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Title: Generation of calcineurin inhibitor resistant EBV-CTLs for the treatment of post transplant lymphoma
Author: Brewin, J. J.
ISNI:       0000 0004 2726 9990
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2010
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Epstein Barr Virus driven post transplant lymphoproliferative disease (PTLD) is a serious complication following either stem cell or solid organ transplantation (SOT), occurring as a result of suppressed cellular immunity. Adoptive transfer of EBV specific cytotoxic T cells (EBV-CTLs) is effective as both prophylaxis and treatment for PTLD following stem cell transplantation, but is less effective when applied to PTLD after SOT. It is likely that the continued pharmacologic immunosuppression designed to prevent graft rejection compromises the function of infused EBV-CTLs. To address this issue, we have generated calcineurin (CN) mutants that do not bind the immunosuppressants Tacrolimus (FK506) and Cyclosporin A (CsA), therefore conferring resistance to these CN inhibitors. A panel of 54 such mutants was designed, generated and screened in a Luciferase expression assay, with identification of those capable of resisting suppression with FK506, CsA or both. Subsequently, in assays utilising both the Jurkat T cell line and primary human EBV-CTL lines, mutant CNa12 conferred resistance to CsA, mutant CNa22 conferred resistance to FK506 and mutant CNb30 rendered cells resistant to both calcineurin inhibitors. EBV-CTL lines transduced with a retroviral vector encoding these mutants retained the ability to both proliferate and secrete normal levels of interferon-\gamma in the presence of therapeutic and supratherapeutic levels of FK506 (CNa12), CsA (CNa22), or both (CNb30). The cytotoxicity, phenotype and antigen dependence of EBV-CTL lines were unaffected by expression of mutant CN. A xenogenic murine model of PTLD was established in the RAG2^{-/-}/\gamma c^{-/-}/C5^{-/-} triple knockout SCID mouse strain and the ability of EBV-CTL lines to induce tumour regression was examined. Administration of EBV-CTLs caused regression of subcutaneous LCL tumours in vivo in some donors. This model will be utilised in further investigations of transduced EBV-CTLs in vivo. The generation of EBV-CTLs that are resistant to CN inhibitors should allow effective immunotherapy in the SOT setting without risking rejection of the graft by reducing immunosuppression. Additionally this represents a generic approach to improving immunotherapy in the face of immunosuppression in other settings.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available