LEA29Y expression in transgenic neonatal porcine islet-like cluster promotes long-lasting xenograft survival in humanized mice without immunosuppressive therapy

Abstract

Genetically engineered pigs are a promising source for islet cell transplantation in type 1 diabetes, but the strong human anti-pig immune response prevents its successful clinical application. Here we studied the efficacy of neonatal porcine islet-like cell clusters (NPICCs) overexpressing LEA29Y, a high-affinity variant of the T cell co-stimulation inhibitor CTLA-4Ig, to engraft and restore normoglycemia after transplantation into streptozotocin-diabetic NOD-SCID IL2rγ−/− (NSG) mice stably reconstituted with a human immune system. Transplantation of INSLEA29Y expressing NPICCs resulted in development of normal glucose tolerance (70.4%) and long-term maintenance of normoglycemia without administration of immunosuppressive drugs. All animals transplanted with wild-type NPICCs remained diabetic. Immunohistological examinations revealed a strong peri- and intragraft infiltration of wild-type NPICCs with human CD45+ immune cells consisting of predominantly CD4+ and CD8+ lymphocytes and some CD68+ macrophages and FoxP3+ regulatory T cells. Significantly less infiltrating lymphocytes and only few macrophages were observed in animals transplanted with INSLEA29Y transgenic NPICCs. This is the first study providing evidence that beta cell-specific LEA29Y expression is effective for NPICC engraftment in the presence of a humanized immune system and it has a long-lasting protective effect on inhibition of human anti-pig xenoimmunity. Our findings may have important implications for the development of a low-toxic protocol for porcine islet transplantation in patients with type 1 diabetes.

Pigs expressing the human inhibitory ligand PD‐L1 (CD 274) provide a new source of xenogeneic cells and tissues with low immunogenic properties

“…It has recently been published that AAV8‐mediated expression of a PD‐L1‐CTLA4.Ig‐vector‐protected islets from rejection in an allogeneic mouse model, suggesting that enhanced inhibitory signaling (PD‐L1) combined with blockade of co‐stimulatory signals (CTLA4.Ig) could be a highly effective strategy. Transfer of the combinatory approach to xenotransplantation and testing the hypothesis could readily be conducted because in addition to hPD‐L1 transgenic pigs described here pigs expressing soluble LEA29Y (a more potent derivate of human CTLA4.Ig) are available …”

“…Transfer of the combinatory approach to xenotransplantation and testing the hypothesis could readily be conducted because in addition to hPD-L1 transgenic pigs described here pigs expressing soluble LEA29Y (a more potent derivate of human CTLA4.Ig) are available. 65,66 Hyperacute and acute vascular rejections are powerful immunological hurdles on the way to clinical xenotransplantation. 2 It is therefore understandable that genetic engineering strategies so far were initially designed to overcome these early immune responses.…”

Integration of nano‐ and biotechnology for beta‐cell and islet transplantation in type‐1 diabetes treatment

“…In order to build up a breeding colony, pre‐existing genetically modified pig lines were introduced into a specified pathogen‐free pig facility —CiMM (Centre for Innovative Medical Models) by embryo transfer into specified pathogen‐free recipient sows, where embryos were generated by somatic cell nuclear transfer (SCNT) or in vitro fertilization . Thereby, provision of genetically optimized porcine pancreatic islets expressing LEA29Y or INS‐eGFP expressing reporter islets suitable for human xenotransplantation processes is enabled. The iRFP720 expression of transgenic animals was verified by FACS analysis and subsequently by in vivo imaging using MSOT technology in transgenic pigs (Figure ).…”

Humanizing the mouse immune system to study splanchnic organ inflammation

“…have recently generated a new transgenic pig that overexpresses a high affinity, immunoglobulin fusion protein (called LEA29Y) under the control of the porcine insulin gene promoter (Buerck et al . ). LEA29Y was developed as a high affinity analogue of CTLA‐4 Ig that binds tightly to the B‐7 receptors (CD80 and CD86) expressed on antigen‐presenting cells thereby suppressing T cell activation and proliferation (Larsen et al .…”

“…Buerck and coworkers demonstrated that engraftment of neonatal porcine islet‐like cell clusters (NPICCs) overexpressing LEA29Y restored glucose homeostasis in streptozotocin‐treated (diabetic) Hu‐HSC‐IL2rγ −/− mice (Buerck et al . ). They found that transplantation of transgenic but not wild type NPICCs into these diabetic humanized mice resulted in long‐term control of hyperglycaemia in the absence of xenogeneic GVHD.…”