Xenotransplantation has great potential to provide beta cell replacement and thereby provide a cure for large numbers of people with type 1 diabetes. Crucial to the success of xenotransplantation is establishment of the most viable sites for transplantation.

Porcine neonatal islet‐like cell clusters (NICC) are being considered as a source of β‐cell replacement. However, the lag time to full function due to hormonal immaturity remains a problem. This study aimed to determine whether time in culture was important for NICC function in vivo.

Objective. Identifying a limitless source of β-cells that survive transplantation into a neovascularised site and provide normal blood glucose control remains an important goal in the development of pancreatic islet xenotransplantation. It was our hypothesis that fetal porcine pancreas fragments could achieve these objectives, and this was tested in a large preclinical animal model. Research Design and Methods. Inbred “Westran Pig” fetal porcine pancreas fragments were transplanted beneath the splenic capsule into syngeneic Westran Pig recipients without immunosuppression, and 3 months later, a total native pancreatectomy was performed to demonstrate function. Results. Histologic analysis showed appropriate development of islet-like structures up to and beyond 120 days after transplantation. After native pancreatectomy, recipients survived more than 100 days without exogenous insulin and with normal glucose homeostasis as assessed by normal glucose tolerance tests, K values, and normal glucagon secretion. Conclusions. This study confirms that fetal pig islet tissue has the potential to mature and function normally in a neovascularised site, hence, avoiding the innate immune destruction that occurs when islet tissue is exposed directly to the circulation.

Hawthorne WJ, Simond DM, Stokes R, Patel AT, Walters S, Burgess J, O’Connell PJ. Pre‐clinical model of composite foetal pig pancreas fragment/renal xenotransplantation to treat renal failure and diabetes. Xenotransplantation 2011; 18: 390–399. © 2011 John Wiley & Sons A/S. Abstract: Background: Development of a limitless source of β cells for xenotransplantation into patients suffering type 1 diabetes and renal failure that can control their diabetes and provide normal renal function in one procedure would be a major achievement. For the islet tissue to survive transplantation, as an islet‐kidney composite graft this would have significant advantages. It would simplify the surgical procedure; remove the complications caused by the exocrine pancreas whilst reversing diabetes and uraemia. It was our hypothesis that a composite foetal porcine pancreas fragment (FPPF)/renal graft could achieve these objectives in a large pre‐clinical animal model as a means to establish whether this would be feasible before moving to the clinic. Methods: Inbred ‘Westran’ pig FPPF were transplanted under the kidney capsule of syngeneic Westran pig recipients without immunosuppression. Following maturation of the FPPF under the renal subcapsular space of this recipient, this kidney bearing the composite FPPF piggyback graft was removed and transplanted into another nephrectomized and pancreatectomized recipient to demonstrate function. Results: Under the kidney capsule of the first transplant group (n = 6), the FPPF‐transplanted tissue developed and matured to form islet cell nests. These composite FPPF/renal grafts were then successfully removed and transplanted into the second functional assessment recipient group. This second group of six composite FPPF/renal‐grafted pigs had normal renal function for more than 44 days and normal glucose homoeostasis without exogenous insulin as assessed by normal glucose tolerance tests, K values and normal glucagon secretion. Histological analysis showed despite the ischaemic insult during the composite kidney transplant procedure, there was appropriate development of islet‐like structures up to and beyond 224 days after the original transplantation under the kidney capsule. Conclusions: This study shows that the use of composite FPPF/renal grafts can cure both diabetes and renal failure with a single‐transplant procedure. Using such composite grafts for xenotransplantation would simplify the surgical procedure and protect the islet graft from the immediate innate immune response.

Background. Macrophages are important effector cells in T cell-mediated xenograft rejection. The aim of this study was to determine whether CD4+CD25+ regulatory T cells (Tregs) were capable of suppressing macrophage activation in vitro. Methods. Porcine cell or xenoantigen-primed human peripheral blood mononuclear cells, CD4+ T cell-depleted peripheral blood mononuclear cells, or CD14+ macrophages plus autologous CD4+CD25− T cells were cultured with or without expanded autologous Tregs. Transwell cultures were used to separate the various components to determine the need for cell-cell contact. Results. Pig cell primed CD14+ macrophages required the presence of CD4+CD25− T cells for activation and increased expression of CD40, interleukin-12, and tumor necrosis factor-&agr;. This up-regulated expression of macrophage activation markers was reduced substantially in the presence of autologous Tregs. Coculture with Tregs did not alter macrophage viability but reduced the capacity of macrophages to stimulate proliferation of responder T cells. Tregs required direct contact with CD4+CD25− T cells to inhibit macrophage activation but activated macrophage phenotype was not altered by separating the stimulated human peripheral blood mononuclear cells or CD14+ macrophages from Tregs in a transwell system. Macrophages did not require direct cell contact with porcine stimulator cells for full activation by CD4+CD25− T cells. Conclusions. Human Tregs were able to suppress xenoantigen-primed and CD4+ T-cell-mediated macrophage activation and antigen-presenting cell function. However, Tregs had no direct effect on macrophages in vitro.

Background. Cellular rejection of xenografts is predominantly mediated by CD4(+) T cells. Little is known of the effectiveness of CD4(+)CD25(+) T regulatory (Treg) cells at suppressing this strong T-cell mediated immune response. In this study, we evaluated the activity of fresh Treg cells and expanded Treg cells to suppress the xeno immune response in vitro.Methods. Human Treg cells were preferentially expanded by CD3/CD28 expand beads, interleukin (IL)-2, and rapamycin. Human CD4(+)CD25(-) T cells were stimulated with irradiated porcine peripheral blood mononuclear cells in the presence or absence of fresh or expanded human Treg cells for 5 days before proliferation assay. In a separate experiment, the porcine xenoantigen-stimulated CD4(+)CD25(-) T cells were separated from Treg cells by transwells and assessed for cytotoxicity of porcine peripheral blood mononuclear cells target cells. Cytokine-producing cells and cytokine release in the cocultures were examined by enzyme-linked immunosorbent spot and enzyme-linked immonosorbent assay, respectively.Results. Human Treg were expanded up to 3500-fold after 14 days in culture. The addition of fresh Treg suppressed the T-cell mediated xenoimmune response. Compared with fresh Treg cells, expanded Treg cells were more Potent at suppressing CD4(+)CD25(-) T-cell-mediated antiporcine xenogeneic responses. This suppression required cell contact. However, the enhanced suppression by expanded Treg cells was associated with increased secretion of IL-4 and IL-10 when compared with their nonexpanded Treg counterparts.Conclusion. This study shows that expanded human Treg cells were capable of suppressing antiporcine xenogeneic responses in vitro and involve both contact dependent and cytokine mediated mechanisms.