title: Preparation of genetically homogeneous antigen-specific thymidine kinase positive T-lymphocyte clones for the control of alloreactivity post-bone marrow transplantation.
abstract: We have previously proposed the use of HLA-specific T-cell clones transduced with a suicide gene to produce an allogeneic effect that can be controlled after allogeneic bone marrow transplantation. Procedures described so far to obtain specific T-cells transduced with a suicide gene have led to the recovery of heterogeneous polyclonal T-cells with a limited level of purity. We have therefore developed an approach to select specific T-cell clones in which the suicide transgene is inserted at a unique site of the genome, and used it to produce CD(+)-cytotoxic HLA-DP-specific T-cell clones. Immunization was performed by a one-way mixed lymphocyte culture and responder T lymphocytes were transduced at day 16, 6 days after the second stimulation. Transductions were carried out using gibbon ape leukemia virus (GALV)-pseudotyped retroviral particles harboring a bicistronic Thy-1/TK vector produced by TEFLY GA16-pKM4 clone 34 packaging cells. Three to 5 days later, CD90 immunomagnetic selection and cloning were performed on the transduced T cells. Our results demonstrate that this procedure led to the recovery of T-cell clones, the majority of which had the expected specificity and a single site of transgene insertion. Such clonotransgenic T-cell populations represent suitable tools to drive a defined alloreaction that can be controlled after bone marrow transplantation.
authors.affiliation: INSERM U463, Centre Hospitalier Universitaire de Nantes, 9 Quai Moncousu, 44093 Nantes cedex, France.
authors.forename: Béatrice
authors.initials: B
authors.lastname: Clémenceau
authors.forename: Géraldine
authors.initials: G
authors.lastname: Gallot
authors.forename: François M
authors.initials: FM
authors.lastname: Lemoine
authors.forename: David
authors.initials: D
authors.lastname: Klatzmann
authors.forename: Henri
authors.initials: H
authors.lastname: Vié
mesh_terms: D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism; D016026:Bone Marrow Transplantation; D002478:Cells, Cultured / Q000637:transplantation; D002999:Clone Cells; D041722:Genes, Transgenic, Suicide / Q000502:physiology*; D006682:HLA-DP Antigens / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007114:Immunization; D018189:Immunomagnetic Separation; D007167:Immunotherapy; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics; D008213:Lymphocyte Activation; D012190:Retroviridae / Q000235:genetics; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology; D018800:Thy-1 Antigens / Q000276:immunology / Q000378:metabolism; D013937:Thymidine Kinase / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D015703:Antigens, CD; D006682:HLA-DP Antigens; D018800:Thy-1 Antigens; D013937:Thymidine Kinase