection of an avian retrovirus encoding polyoma middle T (PyMT), an oncogene that transforms endothelial cells, caused rapid lethality in all SCL-TVA mice but not in control TVA(-) littermates. The infected animals exhibited hemorrhagic foci in several organs. Histopathologic analysis confirmed the presence of hemangiomas and the endothelial origin of the PyMT-transformed cells. Surprisingly, the transformed endothelial cells contained readily detectable numbers of TVA(+) cells. By contrast, normal blood vessels had very few of these cells. The presence of TVA(+) cells in the lesions suggests that the cells originally infected by PyMT retained stem cell characteristics. Further analysis showed that the tumor cells exhibited activation of the phosphatidylinositol 3-kinase/Akt and S6/mammalian target of rapamycin pathways, suggesting a mechanism used by PyMT to transform endothelial progenitors in vivo. CONCLUSIONS: We conclude that this experimental system can specifically deliver oncogenes to vascular endothelial progenitors in vivo and cause a fatal neoplastic disease. This animal model should allow the generation of endothelial cancer stem cells in the natural environment of an immunocompetent animal, thereby enabling the recapitulation of genetic alterations that are responsible for the initiation and progression of human malignancies of endothelial origin.
Assuntos
Antígenos Transformantes de Poliomavirus/administração & dosagem , Proteínas Aviárias/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Endoteliais/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Hemangioma/genética , Proteínas Proto-Oncogênicas/genética , Receptores Virais/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Transformação Celular Neoplásica/genética , Células Cultivadas , Galinhas , Hemangioma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Células NIH 3T3 , Oncogenes/fisiologia , Células-Tronco/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , TransgenesRESUMO
Self-reactive T cells that survive the process of positive and negative selection during thymocyte development represent potential effector cells against tumors that express these same self-Ags. We have previously shown that CD8+ T lymphocytes (T(CD8)) specific for an immunorecessive epitope, designated epitope V, from the SV40 large T Ag (Tag) escape thymic deletion in line SV11 Tag-transgenic mice. In contrast, these mice are tolerant to the three most dominant Tag epitopes. The majority of the residual epitope V-specific T(CD8) have a low avidity for the target epitope, but a prime/boost regimen can expand higher avidity clones in vivo. Whether higher avidity T(CD8) targeting this epitope are affected by Tag-expressing tumors in the periphery or can be recruited for control of tumor progression remains unknown. In the current study, we determined the fate of naive TCR-transgenic T(CD8) specific for Tag epitope V (TCR-V cells) following transfer into SV11 mice bearing advanced-stage choroid plexus tumors. The results indicate that TCR-V cells are rapidly triggered by the endogenous Tag and acquire effector function, but fail to accumulate within the tumors. Primary immunization enhanced TCR-V cell frequency in the periphery and promoted entry into the brain, but a subsequent booster immunization caused a dramatic accumulation of TCR-V T cells within the tumors and inhibited tumor progression. These results indicate that epitope V provides a target for CD8+ T cells against spontaneous tumors in vivo, and suggests that epitopes with similar properties can be harnessed for tumor immunotherapy.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Imunização Secundária , Infecções por Polyomavirus/prevenção & controle , Infecções Tumorais por Vírus/prevenção & controle , Transferência Adotiva , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Antígenos Transformantes de Poliomavirus/biossíntese , Antígenos Transformantes de Poliomavirus/genética , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/prevenção & controle , Linfócitos T CD8-Positivos/transplante , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/imunologia , Plexo Corióideo/imunologia , Plexo Corióideo/patologia , Testes Imunológicos de Citotoxicidade , Progressão da Doença , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/biossíntese , Epitopos de Linfócito T/genética , Feminino , Genes Recessivos/imunologia , Imunização Secundária/métodos , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estadiamento de Neoplasias , Infecções por Polyomavirus/imunologia , Infecções por Polyomavirus/mortalidade , Infecções por Polyomavirus/patologia , Estrutura Terciária de Proteína/genética , Receptores de Antígenos de Linfócitos T/genética , Análise de Sobrevida , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/mortalidade , Infecções Tumorais por Vírus/patologiaRESUMO
