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Results for “Exosomes”

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At least 127 records · Page 7Linked to original sources

PubMed2006

Efficient induction of antitumor T cell immunity by exosomes derived from heat-shocked lymphoma cells.

Exosomes secreted by tumor cells could serve as a promising immunotherapeutic tumor vaccine. Heat shock proteins (HSP) induced in tumor cells by heat shock are molecular chaperones with potent adjuvant activity in the induction of antigen-specific T cell responses. To improve exosome-based tumor vaccines, we have investigated the efficacy of exosomes derived from heat-shocked mouse B lymphoma cells (HS-Exo) in the induction of antitumor immune responses. We found that HS-Exo, compared with control exosomes derived from the same cells (Exo), contain more HSP60 and HSP90 and increased amounts of molecules involved in immunogenicity including MHC class I, MHC class II, CD40, CD86, RANTES and IL-1beta. Furthermore, HS-Exo induce both phenotypic and functional maturation of dendritic cells more efficiently. HS-Exo immunization activates T cell responses more potently. Importantly, HS-Exo induce dramatically increased antitumor immune responses compared to control exosomes from the same cells in prophylaxis and therapeutic in vivo lymphoma models. We further demonstrate that CD8(+) T cells are the predominant T cell subset responsible for the antitumor effect of HS-Exo and that CD4(+) T cells are necessary in the induction phase of tumor rejection in a prophylaxis model. These findings provide a novel strategy to improve the efficacy of exosome-based tumor vaccines.

Animals↗

PubMed2003

Antigen-presenting cell exosomes are protected from complement-mediated lysis by expression of CD55 and CD59.

Exosomes are secreted nanometer-sized vesicles derived from antigen-presenting cells, which have attracted recent interest as they likely play important roles in immune regulation, and their use as cell-free tools for immunotherapy has been proposed. Liposomes used clinically as transport vehicles can activate the complement system, leading to their rapid degradation and significant inflammatory toxicity. The use of isolated exosomes in therapy, therefore, may also elicit complement activation, reducing their potential efficacy. We have examined the expression and functional roles of the membrane regulators of complement (CD46, CD55 and CD59) on antigen-presenting cell-derived exosomes. Exosomes express the glycosylphosphatidylinositol (GPI)-anchored regulators CD55 and CD59, but not the transmembrane protein CD46. Antibody blocking of CD55 in the presence of sensitizing antibody (w6/32) and human serum resulted in increased C3b deposition and significantly increased exosome lysis. Blockade of CD59 also resulted in significant lysis, while blocking both CD55 and CD59 increased lysis still further. We conclude that exosomes express GPI-anchored complement regulators in order to permit their survival in the extracellular environment.

Antibodies, Monoclonal↗

PubMedDate not supplied

Phenotypic and functional characterization of intestinal epithelial exosomes.

Intestinal epithelial cells (IEC) are located at a strategic position between the external environment and the most extended lymphoid tissue in the body. Besides their central role in the absorption of nutrients, IEC also provide antigenic information to the immune system and are involved in the balance tolerance/allergy to food antigens. Like professional antigen presenting cells, IEC have been shown to secrete 30- to 90-nm diameter vesicles named exosomes, in a polarized way, either from their apical or basolateral side. These vesicles carry molecules involved in adhesion and antigen presentation, comprising major histocompatibility complex (MHC) class I and class II molecules, tetraspan proteins, CD26/dipeptidyl-peptidase IV, and A33 antigen, a molecule essentially restricted to the intestinal epithelium. Invariant chain, transferrin receptor, and Na-K-ATPase are not expressed on epithelial exosomes. In vivo, in mice, epithelial exosomes carrying MHC/ovalbumin peptide complexes induce specific immune responses when injected intraperitoneally. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells and enriched in epithelial exosomes, is found at the surface of cells entering mesenteric lymph nodes suggesting exosome migration from the epithelial layer to the gut associated lymphoid system. Taken together, intestinal epithelial exosomes released at the basolateral surface of enterocytes could be antigen-carrying structures constituting a link between luminal antigens and the local immune system and acting as sensors of the antigenic information present in the intestinal lumen.

Animals↗

PubMed2005

RNA degradation by the exosome is promoted by a nuclear polyadenylation complex.

The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Myocilin-associated exosomes in human ocular samples.

Mutations in myocilin result in ocular hypertension, likely due to decreased drainage of aqueous humor through the trabecular meshwork. Since less myocilin is found in the aqueous humor of those with disease-causing mutations, understanding myocilin's role in the aqueous humor is of clinical importance. Recently, myocilin was shown to exit cultured trabecular meshwork cells in association with shed vesicles called exosomes. To examine relevance of this finding in a physiological setting, the present study examined three different types of ocular samples for the presence of myocilin-associated exosomes. Using differential centrifugation steps, we found myocilin associated with exosomes isolated from effluent collected from human anterior segments in organ culture and aqueous humor obtained from human cadaveric eyes or from patients undergoing excisional surgery. Similar to results with cultured cells, myocilin associated predominately with exosomes in fresh samples, appeared mostly soluble at later times, and had biochemical properties (density of 1.13-1.19 g/ml in linear sucrose gradient) similar to those characteristics of exosomes. These data indicate that exosomes are present and may facilitate the transport of myocilin into the extracellular space of human ocular cells.

Aqueous Humor↗

PubMed2002

Protein-protein interactions between human exosome components support the assembly of RNase PH-type subunits into a six-membered PNPase-like ring.

The exosome is a complex of 3'-->5' exoribonucleases, which functions in a variety of cellular processes, all requiring the processing or degradation of RNA. Here we present a model for the assembly of the six human RNase PH-like exosome subunits into a hexameric ring structure. In part, this structure is on the basis of the evolutionarily related bacterial degradosome, the core of which consists of three copies of the PNPase protein, each containing two RNase PH domains. In our model three additional exosome subunits, which contain S1 RNA-binding domains, are positioned on the outer surface of this ring. Evidence for this model was obtained by the identification of protein-protein interactions between individual exosome subunits in a mammalian two-hybrid system. In addition, the results of co-immunoprecipitation assays indicate that at least two copies of hRrp4p and hRrp41p are associated with a single exosome, suggesting that at least two of these ring structures are present in this complex. Finally, the identification of a human gene encoding the putative human counterpart of the bacterial PNPase protein is described, which suggests that the exosome is not the eukaryotic equivalent of the bacterial degradosome, although they do share similar functional activities.

Amino Acid Sequence↗

PubMed2000

Musing on the structural organization of the exosome complex.

The exosome complex of 3'-->5' exoribonucleases functions in both the precise processing of 3' extended precursor molecules to mature stable RNAs and the complete degradation of other RNAs. Both processing and degradative activities of the exosome depend on additional cofactors, notably the putative RNA helicases Mtr4p and Ski2p. It is not known how these factors regulate exosome function or how the exosome distinguishes RNAs destined for processing events from substrates that are to be completely degraded. Here we review the available data concerning the modes of action of the exosome and relate these to possible structural arrangements for the complex. As no detailed structural data are yet available for the exosome complex, or any of its constituent enzymes, this discussion will rely heavily on rather speculative models.

Allosteric Regulation↗

PubMed2003

An exosome-like complex in Sulfolobus solfataricus.

We present the first experimental evidence for the existence of an exosome-like protein complex in Archaea. In Eukarya, the exosome is essential for many pathways of RNA processing and degradation. Co-immunoprecipitation with antibodies directed against the previously predicted Sulfolobus solfataricus orthologue of the exosome subunit ribosomal-RNA-processing protein 41 (Rrp41) led to the purification of a 250-kDa protein complex from S. solfataricus. Approximately half of the complex cosediments with ribosomal subunits. It comprises four previously predicted orthologues of the core exosome subunits from yeast (Rrp41, Rrp42, Rrp4 and Csl4 (cepl synthetic lethality 4; an RNA-binding protein and exosome sub-unit)), whereas other predicted subunits were not found. Surprisingly, the archaeal homologue of the bacterial DNA primase DnaG was tightly associated with the complex. This suggests an RNA-related function for the archaeal DnaG-like proteins. Comparison of experimental data from different organisms shows that the minimal core of the exosome consists of at least one phosphate-dependent ribonuclease PH homologue, and of Rrp4 and Csl4. Such a protein complex was probably present in the last common ancestor of Archaea and Eukarya.

Macromolecular Substances↗

PubMed1995

Hsp-70 is closely associated with the transferrin receptor in exosomes from maturing reticulocytes.

The presence of the heat shock protein (hsp-70) has been detected in exocytosed vesicles (which are named exosomes) from mammalian and avian immature red cells (i.e. reticulocytes) as well as from a differentiating avian erythroleukaemic cell line. The close, but non-covalent, association of hsp-70 with the transferrin receptor (TFR) in exosomes is demonstrated by: (1) the ability to cross-link hsp-70 to TFR; (2) the co-immunoprecipitation of hsp-70 and TFR with an antibody against TFR, and the co-immunoprecipitation of TFR and hsp-70 with antibody against hsp-70; and (3) the retention of TFR by hsp-70 bound to ATP-agarose and the simultaneous elution of both proteins by excess ATP. Semi-quantitative analysis of the relative efficiency of cross-linking of these proteins in exosomes versus plasma membranes shows that TFR in exosomes is preferentially bound to hsp-70. From an analysis of the relative amounts of hsp-70 and TFR regenerated from the cross-linked complex, the ratio of TFR monomer bound to hsp-70 is approximately 1.5 to 1. Given the presence of hsp-70 in exosomes from several species and its close association with TFR (the major protein lost during reticulocyte maturation) it is proposed that hsp-70 plays a role in exosome formation and/or release in immature red cells.

Animals↗

PubMed2001

Intestinal epithelial cells secrete exosome-like vesicles.

BACKGROUND & AIMS: Given the observations that intestinal epithelial cells (IECs) can present antigens to CD4(+) T lymphocytes and that professional antigen-presenting cells secrete exosomes (antigen-presenting vesicles), we hypothesized that IECs may secrete exosomes carrying molecules implicated in antigen presentation, which may be able to cross the basement membrane and convey immune information to noncontiguous immune cells. METHODS: Human IEC lines HT29-19A and T84-DRB1*0401/CIITA were grown on microporous filters. Release of exosomes under basal or inflammatory conditions was evaluated in conditioned apical and basolateral media after differential ultracentrifugations. Morphologic and biochemical characterization of exosomes was performed using immunoelectron microscopy, Western blotting, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS: The intestinal cell lines released 30-90-nm-diameter vesicles from the apical and basolateral sides, and this release was significantly increased in the presence of interferon gamma. MHC class I, MHC class II, CD63, CD26/dipeptidyl-peptidase IV, and A33 antigen were present in epithelial-derived exosomes. CONCLUSIONS; Human IEC lines secrete exosomes bearing accessory molecules that may be involved in antigen presentation. These data are consistent with a model in which IECs may influence antigen presentation in the mucosal or systemic immune system independent of direct cellular contact with effector cells.

Antigens, CD↗

PubMed2003

Evidence that HIV budding in primary macrophages occurs through the exosome release pathway.

Lipid rafts are specialized regions of cell membranes enriched in cholesterol and sphingolipids that are involved in immune activation and signaling. Studies in T-cells indicate that these membrane domains serve as sites for release of human immunodeficiency virus (HIV). By budding through lipid rafts in T-cells, HIV selectively incorporates raft markers and excludes non-raft proteins. This process has been well studied in T-cells, but it is unknown whether lipid rafts serve as budding sites for HIV in macrophages. Recently, we proposed a new model of retroviral biogenesis called the Trojan exosome hypothesis (Gould, S. J., Booth, A., and Hildreth, J. E. K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10592-10597). This model proposes that retroviruses coopt the existing cellular machinery for exosomal release. Here, we performed the first test designed to differentiate between the lipid raft hypothesis of retroviral biogenesis and the Trojan exosome hypothesis. Using macrophages, we examined the relative abundance of several host proteins on the cell surface, in lipid rafts, and on both HIV particles and exosomes derived from these cells. Our results show significant differences in the abundance of host proteins on the cell surface and in HIV. Moreover, our data demonstrate discordance in the abundance of some proteins in lipid rafts and in HIV. Finally, our data reveal a strong concordance between the host cell protein profile of exosomes and that of HIV. These results strongly support the Trojan exosome hypothesis and its prediction that retroviral budding represents exploitation of a pre-existing cellular pathway of intercellular vesicle trafficking.

Antigens, CD↗

PubMed2005

Diacylglycerol kinase alpha regulates the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes.

