Foot and mouth disease (FMD) is a highly contagious infection caused by FMD-virus (FMDV) that affects livestock worldwide with significant economic impact. The main strategy for the control is vaccination with FMDV chemically inactivated with binary ethylenimine (FMDVi). In FMDV infection and vaccination, B cell response plays a major role by providing neutralizing/protective antibodies in animal models and natural hosts. Extracellular vesicles (EVs) and small EVs (sEVs) such as exosomes are important in cellular communication. EVs secreted by antigen-presenting cells (APC) like dendritic cells (DCs) participate in the activation of B and T cells through the presentation of native antigen membrane-associated to B cells or by transferring MHC-peptide complexes to T cells and even complete antigens from DCs. In this study, we demonstrate for the first time that APC activated with the FMDVi O1 Campos vaccine-antigens secrete EVs expressing viral proteins/peptides that could stimulate FMDV-specific immune response. The secretion of EVs-FMDVi is a time-dependent process and can only be isolated within the first 24 h post-activation. These vesicles express classical EVs markers (CD9, CD81, and CD63), along with immunoregulatory molecules (MHC-II and CD86). With an average size of 155 nm, they belong to the category of EVs. Studies conducted in vitro have demonstrated that EVs-FMDVi express antigens that can stimulate a specific B cell response against FMDV, including both marginal zone B cells (MZB) and follicular B cells (FoB). These vesicles can also indirectly or directly affect T cells, indicating that they express both B and T epitopes. Additionally, lymphocyte expansion induced by EVs-FMDVi is greater in splenocytes that have previously encountered viral antigens in vivo. The present study sheds light on the role of EVs derived from APC in regulating the adaptive immunity against FMDV. This novel insight contributes to our current understanding of the immune mechanisms triggered by APC during the antiviral immune response. Furthermore, these findings may have practical implications for the development of new vaccine platforms, providing a rational basis for the design of more effective vaccines against FMDV and other viral diseases.

ABSTRACTCells secrete membrane-enclosed extracellular vesicles (EVs) and non-vesicular nanoparticles (ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed either to induce immune priming of antigen presenting cells, or, to be immuno-suppressive agents promoting tumor immune escape. We suspect that such disparate functions are due to variable composition in EV subtypes and ENPs of the analyzed EV preparations. We aimed to exhaustively characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by tumor cells. We established a robust protocol to separate small (s)EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells (DCs). ENPs were poorly captured and did not affect DCs. sEVs specifically induced DC death. A mixed EV/VLP preparation was the most efficient to induce DC maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by tumors and their contribution to anti-tumor immune responses and to tumor progression.

The tetraspanins CD9, CD81 and CD63 are major components of extracellular vesicles (EVs). Yet, their impact on EV composition remains under-investigated. In the MCF7 breast cancer cell line CD63 was as expected predominantly intracellular. In contrast CD9 and CD81 strongly colocalized at the plasma membrane, albeit with different ratios at different sites, which may explain a higher enrichment of CD81 in EVs. Absence of these tetraspanins had little impact on the EV protein composition as analysed by quantitative mass spectrometry. We also analysed the effect of concomitant knock-out of CD9 and CD81 because these two tetraspanins play similar roles in several cellular processes and associate directly with two Ig domain proteins, CD9P-1/EWI-F/PTGFRN and EWI-2/IGSF8. These were the sole proteins significantly decreased in the EVs of double CD9- and CD81-deficient cells. In the case of EWI-2, this is primarily a consequence of a decreased cell expression level. In conclusion, this study shows that CD9, CD81 and CD63, commonly used as EV protein markers, play a marginal role in determining the protein composition of EVs released by MCF7 cells and highlights a regulation of the expression level and/or trafficking of CD9P-1 and EWI-2 by CD9 and CD81.

Détermination du potentiel des sous-types de vésicules et de particules extracellulaires comme porteurs de protéines en vue de futures applications thérapeutiques Les vésicules extracellulaires (VEs) et autres nanoparticules extracellulaires (ENP) sont sécrétées par les cellules dans le milieu extracellulaire et ont été identifiées comme des médiateurs importants de la communication avec d'autres cellules et la matrice extracellulaire qui les entoure. Les VEs contiennent diverses molécules, y compris des protéines, qui peuvent délivrer des signaux pour induire des changements physiologiques chez les cellules réceptrices. Du fait de cette propriété de communication intercellulaire particulière, les VEs sont activement étudiées pour éventuellement être utilisées à des fins thérapeutiques. Dans ce travail, nous avons cherché à comparer les fonctions de différents sous-types de VEs, des particules extracellulaires non VEs, et des protéines dans deux contextes pathologiques différents : l'infection virale et les réponses immunitaires anti-tumorales. Premièrement, nous avons évalué les VEs exprimant à leur surface une protéine recombinante reconnue par un virus, le SARS-CoV-2, par rapport à la même protéine non associée aux VEs, pour servir de leurre au virus, et ainsi réduire l'infection. Deuxièmement, nous avons séparé et comparé les sous-types de VEs dérivées de tumeurs, y compris les rétrovirus endogènes, et d'autres particules extracellulaires, pour leur capacité à transférer un antigène et à moduler l'activation des cellules présentatrices d'antigènes. Nos résultats illustrent l'importance d'identifier le(s) sous-type(s) de particule(s) portant la fonction d'intérêt, dans l'optique d'optimiser de futures utilisations de VEs à visée thérapeutique.

