Retrovirus-mediated gene transfer in human primary T lymphocytes induces an activation- and transduction/selection-dependent TCR-B variable chain repertoire skewing of gene-modified cells.
In a clinical trial that we recently reported, a suicide gene transfer in human primary T cells required 12 days of ex vivo culture, including activation of peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody (CD3 mAb), retrovirus-mediated transduction, and selection of gene-modified cells (GMC) by G418. The aim of the present study was to determine the impact of the initial T cell activation and of the transduction/selection on T cell receptor beta variable chain (TCRBV) repertoire of GMC by using the spectratyping method. The TCRBV repertoires of nontransduced, nonselected control (Co) cells and of GMC generated after an initial stimulation with CD3 mAb, CD3/CD28 beads, or allogeneic PBMC or Epstein-Barr virus-transformed B (B-EBV) cells were compared to the ones of their corresponding PBMC. The TCRBV repertoires were skewed in Co cells generated after CD3 mAb or after allogeneic stimulation, and even more so in their corresponding GMC, demonstrating that both culture-dependent and transduction/selection-dependent events led to TCRBV repertoire alterations. However, TCRBV repertoires were not altered, or to a lesser extent, in Co cells or GMC produced after CD3/CD28 bead activation, demonstrating a protective effect on both culture-dependent and transduction/selection-dependent repertoire alterations. Thus, we suggest to replace the initial CD3 mAb stimulation by CD3/CD28 beads for the production of clinical-grade GMC in the setting of future gene therapy trials.
Coito
Sylvie
S
Laboratoire Thérapeutique Immuno-Moléculaire, INSERM E-0119/UPRES EA-2284 130, 25020 Besançon cedex, France.
Sauce
Delphine
D
Duperrier
Anne
A
Certoux
Jean-Marie
JM
Bonyhadi
Mark
M
Collette
Alexis
A
Kuehlcke
Klaus
K
Hervé
Patrick
P
Tiberghien
Pierre
P
Robinet
Eric
E
Ferrand
Christophe
C
D000911:Antibodies, Monoclonal / Q000737:chemistry; D001402:B-Lymphocytes / Q000378:metabolism / Q000821:virology; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D015704:CD4 Antigens / Q000096:biosynthesis; D016827:CD8 Antigens / Q000096:biosynthesis; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D009841:Oligonucleotides / Q000737:chemistry; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes
D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; D018076:DNA, Complementary; D009841:Oligonucleotides; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta