ABSTRACT
BACKGROUND AND OBJECTIVE: Gene transfer and expression of exogenous genetic information coding for an immunogenic protein in antigen presenting cells (APCs) can promote an immune response. This was investigated by retroviral transfer of a marker gene into CD34+ derived APCs. DESIGN AND METHODS: To achieve long term expression of a specific transgene in APCs, G-CSF mobilized peripheral blood CD34+ cell populations were retrovirally transduced with the bacterial nlsLacZ, a marker gene used here as a model, in the presence of IL-3, IL-6, GM-CSF and SCF prior to being induced to differentiate into dendritic and macrophage cells by GM-CSF and TNF-a. RESULTS: Addition of IL-4 was found to induce dendritic differentiation preferentially by inhibiting proliferation and differentiation of the macrophage lineage. As assessed by X-Gal staining, LacZ gene expression was observed in cells from both the dendritic lineage (CD1a+/CD14-) which still exhibits the highest immunostimulatory activity in mixed lymphocyte reaction and from the macrophage lineage (CD1a-/ CD14+). INTERPRETATION AND CONCLUSIONS: This study sets out the possibility of transducing dendritic and macrophage progenitors present in the CD34+ cell population and in using a marker gene such as nlsLacZ to study gene expression in antigen presenting cell compartments.
Subject(s)
Dendritic Cells/enzymology , Genetic Vectors/genetics , Hematopoietic Stem Cells/cytology , Lac Operon , Retroviridae/genetics , beta-Galactosidase/biosynthesis , Antigen Presentation , Antigens, CD1/analysis , Antigens, CD34/analysis , Cell Differentiation/drug effects , Cell Lineage , Dendritic Cells/virology , Enzyme Induction , Genes, Reporter , Hematopoietic Cell Growth Factors/pharmacology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/virology , Humans , Interleukin-4/pharmacology , Lipopolysaccharide Receptors/analysis , Lymphocyte Culture Test, Mixed , Macrophages/drug effects , Macrophages/enzymology , Macrophages/virology , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Transfection , Tumor Necrosis Factor-alpha/pharmacology , beta-Galactosidase/geneticsABSTRACT
Relative transduction efficiency with retroviral vector-producing clones was assayed by cocultivating TF-1, a human CD34+ hematopoietic cell line and YR-2, a sheep B-lymphoid cell line, with LacZ containing vector-producing cells, and then by scoring the percentage of X-Gal+ cells. At the same time, viral titer was estimated by titration assay with murine fibroblasts. Results clearly demonstrated a lack of correlation between viral titer and efficiency of transduction into hematopoietic cells, which depends neither on the type of packaging cell line, PG-13 and GP-envAM12 in this study, nor on the type of LacZ containing retroviral vector. These results strongly favor consideration of interactions between producers and target cells of the study for the screening of producing cell lines.