Dendritic Cell-derived Exosomes Research Articles

A prospective highlight on exosomal nanoshuttles and cancer immunotherapy and vaccination.

Introduction: Exosomes (EXOs) and ectosomes (ECTOs) are nanoscale membranous extracellular vesicles (EVs) derived from different cells mediating various cellular communications. EXOs are liberated based on the exocytosis of multivesicular bodies, while ECTOs are ubiquitously released from the plasma membranes. Methods: Here, in this paper, we go over the extracellular vesicular machineries and concisely highlight their clinical importance in solid tumors and their possible applications in cancer immunotherapy/vaccination. Results: In various types of cancers, these vesicles play central roles delivering cancer cell messages to the target cells, as a result both of them seem to provide a novel useful means for diagnosis and therapy of malignancies. Dendritic cell-derived exosomes (DEXOs) are able to activate the tumor antigen-specific CD8+ cytotoxic T-lymphocytes (CTLs) and hence induce antitumor responses in vivo. Within the tumor microenvironment (TME), however, tumor cells seem to generate exosomes (the so-called oncosoems) that may act in favor of tumor progression. Conclusions: As complex systems, these vesicular micro-/nano-machines convey important cellular messages dependent upon the cells/tissue setting(s). In addition to their potential in diagnosis of cancers, they have been exploited for cancer immunotherapy/vaccination. However, such treatment strategies need to be carefully designed to attain desired clinical outcomes.

Tetraspanin-3 regulates protective immunity against Eimeria tenella infection following immunization with dendritic cell-derived exosomes

The effects of immunization with dendritic cell (DC) exosomes, which had been incubated with a tetraspanin-3 (Tspan-3) blocking antibody (Ab) or with an isotype-matched non-immune IgG, were studied using an experimental model of Eimeria tenella avian coccidiosis. Purified exosomes from cecal tonsil and splenic DCs expressed Tspan-3 protein. Chickens injected with exosomes incubated with the control IgG and derived from cecal tonsil DCs preloaded in vitro with E. tenella Ag had Ag-immunostaining cells in the ceca, but not the spleen. Conversely, Ag-containing cells were found only in the spleen, but not the ceca, of chickens given IgG treated splenic DC exosomes. Interestingly, chickens that received exosomes incubated with Tspan-3 Ab had Ag-containing cells observed in both lymphoid organs following administration of exosomes from either DC population. After injection of exosomes non-incubated with Tspan-3 Ab, greater numbers of cells secreting interleukin-2 (IL-2), IL-16, interferon-γ, and E. tenella-reactive Abs were observed in the cecal tonsils of chickens immunized with cecal DC exosomes compared with the spleen. By contrast, more cytokine-and Ab-producing cells were present in the spleen of chickens given splenic DC exosomes compared with the ceca. Incubation with Tspan-3 Ab gave similar numbers of cytokine- and Ab-producing cells in the cecal tonsils and spleen regardless of the source of exosomes. Immunization with E. tenella Ag-loaded cecal tonsil DC exosomes increased in vivo resistance against subsequent E. tenella infection. Increased protection against infection following cecal DC exosome immunization was partially blocked by incubation of exosomes with Tspan-3 Ab. These results suggest that Tspan-3 is involved in the tissue distribution, as well as cytokine and Ab production, following DC exosome administration, and that Tspan-3 contributes to in vivo protection against experimental E. tenella challenge infection following exosomal immunization.

Erratum to: Immature Dendritic Cell-Derived Exosomes: a Promise Subcellular Vaccine for Autoimmunity

1 Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China 410008 2 Department of Neurology, The First Hospital in Changsha City, Changsha, Hunan, People’s Republic of China 3 To whom correspondence should be addressed at Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China 410008. E-mail: yangh69@126.com

Mechanism of Evenness Interrupted (Evi)-Exosome Release at Synaptic Boutons

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release.

Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases

Exosomes are 40–100 nm membrane vesicles secreted into the extracellular space by numerous cell types. These structures can be isolated from body fluids including urine and plasma. Exosomes contain proteins, mRNAs, miRNAs, and signaling molecules that reflect the physiological state of their cells of origin and consequently provide a rich source of potential biomarker molecules. Aside from diagnostic uses, exosome-mediated transfer of proteins, mRNAs, miRNAs, and signaling molecules offer the promise that they may be used for therapeutic purposes. In this review, we integrate new knowledge about exosomes from outside the field of nephrology with recent progress by renal researchers in order to provide a basis for speculation about how the study of exosomes may affect the fields of nephrology and renal physiology in the next few years.

