Human A2-CAR T Cells Reject HLA-A2 + Human Islets Transplanted Into Mice Without Inducing Graft-versus-host Disease.

BACKGROUND: Type 1 diabetes is an autoimmune disease characterized by T-cell-mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include the use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft-versus-host disease (xGVHD). METHODS: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4 + and CD8 + T cells and tested their ability to reject HLA-A2 + islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T-cell engraftment, islet function, and xGVHD were assessed longitudinally. RESULTS: The speed and consistency of A2-CAR T-cell-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of coinjected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, coinjection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2 + human islets within 1 wk and without xGVHD for 12 wk. CONCLUSIONS: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of islet-replacement therapies.

Human A2-CAR T cells reject HLA-A2+ human islets transplanted into mice without inducing graft versus host disease.

Background: Type 1 diabetes (T1D) is an autoimmune disease characterised by T cell mediated destruction of pancreatic beta-cells. Islet transplantation is an effective therapy, but its success is limited by islet quality and availability along with the need for immunosuppression. New approaches include use of stem cell-derived insulin-producing cells and immunomodulatory therapies, but a limitation is the paucity of reproducible animal models in which interactions between human immune cells and insulin-producing cells can be studied without the complication of xenogeneic graft- versus -host disease (xGVHD). Methods: We expressed an HLA-A2-specific chimeric antigen receptor (A2-CAR) in human CD4+ and CD8+ T cells and tested their ability to reject HLA-A2+ islets transplanted under the kidney capsule or anterior chamber of the eye of immunodeficient mice. T cell engraftment, islet function and xGVHD were assessed longitudinally. Results: The speed and consistency of A2-CAR T cells-mediated islet rejection varied depending on the number of A2-CAR T cells and the absence/presence of co-injected peripheral blood mononuclear cells (PBMCs). When <3 million A2-CAR T cells were injected, co-injection of PBMCs accelerated islet rejection but also induced xGVHD. In the absence of PBMCs, injection of 3 million A2-CAR T cells caused synchronous rejection of A2+ human islets within 1 week and without xGVHD for 12 weeks. Conclusions: Injection of A2-CAR T cells can be used to study rejection of human insulin-producing cells without the complication of xGVHD. The rapidity and synchrony of rejection will facilitate in vivo screening of new therapies designed to improve the success of isletreplacement therapies.

The Impact of Different Implantation Sites and Sex on the Differentiation of Human Pancreatic Endoderm Cells Into Insulin-Secreting Cells In Vivo.

Few studies have examined the differentiation of human embryonic stem cell (hESC)-derived pancreatic endoderm cells (PECs) in different implantation sites. Here, we investigate the influence of implantation site and recipient sex on the differentiation of hESC-derived PECs in vivo. Male and female mice were implanted with 5 × 106 hESC-derived PECs under the kidney capsule, in the gonadal fat pad, or subcutaneously within macroencapsulation (TheraCyte) devices. PECs implanted within TheraCyte devices developed glucose-stimulated human C-peptide secretion faster than cells implanted under the kidney capsule or in the gonadal fat pad. Interestingly, hESC-derived PECs implanted under the kidney capsule in females developed glucose-stimulated human C-peptide faster than in males and secreted higher levels of arginine-stimulated glucagon and glucagon-like peptide 1 than other implantation sites. Furthermore, hESC-derived grafts collected from the kidney capsule and gonadal fat pad sites displayed a mix of endocrine and ductal cells as well as contained cysts, whereas TheraCyte device grafts displayed mostly endocrine cells and cysts were not observed. Here we demonstrate that the macroencapsulated subcutaneous site and the female recipient can promote faster differentiation of hESC-derived PECs to endocrine cells in mice. ARTICLE HIGHLIGHTS: Few studies have directly compared the differentiation of human embryonic stem cell-derived progenitors in different implantation sites in male and female recipients. We investigated whether the site of implantation and/or the sex of the recipient influenced the differentiation of pancreatic progenitors in vivo in mice. Mice implanted with cells in macroencapsulation devices contained fewer off-target structures and developed stimulated insulin release faster than other implant sites, while females implanted with cells under the kidney capsule developed stimulated insulin release before males. Macroencapsulation devices reduced the formation of off-target cells from human embryonic stem cell-derived progenitors, a useful characteristic for clinical applications.

