Taylor Cormack

Porcine cytomegalovirus infection is associated with early rejection of kidney grafts in a pig to baboon xenotransplantation model.

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 &agr;1,3-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 &agr;1,3-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.

High Incidence of Xenogenic Bone Marrow Engraftment in Pig-to-Baboon Intra-Bone Bone Marrow Transplantation

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.

The Induction of Tolerance of Renal Allografts by Adoptive Transfer in Miniature Swine

Our previous in vitro data have demonstrated that regulatory mechanisms are involved in tolerance of class I‐mismatched renal allografts in miniature swine treated with 12 days of high dose Cyclsporin A. In this study, we attempted to induce tolerance of class I‐mismatched kidneys by adoptive transfer of cells and/or kidneys from long‐term tolerant animals. Fifteen SLAdd miniature swine received 1.5 Gy whole body irradiation and class I‐mismatched (SLAgg) kidneys from naïve pigs with or without cotransplanted kidneys and/or adoptively transferred cells from long‐term tolerant (LTT) SLAdd recipients of SLAgg grafts. In addition, three SLAdd miniature swine received class I mismatched kidney with adoptively transferred cells from LTT SLAdd recipients. Naïve kidneys transplanted without a LTT kidney were rejected within 9 days. All recipients of naive kidneys along with cells and kidney grafts from LTT animals showed markedly prolonged survival of the naive renal grafts (day 28, >150 and >150 days). These studies suggest that (1) tolerated kidneys have potent regulatory effects and (2) cells from LTT animals infused in conjunction with kidney grafts augment these regulatory effects. To our knowledge, these studies represent the first demonstration of successful adoptive transfer of tolerance in large animals.

Abrogation of Renal Allograft Tolerance in MGH Miniature Swine: The Role of Intra-Graft and Peripheral Factors in Long-Term Tolerance

We have previously demonstrated that long‐term tolerance (LTT) of an MHC class‐I mismatched renal allograft can be achieved with a short course of cyclosporine. In order to examine regulatory mechanisms underlying tolerance in this model, we assessed the contributions of factors within the graft and in the peripheral blood for their relative roles in the maintenance of stable tolerance. Twelve LTT recipients of MHC class‐I mismatched primary kidneys were subjected to a treatment consisting of donor‐specific transfusion followed by leukapheresis, in order to remove peripheral leukocytes, including putative regulatory T cells (Tregs). Following treatment, 2 controls were followed clinically and 10 animals had the primary graft removed and received a second, donor‐MHC‐matched kidney. Neither control animal showed evidence of rejection, while 8 of 10 retransplanted animals developed either rejection crisis or full rejection of the second transplant. In vitro assays confirmed that the removed leukocytes were suppressive and that CD4+Foxp3+ Treg reconstitution in blood and kidney grafts correlated with return to normal renal function in animals experiencing transient rejection crises. These data indicate that components of accepted kidney grafts as well as peripheral regulatory components both contribute to the tolerogenic environment required for tolerance of MHC class‐I mismatched allotransplants.

VASCULARIZED COMPOSITE ALLOGRAFT TRANSPLANT SURVIVAL IN MINIATURE SWINE: IS MHC TOLERANCE SUFFICIENT FOR ACCEPTANCE OF EPIDERMIS?

Background We have previously reported that Massachusetts General Hospital miniature swine, which had accepted class I–mismatched kidneys long-term after 12 days of high-dose cyclosporine A, uniformly accepted donor-major histocompatibility complex (MHC)–matched kidneys without immunosuppression but rejected donor MHC-matched split-thickness skin grafts by day 25, without changes in renal graft function or antidonor in vitro responses. We have now tested whether this “split tolerance” would also be observed for the primarily vascularized skin of vascularized composite allografts (VCAs). Methods Group 1 animals (n=3) received donor MHC-matched VCAs less than 70 days after primary kidney transplant (KTx). Group 2 animals (n=3) received a second donor-matched kidney transplant followed by a donor-matched VCA more than 200 days after primary KTx. Results Animals in Group 1 lost the epidermis on days 28, 30, and 40, with all other components of the VCAs remaining viable. Histology showed cellular infiltration localized to dermal-epidermal junction. One of three recipients of VCAs in Group 2, accepted all components of the VCA, including epidermis (>200 days). The other two recipients lost only the epidermis on days 45 and 85, with survival of the remainder of the VCA long-term. Conclusions All tissues of a VCA are accepted long-term on animals tolerant of class I–mismatched kidneys, with the exception of epidermis, the survival of which is markedly prolonged compared with split-thickness skin grafts but not indefinite. Exposure of tolerant animals to second donor-matched kidneys before VCA increases the longevity of the VCA epidermis, suggesting an increase in the immunomodulatory mechanisms associated with tolerance of the kidney.

