RESUMEN
Adoptive T cell tumor immunotherapy potentially consists of two protective components by the transferred effector cells, the immediate immune response and the subsequent development of memory T cells. The extent by which adoptively transferred CD8(+) CTL are destined to become memory T cells is ambiguous as most studies focus on the acute effects on tumor shortly following adoptive transfer. In this study we show that a substantial fraction of the input CTL develop into memory cells that reject a s.c. tumor challenge. The use of exogenous IL-2 or a combination of IL-2 and IL-4, but not solely IL-4, during the ex vivo culture for the CTL inoculation was necessary for efficient development of CD8(+) memory T cells. Thus, an important component of adoptive immunotherapy using CTL is the production of CD8(+) Ag-specific memory cells which is primarily favored by IL-2 receptor signaling during ex vivo generation of the effector CTL.
Asunto(s)
Linfocitos T CD8-positivos/trasplante , Memoria Inmunológica , Inmunoterapia Adoptiva/métodos , Interleucina-2/farmacología , Linfocitos T Citotóxicos/trasplante , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Interleucina-2/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/trasplanteRESUMEN
In the present report, we have studied the potential of naive and activated effector CD8(+) T cells to function as anti-tumor T cells to a solid tumor using OVA-specific T cells from TCR-transgenic OT-I mice. Adoptive transfer of naive OT-I T cells into tumor-bearing syngeneic mice did not inhibit tumor cell growth. The adoptively transferred OT-I T cells did not proliferate in lymphoid tissue of tumor-bearing mice and were not anergized by the tumor. In contrast, adoptive transfer of preactivated OT-I CTL inhibited tumor growth in a dose-dependent manner, indicating that E.G7 was susceptible to immune effector cells. Importantly, naive OT-I T cells proliferated and elicited an anti-tumor response if they were adoptively transferred into normal or CD4-deficient mice that were then vaccinated with GM-CSF-induced bone marrow-derived OVA-pulsed APC. Collectively, these data indicate that even though naive tumor-specific T cells are present at a relatively high fraction they remain ignorant of the tumor and demonstrate that a CD8-mediated anti-tumor response can be induced by Ag-pulsed APC without CD4 T cell help.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Proteínas del Huevo/inmunología , Activación de Linfocitos , Ovalbúmina/inmunología , Timoma/inmunología , Timoma/prevención & control , Animales , Presentación de Antígeno/genética , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/trasplante , División Celular/inmunología , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad , Proteínas del Huevo/metabolismo , Proteínas del Huevo/farmacología , Femenino , Inmunoterapia Adoptiva , Inyecciones Intravenosas , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Ovalbúmina/metabolismo , Ovalbúmina/farmacología , Fragmentos de Péptidos , Subgrupos de Linfocitos T/trasplante , Timoma/genética , Timoma/patología , Células Tumorales CultivadasRESUMEN
SV40-based vectors are very efficient in gene delivery into human hematopoietic cells. In the present work, we investigated the expression of constructs carrying the human beta-globin gene that were delivered as beta-globin pseudovirions. Expression studies were performed by RNA analysis of primary human erythroid progenitors cultivated from peripheral blood of beta(0)-thalassemia patients who are unable to produce normal beta-globin RNA. This erythroid culture system recapitulates in vitro the process of growth, differentiation, and maturation of authentic erythroid precursors. The progenitors were induced to differentiate by the addition of erythropoietin (EPO). Five days later, the cells were infected with pseudovirions containing the normal beta-globin gene, and RNA was harvested on day 8. The results showed significant levels of normal beta-globin gene mRNA. A small DNA fragment derived from the 5'-region of the HSII element of the human beta-globin locus control region (LCR) enhanced expression of the linked beta-globin gene 20-30-fold. Normal beta-globin mRNA expression was in direct correlation to the multiplicity of infection. These studies suggest the potential feasibility of using the beta-globin delivery system for gene therapy of beta-thalassemia.
