INVESTIGATING THE EFFECTS OF NOREPINEPHRINE ON AUTOREACTIVE CD8+ T CELLS USING IN-VITRO AND IN-VIVO EXPERIMENTS

Sammanfattning

Type 1 diabetes is an autoimmune condition where the immune system targets the insulin cells, mainly beta cells, resulting in the destruction of these insulin-producing cells. There is no treatment for type 1 diabetes. Since factors such as genetic predisposition can be a major influence in the progression of type 1 diabetes. The main goal of this study is to understand the effect of norepinephrine (NE) on the autoreactive CD8+ T cells. NE is a commonly known catecholamine present in the microenvironment of type 1 diabetes individuals. The effect of the NE was studied in vitro using live imaging performed by a confocal microscope, and analysis was carried out using IMARIS. Naïve CD8+ T cells are known to be motile and scan for antigens presented by APCs to interact with them to become activated. These CD8+ T cells become activated, becoming cytotoxic, causing beta cell destruction. To study the effect, it had on type 1 diabetes, the Ins2-OVA mouse model was used. This mouse model was used because of the beta cells presenting Ovalbumin (OVA) membrane-bound OVA in pancreatic β-cells where the ovalbumin (OVA) gene is expressed under the control of the rat insulin II promoter (Ins2). The OT-1 T cells specifically target these OVA-expressing beta cells, enabling us to study further about type 1 diabetes. Most of the studies were performed in vitro, where we visualized the effects of NE on T1D induced islets harvested after day 6, and also OT-1 T cells from day 6, as well as day 8 OT-1 T cell culture. Since we know that activated OT-1 T cells expressed adrenergic receptors thus we hypothesized that is how NE affected the OT-1 T cell motility. We also used qPCR to study the OT-1 T cell gene expression from day 0 to day 8 after stimulating these cells with OVA, which showed cytotoxic potential. We investigated the effects of two norepinephrine (NE) antagonists, phentolamine and propranolol. Phentolamine was used to block NE's effects, while propranolol was used to assess the phenotypic changes of OT-1 T cells. Treatment with NE increased early activation markers, whereas propranolol treatment led to a significant decrease, indicating promising results.