Representative data for the expression of CD69 on Exo?, Exoint or Exohi CD3+ cells is usually shown in (B). conclude that tumor-associated exosomes are immunosuppressive, and represent a therapeutic target blockade of which would enhance the antitumor response of quiescent tumor-associated T cells and prevent the functional arrest of adoptively transferred tumor-specific T cells or chimeric antigen receptor (CAR) T cells. value higher than 0.05 was not significant (NS), whereas * 0.05; ** 0.01 and *** 0.001 were considered significant. Results Characterization of immunosuppressive vesicles from ovarian tumor ascites fluids Vesicles isolated from ovarian malignancy patients tumor ascites fluid by ultracentrifugation were examined for ultrastructural morphology and size by transmission electron microscopy (TEM). Uranyl oxalate stained vesicles were homogeneously spherical, membrane bound particles consistent with the morphology of exosomes (Fig. 1A). Open in a separate windows Fig. 1 Characterization of extracellular vesicles isolated from human ovarian ascites fluidElectron microscope images of vesicles isolated from ovarian tumor ascites fluids using ultracentrifugation (A). Size distribution of the vesicles was decided using nanoparticle tracking analysis (B) and phase transition study of vesicles isolated from ovarian Tshr tumor ascites fluid by ultracentrifugation was carried out using anisotropy measurements (C). The composition of vesicles isolated AUY922 (Luminespib, NVP-AUY922) from ovarian tumor ascites fluid by ultracentrifugation was decided using an Exosome Antibody Array (D). Dark spots indicate presence of the marked protein. Absence of a spot for GM130 indicates absence of cellular contaminants in the preparation. Data shown are representative of 3 impartial experiments. Orthogonal biophysical techniques such as nanoparticle tracking analysis (NTA) and fluorescence anisotropy were employed to determine size and lamellarity of the vesicles. NTA analysis of the vesicles revealed a size distribution of 50C200 nm with a modal diameter of 60C80 nm (Fig. 1B). The lamellarity of these vesicles was analyzed by labeling these vesicles with diphenyl hexatriene (DPH); lipid order and dynamics were measured at numerous temperatures using fluorescence anisotropy (Fig. 1C). At lesser temperatures, anisotropy values were higher, consistent with a rigid acyl chain packing, but anisotropy values decreased with higher temperatures due to increased acyl chain mobility. The anisotropy values as a function of heat showed a broad transition centered around 37C suggesting lamellarity in lipid business. We conclude that this vesicles present within ovarian tumors are surrounded by a lipid bilayer. Vesicles isolated by ultracentrifugation from ovarian tumor ascites fluids were assayed for the presence of marker proteins that are typically found on exosomes (30) using a commercially available antibody platform called Exosome Antibody Array. Five of the exosome marker proteins (CD81, Tsg-101, Flotillin-1, EpCAM, and Annexin V) were found to be abundant in the vesicles; two other markers, CD63 and Alix, were detected but less abundant (Fig. 1D). The absence of a positive spot for GM130 indicated that our exosome preparations were not contaminated with cellular material. As we as well as others have previously reported, tumor-associated exosomes also express a negatively charged glycerophospholipid, phosphatidylserine (PS) (31), representing a lipid marker expressed on the surface of exosomes. Based upon the morphology, size, and presence of relevant protein and lipid markers, we conclude that this extracellular vesicles we are isolating from ovarian malignancy patients tumor ascites fluids AUY922 (Luminespib, NVP-AUY922) are exosomes. Exosomes inhibit nuclear translocation of AUY922 (Luminespib, NVP-AUY922) NFAT and NFB following activation Extracellular vesicles derived from malignancy patients sera/plasma (32) or from patients ovarian tumor ascites fluids (31) have been reported to inhibit the activation of T cells. However, those studies used a method to active the T cells that depended on antibodies to CD3 and CD28 immobilized on antibody-coated beads (32). Such a protocol represents an artificial stimulus for T cells of unknown specificity. Because exosomes may just block CD3 and/or CD28 antibody binding to T cells, we asked whether tumor ascites-derived exosomes would similarly inhibit an antigen-induced activation of T cells. To address this question, we utilized Class I MHC multimers (dextramers) loaded with peptides known to bind to antigen receptors on either EBV- or CMV-specific T cells and activate them (25). T cell activation is determined by a translocation of NFAT from your cytosol into the nucleus and has been confirmed by cells production of cytokines (25). Peripheral blood from HLA-A2 donors known to have EBV- or CMV-specific T cells were incubated either on ice (non-permissive for activation) or at room heat (permissive for activation) with EBV peptide (Fig. 2A, B) or CMV peptide (Fig. 2C, D) loaded.