Background. Pigs are potential organ donors for humans. Some proposed xenotransplant tolerance regimens require genetically identical cells from different animals (eg, juvenile bone marrow and mature organs or tissues). We therefore sought to develop a highly inbred line of miniature swine for this purpose. The aim of this study was to test histocompatibility in a new subline of highly inbred miniature swine. Methods. Pigs from 2 generations with coefficient of inbreeding (COI) of 92% (n = 6) and 94% (n = 4) each received 2 split-thickness skin grafts (STSGs): an autograft and an allograft from the same generation. This was repeated in a group of 4 pigs from the generation with COI 92%. STSGs were followed for 28–35 d (COI 92%) or >380 d (COI 94%). Results. For the pigs with COI of 92%, 1 pig rejected the first allograft on day 9. All other pigs showed prolonged (>24 d) STSG survival. All subsequently rejected a second matched allograft in <14 d, indicating sensitization to minor histocompatibility antigens still segregating in the herd. For the pigs with COI of 94%, 1 pig rejected its allograft at day 9 while the other 3 accepted their allografts >386 d. Conclusions. At COI of 92%, highly inbred swine experienced prolonged STSG survival, but persistent minor histocompatibility antigen disparities caused delayed skin graft rejection. Most pigs with COI of 94% accepted reciprocal skin grafts long-term without immunosuppression, indicating homozygosity of the skin graft donors for all relevant histocompatibility loci. Organ transplants within this new inbred line are expected to be accepted indefinitely without a requirement for exogenous immunosuppression, facilitating experiments requiring genetically identical cells from different animals.

Background Recent advances in gene editing technology have enabled the production of multi-knockout (KO) and transgenic pigs in order to overcome immunologic barriers in xenotransplantation (XTx). However, the genetic manipulations required to produce these changes may have the unintended consequence of producing or revealing neoantigens reactive with natural antibodies present in baboons. In this study, we examined whether the neoantigens that develop in multi-transgenic (mTg) GalT, Cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), beta-1,4-N-acetyl-galactosaminyl transferase 2 (B4) KO pigs can cause rejection of xenografts in baboons. Methods Five baboons that had <35% cytotoxicity against GalT-KO peripheral blood mononuclear cells (PBMCs) in a pre-screening assay received pig kidneys and vascularized thymic grafts (VT + K) from multi-transgenic hCD47, human thrombomodulin (hTBM), human endothelial protein C receptor (EPCR) with/without hCD46 and hCD55 with GalT-KO/NeuGC-KO/B4-KO (mTg Tri-KO) swine. In order to further examine the effects of anti-donor non-Gal natural antibody (nAb), anti-pig preformed IgM and IgG nAb binding against the GalT-KO PBMCs was compared with the donor-type PBMCs using donor pretransplant sera as well as 5 additional naive baboon sera by flow cytometric analysis. Results Five baboons that received VT + K grafts had stable renal function in the first 11 days (serum creatinine < 1.5 mg/dL). Two of the five baboons had higher binding of preformed IgG to mTg Tri-KO PBMCs than to GalT-KO PBMCs (mTg Tri-KO > GalT-KO), and they rejected their grafts at POD 20. In contrast, the other three baboons demonstrated either mTg Tri-KO = GalT-KO or mTg Tri-KO < GalT-KO, and they maintained renal function 43, 52, and 154 days without rejection. Among 10 baboon sera, two had less antibody binding against PBMCs that were syngeneic to the mTg Tri-KO than against GalT-KO PBMCs (mTg Tri-KO < GalT-KO); three had similar binding to mTg Tri-KO and GalT-KO PBMCs (mTg Tri-KO = GalT-KO); and five had higher binding to m Tg Tri-KO than to GalT-KO PBMCs (mTg Tri-KO > GalT-KO). Conclusions These data suggest that neoantigens associated with mTg Tri-KO promote acute xenograft rejection in a pig-to-baboon VT + K XTx model. The screening assays may be useful to select "safe" recipients to receive mTg Tri-KO kidneys.