Significance Our work uncovers mechanisms by which tumor cells impact tumor-associated macrophages in human triple-negative breast cancer. Via extracellular vesicles (EVs), these tumors promote macrophages with proinflammatory features, correlated with better clinical outcome. Our results suggest exploration of these EVs as tools, alone, or in combination with other therapies, to promote a favorable environment for the generation of anti-tumor immune responses.

SARS-CoV-2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2-EVs are efficient decoys for SARS-CoV-2 S protein-containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2.

Cells release a variety of extracellular vesicles (EVs; including exosomes, microvesicles, and many others) into their environment. EVs can bud in endosomes or directly at the plasma membrane, carrying a selection of components from the cell and displaying various functional properties. Different techniques can be used to separate EV subtypes and EVs from co-isolated components, resulting in preparations of different abundance and purity.

Exosomes are 60-150 nm small extracellular vesicles (EVs) released by most cells. Tumor-cell-derived exosomes, used as a vaccine, elicit a specific cytotoxic response against tumor cells, usually with a greater immunogenicity than tumor-cell lysates. However, the number of exosomes isolated from culture cells is limited. In recent studies, it was observed that cells respond to different stressor stimuli such as cytotoxic drugs, hypoxia, acidosis, or radiation by increasing the release of EVs. In this study, using the murine LBC T-cell lymphoma, we found that cyclophosphamide significantly increased EVs yield. These EVs express exosome marker proteins such as TSG-101, CD9, CD81, and CD63. Furthermore, similar humoral and cellular immune responses were induced in vivo by EVs isolated from LBC-tumor cells whether they were grown under normal culture conditions (EVs C) or in the presence of cyclophosphamide (EVs CTX). Mice vaccinated either with EVs C or EVs CTX were similarly protected against an intraperitoneal challenge with LBC tumor cells. CD4+ and CD8+ IFN-gamma secreting cells were induced in immunized mice and a specific cytotoxic cellular immune response was elicited in vitro. These results demonstrate that a Th1 response was induced by immunization with the EVs. Our findings suggest that treatment of tumor cells with cyclophosphamide is a useful method to enhance the secretion of EVs in sensitive cell lines without altering their antitumor properties and thus may be used to produce antigens for future design of cancer vaccines. (C) 2019 Elsevier Ltd. All rights reserved.

T-cell lymphomas include diverse malignancies. They are rare, some have low survival rates and they lack curative therapies. The aim of this work was to assess whether employing the TLR7 agonist imiquimod and the T-cell costimulatory molecule CD40 or the combination of both as adjuvants of a cell lysate vaccine could enhance the antitumor immune response using a murine T-cell lymphoma model. Immunization with LBC-lysate and imiquimod protected almost all vaccinated animals. A specific humoral and a Thl-type cellular immunity were induced in mice that rejected the lymphoma, characterized by an elevated number of CD4 + T-cells and secretion of IFN-gamma, locally and systemically. In contrast, CD40 alone or in combination with imiquimod did not improve the protective response obtained with LBC-lysate and imiquimod. Systemic administration of imiquimod proved to have high potential to serve as a vaccine adjuvant for the treatment of T-cell lymphomas and was effective in this immunotherapy model.

Extracellular vesicles (EVs), including endosome-derived nanovesicles (exosomes), are involved in cell-cell communication. Through transfer of their molecular contents, extracellular nanovesicles can alter the function of recipient cells. Due to these characteristics, EVs have shown potential as a new alternative for cancer immunotherapy. Tumor exosomes isolated from malignant ascites can activate dendritic cells, thereby priming the immune system to recognize and kill cancer cells. However, a suppressive role on tumor immune response has also been reported, suggesting that the neoplastic stage of carcinogenesis and the microenvironment where tumor cells grow may influence the amount of EVs released by the cell. This neoplastic stage and microenvironment may also impact EVs' components such as proteins and miRNA, determining their biological behavior. Most T-cell lymphomas have an aggressive clinical course and poor prognosis. Consequently, complementary alternative therapies are needed to improve the survival rates achieved with conventional treatments. In this work, we have characterized EVs isolated from ascites of mice bearing a very aggressive murine T-cell lymphoma and have studied their immunogenic properties. Small EVs were isolated by differential centrifugation, ultrafiltration, and ultracentrifugation at 100,000 x g on a sucrose cushion. The EVs were defined as exosomes by their morphology and size analyzed by electron microscopy, their floating density on a sucrose gradient, as well as their expression of endosome marker proteins ALIX, TSG-101; the tetraspanins CD63, CD9, and CD81. In addition, they contain tumor antigens, the marker for malignancy CD24, the heat shock protein HSP-70, and an unusual surface expression of HSP-90 was demonstrated. The administration of EVs isolated from ascites (EVs A) into naive-syngeneic mice induced both humoral and cellular immune responses that allowed the rejection of subsequent tumor challenges. However, the immunization had no effect on a non-related mammary adenocarcinoma, demonstrating that the immune response elicited was specific and also it induced immune memory. In vitro analysis demonstrated that T-cells from EVs A-immunized mice secrete IFN-.in response to tumor stimulation. Furthermore, tumor-specific CD4+ and CD8+ IFN-gamma secreting cells could be efficiently expanded from mice immunized with EVs A, showing that a T helper 1 response is involved in tumor rejection. Our findings confirm exosomes as promising defined acellular tumor antigens for the development of an antitumor vaccine.

Event Abstract Back to Event EXOSOMES DERIVED FROM THE ASCITIC FLUID OF MICE BEARING A T-CELL LYMPHOMA EXPRESS TUMOR ASSOCIATED ANTIGENS Florencia Menay1, Federico Cocozza1, Alejandrina Vendrell1, Claudia Waldner1 and Claudia Mongini1* 1 National Research Council of Argentina (CEFYBO-CONICET), Argentina Exosomes are extracellular nanovesicles of 60-100 nm (EVs), released by almost all cell types, including tumor cells. EVs are involved in cell-cell communication. Through transfer of their molecular contents, EVs are capable of altering the function of recipient cells. Tumor derived exosomes may have either immunostimulating or immunosuppressant properties. Previous works demonstrated that the administration of exosomes derived from tumor cells lines into syngeneic animals produced both humoral and cellular immune response in immunized host, capable of rejecting tumor. These immunological functions have led to the development of anti-tumor vaccines based on exosomes, which are currently in early clinical development. The aim of this work was to isolate and define the immuno-modulating characteristics of exosomes derived from the ascitic fluid obtained from T-cell lymphoma bearing mice. BALB/c mice were inoculated ip. with 1,00E+06 tumor LBC cells and after 20 ± 2 days ascitic fluid was collected by ventral puncture. Exosomes were isolated by a serial of differential centrifugation, ultrafiltration and ultracentrifugation at 100,000xg. The microvesicles were characterized by their floating density in a sucrose gradient and the presence of marker proteins Alix, Tsg101, CD63, and Hsp70 was determined by Western blotting and flow cytometry. BALB/c mice were immunized twice with 20 ug of exosomes/mouse with an interval of 7 days. The humoral immune response induced was studied by dot and Western blot, in sera obtained from immunized mice and compared with those from naïve mice. All mice studied had high titre of serum antibodies against both exosomes and LBC cells (1:12800) and recognized proteins of 45 and 50 kDa present in the exosomes and tumor cell lysate, as determined by Western blot. To study the immune properties in vivo immunized mice were challenged with 1,00E+06 LBC tumor cells. Fifty percent of the mice immunized with exosomes did not develop tumors, while the tumor incidence in control mice (non-immunized and challenged with the LBC cells) was 100% (n = 8).To assess the specificity of the immune response generated on mice that rejected LBC tumor, mice were re-challenging with 2,00E+05 cells of a non related breast adenocarcinoma (LM3). All LBC tumor free mice that were re-challenged with tumor cells developed the LM3 breast tumor. We conclude that exosomes present in the ascitic fluid of LBC tumor bearing mice express tumor antigens and have immune-stimulating properties. The immune response elicited in hosts was specific as it was sufficient to prevent tumor development in syngeneic mice but failed to prevent the progression of a non-related tumor. These findings reveal that exosomes are potent immune regulators and are relevant for the development of an "acellular" immunotherapy approach. Keywords: Exosomes, T-cell lymphoma, tumor vaccines, Tumor immunotherapy, Ascitic Fluid Conference: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015. Presentation Type: Poster Presentation Topic: Tumor immunology Citation: Menay F, Cocozza F, Vendrell A, Waldner C and Mongini C (2015). EXOSOMES DERIVED FROM THE ASCITIC FLUID OF MICE BEARING A T-CELL LYMPHOMA EXPRESS TUMOR ASSOCIATED ANTIGENS. Front. Immunol. Conference Abstract: IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología. doi: 10.3389/conf.fimmu.2015.05.00063 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 29 May 2015; Published Online: 14 Sep 2015. * Correspondence: Dr. Claudia Mongini, National Research Council of Argentina (CEFYBO-CONICET), Buenos Aires, Buenos Aires, 1121, Argentina, cmongini@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract Supplemental Data The Authors in Frontiers Florencia Menay Federico Cocozza Alejandrina Vendrell Claudia Waldner Claudia Mongini Google Florencia Menay Federico Cocozza Alejandrina Vendrell Claudia Waldner Claudia Mongini Google Scholar Florencia Menay Federico Cocozza Alejandrina Vendrell Claudia Waldner Claudia Mongini PubMed Florencia Menay Federico Cocozza Alejandrina Vendrell Claudia Waldner Claudia Mongini Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.