Final Report Summary - DNGR-1 IN DCS (The dendritic cell receptor DNGR-1: Modulation of endosomal dynamics upon recognition of necrotic cells) The overall aim of the project was to study the pattern recognition receptor, DNGR-1, and its proposed role in cross-presentation of dead-cell associated antigens. DNGR-1 (also known as CLEC9A) is a C-type lectin receptor that is specifically expressed on dendritic cells (DCs). These cells play an essential function in initiating adaptive immunity. Key to DC function are receptors for recognizing and capturing pathogens and damaged cells and for controlling subsequent antigen extraction, processing and presentation. DNGR-1 recognizes the actin cytoskeleton exposed on necrotic cells. Interestingly, DNGR-1 is not required for internalization of cell corpses by DCs but regulates their subsequent ability to cross-present corpse-associated antigens to CD8+ T-cells. The mechanisms underlying DNGR-1 function are unknown but require receptor signaling upon ligand engagement and are though to relate to modulation of endocytic traffic. Thus, the overall goals of this project were to gain a deeper understanding into the intracellular spatio-temporal dynamics of the receptor, DNGR-1, and to ultimately identify molecular mechanism(s) by which this receptor promotes cross-presentation. As part of the first objective to monitor receptor trafficking, I have established dendritic cell lines expressing fluorescently labeled DNGR-1. I have used these cell lines to study the intracellular localization of the receptor in steady state and in ligand-stimulated conditions using fluorescence microscopy of fixed as well as live cells using time-lapse imaging. In addition, I have established an assay to measure receptor internalization by a FACS based assay. To monitor the fate of internalized receptor, I have established cell lines with extracellular tagging of the receptor using a sortase-based approach. In parallel, I have been working on developing assays to monitor changes in the endo/phagosomal environment to study if DNGR-1 is affecting any of the reported intracellular events linked to cross-presentation. These include assays measuring changes in endosomal pH, reactive oxygen species, leakiness or in lysosomal degradation.My work has revealed unusual and novel observations in the endosomal dynamics of a ligand activated DC receptor. Upon stimulation of cells expressing DNGR-1 with its ligand, F-actin, the receptor is internalized together with its ligand. Interestingly, internalized receptor/ligand structures appear to form unusually large vesicles. Upon further inspection, these vesicles were found to bear markers of early and late endosomes. They did not appear to be acidic and lysosomal in nature. Correlative light and electron microscopy of these vesicles revealed them to be electron lucent and largely devoid of intraluminal vesicles. This phenotype resembles that in which endosomal maturation is perturbed. A well-known example of this phenomenon is one in which the GTPase Rab5, a fundamental regulator of endosomal maturation, is inhibited to undergo deactivation. This results in formation of enlarged endosomes, with an early/late endosome hybrid phenotype, formed through homotypic fusion reactions. Lysosomal degradation of endosomal content is delayed and receptor signaling is prolonged. Delayed endosomal maturation has in the past been often associated with increased cross-presentation ability, explained as a way to preserve antigenic information. Therefore, these results suggest that by activating the receptor DNGR-1, endosomal maturation is prevented, which could help to preserve receptor associated antigenic material for presentation on MHC I. With these studies we have been able to increase our understanding of the intracellular trafficking of DNGR-1 in the context of its ligand, F-actin. Previous work in the group has revealed that artificial antigen targeting of the receptor can promote cross-presentation to CD8+ cytolytic T-cells. Moreover, when given together with adjuvants, this targeting has been shown to lead to efficient cytolytic T-cell responses in mice and a subsequent decrease in burden of tumours expressing this foreign antigen. Being therefore a potential candidate for immunotherapy regimes that could be used for cancer and infectious diseases, we expect this work to aid in the future development of DC-mediated immunotherapies and vaccination strategies.
