No abstract

Background Hematopoietic chimerism induces transplantation tolerance across allogeneic and xenogeneic barriers, but has been difficult to achieve in the pig-to-primate model. We have now utilized swine with knockout of the gene coding for α-1,3-galactosyltransferase (GalT-KO pigs) as bone marrow donors in an attempt to achieve chimerism and tolerance by avoiding the effects of natural antibodies to Gal determinants on pig hematopoietic cells. Methods Baboons (n = 4; Baboons 1 to 4 = B156, B158, B167, and B175, respectively) were splenectomized and conditioned with TBI (150 cGy), thymic irradiation (700 cGy), T cell depletion with rabbit anti-thymocyte globulin (rATG) and rat anti-primate CD2 (LoCD2b), and received FK506 and supportive therapy for 28 days. All animals received GalT-KO bone marrow (1 to 2 × 109 cells/kg) in two fractions on days 0 and 2, and were thereafter monitored for the presence of pig cells by flow cytometry, for porcine progenitor cells by PCR of BM colony-forming units, and for cellular reactivity to pig cells by mixed lymphocyte reaction (MLR). In vitro antibody formation to LoCD2b and rATG was tested by ELISA; antibody reactivity to GalT-KO pig cells was tested by flow cytometry and cytotoxicity assays. Additionally, Baboons 3 and 4 received orthotopic kidney transplants on days 17 and 2, respectively, to test the potential impact of the protocol on renal transplantation. Results None of the animals showed detectable pig cells by flow cytometry for more than 12 h post-BM infusion. However, porcine progenitor cell engraftment, as evidenced by pig-derived colony forming units in the BM, as well as peripheral microchimerism in the thymus, lymph node, and peripheral blood was detected by PCR in baboons 1 and 2 for at least 28 days post-transplant. ELISA results confirmed humoral immunocompetence at time of transplantation as antibody titers to rat (LoCD2b) and rabbit (ATG) increased within 2 weeks. However, no induced antibodies to GalT-KO pig cells or increased donor specific cytotoxicity was detectable by flow cytometry. In contrast, baboons 3 and 4 developed serum antibodies to pig cells as well as to rat and rabbit immunoglobulin by day 14. Retrospective analysis revealed that although all four baboons possessed low levels of antibody-mediated cytotoxicity to GalT-KO cells prior to transplantation, the two baboons (3 and 4) that became sensitized to pig cells (and rejected pig kidneys) had relatively high pre-transplantation titers of anti–non-Gal IgG detectable by flow cytometry, whereas baboons 1 and 2 had undetectable titers. Conclusions Engraftment and specific non-responsiveness to pig cells has been achieved in two of four baboons following GalT-KO pig-to-baboon BMT. Engraftment correlated with absence of preformed anti–non-Gal IgG serum antibodies. These results are encouraging with regard to the possibility of achieving transplantation tolerance across this xenogeneic barrier.

Clinical composite tissue allotransplantation can adequately reconstruct defects that are not possible by other means. However, immunosuppressant toxicity limits the use of these techniques. Clinical attempts to reduce the amount of immunosuppression required by induction of an immunologically permissive state have so far been unsuccessful. The aim of this study was to induce tolerance in a preclinical large animal model. Donor hematopoietic stcm cell (HSC) engraftment was induced by T-cell depletion, irradiation, and a short course of cyclosporine administered to the recipient, along with a hematopoietic cell infusion from a single haplotype major histocompatibility complex (MHC) mismatched donor. Skin was then allotransplanted from the donor. Both primarily vascularized skin flaps and secondarily vascularized conventional skin grafts were allotransplanted to investigate if the mode of transplantation affected outcome. Control animals received the skin allotransplants without conditioning. Tolerance was defined as no evidence of rejection at 90 days following transplantation. Conventional skin grafts only achieved prolonged survival (<65 days) in HSCengrafted animals (P < .01). In contrast, there was indefinite skin flap survival with the achievement of tolerance in HSC-engrafted animals; this was confirmed on histology with donor-specific unresponsiveness on MLR and CML. Furthermore, a conventional skin donor graft subsequently applied to an animal tolerant to a skin flap was not rejected and did not trigger skin flap rejection. To our knowledge, this is the first time skin tolerance has been achieved across a MHC barrier in a large animal model. This is a significant step toward thc goal of clinical skin tolerance induction.

Background Cellular treatments for repairing diseased tissues represent a promising clinical strategy. Umbilical cord tissue-derived cells (UTC) are a unique source of cells with a low immunogenic profile and potential for tissue repair. By using UTC from miniature swine, we previously demonstrated that despite their low immunogenic phenotype, UTC could induce an immune response under certain inflammatory conditions and after multiple subcutaneous (SC) injections. Given that repeat dosing of cells may be necessary to achieve a lasting therapeutic benefit, in this study, we examined approaches to avoid an immune response to multiple SC injections of UTC. Methods By using in vitro and in vivo measures of sensitization to SC cellular injections, we assessed the effects of varying the location of administration site, prolongation of timing between injections, and use of immunosuppressive treatments on repeated cellular injections in Massachusetts General Hospital major histocompatibility complex-defined miniature swine. Results Although under normal conditions, a single SC injection of major histocompatibility complex-mismatched UTC did not induce a detectable immune response, multiple SC injections of UTC demonstrated rapid humoral and cell-mediated immune responses. Avoidance of an immune response to repeat SC injection was achieved by concurrent immunosuppression with each dose of UTC. Conclusions UTC and other similar cell types believed to be nonimmunogenic have the potential to induce immune responses under certain conditions. These studies provide important considerations and guidelines for preclinical studies investigating allogeneic cellular therapies.

SummaryThe problem of allogeneic skin rejection is a major limitation to more widespread application of clinical composite tissue allotransplantation (CTA). Previous research examining skin rejection has mainly studied rejection of conventional skin grafts (CSG) using standard histological techniques. The aim of this study was to objectively assess if there were differences in the immune response to CSG and primarily vascularized skin in composite tissue allotransplants (SCTT) using in vivo techniques in order to gain new insights in to the immune response to skin allotransplants.CSG and SCTT were transplanted from standard Lewis (LEW) ad Wistar Furth (WF) to recipient transgenic green fluorescent Lewis rats (LEW-GFP). In vivo confocal microscopy was used to observe cell trafficking within skin of the transplants. In addition, immunohistochemical staining was performed on skin biopsies to reveal possible expression of class II major histocompatibility antigens.A difference was observed in the immune response to SCTT compared to CSG. SCTT had a greater density cellular infiltrate than CSG (p < 0.03) that was focused more at the center of the transplant (p < 0.05) than at the edges, likely due to the immediate vascularization of the skin. Recipient dendritic cells were only observed in rejecting SCTT, not CSG. Furthermore, dermal endothelial class II MHC expression was only observed in allogeneic SCTT. The immune response in both SCTT and CSG was focused on targets in the dermis, with infiltrating cells clustering around hair follicles (CSG and SCTT; p < 0.01) and blood vessels (SCTT; p < 0.01) in allogeneic transplants. This study suggests that there are significant differences between rejection of SCTT and CSG that may limit the relevance of much of the historical data on skin graft rejection when applied to composite tissue allotransplantation. Furthermore, the use of novel in vivo techniques identified

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.

No abstract

These results suggest that it is possible to retransplant after rejection of a musculocutaneous transplant while on immunosuppression. Furthermore, the second transplant will not be limited in form or function by recipient tissue bed damage.