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.
Record created on 2005-09-05, modified on 2016-08-08