Mechanistic insights into novel cellular therapy strategies for the treatment of corneal allograft rejection
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It has previously been demonstrated by our lab that tolerogenic dendritic cells (tDCs) prolong corneal allograft survival. This study revealed that tDCs had significantly higher levels of sialic acid on the cell surface compared to immature DCs (iDCs). The first part of this work focused on investigating how the generation of tDCs by dexamethasone changed the glycosylation profile of tDCs. Dendritic cellular therapies and dendritic cell vaccines show promise for the treatment of autoimmune diseases, the prolongation of graft survival in transplantation and in educating the immune system to fight cancers. Cell surface glycosylation plays a crucial role in the cell-cell interaction, uptake of antigens, migration, and homing of DCs. Glycosylation is known to change with environment and the functional state of DCs. tDCs are commonly generated using corticosteroids including dexamethasone, however, to date little is known on how corticosteroid treatment alters glycosylation and what functional consequences this may have. Here, we present a comprehensive profile of rat bone marrow derived dendritic cells, examining their cell surface glycosylation profile before and after Dexa treatment as resolved by both lectin microarrays and lectin-coupled flow cytometry. We further examine the functional consequences of altering cell surface glycosylation on immunogenicity and tolerogenicity of DCs. Dexa treatment of rat DCs leads to profoundly reduced expression of markers of immunogenicity (MHC I/II, CD80, CD86) and pro-inflammatory molecules (IL-6, IL-12p40, iNOS) indicating a tolerogenic phenotype. Moreover, by comprehensive lectin microarray profiling and flow cytometry analysis we show that sialic acid (Sia) is significantly up regulated on tDCs after Dexa treatment and that this may play a vital role in the therapeutic attributes of these cells. Interestingly, removal of Sia by neuraminidase treatment increases the immunogenicity of iDCs and also leads to increased expression of pro-inflammatory cytokines while tDCs are protected from this increase in immunogenicity. These findings may have important implications in strategies aimed at increasing tolerogenicity where it is advantageous to reduce immune activation over prolonged periods. These findings are also relevant in therapeutic strategies aimed at increasing the immunogenicity of cells, for example, in the context of tumour specific immunotherapies. It has previously been demonstrated by our lab that the i.v. infusion of allogeneic MSCs prolong corneal allograft survival, however, syngeneic MSCs fail to do so. The - V - second part of this study focused on the optimisation of a pre-activation strategy to enhance the immunomodulatory properties of syngeneic MSCs. While TNF-α + IL-1β MCSs showed enhanced potency in vitro, they were not successfully in prolonging corneal allograft survival. It was determined that TGF-β1 pre-activated MSCs (TGF-β MSCs) were the most potently immunosuppressive. TGF-β MSCs potently suppress both CD4+ and CD8+ proliferation, increase the numbers of Tregs in co-cultures and significantly modulated MHC I, MHC II, CD80 and CD86 on activated macrophages. Further characterisation of TGF-β MSCs demonstrated increased expression of CD73 on the cell surface with increases in mRNA for ptgs2 (Cox2) and in addition increased levels of PGE2 were detected in the supernatants of T lymphocyte co-cultures. As a result, TGF-β MSCs were chosen for in vivo administration. The third part of this study focused on how in vitro pre-activated TGF-β MSCs prolonged rejection free survival of corneal allografts after administration. TGF-β MSC treated animals presented with a survival rate of 70% (n=13) compared to 25% (n=14) in the untreated MSC group. Prolongation of graft survival was associated with; (i)increased Treg populations in the draining lymphocyte nodes (DLNs) and lungs of TGF-β MSC treated animals, (ii) increases in regulatory B cell populations in the DLNs and spleens of TGF-β MSC treated animals and (iii) decreases in APC populations in the DLNs, lungs and spleens of TGF-β MSC treated animals. Finally, we confirmed that TGF-β MSCs mediated their effects via canonical SMAD2/3 signalling, that the potent immunosuppressive effects mediated by TGF-β MSCs was contact dependent and that CD73 and more significantly PGE2 (via EP4) play a vital role in TGF-β MSC mediated immunosuppression of T lymphocytes. In summary, this study demonstrates that TGF-β enhances the ability of syngeneic MSCs to prolong corneal allograft survival. In vivo, TGF-β MSC therapy was associated with increased proportion of regulatory cells which resulted in prolongation of graft survival. The results in this study point toward PGE2 as the main mediator of TGF-β MSC immunosuppression. Therefore, this study identifies the anti-inflammatory cytokine TGF-β as a pre-activation strategy to enhance syngeneic
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