The goal of this proposed study is to develop an effective strategy for induction of durable human organ transplant tolerance using a renal transplant model to serve as a preclinical bridge with direct relevance to clinical organ transplantation. Our preliminary studies in a novel posttransplant strategy (posttransplant rabbit antithymocyte globulin (RATG), subtotal fractionated limited lymphoid irradiation and splenectomy (LLlI/splx), followed by infusion of a subpopulation of donor bone marrow cells (DBMC)) have demonstrated development of specific donor unresponsiveness, chimerism, and acceptance of life-sustaining, MHC class I and II disparate renal allografts (KTx) in rhesus monkeys for >3.5 years with no immunosuppression given before or after the first week posttransplant. Although the results are compelling, preclinical issues persist that require resolution to promote safe application to human transplantation. The proposed studies will examine a hypothesis that early, effective inhibition of direct and indirect pathways of alloantigen presentation by a combined low morbidity therapeutic approach using pluripotential DBM stem cells and short-term recipient immunosuppression will facilitate stable chimerism and tolerance without chronic immunosuppressive therapy.
The specific aims of the proposal are: (1) To investigate the capacity of DR-/dim CD3-/dim CD34+ hematopoietic stem cells purified from donor bone marrow (DBM-HSC) to induce stable chimerism and tolerance in short-term immunosuppressed KTx recipients: To accomplish this we will use colony stimulating factors to establish optimal DBM-HSC recovery and culture conditions for ex vivo propagation of DBM-HSC; examine the activity of freshly isolated and precultured DBM-HSC preparations for induction of chimerism and KTx tolerance; determine if stable tolerance induction will require confusion of pure CD34+ DBM-HSC with the CD8+ DBM fraction that mediates veto activity in rhesus monkeys. (2) To examine the role of DBM-HSC derived chimerism in maintenance of tolerance: These studies will (A) examine the lineage and levels of chimeric cells in various tissues in relation to immunological unresponsiveness; (B) directly examine the role of DBM-HSC derived chimerism in maintenance of tolerance using DBM-HSC that are transduced with HSV-1 thymidine kinase before infusion to render them susceptible to subsequent destruction by ganciclovir in the tolerant recipient; (3) To assist transition of this tolerance strategy to clinical organ transplantation: Using the optimal DBM-HSC protocol, we will examine the effects of recipient cytokine pretreatment, timing of DBM-HSC infusion, adjunctive short term immunosuppressive drugs, and mobilized PBLHSC to facilitate clinical application. The information gained in these studies is expected to guide successful application of tolerance induction to human transplantation.
|Goldstein, D R; Thomas, J M; Kirklin, J K et al. (2001) An essential role for natural killer cells in augmentation of allograft survival mediated by donor spleen cells. Transplantation 72:954-6|
|Goldstein, D R; Thomas, J M; Kirklin, J K et al. (2001) Indefinite allograft survival mediated by donor bone marrow is dependent on the presence of a functional CD95 (Fas) gene in recipients. J Heart Lung Transplant 20:1132-5|
|Goldstein, D R; Chang, T; Sweeney, S D et al. (2000) Enhanced allograft survival induced by posttransplant donor spleen cell infusion occurs via a mechanism that is distinct from the mechanism of enhancement by donor bone marrow. Transplantation 69:1020-2|
|Contreras, J L; Eckhoff, D E; Cartner, S et al. (2000) Long-term functional islet mass and metabolic function after xenoislet transplantation in primates. Transplantation 69:195-201|
|Goldstein, D R; Chang, T; Sweeney, S D et al. (2000) A differential requirement for CD8+ donor cells in the augmentation of allograft survival by posttransplantation administration of donor spleen cells and donor bone marrow cells. Transplantation 70:1068-73|
|Pittman, K; Berzina, P; Contreras, J et al. (1999) Synergy of class I and class II MHC deficiency (C1D and C2D) islets and early short-term suppression in achieving long-term islet graft survival and tolerance induction. Transplant Proc 31:635-6|
|Thomas, F; Contreras, J; Bilbao, G et al. (1999) An improved technique for isolating pig islets: the importance of tensegrity in islet long-term culture viability. Transplant Proc 31:633-4|
|Llyung, T; Contreras, J; Thomas, F et al. (1999) Long-term (10-month) pig islet xenograft tolerance using measured total lymphoid irradiation, splenectomy, short-term rabbit antithymocyte globulin, and deoxyspergualin without long-term immunosuppression. Transplant Proc 31:962-3|
|Thomas, F T; Ricordi, C; Contreras, J L et al. (1999) Reversal of naturally occuring diabetes in primates by unmodified islet xenografts without chronic immunosuppression. Transplantation 67:846-54|
|Thomas, F; Contreras, J; Ricordi, C et al. (1999) Reversal of naturally occurring insulinopenic diabetes and induction of isolated islet xenograft tolerance in a preclinical study. Transplant Proc 31:637-8|
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