Publications by authors named "Thomas W Kay"

124 Publications

Tolerance to Proinsulin-1 Reduces Autoimmune Diabetes in NOD Mice.

Front Immunol 2021 25;12:645817. Epub 2021 Mar 25.

St. Vincent's Institute, Fitzroy, VIC, Australia.

T-cell responses to insulin and its precursor proinsulin are central to islet autoimmunity in humans and non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes. Mice have two proinsulin genes proinsulin -1 and 2 that are differentially expressed, with predominant proinsulin-2 expression in the thymus and proinsulin-1 in islet beta-cells. In contrast to proinsulin-2, proinsulin-1 knockout NOD mice are protected from autoimmune diabetes. This indicates that proinsulin-1 epitopes in beta-cells maybe preferentially targeted by autoreactive T cells. To study the contribution of proinsulin-1 reactive T cells in autoimmune diabetes, we generated transgenic NOD mice with tetracycline-regulated expression of proinsulin-1 in antigen presenting cells (TIP-1 mice) with an aim to induce immune tolerance. TIP-1 mice displayed a significantly reduced incidence of spontaneous diabetes, which was associated with reduced severity of insulitis and insulin autoantibody development. Antigen experienced proinsulin specific T cells were significantly reduced in in TIP-1 mice indicating immune tolerance. Moreover, T cells from TIP-1 mice expressing proinsulin-1 transferred diabetes at a significantly reduced frequency. However, proinsulin-1 expression in APCs had minimal impact on the immune responses to the downstream antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and did not prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, boosting immune tolerance to proinsulin-1 partially prevents islet-autoimmunity. This study further extends the previously established role of proinsulin-1 epitopes in autoimmune diabetes in NOD mice.
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http://dx.doi.org/10.3389/fimmu.2021.645817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027244PMC
March 2021

Deficiency of the innate immune adaptor STING promotes autoreactive T cell expansion in NOD mice.

Diabetologia 2021 Apr 23;64(4):878-889. Epub 2021 Jan 23.

St Vincent's Institute, Fitzroy, VIC, Australia.

Aims/hypothesis: Stimulator of IFN genes (STING) is a central hub for cytosolic nucleic acid sensing and its activation results in upregulation of type I IFN production in innate immune cells. A type I IFN gene signature seen before the onset of type 1 diabetes has been suggested as a driver of disease initiation both in humans and in the NOD mouse model. A possible source of type I IFN is through activation of the STING pathway. Recent studies suggest that STING also has antiproliferative and proapoptotic functions in T cells that are independent of IFN. To investigate whether STING is involved in autoimmune diabetes, we examined the impact of genetic deletion of STING in NOD mice.

Methods: CRISPR/Cas9 gene editing was used to generate STING-deficient NOD mice. Quantitative real-time PCR was used to assess the level of type I IFN-regulated genes in islets from wild-type and STING-deficient NOD mice. The number of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific CD8 T cells was determined by magnetic bead-based MHC tetramer enrichment and flow cytometry. The incidence of spontaneous diabetes and diabetes after adoptive transfer of T cells was determined.

Results: STING deficiency partially attenuated the type I IFN gene signature in islets but did not suppress insulitis. STING-deficient NOD mice accumulated an increased number of IGRP-specific CD8 T cells (2878 ± 642 cells in NOD.STING mice and 728.8 ± 196 cells in wild-type NOD mice) in peripheral lymphoid tissue, associated with a higher incidence of spontaneous diabetes (95.5% in NOD.STING mice and 86.2% in wild-type NOD mice). Splenocytes from STING-deficient mice rapidly induced diabetes after adoptive transfer into irradiated NOD recipients (median survival 75 days for NOD recipients of NOD.STING mouse splenocytes and 121 days for NOD recipients of NOD mouse splenocytes).

Conclusions/interpretation: Data suggest that sensing of endogenous nucleic acids through the STING pathway may be partially responsible for the type I IFN gene signature but not autoimmunity in NOD mice. Our results show that the STING pathway may play an unexpected intrinsic role in suppressing the number of diabetogenic T cells.
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http://dx.doi.org/10.1007/s00125-020-05378-zDOI Listing
April 2021

The JAK1 Selective Inhibitor ABT 317 Blocks Signaling Through Interferon-γ and Common γ Chain Cytokine Receptors to Reverse Autoimmune Diabetes in NOD Mice.

Front Immunol 2020 4;11:588543. Epub 2020 Dec 4.

Immunology and Diabetes Unit, St Vincent's Institute, Fitzroy, VIC, Australia.

