Mature dendritic cells secrete exosomes with strong ability to induce antigen-specific effector immune responses

Exosomes are secreted vesicles formed in late endocytic compartments. Immature dendritic cells (DCs) secrete exosomes which transfer functional MHC-peptide complexes to other DCs. Since immature and mature DCs induce different functional T cell responses (i.e., tolerance versus priming), we asked whether DC maturation also influenced the priming abilities of their exosomes. We show that immature and mature murine DCs secrete morphologically similar exosomes. Extensive proteomic analysis of the two exosome populations showed identical overall protein composition, and provided an exhaustive image of the protein composition of DC-derived exosomes. By quantitative analysis, however, exosomes from mature DCs proved enriched in MHC class II, B7.2, ICAM-1, and depleted in MFG-E8, as compared to immature exosomes. In functional T cell stimulation assays, exosomes secreted by mature DCs were 50- to 100-fold more potent than exosomes from immature DCs, both in vitro and in vivo. MHC class II and ICAM-1 were necessary for the increased immune activity of exosomes secreted by mature DCs. Therefore, changes in protein composition and priming abilities of exosomes reflect the maturation signals received by DCs.     

ICAM-1 on exosomes from mature dendritic cells is critical for efficient naive T-cell priming

Exosomes are secreted vesicles formed in late endocytic compartments. Immature dendritic cells (DCs) secrete exosomes, which transfer functional major histocompatibility complex (MHC)-peptide complexes to other DCs. Since immature and mature DCs induce different functional T-cell responses (ie, tolerance versus priming), we asked whether DC maturation also influenced the priming abilities of their exosomes. We show that exosomes secreted by lipopolysaccharide (LPS)-treated mature DCs are 50- to 100-fold more potent to induce antigen-specific T-cell activation in vitro than exosomes from immature DCs. In vitro, exosomes from mature DCs transfer to B lymphocytes the ability to prime naive T cells. In vivo, only mature exosomes trigger effector T-cell responses, leading to fast skin graft rejection. Proteomic and biochemical analyses revealed that mature exosomes are enriched in MHC class II, B7.2, intercellular adhesion molecule 1 (ICAM-1), and bear little milk-fat globule-epidermal growth factor-factor VIII (MFG-E8) as compared with immature exosomes. Functional analysis using DC-derived exosomes from knock-out mice showed that MHC class II and ICAM-1 are required for mature exosomes to prime naive T cells, whereas B7.2 and MFG-E8 are dispensable. Therefore, changes in protein composition and priming abilities of exosomes reflect the maturation signals received by DCs.     

Accumulation of MFG-E8/lactadherin on exosomes from immature dendritic cells

Exosomes are vesicles of endocytic origin secreted spontaneously by dendritic cells (DCs). We have shown previously that exosomes can transfer antigen or MHC-peptide complexes between DCs, thus potentially amplifying the immune response. We had also identified milk fat globule EGF/factor VIII (MFG-E8), also called lactadherin, as one of the major exosomal proteins. MFG-E8 has two domains: an Arg-Gly-Asp sequence that binds integrins alphavbeta3 and alphavbeta5 (expressed by human DCs and macrophages) and a phosphatidyl-serine (PS) binding sequence through which it associates to PS-containing membranes (among which exosomes). MFG-E8 is thus a good candidate molecule to address exosomes to DCs. Here, we show that MFG-E8 is expressed by immature bone-marrow-derived DCs (BMDCs) and secreted in association with exosomes in vitro. We have generated mice expressing an inactive form of MFG-E8, fused to beta-galactosidase. Analyzing these mice, we demonstrate that MFG-E8 is expressed in vivo in splenic DCs. In a mouse DC-dependent, antigen-specific, CD4 T cell-stimulation assay, exosomes produced by MFG-E8-deficient BMDCs were barely less efficient than exosomes bearing MFG-E8. We conclude that MFG-E8 is efficiently targeted to exosomes but is not essential to address exosomes to mouse BMDCs. Involvement of MFG-E8/lactadherin in exosome targeting to other DC subpopulations, or to human DCs, is still possible.     

Endocytosis, intracellular sorting, and processing of exosomes by dendritic cells

Exosomes are nanovesicles released by leukocytes and epithelial cells. Although their function remains enigmatic, exosomes are a source of antigen and transfer functional major histocompatibility complex (MHC)-I/peptide complexes to dendritic cells (DCs) for CD8(+) T-cell activation. Here we demonstrate that exosomes also are internalized and processed by immature DCs for presentation to CD4(+) T cells. Endocytosed exosomes are sorted into the endocytic compartment of DCs for processing, followed by loading of exosome-derived peptides in MHC-II molecules for presentation to CD4(+) T cells. Targeting of exosomes to DCs is mediated via milk fat globule (MFG)-E8/lactadherin, CD11a, CD54, phosphatidylserine, and the tetraspanins CD9 and CD81 on the exosome and alpha(v)/beta(3) integrin, and CD11a and CD54 on the DCs. Circulating exosomes are internalized by DCs and specialized phagocytes of the spleen and by hepatic Kupffer cells. Internalization of blood-borne allogeneic exosomes by splenic DCs does not affect DC maturation and is followed by loading of the exosome-derived allopeptide IEalpha(52-68) in IA(b) by host CD8alpha(+) DCs for presentation to CD4(+) T cells. These data imply that exosomes present in circulation or extracellular fluids constitute an alternative source of self- or allopeptides for DCs during maintenance of peripheral tolerance or initiation of the indirect pathway of allorecognition in transplantation.     

胃腺癌BGC-823细胞总RNA转染人树突状细胞分泌exosomes的抗肿瘤效应研究

目的研究人外周血树突状细胞（DC）体外经人胃腺癌BGC-823细胞系总RNA转染后,提取培养上清液中DC分泌的外泌体（exosomes）,诱导出特异性抗胃癌效应。方法分离外周血单核细胞,经GM-CSF、IL-4培养5d后,获得未成熟DC（imDC）;体外以脂质体转染BGC-823细胞总RNA。第7天收集上清,利用超速离心法提取exosomes。分别将DC以及exosomes与T淋巴细胞共培养3d,获得细胞毒T淋巴细胞（CTL）,检测CTL对BGC-823细胞的杀伤作用。结果与未转染组相比,转染BGC-823总RNADC来源的exosomes明显促进T细胞对BGC-823的杀伤活性（P〈0．05）。结论应用BGC-823总RNA转染DC分泌exosomes能够诱导出强烈的抗肿瘤免疫反应。     

肺癌细胞裂解物负载对树突状细胞分泌的exosome诱导抗肿瘤作用的影响

目的为制备高效的胞外体（exosome）肿瘤疫苗提供理论依据。方法用细胞因子诱导培养树突状细胞（DC）,将肺癌细胞裂解物负载DC,提取exosome用exosome活化T细胞（负载组）,以未负载DC的exosome（未负载组）及肺癌细胞裂解物负载DC（DC组）活化的T细胞为对照,MTr法检测三组肺癌细胞的杀伤率。结果exosome中有HSP70、HLA及CEA表达。活化T细胞／肺癌细胞为25：1、10：1、5：1时负载组杀伤率均明显高于未负载组及DC组（P均〈0．05）。结论肺癌细胞裂解物负载能增强DC分泌的exosome诱导的抗肿瘤作用;本研究为制备高效的exosome肿瘤疫苗提供了理论依据。     

Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in a src-dependent fashion

Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length (P = 0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release (P < 0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes (P < 0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment (P < 0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment.     