Background We have recently demonstrated that human-CD47 (hCD47) expressed on endothelial cells of porcine lung xenografts extended median graft survival from 3.5 days to 8.7 days in baboons. Intra-bone bone marrow transplantation (IBBMTx) in a pig-to-baboon model was previously shown to markedly prolong the duration of macrochimerism up to 21 days from 1 to 4 days by intravenous BMTx. We now examined whether the use of hCD47 transgenic (Tg) BM further prolonged the duration of chimerism following IBBMTx. We then tested if lung xenograft survival was prolonged following IBBMTx. Methods Baboons received GalTKO-hCD47/hCD55Tg (n = 5) or -hCD55Tg (n = 1) or -hCD46/HLA-E Tg (n = 1) pig IBBMTx. Macrochimerism, anti-pig T cells and antibody responses were assessed. Animals received lung xenografts from either hCD47+ or hCD47- porcine lungs 1-3 months later. Results All baboons that received hCD47Tg porcine IBBM maintained durable macrochimerism >30 days, and two maintained chimerism for >8 weeks. Notably, anti-pig antibody levels decreased over time and anti-pig cellular unresponsiveness developed following IBBMTx. Lungs from hCD47Tg IBBMTx matched pigs were transplanted at day 33 or day 49 after IBBMTx. These animals showed extended survival up to 13 and 14 days, while animals that received lungs from hCD47 negative pigs displayed no prolonged survival (1-4 days). Conclusion This is the first report demonstrating durable macrochimerism beyond 8 weeks, as well as evidence for B cell tolerance in large animal xenotransplantation. Using hCD47Tg pigs as both IBBMTx and lung donors prolongs lung xenograft survival. However, additional strategies are required to control the acute loss of lung xenografts.

Background The early development of proteinuria following pig renal xenotransplantation (XKTx) was a major obstacle to prolonged life-supporting GalTKO XKTx in baboons. We have recently shown that CTLA-4Ig and Rituximab reduce proteinuria in association with downregulatation of the inflammatory response of CD80/SMPDL-3 in porcine glomeruli, allowing GalTKO thymokidneys (TK) without other transgenes (Tg) to function for >6 months. In this study, we addressed the hypothesis that failure of porcine CD47 to provide inhibitory signals to baboon macrophages via SIRPα triggers an inflammatory cascade that promotes proteinuria. We addressed this hypothesis by using hCD47 Tg porcine GalTKO animals as the source of TK. Methods Study 1 (in vivo study to assess the effect of hCD47 Tg on GalTKO TK donor on post-XKTx proteinuria): Two baboons received hCD47+/hCD55+ GalTKO kidneys (Group 1). One received hCD55+ GalTKO kidney (Group 2). As a control (Group 3), 6 baboons received GalTKO kidneys without additional Tg. All animals received an anti-CD154 mAb-based immunosuppressive regimen without CTLA4-Ig. Expression of hCD47 or hCD55 by donor kidneys was assessed histologically. All recipients had anti-non-Gal NAb antibody-complement mediated cytotoxicity lower than 35%. Study 2 (in vitro study): Expression of SIRPα and hCD47 in porcine glomeruli following XKTx was investigated. Phagocytosis of porcine endothelial cells (EC) as well as podocytes with/without hCD47 was assessed in co-culture assays. Results Study 1: No elicited anti-pig ab developed following hCD47/CD55 Tg XKTx in any recipients. All of the recipients in Groups 2 and 3 had to be euthanized before POD70 in association with the development of massive proteinuria (Fig 1A-2, A-1). In contrast, of the two baboons in Group 1, the animal that expressed hCD47 highly in glomeruli (Fig. 2 left) developed only minimal proteinuria (>120 days). The other animal, which expressed hCD47 at a lower level (Fig 2 right) developed only 1+ and occasionally 2+ proteinuria and was euthanized at POD 69 due to graft growth (Fig.1A-3). Study 2: Expression of SIRPα on glomerular cells markedly decreased in GalTKO kidneys following XKTx, while no change in SIRPα was seen in hCD47+ GalTKO kidneys. Statistically significant reductions of phagocytosis of both porcine EC and podocytes were observed when hCD47 was expressed on porcine ECs (top in Fig 3) or podocytes (bottom in Fig 3).

BackgroundDespite recent progress in survival times of xenografts in non-human primates, there are no reports of survival beyond 5days of histologically well-aerated porcine lung grafts in baboons. Here, we report our initial results of pig-to-baboon xeno-lung transplantation (XLTx). MethodsEleven baboons received genetically modified porcine left lungs from either GalT-KO alone (n=3), GalT-KO/humanCD47(hCD47)/hCD55 (n=3), GalT-KO/hD47/hCD46 (n=4), or GalT-KO/hCD39/hCD46/hCD55/TBM/EPCR (n=1) swine. The first 2 XLTx procedures were performed under a non-survival protocol that allowed a 72-hour follow-up of the recipients with general anesthesia, while the remaining 9 underwent a survival protocol with the intention of weaning from ventilation. ResultsLung graft survivals in the 2 non-survival animals were 48 and >72hours, while survivals in the other 9 were 25 and 28hours, at 5, 5, 6, 7, >7, 9, and 10days. One baboon with graft survival >7days, whose entire lung graft remained well aerated, was euthanized on POD 7 due to malfunction of femoral catheters. hCD47 expression of donor lungs was detected in both alveoli and vessels only in the 3 grafts surviving >7, 9, and 10days. All other grafts lacked hCD47 expression in endothelial cells and were completely rejected with diffuse hemorrhagic changes and antibody/complement deposition detected in association with early graft loss. ConclusionsTo our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7days in baboons. Our results indicate that GalT-KO pig lungs are highly susceptible to acute humoral rejection and that this may be mitigated by transgenic expression of hCD47.

