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Updated: Aug 8, 2026
Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines
Published on: November 8, 2011
Nucleofection of non-B cells with mini-Epstein-Barr virus DNA
Jürgen Radons1, Catharina Gross, Stefan Stangl
1Abteilung Hämatologie und Internistische Onkologie, Universität Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany.
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.
Insights
This study developed a novel nucleofection technique for gene transfer into non-B cells, enhancing natural killer (NK) cell activity against heat shock protein 70 (Hsp70)-positive tumors. The method enables the generation of Hsp70-reactive NK cell lines for cancer therapy.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Research
Background:
- Heat shock protein 70 (Hsp70) on tumor cells is recognized by NK cells.
- Hsp70-positive tumors are sensitive to NK cell-mediated lysis when enhanced by IL-2 and Hsp70-peptide.
- Generating Hsp70-reactive NK cell lines requires efficient gene transfer into non-B cells.
Purpose of the Study:
- To establish a rapid and efficient gene transfer method for non-B cells.
- To provide an experimental basis for generating Hsp70-reactive NK cell lines.
Main Methods:
- A modified nucleofection technique using a mini-Epstein-Barr virus (mini-EBV) plasmid (p2667) was employed for gene transfer.
- Transfection efficiency and cell population dynamics were assessed using green fluorescent protein (GFP) as a marker.
- Cytolytic activity of transfected NK cells against Hsp70-positive tumor cells was evaluated.
Main Results:
- The mini-EBV nucleofection technique successfully introduced genes into non-B cells, with GFP expression observed by day 18.
- Post-transfection, cell populations comprised NKT, T cells, and NK cells, with no detectable B cells.
- Transfected NK cells maintained cytolytic activity against Hsp70-positive tumor cells, comparable to non-transfected cells.
Conclusions:
- Mini-EBV facilitates efficient gene transfer into non-B cells, including NK cells.
- This nucleofection method supports the generation of effector cell lines with conserved Hsp70-reactivity.
- The findings lay the groundwork for developing novel NK cell-based immunotherapies for Hsp70-positive cancers.
