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dc.contributor.advisorRitter, Thomasen
dc.contributor.authorMaenz, Martinen
dc.date.accessioned2011-06-14T11:33:57Zen
dc.date.available2011-06-14T11:33:57Zen
dc.date.issued2011-05-27en
dc.identifier.urihttp://hdl.handle.net/10379/2001en
dc.description.abstractThis project investigated the therapeutic potential of allo-antigen specific regulatory T-cells in the context of penetrating keratoplasty. Two methods of ex-vivo generation of T regulatory cells have been explored in this study. Using recombinant retroviruses gene transfer of the transcription factor foxp3 was achieved into allo-antigen specific primary rat T-cells. However, expansion of foxp3 gene modified cells necessary for in-vitro and in-vivo studies, was found to be too cumbersome. The second approach exploited a non-depleting anti-CD4 antibody. Allo-primed mixed lymphocyte cultures were treated with low-dose anti-CD4 antibody resulting in an outgrowth of foxp3 positive T-cells. CFSE labelled responder T-cells targeted with anti-CD4 treatment indeed confirmed preferential growth of regulatory T-lymphocytes. Tregs generated by this procedure were studied by flowcytometry and found to express high levels of CD25, Ox-40 and ICOS, confirming their suppressor T-cell phenotype. To study Treg effects on the outcome of allo-graft survival, a full allogeneic cornea transplant model was established. Two different strain combinations were tested. First, a high responder LEW-DA model was set-up but was found to be too fragile and unreliable to serve as a preclinical model. A second novel strain combination was tested and discovered to be of low-responder characteristics and remarkably robust and highly suitable for in-vivo applications. The rejection process of the BN-PVG model was comprehensively studied using flowcytometric analysis of draining lymph nodes. Moreover, a gentle digestion protocol was established to isolate viable graft infiltration lymphocytes. Applying multi-parameter FACS six distinct cell populations were observed among which were CD8+ cytotoxic T-cells, CD4+ T-cells, CD3- CD8+/- CD161high NK, CD3+ CD8+ CD161dull NK-T-cells, CD161dull large granular lymphocytes and MHC-2 positive cells. Additionally a serum analysis found evidence for IgM, IgG1 and IgG2a allo-antibodies in rejection animals. Finally ex-vivo generated Tregs were tested towards their ability to prevent allo-graft rejection. CD3 sorted regulatory T-cell preparations with direct allo-antigen specificity did not prevent or delay rejection of allogeneic corneal grafts. The implications of this finding for the clinical application of adoptive Treg therapies have been discussed in detail.en
dc.rightsAttribution-NonCommercial-NoDerivs 3.0 Ireland
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/3.0/ie/
dc.subjectImmunologyen
dc.subjectCorneaen
dc.subjectTransplantationen
dc.subjectImmune-modulationen
dc.subjectRegulatory T-cellsen
dc.titleEx-vivo generation of regulatory T-cells expressing transcription factor foxp3 and their application in cornea transplantationen
dc.typeThesisen
dc.contributor.funderIRCSETen
dc.contributor.funderSFIen
dc.local.noteThis project investigated the possibility to treat transplant rejection using a specialised subset of white blood cells (regulatory T-cells). These cells were generated in a culture dish out side a mammalian body (ex-vivo) and subsequently tested in a pre-clinical model of cornea transplantation, which is the most frequently performed transplantation procedure in humans.en
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