RESUMO
Only so-called "professional" antigen-presenting cells (APCs) of hematopoietic origin are believed capable of inducing T lymphocyte responses. However, fibroblasts transfected with viral proteins directly induced antiviral cytotoxic T lymphocyte responses in vivo, without involvement of host APCs. Fibroblasts induced T cells only in the milieu of lymphoid organs. Thus, antigen localization affects self-nonself discrimination and cell-based vaccine strategies.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Fibroblastos/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Tecido Linfoide/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas Virais/imunologia , Animais , Citotoxicidade Imunológica , Glicoproteínas/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células L , Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
To identify the plasma membrane (PM) structures implicated in T-cell activation, we studied the distribution of interleukin-2 receptors (IL-2R) and the surface topography of lymphocytes by affinity labeling in electron microscopy (EM). In particular, we analyzed the distribution of the IL-2R alpha-chain on CTLL-2 cells (a murine cytotoxic T-cell lymphoma line). Some of our experiments were extended to the functionally and morphologically distinct cell line EL4 (a routine helper T-cell lymphoma line). As affinity ligands we used a rat monoclonal antibody (clone 7D4) reactive with the routine alpha-chain of IL-2R and recombinant mouse IL-2 (rIL-2). The distribution of IL-2R was visualized on the cell surface by ligands coupled to colloidal gold particles of different sizes. Unfixed cells were labeled with gold probes and attached to concanavalin A (ConA)-pretreated coverslips. Subsequently, the cells were prepared for EM. Examination of ultrathin sections and large surface replicas revealed a high degree of variability in cell morphology and in the density of the randomly distributed gold-labeled ligands among CTLL cells. According to their typical appearance, lymphocytes with strong receptor expression can be easily identified within the cell population. In contrast, the label on many mitogen-activated EL4 cells showed a cap-like polar distribution. The results suggest the existence of diverse distribution patterns of IL-2R on CTLL and EL4 cells. These differences are believed to reflect the different physiological roles played by T-cell subsets in the immune system.
Assuntos
Receptores de Interleucina-2/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Coloide de Ouro , Ligantes , Camundongos , Ratos , Linfócitos T Citotóxicos/ultraestrutura , Células Tumorais CultivadasRESUMO
Mobilized peripheral blood progenitor cells (PBPC) are an attractive target for the retrovirus-mediated transfer of cytostatic drug resistance genes. We analyzed NOD/SCID mouse repopulating CD34+ PBPC from cancer patients following retroviral Transwell transduction in various cytokine combinations with the FMEV-based (Friend-mink cell focus forming/murine embryonic stem cell virus) hybrid vector SF-MDR carrying the human multidrug resistance-1 (MDR1) gene. Five to 10 weeks following transplantation of 2.0 x 10(6) CD34+ PBPC into NOD/SCID mice we observed medium to high levels of human cell engraftment with up to 33%. The extent of vector-marked human cells was assessed by a quantitative real-time polymerase chain reaction (PCR). SF-MDR gene transfer into long-term in vivo repopulating human hematopoietic cells was optimal in the presence of either IL-3/IL-6/SCF/FL or FL/TPO/SCF resulting in three-fold (12.4% +/- 1.7%) or four-fold (16.5% +/- 6.8%) higher average proportions of gene-marked human cells in NOD/SCID mice as compared to IL-3 alone (P < 0.01). In conclusion, we could optimize the engraftment capacity and the retroviral gene transfer to CD34+ PBPC using cocktails of early acting cytokines in combination with the recombinant fibronectin fragment CH-296. Our data suggest that the NOD/SCID model provides a valid assay to estimate the gene transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation settings.