Adoptive Transfer of Renal Allograft Tolerance in a Large Animal Model

Our recent studies in an inbred swine model demonstrated that both peripheral and intra‐graft regulatory cells were required for the adoptive transfer of tolerance to a second, naïve donor‐matched kidney. Here, we have asked whether both peripheral and intra‐graft regulatory elements are required for adoptive transfer of tolerance when only a long‐term tolerant (LTT) kidney is transplanted. Nine highly‐inbred swine underwent a tolerance‐inducing regimen to prepare LTT kidney grafts which were then transplanted to histocompatible recipients, with or without the peripheral cell populations required for adoptive transfer of tolerance to a naïve kidney. In contrast to our previous studies, tolerance of the LTT kidney transplants alone was achieved without transfer of additional peripheral cells and without strategies to increase the number/potency of regulatory T cells in the donor. This tolerance was systemic, since most subsequent, donor‐matched challenge kidney grafts were accepted. These results confirm the presence of a potent tolerance‐inducing and/or tolerance‐maintaining cell population within LTT renal allografts. They suggest further that additional peripheral tolerance mechanisms, required for adoptive transfer of tolerance to a naïve donor‐matched kidney, depend on peripheral cells that, if not transferred with the LTT kidney, require time to develop in the adoptive host.

The rejuvenating effects of leuprolide acetate on the aged baboon's thymus

BACKGROUND We have previously demonstrated that the juvenile thymus plays an essential role in tolerance induced by both renal transplantation and a short course of calcineurin inhibitors. Aged thymi have a decreased ability to induce tolerance. Luteinizing hormone-releasing hormone (LHRH) is known to pharmacologically rejuvenate the thymus in rodents. In order to develop a clinically applicable regimen of transplantation tolerance in adults, we sought to determine if thymic rejuvenation would occur with LHRH agonism in non-human primates. METHODS AND RESULTS Thymic rejuvenation was evaluated by magnetic resonance imaging (MRI), histology, as well as in-vitro cellular and molecular tests. Four aged male hamadryas baboons underwent subcutaneous injection of a 3-month depot of Lupron (11.25mg; LI) and were followed for 3 months. Thymi increased volumetrically by MRI. After LI, thymic cellularity markedly increased within the cortical and medullary thymus. Additionally, a significant increase in the CD4(+)/CD45RA(hi+) population in the peripheral blood occurred for 50 days after LI, and flow cytometry of thymic tissue revealed a large increase in the percentage of CD4(+)/CD8(+) cells. TREC assay corroborated enhancement in thymic function. CONCLUSION These data indicate that LI is associated with thymic rejuvenation in baboons, and further confirm that extrinsic factors play an important role in thymic rejuvenation in a non-human primate model.

Increased levels of anti-non-Gal IgG following pig-to-baboon bone marrow transplantation correlate with failure of engraftment

The development of genetically modified pigs, which lack the expression of alpha 1‐3 galactosyl transferase, (GalT‐KO pigs) has facilitated the xenogeneic transplantation of porcine organs and tissues into primates by avoiding hyperacute rejection due to pre‐existing antibodies against the Gal epitope. However, antibodies against other antigens (anti‐non‐Gal antibodies), are found at varying levels in the pre‐transplant sera of most primates. We have previously found that baboons with high levels of pre‐transplant anti‐non‐Gal IgG, conditioned with a non‐myeloablative conditioning regimen, failed to engraft following pig‐to‐baboon bone marrow transplantation (Xenotransplantation, 17, 2010 and 300). Two baboons with low levels of pre‐transplant anti‐non‐Gal IgG, conditioned with the same regimen, showed porcine bone marrow progenitors at 28 days following transplantation, suggesting engraftment. These baboons also showed evidence of donor‐specific hyporesponsiveness. This observation led us to investigate the hypothesis that selecting for baboon recipients with low pre‐transplant anti‐non‐Gal IgG levels might improve engraftment levels following GalT‐KO pig‐to‐baboon bone marrow transplantation.