BACKGROUND: The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general. METHODS: In an attempt to explore how naked DNA enters hepatocytes the fate of a variety of molecules and particles was followed over a 24-h time frame using fluorescence microscopy. The uptake of some of these compounds was correlated with marker gene expression from a co-injected plasmid DNA. In addition, the uptake of the injected compounds was correlated with the histologic appearance of hepatocytes. RESULTS: Out of the large number of nucleic acids, peptides, proteins, inert polymers and small molecules that we tested, most were efficiently delivered into hepatocytes independently of their size and charge. Even T7 phage and highly charged DNA/protein complexes of 60-100 nm in size were able to enter the cytoplasm. In animals co-injected with an enhanced yellow fluorescent protein (EYFP) expression vector and fluorescently labeled immunoglobulin (IgG), hepatocytes flooded with large amounts of IgG appeared permanently damaged and did not express EYFP-Nuc. Hepatocytes expressing EYFP had only slight IgG uptake. In contrast, when an EYFP expression vector was co-injected with a fluorescently labeled 200-bp linear DNA fragment, both were mostly (in 91% of the observed cells) co-localized to the same hepatocytes 24 h later. CONCLUSIONS: The appearance of permanently damaged cells with increased uptake of some molecules such as endogenous IgG raised the possibility that a molecule could be present in a hepatocyte but its transport would not be indicative of the transport process that can lead to foreign gene expression. The HTV procedure enables the uptake of a variety of molecules (as previous studies also found), but the uptake process for some of these molecules may be associated with a more disruptive process to the hepatocytes that is not compatible with successful gene delivery.
Assuntos
Técnicas de Transferência de Genes , Hepatócitos/metabolismo , Plasmídeos/administração & dosagem , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/administração & dosagem , Antígenos Transformantes de Poliomavirus/administração & dosagem , Antígenos Transformantes de Poliomavirus/genética , Proteínas de Bactérias/genética , Bacteriófago T7/genética , Sequência de Bases , Transporte Biológico Ativo , Corantes Fluorescentes/administração & dosagem , Injeções Intravenosas , Proteínas Luminescentes/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos ICR , Microscopia de Fluorescência , Dados de Sequência Molecular , Sinais de Localização Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Plasmídeos/genética , Proteínas Recombinantes/genética , Estreptavidina/administração & dosagem , Cauda/irrigação sanguíneaRESUMO
In a transgenic mouse model expressing SV40 T Ag (Tag) as a de novo tumor Ag, immune surveillance fails and islet cell carcinomas grow progressively. To develop an anticancer strategy that would be effective in eradicating solid, autochthonously growing tumors, we evaluated the effectiveness of immunostimulatory oligodeoxynucleotides (ODN) with cytosine-guanine-rich (CpG) motifs (CpG-ODN). In a classical vaccination protocol, Tag was administered with CpG-ODN as adjuvant. The antitumor vaccination, however, was only effective in a prophylactic setting, despite the successful activation of a Tag-specific CTL response in vivo. Histological examination demonstrated that even primed immune cells failed to infiltrate tumors once a malignant environment was established. To ensure that effector cells were not limiting, highly activated tumor Ag-specific T cells were transferred into tumor-bearing mice. However, this treatment also failed to result in tumor infiltration and rejection. Therefore, we further tested the efficacy of CpG-ODN as a proinflammatory agent in combination with the transfer of preactivated Tag-specific CD4(+) and CD8(+) T cells. Indeed, this combination therapy proved to be highly effective, because CpG-ODN rendered insulinomas permissive for massive infiltration and destruction. The opening of tumor tissue correlated with uptake of CpG-ODN by tissue-resident macrophages and a strong up-regulation of adhesion molecules such as ICAM and VCAM on blood vessel endothelia. These data demonstrate that systemic application of proinflammatory reagents drastically enhances extravasation of effector cells into tumor tissue, an observation that is of general importance for immunotherapy of solid tumors in a clinical setting.