Background Recent data have suggested that low levels of preformed anti-non-Gal natural antibody (nAb) limit xenograft survival. Durable mixed xenogeneic chimerism resulted in tolerance of human T cell-independent B cells that produce xenoantibodies, including anti-non-Gal nAb, in mouse models. However, previous attempts at intravenous (IV) α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into non-human primates have been associated with very short-lived chimerism (<24 hours in most cases). In order to achieve improved engraftment with more persistent chimerism, we have recently developed an intra-bone BM (IBBM) Tx strategy in a pig-to-baboon model. We have reported continuous peripheral blood macrochimerism for up to 13 days (mean 7.7 days; range 3–13) post-IBBM/BM-Tx (n=6) and in three animals, macrochimerism reappeared at days 10, 14 and 21 (Am J Transplant 2015). Recent data has suggested that incompatibility between CD47 and SIRPα causes phagocytosis of porcine cells. In this study, we examined whether the use of BM cells from human CD47 (hCD47) transgenic pigs further improves durable chimerism and assessed anti-pig antibody responses following BM Tx,Methods: Four baboons received GalTKO/human CD47(hCD47)/hCD55 pig BM cells, and one received GalTKO/hCD55 porcine BM cells with a conditioning regimen consisting of whole body irradiation thymic, irradiation, ATG, and Rituximab followed by anti-CD40L antibody and MMF. Approximately 2/3 of BM cells were infused intraosseously and the remainder intravenously. Macrochimerism, anti-pig T cells responses, and anti-pig antibody responses were assessed following IBBM Tx. Results were compared with those from baboons that received GalTKO porcine BM cells without any other gene modification (n=6). Results As shown in the Figure (left panel), all four baboons that received hCD47+/hCD55+ porcine BM using the IBBM Tx method maintained durable macrochimerism for >50 days, and two of the 4 maintained blood levels above 1.6%, even at 8 weeks following IBBMTx (red line in the left panel of Fig). We also confirmed macrochimerism in recipients’ BM. A baboon that received hCD55+ but hCD47-negative porcine BM cells also had a slightly higher % of chimerism than GalTKO alone porcine BM cells in the first 3 weeks, but the duration was similar to GalTKO alone porcine BM cells (Blue line vs black line in the left panel of Fig). All recipients of IBBM Tx lost their anti-pig T cell responses in vitro and showed no development of elicited anti-pig ab. Notably, anti-pig nonGal nAb levels in three of 4 recipients of hCD47+/hCD55+ porcine BM cells declined markedly following IBBM Tx (right panel of Fig). Conclusion To our knowledge, this is the first demonstration of (1) durable macrochimerism beyond 8 weeks and (2) reduction of anti-non Gal nAb levels following hCD47+/hCD55+ GalTKO porcine IBBMTx in preclinical pig-to-non-human primate models. The results suggest that IBBMTx using hCD47+ porcine BM is a potent strategy to induce pig-specific T and B cell tolerance across xenogeneic barriers..NIH P01AI45897.:

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3-13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

Background Recent survivals of our pig-to-baboon kidney xenotransplants have been markedly shorter than the graft survivals we previously reported. The discovery of high levels of porcine cytomegalovirus (pCMV) in one of the rejected xenografts led us to evaluate whether this reduction in graft survival might be because of the inadvertent introduction of pCMV into our α1,3-galactosyltransferase gene knockout swine herd. Methods Archived frozen sections of xeno-kidney grafts over the past 10 years were analyzed for the presence of pCMV, using real-time polymerase chain reaction. Three prospective pig-to-baboon renal transplants using kidneys from swine delivered by cesarean section (C-section) and raised in isolation were likewise analyzed. Results Kidney grafts, from which 8 of the 18 archived samples were derived were found to be pCMV-negative, showed a mean graft survival of 48.3 days and were from transplants performed before 2008. None showed signs of disseminated intravascular coagulopathy and were lost because of proteinuria or infectious complications. In contrast, 10 of the archived samples were pCMV positive, were from kidney transplants with a mean graft survival of 14.1 days, had been performed after 2008, and demonstrated early vascular changes and decreased platelet counts. Three prospective xenografts from swine delivered by C-section were pCMV negative and survived an average of 53.0 days. Conclusions Decreased survivals of α1,3-galactosyltransferase gene knockout renal xenografts in this laboratory correlate temporally with latent pCMV in the donor animals and pCMV in the rejected xeno-kidneys. Transmission of pCMV to swine offspring may be avoided by C-section delivery and scrupulous isolation of donor animals.

