Nucleofection of non-B cells with mini-Epstein-Barr virus DNA.
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.
Radons
Jürgen
J
Abteilung Hämatologie und Internistische Onkologie, Universität Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany.
Gross
Catharina
C
Stangl
Stefan
S
Multhoff
Gabriele
G
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics* / Q000378:metabolism / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D017930:Genes, Reporter; D053804:Granzymes; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007694:Killer Cells, Natural / Q000201:enzymology / Q000378:metabolism* / Q000821:virology; D012697:Serine Endopeptidases / Q000096:biosynthesis; D016176:T-Lymphocyte Subsets / Q000201:enzymology / Q000378:metabolism / Q000821:virology; D014162:Transfection / Q000379:methods*
D004279:DNA, Viral; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases