This project aims to test the hypothesis that tolerance to MHC mismatched living related kidney transplant can be effectively and safely achieved by establishing a stable immune mixed chimeric state in non-human primates using a novel non-myeloablative, helical tomotherapy-based total lymphoid irradiation (TLI) conditioning regimen followed by Mozobil + G-CSF mobilized donor hematopoietic cell infusions. Furthermore, we will test the hypotheses that the success of this protocol will depend upon "natural" maternal-fetal preconditioning of donor and recipient, and a cytokine bias of host Treg cells toward increased production of IL-10 and IL-4 in TLI/ATG recipients of HSCs. We propose to test these hypotheses by means of 2 specific aims: 1) Combined Hematopoietic Cell/Kidney Transplants: to determine the proportion of Rhesus macaques that can be withdrawn from all immunosuppressive drugs while maintaining normal graft function of MHC 1-haplotype mismatched living related donor kidney transplants. We will assess tolerance by stable kidney function and donor chimerism as a function of time after transplantation in recipients conditioned with helical tomotherapy-based TLI and rabbit anti-thymocyte globulin (ATG) and given purified donor CD34+ hematopoietic stem cells (HSC) and specified doses of donor T cells. 2) Immune Monitoring, Immunopathology and Immunocompetence: a) to determine if serial monitoring of intracellular cytokine expression including IL-4, IL-10 IFN-?, and TGF? in activated NK T cells, conventional CD4+ T cells, CD4+CD25+ Treg cells in the peripheral blood mononuclear cells (PBMCs) of recipients of combined kidney and HSC transplants supports a Th2 bias in animals with chimerism and no GVHD that allows successful withdrawal of immunosuppressive drugs after transplantation; b) to determine whether serial trans-vivo Delayed Type Hypersensitivity (tvDTH) and mixed lymphocyte reaction (MLR) analysis of tolerance on indirect and direct pathways of alloreactivity, respectively, beginning pre-transplant, can be used to predict recipients with chimerism and no GVHD allowing successful withdrawal of immunosuppressive drugs. We will also test the phenotype and function of host and donor dendritic cells (DCs) obtained after withdrawal of immunosuppressive drugs, and after a challenge with a donor and third party skin graft to assess the role of tolerogenic versus immunogenic antigen presenting cells (APCs) in graft outcome. c) to determine the degree of recipient immune competency after immunosuppressive drug withdrawal, by monitoring the incidence of microbial infection, testing the recall T cel and antibody responses to influenza virus vaccination after transplantation, as well as recall T cell responses to herpes zoster, herpes simplex, cytomegalovirus antigens, and tetanus toxoid. In addition, we will determine T cell recovery and thymic function after transplantation by measuring the levels of T cell excision circles, and naive, memory, and regulatory CD4+ and CD8+ T cells in the blood at serial time points. To test for donor-specific tolerance and immunocompetence to donor third party allo-antigens, donor skin grafts will be placed on the recipient.
Functional tolerance in human solid organ transplant recipients has not been consistently achieved. There is, however, great opportunity to utilize several immune tolerance mechanisms to promote this challenging objective. One of the most promising is creation of a stable mixed chimeric host immune environment, comprising both recipient and donor immune elements resulting from infusion of donor hematopoietic stem cells (HSC) and T cells, to create tolerance to the transplanted solid organ.
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