Cytokines that signal through the JAK-STAT pathway, such as interferon-γ (IFN-γ) and common γ chain cytokines, contribute to the destruction of insulin-secreting β cells by CD8 T cells in type 1 diabetes (T1D). We previously showed that JAK1/JAK2 inhibitors reversed autoimmune insulitis in non-obese diabetic (NOD) mice and also blocked IFN-γ mediated MHC class I upregulation on β cells. Blocking interferons on their own does not prevent diabetes in knockout NOD mice, so we tested whether JAK inhibitor action on signaling downstream of common γ chain cytokines, including IL-2, IL-7 IL-15, and IL-21, may also affect the progression of diabetes in NOD mice. Common γ chain cytokines activate JAK1 and JAK3 to regulate T cell proliferation. We used a JAK1-selective inhibitor, ABT 317, to better understand the specific role of JAK1 signaling in autoimmune diabetes. ABT 317 reduced IL-21, IL-2, IL-15 and IL-7 signaling in T cells and IFN-γ signaling in β cells, but ABT 317 did not affect GM-CSF signaling in granulocytes. When given to NOD mice, ABT 317 reduced CD8 T cell proliferation as well as the number of KLRG effector and CD44CD62L effector memory CD8 T cells in spleen. ABT 317 also prevented MHC class I upregulation on β cells. Newly diagnosed diabetes was reversed in 94% NOD mice treated twice daily with ABT 317 while still on treatment at 40 days and 44% remained normoglycemic after a further 60 days from discontinuing the drug. Our results indicate that ABT 317 blocks common γ chain cytokines in lymphocytes and interferons in lymphocytes and β cells and are thus more effective against diabetes pathogenesis than IFN-γ receptor deficiency alone. Our studies suggest use of this class of drug for the treatment of type 1 diabetes.
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http://dx.doi.org/10.3389/fimmu.2020.588543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746546PMC
December 2020

IL-17F induces inflammation, dysfunction and cell death in mouse islets.

Sci Rep 2020 08 4;10(1):13077. Epub 2020 Aug 4.

St. Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy, Melbourne, VIC, 3065, Australia.

Type 17 immune responses, typified by the production of the cytokines IL-17A and IL-17F, have been implicated in the development of type 1 diabetes in animal models and human patients, however the underlying pathogenic mechanisms have not been clearly elucidated. While previous studies show that IL-17A enhances inflammatory gene expression and cell death in mouse β-cells and human islets, the function of IL-17F in pancreatic β-cells is completely untested to date. Here we show that IL-17F exhibits potent pathogenic effects in mouse β-cell lines and islets. IL-17F signals via the IL-17RA and -RC subunits in β-cells and in combination with other inflammatory cytokines induces expression of chemokine transcripts, suppresses the expression of β-cell identity genes and impairs glucose stimulated insulin secretion. Further IL-17F induces cell death in primary mouse islets. This occurs via Jnk, p38 and NF-κB dependent induction of Nos2 and is completely ablated in the presence of an inducible nitric oxide synthase (iNOS) inhibitor. Together these data indicate that IL-17F possesses similar pathogenic activities to IL-17A in mouse β-cell lines and islets and is likely to be a type 17 associated pathogenic factor in type 1 diabetes.
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http://dx.doi.org/10.1038/s41598-020-69805-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403586PMC
August 2020

IL-21 regulates SOCS1 expression in autoreactive CD8 T cells but is not required for acquisition of CTL activity in the islets of non-obese diabetic mice.

Sci Rep 2019 10 25;9(1):15302. Epub 2019 Oct 25.

St Vincent's Institute of Medical Research, Melbourne, Australia.

In type 1 diabetes, maturation of activated autoreactive CD8 T cells to fully armed effector cytotoxic T lymphocytes (CTL) occurs within the islet. At present the signals required for the maturation process are poorly defined. Cytokines could potentially provide the necessary "third signal" required to generate fully mature CTL capable of killing insulin-producing β-cells. To determine whether autoreactive CTL within islets respond to cytokines we generated non-obese diabetic (NOD) mice with a reporter for cytokine signalling. These mice express a reporter gene, hCD4, under the control of the endogenous regulatory elements for suppressor of cytokine signalling (SOCS)1, which is itself regulated by pro-inflammatory cytokines. In NOD mice, the hCD4 reporter was expressed in infiltrated islets and the expression level was positively correlated with the frequency of infiltrating CD45 cells. SOCS1 reporter expression was induced in transferred β-cell-specific CD8 8.3T cells upon migration from pancreatic draining lymph nodes into islets. To determine which cytokines induced SOCS1 promoter activity in islets, we examined hCD4 reporter expression and CTL maturation in the absence of the cytokine receptors IFNAR1 or IL-21R. We show that IFNAR1 deficiency does not confer protection from diabetes in 8.3 TCR transgenic mice, nor is IFNAR1 signalling required for SOCS1 reporter upregulation or CTL maturation in islets. In contrast, IL-21R-deficient 8.3 mice have reduced diabetes incidence and reduced SOCS1 reporter activity in islet CTLs. However IL-21R deficiency did not affect islet CD8 T cell proliferation or expression of granzyme B or IFNγ. Together these data indicate that autoreactive CD8 T cells respond to IL-21 and not type I IFNs in the islets of NOD mice, but neither IFNAR1 nor IL-21R are required for islet intrinsic CTL maturation.
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http://dx.doi.org/10.1038/s41598-019-51636-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6814838PMC
October 2019

Soluble FAS ligand is not required for pancreatic islet inflammation or beta-cell destruction in non-obese diabetic mice.

Cell Death Discov 2019 23;5:136. Epub 2019 Sep 23.

1St. Vincent's Institute, Fitzroy, Victoria 3065 Australia.