Role of bone marrow maturity, insulin‐like growth factor 1 receptor and forkhead box protein N1 in thymic involution and rejuvenation

Thymic involution is associated with age‐related changes of the immune system. Utilizing our innovative technique of transplantation of a thymus as an isolated vascularized graft in MHC‐inbred miniature swine, we have previously demonstrated that aged thymi are rejuvenated after transplantation into juvenile swine. Here we have studied the role of insulin‐like growth factor (IGF) and forkhead‐box protein‐N1 (FOXN1) as well as bone marrow (BM) in thymic rejuvenation and involution. We examined thymic rejuvenation and involution by means of histology and flow cytometry. Thymic function was assessed by the ability to induce tolerance of allogeneic kidneys. Aged thymi were rejuvenated in a juvenile environment, and successfully induced organ tolerance, while juvenile thymi in aged recipients involuted and had a limited ability to induce tolerance. However, juvenile BM inhibited the involution process of juvenile thymi in aged recipients. An elevated expression of both FOXN1 and IGF1 receptors (IGF‐1R) was observed in juvenile thymi and rejuvenated thymi. Juvenile BM plays a role in promoting the local thymic milieu as indicated by its ability to inhibit thymic involution in aged animals. The expression of FOXN1 and IGF‐1R was noted to increase under conditions that stimulated rejuvenation, suggesting that these factors are involved in thymic recovery.

Prevention of Early Development of Proteinuria by Rituximab in a Pig to Baboon Thymokidney Model Likely Associated with Modulating Podocyte Function in An SMPDL-3b-Dependent Manner: 1259

Background: Signal regulatory protein (SIRP)α is a critical immunoinhibitory receptor expressed on macrophages, and its interaction with CD47, a ligand for SIRPα, prevents autologous phagocytosis. We have previously proven that the interspecies incompatibility of CD47 causes in vitro phagocytosis of porcine xenogeneic cells by human macrophages. In this study, we used an in vivo mouse model to investigate whether genetic manipulation of rat insulinoma cells for mouse CD47 expression could inhibit macrophage-mediated xenograft rejection. Methods: To determine whether rat CD47 can interact with mouse SIRPa, we compared SIRPa tyrosine phosphorylation in mouse macrophages after interaction of the macrophages with mouse or rat red blood cells (RBCs). To determine whether expression of mouse CD47 on rat insulinoma (INS-1E) cells could confer protection against phagocytosis by mouse macrophages, we generated mouse CD47expressing INS-1E (mCD47-INS-1E) cells. The phagocytotic activity of mouse macrophages towards INS-1E cells was evaluated by in vivo assays. Carboxyfluorescein succinimidyl ester (CFSE)-labeled mCD47-INS-1E cells and control vector-transfected INS-1E (cont-INS1E) cells were injected into the peritoneal cavity of Rag2gc -/mice that had streptozotocin-induced diabetes and lacked T, B, and natural killer (NK) cells. The blood glucose levels were intermittently monitored using a blood glucose test meter. To further investigate whether inhibition of CD47-SIRPα signaling would abrogate the effect of genetic manipulation of CD47 expression, we injected anti-mouse SIRPa monoclonal antibody (mAb) into the peritoneal cavity of Rag2gc -/mice that had streptozotocininduced diabetes. Results: Western blotting showed that incubation of mouse macrophages with mouse RBCs and mCD47-INS-1E cells caused significant SIRPa tyrosine phosphorylation. However, SIRPa tyrosine phosphorylation was not induced in mouse macrophages incubated with rat RBCs and cont-INS-1E cells, similar to that observed in the control macrophages incubated with medium alone. Mouse CD47 expression on INS-1E cells radically reduced the susceptibility of these cells to phagocytosis by mouse macrophages. Diabetic Rag2gc -/mice became normoglycemic immediately after receiving mCD47-INS-1E cells. In contrast, the mice that received cont-INS-1E cells failed to achieve normoglycemia. However, mice that had received mCD47-INS-1E cells and were injected with anti-mouse SIRPa mAb failed to achieve normoglycemia, similar to that observed in mice that had received cont-CD47-INS-1E cells. Conclusions: These results show that interspecies incompatibility of CD47 significantly contributes to in vivo rejection of xenogeneic cells by macrophages. Thus, genetic induction of recipient CD47 on donor cells for generating SIPRα-inhibitory signals on recipient macrophages is necessary to prevent macrophage-mediated xenograft rejection. 1259