Assuntos
Genes MDR , Células-Tronco Hematopoéticas/metabolismo , Transdução Genética , Animais , Sequência de Bases , Primers do DNA/genética , Terapia Genética , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Retroviridae/genética , Transplante HeterólogoRESUMO
Transplantation of suicide gene modified allogeneic T lymphocytes is an approach to prevent T cell mediated GVHD while preserving the 'graft-versus-leukemia' (GVL) effect of an allograft. A prerequisite for such a therapy is the efficient transduction of T cells with suitable vectors. Since existing techniques allow only insufficient transduction of T cells, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 10(6) cells/ml) by retroviral infection. The presented protocol allowed us to obtain transduction rates of more than 70% of CD3+ cells after two cycles of infection. It is based on the use of FBS-free media for both the production of retrovirus-containing supernatant, as well as the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large scale conditions, it may easily be adapted to clinical needs and 'good manufacturing practice' (GMP) standards.
Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Retroviridae/genética , Linfócitos T/virologia , Transdução Genética , Fibronectinas , Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia , Humanos , Técnicas In Vitro , Transplante HomólogoRESUMO
Retroviral vectors are currently the most important and best characterized tools for ex vivo genetic modification of hematopoietic progenitor/stem cells. As a prerequisite for clinical applications, large volumes of high-titer vector supernatants have to be generated in compliance with 'GMP' guidelines. This goal can be reached using a carefully selected producer cell clone and a conventional large-scale cell culture system. The retroviral vector SF1m provides efficient expression of the human multidrug resistance 1 (MDR1) gene in hematopoietic progenitor/stem cells in vitro and in NOD/SCID mouse repopulating human cells in vivo. Currently, a clinical phase I/II study is in preparation to test whether intensified consolidation chemotherapy is enabled by autologous transplantation of peripheral blood progenitor/stem cells that have been genetically modified with SF1m. Using multi-tray cell factories >19 l of serum-free vector containing supernatant were generated from cells of a previously established SF1m-producer clone, based on the PG13 packaging cell line. Testing of the final samples revealed sufficient quality (>1.5 x 10(6) infectious particles/ml) for clinical scale transduction of CD34+ cells. Results from the production runs and the applied biosafety concept are described.
Assuntos
Genes MDR , Terapia Genética/métodos , Vetores Genéticos , Retroviridae/genética , Animais , Antineoplásicos/administração & dosagem , Biotecnologia , Linhagem Celular , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Segurança , Transplante AutólogoRESUMO
The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37-75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O(6)-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.
Assuntos
Vacinas contra a AIDS/administração & dosagem , Terapia Genética/métodos , Inibidores da Fusão de HIV/uso terapêutico , Infecções por HIV/prevenção & controle , HIV-1 , Células-Tronco Hematopoéticas/metabolismo , Vacinas contra a AIDS/genética , Animais , Citometria de Fluxo , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Infecções por HIV/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , O(6)-Metilguanina-DNA Metiltransferase/análise , Retroviridae/genética , Transdução Genética/métodosRESUMO
In (B10.BR----B10) chimeras infected with lymphocytic choriomeningitis (LCM) virus higher titers were attained in spleens and livers than in organs of the mice used for their construction, and the subsequent elimination was retarded, but eventually the virus was cleared. The numbers of LCM virus-specific CTL and their precursors as quantitated with chromium-release assay and limiting dilution method, respectively, were lower in chimeras than in B10.BR or C57BL/10J mice, and fewer were restricted for the haplotypes of the donors than of the recipients. The same was true with regard to antiviral effector cells, which were determined by adoptive immunization. The numbers of spleen cells releasing IgM and IgG antiviral antibodies were virtually as high in chimeras as they were in C57BL/10J and B10.BR mice. Transfer of immune splenocytes from either B10.BR or C57BL/10J mice resulted in incomplete virus elimination from the spleens of infected chimeras, whereas injection of a mixture of the two types of immune cells led to efficient clearance. We conclude that in the chimeras cells of both donor and recipient haplotypes participate in the infection, which is terminated by H-2k- and H-2b-restricted T lymphocytes that these animals are capable of generating. We conclude, furthermore, that clearance of the LCM virus from the tissues requires contact between effector and target cells.