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig-to-primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell-surface molecule that interacts in a species-specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT-KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High-level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable chimerism levels were observed in the peripheral blood of these recipients. In contrast, transplantation of WT progenitor cells resulted in little or no bone marrow engraftment and no detectable peripheral chimerism. These results demonstrate a substantial protective effect of hCD47 expression on engraftment and persistence of porcine cells in this model, presumably by modulation of macrophage phagocytosis.

Background and aims of study: We have previously demonstrated a requirement for the presence of a juvenile thymus for the induction of transplantation tolerance to renal allografts by a short-course of calcineurin inhibition in miniature swine. We have also shown that aged, involuted thymi can be rejuvenated when transplanted as vascularized thymic lobes into juvenile swine recipients. The present studies were aimed at elucidating the extrinsic factors facilitating this restoration of function in the aged thymus. In particular, we tested the impact of sex steroid blockade by Luteinizing Hormone-Releasing Hormone (LHRH).Materials and methods: 30 naive animals (25 males and 5 females) were used for measurement of serum testosterone levels. 3 mature male pigs (aged at 22, 22 and 29 months old) were used to test the effects of Lupron (LHRH analog) injection at 45 mg (per 70-80 kg body weight) as a 3-month depot on testosterone levels and thymic rejuvenation. Thymic rejuvenation was assessed by histology, flow cytometric analysis, morphometric analysis and TREC assays.Results: Hormonal alterations were induced by Lupron and resulted in macroscopic and histologic regeneration of the thymus of aged animals within 2 months, as evidenced by restoration of juvenile thymus architecture and increased cellularity. Two animals that were evaluated for TREC both showed increased levels in the periphery following Lupron treatment.Conclusion: Treatment of aged animals with Lupron leads to thymic rejuventaion in adult miniature swine. This result could expand the applicability of thymus-dependent tolerance-inducing regimens to adult recipients. (C) 2010 Elsevier B.V. All rights reserved.

This manuscript reports on five cases of spontaneous myelogenous leukemia, similar to human disease, occurring within highly inbred, histocompatible sublines of Massachusetts General Hospital (MGH) MHC-defined miniature swine. In cases where a neoplasm was suspected based on clinical observations, samples were obtained for complete blood count, peripheral blood smear, and flow cytometric analysis. Animals confirmed to have neoplasms were euthanized and underwent necropsy. Histological samples were obtained from abnormal tissues and suspect lesions. The phenotype of the malignancies was assessed by flow cytometric analysis of processed peripheral blood mononuclear cells and affected tissues. Five cases of spontaneous myeloid leukemia were identified in adult animals older than 30 months of age. All animals presented with symptoms of weight loss, lethargy, and marked leukocytosis. At autopsy, all animals had systemic disease involvement and presented with severe hepatosplenomegaly. Three of the five myelogenous leukemias have successfully been expanded in vitro. The clustered incidence of disease in this closed herd suggests that genetic factors may be contributing to disease development. Myelogenous leukemia cell lines established from inbred sublines of MGH MHC-defined miniature swine have the potential to be utilized as a model to evaluate therapies of human leukemia.

Crossreactivity of anti-HLA antibodies with SLA alleles may limit the use of pig xenografts in some highly sensitized patients. An understanding of the molecular basis for this crossreactivity may allow better selection of xenograft donors. We have tested 68 human monoclonal HLA class I antibodies (mAbs) for reactivity with pig lymphocytes from SLA defined pigs and found nine to be crossreactive. Eight of nine were broadly HLA reactive IgM-mAbs. The putative HLA epitopes for seven mAbs. were conserved in the aminoacid sequence of the SLA alleles studied. The lack of reactivity of a large number of mAbs largely correlated with the absence of the putative epitopes in the SLA alleles studied. We conclude that most patients with anti-HLA class I antibodies should be able to find pig donors lacking SLA antigens that cross react with their antibodies and that many of the crossreacting epitopes can be defined by analysis of shared epitopes in the aminoacid sequence of human and pig MHC antigens.

