Induction of mixed chimerism has the potential to induce T cell tolerance to allografts and xenografts. Studies in Project 4 in the funded Program Project have demonstrated that, in addition to T cells, B cells and NK cells are tolerized by induction of mixed chimerism. T cell and antibody-mediated cardiac allograft and xenograft rejection, including hyperacute rejection and delayed vascular rejection, are avoided by induction of mixed chimerism. Using alpha1,3-galactosyltransferase (GaIT) knockout mice, we have demonstrated that both naive and presensitized B cells recognizing alphaGal are tolerized by induction of mixed chimerism. The combined use of fluorochrome-conjugated Gal-BSA and ELISPOT analyses has permitted us to identify the phenotype of natural antibody-producing cells,and to obtain evidence that both deletion/receptor editing and anergy are important mechanisms involved in their tolerization by induction of mixed chimerism. The ability to tolerize B cells by induction of mixed chimerism has important implications for the ability to perform cardiac allografts across ABO blood group barriers and in presensitized recipients, and for the extension of the mixed chimerism approach to xenotransplantation. In this proposal, we plan to extend the studies of B cell tolerance in this model. We will perform studies to determine the molecular mechanisms by which non-myeloablative induction of mixed hematopoietic chimerism leads to tolerance of pre-existing anti-Gal-producing cells in naive and pre-sensitized mice. We will also tackle the difficult problem of T cell presenstization to the donor, and will attempt to simultaneously induce T cell and B cell tolerance across full allogeneic barriers in presensitized mice using a non-myeloablative conditioning regimen involving CD4 and CD8 T cell-depleting mAbs. Finally, in an effort to maximize the clinical applicability of the mixed chimerism approach to cardiac allografting in the presensitized host, we aim to develop an effective non-myeloablative approach to inducing mixed allogeneic chimerism and tolerance using costimulatory blockade in mice that are pre-sensitized to donor antigens.