Pathogens map the way; Nano-domains as pathogen docking and signaling platforms on antigen presenting cells.

During evolution pathogens have constantly adapted to escape eradication and sometimes even exploit host defense mechanisms to do so. Antigen presenting cells (APCs), which are among the first cells of the immune system to encounter pathogens, are equipped with a myriad of pathogen recognition- and uptake- receptors. Our recent work now demonstrates that long before APCs contact pathogens, a clear organization of cellular pathogen recognition receptors at the surface already exists in the form of sub-micron sized domains. Moreover, preliminary proteomic analyses of these nano-domains indicate that distinct pathogen recognition receptors co-localize, suggesting that the composition of nano-domains may differ. This level of pre-organization is intriguing and appears directly related to cell function. How this level of receptor organization is established and maintained is unknown. HYPOTHESIS. We hypothesize that the composition of pathogen recognition receptors origanised in nano-domains is an essential component in endosome routing and signaling, and as such contributes importantly to the outcome of an immune response (Figure 1). AIM. Therefore the aim of this project is to unravel the mechanisms that lead to the formation of such nano-domains, the dynamics of its composition and the functional consequences thereof, exploiting dynamic high-resolution molecular imaging, and proteomics approaches. Thus we expect to get detailed insight in the molecular mechanisms that control endosome routing and signaling dependent on composition and surface organization of pathogen recognition receptors after ligation of model pathogens or pathogen mimics (nano-beads coated with pathogen derived ligands). APROACH. Within this program we shall focus on two pathogen recognition receptor families, C-type lectin- and Toll- like receptor families (CLRs, TLRs). While CLRs are generally recognized as pathogen uptake receptors, TLRs are known to transmit signals to alert the cell of danger. Since many pathogens contain ligands for both type of receptors it is highly likely that both TLRs and CLRs act in concert. Initial studies indicate that this is indeed the case. This, and our findings that CLRs are organized at the cell surface in nano-clusters and that this organization is essential for function, strongly support the idea that the composition of these nano-domains may directly affect intracellular routing and signaling. Initial proteomic analyses by co-immunoprecipitation experiments now for the first time show that different types of CLRs are indeed associated and can also interact with tetraspanin molcules, providing direct evidence that nano-domains may differ in composition and function. Exploiting high resolution fluorescence and electron microscopy we shall compare cells expressing different compositions of wild type and mutant forms of CLRs and TLRs in their response to prototype pathogens or pathogen mimics and analyse endosomal routing and signaling. The plan of investigation consists of three parts: Part A is aimed at getting quantitative insight in the composition of CLR containing nano-domains both by biochemical methods and high resolution microscopy. Part B will focus on the dynamics of CLR containing nano-domains, in particular in response to APC activation or maturation signals. Part C will focus on down stream signals from CLRs and how these can act in concert with TLR signals to affect cell function. ELEMENTS OF INNOVATION. (1)For the first time we are able to determine the topography of receptors at the cell surface by high resolution microscopical techniques developed by this team. (2) Initial proteome analyses of CLR containing nano-domains show that different CLRs can associate with each other, or with members of the Tetraspanin family of scaffolding proteins. The latter may play an important role in the formation and stability of nano-domains.(3) Highly purified CLR and TLR ligands will be linked to so called Quantum Dots, fluorescent nano-particles of extreme photo stability, to visualize intracellular routing of pathogens. OUTCOME AND CLINICAL RELEVANCE. This project will provide in depth insight in the mechanisms that control nano-domain organization on APCs. It will allow us to understand how pathogens have learned during evolution to exploit these domains to become phagocytosed, and by which signals they are rerouted either towards survival- or to degradation- pathways. We expect that this knowledge will lead to development of novel vaccination strategies by targeting viral-, bacterial-, or tumor- antigens, to APCs in vivo, thus exploiting these specific pathogen docking sites for antigen entry. In the near future we may make use of these endocytic receptors to control DCs in vivo and direct the immune response to either immunity or tolerance, to fight cancer, infectious diseases or autoimmune diseases.