Asunto(s)
Células Precursoras Eritroides/metabolismo , Eritropoyesis/genética , Regulación del Desarrollo de la Expresión Génica , Terapia Genética , Vectores Genéticos/genética , Globinas/genética , Virus 40 de los Simios/genética , Talasemia beta/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Células Precursoras Eritroides/virología , Eritropoyetina/farmacología , Estudios de Factibilidad , Globinas/biosíntesis , Humanos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Talasemia beta/patologíaRESUMEN
Transduction of MDR1 may be of use in chemoprotection of normal bone marrow (BM) cells during treatment of malignancies, or as a selectable marker for the transfer of other genes into the BM, a critical target for the cure of many diseases. To that aim, the human multidrug resistance gene MDR1 was cloned into an SV40 pseudoviral vector containing the SV40 origin of replication (ori) and encapsidation signal (ses), and the plasmid was encapsidated in COS cells as SV40/MDR1 pseudovirions. Expression of the human MDR1 gene was demonstrated in murine MEL cells infected with SV40/MDR1 pseudovirions, using a monoclonal antibody (MPK16) specific for the human 170-kD P-glycoprotein. Functional P-glycoprotein was demonstrated by resistance to colchicine in NIH-3T3 cells infected with SV40/MDR1 pseudovirions. Activity of P-glycoprotein was assayed by rhodamine-123 dye exclusion and fluorescence-activated cell sorter analysis (FACS) in various cell types including hematopoietic cells. Highly efficient gene transfer and expression was demonstrated in all murine and human cell types tested, including primary human BM cells. Using multiplicities of infection (moi) of 1-2, over 95% of cells were found to become MDR1+. The percent of MDR1+ cells was proportional to the moi. We conclude that the SV40 pseudoviral vector is efficient for gene transmission into human hematopoietic cells.
Asunto(s)
Técnicas de Transferencia de Gen , Genes MDR , Vectores Genéticos , Células Madre Hematopoyéticas , Virus 40 de los Simios/genética , Células 3T3 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Animales , Células COS , Células Cultivadas , Colchicina/farmacología , Resistencia a Medicamentos/genética , Fibroblastos , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Ratones , Plásmidos , Reacción en Cadena de la Polimerasa , Células Tumorales CultivadasRESUMEN
SV40 is an attractive potential vector with high-efficiency gene transfer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases. In the present study, the three SV40 capsid proteins, VP1, VP2, and VP3, were produced in Spodoptera frugiperda (Sf9) insect cells. Their co-production led to spontaneous assembly of SV40-like particles. Nuclear extracts containing the three proteins were allowed to interact with purified SV40 DNA, or with plasmid DNA produced and purified from Escherichia coli. The experiments demonstrated a physical association between the DNA and capsid proteins, protection from DNase I digestion, and the formation of infectious particles. The results indicate that intact, supercoiled DNA is being packaged and transmitted into the target cells. The transmitted DNA is biologically functional in gene expression and replication. The process, which utilizes naked DNA, is not dependent on the SV40 packaging signal ses. The procedure allows packaging of plasmids significantly larger than SV40 and permits the inclusion of potent regulatory signals, such as beta-globin locus control region (LCR) elements. These studies are the first step in the development of purified, in vitro-constructed pseudovirions for experimental and medical use.
Asunto(s)
Cápside/genética , Vectores Genéticos , Virus 40 de los Simios/fisiología , Animales , Proteínas de la Cápside , Extractos Celulares , Línea Celular , Núcleo Celular , Chlorocebus aethiops , ADN Viral , Genes Virales , Terapia Genética , Humanos , Plásmidos , Proteínas Recombinantes de Fusión/genética , Virus 40 de los Simios/genética , Spodoptera/citología , Virión , Ensamble de VirusRESUMEN
Using the experimental system of simian virus 40 (SV40) pseudovirions we have previously shown that SV40 requires a specific DNA element for packaging, ses, which was mapped to the SV40 regulatory region. ses was previously found to play a role in facilitating the nucleosomal rearrangement required for chromatin condensation and viral packaging. Here, the fine structure of ses was investigated by genetic studies. Analyses of ses+ revertants indicated that in order to function, ses must be present in close proximity to the origin of replication (ori), supporting a role in the regulation of the viral life cycle. Fine dissection of ses was performed using a series of plasmids carrying mutations and deletions in this region. The results suggest that multiple DNA elements participate in the SV40 packaging process, including the GC-boxes and elements derived from the enhancer. The elements are redundant, and they can function in various combinations. Packaging efficiency correlated with the number of GC-boxes, known to bind Sp1. In addition, AP-2 binding elements appeared to more important than others. These findings were supported by experiments which showed that packaging was significantly enhanced by adding AP-2 binding sites to plasmids with large deletions and lacking those sites. The results imply that binding of Sp1 and/or AP-2 may participate in the packaging process.