Browsing by Subject "RAB GTPase"

Adaptive immunity is a specialized immune system, responsible for pathogen-specific immunity and the creation of memory cells. This system is activated in secondary lymphoid organs, such as lymph nodes, by antigen presentation conducted by professional antigen-presenting cells (APCs) to T cells. The activated T cells migrate to inflamed tissue for the elimination of pathogens. Recently, it has been shown that lymphatic endothelial cells (LECs) of the lymphatic system also partake in immune regulation alongside professional APCs. LECs accomplish this by internalizing soluble antigens from the lymph. The internalized antigens can be either presented on the LEC plasma membrane with major histocompatibility complex I for induction of T cell anergy, or transferred to migrating professional APCs for complete T cell activation. Outside of direct immune regulation, the antigens can also be transcellularly transported through the LECs, which contributes to the transport of soluble antigens to lymph nodes. Through all these functions, LECs have been shown to play a role in the maintenance of peripheral T cell tolerance, impairment of tumor-specific immune responses, and conversely, the creation of stronger secondary immune response due to stimulation of T memory cell generation. However, the mechanisms of antigen internalization in LECs remain highly uncharacterized. To further study the role of LECs in adaptive immunity regulation, this thesis investigates the intracellular routes of antigen internalization and trafficking, in LECs. In this thesis, I set up antigen feeding assay on two primary cell culture models, and identify vesicular compartments involved in antigen internalization. To study antigen internalization, I set up extraction and cell culture methods for mouse lymph node primary cells and performed antigen feeding on the cultured cells. In addition, I optimized antigen feeding assay and identified means to increase antigen internalization in primary human dermal LECs (HDLECs). As LECs internalize antigens in both cell culture models, both models offer an opportunity to study antigen internalization and fates of antigens, in the context of LEC-performed adaptive immunity regulation. To investigate the pathways involved in antigen internalization and trafficking in LECs, I utilized RAB GTPases, major regulators of vesicular traffic, each specific for a distinct vesicular compartment. By observing and quantifying the colocalization between antigens and fluorescently stained RAB GTPases, I identified the antigen-associated vesicular compartments. The identification of the antigen trafficking pathways was successful, as functional validation of the results by inhibition of the antigen colocalizing RAB GTPases led to decreased antigen internalization. In conclusion, in this thesis, I optimized antigen feeding assay in LECs and identified pathways involved in antigen internalization and trafficking. The results of this thesis provide ground for further identification of molecules involved in LEC-performed adaptive immunity regulation. Manipulation of these control molecules would provide opportunities to modify the role of LECs in adaptive immunity regulation, to a desired direction.