Fas ligand (FasL) mediates both apoptotic and inflammatory responses in the immune system. FasL function critically depends on the different forms of FasL; soluble Fas ligand lacking the transmembrane and cytoplasmic domains is a poor mediator of apoptosis, whereas full-length, membrane-associated FasL (mFasL) is pro-apoptotic. mFasL can be released from T lymphocytes, via the secretion of mFasL-bearing exosomes. mFasL in exosomes retains its activity in triggering Fas-dependent apoptosis, providing an alternative mechanism of cell death that does not necessarily imply cell-to-cell contact. Diacylglycerol kinase alpha (DGKalpha), a diacylglycerol (DAG)-consuming enzyme, is involved in the attenuation of DAG-derived responses initiated at the plasma membrane that lead to T lymphocyte activation. Here we studied the role of DGKalpha on activation-induced cell death on a T cell line and primary T lymphoblasts. The inhibition of DGKalpha increases the secretion of lethal exosomes bearing mFas ligand and subsequent apoptosis. On the contrary, the overactivation of the DGKalpha pathway inhibits exosome secretion and subsequent apoptosis. DGKalpha was found associated with the trans-Golgi network and late endosomal compartments. Our results support the hypothesis that the DGKalpha effect on apoptosis occurs via the regulation of the release of lethal exosomes by the exocytic pathway, and point out that the spatial orchestration of the different pools of DAG (plasma membrane and Golgi membranes) by DGKalpha is crucial for the control of cell activation and also for the regulation of the secretion of lethal exosomes, which in turn controls cell death.

Antigens, CD↗

PubMed2005

Exosome-dependent trafficking of HSP70: a novel secretory pathway for cellular stress proteins.

The heat shock proteins (HSPs) are a family of intracellular proteins found in all eukaryotes and prokaryotes. Their functions are well characterized and are central to maintaining cellular homeostasis and in promoting cell survival in response to stressful cellular conditions. However, several studies provide evidence that specific members of the HSP family might be secreted via an unidentified exocytotic pathway. Here we show that exosomes, small membrane vesicles that are secreted by numerous cell types, contribute to the release of HSP70 from human peripheral blood mononuclear cells (PBMCs) in both basal and stress-induced (heat shock at 40 or 43 degrees C for 1 h) states. HSP70 release from PBMCs is independent of the common secretory pathway because Brefeldin A, an inhibitor of the classical protein transport pathway, did not block HSP70 release. Furthermore, we show that HSP70 release from PBMCs does not occur via a lipid raft-dependent pathway, because treatment with methyl-beta-cyclodextrin, a raft-disrupting drug, had no affect on HSP70 release. To examine whether exosomes contributed to HSP70 release from PBMCs, exosomes were purified from PBMC cultures, and exosomal number and HSP70 content were determined. We demonstrate that although heat shock does not influence the exosomal secretory rate, the HSP70 content of exosomes isolated from heat shocked PBMCs is significantly higher than control. These data identify a novel secretory pathway by which HSP70 can be actively released from cells in both the basal and stress-induced state.

3T3-L1 Cells↗

PubMed2006

Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane.

Exosomes are secreted, single membrane organelles of approximately 100 nm diameter. Their biogenesis is typically thought to occur in a two-step process involving (1) outward vesicle budding at limiting membranes of endosomes (outward = away from the cytoplasm), which generates intralumenal vesicles, followed by (2) endosome-plasma membrane fusion, which releases these internal vesicles into the extracellular milieu as exosomes. In this study, we present evidence that certain cells, including Jurkat T cells, possess discrete domains of plasma membrane that are enriched for exosomal and endosomal proteins, retain the endosomal property of outward vesicle budding, and serve as sites of immediate exosome biogenesis. It has been hypothesized that retroviruses utilize the exosome biogenesis pathway for the formation of infectious particles. In support of this, we find that Jurkat T cells direct the key budding factor of HIV, HIV Gag, to these endosome-like domains of plasma membrane and secrete HIV Gag from the cell in exosomes.

Antigens, CD↗

PubMed2005

Exosomal-like vesicles are present in human blood plasma.

