Search

The β-cell plays a crucial role in the pathogenesis of type 1 diabetes, in part through the posttranslational modification of self-proteins by biochemical processes such as deamidation. These neoantigens are potential triggers for breaking immune tolerance. We report the detection by LC-MS/MS of 16 novel Gln and 27 novel Asn deamidations in 14 disease-related proteins within inflammatory cytokine-stressed human islets of Langerhans. T-cell clones responsive against one Gln- and three Asn-deamidated peptides could be isolated from peripheral blood of individuals with type 1 diabetes. Ex vivo HLA class II tetramer staining detected higher T-cell frequencies in individuals with the disease compared with control individuals. Furthermore, there was a positive correlation between the frequencies of T cells specific for deamidated peptides, insulin antibody levels at diagnosis, and duration of disease. These results highlight that stressed human islets are prone to enzymatic and biochemical deamidation and suggest that both Gln- and Asn-deamidated peptides can promote the activation and expansion of autoreactive CD4+ T cells. These findings add to the growing evidence that posttranslational modifications undermine tolerance and may open the road for the development of new diagnostic and therapeutic applications for individuals living with type 1 diabetes., (© 2024 by the American Diabetes Association.)

Post-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2 , the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1β and IFNγ in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD β-cells underscores the role of deamidation in amplifying T-cell reactivity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Callebaut, Bruggeman, Zamit, Sodré, Irla, Mathieu, Buitinga and Overbergh.)

Glucagon-like peptide 1 receptor (GLP-1R) imaging with radiolabeled exendin has proven to be a powerful tool to quantify β-cell mass (BCM) in vivo. As GLP-1R expression is thought to be influenced by glycemic control, we examined the effect of blood glucose (BG) levels on GLP-1R-mediated exendin uptake in both murine and human islets and its implications for BCM quantification. Periods of hyperglycemia significantly reduced exendin uptake in murine and human islets, which was paralleled by a reduction in GLP-1R expression. Detailed mapping of the tracer uptake and insulin and GLP-1R expression conclusively demonstrated that the observed reduction in tracer uptake directly correlates to GLP-1R expression levels. Importantly, the linear correlation between tracer uptake and β-cell area was maintained in spite of the reduced GLP-1R expression levels. Subsequent normalization of BG levels restored absolute tracer uptake and GLP-1R expression in β-cells and the observed loss in islet volume was halted. This manuscript emphasizes the potency of nuclear imaging techniques to monitor receptor regulation noninvasively. Our findings have significant implications for clinical practice, indicating that BG levels should be near-normalized for at least 3 weeks prior to GLP-1R agonist treatment or quantitative radiolabeled exendin imaging for BCM analysis., (© 2020 by the American Diabetes Association.)

The β-cell has become recognized as a central player in the pathogenesis of type 1 diabetes with the generation of neoantigens as potential triggers for breaking immune tolerance. We report that posttranslationally modified glucose-regulated protein 78 (GRP78) is a novel autoantigen in human type 1 diabetes. When human islets were exposed to inflammatory stress induced by interleukin-1β, tumor necrosis factor-α, and interferon-γ, arginine residue R510 within GRP78 was converted into citrulline, as evidenced by liquid chromatography-tandem mass spectrometry. This conversion, known as citrullination, led to the generation of neoepitopes, which effectively could be presented by HLA-DRB1*04:01 molecules. With the use of HLA-DRB1*04:01 tetramers and ELISA techniques, we demonstrate enhanced antigenicity of citrullinated GRP78 with significantly increased CD4 + T-cell responses and autoantibody titers in patients with type 1 diabetes compared with healthy control subjects. Of note, patients with type 1 diabetes had a predominantly higher percentage of central memory cells and a lower percentage of effector memory cells directed against citrullinated GRP78 compared with the native epitope. These results strongly suggest that citrullination of β-cell proteins, exemplified here by the citrullination of GRP78, contributes to loss of self-tolerance toward β-cells in human type 1 diabetes, indicating that β-cells actively participate in their own demise., (© 2018 by the American Diabetes Association.)

