In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Biological properties of extracellular vesicles and their physiological functions

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Principles Of Fluorescence Spectroscopy

Flow cytometry is an essential tool for dissecting the functional complexity of hematopoiesis. We used single-cell "mass cytometry" to examine healthy human bone marrow, measuring 34 parameters simultaneously in single cells (binding of 31 antibodies, viability, DNA content, and relative cell size). The signaling behavior of cell subsets spanning a defined hematopoietic hierarchy was monitored with 18 simultaneous markers of functional signaling states perturbed by a set of ex vivo stimuli and inhibitors. The data set allowed for an algorithmically driven assembly of related cell types defined by surface antigen expression, providing a superimposable map of cell signaling responses in combination with drug inhibition. Visualized in this manner, the analysis revealed previously unappreciated instances of both precise signaling responses that were bounded within conventionally defined cell subsets and more continuous phosphorylation responses that crossed cell population boundaries in unexpected manners yet tracked closely with cellular phenotype. Collectively, such single-cell analyses provide system-wide views of immune signaling in healthy human hematopoiesis, against which drug action and disease can be compared for mechanistic studies and pharmacologic intervention.

Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum

/pdf/pathology-and-genetics-of-tumours-of-the-digestive-system-3xy9cwrc3c.pdf

Pathology and genetics of tumours of the digestive system

Extracellular vesicles, including exosomes, are small membrane vesicles derived from multivesicular bodies or from the plasma membrane. Most, if not all, cell types release extracellular vesicles, which then enter the bodily fluids. These vesicles contain a subset of proteins, lipids and nucleic acids that are derived from the parent cell. It is thought that extracellular vesicles have important roles in intercellular communication, both locally and systemically, as they transfer their contents, including proteins, lipids and RNAs, between cells. Extracellular vesicles are involved in numerous physiological processes, and vesicles from both non-immune and immune cells have important roles in immune regulation. Moreover, extracellular vesicle-based therapeutics are being developed and clinically tested for the treatment of inflammatory diseases, autoimmune disorders and cancer. Given the tremendous therapeutic potential of extracellular vesicles, this Review focuses on their role in modulating immune responses, as well as their potential therapeutic applications.

Regulation of immune responses by extracellular vesicles

Highly multiparametric analysis by mass cytometry.

During mammalian pregnancy maternal-fetal tolerance involves a number of immunosuppressive factors produced by placenta. Recently, placenta-derived exosomes have emerged as new immune regulators in the maternal immune tolerance. Exosomes are membrane nanovesicles with defined morphology, which are secreted from endosomal multivesicular bodies (MVB) upon fusion with the plasma membrane. Previously, we reported that the MHC class I chain-related (MIC) proteins A and B, human ligands of the activating NK cell receptor NKG2D, are expressed by placenta, sorted to MVB of syncytiotrophoblast and probably released via MIC-bearing exosomes. In this report, we show that the second family of human NKG2D ligands, the UL-16 binding proteins (ULBP), is also expressed by placenta. Importantly, this expression was not due to placental CMV infection. Immunoelectron microscopy disclosed that ULBP1-5 are produced and retained in MVB of the syncytiotrophoblast on microvesicles/exosomes. Using human placenta explant cultures and different assays, we demonstrate that exosomes bearing NKG2D ligands are released by human placenta. Isolated placental exosomes carried ULBP1-5 and MIC on their surface and induced down-regulation of the NKG2D receptor on NK, CD8(+), and gammadelta T cells, leading to reduction of their in vitro cytotoxicity without affecting the perforin-mediated lytic pathway. Release of placental NKG2D ligands via exosomes is an alternative mechanism for generation of bioactive soluble form of these ligands. These findings highlight a role for NKG2D ligand-bearing placental exosomes in the fetal immune escape and support the view of placenta as a unique immunosuppressive organ.

Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function

To identify a rare (e.g., diseased or foreign) cell in a complex mixture, or to understand the proteomic complexity[1,2] of cells, one needs to be able to measure simultaneously and quantitatively a large number of proteins or other biomarkers that may be present in a complex sample. This is a difficult task and is beyond the reach of current capabilities. To address a problem of this complexity, we have begun to develop a high-sensitivity assay[3–6] based upon elemental tags that will enable the simultaneous measurement of many proteins in a single sample. The advantage of this approach lies in the large number of available elements and isotopes (potentially greater than 79) found in low abundance in biological systems, which will allow multiple tags to be used simultaneously. Inductively coupled plasma mass spectrometry (ICP-MS) is an ideal technique for detecting and quantifying these tags, as ICP-MS provides excellent resolution between the tag masses and an exceptional dynamic range (nine orders of magnitude).[7] This method allows one to overcome some of the limitations of currently available fluorescent tagging approaches.[8] These limitations arise from the spectral overlap of different dyes and the difficulty in measuring simultaneously targets that differ in abundance by an order of magnitude or more. Other benefits of ICP-MS detection include the high sensitivity, which is comparable to that of radioimmunoassays or chemiluminescent assays,[3] insensitivity of elemental tags to photobleaching and storage time, as well as the stability of the tagged sample so that it can be stored or shipped for analysis. We discuss herein the development of a new class of elemental tags for ICP-MS detection and their use for tagging of antibodies chosen to allow specific recognition of distinguishing cell surface markers. By using this technique it should be possible to achieve detection limits on the order of parts per billion, which will allow the detection of cell surface markers with copy numbers as low as 100.

Polymer-Based Elemental Tags for Sensitive Bioassays†

We report a set of novel immunoassays in which proteins of interest can be detected using specific element-tagged antibodies. These immunoassays are directly coupled with an inductively coupled plasma mass spectrometer (ICPMS) to quantify the elemental (in this work, metal) component of the reacted tagged antibodies. It is demonstrated that these methods can detect levels of target proteins as low as 0.1-0.5 ng/mL and yield a linear response to protein concentration over 3 orders of magnitude.

A sensitive and quantitative element-tagged immunoassay with ICPMS detection.

The human gut epithelium is a unique immunological compartment, containing substantial amounts of intra-epithelial lymphocytes (IEL) with unknown functions. In this study we show that distinct and ...

Vladimir Baranov

Papers

Highly multiparametric analysis by mass cytometry.

Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function

Polymer-Based Elemental Tags for Sensitive Bioassays†

A sensitive and quantitative element-tagged immunoassay with ICPMS detection.

Intra-epithelial lymphocytes. Evidence for regional specialization and extrathymic T cell maturation in the human gut epithelium