Clotilde Théry

Exosomes: composition, biogenesis and function

Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells in culture. Interest in exosomes has intensified after their recent description in antigen-presenting cells and the observation that they can stimulate immune responses in vivo. In the past few years, several groups have reported the secretion of exosomes by various cell types, and have discussed their potential biological functions. Here, we describe the physical properties that define exosomes as a specific population of secreted vesicles, we summarize their biological effects, particularly on the immune system, and we discuss the potential roles that secreted vesicles could have as intercellular messengers.

Membrane vesicles as conveyors of immune responses

In multicellular organisms, communication between cells mainly involves the secretion of proteins that then bind to receptors on neighbouring cells. But another mode of intercellular communication — the release of membrane vesicles — has recently become the subject of increasing interest. Membrane vesicles are complex structures composed of a lipid bilayer that contains transmembrane proteins and encloses soluble hydrophilic components derived from the cytosol of the donor cell. These vesicles have been shown to affect the physiology of neighbouring recipient cells in various ways, from inducing intracellular signalling following binding to receptors to conferring new properties after the acquisition of new receptors, enzymes or even genetic material from the vesicles. This Review focuses on the role of membrane vesicles, in particular exosomes, in the communication between immune cells, and between tumour and immune cells.

Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids

Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell‐culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high‐quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.

Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.

In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

Proteomic Analysis of Dendritic Cell-Derived Exosomes: A Secreted Subcellular Compartment Distinct from Apoptotic Vesicles

Dendritic cells constitutively secrete a population of small (50–90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1α) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.

Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming

The initiation of T-cell–mediated antitumor immune responses requires the uptake and processing of tumor antigens by dendritic cells and their presentation on MHC-I molecules. Here we show in a human in vitro model system that exosomes, a population of small membrane vesicles secreted by living tumor cells, contain and transfer tumor antigens to dendritic cells. After mouse tumor exosome uptake, dendritic cells induce potent CD8+ T-cell–dependent antitumor effects on syngeneic and allogeneic established mouse tumors. Therefore, exosomes represent a novel source of tumor-rejection antigens for T-cell cross priming, relevant for immunointerventions.

Rab27a and Rab27b control different steps of the exosome secretion pathway

Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.

Standardization of sample collection, isolation and analysis methods in extracellular vesicle research

The emergence of publications on extracellular RNA (exRNA) and extracellular vesicles (EV) has highlighted the potential of these molecules and vehicles as biomarkers of disease and therapeutic targets. These findings have created a paradigm shift, most prominently in the field of oncology, prompting expanded interest in the field and dedication of funds for EV research. At the same time, understanding of EV subtypes, biogenesis, cargo and mechanisms of shuttling remains incomplete. The techniques that can be harnessed to address the many gaps in our current knowledge were the subject of a special workshop of the International Society for Extracellular Vesicles (ISEV) in New York City in October 2012. As part of the “ISEV Research Seminar: Analysis and Function of RNA in Extracellular Vesicles (evRNA)”, 6 round-table discussions were held to provide an evidence-based framework for isolation and analysis of EV, purification and analysis of associated RNA molecules, and molecular engineering of EV for therapeutic intervention. This article arises from the discussion of EV isolation and analysis at that meeting. The conclusions of the round table are supplemented with a review of published materials and our experience. Controversies and outstanding questions are identified that may inform future research and funding priorities. While we emphasize the need for standardization of specimen handling, appropriate normative controls, and isolation and analysis techniques to facilitate comparison of results, we also recognize that continual development and evaluation of techniques will be necessary as new knowledge is amassed. On many points, consensus has not yet been achieved and must be built through the reporting of well-controlled experiments.

Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles

Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.

Indirect activation of naïve CD4+ T cells by dendritic cell-derived exosomes.

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.