CD8 T cells play a central role in beta-cell destruction in type 1 diabetes. CD8 T cells use two main effector pathways to kill target cells, perforin plus granzymes and FAS ligand (FASL). We and others have established that in non-obese diabetic (NOD) mice, perforin is the dominant effector molecule by which autoreactive CD8 T cells kill beta cells. However, blocking FASL pharmacologically was shown to protect NOD mice from diabetes, indicating that FASL may have some role. FASL can engage with its receptor FAS on target cells either as membrane bound or soluble FASL. It has been shown that membrane-bound FASL is required to stimulate FAS-induced apoptosis in target cells, whereas excessive soluble FASL can induce NF-κB-dependent gene expression and inflammation. Because islet inflammation is a feature of autoimmune diabetes, we tested whether soluble FASL could be important in disease pathogenesis independent of its cell death function. We generated NOD mice deficient in soluble FASL, while maintaining expression of membrane-bound FASL due to a mutation in the FASL sequence required for cleavage by metalloproteinase. NOD mice lacking soluble FASL had normal numbers of lymphocytes in their spleen and thymus. Soluble FASL deficient NOD mice had similar islet inflammation as wild-type NOD mice and were not protected from diabetes. Our data indicate that soluble FASL is not required in development of autoimmune diabetes.
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http://dx.doi.org/10.1038/s41420-019-0217-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755132PMC
September 2019

T-Cell-Specific PTPN2 Deficiency in NOD Mice Accelerates the Development of Type 1 Diabetes and Autoimmune Comorbidities.

Diabetes 2019 06 1;68(6):1251-1266. Epub 2019 Apr 1.

Monash Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

Genome-wide association studies have identified as an important non-MHC gene for autoimmunity. Single nucleotide polymorphisms that reduce expression have been linked with the development of various autoimmune disorders, including type 1 diabetes. The tyrosine phosphatase PTPN2 attenuates T-cell receptor and cytokine signaling in T cells to maintain peripheral tolerance, but the extent to which PTPN2 deficiency in T cells might influence type 1 diabetes onset remains unclear. NOD mice develop spontaneous autoimmune type 1 diabetes similar to that seen in humans. In this study, T-cell PTPN2 deficiency in NOD mice markedly accelerated the onset and increased the incidence of type 1 diabetes as well as that of other disorders, including colitis and Sjögren syndrome. Although PTPN2 deficiency in CD8 T cells alone was able to drive the destruction of pancreatic β-cells and the onset of diabetes, T-cell-specific PTPN2 deficiency was also accompanied by increased CD4 T-helper type 1 differentiation and T-follicular-helper cell polarization and increased the abundance of B cells in pancreatic islets as seen in human type 1 diabetes. These findings causally link PTPN2 deficiency in T cells with the development of type 1 diabetes and associated autoimmune comorbidities.
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http://dx.doi.org/10.2337/db18-1362DOI Listing
June 2019

Replica moulded poly(dimethylsiloxane) microwell arrays induce localized endothelial cell immobilization for coculture with pancreatic islets.

Biointerphases 2019 01 30;14(1):011002. Epub 2019 Jan 30.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

PolyJet three-dimensional (3D) printing allows for the rapid manufacturing of 3D moulds for the fabrication of cross-linked poly(dimethylsiloxane) microwell arrays (PMAs). As this 3D printing technique has a resolution on the micrometer scale, the moulds exhibit a distinct surface roughness. In this study, the authors demonstrate by optical profilometry that the topography of the 3D printed moulds can be transferred to the PMAs and that this roughness induced cell adhesive properties to the material. In particular, the topography facilitated immobilization of endothelial cells on the internal walls of the microwells. The authors also demonstrate that upon immobilization of endothelial cells to the microwells, a second population of cells, namely, pancreatic islets could be introduced, thus producing a 3D coculture platform.
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http://dx.doi.org/10.1116/1.5087737DOI Listing
January 2019

IFNγ-Induced MHC Class II Expression on Islet Endothelial Cells Is an Early Marker of Insulitis but Is Not Required for Diabetogenic CD4 T Cell Migration.

Front Immunol 2018 28;9:2800. Epub 2018 Nov 28.

St. Vincent's Institute, Fitzroy, VIC, Australia.

Diabetogenic T cells infiltrate the pancreatic islets by transmigrating across the microcapillaries residing close to, or within, the pancreatic islets. Deficiency in IFNγ signaling prevents efficient migration of T cells into the pancreatic islets, but the IFNγ-regulated molecules that mediate this are uncertain. Homing of autoreactive T cells into target tissues may require antigen specificity through presentation of cognate antigen by MHC expressed on the vascular endothelium. We investigated the hypothesis that IFNγ promotes the migration of islet antigen-specific CD4 T cells by upregulating MHC class II on islet endothelial cells (IEC), thereby providing an antigen-specific signal for islet infiltration. Upon IFNγ stimulation, MHC class II, which is not constitutively expressed on IEC, was induced. IFNγ-dependent upregulation of MHC class II was detected in IEC isolated from prediabetic NOD mice at the earliest stages of insulitis, before other markers of inflammation were present. Using a CD4 T cell-mediated adoptive transfer model of autoimmune diabetes we observed that even though diabetes does not develop in recipient mice lacking IFNγ receptors, mice with MHC class II-deficient IEC were not protected from disease. Thus, IFNγ-regulated molecules, but not MHC class II or antigen presentation by IECs is required for the early migration of antigen-specific CD4 T cells into the pancreatic islets.
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http://dx.doi.org/10.3389/fimmu.2018.02800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6282031PMC
October 2019

Proinsulin C-peptide is an autoantigen in people with type 1 diabetes.

