RESUMEN
X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.
Asunto(s)
Catepsina G/genética , Enfermedad Granulomatosa Crónica/genética , Enfermedad Granulomatosa Crónica/terapia , Células Mieloides/fisiología , Proteínas Proto-Oncogénicas c-fes/genética , Proteínas Recombinantes de Fusión/genética , Transgenes , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Genes Ligados a X , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Granulocitos/metabolismo , Enfermedad Granulomatosa Crónica/enzimología , Enfermedad Granulomatosa Crónica/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis/genética , Células Mieloides/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Virus Formadores de Foco en el Bazo/genética , Virus Formadores de Foco en el Bazo/metabolismo , Células Madre/metabolismo , Secuencias Repetidas Terminales , Transactivadores/metabolismoRESUMEN
Background: Mutations of the common cytokine receptor gamma chain (γc) cause Severe Combined Immunodeficiency characterized by absent T and NK cell development. Although stem cell therapy restores these lineages, residual immune defects are observed that may result from selective persistence of γc-deficiency in myeloid lineages. However, little is known about the contribution of myeloid-expressed γc to protective immune responses. Here we examine the importance of γc for myeloid dendritic cell (DC) function. Methods: We utilize a combination of in vitro DC/T-cell co-culture assays and a novel lipid bilayer system mimicking the T cell surface to delineate the role of DC-expressed γc during DC/T-cell interaction. Results: We observed that γc in DC was recruited to the contact interface following MHCII ligation, and promoted IL-15Rα colocalization with engaged MHCII. Unexpectedly, trans-presentation of IL-15 was required for optimal CD4+T cell activation by DC and depended on DC γc expression. Neither recruitment of IL-15Rα nor IL-15 trans-signaling at the DC immune synapse (IS), required γc signaling in DC, suggesting that γc facilitates IL-15 transpresentation through induced intermolecular cis associations or cytoskeletal reorganization following MHCII ligation. Conclusions: These findings show that DC-expressed γc is required for effective antigen-induced CD4+ T cell activation. We reveal a novel mechanism for recruitment of DC IL-15/IL-15Rα complexes to the IS, leading to CD4+ T cell costimulation through localized IL-15 transpresentation that is coordinated with antigen-recognition.