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Impact of Retroviral Gene Transfer on T Cell Phenotype and Function

, : Impact of Retroviral Gene Transfer on T Cell Phenotype and Function. Blood 100(11): Abstract No 2562, November 16

T cell suicide gene therapy has been used to treat leukaemic relapse and lymphoproliferative disease in patients undergoing allogeneic haematopoietic stem cell transplantation for haematological malignancies. Initial clinical trials supported the notion that T cells could be genetically modified to express the herpes simplex thymidine kinase (HSVTK) and used for their beneficial anti-tumour effects, but safely eliminated by the administration of Ganciclovir in the event of graft versus host disease. More recently, there have been concerns about the functional potential of T cells following the process of retroviral transduction and extended periods of ex-vivo manipulation. The incidence of GVHD following the infusion of such T cells has been lower than anticipated, and the risk of viral complications increased. It has been suggested that shortened periods of ex-vivo culture, and more physiological pre-activation may help preserve function. We have assessed the impact of an optimised transduction procedure on primary T cells pre-activated for 48-72 hours with a combination of anti-CD3 and anti-CD28 monoclonal antibodies and low levels of IL2. Cells were exposed to two rounds of exposure to retroviral vector encoding a mutant HSVTK and the truncated low affinity nerve growth factor receptor gene (under the control of the SFFV 5'LTR). Transduced cells were readily enriched to >95% purity by a single round of selection using magnetic beads, and the procedure was completed in under 7days. Tracking with CFSE staining showed that around 7 cell divisions occurred over this period and phenotype analysis revealed that the T cells were highly activated, and predominantly of the CD45RO memory phenotype. There was no skewing of the T cell receptor Vbeta repertoire and previously expanded clones were preserved. Suicide gene function was confirmed and the cells were highly sensitive to both Ganciclovir and Aciclovir. However, when transduced T cells from CMV seropostive donors were cultured with autologous CMV pulsed dentritic cells, rapid proliferation was followed by extensive apoptosis and loss of responder populations by the second week of culture. Recovery of sufficient viable CTLs allowed comparisons between transduced and non-transduced effectors in cytotoxicity assays. It appears that although the T cell repertoire remains intact using optimised pre-stimulation conditions the survival of previously activated cells is markedly reduced in extended culture.


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