Creation of Chimeric Hearts: A Tool for Testing the “Passenger Leukocyte” Hypothesis

Abstract

Background. Bone marrow-derived antigen-presenting cells (APCs) are thought to be a migratory component of organ allografts that activate the rejection response, and recently they have been postulated to play a critical role in tolerance induction. Our goal was to create chimeric hearts (organs with parenchyma and APCs of differing genotype) for use in models of transplantation to test the “passenger leukocyte” theory. Methods. Murine bone marrow transplantations were performed in two fully major histocompatibility complex (MHC) mismatched strain combinations: C3H → B10 and CBA → BALB/c. Recipients were lethally irradiated (10 Gy) and then received 1 × 10 7 bone marrow cells intravenously. Bone marrow transplant survivors had their organ APCs isolated by digestion with collagenase D, followed by density gradient centrifugation. The APC-enriched fraction was stained with fluorescein-labeled monoclonal antibodies specific for either donor (I-A k) or recipient (I-A b/d) class II MHC antigens, which are expressed by all APCs but not by parenchymal cells. Donor and recipient class II expression was determined by flow cytometry. Results. Sixty-nine of 100 (69.0%) of C3H → B10 and 52/107 (48.6%) of CBA → BALB/c bone marrow transplant recipients survived more than 100 days, whereas all B10 (n = 12) and BALB/c (n = 10) irradiation controls died within 14 days. Mortality appeared to be caused by engraftment failure as most recipients died before day 20. Flow cytometry demonstrated complete APC replacement in hearts (n = 17) and spleens (n = 40), as APC-enriched fractions stained only for donor class II MHC antigens. Conclusion. Bone marrow transplantation leads to replacement of heart APCs in two murine models. Chimeric hearts are now being used to test the role of APCs in allograft rejection and in tolerance induction.