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      Unidirectional transfer of microRNA-loaded exosomes from T cells to antigen-presenting cells

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          Abstract

          The immune synapse is an exquisitely evolved means of communication between T cells and antigen-presenting cells (APCs) during antigen recognition. Recent evidence points to the transfer of RNA via exosomes as a novel mode of intercellular communication. Here we show that exosomes of T, B and dendritic immune cells contain microRNA (miRNA) repertoires that differ from those of their parent cells. We investigate whether miRNAs are exchanged during cognate immune interactions, and demonstrate the existence of antigen-driven unidirectional transfer of miRNAs from the T cell to the APC, mediated by the delivery of CD63+ exosomes on immune synapse formation. Inhibition of exosome production by targeting neutral sphingomyelinase-2 impairs transfer of miRNAs to APCs. Moreover, miRNAs transferred during immune synapsis are able to modulate gene expression in recipient cells. Thus, our results support a mechanism of cellular communication involving antigen-dependent, unidirectional intercellular transfer of miRNAs by exosomes during immune synapsis.

          Abstract

          Exosomes released from cells can transfer RNA to recipient cells. In this study, the authors demonstrate that microRNAs in exosomes from T cells can be transferred to antigen-presenting cells during immune synapsis, and that this can alter gene expression, suggesting a new form of cellular communication.

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          Most cited references 57

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          Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.

          Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called "exosomal shuttle RNA" (esRNA).
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            Glioblastoma microvesicles transport RNA and protein that promote tumor growth and provide diagnostic biomarkers