Assuntos
Medula Óssea/imunologia , Coriomeningite Linfocítica/imunologia , Quimera por Radiação , Animais , Anticorpos Antivirais/biossíntese , Formação de Anticorpos , Transplante de Medula Óssea/imunologia , Citotoxicidade Imunológica , Antígenos H-2/imunologia , Imunidade Celular , Imunização Passiva , Coriomeningite Linfocítica/microbiologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos , Baço/citologia , Baço/microbiologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Interleukin 2 is thought to be active as a monomeric protein. As the nonessential Cys-140 of murine interleukin 2 (mIL2) is located in the hydrophobic interface of the amphiphilic F domain it was successfully used to stabilize hydrophobic amino acid contacts between two mIL2 cores yielding biologically active cystine-bonded dimeric mIL2. (3H) thymidine incorporation assays with intermolecular cystine-bonded or monomeric mIL2 revealed almost identical median effective concentrations (EC50) and high-affinity dissociation constants (Kdh), respectively. Comparative binding and internalization assays suggest that one cystine-bonded dimeric or two monomeric mIL2 molecules bind to the high-affinity receptor complex. Furthermore, DSS concentration-dependent crosslinking studies using monomeric mIL2 revealed four membrane-derived protein-complexes with apparent molecular weights of about 70 kDa, 85 kDa, 95 kDa and 100 kDa, respectively, showing that both mIL2 receptor chains may be crosslinked to a monomeric or dimeric ligand molecule, respectively. We therefore propose that dimerization of murine interleukin 2 occurring either in solution at concentrations above the low-affinity dissociation constant or at the low-affinity receptor is important for regulation of high-affinity complex formation and signal transduction.
Assuntos
Cistina/metabolismo , Interleucina-2/metabolismo , Receptores de Interleucina-2/metabolismo , Sequência de Aminoácidos , Animais , Divisão Celular , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Interleucina-2/química , Cinética , Ligantes , Camundongos , Dados de Sequência Molecular , Succinimidas/farmacologiaRESUMO
BACKGROUND: For many inherited and acquired diseases of the blood system, gene transfer into hematopoietic cells is a promising strategy to alleviate disease-related symptoms or even correct genetic alterations. In clinical gene therapy applications, low transduction efficiencies have been a major limitation mainly because of insufficient effective titers of the retroviral supernatants used. Thus, optimization of clinical-grade vector production under current 'Good Manufacturing Practice' (GMP) conditions is a prerequisite for successful gene therapy trials. METHODS: We established stable retroviral producer clones with single integrations of a retroviral vector encoding for the multidrug-resistance gene 1 (MDR1). Optimization of vector production in multi-tray cell factories (MTCFs) was studied with particular regard to harvest medium, cell density and harvest time point. RESULTS: We demonstrated that high-titer vector stocks could be produced in serum-free medium. By reducing the volume of harvest medium, titers could be increased up to four-fold. Plating optimal cell densities of 1 x 10(4) cells/cm2, repetitive harvests of vector supernatant were feasible over four consecutive days. Combining the most advantageous culture and harvest parameters tested, we were able to produce large quantities of serum-free vector supernatant in 40-tray MTCFs. Highly efficient gene transfer into primary human CD34+ progenitor cells demonstrated the quality of these vector stocks. CONCLUSION: The large-scale vector-production protocol in MTCFs described here is easy to handle, is applicable to a wide range of adherent producer cell lines and, most importantly, complies with current GMP guidelines.
Assuntos
Terapia Genética/métodos , Retroviridae/genética , Antígenos CD34/análise , Biotecnologia , Linhagem Celular , Meios de Cultura Livres de Soro , Genes MDR , Vetores Genéticos , HumanosRESUMO
Homodimeric murine interleukin 3 (mIL-3) agonists were generated by intermolecular cystine-bonding. Steady-state binding assays and association kinetics performed at 4 degrees C using these agonists revealed specific binding to both the high- and low-affinity receptor. DSS-mediated crosslinking studies performed at 4 degrees C with agonist concentrations compatible with high-affinity receptor complex formation allowed to detect protein complexes of the alpha chain, the beta chain(s) and the high-affinity receptor complex migrating with apparent molecular weights of 90 kDa, 140 kDa, and above 180 kDa, respectively. In contrast, monomeric mIL-3 was crosslinked to the alpha chain receptor only unless high concentrations were used. Binding studies performed at 4 degrees C revealed a positive cooperative interaction of monomeric mIL-3 with the low-affinity receptor. Proliferation studies and association kinetics performed at 37 degrees C showed that under physiological conditions these agonists were at least 2- to 3-fold more potent than monomeric mIL-3. We therefore propose that dimerization of mIL-3 may be involved in high-affinity receptor complex formation.
