Abstract
Background: The induction of stable hematopoietic cell chimerism through bone marrow transplantation (BMT) has been demonstrated to induce donor-specific tolerance in rodent, porcine, nonhuman primate, and human clinical allogenic models, and has also been successful in concordant rodent and nonhuman primate xenogeneic models, as well as in the pig-to-NOD/SCID humanized mouse xenogenic model. However, stable chimerism and tolerance has been difficult to achieve in the discordant pig-to-baboon xenotransplantation model, possibly due in part to the presence in baboons of pre-formed natural xeno-reactive antibodies to a1,3-galactose (Gal) determinants expressed in pigs, but not in Old World primates and humans. The recent availability of miniature swine homozygous for a disruption in the gene encoding a1,3-galactosyltransferase (GalT-KO pigs) has now made it possible to study pig-to-baboon xenografts in the absence of effects of anti-Gal antibodies. We have investigated the GalT-KO pig-to-baboon model further by modifying the conditioning and immunosuppression regimen to facilitate engraftment and tolerance through bone marrow transplantation.
Methods: BM was harvested from GalT-KO swine (n=3). Baboons (n=3) were pre-treated with whole body (3 Gy) and thymic (7 Gy) irradiation, Sangstat rabbit anti-thymocyte globulin (ATG), LoCD2b (rat IgG2b anti-primate CD2) and splenectomy, and received FK506 immunosuppressive and supportive therapy for 28 days. The baboons were monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells in the bone marrow by porcine cytochrome b specific PCR of colony-forming units (CFUs), and for cellular reactivity to pig cells by MLR and CML. Antibody formation to LoCD2b and ATG was tested by enzyme-linked immunosorbent assay (ELISA), and antibody reactivity to GalT-KO pig cells was tested by flow cytometry and antibody mediated cytotoxicity assay.
Results: A mean of 1.4 × 109 BM cells/kg was infused into each baboon. Although pig cells were undetectable in the peripheral blood of the baboons by flow cytometry, porcine progenitor cell engraftment as well as chimerism in the bone marrow and thymus was detected by PCR in the first baboon on day 28. ELISA results indicated the presence of antibodies to rat (LoCD2b) and rabbit (ATG) immunoglobulin within two weeks; however, no antibodies to pig cells could be detected by flow cytometry or cytotoxicity assay. The second baboon had undetectable serum antibodies to pig cells for 60 days despite the presence of induced antibodies to rat LoCD2b and rabbit ATG. Porcine progenitor cell engraftment was confirmed by PCR of CFUs on day 60 and MLR showed no response to pig although the animal regained alloresponses by this time. The third baboon, in contrast, had detectable induced serum antibodies to pig cells as well as rat and rabbit immunoglobulin by day 14 following BMT.
Conclusions: Engraftment has been achieved following GalT-KO pig-to-baboon BMT with evidence of specific humoral and cellular non-responsiveness to pig cells (2/3 baboons), suggesting the possibility that this protocol may facilitate xenograft tolerance.
Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model