Exosomes are small membrane vesicles (50-90 nm in diameter) secreted by most hematopoietic cells. We provide here the first evidence for the presence of exosomes in vivo, in the blood. Plasma samples of all healthy donors tested (n = 15) contain vesicles that are similar in shape, size and density to the previously described exosomes. They were clearly identified by electron microscopy after isolation by differential ultracentrifugation or immunoisolation with CD63-coated latex beads. We performed their biochemical characterization by western blot analysis and by flow cytometry after vesicle adsorption onto latex beads using a panel of mAbs. We observed that these plasma-derived vesicles contain tetraspanin molecules such as CD63, CD9, CD81 as well as class I and class II MHC molecules and Lamp-2 (i.e. proteins that are known to be enriched in exosomes). In addition, these vesicles float on sucrose gradient at a density similar to exosomes. Our results demonstrate that blood is a physiological fluid for exosome circulation in the body, suggesting their role in cell-cell or organ-organ communications as carriers for molecules that need to reach distant cell targets.

Adult↗

PubMed2000

Degradation of ribosomal RNA precursors by the exosome.

The yeast exosome is a complex of 3'-->5' exonucleases involved in RNA processing and degradation. All 11 known components of the exosome are required during 3' end processing of the 5.8S rRNA. Here we report that depletion of each of the individual components inhibits the early pre-rRNA cleavages at sites A(0), A(1), A(2)and A(3), reducing the levels of the 32S, 20S, 27SA(2)and 27SA(3)pre-rRNAs. The levels of the 27SB pre-rRNAs were also reduced. Consequently, both the 18S and 25S rRNAs were depleted. Since none of these processing steps involves 3'-->5' exonuclease activities, the requirement for the exosome is probably indirect. Correct assembly of trans -acting factors with the pre-ribosomes may be monitored by a quality control system that inhibits pre-rRNA processing. The exosome itself degrades aberrant pre-rRNAs that arise from such inhibition. Exosome mutants stabilize truncated versions of the 23S, 21S and A(2)-C(2)RNAs, none of which are observed in wild-type cells. The putative helicase Dob1p, which functions as a cofactor for the exosome in pre-rRNA processing, also functions in these pre-rRNA degradation activities.

Hydrolysis↗

PubMed2004

Adhesion and signaling by B cell-derived exosomes: the role of integrins.

Exosomes are nanometer-sized vesicles secreted by various cells, with potentially diverse roles in physiology. Although emphasis has been placed on their involvement in immune modulation, their potential for more wide-ranging biological effects has not been appreciated. A common exosome feature is the expression of adhesion molecules, which include the integrin family. We have for the first time addressed the possible function of B cell-derived exosome-integrins by examining adhesive interactions of exosomes (immobilized onto beads) with extracellular matrix (ECM) components and cytokine-treated fibroblasts. Integrin (beta1 and beta2) expression was demonstrated by Western blotting and flow cytometry. Binding studies (with blocking antibodies) demonstrated their function in adhesion to collagen-I, fibronectin, and tumor necrosis factor (TNF)-alpha-activated fibroblasts. Exosome adhesion to TNF-alpha-activated fibroblasts also triggered integrin-dependent changes in cytosolic calcium, measured by single cell imaging. Thus, B cell-derived exosomes express functional integrins, which are capable of mediating anchorage to ECM and cell-surface adhesion molecules, and may be a novel mode of delivering adhesion signals at distances beyond that of direct cell-cell contact during inflammation.

B-Lymphocytes↗

PubMedDate not supplied

Exosomes from plasmacytoma cells as a tumor vaccine.

Exosomes are membrane-bound vesicles derived from multivesicular bodies that are externalized by cells through fusion with the plasma membrane. Exosomes have been implicated in cell-to-cell signaling, and those derived from immunologic cells may be involved in both direct and cross-presentation of antigens to T cells. The research presented here evaluated their efficacy as a prophylactic cancer vaccine in a mouse plasmacytoma model. Plasmacytoma cells were shown to release exosomes in vitro, and vaccination with a single dose (5 microg) of exosome protein protected 80% of mice against challenge with wild-type tumors. Protection could be linked to the immune system since vaccinated mice generated specific cytotoxic T lymphocytes, the effects were not seen in SCID mice, and immunity was tumor-specific. Several proteins involved in immunity, including two potential tumor antigens (P1A and intracisternal A particle protein) as well as Hsp70, were demonstrated to be present in exosomes. The authors conclude that exosomes can induce tumor-specific immunity and prevent tumor development and are a potential strategy for future therapeutic tumor vaccination.

Animals↗