Back to Search
Start Over
Nucleofection of non-B cells with mini-Epstein-Barr virus DNA.
- Source :
-
Journal of immunological methods [J Immunol Methods] 2005 Aug; Vol. 303 (1-2), pp. 135-41. - Publication Year :
- 2005
-
Abstract
- A tumor-specific cell surface localization of heat shock protein 70 (Hsp70) on CX+ colon carcinoma cells provides a recognition structure for NK cells but not for NKT and T cells. Incubation with low-dose IL-2 plus Hsp70-peptide TKD enhances production and release of granzyme B by NK cells and thus renders Hsp70-positive tumors more sensitive to their cytolytic attack. To provide the experimental basis for the generation of Hsp70-reactive NK cell lines we established a modified nucleofection technique as a rapid and efficient method for gene transfer into non-B cells. Therefore, TKD-stimulated, CD3/CD19-depleted effector cells, consisting of 85% CD3- CD16/56+ NK cells, 1.4% CD3+ CD16/56+ NKT cells, and 0.3% CD3+ CD16/56- T cells were nucleofected with the green fluorescent protein (GFP)-containing mini-Epstein-Barr virus (mini-EBV) plasmid p2667 (1478.A d2GFP). GFP, a marker for the expression of EBV-associated genes, became visible for the first time on day 18 after transfection. On day 28 mini-EBV-transfected cells consisted of 49% NKT, 38% T cells, and 13% NK cells; no contaminating B cells were detectable. Even 1.5 years after transfection GFP and CD94 were found to be co-expressed on transfectants. These data indicated that mini-EBV provides a useful tool for the nucleofection of non-B cells. The cytolytic activity of NK-transfectants towards Hsp70 membrane-positive CX+ tumor cells was comparable to that of non-transfected effector cells. In summary, our results might provide the basis for the generation of non-B effector cell lines including NK cells with conserved Hsp70-reactivity.
- Subjects :
- Cell Line, Tumor
Cell Nucleus metabolism
DNA, Viral metabolism
Flow Cytometry methods
Genes, Reporter
Granzymes
Herpesvirus 4, Human metabolism
Humans
Killer Cells, Natural enzymology
Killer Cells, Natural virology
Serine Endopeptidases biosynthesis
T-Lymphocyte Subsets enzymology
T-Lymphocyte Subsets metabolism
T-Lymphocyte Subsets virology
Cell Nucleus genetics
Cell Nucleus virology
DNA, Viral genetics
Herpesvirus 4, Human genetics
Killer Cells, Natural metabolism
Transfection methods
Subjects
Details
- Language :
- English
- ISSN :
- 0022-1759
- Volume :
- 303
- Issue :
- 1-2
- Database :
- MEDLINE
- Journal :
- Journal of immunological methods
- Publication Type :
- Academic Journal
- Accession number :
- 16055146
- Full Text :
- https://doi.org/10.1016/j.jim.2005.06.007