Identification of vesicular SNARE proteins involved in the secretoryintracellular trafficking of IL-12 in dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells. They have the unique ability of recognizing antigens, internalizing and processing them for presentation on MHC complexes. DCs also express pattern-recognition receptors like Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns and induce DCs maturation. As a consequence DCs increase their antigens presenting ability, their levels of co-stimulatory molecules on the cell surface, and their production of cytokines, which are critical for inducing specific T cell activation. All these functions make DCs key elements in the immunological response against pathogens, viruses and tumors. One of the most important cytokines in T cell priming, interleukine-12 (IL-12), begins to be largely transcribed immediately after TLR activation. We have previously shown that in DCs engaged in antigen specific immunological synapses (IS), IL-12 is found enriched in the proximity of the contact site between DC and T cells, and several evidences suggest that its secretion is polarized towards the T cell. These data indicate that polarized trafficking of cytokines containing vesicles in DCs is a mechanism to optimize T cell priming. Polarized trafficking of soluble mediators through defined subcellular areas has been described as fundamental for the function of several other immune cells, but at present a clear characterization of the pathways and organelles implicated in the regulation of cytokine secretion in dendritic cells is still missing. The family of SNARE (soluble-N ethylmaleimide sensitive-factor accessory-protein (SNAP) receptor) proteins, which regulate the membrane fusion process during the intracellular trafficking of soluble mediators, has emerged as the most informative for the identification of such pathways. Indeed different SNAREs regulate diverse mechanisms such as the constitutive secretion of immune mediators, phagocytosis and endocytosis, or also the release of inflammatory mediators and products from secretory granules. During my thesis I have investigated the role of SNAREs in IL-12 secretion by DCs. I have analyzed the expression and modulation during maturation of three SNAREs, Syntaxin6, VAMP3 and VAMP7, that were previously shown to play a role in other immune cells. The subcellular localization and co-localization with the IL-12 subunits were analyzed in DCs alone and in the context of IS by immunofluorescence. I performed functional analyses by silencing SNAREs with the siRNA strategy to detect impairment in DC cytokines secretion and in T cell priming. These studies led to the identification of the lysosomal-late endosomal VAMP7 as a specific regulator in the polarized secretion of IL-12 during T cell priming. This suggest a role, in the secretion of soluble newly synthesized mediators, of late endosomal organelles, until now thought to be implicated only in the regulated secretion of pre-constituted mediators such as cytotoxic granules. Furthermore VAMP3 was found to downregulate the production of all the cytokines tested; we propose this SNARE as regulator of the TLR9 signaling pathway. The second part of the work stems from the concept that DCs properties become suppressed by the tumor microenvironment and many evidences suggest that tolerogenic DCs may be a crucial element in the development of tumor-mediated immune anergy. Different steps in the antigen presentation process are impaired, but also the pattern of secreted cytokines is skewed. In particular, how secretion of inflammatory cytokines becomes impaired in tumor-exposed DCs has not been investigated. I set up a model of mice bearing Lewis lung carcinoma (3LL) to analyze DCs in vitro (co-colture) and in vivo (spleen and tumor-bearing lungs). In all the models I observed a strong impairment in IL-12 production, upon restimulation with TLR agonists. In spleen DCs subtypes remained unchanged during tumor progression, whereas in tumor-bearing lungs the DC composition varied, with an expansion of CD11bhi DCs and a reduction in CD103+ DCs. DCs were isolated from all the three models to analyze the expression of trafficking proteins, and I found that tumor-exposed DCs downregulate all the SNAREs and in particular VAMP3. Moreover Rab27a, a GTPase implicated in the regulation of the exocytosis of secretory granules and lysosome-related organelles is also downregulated at protein level. These preliminary observations suggest that regulation of trafficking proteins in DCs may represent a novel pathway targeted during immunosuppression.