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Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti–Epstein-Barr virus potential through both culture-dependent and selection process–dependent mechanisms

2002; Elsevier BV; Volume: 99; Issue: 4 Linguagem: Inglês

10.1182/blood.v99.4.1165

1528-0020

Delphine Sauce, Marie Bodinier, Marina I. Garín, Bruno Petracca, Nicolas Tonnelier, Anne Duperrier, Junia V. Melo, Jane F. Apperley, Christophe Ferrand, Patrick Hervé, François Lang, Pierre Tiberghien, Éric Robinet,

Immune Cell Function and Interaction

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-γ 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.