Deletion of pancreas-specific miR-216a reduces beta-cell mass and inhibits pancreatic cancer progression in mice.

miRNAs have crucial functions in many biological processes and are candidate biomarkers of disease. Here, we show that miR-216a is a conserved, pancreas-specific miRNA with important roles in pancreatic islet and acinar cells. Deletion of miR-216a in mice leads to a reduction in islet size, ß-cell mass, and insulin levels. Single-cell RNA sequencing reveals a subpopulation of ß-cells with upregulated acinar cell markers under a high-fat diet. miR-216a is induced by TGF-ß signaling, and inhibition of miR-216a increases apoptosis and decreases cell proliferation in pancreatic cells. Deletion of miR-216a in the pancreatic cancer-prone mouse line KrasG12D;Ptf1aCreER reduces the propensity of pancreatic cancer precursor lesions. Notably, circulating miR-216a levels are elevated in both mice and humans with pancreatic cancer. Collectively, our study gives insights into how ß-cell mass and acinar cell growth are modulated by a pancreas-specific miRNA and also suggests miR-216a as a potential biomarker for diagnosis of pancreatic diseases.

AAV GCG-EGFP, a new tool to identify glucagon-secreting α-cells.

The study of primary glucagon-secreting α-cells is hampered by their low abundance and scattered distribution in rodent pancreatic islets. We have designed a double-stranded adeno-associated virus containing a rat proglucagon promoter (700 bp) driving enhanced green fluorescent protein (AAV GCG-EGFP), to specifically identify α-cells. The administration of AAV GCG-EGFP by intraperitoneal or intraductal injection led to EGFP expression selectively in the α-cell population. AAV GCG-EGFP delivery to mice followed by islet isolation, dispersion and separation by FACS for EGFP resulted in an 86% pure population of α-cells. Furthermore, the administration of AAV GCG-EGFP at various doses to adult wild type mice did not significantly alter body weight, blood glucose, plasma insulin or glucagon levels, glucose tolerance or arginine tolerance. In vitro experiments in transgene positive α-cells demonstrated that EGFP expression did not alter the intracellular Ca2+ pattern in response to glucose or adrenaline. This approach may be useful for studying purified primary α-cells and for the in vivo delivery of other genes selectively to α-cells to further probe their function or to manipulate them for therapeutic purposes.

Cotransplantation of Mesenchymal Stem Cells With Neonatal Porcine Islets Improve Graft Function in Diabetic Mice.

Mesenchymal stem cells (MSCs) possess immunoregulatory, anti-inflammatory, and proangiogenic properties and, therefore, have the potential to improve islet engraftment and survival. We assessed the effect human bone marrow-derived MSCs have on neonatal porcine islets (NPIs) in vitro and determined islet engraftment and metabolic outcomes when cotransplanted in a mouse model. NPIs cocultured with MSCs had greater cellular insulin content and increased glucose-stimulated insulin secretion. NPIs were cotransplanted with or without MSCs in diabetic B6.129S7-Rag1tm1Mom/J mice. Blood glucose and weight were monitored until reversal of diabetes; mice were then given an oral glucose tolerance test. Islet grafts were assessed for the degree of vascularization and total cellular insulin content. Cotransplantation of NPIs and MSCs resulted in significantly earlier normoglycemia and vascularization, improved glucose tolerance, and increased insulin content. One experiment conducted with MSCs from a donor with an autoimmune disorder had no positive effects on transplant outcomes. Cotransplantation of human MSCs with NPIs demonstrated a beneficial metabolic effect likely as a result of earlier islet vascularization and improved islet engraftment. In addition, donor pathology of MSCs can influence the functional capacity of MSCs.