Assuntos
Interleucina-3/metabolismo , Receptores de Interleucina-3/metabolismo , Animais , Bioensaio , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Transformada , Cistina , Eletroforese em Gel de Poliacrilamida , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-3/química , Interleucina-3/farmacologia , Interleucina-5/farmacologia , Cinética , Ligantes , Substâncias Macromoleculares , Camundongos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Timidina/metabolismoRESUMO
Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% [mean +/- SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P <.01). A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol. (Blood. 2000;95:1237-1248)
Assuntos
Adipócitos/citologia , Genes MDR , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Retroviridae , Transfecção/métodos , Transplante Heterólogo/imunologia , Animais , Antígenos CD34 , Células da Medula Óssea/citologia , Linhagem Celular , Vetores Genéticos , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Antígenos Comuns de Leucócito/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias/sangue , Reação em Cadeia da Polimerase , Células Estromais/citologiaRESUMO
Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.
Assuntos
Vetores Genéticos/genética , Recombinação Genética , Retroviridae/genética , Ativação Viral , Integração Viral , Animais , Sequência de Bases , Southern Blotting , Células Cultivadas , Clonagem Molecular , DNA Viral/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Sequências Repetitivas de Ácido Nucleico/genética , Seleção GenéticaRESUMO
We have developed a technique for the rapid cloning of unknown flanking regions of transgenic DNA. We complemented a truncated kanamycin resistance gene of a bacterial plasmid with a neomycin resistance gene fragment from a gene transfer vector. Optimized transformation conditions allowed us to directly select for kanamycin-resistant bacteria. We cloned numerous proviral flanking fragments from growth factor-independent cell mutants that were obtained after infection with a replication incompetent retroviral vector and identified integrations into the cyclin D2 and several unknown genomic sequences. We anticipate that our method could be adapted to various vector systems that are used to tag and identify genes and to map genomes.
Assuntos
Clonagem Molecular/métodos , Teste de Complementação Genética , Resistência a Canamicina/genética , Provírus/genética , Retroviridae/genética , Ciclina D2 , Ciclinas/genética , Células-Tronco Hematopoéticas/virologia , HumanosRESUMO
Current gene therapeutic protocols directed towards the treatment of inherited disorders (eg ADA-SCID) and viral infections (eg AIDS), as well as adoptive immunotherapy approaches are based on the use of genetically modified lymphocytes. Since only insufficient transduction of T cells is obtained using existing techniques, the development of more efficient gene transfer protocols into these cells is of great importance. We present here a protocol for the highly efficient transduction of human primary T cells at high densities (1 x 106/ml) by retroviral infection. Using retroviral vectors encoding a truncated human low-affinity nerve growth factor receptor (DeltaLNGFR) as a gene transfer marker, we obtained transduction frequencies of more than 70% of CD3+ cells after two cycles of infection. Our protocol is based on the use of FBS-free media for both the production of retrovirus-containing supernatant and the cultivation of the primary T cells. Since the protocol presented here works just as efficiently under large-scale conditions, it may be easily adapted to clinical needs and 'good manufacturing practice' (GMP) standards.
