Different types of in vitro generated human monocyte-derived dendritic cells release exosomes with distinct phenotypes (original) (raw)
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Indirect activation of naïve CD4+ T cells by dendritic cell–derived exosomes
Nature Immunology, 2002
Dendritic cells (DCs) secrete vesicles of endosomal origin, called exosomes, that bear major histocompatibility complex (MHC) and T cell costimulatory molecules. Here, we found that injection of antigen-or peptide-bearing exosomes induced antigen-specific naïve CD4 + T cell activation in vivo. In vitro, exosomes did not induce antigen-dependent T cell stimulation unless mature CD8α -DCs were also present in the cultures. These mature DCs could be MHC class II-negative, but had to bear CD80 and CD86. Therefore, in addition to carrying antigen, exosomes promote the exchange of functional peptide-MHC complexes between DCs. Such a mechanism may increase the number of DCs bearing a particular peptide, thus amplifying the initiation of primary adaptive immune responses.
Production and characterization of clinical grade exosomes derived from dendritic cells
Journal of Immunological Methods, 2002
We describe methods for the production, purification, and characterization of clinical grade (cGMP) exosomes derived from antigen presenting cells (APCs). Exosomes have been shown to have immunotherapeutic properties through their presentation of biologically relevant antigens [Nat. Med. 4 (1998) 594] and are being developed as an alternative to cellular therapies. Exosomes are 50 -90-nm-diameter vesicles secreted from multivesicular bodies (MVBs) found in a variety of both hematopoietic and tumor cells. These particles contain antigen presenting molecules (MHC class I, MHC class II, and CD1), tetraspan molecules (CD9, CD63, CD81), adhesion molecules (CD11b and CD54), and costimulatory molecules (CD86); hence, providing them the necessary machinery required for generating a potent immune response [J. 7309]. Exosomes from monocyte-derived dendritic cells (MDDCs) were rapidly purified (e.g. 4 -6 h of a 2 -3 l culture) based on their unique size and density. Ultrafiltration of the clarified supernatant through a 500-kDa membrane and ultracentrifugation into a 30% sucrose/ deuterium oxide (D 2 O) (98%) cushion (density 1.210 g/cm 3 ) reduced the volume and protein concentration approximately 200and 1000-fold, respectively. The percentage recovery of exosomes ranged from 40% to 50% based on the exosome MHC class II concentration of the starting clarified supernatant. This methodology was extended to a miniscale process with comparable results. Conversely, the classical differential centrifugation technique is a more lengthy and variable process resulting in exosomes being contaminated with media proteins and containing only 5 -25% of the starting exosome MHC class II concentration; hence, making it difficult for their use in clinical development. Lastly, we developed the following quality control assays to standardize the exosome vaccine: quantity (concentration of MHC class II) and protein characterization (FACS). The combination of a rapid and reproducible purification method and quality control assays for exosomes has allowed for its evaluation as a cancer vaccine in clinical trials [Proc. Am. Soc. Oncol. 21 (2002) 11a]. : S 0 0 2 2 -1 7 5 9 ( 0 2 ) 0 0 3 3 0 -7
Exosomes bearing HLA‐DR1 molecules need dendritic cells to efficiently stimulate specific T cells
International …, 2002
Exosomes are small vesicles (60±100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen-presenting cells (APC), such as B lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC-derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of speci®c T cells by MHC class II-bearing exosomes in vitro. The rat mast cell line, RBL-2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide [HLA-DR1±hemagglutinin (HA)]. These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross-linking. Exosomes bearing DR1±HA(306±318) complexes activated HA/DR1-speci®c T cells only weakly, whereas the cross-linking of such exosomes to latex beads increased stimulation of speci®c T cells. By contrast, the incubation of free exosomes with DC resulted in the highly ef®cient stimulation of speci®c T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for ef®cient T cell activation.
Cellular & molecular immunology, 2011
T cells secrete bioactive exosomes (EXO), but the potential immunoregulatory effect of T-cell EXO is largely unknown. In this study, we generated activated ovalbumin (OVA)-specific CD4(+) T cells in vitro via coculture of OVA-pulsed dendritic cells (DC(OVA)) with naive CD4(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTII mice. CD4(+) T-cell EXO were then purified from the CD4(+) T-cell culture supernatants by differential ultracentrifugation. CD4(+) T-cell EXO exhibited the 'saucer' shape that is characteristic of EXO with a diameter between 50 and 100 nm, as assessed by electron microscopy, and contained the EXO-associated proteins LAMP-1, TCR and lymphocyte function associated antigen-1 (LFA-1), as determined by western blot. Flow cytometric analysis showed that CD4(+) T-cell EXO expressed CD4(+) T-cell markers (CD4, TCR, LFA-1, CD25 and Fas ligand), but to a lesser extent than CD4(+) T cells. We demonstrated that DC(OVA) took up CD4(+) T-cell EXO...
