The use of polyclonal antilymphocyte serum (ALS) treatment and donor specific bone marrow infusion (BM) has been extremely effective in inducing specific tolerance to tissue allografts in rodents. This model has also been successful in inducing tolerance to immediately vascularized whole organ renal allografts in dogs, non-human primates and possibly man. The basic premise of this proposal is that multiple tolerance mechanisms are involved which appear sequentially and are interrelated as tolerance evolves and is stabilized. FACS analysis will be used to identify the sequential appearance of peripheral and central (thymic) lymphoid chimerism. Limiting dilution analysis (LDA) will be performed serially to identify the frequency of precursor cytotoxic lymphocytes and FACS analysis will be done to identify recipient T cells with TcR specific for the donor. These studies will provide evidence for clonal deletion or reduction as a mechanism in tolerance. Standard suppressor cell adoptive transfer assays will be done serially to identify active suppressor cell mechanisms. Depletion experiments using specific monoclonal antibodies prior to adoptive transfer will be employed to identify the source and phenotype of the putative suppressor cells. Serial lymphokine production in vitro by lymphocytes from tolerant animals (IL-2, IL-4, IL-10, IFN-g) will be performed to determine if tolerance is associated with altered lymphokine production at any time. Parallel studies in tolerant recipients will determine the total T helper cell, Th1 and Th2 helper cell frequencies by modified LDA analysis to see if tolerance is associated with differentiated T helper cell activation. Related studies will seek to modify tolerance by appropriately timed exogenous lymphokine (IL-2, IFN-g) administration based on deficiencies of lymphokine production identified in in vitro studies. Since the active cells in BM that induce tolerance are of stem cell lineage, experiments will be performed to attempt to augment BM tolerogenicity by treatment with lymphokines in vivo and in vitro (IL-3, GM-CSF) known to modify stem cells. Since rapamycin markedly augments the tolerance induced by ALS and BM, experiments will be done to examine if and how the mechanism(s) associated with tolerance in ALS, BM mice are altered in ALS, BM mice given rapamycin. The focus of this grant proposal is elucidation of the mechanisms involved in the ALS, BM tolerance model. Detailed understanding of the mechanism(s) should provide important information to permit successful application of this tolerance protocol to clinical transplantation.
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