Assuntos
Síndrome da Imunodeficiência Adquirida/terapia , Técnicas de Transferência de Genes , Imunoterapia Adotiva/métodos , Imunodeficiência Combinada Severa/terapia , Linfócitos T , Complexo CD3 , Células Cultivadas , Centrifugação , Meios de Cultura Livres de Soro , Fibronectinas , Citometria de Fluxo , Expressão Gênica , Vetores Genéticos/administração & dosagem , Humanos , Células Jurkat , Ativação Linfocitária , Receptor de Fator de Crescimento Neural/genética , Retroviridae/genética , Estatísticas não Paramétricas , Linfócitos T/imunologiaRESUMO
We have adapted a recently published protocol for retroviral gene transfer into hematopoietic cells [A. J. Schilz et al. (1998) Blood 92: 3163-3171] with respect to clinical requirements such as large-volume vector stock generation, adequate cell source, high cell numbers, and serum-free conditions. We present data on transduction efficacy and expression of the multidrug resistance 1 (MDR1) gene in human CD34(+) cells from mobilized peripheral blood (PB) mediated by a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector. Using a 1-day cytokine-mediated prestimulation, consisting of human interleukin (IL)-3, IL-6, stem cell factor (SCF), Flt-3 ligand (FL), and thrombopoietin (TPO), followed by a 3-day transduction procedure, we were able to detect up to 51% CD34(+) cells expressing MDR1. Xenotransplantation of transduced cells into NOD/LtSz-scid/scid (NOD/SCID) mice resulted in a mean engraftment level of 23% (0.1 to 87%). As shown by quantitative PCR analysis, a mean of 12.7% (range 0.3 to 55%) of the engrafted human cells in the bone marrow of chimeric mice contained the MDR1 cDNA. Furthermore, enhanced expression of MDR1 above control levels was detected in up to 15% of the engrafted human cell population. Our data suggest that NOD/SCID repopulating cells derived from mobilized PB can be transduced efficiently with existing retroviral vector systems under clinically applicable conditions.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Expressão Gênica , Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas/citologia , Retroviridae/genética , Animais , Antígenos CD34/análise , Sequência de Bases , Divisão Celular , Meios de Cultura Livres de Soro , Primers do DNA , Feminino , Células-Tronco Hematopoéticas/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Retroviral suicide gene vectors have successfully been used in clinical studies to improve the safety of adoptive immunotherapy with allogeneic T lymphocytes in the treatment of malignant and viral diseases. At the same time these studies have revealed several problems that are yet to be resolved including impaired T cell function due to long ex vivo culture. Here we present new retroviral vectors co-expressing truncated CD34, a gene transfer marker which ensures rapid enrichment of transduced cells using commercially available GMP-approved devices, and a splice-corrected variant of Herpes simplex virus thymidine kinase (scHSVtk) which confers high sensitivity to the prodrug ganciclovir. We show that a retroviral hybrid vector, MP71, based on the myeloproliferative sarcoma virus (MPSV) and the murine embryonic stem cell virus (MESV), encoding a tCD34/scHSVtk fusion protein mediates high expression of the 'sort-suicide' selection marker, thereby allowing for highly efficient purification and selective elimination of transduced cells.
Assuntos
Imunoterapia Adotiva/métodos , Depleção Linfocítica/métodos , Linfócitos T/transplante , Antígenos CD34/genética , Ganciclovir/farmacologia , Marcadores Genéticos , Vetores Genéticos , Humanos , Células Jurkat , Microscopia Confocal , Retroviridae/genética , Linfócitos T/efeitos dos fármacos , Transdução GenéticaRESUMO
Y chromosome-specific sequences can be used to detect remaining male cells after sex-mismatched allogeneic blood stem cell transplantation (HSCT) involving a male patient and female donor, which represents approximately 25% of all cases. We developed a quantitative Y chromosome-specific PCR assay (QYCS-PCR) based on the DFFRY gene for the determination of hematopoietic donor chimerism. We analyzed blood and marrow samples from more than 40 patients at various time points after both standard and nonmyeloablative allogeneic HSCT. We found that real-time PCR combines extreme sensitivity, with a detection level of less than 1 male in 100,000 female cells (<0.001%), with very good reproducibility, especially in the important range of minor host chimerism. QYCS-PCR results were in close agreement with data from other techniques as bcr/abl-PCR and/or fluorescent in situ hybridization (FISH) analysis. In two relapsed patients, increasing numbers of Y-positive hematopoietic cells indicated recurrence of malignant disease prior to clinical confirmation. In conclusion, quantitative Y chromosome-specific PCR is a promising approach for monitoring the extent of chimerism in blood and other tissues after sex-mismatched hematopoietic stem cell transplantation (HSCT) or organ transplantation.