Dendritic cell derived-exosomes: biology and clinical implementations
Journal of Leukocyte Biology, 2006
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. J. Leukoc. Biol. 80: 000 -000; 2006.
Exosomes as Immunotheranostic Nanoparticles
Clinical Therapeutics, 2014
Background: Exosomes are small biological membrane vesicles that measure 30 to 100 nm in diameter. They are involved in a wide array of biological activities, such as cell-cell communication, signal transduction, transport of genetic materials, and modulation of immune response. Evidence indicates that they can be used as not only therapeutic agents targeted against disease but also diagnostic biomarkers for pathologic conditions. Objective: In this review, we endeavor to present exosomes as immunologic agents that can be used as pioneering cancer vaccines to prime the immune system and explicate their therapeutic and diagnostic capabilities. Methods: An extensive literature search for studies that involved the use of exosomes as immunotheranostic nanoparticles was conducted using PubMed, ISI Web of Knowledge, and Google Scholar. Clinical trials that involved exosomes were also compiled by searching the clinicaltrials.gov database. Results: In its therapeutic facet of application, exosomes can be used as vehicles for drug or gene delivery. These biological vesicles have been found to have excellent host biodistribution and biocompatibility, issues often presented with gene delivery vehicles. Diagnostically, exosomes may prove to be useful biomarkers that are able to surpass current setbacks of modern diagnostic testing, which include invasive methods. Finally, current evidence has implied that the use of exosomes could form the basis for the development of future cell-free cancer vaccines. Conclusion: Exosomes have numerous functions, and their double-edged features make the scope of their clinical applications, as both a diagnostic and therapeutic tool, immense.
Journal of Extracellular Vesicles, 2013
Background: In 2001, it was postulated that tumour-derived exosomes could be a potent source of tumourassociated antigens (TAA). Since then, much knowledge is gained on their role in tumorigenesis but only very recently tumour-derived exosomes were used in dendritic cell (DC)-based immunotherapy. For this, DCs were cultured ex-vivo and loaded with exosomes derived from immunogenic tumours such as melanoma or glioma and re-administrated to induce anti-tumour responses in primary and metastatic tumour mouse models. In contrast, malignant mesothelioma (MM) is a non-immunogenic tumour and because only a few mesothelioma-specific TAA are known to date, we investigated whether mesothelioma-derived exosomes could be used as antigen source in DC-based immunotherapy. Methods: Mouse MM AB1 cells were used to generate tumour lysate and tumour-derived exosomes. Tumour lysate was generated by 5 cycles of freezeÁthawing followed by sonication of AB1 cells. Tumour exosomes were collected from the AB1 cell culture supernatant and followed a stepwise ultracentrifugation. Protein quantification and electron microscopy were performed to determine the protein amount and to characterise their morphology. To test whether MM derived exosomes are immunogenic and able to stimulate an antitumoral response, BALB/c mice were injected with a lethal dose of AB1 tumour cells at day 0, followed by intraperitoneal injection of a single dose of DCs loaded with tumour exosomes, DCs loaded with tumour lysate, or phosphate buffered saline (PBS), at day 7. Results: Mice which received tumour exosome-loaded DC immunotherapy had an increased median and overall survival compared to mice which received tumour lysate-loaded DC or PBS. Conclusion: In this study, we showed that DC immunotherapy loaded with tumour exosomes derived from non-immunogenic tumours improved survival of tumour bearing mice.
The application of exosomes as a nanoscale cancer vaccine
International journal of nanomedicine, 2010
Cancer is a leading cause of death globally, and it is predicted and projected to continue rising as life expectancy increases. Although patient survival rates for some forms of cancers are high due to clinical advances in treatment protocols, the search for effective cancer vaccines remains the ultimate Rosetta Stone in oncology. Cervarix(®), Gardasil(®), and hepatitis B vaccines are currently employed in preventing certain forms of viral cancers. However, they are, strictly speaking, not 'true' cancer vaccines as they are prophylactic rather than therapeutic, are only effective against the oncogenic viruses, and do not kill the actual cancer cells. On April 2010, a new prostate cancer vaccine Provenge(®) (sipuleucel-T) was approved by the US FDA, and it is the first approved therapeutic vaccine that utilizes antigen-presenting cell technology involving dendritic cells in cancer immunotherapy. Recent evidence suggests that the use of nanoscale particles like exosomes in imm...