Durable Mixed Chimerism May Permit Subsequent Immunosuppression-Free Intestinal Transplantation - a Proof-of-Principle Study.

Intestinal transplantation (ITx) is the definitive treatment for intestinal failure but has the highest rejection rate among solid organ transplants, requiring high doses of immunosuppression with high rates of infection, graft-versus-host disease, and malignancy. Transplant tolerance would overcome the need for long-term immunosuppression. Using non-myeloablative conditioning, our laboratory has developed a novel swine model of hematopoietic stem cell transplantation (HSCT) that produces durable mixed chimerism (MC) and immune tolerance without toxicity. We investigated whether durable MC would promote tolerance of subsequently transplanted donor-matched intestinal allografts without immunosuppression. Using miniature swine with defined MHC, we performed HSCT across an MHC-Class-I haplotype mismatch. Immunosuppression was stopped by day 45. MC was evaluated by flow cytometry, and mixed lymphocyte reaction (MLR) assays were used to evaluate cellular responses. Subsequently, orthotopic ITx was performed without immunosuppression using a donor that was MHC-matched to the HSCT donor. Recipients were observed for four weeks and euthanized for tissue collection and mechanistic assays. After HSCT, the recipients developed durable multilineage MC and apparent deletional tolerance. After ITx, recipients showed no clinical or histological signs of rejection, and chimerism was unchanged. These results demonstrate the potential value of generating durable MC to achieve transplant tolerance.

Expression of human CD47 in pig glomeruli prevents proteinuria and prolongs graft survival following pig-to-baboon xenotransplantation.

BACKGROUND: Nephrotic syndrome is a common complication of pig-to-baboon kidney xenotransplantation (KXTx) that adversely affects outcomes. We have reported that upregulation of CD80 and down-regulation of SMPDL-3b in glomeruli have an important role in the development of proteinuria following pig-to-baboon KXTx. Recently we found induced expression of human CD47 (hCD47) on endothelial cells and podocytes isolated from hCD47 transgenic (Tg) swine markedly reduced phagocytosis by baboon and human macrophages. These observations led us to hypothesize that transplanting hCD47 Tg porcine kidneys could overcome the incompatibility of the porcine CD47-baboon SIRPα interspecies ligand-receptor interaction and prevent the development of proteinuria following KXTx. METHODS: Ten baboons received pig kidneys with vascularized thymic grafts (n = 8) or intra-bone bone marrow transplants (n = 2). Baboons were divided into three groups (A, B, and C) based on the transgenic expression of hCD47 in GalT-KO pigs. Baboons in Group A received kidney grafts with expression of hCD47 restricted to glomerular cells (n = 2). Baboons in Group B received kidney grafts with high expression of hCD47 on both glomerular and tubular cells of the kidneys (n = 4). Baboons in Group C received kidney grafts with low/no glomerular expression of hCD47, and high expression of hCD47 on renal tubular cells (n = 4). RESULTS: Consistent with this hypothesis, GalT-KO/hCD47 kidney grafts with high expression of hCD47 on glomerular cells developed minimal proteinuria. However, high hCD47 expression in all renal cells including renal tubular cells induced an apparent destructive inflammatory response associated with upregulated thrombospondin-1. This response could be avoided by a short course of weekly anti-IL6R antibody administration, resulting in prolonged survival without proteinuria (mean 170.5 days from 47.8 days). CONCLUSION: Data showed that transgenic expression of hCD47 on glomerular cells in the GalT-KO donor kidneys can prevent xenograft nephropathy, a significant barrier for therapeutic applications of xenotransplantation. The ability to prevent nephrotic syndrome following KXTx overcomes a critical barrier for future clinical applications of KXTx.

Antibody reactivity with new antigens revealed in multi-transgenic triple knockout pigs may cause early loss of pig kidneys in baboons.

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), ß-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 naïve 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.

Intra-bone bone marrow transplantation from hCD47 transgenic pigs to baboons prolongs chimerism to >60 days and promotes increased porcine lung transplant survival.

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.

Safety and pharmacodynamics of anti-CD2 monoclonal antibody treatment in cynomolgus macaques - an experimental study.

