This proposal is designed to develop new methods using dendritic cells (DC) to induce allotolerance in a mouse model. The proposal is based on the observation that i.v. infused thymic DCs selectively home to thymus and induce alloantigenic tolerance as compared to splenic DCs that show impaired thymic homing capabilities and do not have the ability to induced tolerance. These data are corroborated by the ability of thymic DCs to selectively home in the thymus after adoptive transfer leading to tolerance. The overall goals of this proposal are to 1) understand the mechanism(s) of the tolerance induced by allogeneic DCs, 2) define the DC subpopulation with the highest tolerogenic capability, and 3) develop the technical methodologies designed to optimize the use of adoptive transfer of DCs to induce allotolerance. The focus of this proposal is to further develop methodologies that will render DC tolerogen therapies clinically feasible. In particular, they propose to fully delineate mechanisms of effects in this system, improve methods for identification and isolation of DC with therapeutic potential, increase the duration of tolerance, explore the phenotypes necessary for unique thymic homing, and develop in vitro techniques to propagate or immortalize these cells. In addition to basic implications, they propose that their findings may ultimately have significant clinical applications, with considerable practical advantages over other methods for intrathymic introduction of neo-antigens to obviate allograft rejection and other T-cell-mediated disease processes.