Investigations relating to the induction of immunological tolerance through spleen transplantation in miniature swine

The ultimate goal in transplantation is to achieve donor-specific immunological tolerance. The induction of tolerance would result in the long-term survival of a transplanted organ without the need for continuous immunosuppressive therapy, thus avoiding its many attendant risks and complications. In this dissertation, spleen transplantation will be investigated as a method to induce immunological tolerance. In the literature, there is evidence for a tolerogenic effect of spleen transplantation in rodents. However, the relevance of this phenomenon remained to be demonstrated in large animal models and in humans. To investigate whether the approach of spleen transplantation would be clinically applicable, we developed a model in pigs. The Massachusetts General Hospital miniature swine model was chosen, based on the fact that the Major Histocompatibility Complex (MHC) is well-defined, enabling clinically-relevant donor-recipient combinations to be investigated. During the last 30 years, considerable experience in transplantation immunology has been gained in this model. In miniature swine, a transplanted spleen (a highly immunogenic organ) is rejected when no immunosuppressive therapy is given. However, even in the presence of a full MHC-mismatch between donor and recipient, the transplanted spleen is accepted when a short course of cyclosporine is administered with pre-transplant low-dose whole body (100cGy) and thymic (700cGy) irradiation. After successful spleen transplantation, mixed hematopoietic cell chimerism is induced in the blood and lymphoid organs of the recipient. Donor cells migrate from the spleen to recipient lymph nodes, thymus, and bone marrow, in the absence of graft-versus-host-disease. Meanwhile, the donor spleen is repopulated with recipient cells. Creating a state of mixed chimerism is known to be a highly-effective means of inducing immunological tolerance. When the spleen is successfully transplanted, the recipient demonstrates donor-specific T cell tolerance in vitro. After discontinuation of immunosuppression, T cells remain unresponsive to donor antigen (on CML and MLR). Regulatory T cells are detected in the circulation of the recipient, and suppress naïve T cell responses in a donor- specific manner. Thus, the recipient remains immunocompetent. We investigated whether in vivo donor-specific tolerance develops. In pigs tolerized by spleen transplantation, donor-specific kidneys survived for many months without exogenous immunosuppression, while in control pigs (without spleen transplants) the grafts were rejected within 2 weeks. The fact that donor cells engrafted in recipient bone marrow suggests that hematopoietic stem cells were present in the donor spleen. We therefore investigated the presence of hematopoietic stem cells in adult naïve spleens, using flow cytometry and a variety of functional stem cell assays. We were able to demonstrate that the spleens of pigs, baboons, and humans harbor very primitive hematopoietic cells, in frequencies comparable to those in the bone marrow. This observation had not been described previously. We conclude that spleen transplantation has the capacity to induce donor-specific immunological tolerance. Furthermore, the spleen is a relatively rich source of primitive hematopoietic progenitor cells that may prove of clinical relevance. More studies are underway to investigate whether spleen transplantation can be introduced into clinical practise as a means of inducing tolerance to a donor- specific organ.

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