Proc Natl Acad Sci U S A 2018 10 1;115(42):10732-10737. Epub 2018 Oct 1.

Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia;

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells, found within the islets of Langerhans in the pancreas, are destroyed by islet-infiltrating T cells. Identifying the antigenic targets of beta-cell reactive T cells is critical to gain insight into the pathogenesis of T1D and develop antigen-specific immunotherapies. Several lines of evidence indicate that insulin is an important target of T cells in T1D. Because many human islet-infiltrating CD4 T cells recognize C-peptide-derived epitopes, we hypothesized that full-length C-peptide (PI), the peptide excised from proinsulin as it is converted to insulin, is a target of CD4 T cells in people with T1D. CD4 T cell responses to full-length C-peptide were detected in the blood of: 14 of 23 (>60%) people with recent-onset T1D, 2 of 15 (>13%) people with long-standing T1D, and 1 of 13 (<8%) HLA-matched people without T1D. C-peptide-specific CD4 T cell clones, isolated from six people with T1D, recognized epitopes from the entire 31 amino acids of C-peptide. Eighty-six percent (19 of 22) of the C-peptide-specific clones were restricted by HLA-DQ8, HLA-DQ2, HLA-DQ8, or HLA-DQ2, HLA alleles strongly associated with risk of T1D. We also found that full-length C-peptide was a much more potent agonist of some CD4 T cell clones than an 18mer peptide encompassing the cognate epitope. Collectively, our findings indicate that proinsulin C-peptide is a key target of autoreactive CD4 T cells in T1D. Hence, full-length C-peptide is a promising candidate for antigen-specific immunotherapy in T1D.
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http://dx.doi.org/10.1073/pnas.1809208115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196477PMC
October 2018

Defining Outcomes for β-cell Replacement Therapy in the Treatment of Diabetes: A Consensus Report on the Igls Criteria From the IPITA/EPITA Opinion Leaders Workshop.

Transplantation 2018 09;102(9):1479-1486

Institute of Transplantation, The Freeman Hospital and Newcastle University, Newcastle upon Tyne, United Kingdom.

β-cell replacement therapy, available currently as pancreas or islet transplantation, has developed without a clear definition of graft functional and clinical outcomes. The International Pancreas and Islet Transplant Association and European Pancreas and Islet Transplantation Association held a workshop to develop consensus for an International Pancreas and Islet Transplant Association and European Pancreas and Islet Transplant Association Statement on the definition of function and failure of current and future forms of β-cell replacement therapy. There was consensus that β-cell replacement therapy could be considered as a treatment for β-cell failure, regardless of etiology and without requiring undetectable C-peptide, accompanied by glycemic instability with either problematic hypoglycemia or hyperglycemia. Glycemic control should be assessed at a minimum by glycated hemoglobin (HbA1c) and the occurrence of severe hypoglycemia. Optimal β-cell graft function is defined by near-normal glycemic control (HbA1c ≤6.5% [48 mmol/mol]) without severe hypoglycemia or requirement for insulin or other antihyperglycemic therapy, and with an increase over pretransplant measurement of C-peptide. Good β-cell graft function requires HbA1c less than 7.0% (53 mmol/mol) without severe hypoglycemia and with a significant (>50%) reduction in insulin requirements and restoration of clinically significant C-peptide production. Marginal β-cell graft function is defined by failure to achieve HbA1c less than 7.0% (53 mmol/mol), the occurrence of any severe hypoglycemia, or less than 50% reduction in insulin requirements when there is restoration of clinically significant C-peptide production documented by improvement in hypoglycemia awareness/severity, or glycemic variability/lability. A failed β-cell graft is defined by the absence of any evidence for clinically significant C-peptide production. Optimal and good function are considered successful clinical outcomes.
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http://dx.doi.org/10.1097/TP.0000000000002158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6408213PMC
September 2018

NF-κB is weakly activated in the NOD mouse model of type 1 diabetes.

Sci Rep 2018 03 9;8(1):4217. Epub 2018 Mar 9.

Immunology and Diabetes Unit, St Vincent's Institute, Fitzroy, Victoria, Australia.

Type 1 diabetes is an autoimmune disease characterised by selective destruction of pancreatic beta cells by the immune system. The transcription factor nuclear factor-kappa B (NF-κB) regulates innate and adaptive immune responses. Using gene targeting and in vitro analysis of pancreatic islets and immune cells, NF-κB activation has been implicated in type 1 diabetes development. Here we use a non-obese diabetic (NOD) mouse model that expresses a luciferase reporter of transcriptionally active NF-κB to determine its activation in vivo during development of diabetes. Increased luciferase activity was readily detected upon treatment with Toll-like receptor ligands in vitro and in vivo, indicating activation of NF-κB. However, activated NF-κB was detectable at low levels above background in unmanipulated NOD mice, but did not vary with age, despite the progression of inflammatory infiltration in islets over time. NF-κB was highly activated in an accelerated model of type 1 diabetes that requires CD4 T cells and inflammatory macrophages. These data shed light on the nature of the inflammatory response in the development of type 1 diabetes.
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http://dx.doi.org/10.1038/s41598-018-22738-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844878PMC
March 2018

Oxygen-permeable microwell device maintains islet mass and integrity during shipping.