Natural nanoparticules against cancer: mature dendritic cell-derived exosomes

Deep insight on Natural nanoparticules against cancer: mature dendritic cell-derived exosomes.

Updated Technology to Produce Highly Immunogenic Dendritic Cell-derived Exosomes of Clinical Grade

Dendritic cell-derived exosomes (Dex) are nanovesicles bearing major histocompatibility complexes promoting T-cell-dependent antitumor effects in mice. Two phase I clinical trials aimed at vaccinating cancer patients with peptide-pulsed Dex have shown the feasibility and safety of inoculating clinical-grade Dex, but have failed to show their immunizing capacity. These low immunogenic capacities have led us to develop second-generation Dex with enhanced immunostimulatory properties. Here, we show that interferon-γ is a key cytokine conditioning the dendritic cell to induce the expression of CD40, CD80, CD86, and CD54 on Dex, endowing them with direct and potent peptide-dependent CD8(+) T-cell-triggering potential in vitro and in vivo. In this study, we describe the clinical grade process to manufacture large-scale interferon-γ-Dex vaccines and their quality control parameters currently used in a phase II trial.

Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins

Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA(2)-IVA, the calcium-independent iPLA(2)-VIA, and the secreted sPLA(2)-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPgammaS triggered activation of phospholipase A(2) (PLA(2))and PLD(2). A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E(2) (PGE(2)) and 15-deoxy-Delta(12,14)-prostaglandinJ(2) (15-d PGJ(2)), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell.

Proteomics Analysis of Bladder Cancer Exosomes

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.

Dendritic Cell-Derived Exosomes for Cancer Immunotherapy: What's Next?

Exosomes are nanovesicles originating from late endosomal compartments and secreted by most living cells in ex vivo cell culture conditions. The interest in exosomes was rekindled when B-cell and dendritic cell-derived exosomes were shown to mediate MHC-dependent immune responses. Despite limited understanding of exosome biogenesis and physiological relevance, accumulating evidence points to their bioactivity culminating in clinical applications in cancer. This review focuses on the preclinical studies exploiting the immunogenicity of dendritic cell-derived exosomes (Dex) and will elaborate on the past and future vaccination trials conducted using Dex strategy in melanoma and non-small cell lung cancer patients.

Immature dendritic cell-derived exosomes rescue septic animals via milk fat globule epidermal growth factor VIII

Miksa, M., R. Wu, W. Dong, H. Komura, D. Amin, Y. Ji, Z. Wang, H. Wang, T. S. Ravikumar, K. J. Tracey, and P. Wang. 2009. Immature dendritic cell-derived exosomes rescue septic animals via milk fat globule epidermal growth factor VIII. [ J. Immunol. 183: 5983–5990][1] . The title of this article

Immature dendritic cell-derived exosomes rescue septic animals via milk fat globule epidermal growth factor-factor VIII [corrected

Sepsis, a highly lethal systemic inflammatory syndrome, is associated with increases of proinflammatory cytokines (e.g., TNF-alpha, HMGB1) and the accumulation of apoptotic cells that have the potential to be detrimental. Depending on the timing and tissue, prevention of apoptosis in sepsis is beneficial; however, thwarting the development of secondary necrosis through the active removal of apoptotic cells by phagocytosis may offer a novel anti-sepsis therapy. Immature dendritic cells (IDCs) release exosomes that contain milk fat globule EGF factor VIII (MFGE8), a protein required to opsonize apoptotic cells for phagocytosis. In an experimental sepsis model using cecal ligation and puncture, we found that MFGE8 levels decreased in the spleen and blood, which was associated with impaired apoptotic cell clearance. Administration of IDC-derived exosomes promoted phagocytosis of apoptotic cells and significantly reduced mortality. Treatment with recombinant MFGE8 was equally protective, whereas MFGE8-deficient mice suffered from increased mortality. IDC exosomes also attenuated the release of proinflammatory cytokines in septic rats. Liberation of HMGB1, a nuclear protein that contributes to inflammation upon release from unengulfed apoptotic cells, was prevented by MFGE8-mediated phagocytosis in vitro. We conclude that IDC-derived exosomes attenuate the acute systemic inflammatory response in sepsis by enhancing apoptotic cell clearance via MFGE8.

Active Uptake of Dendritic Cell-Derived Exovesicles by Epithelial Cells Induces the Release of Inflammatory Mediators through a TNF-α-Mediated Pathway

Dendritic cells (DCs) can release hundreds of membrane vesicles, called exovesicles, which are able to activate resting DCs and distribute antigen. Here, we examined the role of mature DC-derived exovesicles in innate and adaptive immunity, in particular their capacity to activate epithelial cells. Our analysis of exovesicle contents showed that exovesicles contain major histocompatibility complex-II, CD40, and CD83 molecules in addition to tumor necrosis factor (TNF) receptors, TNFRI and TNFRII, and are important carriers of TNF-alpha. These exovesicles are rapidly internalized by epithelial cells, inducing the release of cytokines and chemokines, but do not transfer an alloantigen-presenting capacity to epithelial cells. Part of this activation appears to involve the TNF-alpha-mediated pathway, highlighting the key role of DC-derived exovesicles, not only in adaptive immunity, but also in innate immunity by triggering innate immune responses and activating neighboring epithelial cells to release cytokines and chemokines, thereby amplifying the magnitude of the innate immune response.

17. Targeted Delivery of Plasmid DNA and siRNA with Modified Dendritic Cell-Derived Exosomes

17. Targeted Delivery of Plasmid DNA and siRNA with Modified Dendritic Cell-Derived Exosomes

Dendritic Cell-Derived Exosomes Promote Natural Killer Cell Activation and Proliferation: A Role for NKG2D Ligands and IL-15Rα

Dendritic cell (DC) derived-exosomes (Dex) are nanomeric vesicles harboring functional MHC/peptide complexes promoting T cell-dependent tumor rejection. In the first Phase I trial using peptide-pulsed Dex, the observation of clinical regressions in the absence of T cell responses prompted the search for alternate effector mechanisms. Mouse studies unraveled the bioactivity of Dex on NK cells. Indeed, Dex promoted an IL-15Rα- and NKG2D-dependent NK cell proliferation and activation respectively, resulting in anti-metastatic effects mediated by NK1.1+ cells. In humans, Dex express functional IL-15Rα which allow proliferation and IFNγ secretion by NK cells. In contrast to immature DC, human Dex harbor NKG2D ligands on their surface leading to a direct engagement of NKG2D and NK cell activation ex vivo. In our phase I clinical trial, we highlight the capacity of Dex based-vaccines to restore the number and NKG2D-dependent function of NK cells in 7/14 patients. Altogether, these data provide a mechanistic explanation on how Dex may stimulate non MHC restricted-anti-tumor effectors and induce tumor regression in vivo.

No Significant CTL Cross-Priming by Dendritic Cell-Derived Exosomes during Murine Lymphocytic Choriomeningitis Virus Infection

Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells in culture, but are also present in serum. They contain a wide array of protein ligands on their surface, which has led to the hypothesis that they might mediate intercellular communication. Indeed, data support that exosomes can transfer Ags to dendritic cells (DC), and, interestingly, that these DC can subsequently induce T cell priming or tolerance. We have investigated whether this concept can be expanded to antiviral immunity. We isolated exosomes from supernatant of cultured bone marrow-derived DC (BMDC) that were infected with lymphocytic choriomeningitis virus (LCMV) or loaded with an immunodominant LCMV peptide, and characterized them by flow cytometry upon binding to beads. We then incubated the exosome preparations with BMDC and looked at their potential to activate LCMV gp33-specific naive and memory CD8 T cells. We found that exosomes do not significantly contribute to CD8 T cell cross-priming in vitro. Additionally, exosomes derived from in vitro-infected BMDC did not exhibit significant in vivo priming activity, as evidenced by the lack of protection following exosome vaccination. Thus, DC-derived exosomes do not appear to contribute significantly to CTL priming during acute LCMV infection.

