Transcriptome of retrovirally transduced CD8+ lymphocytes: influence of cell activation, transgene integration, and selection process.

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.

General practice and the Internet revolution. Use of an Internet social network to communicate information on prevention in France.

The popularity of social networks and the huge number of exchanges have made them immensely important for the communication of information. This French study explored prevention in hereditary breast cancer using a social Internet network to communicate information. The principal objective was to inform French women aged from 20 to 50 years, using the social network Facebook, about the warning signs of breast cancer in cases of a predisposition to the disease due to a genetic mutation. The secondary objectives were to inform people about screening. An information page entitled "hereditary breast cancer: and if I was concerned?" was distributed in 3 different ways: from friend to friend, via groups of persons, and by targeted advertising. Four articles and 11 messages were distributed over 27 days. The total number of visits for this period amounted to 1019. A total of 81 percent of the Internauts were women and 55 percent of the visitors were aged between 25 and 44 years. Other information campaigns concerning public health issues could be conducted using this tool. A legal framework is necessary to preserve the quality of the medical information provided. This new means of communication, used for prevention purposes, will add to other frequently used methods of communication.

IL-6 promoter polymorphism -174 is associated with new-onset diabetes after transplantation.

New-onset diabetes after transplantation (NODAT) is a serious complication of transplantation. This study tested whether IL-6 production capacity may influence the development of NODAT in two different groups of patients. The occurrence of NODAT was analyzed with respect to IL-6 gene promoter polymorphism at position -174 (G-->C) and other relevant risk factors retrospectively in 217 renal transplant recipients and prospectively in 132. A linear increase in both circulating IL-6 (P = 0.09) and C-reactive protein (an indicator of basal IL-6 secretion; P = 0.03) concentrations from the CC genotype to the GG genotype was observed. In the multivariate model, the CC genotype was associated with a decreased risk for NODAT compared with the GG genotype in the two cohorts. Homeostasis Model Assessment for Insulin Resistance also revealed lesser insulin sensitivity in the GG carriers than in the CC carriers (2.15 +/- 2 versus 1.32 +/- 1.03; P = 0.03). Subgroup analysis showed that the influence of IL-6 gene promoter polymorphism on the development of NODAT was restricted mostly to overweight patients. These results highly suggest that IL-6 production capacity influences the development of NODAT and that diabetes-inducing drug administration should be limited in overweight patients who carry the GG genotype.

Retrovirus-mediated gene transfer in polyclonal T cells results in lower apoptosis and enhanced ex vivo cell expansion of CMV-reactive CD8 T cells as compared with EBV-reactive CD8 T cells.

To modulate alloreactivity after hematopoietic stem cell transplantation, "suicide" gene-modified donor T cells (GMCs) have been administered with an allogeneic T-cell-depleted marrow graft. We previously demonstrated that such GMCs, generated after CD3 activation, retrovirus-mediated transduction, and G418 selection, had an impaired Epstein-Barr virus (EBV) reactivity, likely to result in an altered control of EBV-induced lymphoproliferative disease. To further characterize the antiviral potential of GMCs, we compared the frequencies of cytomegalovirus (CMV)-specific CD8+ T (CMV-T) cells and EBV-specific CD8+ T (EBV-T) cells within GMCs from CMV- and EBV-double seropositive donors. Unlike anti-EBV responses, the anti-CMV responses were not altered by GMC preparation. During the first days of culture, CMV-T cells exhibited a lower level of CD3-induced apoptosis than did EBV-T cells. In addition, the CMV-T cells escaping initial apoptosis subsequently underwent a higher expansion rate than EBV-T cells. The differential early sensitivity to apoptosis could be in relation to the "recent activation" phenotype of EBV-T cells as evidenced by a higher level of CD69 expression. Furthermore, EBV-T cells were found to have a CD45RA-CD27+CCR7- effector memory phenotype, whereas CMV-T cells had a CD45RA+CD27-CCR7- terminal effector phenotype. Such differences could be contributive, because bulk CD8+CD27- cells had a higher expansion than did bulk CD8+CD27+ cells. Overall, ex vivo T-cell culture differentially affects apoptosis, long-term proliferation, and overall survival of CMV-T and EBV-T cells. Such functional differences need to be taken into account when designing cell and/or gene therapy protocols involving ex vivo T-cell manipulation.