Establishment of stable multilineage hematopoietic chimerism and donor-specific tolerance without irradiation

Background. Induction of tolerance to organ transplants will increase graft survival and decrease patient mortality and morbidity. Radiation-induced cytoreduction/ablation followed by donor hematopoietic cell reconstitution has been the most consistently successful approach to experimental tolerance induction. However, reluctance of clinicians to expose recipients to radiation has hampered its clinical application. Methods. In the studies described, administration of polyclonal antilymphocyte serum (ALS), donor-specific bone marrow (DSBM) (150x10(6) cells), and sirolimus (24 mg/kg) in a completely mismatched murine model (B10.A donor, C57B/10 recipient) produced 100% indefinite (>250 days) skin graft survival. The level and character of donor-specific chimerism was evaluated with flow cytometry. Results. Specific tolerance was confirmed by continued acceptance of primary and secondary donor-specific skin allografts and rejection of third-party grafts. The level and duration of chimerism induced was directly related to the dose of DSBM administered. Mice given 150x106 DSBM cells showed levels of 8-10% donor peripheral blood mononuclear cell chimerism by 30 days, and these levels persisted indefinitely (>250 days) in association with permanent tolerance of donor grafts. Eighty percent of donor chimeric cells were B lymphocytes (MHC class I and II positive, Fc receptor positive, CD45/B220 positive but negative for CD4, CD8 and Thy 1.2) and 20% were sorted in the macrophage monocyte population. Conclusions. These studies demonstrate for the first time that cytoreduction/ablation with ALS combined with sirolimus and reconstitution with donor bone marrow induces tolerance and chimerism in a completely mismatched murine combination. The use of ALS and sirolimus, currently employed therapies in clinical transplantation, and the lack of requirement for radiation make this tolerance protocol attractive for clinical application.