Allogeneic islet transplantation into the liver in combination with immune suppressive drug therapy is widely regarded as a potential cure for type 1 diabetes. However, the intrahepatic system is suboptimal as the concentration of drugs and nutrients there is higher compared to pancreas, which negatively affects islet function. Islet encapsulation within semipermeable membranes is a promising strategy that allows for the islet transplantation outside the suboptimal liver portal system and provides environment, where islets can perform their endocrine function. In this study, we develop a macroencapsulation device based on thin microwell membranes. The islets are seeded in separate microwells to avoid aggregation, whereas the membrane porosity is tailored to achieve sufficient transport of nutrients, glucose and insulin. The non-degradable, microwell membranes are composed of poly (ether sulfone)/polyvinylpyrrolidone and manufactured via phase separation micro molding. Our results show that the device prevents aggregation and preserves the islet's native morphology. Moreover, the encapsulated islets maintain their glucose responsiveness and function after 7 days of culture (stimulation index above 2 for high glucose stimulation), demonstrating the potential of this novel device for islet transplantation.

Pancreatic islet transplantation is a promising therapy for patients with type 1 diabetes. However, the duration of long-term graft survival is limited due to inflammatory as well as non-inflammatory processes and routine clinical tests are not suitable to monitor islet survival. 111 In-exendin-SPECT (single photon emission computed tomography) is a promising method to non-invasively image islets after transplantation and has the potential to help improve the clinical outcome. Whether 111 In-exendin-SPECT allows detecting small differences in beta-cell mass (BCM) and measuring the actual volume of islets that were successfully engrafted has yet to be demonstrated. Here, we evaluated the performance of 111 In-exendin-SPECT using an intramuscular islet transplantation model in C3H mice. In vivo imaging of animals transplanted with 50, 100, 200, 400 and 800 islets revealed an excellent linear correlation between SPECT quantification of 111 In-exendin uptake and insulin-positive area of islet transplants, demonstrating that 111 In-exendin-SPECT specifically and accurately measures BCM. The high sensitivity of the method allowed measuring small differences in graft volumes, including grafts that contained less than 50 islets. The presented method is reliable, convenient and holds great potential for non-invasive monitoring of BCM after islet transplantation in humans.

While subcutaneous tissue has been proposed as a clinically relevant site for pancreatic islet transplantation, a major issue of concern remains, which is its poor vascular state. In an effort to overcome this limitation, we present an efficient and reproducible method to form human composite islets (CIs) with proangiogenic cell types in a controlled manner using nonadherent agarose microwell templates. In this study, we assessed the three-dimensional structure, function, and angiogenic potential of human CIs with human mesenchymal stromal cells (hMSCs), with or without human umbilical vein endothelial cells (HUVECs), and preconditioned hMSCs (PC-hMSCs) in EGM-2 under shear stress. Distinct cellular rearrangements could be observed in CIs, but islet functionality was maintained. In vitro angiogenesis assays found significantly enhanced sprout formation in case of CIs. In particular, the number of sprouts emanating from CIs with PC-hMSCs was significantly increased compared to other conditions. Subsequent in vivo assessment confirmed the proangiogenic potential of CIs. However, in contrast to our in vitro angiogenesis assays, CIs with hMSCs and HUVECs exhibited a higher in vivo angiogenic potential compared to control islets or islets combined with hMSCs or PC-hMSCs. These findings highlight the importance and necessity of verifying in vitro studies with in vivo models to reliably predict, in this case, revascularization outcomes. Regardless, we demonstrate here the therapeutic potential of CIs with proangiogenic support cells to enhance islet revascularization at a clinically relevant, although poorly vascularized, transplantation site.

Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islet of Langerhans. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymer (composition: 4000PEOT30PBT70) and were imprinted with microwells, ∼400 µm in diameter and ∼350 µm in depth. The water contact angle and water uptake were 39±2° and 52.1±4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than for thin film scaffolds, indicating enhanced nutrient diffusion. Human islets cultured in microwell scaffolds for seven days showed insulin release and insulin content comparable to those of free-floating control islets. Islet morphology and insulin and glucagon expression were maintained during culture in the microwell scaffolds. Our results indicate that the microwell scaffold platform prevents islet aggregation by confinement of individual islets in separate microwells, preserves the islet's native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation.