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Title: In vitro expansion of donor-derived CMV-specific T lymphocytes for adoptive cellular therapy : optimisation of antigen presentation by dendritic cells, characterisation of the culture output, and adaptation of culture conditions to CMV-naïve donors
Author: Verfuerth, Stephanie
ISNI:       0000 0001 3546 2027
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2006
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Cytomegalovirus infection adversely affects the survival of haemopoietic stem cell transplant recipients, despite antiviral chemotherapy. As an alternative treatment, adoptive cellular therapy can transfer specific immunity from donor to recipient, avoiding drug side effects. A range of methods for the isolation and/or enrichment of CMV-specific donor- derived T cells have been explored. Our cell product comprises donor-derived CMV-specific T cells, enriched through co- culture with CMV antigen-presenting MoDC. These cells are currently assessed in two ongoing clinical studies. To further optimise the co-culture procedure, the effects of various mediators of DC maturation on DC immunophenotype and their capacity for induction of antigen-specific T cell proliferation were evaluated. Whereas DC matured with a cytokine cocktail appeared to be the most mature as defined by surface maturation marker expression, only exposure to CD40L gave rise to the functionally most effective MoDC, leading to a reduction in the required co-culture time. Antigen loading and timing of cryopreservation steps were also optimised experimentally. Knowing the composition of cultured cell products may result in improved protocols, and could help interpret data from clinical studies. The proportion of T cells binding HLA- CMV-peptide tetramers and the proportion of T cells secreting IFN-y in response to restimulation increased form pre- to post-culture. However, in spite of the occurrence of massive T cell proliferation in response to CMV antigen, the majority of post-culture T cells neither bound tetramer nor secreted IFN-y. There was partial overlap between tetramer-binding and IFN-y-secreting CD8+ T cell populations, as indicated by TCR CDR3 spectratyping. IFN-y secreting post-culture T cells were capable of further divisions, as verified through non-specific cytokine-induced proliferation. Induction of cell products from CMV-naive donors continues to remain a challenge. Altered culture conditions resulted in proliferation in some CMV seronegative donor cultures. Culture output did not bind tetramers, but IFN-y secretion in response to CMV antigen, and skewing of post-culture spectratypes were demonstrated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available