            Glioblastoma tumor cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles we demonstrate that microvesicle-delivered messages are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and elicit tubule formation by endothelial cells. Tumor-derived microvesicles therefore serve as a novel means of delivery of genetic information as well as proteins to recipient cells in the tumor environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and microRNAs characteristic of gliomas can be detected in serum microvesicles of glioblastoma patients. The tumor-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumor-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test. Glioblastomas are highly malignant brain tumors with a poor prognosis despite intensive research and clinical efforts1. These tumors as well as many others have a remarkable ability to mold their stromal environment to their own advantage. Tumor cells alter surrounding normal cells to facilitate tumor cell growth, invasion, chemoresistance, immune evasion and metastasis 2–4. The tumor cells also hijack the normal vasculature and stimulate rapid formation of new blood vessels to supply tumor nutrition 5. Although the immune system can initially suppress tumor growth, it is often progressively blunted by tumor activation of immunosuppressive pathways 6. Recent studies show the importance of communication between tumor cells and their environment through shedding of membrane microvesicles which can fuse to cells in the vicinity 7. Microvesicles are 30–100 nm in diameter and shed from many different cell types under both normal and pathological conditions 8. These exosomes can be formed through inward budding of endosomal membranes giving rise to intracellular multivesicular bodies (MVB) that later fuse with the plasma membrane, releasing the exosomes to the exterior 8,9. They can also be shed directly by outward budding of the plasma membrane, as shown for Jurkat T-cells 10. Microvesicles in Drosophila, termed argosomes, contain morphogens such as Wingless protein and move throughout the imaginal disc epithelium in the developing embryos 11. Microvesicles found in semen, known as prostasomes, can promote sperm motility, stabilize the acrosome reaction, facilitate immunosuppression and inhibit angiogenesis 12. On the other hand, prostasomes released by malignant prostate cells promote angiogenesis. It has been shown that microvesicles can transfer some of their contents to other cell types 13–16. The content of microvesicles and their biological function depends on the cell of origin. Microvesicles derived from B-cells and dendritic cells have potent immuno-stimulatory and antitumor effects in vivo and have been used as antitumor vaccines 17. Dendritic cell-derived microvesicles contain co-stimulatory proteins necessary for T-cell activation, whereas most tumor cell-derived microvesicles do not. Instead they act to suppress the immune response and accelerate tumor growth and invasiveness 18–21. Breast cancer microvesicles stimulate angiogenesis, and platelet-derived microvesicles promote tumor progression and metastasis of lung cancer cells 22,23. Human glioblastoma tissues were obtained from surgical resections and tumor cells were dissociated and cultured as monolayers in medium using fetal bovine serum (FBS) depleted for microvesicles (dFBS). Cultured primary cells obtained from three glioblastoma tumors were found to produce microvesicles at early and later passages (1–15 passages). Tumor cells were covered with microvesicles varying in size from about 50 – 500 nm (Fig. 1a and b). The microvesicles contained RNA and protein in an approximate ratio of 1:80. To evaluate whether the RNA was contained inside the microvesicles, they were either exposed to RNase A or left untreated before RNA extraction (Fig. 1c). There was always less than a 7% decrease in RNA content following RNase treatment. Thus, it appears that almost all of the RNA is contained within the vesicles and is thereby protected from external RNases by the surrounding membrane. Bioanalysis of RNA from microvesicles and their donor cells revealed that the microvesicles contain a broad range of RNA sizes consistent with a variety of mRNAs and miRNAs, but lack the ribosomal RNA peaks characteristic of cellular RNA (Fig. 1d and e). Microarray analysis of mRNA populations in microvesicles and their donor glioblastoma cells was performed using the Agilent 44K whole genome microarray. Approximately 22,000 gene transcripts were found in the cells and 27,000 transcripts in the microvesicles (detected at well above background levels, 99% confidence interval) on both arrays. Approximately 4,700 different mRNAs were detected exclusively in microvesicles on both arrays, indicating a selective enrichment process within the microvesicles (Supplementary Table 1). Consistent with this, there was a poor overall correlation in levels of mRNAs in the cells as compared to microvesicles from two tumor preparations (Fig. 2a and b), supporting selective enrichment of some cellular mRNAs in microvesicles. In contrast, a comparison of levels of specific mRNAs in different preparations of donor cells or of microvesicles showed a strong correlation, indicating a consistent distribution within these distinct cellular compartments (Fig. 2c and d). We found 3426 transcripts differentially distributed more than 5-fold (p-value 95%. The cells were stably transduced and microvesicles generated during the subsequent passages (2–10) were isolated and purified as above. Microvesicles (50 μg) were added to 50,000 HBMVEC and incubated for 24 hrs. The Gluc activity in the supernatant was measured directly after microvesicle addition (0 hrs), and 15 hrs and 24 hrs later and normalised to the Gluc activity in the microvesicles. The results are presented as the mean ± SEM (n = 4). PKH67 labelled microvesicle Purified glioblastoma microvesicles were labelled with PKH67 Green Fluorescent labelling kit (Sigma-Aldrich, St Louis, MO, USA) as described 21. The labelled microvesicles were incubated with HBMVEC in culture (5 μg/50,000 cells). Microvesicles were allowed to bind for 20 min at 4°C and cells were then washed and incubated at 37°C for 1 hr. RT PCR and nested PCR RNA was extracted using the MirVana RNA isolation kit. RNA was converted to cDNA using the Omniscript RT kit (if starting material was >50 ng) or Sensiscript RT kit (if starting material was <50 ng) (Qiagen Inc., Valencia, CA, USA) using a mix of oligo dT and random hexamer primer according to manufacturer’s recommendation. The following PCR primers were used: GAPDH primers; Forw 5′-GAA GGT GAA GGT CGG AGT C-3′, Reverse 5′-GAA GAT GGT GAT GGG ATT TC-3′. EGFR/EGFRvIII PCR1; Forw 5′-CCAGTATTGATCGGGAGAGC-3′, Reverse 5′-TCAGAATATCCAGTTCCTGTGG-3′, EGFR/EGFRvIII PCR2; Forw 5′-ATG CGA CCC TCC GGG ACG-3′, Reverse 5′-GAG TAT GTG TGA AGG AGT-3′. The Gluc primers have been described previously 24. PCR protocol: 94°C 3 min; 94°C 45 s, 60°C 45 s, 72°C 2 min × 35 cycles; 72°C 7 min. Angiogenesis antibody array One mg total protein from either primary glioblastoma cells or purified microvesicles isolated from the same cells were lysed in Promega lysis buffer (Promega, Madison, WI, USA) and then added to the human angiogenesis antibody array (Panomics, Fremont, CA, USA) according to manufacturer’s recommendations. The arrays were scanned and analysed with the ImageJ software (NIH). Statistics The statistical analyses were performed using Students t-test. Supplementary Material 1
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              Origins and Mechanisms of miRNAs and siRNAs.

              Over the last decade, approximately 20-30 nucleotide RNA molecules have emerged as critical regulators in the expression and function of eukaryotic genomes. Two primary categories of these small RNAs--short interfering RNAs (siRNAs) and microRNAs (miRNAs)--act in both somatic and germline lineages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA- and miRNA-based regulation has direct implications for fundamental biology as well as disease etiology and treatment.
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                Author and article information

                Journal
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                April 2011
                19 April 2011
                : 2
                : 282
                Affiliations
                [1 ]simpleCentro Nacional de Investigaciones Cardiovasculares, Melchor Fernández Almagro , 3. 28029, Madrid, Spain.
                [2 ]simpleServicio de Inmunología, Hospital Universitario de la Princesa, Instituto de Investigación Sanitaria Princesa, Diego de León , 62. 28006 Madrid, Spain.
                [3 ]These authors contributed equally to this work.
                Author notes
                Article
                ncomms1285
                10.1038/ncomms1285
                3104548
                21505438
                Copyright © 2011, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

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