| Summary: | The adoptive transfer of human regulatory T cells (Tregs) in transplantation offers an attractive therapeutic alternative in the current struggle to improve long-term outcomes. CD4+CD25+FOXP3+ (Tregs) play an important role in immunoregulation and have been shown in animal models to promote transplantation tolerance. Phase I trials in bone marrow transplantation and type I diabetes have already shown that ex vivo expanded Tregs have an excellent safety profile, which is encouraging for the broader application of these cells. The clinical trials initiated at King’s College London, ThRIL and the ONE study, are the leading trials of autologous Treg immunotherapy worldwide in the setting of liver and kidney transplantation, respectively. The success of these trials is reliant on the implementation of protocols that comply with Good Manufacturing Practice (GMP) guidelines centered on the successful isolation and expansion of a functional and stable human Treg population from prospective transplant recipients. The main focus of this thesis has been the adoptive cell therapy of Tregs in the context of liver transplantation. In this regard, it was first pertinent to study the biology of Tregs from patients with alcohol related cirrhosis (ARC), representing the majority of patients on the liver transplant waiting list. As such, an in-depth phenotypic and functional characterisation of the isolated Tregs from these patients was carried out. The results shown herein demonstrate that Tregs from ARC patients display impaired suppressive function. Based on this finding, a series of experiments were conducted in order to delineate the mechanism behind the apparent defect in Treg suppressive function. This led to a novel discovery of a defect in the expression of the cytoprotective enzyme, heme oxgenase-1 (HO-1), by patient Tregs, in correlation with the apparent Treg dysfunction. Subsequently, adherence to a GMP compatible 36-day expansion protocol resulted in the enrichment of a pure population of Tregs (91.3% CD4+CD25+ and 0.153% CD8+ cells), reaching numbers needed for their clinical translation. In addition, the protocol ensured the maintenance of FOXP3 expression (94.6% of the CD4+CD25+ cells expressed FOXP3 at the end of expansion) with an increase in the frequency of FOXP3Hi cells throughout expansion. Culture in the presence of rapamycin also confirmed the stability of the expanded Tregs, whereby the cells did not convert to Th17 cells when cultured in the presence of pro-inflammatory stimuli.
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