Studies of clinically applicable human tolerogenic dendritic cells and PD-L2 genetic modification of human islet allograft to promote graft tolerance.

Islet transplantation is a developing therapy for type 1 diabetic patients (T1D), which has been limited by problems associated with hypoxia, poor revascularisation and allograft rejection. Immunosuppressive agents used to prevent rejection are associated with severe side effects including islet toxicity, increased susceptibility to the development of cancer, infections and cardio-vascular problems. In order for islet transplantation to be used widely as a potentially curative treatment for T1D there is a need to develop novel therapies to treat allograft rejection without the use of immunosuppressive agents. In chapter 3, the immunomodulatory effects of IFN-γ on human monocyte-derived DC were investigated, using a standard 7-day in vitro DC propagation protocol. IFN-γ was shown to exert its immunomodulatory function on monocytes early during DC differentiation (IFNγ-DC[subscript]D0), resulting in an immature DC (iDC) phenotype with reduced expression of maturation markers CD83 and RelB. IFNγ-DC[subscript]D0 induced a state of T-cell hyporesponsiveness in a MLR, whilst IFN-γ treatment at day 5 (IFNγ-DC[subscript]D5) did not modulate DC function. The ability of IFN-γ to promote the generation of maturation arrested DC, could potentially serve as a cellular therapy for transplant rejection. However DC propagation using the standard 7-10 day protocol is not clinically applicable in the islet transplant setting. In chapter 4, a 'FAST-DC' protocol to promote the rapid generation of tolerogenic DC was investigated and used to generate IFNγ modulated DC in 48h. These IFNγ-DC featured an iDC phenotype similar to that seen in chapter 3. Maturation arrested IFNγ-DC caused significant T-cell hyporesponsiveness and promoted a higher frequency of CD4+CD25+ Foxp3[superscript]HI T-regulatory cells. IFNγ-DC primed T-cells were shown to be functionally suppressive in an antigen specific manner. It was also confirmed that IFN-γ reduced the phosphorylation of IL-4 activated STAT-6, which in turn affected the downstream gene expression of Interferon regulatory factor 4 (IRF4). IFNγ-DC were also investigated in vivo, where a humanised model of islet allo-transplantation model was developed. Diabetic NOD-SCID mice were transplanted with human islets and challenged with donor-derived DC and allogeneic PBMNC. After 21 days post transplantation, there was no significant change to euglycaemic state, between the tested groups. Genetic modification of the allograft is an alternative therapy to protecting the graft from the recipient‟s immune system. In chapter 5, human islets were genetically modified with programmed cell death ligand 2 (PD-L2), an inhibitory molecule known inhibit T-cell immune responses. Two recombinant adenovirus constructs carrying the PD-L2 gene were generated. One construct encoded a soluble isoform, while the other expressed a full transmembrane PD-L2 molecule. Adenoviral transduction did not affect the viability or insulin producing capacity of islets. Interestingly, soluble PD-L2 was more efficient at inducing signalling by 1000 fold, compared to the transmembrane isoform. In summary, this thesis demonstrated the timing of IFN-γ exposure is crucial in determining the function of DC and their maturational state, where IFN-γ exposure only during DC differentiation resulted in the inhibition of DC maturation. Secondly, the combination of IFN-γ and a FAST-DC protocol, enabled the generation of tolerogenic DC in 48h, making DC therapy more clinically applicable. Finally, the induced expression of soluble PD-L2 by human islets potently signals through human PD-1, which may provide the basis for the protection of islets from allo- and auto T-cell responses.Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 201

Development of a Coaxial 3D Printing Platform for Biofabrication of Implantable Islet-Containing Constructs

Over the last two decades, pancreatic islet transplantations have become a promising treatment for Type I diabetes. However, although providing a consistent and sustained exogenous insulin supply, there are a number of limitations hindering the widespread application of this approach. These include the lack of sufficient vasculature and allogeneic immune attacks after transplantation, which both contribute to poor cell survival rates. Here, these issues are addressed using a biofabrication approach. An alginate/gelatin-based bioink formulation is optimized for islet and islet-related cell encapsulation and 3D printing. In addition, a custom-designed coaxial printer is developed for 3D printing of multicellular islet-containing constructs. In this work, the ability to fabricate 3D constructs with precise control over the distribution of multiple cell types is demonstrated. In addition, it is shown that the viability of pancreatic islets is well maintained after the 3D printing process. Taken together, these results represent the first step toward an improved vehicle for islet transplantation and a potential novel strategy to treat Type I diabetes

Desmoglein-2 is Important for Islet Function and β-Cell Survival

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg

IGF-2 coated porous collagen microwells for the culture of pancreatic islets

Islet transplantation, the only curative therapy for type I diabetes, requires isolation of the graft in highly specialized facilities for its later dispatch to remote transplantation centres. During transport and culture, many valuable cells are lost due to several factors such as mechanical stress, islet aggregation and dissociation. Here, we evaluate a porous microwell array sheet made of natural collagen type I extracellular matrix (ECM) protein as a novel islet culture substrate. This culture platform can be coated with IGF-2, a growth factor favorable for islet survival, and allows segregation of the islets within the porous microwell sheet, preventing aggregation. This design shows promising results for improving human pancreatic islets viability and function during culture and could form a novel paradigm for the transport of islets between isolation and transplantation centres

A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response

SARS-CoV-2 produces a microRNA CoV2-miR-O8 in patients with COVID-19 infection

Summary: Many viruses produce microRNAs (miRNAs), termed viral miRNAs (v-miRNAs), with the capacity to target host gene expression. Bioinformatic and cell culture studies suggest that SARS-CoV-2 can also generate v-miRNAs. This patient-based study defines the SARS-CoV-2 encoded small RNAs present in nasopharyngeal swabs of patients with COVID-19 infection using small RNA-seq. A specific conserved sequence (CoV2-miR-O8) is defined that is not expressed in other coronaviruses but is preserved in all SARS-CoV-2 variants. CoV2-miR-O8 is highly represented in nasopharyngeal samples from patients with COVID-19 infection, is detected by RT-PCR assays in patients, has features consistent with Dicer and Drosha generation as well as interaction with Argonaute and targets specific human microRNAs

Desmoglein-2 is important for islet function and β-cell survival

Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lacking Dsg2 were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal that DSG2 is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In a Dsg2 loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster. Dsg2lo/lo mice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated from Dsg2lo/lo mice were more susceptible to cytokine-induced β-cell apoptosis. Following transplantation into diabetic mice, islets isolated from Dsg2lo/lo mice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine β-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of β-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes