First Symposium of "Grupo Español de Investigación en Vesículas Extracelulares (GEIVEX)", Segovia, 8-9 November 2012.

The pivotal role played by extracellular vesicles in immune responses was presented in this session chaired by Francisco Sanchez-Madrid. Exosomes present antigen and regulate the activation and function of NK cells and macrophages. In addition, they may be suitable biomarkers for atherosclerosis of therapeutic agents for autoimmune diseases. Francesc E. Borràs (IVECAT-IGTP, Badalona, Barcelona) presented data demonstrating the higher capacity of mature monocyte-derived dendritic cells (DCs) to internalize exosomes, despite being less phagocytic than immature DCs. As the capture of exosomes by DCs is of great relevance in transplantation, since exosomes bear MHCI and MHCII and can therefore present antigens, Dr. Borràs’ group studied the capacity of blood-borne DCs to capture exosomes. They showed that both conventional DCs and plasmacytoid DCs are capable of capturing exosomes, although the latter at a slower rate than conventional DCs. They also showed that pDCs can present exosomal antigens to autologous T cells. As this specialized DC subset has been previously implicated in the tolerogenic response to aloantigens, this study pointed to exosomes and other microvesicles as a source of antigen for DCs to induce tolerance. Carlos Ernesto Fernandez, working in the IIS-FJD in the group of Jose Luis Martin-Ventura, presented novel data regarding the involvement of exosomes as a mechanism of secretion of novel biomarkers of atherosclerosis. Interestingly, the secretion of exosomes by vascular cells seems to be modulated by antioxidants, thus linking pathological pathways of atherosclerosis such as oxidative stress with the production of exosomes. Further studies would delineate the potential use of exosomes as biomarkers of atherosclerosis. María del Mar Valés-Gómez (CNB, Madrid) centres her research on natural killer (NK) cells, which are a first line of defence to tumour and infection, whose cytotoxic activity is regulated by a balance of inhibitory and activating receptors. The activating receptor NKG2D is present in all human NK and T cells and mediates recognition of pathogen-infected and cancer cells. However, tumour cells can evade the immune system by releasing NKG2D ligands to induce down-regulation of the receptor. Some NKG2D-ligands can be recruited to exosomes and potently modulate receptor expression, while other are more susceptible to metalloprotease cleavage and shed as soluble molecules. The presence of NKG2D ligands in cancer patient sera can be used as a marker of tumour progression; for example, ULBP2 is found in advanced stages of melanoma. Nevertheless, the route of NKG2D-ligand release to the supernatant and their polymorphism are important factors to consider in the use of these proteins as cancer biomarkers. Luis Martínez Lostao (Universidad de Zaragoza) showed the impressive potential of artificial liposomes tailored to bear TNF-related apoptosis inducing ligand (TRAIL) on their surface (LUVs-Apo2L/TRAIL). Apo2L/TRAIL is a death ligand found on exosomes secreted by activated T-lymphocytes. The authors constructed liposomes bearing a Ni 2+ quellating molecule to bind in a correct conformation the His-tagged TRAIL recombinant protein to resemble natural exosomes. LUVs-Apo2L/TRAIL was shown to be a more effective treatment in rheumatoid arthritis than soluble Apo2L/TRAIL without showing hepatotoxic effects. Agnieszka Koziol (CNIC, Madrid) showed that the macrophage secretome contained 425 proteins, 71% of which were of exosomal origin. Among them, lactadherin (milk fat globule-EGF factor 8, or MFGE8) appeared to be a putative substrate of MT1-MMP proteolytic activity, which was confirmed by in vitro digestion assay. A MT1-MMP-dependent cleavage site is located at the phosphatidilserine-binding motif responsible for binding to exosomes and apoptotic cells so that MT1-MMP may modify the binding properties of MFGE8. MFGE8 KO presents a defect in the clearance of apoptotic cells and immune resolution leading to the development of autoimmune diseases. To determine whether MT1-MMP is involved in this function of MFGE8, they analysed the efficiency of MT1 −/− and MT1 +/− mice in apoptotic cell clearance. Both MT1−/− and MT1 + /− displayed defects in phagocytosis of apoptotic cells. MT1 + /− mice also showed reduced survival in an experimental sepsis model and developed an autoimmune disease because of impaired immune response resolution.

The first scientific symposium of GEIVEX bringing together Spanish groups working on different aspects of extracellular vesicles surpassed all expectations as judged by the evaluation made by the participants (see Figure 1 ). Overall, the meeting, the venue, the organisation, the format, the quality and the topics were all highly appreciated. In addition, several collaborations and future plans were, respectively, created and discussed. Among them, it is worth mentioning the next two key actions of GEIVEX: (1) an international workshop on extracellular vesicles to be held at the “Menéndez Pelayo” International University on 18–20 September 2013 (see http://geivex.wordpress.com/3-courses/uimp-course-vesiculas-extracelulares-implicaciones-en-biomedicina/ ); and (2) the next meeting which will be hosted in in Catalunya in the first or second trimester of 2014. Finally, the organizers would like to thank all of the speakers, participants and sponsors for making this first meeting of GEIVEX possible.

Our sponsors presented their own claimed solutions for extracellular vesicle research in this appealing session chaired by Maria Yañez-Mo. Ben Owen, from NanoSight, introduced their already widely used equipment, based on nanoparticle tracking, which, based on their Brownian coefficient, determines the size of particles in solution and their concentration. This technology also allows for the fluorescent labelling of the vesicles. Miltenyi specialist Dr. Alexander Adan commented on the special characteristics of their cytometer, which make it particularly well suited for the study of extracellular vesicles. Other than its small size, it has a volumetric counter and can determine the relative size of nanoparticles in the side scatter, which has a broader angle (15°) than other cytometers. Moreover, they are currently working on adapting their famous MACs system to the isolation and purification of extracellular vesicles. Dr. Dimitri Aubert, from Izon Science, presented the qNano system for high accuracy extracellular vesicle characterisation and quantification, based on their patented tunable resistive pulse sensing technology. This non-optical method counts every particle going through a size-tunable nanopore and measures the resulting changes in conductivity, giving very accurate measurements for particles of diameters 40 nm to 10 µm. Finally, Isabel Crespo, responsible of the cytometry platform of IDIBAPS and invited by BD, presented flow cytometry data on microvesicles. She prepared a very comprehensive report on the requirements for accurate detection of these very small particles, such as buffer filtration, low flow rate, and thresholding to discriminate noise (based on fluorescence by annexin labelling in this case, or even FCS/SSC). Flow cytometry of extracellular vesicles is a future challenge to discriminate by specific markers among the heterogeneity of extracellular vesicles in our preparations from biological samples, which is pushed to the limit by the small size and scarcity of some antigens in extracellular vesicles.