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Viral DNA contamination is responsible for Epstein–Barr virus detection in cytotoxic T lymphocytes stimulated in vitro with Epstein–Barr virus B-lymphoblastoid cell line

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Abstract

Epstein–Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCLs) are used to prepare human EBV-specific T lymphocytes (EBV-CTL) in vitro. Within an LCL, up to 5–7% the cells release infectious EBV, and this has fostered safety concerns for therapeutic applications because of the exposure of T cells to EBV. The release of infectious viruses can be prevented by ganciclovir, but this drug may seriously affect LCL growth. In the wake of these concerns, the present work was designed to compile safety data on EBV-CTL preparation for the purpose of submission to a regulatory agency. We showed that further to supernatant exclusion, the number of EBV genome copies (EBVc) associated with the EBV-CTL always made up a constant proportion of the EBVc number detected in the culture supernatant. In addition, such was the case whether infectious virus could be produced by the LCL or not, suggesting that the EBV signal detected was due to a DNA contamination rather than an infection. Furthermore, we demonstrated that the number of EBVc associated with the EBV-CTL was highly sensitive to DNAse treatment, and finally that EBVc could no longer be detected after the EBV-CTL had been amplified in the absence of LCL. Consequently, during in vitro EBV-CTL preparation, either T cells cannot be infected or they die rapidly after EBV infection.

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Acknowledgments

This work was supported by institutional funding from the Institut National de la Santé et de la Recherche Médicale (INSERM).

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Authors and Affiliations

  1. Institut de Recherche Thérapeutique de l’Université de Nantes, UMR INSERM, U892, 8 quai Moncousu, BP 70721, 44007, Nantes Cedex 1, France

    Mathilde Berthomé, Géraldine Gallot, Régine Vivien, Béatrice Clémenceau, Jean-Michel Nguyen & Henri Vié

  2. Univ Nantes, 44000, Nantes, France

    Mathilde Berthomé, Géraldine Gallot, Régine Vivien, Béatrice Clémenceau, Marianne Coste-Burel & Henri Vié

  3. Centre Hospitalo Universitaire de Nantes, 44093, Nantes, France

    Mathilde Berthomé, Géraldine Gallot, Béatrice Clémenceau, Jean-Michel Nguyen, Marianne Coste-Burel & Henri Vié

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  1. Mathilde Berthomé
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  2. Géraldine Gallot
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  3. Régine Vivien
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  4. Béatrice Clémenceau
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  7. Henri Vié
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Correspondence to Henri Vié.

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M. Berthomé and G. Gallot contributed equally to this work.

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Berthomé, M., Gallot, G., Vivien, R. et al. Viral DNA contamination is responsible for Epstein–Barr virus detection in cytotoxic T lymphocytes stimulated in vitro with Epstein–Barr virus B-lymphoblastoid cell line. Cancer Immunol Immunother 59, 1867–1875 (2010). https://doi.org/10.1007/s00262-010-0913-2

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  • DOI: https://doi.org/10.1007/s00262-010-0913-2

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