Preferential retroviral-mediated transduction of EBV- and CMV-specific T cells after polyclonal T-cell activation.

Graft-versus-host disease, resulting from the T cells present in allogeneic hematopoietic stem cell (HSC) inoculums, can potentially be treated if a suicide gene has been introduced into the donor T cells. However, the diversity and functionality of the transfused T-cell population, including EBV- (EBV-T) and CMV-specific (CMV-T) CD8+ T cells, which are particularly important for immunosuppressed individuals undergoing HSC transplants, are often modified by the gene transfer protocol. Here, we show that following polyclonal T-cell activation, EBV-T and CMV-T cells are preferentially transduced by oncoretroviral vectors, as compared to the bulk CD8+ T-cell population. This preferential transduction is associated with higher surface levels of PiT-2, the receptor for the amphotropic envelope with which the virions are pseudotyped. Moreover, EBV-T and CMV-T cells proliferate more extensively as compared to bulk CD8+ T cells. Thus, retroviral-mediated transduction can be biased toward a given antigenic specificity, even under conditions of polyclonal stimulation.

Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene.

Introduction of the Herpes simplex virus thymidine kinase (HSV-tk) gene into target cells renders them susceptible to killing by ganciclovir (GCV). We are studying the use of HSV-tk-transduced T lymphocytes in the context of hematopoietic stem cell transplantation. We have previously shown, in vitro and in vivo, the occurrence of transduced cells resistant to GCV due to a deletion within HSV-tk. This deletion, a consequence of the presence of cryptic splice donor and acceptor sites, originates in the retroviral producer cell. Here we adopt two different methods that introduce third-base degenerate changes at the cryptic splice sites and so prevent splicing. Consequently, the HSV-tk protein is unaltered and the sensitivity of the target cells to GCV is preserved. The use of this mutated HSV-tk should reduce the likelihood of the development of resistant genetically modified cells during clinical trials.

Allogeneic peripheral blood stem cell transplantation results in less alteration of early T cell compartment homeostasis than bone marrow transplantation.

Since low T cell counts evaluated 1 month after allogeneic bone marrow transplantation (BMT) are associated with an increased risk of leukemia relapse (Powles et al., Blood 1998; 91: 3481-3486), we compared, in a randomized multicentric clinical study, the peripheral blood cells obtained 30 days after allogeneic BMT vs allogeneic G-CSF-mobilized peripheral blood stem cell transplantation (BCT) in an HLA-identical setting. T cell counts were higher 30 days after BCT (718+/-142 cells/microl, n = 20) than after BMT (271+/-53 cells/microl, n = 26, P = 0.006). However, T cells were less activated after BCT than after BMT, as demonstrated by a lower expression level of CD25 and a lower percentage of HLA-DR+ and CD95+ T cells. Furthermore, CD4+, CD8+ and CD45RA+ post-BCT T cell counts correlated with the number of cells infused with the PBSC graft, while such a correlation was not observed between post-BMT counts and BM graft cell numbers, suggesting that the intensity of post-transplant peripheral lymphoid expansion and/or deletion differed between BCT and BMT. A comparison of the input of T cells expressing different CD45 isoforms with the post-transplant cell recovery further confirmed that, within the CD4+ T cell subset, post-transplant expansions occurred at a higher level after BMT than after BCT, affecting mainly the CD4+ CD45RO+ subset. Altogether, our data demonstrate for the first time in a randomized setting that homeostasis of the T cell pool is less altered early after BCT than after BMT. This may have a strong impact on the graft-versus-leukemia (GVL) effect and subsequent relapse rate.