Phase I Clinical Trial of Autologous Ascites-derived Exosomes Combined With GM-CSF for Colorectal Cancer

Exosomes are small membrane vesicles that are secreted by a multitude of cell types. The exosomes derived from dendritic cells (Dex), tumor cells (Tex), and malignant effusions demonstrate immunomodulatory functions, and are even under clinical trial for cancer treatments. In this study we report the phase I clinical trial of the ascites-derived exosomes (Aex) in combination with the granulocyte–macrophage colony-stimulating factor (GM-CSF) in the immunotherapy of colorectal cancer (CRC). The Aex isolated by sucrose/D2O density gradient ultracentrifugation are 60–90-nm vesicles that contain the diverse immunomodulatory markers of exosomes and tumor-associated carcinoembryonic antigen (CEA). Totally 40 patients (HLA-A0201+CEA+) with advanced CRC were enrolled in the study, and randomly assigned to treatments with Aex alone or Aex plus GM-CSF. Patients in both groups received a total of four subcutaneous immunizations at weekly intervals. We found that both therapies were safe and well tolerated, and that Aex plus GM-CSF but not Aex alone can induce beneficial tumor-specific antitumor cytotoxic T lymphocyte (CTL) response. Therefore, our study suggests that the immunotherapy of CRC with Aex in combination with GM-CSF is feasible and safe, and thus can serve as an alternative choice in the immunotherapy of advanced CRC.

Fractalkine-Induced MFG-E8 Leads to Enhanced Apoptotic Cell Clearance by Macrophages

Clearance of apoptotic cells is crucial to maintain cellular function under normal and pathological conditions. We have recently shown that administration of immature dendritic cell-derived exosomes to septic animals promotes phagocytosis of apoptotic cells and improves survival by providing milk fat globule epidermal growth factor-factor VIII (MFG-E8). MFG-E8 acts as an opsonin for apoptotic cells to be engulfed by phagocytosis. In the present study we investigated whether the CX(3)C-chemokine fractalkine (CX(3)CL1) promotes apoptotic cell clearance through the induction of MFG-E8 in peritoneal macrophages. Cultured rat peritoneal macrophages (pMphi) and RAW264.7 macrophages were stimulated with LPS and CX(3)CL1. MFG-E8 expression was assessed by Western blot, cytokine secretion was assessed by ELISA, and phagocytosis of apoptotic thymocytes was determined by microscopy. For in vivo studies, cecal ligation and puncture (CLP) was used to induce sepsis in rats and mice. LPS significantly decreased MFG-E8 levels and phagocytosis of apoptotic cells, whereas CX(3)CL1 induced MFG-E8 expression in both nonstimulated and LPS-stimulated pMphi, without affecting TNF-alpha and IL-6 release. Anti-MFG-E8 blocking antibodies completely abrogated the prophagocytic effect of CX(3)CL1. Twenty hours after the induction of sepsis in rats via CLP, plasma CX(3)CL1 levels as well as MFG-E8 production in peritoneal macrophages decreased by 21% and 56%, respectively. Administration of CX(3)CL1 on the other hand induced MFG-E8 and prevented tissue injury. We conclude that CX(3)CL1 induces MFG-E8 in vitro and in vivo and enhances clearance of apoptotic cells in an MFG-E8-dependent manner. These findings suggest a possible novel treatment for patients in sepsis.

Exovesicles from Human Activated Dendritic Cells Fuse with Resting Dendritic Cells, Allowing Them to Present Alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Dendritic cell derived-exosomes: biology and clinical implementations

Exosomes are nanometer-sized membrane vesicles invaginating from multivesicular bodies and secreted from different cell types. They represent an "in vitro" discovery, but vesicles with the hallmarks of exosomes are present in vivo in germinal centers and biological fluids. Their protein and lipid composition is unique and could account for their expanding functions such as eradication of obsolete proteins, antigen presentation, or "Trojan horses" for viruses or prions. The potential of dendritic cell-derived exosomes (Dex) as cell-free cancer vaccines is addressed in this review. Lessons learned from the pioneering clinical trials allowed reassessment of the priming capacities of Dex in preclinical models, optimizing clinical protocols, and delineating novel, biological features of Dex in cancer patients.