title: 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.
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.
authors.affiliation: Institut de Recherche Thérapeutique de l'Université de Nantes, UMR INSERM, U892, 8 quai Moncousu, BP 70721, 44007, Nantes Cedex 1, France.
authors.forename: Mathilde
authors.initials: M
authors.lastname: Berthomé
authors.forename: Géraldine
authors.initials: G
authors.lastname: Gallot
authors.forename: Régine
authors.initials: R
authors.lastname: Vivien
authors.forename: Béatrice
authors.initials: B
authors.lastname: Clémenceau
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Nguyen
authors.forename: Marianne
authors.initials: M
authors.lastname: Coste-Burel
authors.forename: Henri
authors.initials: H
authors.lastname: Vié
mesh_terms: D001402:B-Lymphocytes / Q000821:virology*; D002460:Cell Line; D004279:DNA, Viral / Q000097:blood*; D015774:Ganciclovir / Q000494:pharmacology; D004854:Herpesvirus 4, Human / Q000187:drug effects / Q000302:isolation & purification*; D006801:Humans; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000821:virology*
chemical_list: D004279:DNA, Viral; D015774:Ganciclovir
references.citation: Blood. 2010 Feb 4;115(5):925-35
references.pmid: 19880495
references.citation: Am J Med. 1982 Jul 20;73(1A):18-26
references.pmid: 6285710
references.citation: Blood. 2001 Sep 15;98(6):1882-8
references.pmid: 11535525
references.citation: Blood. 1997 Jun 15;89(12):4521-30
references.pmid: 9192776
references.citation: J Exp Med. 1991 Apr 1;173(4):971-80
references.pmid: 1706754
references.citation: J Virol. 1980 May;34(2):560-8
references.pmid: 6246281
references.citation: Cancer Immunol Immunother. 2002 Nov;51(10):539-46
references.pmid: 12384805
references.citation: Nature. 1988 Jun 2;333(6172):455-7
references.pmid: 2836734
references.citation: Eur J Haematol. 2007 Jul;79(1):72-5
references.pmid: 17532761
references.citation: Proc Natl Acad Sci U S A. 1974 Mar;71(3):863-6
references.pmid: 4274561
references.citation: J Immunol. 1995 Feb 1;154(3):1440-9
references.pmid: 7822809
references.citation: Clin Chem. 1999 Jul;45(7):982-6
references.pmid: 10388473
references.citation: Semin Cancer Biol. 1996 Aug;7(4):197-207
references.pmid: 8946604
references.citation: J Med Virol. 2009 Jan;81(1):90-8
references.pmid: 19031462
references.citation: Blood. 2000 Jul 15;96(2):443-51
references.pmid: 10887104
references.citation: Anticancer Res. 1999 Jul-Aug;19(4B):3007-17
references.pmid: 10652585
references.citation: Blood. 1992 Oct 1;80(7):1804-12
references.pmid: 1327284
references.citation: J Immunol. 1991 Feb 1;146(3):865-9
references.pmid: 1703182
references.citation: J Infect Dis. 2001 Jan 1;183(1):1-7
references.pmid: 11106535
references.citation: J Virol. 1996 Oct;70(10):7341-6
references.pmid: 8794395
references.citation: Eur J Immunol. 1988 Aug;18(8):1299-302
references.pmid: 2970972
references.citation: J Clin Invest. 1993 Sep;92(3):1444-50
references.pmid: 8397226
references.citation: Pediatr Blood Cancer. 2009 Aug;53(2):184-90
references.pmid: 19353621
references.citation: Leuk Lymphoma. 1999 May;33(5-6):451-8
references.pmid: 10342573
references.citation: J Virol. 1984 Apr;50(1):50-5
references.pmid: 6321799
references.citation: Hum Gene Ther. 2000 Jul 1;11(10):1453-63
references.pmid: 10910142
references.citation: J Clin Pathol. 2007 Jan;60(1):101-3
references.pmid: 17213357
references.citation: Hum Gene Ther. 2000 Jul 1;11(10):1465-75
references.pmid: 10910143
references.citation: Blood. 1995 Jan 15;85(2):456-64
references.pmid: 7811999
references.citation: Blood. 1991 Feb 15;77(4):799-808
references.pmid: 1847084
references.citation: J Immunol. 1998 Jun 15;160(12):6204-9
references.pmid: 9637540
references.citation: Cell Immunol. 1979 Jun;45(1):157-66
references.pmid: 313274
references.citation: Expert Opin Biol Ther. 2008 Sep;8(9):1265-94
references.pmid: 18694349
references.citation: Blood. 1993 Jul 1;82(1):217-23
references.pmid: 8391874
references.citation: J Clin Microbiol. 2000 Feb;38(2):712-5
references.pmid: 10655372
references.citation: Blood. 1993 May 15;81(10):2688-95
references.pmid: 8387835
references.citation: J Virol. 1990 Jan;64(1):437-44
references.pmid: 2152830
references.citation: Am J Pathol. 1994 Jun;144(6):1219-25
references.pmid: 8203462
references.citation: J Gen Virol. 1989 May;70 ( Pt 5):1203-15
references.pmid: 2543775
references.citation: N Engl J Med. 1995 Oct 19;333(16):1038-44
references.pmid: 7675046
references.citation: Am J Pathol. 1990 May;136(5):1093-9
references.pmid: 2161617
references.citation: Lancet. 1990 Jan 20;335(8682):128-30
references.pmid: 1967431
references.citation: J Gen Virol. 1990 Feb;71 ( Pt 2):379-86
references.pmid: 2155291
references.citation: Cytotherapy. 2003;5(4):323-35
references.pmid: 12944239