Modulation of T-cell function using pharmacological agents and retroviruses

• Main Author: Breslin, Elaine Mary
• Published: Cardiff University 2005
• Subjects: 616.07
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.583599

Cellular immunotherapy aimed at reconstituting T-cell responses to Epstein-Barr Virus (EBV) has proved extremely effective in the treatment of Post Transplant Lymphoproliferative Disorders. The application of gene therapy to improve the efficacy and survival of T-cells remains a major goal in the development of an optimal immunotherapeutic strategy and current research indicates that the use of genetically modified T-cells in adoptive transfer is a feasible therapy for the clinic. Immunosuppresive drugs such as rapamycin and CsA inhibit T-cell proliferation and may impair the function and efficacy of adoptively transferred T-cells in vivo. Our objective of this project was to enhance the efficacy and survival of Primary Human T-cells in vitro by modification with a retroviral vector containing an immunosuppressive resistant gene. Cell proliferation experiments using a CFSE cell protein dye elucidated the optimal cell stimulation requirements and the optimal day to target Primary T-cells for retroviral infection. Two distinct retroviral infection systems were established in the laboratory and applied to transduce Kit225 T-cells, Primary Human T-cells and EBV- Cytotoxic T-cells. These T-cells were modified with a retrovirus to confer rapamycin resistance (pBabe mTOR S2035T) or a retrovirus to confer CsA resistance (pBabe CNA V314R). The data showed that both the pBabe mTOR S2035T virus and the pBabe CNA V314R virus could confer immunosuppression resistance in the modified T-cell but that this was not demonstrated for every infection. The data also showed that the modification of Primary Human T-cells with these viruses generated cells with a growth advantage and increased cytokine production compared to Primary T-cells modified with control pBabe puro virus. This suggests that the modification of PBLs with the immunosuppressive resistance genes mTOR S2035T or CNA V314R may be a useful strategy to improve the efficacy and function of T-cells. During the course of this project, experiments also showed that the PI3-Kinase inhibitor LY294002 and rapamycin co-operate to inhibit T-cell proliferation. Taken together, the results demonstrated in this project highlight the importance of the TOR and calcineurin pathways in regulating T-cell proliferation, and suggest strategies to improve both immunosuppression and cellular immunotherapeutic regimes.