Endocr Connect 2018 Mar 26;7(3):490-503. Epub 2018 Feb 26.

The Centre for Clinical and Experimental Transplantation (CCET) The Royal Adelaide HospitalAdelaide, South Australia, Australia

Islet transplantation is currently the only minimally invasive therapy available for patients with type 1 diabetes that can lead to insulin independence; however, it is limited to only a small number of patients. Although clinical procedures have improved in the isolation and culture of islets, a large number of islets are still lost in the pre-transplant period, limiting the success of this treatment. Moreover, current practice includes islets being prepared at specialized centers, which are sometimes remote to the transplant location. Thus, a critical point of intervention to maintain the quality and quantity of isolated islets is during transportation between isolation centers and the transplanting hospitals, during which 20-40% of functional islets can be lost. The current study investigated the use of an oxygen-permeable PDMS microwell device for long-distance transportation of isolated islets. We demonstrate that the microwell device protected islets from aggregation during transport, maintaining viability and average islet size during shipping.
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http://dx.doi.org/10.1530/EC-17-0349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861371PMC
March 2018

Defining outcomes for β-cell replacement therapy in the treatment of diabetes: a consensus report on the Igls criteria from the IPITA/EPITA opinion leaders workshop.

Transpl Int 2018 04;31(4):343-352

Institute of Transplantation, The Freeman Hospital, Newcastle University, Newcastle upon Tyne, UK.

β-cell replacement therapy, available currently as pancreas or islet transplantation, has developed without a clear definition of graft functional and clinical outcomes. The International Pancreas & Islet Transplant Association (IPITA) and European Pancreas & Islet Transplantation Association (EPITA) held a workshop to develop consensus for an IPITA/EPITA Statement on the definition of function and failure of current and future forms of β-cell replacement therapy. There was consensus that β-cell replacement therapy could be considered as a treatment for β-cell failure, regardless of etiology and without requiring undetectable C-peptide, accompanied by glycemic instability with either problematic hypoglycemia or hyperglycemia. Glycemic control should be assessed at a minimum by glycated hemoglobin (HbA ) and the occurrence of severe hypoglycemia. Optimal β-cell graft function is defined by near-normal glycemic control [HbA  ≤ 6.5% (48 mmol/mol)] without severe hypoglycemia or requirement for insulin or other antihyperglycemic therapy, and with an increase over pretransplant measurement of C-peptide. Good β-cell graft function requires HbA  < 7.0% (53 mmol/mol) without severe hypoglycemia and with a significant (>50%) reduction in insulin requirements and restoration of clinically significant C-peptide production. Marginal β-cell graft function is defined by failure to achieve HbA  < 7.0% (53 mmol/mol), the occurrence of any severe hypoglycemia, or less than 50% reduction in insulin requirements when there is restoration of clinically significant C-peptide production documented by improvement in hypoglycemia awareness/severity, or glycemic variability/lability. A failed β-cell graft is defined by the absence of any evidence for clinically significant C-peptide production. Optimal and good functional outcomes are considered successful clinical outcomes.
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http://dx.doi.org/10.1111/tri.13138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5867272PMC
April 2018

Loss of intra-islet heparan sulfate is a highly sensitive marker of type 1 diabetes progression in humans.

PLoS One 2018 7;13(2):e0191360. Epub 2018 Feb 7.

Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells in pancreatic islets are progressively destroyed. Clinical trials of immunotherapies in recently diagnosed T1D patients have only transiently and partially impacted the disease course, suggesting that other approaches are required. Our previous studies have demonstrated that heparan sulfate (HS), a glycosaminoglycan conventionally expressed in extracellular matrix, is present at high levels inside normal mouse beta cells. Intracellular HS was shown to be critical for beta cell survival and protection from oxidative damage. T1D development in Non-Obese Diabetic (NOD) mice correlated with loss of islet HS and was prevented by inhibiting HS degradation by the endoglycosidase, heparanase. In this study we investigated the distribution of HS and heparan sulfate proteoglycan (HSPG) core proteins in normal human islets, a role for HS in human beta cell viability and the clinical relevance of intra-islet HS and HSPG levels, compared to insulin, in human T1D. In normal human islets, HS (identified by 10E4 mAb) co-localized with insulin but not glucagon and correlated with the HSPG core proteins for collagen type XVIII (Col18) and syndecan-1 (Sdc1). Insulin-positive islets of T1D pancreases showed significant loss of HS, Col18 and Sdc1 and heparanase was strongly expressed by islet-infiltrating leukocytes. Human beta cells cultured with HS mimetics showed significantly improved survival and protection against hydrogen peroxide-induced death, suggesting that loss of HS could contribute to beta cell death in T1D. We conclude that HS depletion in beta cells, possibly due to heparanase produced by insulitis leukocytes, may function as an important mechanism in the pathogenesis of human T1D. Our findings raise the possibility that intervention therapy with dual activity HS replacers/heparanase inhibitors could help to protect the residual beta cell mass in patients recently diagnosed with T1D.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0191360PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802856PMC
March 2018

Shuffling peptides to create T-cell epitopes: does the immune system play cards?

Immunol Cell Biol 2018 01 7;96(1):34-40. Epub 2017 Dec 7.

Immunology and Diabetes Unit, St. Vincent's Institute of Medical Research, Fitzroy, Vic., Australia.