Assuntos
Movimento Celular/imunologia , Ilhas de CpG/imunologia , Mediadores da Inflamação/administração & dosagem , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Antígenos Transformantes de Poliomavirus/imunologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Movimento Celular/genética , Citotoxicidade Imunológica/genética , Epitopos de Linfócito T/imunologia , Imunidade Inata/genética , Imunoterapia Adotiva , Mediadores da Inflamação/imunologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/imunologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/transplanteRESUMO
A number of drugs are regarded as possessing local activity because their effects take place at an extremely short distance from their location site in the cell. The response of different cellular compartments to these effects is different. Such substances as photosensitizers (PSs), which are used in photodynamic cancer therapy, should be targeted to the cell compartments where their effect is the most pronounced. This study describes the construction and properties of the chimeric modular recombinant transporters (MRTs) expressed in Escherichia coli and used for PS targeting. These constructs include (1) the alpha-melanocyte-stimulating hormone as a ligand module, which is internalized by the target cells (mouse melanoma); (2) the optimized SV40 large T-antigen nuclear localization signal; (3) the hemoglobin-like protein from E. coli as a carrier module; (4) the endosomolytic module, the translocation domain of the diphtheria toxin. These MRTs were used for PS targeting to the mouse melanoma cell nuclei, the most PS-damaged intracellular compartment, which resulted in a PS photocytotoxic effect increase of several orders of magnitude. In our opinion, MRTs, which target locally active drugs into the desired cell compartment and thereby enhance the drug response, represent a new generation of the pharmacological agents.
Assuntos
Antineoplásicos/administração & dosagem , Núcleo Celular/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Fármacos Fotossensibilizantes/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Sequência de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Proteínas de Transporte/administração & dosagem , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Compartimento Celular , Toxina Diftérica/administração & dosagem , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Melanoma Experimental/patologia , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Plasmídeos/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Células Tumorais Cultivadas , alfa-MSH/administração & dosagem , alfa-MSH/genética , alfa-MSH/metabolismoRESUMO
To asses the requirement of interleukin (IL)-10 for peripheral CD4 T cell tolerance, the IL-10 knockout (KO) was introduced into a T cell receptor-transgenic mouse model (TCR1) specific for SV40 T antigen (Tag). IL-10-deficient TCR1-transgenic mice failed to establish antigen-specific T cell tolerance following sequential injections with Tag peptide. Nevertheless, IL-10 was not required for the establishment of CD4 T cell tolerance in double transgenic RT2/TCR1 mice in which Tag is expressed endogenously under control of the insulin promoter. However, in contrast to stable anergy in wild-type RT2/TCR1 mice, tolerant T cells in RT2/TCR1/Il-10KO mice could be driven into vigorous proliferation by exogenous antigenic stimulation in vivo. The observed reactivation of anergic T cell populations in IL-10-deficient mice was only seen after in vivo but not in vitro peptide priming, reflecting an important regulatory function of IL-10 in the context of the living organism. Taken together, these results demonstrate that IL-10 is required to maintain T cell tolerance following exposure to enhanced antigenic stimuli but is not essential for the induction of self-tolerance.
