Functional and molecular characterisation of murine CD4+CD25+ regulatory T cells

CD4+CD2S+ regulatory T cells (Tregs) are naturally occurring lymphocytes that play a central role in tolerance, autoimmune diseases, transplantation, tumour immunology and infectious diseases. Despite the numerous studies carried on this' subpopulation of Tregs, the mechanisms of action of these cells still remain elusive. This project is focused on the functional and molecular characterisation .of murine Tregs with the hypotheses that they mediate suppression by contact-dependent inhibitory signal. A sensitive assay of Treg function in vitro was developed, based on the co-culture of CD2S+ and CD2S- CD4+ T cells with anti-CD3/CD28-coated DynaBeads�®. Potent, titratable suppression of both proliferation and a range of cytokines in culture supernatants - including IL-2, IL-4, IL-S, IFNy and 1NFa. - was demonstrated. This assay has subsequently been used to measure in vitro Treg function using a complex mathematical model - dev~loped in-house - showing that CD4+CD2S+ Tregs restrain the size of a co-cultured CD4+CD2S- T cell population by the simultaneous suppression of cell division and induction of cell death, mediated by both the intrinsic and extrinsic pathways. of apoptosis, using dilution of CFSE as a read-out. These studies have been complemented by the proteomic examination of freshly isolated and anti-CD3/CD28 Dynabead@-activated CD2S+ and CD2S- CD4+ T cells. A number of complex proteomic techniques have been mastered, such as liquid IEF in the first dimension, which were novel in the context of Tregs biology. In addition, new differentially expressed proteins in Tregs were discovered. In particular, a protein, 14.3.3 that may play a hitherto unrecognised role in Treg function, was identified. Overall, these studies suggest several new perspectives on Treg biology and new paths of exploration.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Development of a cross-platform biomarker signature to detect renal transplant tolerance in humans.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients