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      Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

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          Abstract

          A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

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          Most cited references 37

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          Human tumor-derived exosomes selectively impair lymphocyte responses to interleukin-2.

          Exosomes are nanometer-sized vesicles, secreted by normal and neoplastic cells. The outcome following interaction between the cellular immune system and cancer-derived exosomes is not well understood. Interleukin-2 (IL-2) is a key factor supporting expansion and differentiation of CTL and natural killer (NK) cells but can also support regulatory T cells and their suppressive functions. Our study examined whether tumor-derived exosomes could modify lymphocyte IL-2 responses. Proliferation of healthy donor peripheral blood lymphocytes in response to IL-2 was inhibited by tumor exosomes. In unfractionated lymphocytes, this effect was seen in all cell subsets. Separating CD4(+) T cells, CD8(+) T cells, and NK cells revealed that CD8(+) T-cell proliferation was not inhibited in the absence of CD4(+) T cells and that NK cell proliferation was only slightly impaired. Other exosome effects included selective impairment of IL-2-mediated CD25 up-regulation, affecting all but the CD3(+)CD8(-) T-cell subset. IL-2-induced Foxp3 expression by CD4(+)CD25(+) cells was not inhibited by tumor exosomes, and the suppressive function of CD4(+)CD25(+) T cells was enhanced by exosomes. In contrast, exosomes directly inhibited NK cell killing function in a T-cell-independent manner. Analysis of tumor exosomes revealed membrane-associated transforming growth factor beta(1) (TGFbeta(1)), which contributed to the antiproliferative effects, shown by using neutralizing TGFbeta(1)-specific antibody. The data show an exosome-mediated mechanism of skewing IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells. This coordinated "double hit" to cellular immunity strongly implicates the role of exosomes in tumor immune evasion.
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            Tumor-derived microvesicles promote regulatory T cell expansion and induce apoptosis in tumor-reactive activated CD8+ T lymphocytes.

            Sera of patients with cancer contain membraneous microvesicles (MV) able to induce apoptosis of activated T cells by activating the Fas/Fas ligand pathway. However, the cellular origin of MV found in cancer patients' sera varies as do their molecular and cellular profiles. To distinguish tumor-derived MV in cancer patients' sera, we used MAGE 3/6(+) present in tumors and MV. Molecular profiles of MAGE 3/6(+) MV were compared in Western blots or by flow cytometry with those of MV secreted by dendritic cells or activated T cells. These profiles were found to be distinct for each cell type. Only tumor-derived MV were MAGE 3/6(+) and were variably enriched in 42-kDa Fas ligand and MHC class I but not class II molecules. Effects of MV on signaling via the TCR and IL-2R and proliferation or apoptosis of activated primary T cells and T cell subsets were also assessed. Functions of activated CD8(+) and CD4(+) T lymphocytes were differentially modulated by tumor-derived MV. These MV inhibited signaling and proliferation of activated CD8(+) but not CD4(+) T cells and induced apoptosis of CD8(+) T cells, including tumor-reactive, tetramer(+)CD8(+) T cells as detected by flow cytometry for caspase activation and annexin V binding or by DNA fragmentation. Tumor-derived but not dendritic cell-derived MV induced the in vitro expansion of CD4(+)CD25(+)FOXP3(+) T regulatory cells and enhanced their suppressor activity. The data suggest that tumor-derived MV induce immune suppression by promoting T regulatory cell expansion and the demise of antitumor CD8(+) effector T cells, thus contributing to tumor escape.
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              Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.

              Viable tumor cells actively release vesicles into the peripheral circulation and other biologic fluids, which exhibit proteins and RNAs characteristic of that cell. Our group demonstrated the presence of these extracellular vesicles of tumor origin within the peripheral circulation of cancer patients and proposed their utility for diagnosing the presence of tumors and monitoring their response to therapy in the 1970s. However, it has only been in the past 10 years that these vesicles have garnered interest based on the recognition that they serve as essential vehicles for intercellular communication, are key determinants of the immunosuppressive microenvironment observed in cancer and provide stability to tumor-derived components that can serve as diagnostic biomarkers. To date, the clinical utility of extracellular vesicles has been hampered by issues with nomenclature and methods of isolation. The term "exosomes" was introduced in 1981 to denote any nanometer-sized vesicles released outside the cell and to differentiate them from intracellular vesicles. Based on this original definition, we use "exosomes" as synonymous with "extracellular vesicles." While our original studies used ultracentrifugation to isolate these vesicles, we immediately became aware of the significant impact of the isolation method on the number, type, content and integrity of the vesicles isolated. In this review, we discuss and compare the most commonly utilized methods for purifying exosomes for post-isolation analyses. The exosomes derived from these approaches have been assessed for quantity and quality of specific RNA populations and specific marker proteins. These results suggest that, while each method purifies exosomal material, there are pros and cons of each and there are critical issues linked with centrifugation-based methods, including co-isolation of non-exosomal materials, damage to the vesicle's membrane structure and non-standardized parameters leading to qualitative and quantitative variability. The down-stream analyses of these resulting varying exosomes can yield misleading results and conclusions.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                20 January 2017
                2017
                : 12
                : 1
                Affiliations
                [1 ]Department of Medical and Biological Sciences, University of Udine, Udine, Italy
                [2 ]Department of Neurosurgery, University Hospital of Udine, Udine, Italy
                Istituto Superiore Di Sanita, ITALY
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: RD DC BT GG.

                • Formal analysis: RD DC BT FC GG.

                • Funding acquisition: APB.

                • Investigation: RD DC BT EB FC IM GG.

                • Methodology: RD DC EB IM TI MS GG.

                • Project administration: GG.

                • Resources: APB TI MS CDL.

                • Supervision: GG.

                • Validation: RD DC GG.

                • Visualization: RD GG.

                • Writing – original draft: RD.

                • Writing – review & editing: RD DC GG.

                [¤]

                Current address: Genekor Medical S.A, Athens, Greece

                Article
                PONE-D-16-37541
                10.1371/journal.pone.0169932
                5249124
                28107450
                © 2017 Domenis et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 8, Tables: 1, Pages: 23
                Product
                Funding
                Funded by: FIRB 2011
                Award ID: RBAP11Z4Z9
                Funded by: FIRB 2011
                Award ID: Pr. RBAP11ETKA
                Award Recipient :
                Funded by: Interreg Italia-Slovenia
                Award ID: GLIOMA
                Funded by: European Research Council, 7FP SP 2 IDEAS
                Award ID: 269051
                This work was supported by Futuro in Ricerca (FIRB) 2011, Pr. RBAP11Z4Z9, 2012-2014; Futuro in Ricerca (FIRB) 2011, Pr. RBAP11ETKA, 2012-2015 to APB; Interreg Italia-Slovenia, GLIOMA, 2007-2013; Project ERC-7FP SP 2 IDEAS QUIDPROQUO G.A. n. 269051, 2011-2016. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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