title: Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein-Barr virus potential through both culture-dependent and selection process-dependent mechanisms.
abstract: To modulate alloreactivity after hematopoietic stem cell transplantation, suicide gene-expressing donor T cells can be administered with an allogeneic T-cell-depleted bone marrow graft. Immune competence of such cells is a critical issue. The impact of the ex vivo gene transfer protocol (12-day culture period including CD3/interleukin-2 [IL-2] activation, retroviral-mediated gene transfer, and G418-based selection) on the anti-Epstein-Barr virus (EBV) potential of gene-modified cells has been examined. Cytotoxic (pCTL) and helper (pTh) cell precursor limiting dilution assays, interferon-gamma enzyme-linked immunospot, or fluorescence-activated cell sorter analysis after tetrameric HLA-A2/EBV peptide complexes revealed that the frequency of anti-EBV T cells was lower in gene-modified cells (GMCs) than in similarly cultured but untransduced T cells and was even lower than in fresh peripheral blood mononuclear cells, demonstrating both an effect of the culture and of the transduction or selection. The culture-dependent loss of EBV-reactive cells resulted from the preferential induction of activation-induced cell death in tetramer(+) cells. Replacing the initial CD3/IL-2 activation by CD3/CD28/IL-2 partially restored the anti-EBV response of GMCs by reducing the initial activation-induced cell death and enhancing the proliferation of EBV-tetramer(+) cells. Moreover, the G418 selection, and not the transduction, was directly toxic to transduced tetramer(+) cells. Replacing the G418 selection by an immunomagnetic selection significantly prevented the selection-dependent loss of EBV-specific cells. Overall, ex vivo gene modification of primary T cells can result in a significant reduction in EBV-reactive T cells through both culture-dependent and selection-dependent mechanisms. Improving immune functions of GMCs through modifications of the cell culture conditions and transduction/selection processes is critical for further clinical studies.
authors.affiliation: INSERM E-0119/UPRES EA-2284, Etablissement Français du Sang- Bourgogne/Franche-Comté, Besançon, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Sauce
authors.forename: Marie
authors.initials: M
authors.lastname: Bodinier
authors.forename: Marina
authors.initials: M
authors.lastname: Garin
authors.forename: Bruno
authors.initials: B
authors.lastname: Petracca
authors.forename: Nicolas
authors.initials: N
authors.lastname: Tonnelier
authors.forename: Anne
authors.initials: A
authors.lastname: Duperrier
authors.forename: Junia V
authors.initials: JV
authors.lastname: Melo
authors.forename: Jane F
authors.initials: JF
authors.lastname: Apperley
authors.forename: Christophe
authors.initials: C
authors.lastname: Ferrand
authors.forename: Patrick
authors.initials: P
authors.lastname: Hervé
authors.forename: François
authors.initials: F
authors.lastname: Lang
authors.forename: Pierre
authors.initials: P
authors.lastname: Tiberghien
authors.forename: Eric
authors.initials: E
authors.lastname: Robinet
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D002461:Cell Line, Transformed / Q000821:virology; D003601:Cytotoxicity Tests, Immunologic; D018014:Gene Transfer Techniques / Q000009:adverse effects*; D005839:Gentamicins / Q000494:pharmacology; D015789:HLA-A2 Antigen / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D008213:Lymphocyte Activation; D012190:Retroviridae / Q000235:genetics; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000821:virology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology; D014161:Transduction, Genetic / Q000379:methods
chemical_list: D005839:Gentamicins; D015789:HLA-A2 Antigen; C010680:antibiotic G 418