Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors.
Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system. A monocistronic vector with a CMV promoter gave 32 +/- 4.7% green fluorescent protein (GFP)+ cells. A bicistronic vector, encoding IL-4 and GFP in the first and second cistrons, respectively, gave 14.2 +/- 2.1% GFP+ cells and IL-4 secretion of 1.3 +/- 0.2 ng/10(5) B cells/24 h. This was similar to results obtained in CD34+ cells using the elongation factor-1alpha promoter. Activated memory and naive B cells were transducible. After transduction with a bicistronic vector encoding a viral FLIP molecule, vFLIP was detectable by FACS or Western blot in GFP+, but not in GFP-, B cells, and 57% of sorted GFP+ B cells were protected against Fas ligand-induced cell death. This system should be useful for gene function research in primary B cells and development of gene therapies.
Kvell
Krisztian
K
Division of Hematology, Department of Internal Medicine, University Hospitals, 1211 Geneva-14, Switzerland.
Nguyen
Tuan H
TH
Salmon
Patrick
P
Glauser
Frédéric
F
Werner-Favre
Christiane
C
Barnet
Marc
M
Schneider
Pascal
P
Trono
Didier
D
Zubler
Rudolf H
RH
D018952:Antigens, CD34; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D023201:CD40 Ligand; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D018899:CpG Islands; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D015847:Interleukin-4 / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics*; D008977:Molluscum contagiosum virus / Q000235:genetics; D009101:Multiple Myeloma; D014161:Transduction, Genetic
D018952:Antigens, CD34; D023201:CD40 Ligand; D049452:Green Fluorescent Proteins; D015847:Interleukin-4