For a long time, immunologists have believed that classical CD4 and CD8 T cells recognize peptides (referred to as epitopes), derived from protein antigens presented by MHC/HLA class I or II. Over the past 10-15 years, it has become clear that epitopes recognized by CD8, and more recently CD4 T cells, can be formed by protein splicing. Here, we review the discovery of spliced epitopes recognized by tumor-specific human CD8 T cells. We discuss how these epitopes are formed and some of the unusual variants that have been reported. Now, over a decade since the first report, evidence is emerging that spliced CD8 T-cell epitopes are much more common, and potentially much more important, than previously imagined. Recent work has shown that epitopes recognized by CD4 T cells can also be formed by protein splicing. We discuss the recent discovery of spliced CD4 T-cell epitopes and their potential role as targets of autoimmune T-cell responses. Finally, we highlight some of the new questions raised from our growing appreciation of T-cell epitopes formed by peptide splicing.
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http://dx.doi.org/10.1111/imcb.1015DOI Listing
January 2018

Characterization of the Human Pancreas Side Population as a Potential Reservoir of Adult Stem Cells.

Pancreas 2018 01;47(1):25-34

Objectives: The side population (SP) contains cells with stem cell/progenitor properties. Previously, we observed that the mouse pancreas SP expanded after pancreatic injury. We aimed to characterize the SP in human pancreas as a potential source of stem cells.

Methods: Human organ donor pancreata were fractionated into islets and exocrine tissue, enriched by tissue culture and dispersed into single cells. Cells were phenotyped by flow cytometry, and the SP was defined by efflux of fluorescent dye Hoechst 33342 visualized by ultraviolet excitation. Cells were flow sorted, and their colony-forming potential measured on feeder cells in culture.

Results: An SP was identified in islet and exocrine cells from human organ donors: 2 with type 1 diabetes, 3 with type 2 diabetes, and 28 without diabetes. Phenotyping revealed that exocrine SP cells had an epithelial origin, were enriched for carbohydrate antigen 19-9 ductal cells expressing stem cell markers CD133 and CD26, and had greater colony-forming potential than non-SP cells. The exocrine SP was increased in a young adult with type 1 diabetes and ongoing islet autoimmunity.

Conclusions: The pancreatic exocrine SP is a potential reservoir of adult stem/progenitor cells, consistent with previous evidence that such cells are duct-derived and express CD133.
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http://dx.doi.org/10.1097/MPA.0000000000000950DOI Listing
January 2018

Inhibition of Y1 receptor signaling improves islet transplant outcome.

Nat Commun 2017 09 8;8(1):490. Epub 2017 Sep 8.

Neuroscience Division, Garvan Institute of Medical Research, St Vincent's Hospital, Sydney, NSW, 2010, Australia.

Failure to secrete sufficient quantities of insulin is a pathological feature of type-1 and type-2 diabetes, and also reduces the success of islet cell transplantation. Here we demonstrate that Y1 receptor signaling inhibits insulin release in β-cells, and show that this can be pharmacologically exploited to boost insulin secretion. Transplanting islets with Y1 receptor deficiency accelerates the normalization of hyperglycemia in chemically induced diabetic recipient mice, which can also be achieved by short-term pharmacological blockade of Y1 receptors in transplanted mouse and human islets. Furthermore, treatment of non-obese diabetic mice with a Y1 receptor antagonist delays the onset of diabetes. Mechanistically, Y1 receptor signaling inhibits the production of cAMP in islets, which via CREB mediated pathways results in the down-regulation of several key enzymes in glycolysis and ATP production. Thus, manipulating Y1 receptor signaling in β-cells offers a unique therapeutic opportunity for correcting insulin deficiency as it occurs in the pathological state of type-1 diabetes as well as during islet transplantation.Islet transplantation is considered one of the potential treatments for T1DM but limited islet survival and their impaired function pose limitations to this approach. Here Loh et al. show that the Y1 receptor is expressed in β- cells and inhibition of its signalling, both genetic and pharmacological, improves mouse and human islet function.
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http://dx.doi.org/10.1038/s41467-017-00624-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5591241PMC
September 2017

Understanding and preventing type 1 diabetes through the unique working model of TrialNet.

Diabetologia 2017 Nov 2;60(11):2139-2147. Epub 2017 Aug 2.

Department of Immunobiology, Faculty of Life Sciences & Medicine, King's College London, London, SE1 9RT, UK.

Type 1 diabetes is an autoimmune disease arising from the destruction of pancreatic insulin-producing beta cells. The disease represents a continuum, progressing sequentially at variable rates through identifiable stages prior to the onset of symptoms, through diagnosis and into the critical periods that follow, culminating in a variable depth of beta cell depletion. The ability to identify the very earliest of these presymptomatic stages has provided a setting in which prevention strategies can be trialled, as well as furnishing an unprecedented opportunity to study disease evolution, including intrinsic and extrinsic initiators and drivers. This niche opportunity is occupied by Type 1 Diabetes TrialNet, an international consortium of clinical trial centres that leads the field in intervention and prevention studies, accompanied by deep longitudinal bio-sampling. In this review, we focus on discoveries arising from this unique bioresource, comprising more than 70,000 samples, and outline the processes and science that have led to new biomarkers and mechanistic insights, as well as identifying new challenges and opportunities. We conclude that via integration of clinical trials and mechanistic studies, drawing in clinicians and scientists and developing partnership with industry, TrialNet embodies an enviable and unique working model for understanding a disease that to date has no cure and for designing new therapeutic approaches.
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http://dx.doi.org/10.1007/s00125-017-4384-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5838353PMC
November 2017

Granzyme A Deficiency Breaks Immune Tolerance and Promotes Autoimmune Diabetes Through a Type I Interferon-Dependent Pathway.