Presentation of proteins encapsulated in sterically stabilized liposomes by dendritic cells initiates CD8(+) T-cell responses in vivo

Liposomes have been proposed as a vehicle to deliver proteins to antigen-presenting cells (APC), such as dendritic cells (DC), to stimulate strong T cell-mediated immune responses. Unfortunately, because of their instability in vivo and their rapid uptake by cells of the mononuclear phagocyte system on intravenous administration, most types of conventional liposomes lack clinical applicability. In contrast, sterically stabilized liposomes (SL) have increased in vivo stability. It is shown that both immature and mature DC take up SL into neutral or mildly acidic compartments distinct from endocytic vacuoles. These DC presented SL-encapsulated protein to both CD4(+) and CD8(+) T cells in vitro. Although CD4(+) T-cell responses were comparable to those induced by soluble protein, CD8(+) T-cell proliferation was up to 300-fold stronger when DC had been pulsed with SL-encapsulated ovalbumin. DC processed SL-encapsulated antigen through a TAP-dependent mechanism. Immunization of mice with SL-encapsulated ovalbumin led to antigen presentation by DC in vivo and stimulated greater CD8(+) T-cell responses than immunization with soluble protein or with conventional or positively charged liposomes carrying ovalbumin. Therefore, the application of SL-encapsulated antigens offers a novel effective, safe vaccine approach if a combination of CD8(+) and CD4(+) T-cell responses is desired (ie, in anti-viral or anti-tumor immunity).     

Identification of progenitor cells in long-term spleen stromal cultures that produce immature dendritic cells

Dendritic cells (DC) are produced continuously by a unique, long-term culture (LTC) system in which hemopoiesis is supported by a splenic stromal cell layer in the absence of added growth factors. Flow cytometric analysis reveals the production of two distinct cell subsets. The more predominant large-cell subset resembles highly endocytic DC that are large, granular, and possess membrane extensions. They also express high levels of the DC markers CD11c, CD11b, DEC-205, and CD80 on their cell surface. They do not resemble mature DC because they express low levels of MHC type II and CD86 molecules, as well as c-kit and Fc receptor (FcR). These are known characteristics of immature DC. Small cells are smaller and less granular than large cells, with negative to low expression of CD11c, DEC-205, and CD86. A majority of small cells express varying levels of CD11b and CD80. Subpopulations of small cells express low levels of c-kit, FcR, and MHC type II, and only a 20% subpopulation is weakly endocytic. Upon transfer to an irradiated stromal layer, cells within the small subset proliferate and differentiate to resemble the large cells in size, complexity, membrane extensions, and CD11c and CD86 expression. The two cell subsets produced in LTC are developmentally linked, with the heterogeneous small-cell subset containing progenitors of the larger homogeneous, immature DC subset. LTC represent a valuable model system for studying DC development from hemopoietic progenitors.     

Capture and transfer of HIV-1 particles by mature dendritic cells converges with the exosome-dissemination pathway

Exosomes are secreted cellular vesicles that can be internalized by dendritic cells (DCs), contributing to antigen-specific naive CD4(+) T-cell activation. Here, we demonstrate that human immunodeficiency virus type 1 (HIV-1) can exploit this exosome antigen-dissemination pathway intrinsic to mature DCs (mDCs) for mediating trans-infection of T lymphocytes. Capture of HIV-1, HIV-1 Gag-enhanced green fluorescent protein (eGFP) viral-like particles (VLPs), and exosomes by DCs was up-regulated upon maturation, resulting in localization within a CD81(+) compartment. Uptake of VLPs or exosomes could be inhibited by a challenge with either particle, suggesting that the expression of common determinant(s) on VLP or exosome surface is necessary for internalization by mDCs. Capture by mDCs was insensitive to proteolysis but blocked when virus, VLPs, or exosomes were produced from cells treated with sphingolipid biosynthesis inhibitors that modulate the lipid composition of the budding particles. Finally, VLPs and exosomes captured by mDCs were transmitted to T lymphocytes in an envelope glycoprotein-independent manner, underscoring a new potential viral dissemination pathway.     

Exosomes with major histocompatibility complex class II and co-stimulatory molecules are present in human BAL fluid.

Exosomes are 30-100 nm diameter vesicles formed by inward budding of endosomal compartments and are produced by several cell types, including T-cells, B-cells and dendritic cells (DC)s. Exosomes from DCs express major histocompatibility complexes (MHC) class I and II, and co-stimulatory molecules on their surface, and can induce antigen-specific activation of T-cells. The aims of the present study were to investigate for the presence of exosomes in bronchoalveolar lavage fluid (BALF) from healthy individuals, and to establish if these exosomes bear MHC and co-stimulatory molecules. The authors analysed BALF taken from seven healthy volunteers and used exosomes from monocyte-derived DC (MDDC) cultures as a reference. After ultracentrifugation, exosomes were bound to anti-MHC class II coated magnetic beads and analysed by flow cytometry and electron microscopy. The authors report for the first time that exosomes are present in BALF. These exosomes are similar to MDDC derived exosomes as they express MHC class I and II, CD54, CD63 and the co-stimulatory molecule CD86. The results demonstrate that exosomes are present in the lung, and since they contain both major histocompatibility complex and co-stimulatory molecules it is likely that they are derived from antigen presenting cells and might have a regulatory role in local immune defence.     

Interferon-alpha and -beta inhibit the in vitro differentiation of immunocompetent human dendritic cells from CD14(+) precursors

Dendritic cell (DC) precursors and immature DC reside in epithelium where they encounter pathogens and cytokines, which stimulate their differentiation. We hypothesized that type-I interferons (IFN-alpha and -beta), cytokines that are produced early in the innate immune response against viruses and some bacteria, may influence DC differentiation and function. To examine this possibility, we used an in vitro model of DC differentiation in which initial culture of human CD14(+) monocytes with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 generates immature DC, and subsequent culture with tumor necrosis factor (TNF)-alpha drives the final development into mature DC. We found in this model that IFN-alpha/beta, added from the initiation of the culture on, significantly reduced the survival and altered the morphology and differentiation of DC. TNF-alpha-dependent maturation of IFN-beta-treated immature DC led to cells with reduced expression of CD1a, CD40, CD54, and CD80 when compared with mature DC controls. IFN-alpha/beta-treated DC further had a reduced capacity to induce naive Th-cell proliferation through allostimulation or anti-CD3 monoclonal antibody stimulation. In addition, IFN-alpha/beta-treated DC secreted less IL-12 upon stimulation with Staphylococcus aureus Cowan strain or with CD4(+) T cells, and this decrease correlated directly with their inability to support CD4(+) T-cell secretion of IFN-gamma, even though T-cell lymphotoxin production was unaffected. These findings indicate that type-I IFNs can influence the generation of acquired immune responses by modifying T-helper cell differentiation through the regulation of DC differentiation and function.     

Intestinal epithelial exosomes carry MHC class II/peptides able to inform the immune system in mice

BACKGROUND: Intestinal epithelial cells secrete exosome-like vesicles. The aim of this study was to characterise murine intestinal epithelial exosomes and to analyse their capacity to inform the immune system in vivo in mice. METHODS: Epithelial exosomes were obtained from the murine epithelial cell line MODE K incubated in the presence or absence of interferon gamma (IFN-gamma) together with pepsin/trypsin ovalbumin hydrolysate (hOVA) to mimic luminal digestion. Exosomes isolated from MODE K conditioned media (EXO-hOVA and EXO-hOVA-IFN) were characterised by western blot, peptide mapping, and mass spectrometry. They were injected intraperitoneally to C3H/HeN mice to test their immunocompetence. RESULTS: MODE K epithelial exosomes displayed major histocompatibility complex (MHC) class I and class II (upregulated by IFN-gamma) molecules and tetraspan proteins (CD9, CD81, CD82) potentially involved in the binding to target cells. A33 antigen, an Ig-like molecule highly specific for intestinal epithelial cells, was enriched in exosomes and was also found in mice mesenteric lymph nodes, suggesting exosome migration towards the gut associated lymphoid tissues. Intraperitoneal injection of EXO-hOVA or EXO-hOVA-IFN did not induce humoral or cellular tolerance to OVA in mice. In contrast, exosomes obtained after incubation with IFN-gamma (EXO-hOVA-IFN), bearing abundant MHC class II/OVA complexes, induced a specific humoral immune response. CONCLUSIONS: Epithelial exosomes are antigen presenting vesicles bearing MHC class II/peptide complexes that prime for an immunogenic rather than tolerogenic response in the context of a systemic challenge. In the intestine, both the mucosal microenvironment and local effector cells are probably key players in determining the outcome of the immune response to exosome derived epitopes.     

