The widespread clinical implementation of alloislet transplantation as therapy for type 1 diabetes has been hindered by the lack of suitable islet donors. Pig-to-human islet xenotransplantation is one strategy with potential to alleviate this shortage. Long-term survival of porcine islets has been achieved using antibodies targeting CD154; however, this approach lacks translational potential secondary to associated thromboembolic sequelae. As an alternative strategy targeting the CD40/CD154 T cell activation pathway, we evaluate the ability of a chimeric anti-CD40 monoclonal antibody (Chi220) to protect islet xenografts. Neonatal porcine islets (~50,000 IEQ/kg) were transplanted intraportally into surgically-induced diabetic macaques. Immunosuppression consisted of induction therapy with Chi220 and anti-IL-2 receptor (basiliximab), and maintenance therapy with sirolimus and belatacept (a high-affinity CTLA-4Ig variant). Chi220 effectively promoted xenoislet engraftment and survival; five of six treated recipients achieved insulin-independent normoglycemia (mean length of graft survival 90.8 days, maximum survival of 203 days). No thromboembolic phenomena were noted. CD40 represents a promising alternative to CD154 as a therapeutic target in xenoislet transplantation; other potentially translatable anti-CD40 antibodies warrant further investigation in non-human primate models.

Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. While galactose-α1,3-galactose, the gal epitope, has emerged as the principal target of rejection in pig-to-primate models of solid organ transplant, the importance of gal-specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal-expressing wild-type (WT) or gal-deficient galactosyl transferase knock-out (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n=5) or GTKO (n=5) NPIs. Recipient blood glucose, transaminase, and serum xenoantibody levels were used to monitor response to transplant. Four of 5 GTKO versus 1 of 5 WT recipients achieved insulin-independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step towards improved consistency and interpretability of results in future xenoislet studies.

Immunosuppression is currently the treatment of choice to attenuate the chronic deterioration of tissue function as a result of the effector mechanisms of the immunological response in transplant rejection and autoimmune diseases. However, global immunosuppression greatly increases the risk of acquiring life-threatening infections and is associated with organ toxicity when used long-term. Thus, alternative approaches that inhibit only the unwanted immune responses and preserve general immunity are highly desirable. The receptor/ligand pairs involved in the cross-talk between DC and T cells have been the focus of intense and exciting research during the last decade. The HVEM/LIGHT/BTLA/CD160 costimulatory/coinhibitory pathway has emerged as a potential target for the development of immune therapeutic interventions. Herein, we will summarize and discuss how blockade of the costimulatory HVEM/LIGHT interaction or agonist signaling through the inhibitory BTLA and CD160 receptors could contribute to the control of deleterious immune responses.

The International Xenotransplantation Association has updated its original "Consensus Statement on Conditions for Undertaking Clinical Trials of Porcine Islet Products in Type 1 Diabetes," which was published in Xenotransplantation in 2009. This update is timely and important in light of scientific progress and changes in the regulatory framework pertinent to islet xenotransplantation. Except for the chapter on "informed consent," which has remained relevant in its 2009 version, all other chapters included in the initial consensus statement have been revised for inclusion in this update. These chapters will not provide complete revisions of the original chapters; rather, they restate the key points made in 2009, emphasize new and under-appreciated topics not fully addressed in 2009, suggest relevant revisions, and communicate opinions that complement the consensus opinion. Chapter 1 provides an update on national regulatory frameworks addressing xenotransplantation. Chapter 2 a, previously Chapter 2, suggests several important revisions regarding the generation of suitable source pigs from the perspective of the prevention of xenozoonoses. The newly added Chapter 2b discusses conditions for the use of genetically modified source pigs in clinical islet xenotransplantation. Chapter 3 reviews porcine islet product manufacturing and release testing. Chapter 4 revisits the critically important topic of preclinical efficacy and safety data required to justify a clinical trial. The main achievements in the field of transmission of all porcine microorganisms, the rationale for more proportionate recipient monitoring, and response plans are reviewed in Chapter 5. Patient selection criteria and circumstances where trials of islet xenotransplantation would be both medically and ethically justified are examined in Chapter 6 in the context of recent advances in available and emerging alternative therapies for serious and potentially life-threatening complications of diabetes. It is hoped that this first update of the International Xenotransplantation Association porcine islet transplant consensus statement will assist the islet xenotransplant scientific community, sponsors, regulators, and other stakeholders actively involved in the clinical translation of islet xenotransplantation.

