RECIPIENT BONE MARROW ENGRAFTMENT IN DONOR TISSUE FOLLOWING LONG-TERM TOLERANCE TO A HEMOPOIETIC MUSCULOSKELETAL ALLOGRAFT

Abstract

266 Introduction: Our laboratory previously demonstrated long-term tolerance to vascularized musculoskeletal allografts in MHC matched miniature swine treated with 12 days of cyclosporine(1). Previous work in the rodent model of vascularized rat limb allografts suggested that mixed chimerism derived from the continuous presence of bone marrow cells was responsible for the maintenance of tolerance (2). The aim of this study was to evaluate the role of bone marrow engraftment in recipient tissue following long-term tolerance to a hemopoietic musculoskeletal allograft. Methods: Hind limbs of donor swine were heterotopically transplanted into MHC matched, minor antigen mismatched, recipients and a 12-day course of cyclosporine was given. Swine that demonstrated long-term tolerance to their muscloskeletal grafts were sacrificed at day 292 and day 371 respectively. The bone marrow was harvested and processed from the donor grafts as well as from the recipient hindlimbs. Thymus, spleen, and mesenteric lymph nodes were also harvested from the recipient swine. The presence of donor cells in the peripheral blood and hemopoietic tissues (thymus, spleen, lymph node, and bone marrow) were analyzed by flow cytometry, using the Pig Allelic Antibody (PAA) marker to detect donor cells and Propidium Iodide (PI) to gate out dead cells. Results: By flow cytometry, donor lymphocytes in the peripheral blood of recipients were detected from postoperative day 1 until day 19. Despite the disappearance of donor cells, the musculoskeletal allografts were viable at the time of sacrifice by gross and histologic examination. After graft bone marrow harvest and processing at the time of sacrifice, the yield of bone marrow from the grafts was 4 × 108 and 3 × 108 cells, respectively. Donor cells could also not be detected in peripheral blood or hemopoietic tissues. As demonstrated below pre-transplant PAA positive donor bone marrow cells were identified, while bone marrow cells harvested from the same graft after transplant were PAA negative and therefore of recipient origin. Conclusions: This study demonstrates that in the long-term tolerant recipient of musculoskeletal allografts there is no evidence of persistent chimerism in blood or hemopoietic tissues. Maintenance of tolerance therefore, does not appear to depend on continuous presence of donor bone marrow cells from the graft. In fact, it appears that the recipients' bone marrow cells and lymphocytes repopulate the marrow space. (Figure)Figure