Homing of GAD65 specific autoimmunity and development of insulitis requires expression of both DQ8 and human GAD65 in transgenic mice

MHC-class II genes determine susceptibility in human type-1 diabetes. In their context, presentation of target antigen(s) results in autoimmunity and b-cell destruction. An animal model, in which human b-cell autoantigen(s) are presented to effector cells in the context of human MHC-class II diabetessusceptibility genes, would be desirable for studying molecular mechanisms of disease and developing antigen-specific immune-interventions. We report the development of antigen-specific insulitis in double-transgenic mice carrying the HLA-DQ8 diabetes-susceptibility haplotype and expressing the human autoantigen GAD65 in pancreatic b-cells. Immunization with human GAD65 cDNA resulted in severe insulitis and low antibody levels in double-transgenic mice while control mice were mostly insulitis free. CFA/protein immunization resulted in high antibody levels and modest insulitis. Pancreatic lymphocytic infiltration progressed through stages (exocrine pancreas followed by peri- and intrainsulitis). Adoptive transfer of splenocytes from DNA-immunized mice resulted in development of insulitis in recipient transgenics. Our results show that immunization with a clinically relevant, type-1 diabetes human autoantigen, in a humanized genetic setting, results in the development of an immune response that homes to islets of Langerhans. This animal model will facilitate studies of autoimmunity to GAD65 in the context of HLA-DQ8, and development of methods to induce tolerance and prevent insulitis.

Aberrant Accumulation of the Diabetes Autoantigen GAD65 in Golgi Membranes in Conditions of ER Stress and Autoimmunity

Steinunn Baekkeskov Hanahan, Chiara Cianciaruso, Jeffrey Alan Hubbell, Miriella Carmela Pasquier, Edward Allen Phelps

Pancreatic islet β-cells are particularly susceptible to endoplasmic reticulum (ER) stress, which is implicated in β-cell dysfunction and loss during the pathogenesis of type 1 diabetes (T1D). The peripheral membrane protein GAD65 is an autoantigen in human T1D. GAD65 synthesizes γ-aminobutyric acid, an important autocrine and paracrine signaling molecule and a survival factor in islets. We show that ER stress in primary β-cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in trans-Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen-presenting cells and T-cell stimulation compared with the nonpalmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human β-cells in pancreatic sections from GAD65 autoantibody-positive individuals who have not yet progressed to clinical onset of T1D and from patients with T1D with residual β-cell mass and ongoing T-cell infiltration of islets. We propose that aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system after release from stressed and/or damaged β-cells, triggering autoimmunity.

Amer Diabetes Assoc2016

Investigating the Biological Mechanisms underlying the Initiation of Autoimmunity against Beta Cell Antigens in Type 1 Diabetes

Chiara Cianciaruso

Type 1 diabetes (T1D) is an autoimmune disease characterized by circulating autoantibodies, lymphocytic infiltration of pancreatic islets of Langerhans, and cell-specific destruction of beta cells, leading to insulin deficiency and symptomatic hyperglycemia. Some of the major target autoantigens in T1D are intracellular proteins, such as GAD65, IA-2 and proinsulin, whose initial encounter with the immune system is poorly understood. Here we describe a method for culturing monolayers of primary pancreatic islet cells in vitro, which enables detailed observation of proteins and sub-cellular processes in primary human and rat beta cells by super-resolution and live-cell light microscopy. This technical advance is broadly applicable to obtaining novel biological insights as demonstrated by our identification of a mechanism to control proliferation in primary beta cells through growth and disassembly of primary cilia. This protocol also allowed us to identify two novel interrelated mechanisms for how beta cell proteins, such as the antigen GAD65, can become target of autoimmunity and initiate T1D. A triggering factor that these two pathways have in common is represented by endoplasmic reticulum (ER) stress, to which pancreatic islet beta cells are particularly susceptible, and can be induced by inflammatory factors such as Th1 cytokines. In the first mechanism, we show that ER stress in beta cells perturbs the palmitoylation cycle controlling GAD65 endomembrane distribution, resulting in aberrant accumulation of the palmitoylated form in Golgi membranes. The palmitoylated form has heightened immunogenicity, exhibiting increased uptake by antigen presenting cells (APCs) and T cell stimulation compared to the non-palmitoylated form. Similar accumulation of GAD65 in Golgi membranes is observed in human beta cells in pancreatic sections from GAD65 autoantibody positive individuals, who have not yet progressed to clinical onset of T1D, and T1D patients with residual beta cell mass and ongoing T cell infiltration of islets. Thus, aberrant accumulation of immunogenic GAD65 in Golgi membranes facilitates inappropriate presentation to the immune system following release from stressed and/or damaged beta cells, triggering autoimmunity. In the second mechanism, we present that, following a pathway originating from the Golgi, both rat and human pancreatic islets release the intracellular beta cell autoantigens GAD65, IA-2 and proinsulin in a subtype of extracellular vesicles called exosomes. Islet-exosomes are then taken up by and can activate APCs including dendritic cells. Accordingly, anchoring of GAD65 to exosome-mimetic liposomes strongly boosts antigen presentation and T cell activation in the context of the human T1D susceptibility haplotype HLA-DR4. Furthermore, cytokine-induced ER stress enhances exosome secretion by beta cells, induces exosomal release of the immunostimulatory chaperones calreticulin, Gp96 and ORP150 and increases exosomal stimulation of APCs. Therefore, stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in initiation of autoimmune responses in T1D.

EPFL2017

Bioengineering strategies for inducing tolerance in autoimmune diabetes

Steinunn Baekkeskov Hanahan, Jeffrey Alan Hubbell, Edward Allen Phelps

Type 1 diabetes is an autoimmune disease marked by the destruction of insulin-producing beta cells in the pancreatic islets. Strategies to delay onset or prevent the autoimmune recognition of beta cell antigens or T cell-mediated killing of beta cells have mainly focused on systemic immunomodulation and antigen-specific immunotherapy. To bridge the fields of type 1 diabetes immunology and biomaterials engineering, this article will review recent trends in the etiology of type 1 diabetes immunopathology and will focus on the contributions of emerging bioengineered strategies in the fight against beta cell autoimmunity in type 1 diabetes. (C) 2017 Elsevier B.V. All rights reserved.

Elsevier Science Bv2017

Investigating the Biological Mechanisms underlying the Initiation of Autoimmunity against Beta Cell Antigens in Type 1 Diabetes

Chiara Cianciaruso

EPFL2017