Anti-CD2 treatment provides targeted immunomodulatory properties that have demonstrated clinical usefulness to condition the immune system and to treat transplant rejection. The treatment is species-specific due to structural CD2 antigen differences between nonhuman primates and humans. Herein, we report the safety profile and efficacy of two modifications of the same anti-CD2 monoclonal antibody in cynomolgus macaques. Twelve subjects received one i.v. anti-CD2 (of rat or rhesus type) dose each, range 1-4 mg/kg, and were followed for 1-7 days. Treatment effects were evaluated with flow cytometry on peripheral blood and histopathological evaluation of secondary lymphoid organs. In vitro inhibitory activity on primary MHC disparate mixed lymphocyte reactions (MLRs) was determined. Upon anti-CD2 treatment, CD4+ , CD8+ memory subsets were substantially depleted. Naïve T cells and Tregs were relatively spared and exhibited lower CD2 expression than memory T cells. Early immune reconstitution was noted for naïve cells, while memory counts had not recovered after one week. Both antibodies displayed a concentration-dependent MLR inhibition. Lymph node examination revealed no significant lymphocyte depletion. None of the animals experienced any significant study drug-related adverse events. This study outlines the safety and pharmacodynamic profile of primate-specific anti-CD2 treatment, relevant for translation of anti-CD2-based animal models into clinical trials.

GalT-KO pig lungs are highly susceptible to acute vascular rejection in baboons, which may be mitigated by transgenic expression of hCD47 on porcine blood vessels.

BACKGROUND: Despite recent progress in survival times of xenografts in non-human primates, there are no reports of survival beyond 5 days of histologically well-aerated porcine lung grafts in baboons. Here, we report our initial results of pig-to-baboon xeno-lung transplantation (XLTx). METHODS: Eleven 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. RESULTS: Lung graft survivals in the 2 non-survival animals were 48 and >72 hours, while survivals in the other 9 were 25 and 28 hours, at 5, 5, 6, 7, >7, 9, and 10 days. One baboon with graft survival >7 days, 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 10 days. 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. CONCLUSIONS: To our knowledge, this is the first evidence of histologically viable porcine lung grafts beyond 7 days 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.

Development of a large animal orthotopic intestinal transplantation model with long-term survival for study of immunologic outcomes.

Introduction: Intestinal transplantation (ITx) is the last remaining therapy for patients with intestinal failure once parenteral nutrition is no longer an option, however its use is limited by immunological complications, including high rates of rejection and morbidity associated with immunosuppression, such as infection and malignancy. We aimed to develop a large animal model of ITx with which to study the immune response to ITx and to design and test tolerance induction regimens. Methods: Learning from prior complications, we developed and progressively improved both surgical methods for the donor and recipient as well as postoperative management strategies. Methods of stoma generation, bowel positioning, vessel preparation, and fluid management were optimized. The immunosuppression strategy mirrored our clinical regimen. Results: As a result of our modifications, results improved from survival less than 1 month to consistent long-term survival with good graft function. We review several techniques that were developed to avoid pitfalls that were encountered, which can be used to optimize outcomes in this model. Discussion: Achieving long-term survival after swine orthotopic ITx permits immunological analysis and pre-clinical trials in a large animal model of ITx.

Impact of CMV Reactivation, Treatment Approaches, and Immune Reconstitution in a Nonmyeloablative Tolerance Induction Protocol in Cynomolgus Macaques.

BACKGROUND: Cytomegalovirus (CMV) infection is a serious complication in immunosuppressed patients, specifically transplant recipients. Here, we describe the development and use of an assay to monitor the incidence and treatment of CMV viremia in a Cynomolgus macaque model of bone marrow transplantation (BMT) for tolerance induction. We address the correlation between the course of viremia and immune reconstitution. METHODS: Twenty-one animals received a nonmyeloablative conditioning regimen. Seven received cyclosporine A for 28 days and 14 received rapamycin. A CMV polymerase chain reaction assay was developed and run twice per week to monitor viremia. Nineteen recipients were CMV seropositive before BMT. Immune reconstitution was monitored through flow cytometry and CMV viremia was tracked via quantitative polymerase chain reaction. RESULTS: Recipients developed CMV viremia during the first month post-BMT. Two animals developed uncontrollable CMV disease. CMV reactivation occurred earlier in cyclosporine A-treated animals compared with those receiving rapamycin. Post-BMT, T-cell counts remained significantly lower compared with pretransplant levels until CMV reactivation, at which point they increased during the viremic phase and approached pretransplant levels 3 months post-BMT. Management of CMV required treatment before viremia reached 10 000 copies/mL; otherwise clinical symptoms were observed. High doses of ganciclovir resolved the viremia, which could subsequently be controlled with valganciclovir. CONCLUSIONS: We developed an assay to monitor CMV in Cynomolgus macaques. CMV reactivation occurred in 100% of seropositive animals in this model. Rapamycin delayed CMV reactivation and ganciclovir treatment was effective at high doses. As in humans, CD8 T cells proliferated during CMV viremia.