Background. Survival of ABO-mismatched kidneys with stable renal function despite the persistence of anti-ABO antibodies is called accommodation. The mechanism of accommodation is unclear, but may involve complement regulatory proteins such as CD59. The development of alpha-1,3-galactosyltransferase knock-out (GalT-KO) swine that produce anti-Gal antibodies provides a large animal model capable of determining the role of complement regulatory proteins in accommodation. Methods. ELISA and antibody fluorescence-activated cell sorting were used to examine the rate of anti-Gal antibody expression as a function of age. Major histocompatibility complex-matched kidneys were transplanted from Gal-positive MGH miniature swine to MGH GalT-KO swine with systemic immunosuppression. One recipient underwent adsorbtion of anti-Gal antibodies before transplantation. Graft survival, antibody, and complement deposition patterns and CD59 expression were determined. Results. Three animals rejected Gal-positive kidneys by humoral mechanisms. One animal with low titers of anti-Gal antibody displayed spontaneous accommodation and the animal that was treated with antibody adsorbtion also displayed accommodation. Rejected grafts had deposition of IgM, IgG, C3, and C5b-9 with low expression of CD59, whereas accommodated grafts had low deposition of C5b-9 and high expression of CD59. Retransplantation of one accommodated graft to a naïve GalT-KO animal confirmed that changes in the graft were responsible for the lack of C5b-9 deposition. Conclusion. GalT-KO miniature swine produce anti-Gal antibodies and titers increase with age. These anti-Gal antibodies can cause rejection of major histocompatibility complex-matched kidneys unless accommodation occurs. CD59 up-regulation seems to be involved in the mechanism of accommodation by preventing the formation of the membrane attack complex (MAC) on the accommodated graft.

Clinical transplantation for the treatment of end-stage organ disease is limited by a shortage of donor organs. Successful xenotransplantation could immediately overcome this limitation. The development of homozygous α1,3-galactosyltransferase knockout (GalT-KO) pigs removed hyperacute rejection as the major immunologic hurdle to xenotransplantation. Nevertheless, GalT-KO organs stimulate robust immunologic responses that are not prevented by immunosuppressive drugs. Murine studies show that recipient thymopoiesis in thymic xenografts induces xenotolerance. We transplanted life-supporting composite thymokidneys (composite thymus and kidneys) prepared in GalT-KO miniature swine to baboons in an attempt to induce tolerance in a preclinical xenotransplant model. Here, we report the results of seven xenogenic thymokidney transplants using a steroid-free immunosuppressive regimen that eliminated whole-body irradiation in all but one recipient. The regimen resulted in average recipient survival of over 50 days. This was associated with donor-specific unresponsiveness in vitro and early baboon thymopoiesis in the porcine thymus tissue of these grafts, suggesting the development of T-cell tolerance. The kidney grafts had no signs of cellular infiltration or deposition of IgG, and no grafts were lost due to rejection. These results show that xenogeneic thymus transplantation can support early primate thymopoiesis, which in turn may induce T-cell tolerance to solid organ xenografts.

Background. Nuclear transfer has been used as a means of selectively modifying the mammalian genome. One possible consequence of this technology is that the oocytes used in nuclear transfer may provide additional antigens by cytoplasmic inheritance of maternally derived, mitochondrial DNA (mtDNA). These studies examine the potential consequences of such inheritance in a large animal transplantation model. Methods. Renal transplants were performed between major histocompatibility complex (MHC)-identical animals differing only in the source of their maternally derived cytoplasmic DNA, using a protocol, which uniformly leads to tolerance within standard MHC-inbred lines. In an attempt to correlate transplant results with a putative marker for disparities in cytoplasmically inherited minor histocompatibility antigens, we examined one hypervariable region of mtDNA, designated hypervariable region 1 (HV1). Results. The mtDNA sequence of the HV1 region was found to be invariant among MGH miniature swine of different haplotypes, despite 25 years of selective breeding of the sublines of this colony. In contrast, swine derived by nuclear transfer into outbred oocytes differed in the HV1 region sequence from each other and from MGH swine. Renal transplants from standard, inbred MGH swine to their MHC-identical knockout counterparts derived from outbred oocytes were rejected within 2 weeks, whereas transplants in the reverse direction were accepted for over 30 days. Conclusions. The HV1 sequence of mtDNA may serve as a marker for the level of diversity of mtDNA. These transplant data are consistent with the existence of mtDNA-encoded mitochondrial minor antigens with a level of diversity that can influence the outcome of renal transplantation.