Retrovirus-mediated gene transfer in primary T lymphocytes impairs their anti-Epstein-Barr virus potential through both culture-dependent and selection process-dependent mechanisms.
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.
Sauce
Delphine
D
INSERM E-0119/UPRES EA-2284, Etablissement Français du Sang- Bourgogne/Franche-Comté, Besançon, France.
Bodinier
Marie
M
Garin
Marina
M
Petracca
Bruno
B
Tonnelier
Nicolas
N
Duperrier
Anne
A
Melo
Junia V
JV
Apperley
Jane F
JF
Ferrand
Christophe
C
Hervé
Patrick
P
Lang
François
F
Tiberghien
Pierre
P
Robinet
Eric
E
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
D005839:Gentamicins; D015789:HLA-A2 Antigen; C010680:antibiotic G 418