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Title: The generation and characterisation of alloantigen reactive regulatory T cell populations from umbilical cord blood
Author: Srivastava, Saket
ISNI:       0000 0004 7227 5417
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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The use of polyclonal CD4+ CD25+ FoxP3+ regulatory T cells (Treg) have been successful in the management of acute graft versus host disease (aGvHD), a complication associated with allogeneic haematopoietic stem cell transplantation. However, polyclonal Tregs can cause broad immunosuppression of desired responses. To avoid this, the use of alloantigen reactive Tregs has been suggested, with pre-clinical data indicating that allospecific Tregs are more efficient than polyclonal Tregs in suppressing allospecific responses. This thesis demonstrates the generation and characterisation of alloantigen reactive Treg populations from umbilical cord blood (UCB). Culture conditions were established to isolate and expand CD4+ CD25- and CD4+ CD25+ lymphocytes from UCB. Phenotypic characterisation was conducted by flow cytometry, demethylation analysis of the FoxP3 gene, and TCR spectratyping. The suppressive function and allospecificity of regulatory cells was determined by their effects on responder cells stimulated with the same allogeneic or a 3rd party antigen presenting cells. The stimulation of UCB Tregs by allogeneic monocyte derived dendritic cells (MDDC) with IL-2 resulted in a 62±34-fold expansion and decreased TCR diversity. While freshly isolated UCB Tregs exhibited weak activity, expanded cells provided superior suppression of allospecific responses compared to adult Tregs. Analysis revealed the presence of CD25highCD45RO+ CD39+ Tregs that expressed increased FoxP3, CTLA-4 and Helios. CD39+ cells provided improved function compared to total allostimulated Tregs with >50% suppression obtained at a 1:256 ratio. UCB CD4+ CD25- T cells were stimulated with MDDCs, the arising CD25+ population portrayed characteristics similar to Tregs including partial FoxP3 demethylation. The suppressive function of these cells matched allostimulated Tregs, however was lost upon re-stimulation. The results described in this dissertation demonstrate the possibility to establish suppressive cells with alloantigen reactivity from UCB CD4+ T cells. UCB is of interest as a potential source of cells for therapy, and the potential use of alloreactive UCB Tregs for immunotherapy are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available