Primary human and rat beta cells release the intracellular autoantigens GAD65, IA-2 and proinsulin in exosomes together with cytokine-induced enhancers of immunity

The target autoantigens in several organ-specific autoimmune diseases, including type 1 diabetes (T1D), are intracellular membrane proteins, whose initial encounter with the immune system is poorly understood. Here we propose a new model for how these proteins can initiate autoimmunity. We found that rat and human pancreatic islets release the intracellular β-cell autoantigens in human T1D, GAD65, IA-2 and proinsulin, in exosomes, which are taken up by and activate dendritic cells. Accordingly, anchoring of GAD65 to exosome-mimetic liposomes strongly boosted antigen presentation and T cell activation in the context of the human type 1 diabetes susceptibility haplotype HLA-DR4. Cytokine-induced ER-stress enhanced exosome secretion by β-cells, induced exosomal release of the immunostimulatory chaperones calreticulin, Gp96 and ORP150 and increased exosomal stimulation of antigen presenting cells. We propose that stress-induced exosomal release of intracellular autoantigens and immunostimulatory chaperones may play a role in initiation of autoimmune responses in T1D.

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

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

New roles of the lymphatic endothelium in modulating adaptive immunity: implications for immune tolerance and cancer immunotherapy

Lambert Charles François Potin

Lymphatic vessels have traditionally been considered as passive conduits for interstitial fluid drainage and for immune cell trafficking to lymph nodes. In the last two decades, many studies have challenged this classical dogma: lymphatic endothelial cells (LECs) were shown to actively interact with immune cells by secreting chemokines and cytokines, and presenting antigens to T cells. These newly described functions pinpoint a multi-faceted role of LECs in orchestrating the immune response through both passive and active mechanisms. Yet, precise mechanisms leading to antigen-specific fine-tuning of the T cell response remain unexplored. Moreover, although lymphatics have been shown to play important roles in cancer, the contributions of LECs in regulating antitumor immunity have been largely overlooked by the biomedical community. This doctoral thesis aimed at filling these gaps. First, in order to investigate the mechanisms and implications of LEC antigen presentation to CD4+ T cells, we engineered a mouse model where LECs lacked the ability to present antigens to CD4+ T cells. In these mice, antigen-specific responses to vaccination were increased when compared to wild-type controls. Moreover, we demonstrated that LECs were poor inducers of CD4+ T cell activation in vitro, and that they could dampen CD4+ T cell activation by dendritic cells. Taken together, these findings suggest that LECs participate to the peripheral immune tolerance of CD4+ T cells. Second, we investigated the immunological consequences of the development of lymphatic vessels, called lymphangiogenesis, in mouse melanoma. Upon induction of tumor lymphangiogenesis, we observed striking tumor enrichment with immune cells, which resulted in a higher sensitivity of those tumors to immunotherapy. We subsequently demonstrated that inducing lymphangiogenesis in a mouse model of cold tumors, which lack immune infiltrates and do not respond to immunotherapies, could restore their responsiveness to immunotherapy treatments. Third, to predict which cancer types were the most likely to replicate the findings we obtained in mouse melanoma, we mined The Cancer Genome Atlas database for gene expression. We identified a set of cancers, such as colon adenocarcinoma and cholangiocarcinoma, where high expression of the lymphangiogenic growth factor was associated with a strong T cell signature. This doctoral thesis unveiled new immunomodulatory functions of LECs by which antigen-specific immune tolerance is induced. Additionally, it opened new avenues to target lymphatics therapeutically in cancer to potentiate immunotherapies. We believe this work contributes to demonstrating the immunological importance of LECs, and hope that lymphatics will be considered as an important immunomodulatory player of the tumor microenvironment when developing novel immunotherapies.