Norovirus Changes Susceptibility to Type 1 Diabetes by Altering Intestinal Microbiota and Immune Cell Functions.

Environmental factors contribute to Type 1 diabetes (T1D) susceptibility. The gut microbiome, which includes bacteria, viruses, and fungi, contributes to this environmental influence, and can induce immunological changes. The gut viral component of the microbiome, related to T1D has mostly focused on coxsackieviruses and rotavirus. The role of norovirus, another common enteric virus, in susceptibility to T1D was hitherto unknown. Norovirus is highly infectious and encountered by many children. We studied the mouse norovirus 4 (MNV4), related to human noroviruses, in the Non-obese diabetic (NOD) mouse model, to determine its role in influencing susceptibility to T1D. We infected MNV-free NOD mice with MNV4 by exposing the mice to MNV4-positive bedding from an endemically-infected mouse colony to mimic a natural infection. Control MNV-free NOD mice were exposed to MNV-free bedding from the same colony. Interestingly, MNV4 infection protected NOD mice from the development of T1D and was associated with an expansion of Tregs and reduced proinflammatory T cells. We also found MNV4 significantly modified the gut commensal bacteria composition, promoting increased α-diversity and Firmicutes/Bacteroidetes ratio. To elucidate whether T1D protection was directly related to MNV4, or indirectly through modulating gut microbiota, we colonized germ-free (GF) NOD mice with the MNV4-containing or non-MNV4-containing viral filtrate, isolated from filtered fecal material. We found that MNV4 induced significant changes in mucosal immunity, including altered Tuft cell markers, cytokine secretion, antiviral immune signaling markers, and the concentration of mucosal antibodies. Systemically, MNV4-infection altered the immune cells including B cell subsets, macrophages and T cells, and especially induced an increase in Treg number and function. Furthermore, in vitro primary exposure of the norovirus filtrate to naïve splenocytes identified significant increases in the proportion of activated and CTLA4-expressing Tregs. Our data provide novel knowledge that norovirus can protect NOD mice from T1D development by inducing the expansion of Tregs and reducing inflammatory T cells. Our study also highlights the importance of distinguishing the mucosal immunity mediated by bacteria from that by enteric viruses.

Retrospective Review of Surgical Outcomes and Pair-housing Success in Vasectomized Rhesus Macaques (Macaca mulatta).

Providing social housing for adult male macaques can be challenging. One successful strategy for long-term social housing of adult male macaques is to pair them with adult females; however, unwanted breeding must be prevented by sterilization of the male or female. Vasectomy is a simple, highly effective, and minimally invasive method of contraception that is used at our institution to facilitate social housing. We performed a retrospective review to analyze the surgical outcomes and rate of postoperative complications after vasectomy of adult rhesus macaques at our research facility. In addition, we evaluated the success rate of pairing vasectomized macaques with female partners. Over 10 y, 16 macaques were vasectomized, of which 5 developed postoperative complications such as orchitis, epididymitis, or surgical site infection. These complications resolved completely and without incident after antibiotic and analgesic therapy; an additional male had postoperative incisional swelling that resolved quickly after NSAID treatment. This complication rate is consistent with that in humans by surgeons who perform open vasectomies relatively infrequently. In addition, 5 of the vasectomized macaques (31%) developed sperm granulomas, which are a common and generally benign complication in humans and have been reported to develop in 40% of macaques after vasectomy. Successful pair housing with a female partner was achieved for 13 of 16 (81%) of the vasectomized macaques. We conclude that surgical vasectomy is a safe and simple procedure that can be used as a highly effective method to facilitate social housing of adult male rhesus macaques in research facilities.