Diabetes 2017 12 21;66(12):3041-3050. Epub 2017 Jul 21.

St. Vincent's Institute, Fitzroy, Victoria, Australia

Granzyme A is a protease implicated in the degradation of intracellular DNA. Nucleotide complexes are known triggers of systemic autoimmunity, but a role in organ-specific autoimmune disease has not been demonstrated. To investigate whether such a mechanism could be an endogenous trigger for autoimmunity, we examined the impact of granzyme A deficiency in the NOD mouse model of autoimmune diabetes. Granzyme A deficiency resulted in an increased incidence in diabetes associated with accumulation of ssDNA in immune cells and induction of an interferon response in pancreatic islets. Central tolerance to proinsulin in transgenic NOD mice was broken on a granzyme A-deficient background. We have identified a novel endogenous trigger for autoimmune diabetes and an in vivo role for granzyme A in maintaining immune tolerance.
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http://dx.doi.org/10.2337/db17-0517DOI Listing
December 2017

Cognate antigen engagement on parenchymal cells stimulates CD8 T cell proliferation in situ.

Nat Commun 2017 04 12;8:14809. Epub 2017 Apr 12.

Immunology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.

T-cell responses are initiated upon cognate presentation by professional antigen presenting cells in lymphoid tissue. T cells then migrate to inflamed tissues, but further T-cell stimulation in these parenchymal target sites is not well understood. Here we show that T-cell expansion within inflamed tissues is a distinct phase that is neither a classical primary nor classical secondary response. This response, which we term 'the mezzanine response', commences within days after initial antigen encounter, unlike the secondary response that usually occurs weeks after priming. A further distinction of this response is that T-cell proliferation is driven by parenchymal cell antigen presentation, without requiring professional antigen presenting cells, but with increased dependence on IL-2. The mezzanine response might, therefore, be a new target for inhibiting T-cell responses in allograft rejection and autoimmunity or for enhancing T-cell responses in the context of microbial or tumour immunity.
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http://dx.doi.org/10.1038/ncomms14809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394288PMC
April 2017

Repurposed JAK1/JAK2 Inhibitor Reverses Established Autoimmune Insulitis in NOD Mice.

Diabetes 2017 06 14;66(6):1650-1660. Epub 2017 Mar 14.

St. Vincent's Institute, Fitzroy, Victoria, Australia

Recent advances in immunotherapeutics have not yet changed the routine management of autoimmune type 1 diabetes. There is an opportunity to repurpose therapeutics used to treat other diseases to treat type 1 diabetes, especially when there is evidence for overlapping mechanisms. Janus kinase (JAK) 1/JAK2 inhibitors are in development or clinical use for indications including rheumatoid arthritis. There is good evidence for activation of the JAK1/JAK2 and signal transducer and activator of transcription (STAT) 1 pathway in human type 1 diabetes and in mouse models, especially in β-cells. We tested the hypothesis that using these drugs to block the JAK-STAT pathway would prevent autoimmune diabetes. The JAK1/JAK2 inhibitor AZD1480 blocked the effect of cytokines on mouse and human β-cells by inhibiting MHC class I upregulation. This prevented the direct interaction between CD8 T cells and β-cells, and reduced immune cell infiltration into islets. NOD mice treated with AZD1480 were protected from autoimmune diabetes, and diabetes was reversed in newly diagnosed NOD mice. This provides mechanistic groundwork for repurposing clinically approved JAK1/JAK2 inhibitors for type 1 diabetes.
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http://dx.doi.org/10.2337/db16-1250DOI Listing
June 2017

First Report of Successful Total Pancreatectomy and Islet Autotransplant in Australia.

Pancreas 2017 03;46(3):e18-e20

Department of Endocrinology and Diabetes, Women's and Children's Hospital, Adelaide, Australia Central Northern Adelaide Renal and Transplantation Service, Royal Adelaide Hospital and School of Medicine University of Adelaide, Adelaide, Australia; Departments of Paediatric Surgery and Urology, Women's and Children's Hospital and School of Medicine, University of Adelaide, Adelaide, Australia; South Australian Liver Transplant Unit and HepatoPancreatoBiliary Unit, Flinders Medical Centre, Bedford Park, Australia and School of Medicine, Flinders University, Adelaide, Australia; Immunology and Diabetes, St Vincent's Institute and Department of Medicine, University of Melbourne, Melbourne, Australia; Department of Surgery, University of Louisville, Louisville, KY; Department Endocrinology and Diabetes, Women's and Children's Hospital and Discipline of Paediatrics, Robinson Research Institute, University of Adelaide, Adelaide, Australia; Central Northern Adelaide, Renal and Transplantation Service, Royal Adelaide Hospital, Adelaide, Australia; Central Northern Adelaide Renal and Transplantation Service, Royal Adelaide Hospital and School of Medicine University of Adelaide, Adelaide, Australia; Immunology and Diabetes, St Vincent's Institute, Melbourne, Australia; Department of Medical Imaging, Flinders Medical Centre, Bedford Park, Australia; Department of Radiology, Women's and Children's Hospital, Adelaide, Australia; Department of Gastroenterology, Women's and Children's Hospital, Adelaide, Australia.

