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.