Although non-specific immunosuppression is clinically successful, it is associated with serious complications. Thus, tolerance remains an important goal. Tolerance frequently involves multiple sequential mechanisms. Polyclonal antilymphocyte serum (ALS) and donor bone marrow (BM) has induced tolerance in several species and is currently undergoing clinical trial. Rapamycin given after ALS and BM dramatically increases tolerance. This observation provides an opportunity to study the mechanisms involved in tolerance induction and maintenance in a clinically relevant model. Animals (mice) will be rendered tolerant by ALS, BM and Rapamycin and studied sequentially in the central and peripheral lymphoid tissues for chimerism (FACS analysis), the frequency of recipient tolerogen reactive (pCTLs) cells (limiting dilution analysis), the percentage of recipient cells with TcR specific for tolerogen (Mls-1 differences) and suppressor cells (standard transfer assays). Suppressor cell source (host or donor), cell type and phenotype will be identified by depletion steps using specific monoclonal antibodies (mAb) prior to transfer. Differential activation of Th1 and Th2 helper cells will be determined by sequential analysis of specific cytokine secretion (IL-2, INF-g, IL-4, IL-10) in MLC supernatants (ELISA assays) and of specific cytokine mRNA of lymphoid tissues using reverse transcriptase polymerase chain reaction. These studies should demonstrate what mechanisms function at various stages of tolerance in this model. In adult thymectomized recipients given ALS and BM, transplantation of donor thymus (ThyTx) increases tolerance. This observation is unique in that adult thymus is used in association with relatively minimal (ALS) immunosuppression. Experiments will study the type of thymus graft (normal, irradiated, cultured, thymocyte suspension) and the optimal conditions required for tolerance induction with ALS and BM. Sequential analysis of the various mechanisms of tolerance will be performed to define if and how the mechanisms of tolerance are altered in the ThyTx recipients. Additional experiments will attempt to increase tolerance after ALS, BM and Rapamycin with further manipulations of the recipient or donor thymus. These studies include attempts to augment tolerance after ALS, BM and Rapamycin by intrathymic injection of additional donor antigen , by donor ThyTx, and by reinforcing the tolerance with additional sequential injections of BM with or without additional immunosuppression. These experiments are designed to study the mechanism(s) involved in induction and maintenance of tolerance. Knowledge of the sequential mechanisms involved should lead to strategies to augment tolerance with strong potential for clinical application.
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