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Genome-wide association studies revealed CLEC16A as a candidate gene for Type 1 Diabetes and multiple other autoimmune disorders. The function of CLEC16A remains unknown. However, previous work showed that the CLEC16A ortholog ema and the murine Clec16a were both implicated in autophagy, a process partially required for MHC class II loading and antigen presentation. Furthermore, studies could show that autophagy was required in thymic epithelial cells for antigen presentation during T cell selection, suggesting a possible role of CLEC16A in T cell selection in the thymus. Additionally, it was postulated that CLEC16A may function as an expression quantitative trait locus for its neighboring genes and that Clec16a KD was involved in pancreatic islet function and impaired insulin secretion and glucose homeostasis. Prior to this work, Schuster et al. had created a Clec16a KD NOD mouse, which was protected from spontaneous autoimmune diabetes.
For this work it was hypothesized that CLEC16A variation serves as a Type 1 Diabetes risk gene by affecting autophagy in thymic epithelial cells, which modulates antigen presentation and shapes the T cell repertoire. To expand and complement previous findings by Schuster et al., this thesis aimed to investigate how CLEC16A modifies the function of thymic epithelial cells. For this purpose, CLEC16A KD was induced in human cells via RNA interference and autophagy was studied through immunoblotting. Additionally, inflammation of pancreatic tissue in Clec16a KD NOD mice was scored using H.E. stained pancreatic sections. Thymic transplantation experiments were conducted to test whether the effects of Clec16a KD were T cell intrinsic. Also, intraperitoneal glucose tolerance tests were performed to study glucose homeostasis in Clec16a KD NOD animals. Finally, using qPCR, gene expression levels of neighboring genes such as Dexi and Socs1 were measured to study Clec16a as an expression quantitative trait locus.
In combination with the findings of Schuster et al., this thesis demonstrates that Clec16a KD reduces the severity of insulitis and protects from onset of spontaneous diabetes in the NOD mouse. Disease protection is conveyed by impaired autophagy in TEC, which leads to altered T cell selection and hyporeactive CD4+ T cells. The effects of Clec16a KD in the NOD mouse are thymus intrinsic. Glucose homeostasis remains unchanged in the Clec16a KD NOD mouse and plays no role in disease protection. Clec16a and Dexi presented similar expression levels, but further studies are required to investigate a clear link between these two genes. Finally, impaired autophagy could be replicated in human CLEC16A KD cells, which demonstrates a conserved function of CLEC16A and suggests a possible link between CLEC16A variation and risk of autoimmune disease in human.
Pathogenic relevance of autoantibodies to type XVII collagen from pemphigoid gestationis patients
(2007)
Pemphigoid gestationis (PG) and bullous pemphigoid (BP) are subepidermal autoimmune blistering diseases characterized by self-reactive T and B cells specific for the transmembrane hemidesmosomal protein type XVII collagen/BP180. Major T and B cell epitopes are located within the immunodominant 16th non-collagenous domain A (NC16A) of type XVII collagen. It has been suggested that pathogenically relevant autoantibodies also bind to this immunodominant region. The aim of this study was to map the epitopes targeted by blister-inducing human autoantibodies. For this purpose, we used an in vitro model of autoantibody-induced leucocyte-dependent dermal-epidermal separation. In contrast to the majority of patients with BP (7 of 10), preadsorption against a recombinant form of the NC16A region abolished the blister-inducing potential of autoantibodies from all PG patients tested (n=5). Using overlapping synthetic peptides, we demonstrate that PG autoantibodies bind to 2 defined epitopes within the NC16A region (aa 500-514 and aa 511-523). Preadsorption using an affinity matrix containing these two epitopes completely abolished dermal-epidermal separation induced by PG autoantibodies (in 8 of 9 patients). These findings provide new insights into the pathogenesis of pemphigoid diseases and should prove helpful for the development of an antigen-specific immunoadsorption therapy in PG.