Organ allotransplantation is currently limited by chronic rejection and the toxicity of non-specific long-term immunosuppressive therapy, with its increased risks of malignancies and infections. A state of immunological tolerance would obviate the need for chronic immunosuppression and prevent chronic rejection. Mixed hematopoietic chimerism has been shown in animal models to be a reliable means of inducing robust transplantation tolerance. Unfortunately, clinical application of mixed chimerism. for the induction of organ transplant acceptance has been precluded by the toxicity associated with the host conditioning traditionally thought to be necessary to achieve marrow engraftment in adult recipients. We have developed murine and primate models showing that allogeneic bone marrow engraftment can be achieved across MHC barriers, leading to mixed chimerism and tolerance, without the requirement for lethal host conditioning. We have now applied a modification of this approach, involving the induction of mixed chimerism followed by donor leukocyte infusions, to the treatment of humans with advanced hematologic malignancies. These studies have demonstrated for the first time that stable mixed hematopoietic chimerism can be achieved in HLA-mismatched BMT recipients who are conditioned with a relatively non-toxic, non-myeloablative regimen. We have also reliably achieved mixed chimerism in HLA- matched donor-recipient pairs, with minimal GVHD. Several of these transplants have included major and minor ABO mismatches. Preliminary data showing a loss of anti-donor isohemagglutin and the development of mixed erythroid and B cell chimerism suggest that a state of tolerance may be achieved among B cells that produce anti-blood group natural antibodies. If confirmed, this result would be consistent with studies in the murine mixed chimera model showing that natural antibody-producing B cells are rapidly tolerized by the engraftment of donor hematopoietic cells expressing the target antigen. Additional preliminary data are consistent with the presence of a state of T cell tolerance in mixed chimeras. We propose to systematically evaluate the evolution of donor-vs-host (GvH) and host-vs-donor (HvG) T cell tolerance in these mixed chimeras, and to determine its mechanisms. We will also investigate the relationships between B cell chimerism, erythroid chimerism and tolerance of B cell populations that produce anti-donor and anti-host isoagglutinatins in ABO-mismatched BMT recipients. The demonstration of T cell and B cell tolerance in these human mixed chimeras produced with non-myeloablative conditioning would help to determine the potential of this new BMT strategy for the induction of tolerance to organ allografts, and to treat hemoglobinopathies and other non-malignant disorders.