Exosome Biogenesis,Regulation, and Function in Viral Infection

Exosomes are extracellular vesicles released upon fusion of multivesicular bodies (MVBs) with the cellular plasma membrane. They originate as intraluminal vesicles (ILVs) during the process of MVB formation. Exosomes were shown to contain selectively sorted functional proteins, lipids, and RNAs, mediating cell-to-cell communications and hence playing a role in the physiology of the healthy and diseased organism. Challenges in the field include the identification of mechanisms sustaining packaging of membrane-bound and soluble material to these vesicles and the understanding of the underlying processes directing MVBs for degradation or fusion with the plasma membrane. The investigation into the formation and roles of exosomes in viral infection is in its early years. Although still controversial, exosomes can, in principle, incorporate any functional factor, provided they have an appropriate sorting signal, and thus are prone to viral exploitation. This review initially focuses on the composition and biogenesis of exosomes. It then explores the regulatory mechanisms underlying their biogenesis. Exosomes are part of the endocytic system, which is tightly regulated and able to respond to several stimuli that lead to alterations in the composition of its sub-compartments. We discuss the current knowledge of how these changes affect exosomal release. We then summarize how different viruses exploit specific proteins of endocytic sub-compartments and speculate that it could interfere with exosome function, although no direct link between viral usage of the endocytic system and exosome release has yet been reported. Many recent reports have ascribed functions to exosomes released from cells infected with a variety of animal viruses, including viral spread, host immunity, and manipulation of the microenvironment, which are discussed. Given the ever-growing roles and importance of exosomes in viral infections, understanding what regulates their composition and levels, and defining their functions will ultimately provide additional insights into the virulence and persistence of infections.     

Association of citrullinated proteins with synovial exosomes

OBJECTIVE: In addition to releasing proteins and mediators, cells also release membrane vesicles (exosomes and apoptotic blebs) into the extracellular environment. Apoptotic blebs contain multiple autoantigens, but few data are available concerning the protein content of exosomes. Exosomes are formed during an immune response and can directly stimulate T cells or bind to dendritic cells. The aim of this study was to identify the nature of synovial exosomes from patients with different rheumatic diseases and to examine their potential autoantigenic content, which may be involved in the induction of an autoimmune response. METHODS: Synovial exosomes from patients with rheumatoid arthritis (RA), patients with reactive arthritis, and patients with osteoarthritis were purified, analyzed by electron microscopy, and labeled with immunogold to detect IgG and IgM molecules. Autoantigen content was identified by 2-dimensional electrophoresis-immunoblotting and subsequent mass spectrometry. In order to investigate the presence of citrullinated proteins, immunoblotting with anticitrulline antibodies was performed. RESULTS: Citrullinated proteins were observed in all exosome preparations, in contrast to other autoantigenic proteins (e.g., BiP and heterogeneous nuclear RNP A2) that were previously observed in RA and other autoimmune diseases. These citrullinated proteins included the fibrin alpha-chain fragment, fibrin beta-chain, fibrinogen beta-chain precursor, fibrinogen D fragment, and the Sp alpha (CD5 antigen-like protein) receptor. Purification of synovial exosomes led to the detection of citrullinated fibrinogen and citrullinated Sp alpha associated with IgM and IgG. CONCLUSION: Synovial exosomes contain citrullinated proteins, which are known to be autoantigens in RA. Although immune mechanisms in which exosomes carry citrullinated peptides could play an important role in the induction and distribution of citrullinated proteins, there must be a specific recognition of these proteins that is unique to the RA immune system.     

