The major goal in solid organ transplantation is to prevent rejection yet preserve recipient immunocompetence. Bone marrow (BM)-induced tolerance for solid organ and cellular grafts has been demonstrated in neonatal and adult models in several species, including humans, and may provide the means to overcome the current limitations hampering clinical organ transplantation. However, the morbidity and mortality associated with the infusion of unmodified BM into conditioned recipients has prevented clinical application to tolerance induction. The incidence and severity of graft vs. host disease (GVHD) is directly related to the degree of antigenic disparity between donor and recipient and is attributed to donor T cells in the BM inoculum. Clinical trials depleting these primary GVH effector cells, although successful in reducing GVHD, resulted in a concurrent rise in the incidence of engraftment failure. A rare donor BM-derived cell population called the facilitating cell (FC) is necessary and sufficient to permit engraftment of purified murine SC in completely MHC-disparate recipients, without GVHD. Although the FC does express CD8alphabeta and CD3epsilon and can be inadvertently removed as a T cell, the FC lacks the conventional T cell receptor (TCR) complex. Investigation into the dichotomy of CD3-TCR expression has revealed that TCRbeta gene rearrangement and expression does occur in the FC, but only in the context of a unique 33kD glycoprotein, named FCp33. Disulfide linked to the TCR beta chain as a heterodimer, FCp33 is distinct from any known TCR chain and is not present on mature T cells. The expression of the FCp33-TCR beta-CD3 complex is associated with allogeneic engraftment and the induction of donor-specific tolerance in the absence of clinical GVHD. Thus, the global aim of this proposal is to Determine the Mechanism of FC-Mediated Transplantation Tolerance. A number of developmentally-regulated markers have been identified on the cell surface of the FC, which suggest lymphoid ontogeny and recapitulation of lymphoid development. In AIM I, we will Identify the Ontogenic Pathway & Selection Events of FC Development. The co-expression of T cell-like markers and FCp33 on the FC, but absence on any known mature lymphoid lineages, suggests that FCp33 expression may play a transient role in the development of an unidentified progeny. AIM II will Characterize the Development of FC Progeny In Vivo. Whereas GVHD appears to be associated with the conventional T cell population, the responsibility for stem cell (SC) engraftment and the associated donor-specific tolerance rests with the FC, and FCp33 in particular, as donor-specific transplantation tolerance and FC facilitated SC engraftment fail to occur when FC without FCp33 expression are utilized. In AIM Ill, we will Determine the Mechanism of FC-Mediated Transplantation Tolerance.