title: Transcriptome of retrovirally transduced CD8+ lymphocytes: influence of cell activation, transgene integration, and selection process.
abstract: A suicide gene introduced by retroviral means can allow in vivo control of alloreactivity mediated by donor gene-modified T cells (GMTC) after allogeneic hematopoietic stem cell transplantation. The present study establishes the transcriptomic profile of GMTC prepared according to the GMTC production process used in our clinical trial (activation/selection methods, CD3/NeoR), which was previously demonstrated to induce phenotypical and functional alterations. This transcriptomic profile was compared with that of GMTC prepared by a novel process (CD3-CD28/DeltaNGFR-MACS) that limits alterations. Using a human pan-genomic microarray and GeneSpring software, we determined the gene expression profiles of CD8+ T cells from four healthy donors before and after the different steps required for gene modification. This analysis revealed that the gene expression pattern of GMTC is affected mainly by the activation step. Specific analysis of GMTC production processes showed that DeltaNGFR-MACS selection combined with CD3-CD28 activation limits the aberrant expression of genes involved in immunological functions and apoptotic pathways. Furthermore, our results indicate a limited risk of oncogenesis associated with retroviral-mediated gene transfer in CD8+ cells, a lower perturbation of the cell cycle regulation pathway after CD3-CD28 activation than after CD3 activation, and no significant involvement of the DeltaNGFR transduction signaling pathway when DeltaNGFR is used for selection. Moreover, genes that might be targeted to limit T cell functional alterations after ex vivo manipulation and culture were identified. These findings should be relevant to further adoptive T cell immunotherapy trials using ex vivo-expanded, gene-modified or unmodified T cells.
authors.affiliation: INSERM U645, Besançon, France. marina.deschamps@efs.sante.fr
authors.forename: Marina
authors.initials: M
authors.lastname: Deschamps
authors.forename: Eric
authors.initials: E
authors.lastname: Robinet
authors.forename: Jean Marie
authors.initials: JM
authors.lastname: Certoux
authors.forename: Patricia
authors.initials: P
authors.lastname: Mercier
authors.forename: Delphine
authors.initials: D
authors.lastname: Sauce
authors.forename: John
authors.initials: J
authors.lastname: De Vos
authors.forename: Nicolas
authors.initials: N
authors.lastname: Montcuquet
authors.forename: Mark
authors.initials: M
authors.lastname: Bonyhadi
authors.forename: Thierry
authors.initials: T
authors.lastname: Rème
authors.forename: Pierre
authors.initials: P
authors.lastname: Tiberghien
authors.forename: Christophe
authors.initials: C
authors.lastname: Ferrand
mesh_terms: D000328:Adult; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D006801:Humans; D008213:Lymphocyte Activation; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D011110:Polymorphism, Genetic; D012190:Retroviridae / Q000235:genetics*; D019076:Transgenes