Prions and exosomes: from PrPc trafficking to PrPsc propagation

Exosomes are membrane vesicles released into the extracellular environment upon exocytic fusion of multivesicular endosomes with the cell surface. Exosome secretion can be used by cells to eject molecules targeted to intraluminal vesicles of multivesicular bodies, but particular cell types may exploit exosomes as intercellular communication devices for transfer of proteins and lipids among cells. The glycosylphosphatyidylinositol-linked prion protein (PrP) in both its normal (PrPc) and scrappie (PrPsc) conformation is associated with exosomes. Targeting of exosomes containing the normal cellular PrP could confer susceptibility of cells that do not express PrP to prion multiplication. Furthermore, exosomes bearing proteinase-K resistant PrPsc are infectious, suggesting a model in which exosomes secreted by infected cells could serve as vehicles for propagation of prions. Thus, cells may exploit the nature of endosome-derived exosomes to communicate with each other in normal and pathological situations, providing for a novel route of cell-to-cell communication and therefore of pathogen transmission. These findings open the possibility that methods to interfere with trafficking of such unconventional pathogens could be envisioned from insights on the mechanisms involved in exosome formation, secretion and targeting.     

Specificity and efficacy of dendritic cell-based vaccination against tuberculosis with complex mycobacterial antigens in a mouse model

Dendritic cells (DC) likely play important and unique roles in the generation of protective immunity to mycobacteria. In order to clarify their contributions, bone marrow-derived DC loaded with Mycobacterium tuberculosis sonicate antigens were used to stimulate T cell proliferation both in vitro and in vivo and to vaccinate C57BL/6 mice against subsequent challenge with virulent mycobacteria. Antigen-pulsed DC developed in fetal calf serum (FCS-DC), but not DC developed in normal mouse serum (NMS-DC), stimulated significant proliferation of both na?ve and immune T cells in vitro. The difference between cell populations developed in FCS and NMS in the content of CD11c(+) cells and in production of key cytokines indicated that NMS is less supportive for the development of activated DC. However, following adoptive transfer of a single dose of antigen-pulsed DC into naive recipients, NMS-DC induced T cells that proliferated in response to mycobacterial antigen, whereas FCS-DC stimulated strong non-specific proliferation. Vaccination with two doses of antigen-pulsed NMS-DC by the subcutaneous route induced significant protection against intravenous challenge with a moderate dose of virulent M. tuberculosis. DC-vaccinated mice exhibited significant reductions in bacillary loads in the lungs and spleens, and markedly reduced lung pathology. Three doses of antigen-pulsed NMS-DC induced a significant increase in survival time following high dose challenge, which correlated with a significant increase in IFN-gamma-producing cells in both lung and lymphoid tissues, as assessed by the ELISPOT assay. Taken together, these results indicate that DC play a critical role in the induction of protective resistance against virulent mycobacterial challenge accompanied by the development of antigen-reactive, IFN-gamma-producing T cells, and that their antigenic specificity is influenced by the culture conditions under which the DC are developed.     

The potential of exosomes in diagnosis and treatment of inborn errors of metabolism

Extracellular vesicles, in particular exosomes, have gained much attention as potent mediators of intercellular signaling. Exosomes are 50–130 nm intraluminal vesicles of multivesicular bodies (MVB) that are secreted into the extracellular environment upon fusion of MVB with the plasma membrane. Current research on exosomes focuses on their biogenesis, including specific sorting mechanisms, their potential to transfer proteins and RNA from their cells of origin to target cells, specific methods of vesicle isolation, and their possible application as diagnostic and therapeutic devices. Exosomes are vesicles of endocytic origin that contain a portion of the cytoplasm. Their molecular components represent the composition and thereby the physiological state of the cells from which they originate. In this review, we recapitulate the discovery of exosomes and the subsequent expansion of exosome research into a variety of different areas of interest, with a specific focus on how exosomes could prove to be invaluable for both diagnostic and therapeutic applications within the research field of inborn errors of metabolism.