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http://dx.doi.org/10.1097/MPA.0000000000000769DOI Listing
March 2017

Disruption of Serinc1, which facilitates serine-derived lipid synthesis, fails to alter macrophage function, lymphocyte proliferation or autoimmune disease susceptibility.

Mol Immunol 2017 02 19;82:19-33. Epub 2016 Dec 19.

St Vincent's Institute, Fitzroy, Victoria 3065, Australia; Department of Medicine, St Vincent's Hospital, University of Melbourne, Fitzroy, Victoria 3065, Australia. Electronic address:

During immune cell activation, serine-derived lipids such as phosphatidylserine and sphingolipids contribute to the formation of protein signaling complexes within the plasma membrane. Altering lipid composition in the cell membrane can subsequently affect immune cell function and the development of autoimmune disease. Serine incorporator 1 (SERINC1) is a putative carrier protein that facilitates synthesis of serine-derived lipids. To determine if SERINC1 has a role in immune cell function and the development of autoimmunity, we characterized a mouse strain in which a retroviral insertion abolishes expression of the Serinc1 transcript. Expression analyses indicated that the Serinc1 transcript is readily detectable and expressed at relatively high levels in wildtype macrophages and lymphocytes. The ablation of Serinc1 expression in these immune cells, however, did not significantly alter serine-derived lipid composition or affect macrophage function and lymphocyte proliferation. Analyses of Serinc1-deficient mice also indicated that systemic ablation of Serinc1 expression did not affect viability, fertility or autoimmune disease susceptibility. These results suggest that Serinc1 is dispensable for certain immune cell functions and does not contribute to previously reported links between lipid composition in immune cells and autoimmunity.
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http://dx.doi.org/10.1016/j.molimm.2016.12.007DOI Listing
February 2017

Long-term effects of islet transplantation.

Curr Opin Organ Transplant 2016 10;21(5):497-502

aDepartment of Diabetes and Endocrinology, Westmead Hospital, Westmead bSydney University Medical School, Sydney, New South Wales cSt. Vincent's Institute of Medical Research dDepartment of Medicine, St. Vincent's Hospital, The University of Melbourne, Fitzroy, Victoria, Australia.

Purpose Of Review: Islet transplantation has made great progress in recent years. This is a remarkable technical feat but raises the question of what the long-term benefits and risks are for type I diabetes recipients.

Recent Findings: Graft survival continues to improve, and recent multicenter studies show that islet transplantation is particularly effective to prevent hypoglycemic events even in those who do not become insulin-independent and to achieve excellent glycemic control. Concerns include histocompatability leucocyte antigen (HLA) sensitization and other risks including from immunosuppression that islet transplantation shares with other forms of allotransplantation.

Summary: Reversal of hypoglycemia unawareness and protection from severe hypoglycemia events are two of the main benefits of islet transplantation and they persist for the duration of graft function. Islet transplantation compares favorably with other therapies for those with hypoglycemia unawareness, although new technologies have not been tested head-to-head with transplantation. HLA sensitization increases with time after transplantation especially if immunosuppression is ceased and is a risk for those who may require future transplantation as well as being associated with loss of graft function.
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http://dx.doi.org/10.1097/MOT.0000000000000355DOI Listing
October 2016

Rotavirus acceleration of type 1 diabetes in non-obese diabetic mice depends on type I interferon signalling.

Sci Rep 2016 07 13;6:29697. Epub 2016 Jul 13.

Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia.

Rotavirus infection is associated with childhood progression to type 1 diabetes. Infection by monkey rotavirus RRV accelerates diabetes onset in non-obese diabetic (NOD) mice, which relates to regional lymph node infection and a T helper 1-specific immune response. When stimulated ex vivo with RRV, plasmacytoid dendritic cells (pDCs) from naïve NOD mice secrete type I interferon, which induces the activation of bystander lymphocytes, including islet-autoreactive T cells. This is our proposed mechanism for diabetes acceleration by rotaviruses. Here we demonstrate bystander lymphocyte activation in RRV-infected NOD mice, which showed pDC activation and strong upregulation of interferon-dependent gene expression, particularly within lymph nodes. The requirement for type I interferon signalling was analysed using NOD mice lacking a functional type I interferon receptor (NOD.IFNAR1(-/-) mice). Compared with NOD mice, NOD.IFNAR1(-/-) mice showed 8-fold higher RRV titers in lymph nodes and 3-fold higher titers of total RRV antibody in serum. However, RRV-infected NOD.IFNAR1(-/-) mice exhibited delayed pDC and lymphocyte activation, no T helper 1 bias in RRV-specific antibodies and unaltered diabetes onset when compared with uninfected controls. Thus, the type I interferon signalling induced by RRV infection is required for bystander lymphocyte activation and accelerated type 1 diabetes onset in genetically susceptible mice.
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http://dx.doi.org/10.1038/srep29697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942798PMC
July 2016