Targeting innate and adaptive immune responses to achieve long-term allograft acceptance following transplantation

Abstract: Organ transplantation outcomes have gradually improved over the last 50 years. The development of more effective immunosuppressive drugs has been a major contributor to this improvement. However, current immunosuppressive regimens may cause significant side effects such as kidney failure, opportunistic infections, cardiovascular morbidity and increase the risk of tumor formation. These side effects cause increased mortality, morbidity and reduce the patient’s quality of life, which obscures the long-term outcomes following transplantation. As an example, patient survival after liver transplantation has improved significantly since the 1980s, but this is mainly due to a lower mortality rate in the first year. Thus, one-year censored patient survival has not improved, which is thought to be at least partially attributable side effects of chronic immunosuppression. In addition, current immunosuppressive protocols still do not optimally protect transplanted organ grafts from the recipient’s immune responses. When a graft is transplanted, immediate non-specific innate immune reactions occur. These reactions not only could result in direct damage to the graft, but also sequentially potentiate the activation of adaptive immunity that could result in subsequent rejection. Innate reactions are particularly devastating in the field of cell transplantation, for example, pancreatic islet transplantation. When cells are transplanted into the bloodstream, an instant blood-mediated inflammatory reaction (IBMIR) occurs, which includes severe inflammation, platelet and leukocyte infiltration, resulting in thrombus formation around the grafts. Such reactions can result in more than half of the transplanted grafts being lost. Despite the need to mitigate these reactions, they are not fully accounted for in the current arsenal of immunosuppression. Primary concerns about the adaptive immunity relate to its rejection activity. However, the adaptive immune system also contains a regulatory part that may promote acceptance and tolerance of specific antigens. If these regulatory properties could be utilized to the benefit of transplanted grafts, it may be possible to minimize or, in the best case, even abolish the requirement for life-long immunosuppressive treatment. Regulatory cell therapies have recently been evaluated in pilot clinical trials and have enabled immunosuppression dose reduction in living donor kidney recipients and the possibility of complete weaning of immunosuppression in living donor liver transplantation. Further development of these protocols, including the possibility to apply them in deceased donor transplantation setting is desired. This thesis has investigated novel immune modulating transplantation protocols to target innate immunity and harness the potentially beneficial regulatory part of the adaptive immune system. Paper I investigated the efficacy of the non-hematopoietic erythropoietin analogue cibinetide in an mouse allogenic intra-portal pancreatic islet transplantation model. We showed that induction therapy using cibinetide could protect islet grafts from initial damage from the innate immunity, reducing activation of adaptive immunity and significantly prolonging longterm graft survival. As a step towards clinical implementation, Paper II focused on evaluating the protective effect of cibinetide on human islets. The results showed that cibinetide maintained the robustness and function of human islets in a pro-inflammatory environment and that cibinetide tends to mitigate IBMIR related platelet consumption. The results of the first two papers suggests that cibinetide has the potential to abrogate innate immune responses and thereby improve the short and long-term results of pancreatic islet transplantation. In Paper III we performed a safety study of intra-portal co-infusion of autologous regulatory T cells and allogenic pancreatic islets. We concluded that it was safe to deliver regulatory cells together with islets. The latter part of this thesis focused on preparatory studies using Good Manufacturing Practice (GMP) compatible reagents and methods, applying regulatory cell therapy to recipients of deceased donor liver transplantation to minimize, or in the best case, abolish the requirement for immunosuppression. In Paper IV, we evaluated whether the clinically approved drug Cytotoxic T-lymphocyte-associated protein-4-Ig (CTLA-4-Ig, Belatacept) could be used instead of the non-approved and previously used mouse monoclonal antibodies against human CD80 and CD86 (2D10.4/IT2.2) as a costimulatory blockade in the generation of a donor-specific immunomodulatory cell product. We conclude that CTLA-4- Ig or 2D10.4/IT2.2 could be used to produce a cell product with similar cell composition and donor-specific immunomodulatory efficacy. CTLA-4-Ig, which is a clinically approved drug, is therefore preferable to use to produce donor-specific immunomodulatory cells in a GMP-compatible tolerance induction protocol. In Paper V, we evaluated three methods of procuring peripheral blood mononuclear cells from deceased donors to be used as donor antigen stimulator in the production of donor-specific immunomodulatory cells. We showed that all the methods we evaluated were feasible. However, bedside leukapheresis before an organ donation procedure had tendency of higher cell numbers procured and allows the procurement of mononuclear cells in a sterile and controlled manner, compared to the other methods.

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