Assuntos
Tolerância Imunológica , Interleucina-10/fisiologia , Linfócitos T/imunologia , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Anergia Clonal , Genes Codificadores dos Receptores de Linfócitos T , Interleucina-10/deficiência , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Tolerância a Antígenos PrópriosAssuntos
Córtex Suprarrenal/citologia , Transplante de Células , Córtex Suprarrenal/ultraestrutura , Envelhecimento , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Divisão Celular , Proteínas de Ligação a DNA , Regulação Enzimológica da Expressão Gênica , Engenharia Genética , Humanos , Neovascularização Fisiológica , Telomerase/administração & dosagemRESUMO
PURPOSE: To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothelial cells. METHODS: Liposome-mediated delivery of proteins into bovine corneal endothelial cells (BCEC) was utilized in this study. Initially, beta-galactosidase was used as a marker protein for cell delivery and cells were assayed colorimetrically for beta-galactosidase activity. Subsequently, SV40 large T-antigen protein was introduced into BCEC and positive cells were identified by immunohistochemistry 24 hours after liposome-protein treatment. Quiescent BCECs were double-labeled using BrdU as a measure of de novo DNA synthesis and the SV40 large T-antigen was detected by standard immunohistochemical methods. RESULTS: Beta-galactosidase or SV40 large T antigen were introduced into BCECs using liposome transfer methods. The transfer efficiency of beta-galactosidase was > 30% of the cells. SV40 large T antigen was successfully introduced and was localized to the nuclei of BCECs. The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation. Co-labeling confirmed that only cells containing SV40 large T antigen were positive for de novo DNA synthesis. CONCLUSIONS: This study demonstrates that proteins can be inserted directly into corneal endothelial cells. In the case of the SV40 large T-antigen, the protein localized to the nucleus and maintained its bioactivity by inducing DNA synthesis. This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endothelial cell proliferation.
Assuntos
Antígenos Transformantes de Poliomavirus/farmacologia , Endotélio Corneano/citologia , Endotélio Corneano/efeitos dos fármacos , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Antígenos Transformantes de Poliomavirus/metabolismo , Bromodesoxiuridina/farmacocinética , Bovinos , Divisão Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , DNA/biossíntese , Portadores de Fármacos , Endotélio Corneano/enzimologia , Lipossomos , beta-Galactosidase/metabolismoRESUMO
The lack of CD95 in mice is associated with an accumulation of TCR alphabeta+ CD4- CD8- (double-negative (DN)) cells in the lymph nodes (LNs) and other organs. To test the hypothesis that these DN cells arise from TCR alphabeta+ CD8+ cells after activation via the TCR, we have crossed an MHC class I-restricted TCR transgene (tg) onto the lpr genotype to generate two TCR-transgenic experimental groups, TCRtg+ lpr/+ (CD95-intact) and TCRtg+ lpr/lpr (CD95-deficient). Specific peptide administration resulted in peripheral deletion of TCR alphabeta cells from the LNs of CD95-intact and CD95-deficient mice. On day 3 after peptide administration in the CD95-deficient but not the CD95-intact mice, there was a ninefold increase in the percentage of DN cells in the LN; this increase returned to control levels by day 10. Peripheral deletion was associated with an accumulation of TCR alphabeta+ CD8high cells in the livers of mice of both genotypes by day 3, which returned to control levels by day 10 without an increase in the percentage or total number of DN cells. Our data show that the in vivo stimulation of TCR alphabeta+ CD8+ cells in the absence of CD95 results in an initial accumulation and an eventual loss of DN cells. This identifies a role for CD95 after TCR alphabeta stimulation in the efficient removal of TCR alphabeta+ CD8+ cells after the down-regulation of CD8. CD95 is not essential for this process, because other mechanisms can compensate, but such mechanisms are less efficient in the LN.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/administração & dosagem , Linfócitos T CD8-Positivos/imunologia , Deleção Clonal/genética , Antígenos de Histocompatibilidade Classe I/genética , Imunofenotipagem , Injeções Intraperitoneais , Fígado/citologia , Fígado/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Contagem de Linfócitos , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/imunologia , Receptor fas/genéticaRESUMO
The relationship between the polyoma virus tumor-specific transplantation antigen (TSTA) and 2 of the virus proteins coded from the early region of polyomavirus was investigated. Mice were immunized with small T antigen and a truncated mutant of middle T antigen, both purified from genetically engineered Escherichia coli. The 2 proteins induced protective immunity against polyomavirus-induced tumors, but not against non-polyoma tumors, indicating that one or more of the polyoma T antigens are directly involved in a TSTA function.