Immunosuppressive therapies that block the CD40/CD154 costimulatory pathway have proven to be uniquely effective in preclinical xenotransplant models. Given the challenges facing clinical translation of CD40/CD154 pathway blockade, we examined the efficacy and tolerability of CD40/CD154 pathway-sparing immunomodulatory strategies in a pig-to-nonhuman primate islet xenotransplant model. Rhesus macaques were rendered diabetic with streptozocin and given an intraportal infusion of ~50,000 IEQ/kg wild-type neonatal porcine islets. Base immunosuppression for all recipients included maintenance therapy with belatacept and mycophenolate mofetil plus induction with basiliximab and LFA-1 blockade. Cohort 1 recipients (n=3) were treated with the base regimen alone; cohort 2 recipients (n=5) were additionally treated with tacrolimus induction, and cohort 3 recipients (n=5) were treated with alefacept in place of basiliximab, and more intense LFA-1 blockade. Three of 5 recipients in both cohorts 2 and 3 achieved sustained insulin-independent normoglycemia (median rejection-free survivals 60 and 111 days, respectively), compared to 0 of 3 recipients in cohort 1. These data show that CD40/CD154 pathway-sparing regimens can promote xenoislet survival. Further optimization of these strategies is warranted to aid the clinical translation of islet xenotransplantation.

Sertoli cells have long since been recognized for their ability to suppress the immune system and protect themselves as well as other cell types from harmful immune reaction. However, the exact mechanism or product produced by Sertoli cells that affords this immunoprotection has never been fully elucidated. We examined the effect of mouse Sertoli cell-conditioned medium on human granzyme B-mediated killing and found that there was an inhibitory effect. We subsequently found that a factor secreted by Sertoli cells inhibited killing through the inhibition of granzyme B enzymatic activity. SDS-PAGE analysis revealed that this factor formed an SDS-insoluble complex with granzyme B. Immunoprecipitation and mass spectroscopic analysis of the complex identified a proteinase inhibitor, serpina3n, as a novel inhibitor of human granzyme B. We cloned serpina3n cDNA, expressed it in Jurkat cells, and confirmed its inhibitory action on granzyme B activity. Our studies have led to the discovery of a new inhibitor of granzyme B and have uncovered a new mechanism used by Sertoli cells for immunoprotection.

Decellularization significantly reduces the cellular and humoral immune response to allograft tissue. This could prolong the durability of valve allografts and might prevent immunologic sensitization of allograft recipients.

A variety of transient therapies directed against molecules involved in T-cell activation and function result in long-term islet allograft survival. However, there are relatively few examples of durable islet xenograft survival using similar short-term approaches, especially regarding highly phylogenetically disparate xenograft donors. Previous studies demonstrate that combined anti-lymphocyte function-associated antigen-1 (LFA-1) plus anti-CD154 therapy results in a robust form of islet allograft tolerance not observed with either individual monotherapy. Thus, the aim of this study was to determine whether the perturbation of anti-LFA-1, either alone or in combination with targeting CD154 or CD45RB, would promote neonatal porcine islet (NPI) xenograft survival in mice. NPI xenografts are rapidly rejected in wild-type C57BL/6 mice but reproducibly mature and restore durable euglycemia in diabetic, immune-deficient C57BL/6 rag-1 Ϫ/Ϫ recipients. A short course of individual anti-LFA-1, anti-CD154, or anti-CD45RB therapy resulted in long-term (>100 days) survival in a moderate proportion of C57BL/6 recipients. However, simultaneous treatment with anti-LFA-1 plus either anti-CD154 or anti-CD45RB therapy could achieve indefinite xenograft function in the majority of recipient animals. Importantly, prolongation of islet xenograft survival using combined anti-LFA-1/anti-CD154 therapy was associated with little mononuclear cell infiltration and greatly reduced anti-porcine antibody levels. Taken E ndocrine replacement therapy by islet transplantation is an attractive alternative treatment for patients with type 1 diabetes. However, the widespread clinical application of this treatment is currently limited by the shortage of human cadaveric organs available for transplantation. Transplantation of islets derived from pigs may be one approach that could solve the shortage in human islets, provided that formidable xenograft rejection can be prevented. Neonatal pigs in particular are an attractive inexpensive alternative source of insulin-producing tissue for clinical transplantation. Single neonatal pig donors yield ϳ50,000 islet cell aggregates that consist primarily of epithelial cells and pancreatic endocrine cells (1). Importantly, neonatal porcine islets (NPIs) are responsive to glucose challenge in vitro and are capable of maturing and reversing hyperglycemia in immune-deficient animals (1). Although neonatal pig shows promise in providing an abundant number of islets for clinical transplantation, we have recently reported that NPI xenografts are vigorously rejected when transplanted into untreated mice (2). Thus, the potential of neonatal pigs as a source of insulin-producing tissue for clinical transplantation is greatly hampered by a formidable cellular xenograft response. While a number of short-term approaches to blocking/perturbing a variety of cell surface molecules involved in T-cell function can result in longterm islet allograft survival, it is less clear whether T-celldirected therapies can result in prolo...