Ex vivo generation of bone marrow derived regulatory dendritic cells for promotion of corneal allograft survival.

Investigations into cell based therapies for application in organ transplantation has grown significantly. Here, I describe the ex vivo generation of donor bone marrow derived dendritic cells (BMDCs) and examine the therapeutic potential of genetic and pharmaceutical modifications of BMDCs for application in allogeneic transplantation. BMDCs were generated via culturing bone marrow precursor cells with rat granulocyte macrophage-colony stimulating factor and interleukin 4 containing medium. Lentiviral gene transfer of BMDCs was examined and although successful was not a feasible approach. The treatment of BMDC with dexamethasone (Dexa) to induce an immature, maturation resistant phenotype was however, comprehensively examined in this study. BMDC and Dexa BMDC phenotype, antigen presenting cell function and their immunomodulatory properties were examined and fully characterised. Both populations displayed significant immunomodulatory properties relative to freshly isolated OX62+ DCs, including but not limited to, a significant increase in mRNA expression of programmed death-ligand 1 and indoleamine 2,3-dioxygenase. Both BMDCs and Dexa BMDCs displayed a profound impaired capacity to stimulate allogeneic lymphocytes. Moreover, in a fully MHC I/II mismatched rat corneal transplantation model (Dark Agouti (DA) to Lewis (LEW)), injection of donor (DA) derived BMDC or Dexa BMDCs (1x106cells/animal, day -7) significantly prolonged corneal allograft survival without the need for additional immunosuppression. With both cell therapies, we observed a significant reduction in the level of allograft cellular infiltration, a significant increase in the ratio of intragraft FoxP3 expressing regulatory cells and evidence of a donor specific allo-antibody response. Taken together, our comprehensive analysis demonstrates a detailed analysis of the significant therapeutic effect of donor derived BMDCs with and without glucocorticoid treatment in corneal allograft survival by modulation of the allo-immune response at the level of both the allograft and the draining lymph nodes. This body of research represents a novel therapeutic approach for the prevention of corneal allograft rejection.

Funder

Science Foundation Ireland

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Attribution-NonCommercial-NoDerivs 3.0 Ireland

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University of Galway Theses (PhD Theses)