Cristina Gutiérrez-Vázquez

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

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.

Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs

Exosomes are released by most cells to the extracellular environment and are involved in cell-to-cell communication. Exosomes contain specific repertoires of mRNAs, microRNAs (miRNAs) and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe sequence motifs present in miRNAs that control their localization into exosomes. The protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications.

Sorting it out: Regulation of exosome loading

Extracellular vesicles (EVs), a term that includes both exosomes of endocytic origin and vesicles derived from plasma membranes, are continuously secreted by cells to the extracellular environment, and represent a novel vehicle for cell-cell communication. Exosomes contain specific repertoires of proteins and RNAs, indicating the existence of mechanisms that control the sorting of molecules into them. Although the molecular mechanisms that regulate the loading of proteins into exosomes have been studied for years, the sorting of RNA has been elusive until recently. Here we review the molecular mechanisms that control the sorting of molecules into exosomes, with special attention to the sorting of RNA. We also discuss how the cellular context affects the composition of exosomes, and thus the outcome of the communication between the exosome-producer and recipient cells, with particular focus on the communication between tumor cells and with cells of the tumor microenvironment.

The Intracellular Interactome of Tetraspanin-enriched Microdomains Reveals Their Function as Sorting Machineries toward Exosomes

Background: Tetraspanin-enriched microdomains (TEM) are ubiquitous specialized membrane platforms enriched in extracellular vesicles. Results: Intracellular TEM interactome accounts for a great proportion of the exosome proteome. Selected CD81 ligands are depleted from exosomes in CD81-deficient cells. Conclusion: Insertion into TEM may be necessary for protein inclusion into exosomes. Significance: Exosome cargo selection remains largely unexplored. TEM may be specialized platforms to route exosome components. Extracellular vesicles are emerging as a potent mechanism of intercellular communication because they can systemically exchange genetic and protein material between cells. Tetraspanin molecules are commonly used as protein markers of extracellular vesicles, although their role in the unexplored mechanisms of cargo selection into exosomes has not been addressed. For that purpose, we have characterized the intracellular tetraspanin-enriched microdomain (TEM) interactome by high throughput mass spectrometry, in both human lymphoblasts and their derived exosomes, revealing a clear pattern of interaction networks. Proteins interacting with TEM receptors cytoplasmic regions presented a considerable degree of overlap, although some highly specific CD81 tetraspanin ligands, such as Rac GTPase, were detected. Quantitative proteomics showed that TEM ligands account for a great proportion of the exosome proteome and that a selective repertoire of CD81-associated molecules, including Rac, is not correctly routed to exosomes in cells from CD81-deficient animals. Our data provide evidence that insertion into TEM may be necessary for protein inclusion into the exosome structure.

Transfer of extracellular vesicles during immune cell-cell interactions

The transfer of molecules between cells during cognate immune cell interactions has been reported, and recently a novel mechanism of transfer of proteins and genetic material such as small RNA between T cells and antigen‐presenting cells (APCs) has been described, involving exchange of extracellular vesicles (EVs) during the formation of the immunological synapse (IS). EVs, a term that encompasses exosomes and microvesicles, has been implicated in cell‐cell communication during immune responses associated with tumors, pathogens, allergies, and autoimmune diseases. This review focuses on EV transfer as a mechanism for the exchange of molecules during immune cell‐cell interactions.

Endosomal clathrin drives actin accumulation at the immunological synapse.

Antigen-specific cognate interaction of T lymphocytes with antigen-presenting cells (APCs) drives major morphological and functional changes in T cells, including actin rearrangements at the immune synapse (IS) formed at the cell–cell contact area. Here we show, using cell lines as well as primary cells, that clathrin, a protein involved in endocytic processes, drives actin accumulation at the IS. Clathrin is recruited towards the IS with parallel kinetics to that of actin. Knockdown of clathrin prevents accumulation of actin and proteins involved in actin polymerization, such as dynamin-2, the Arp2/3 complex and CD2AP at the IS. The clathrin pool involved in actin accumulation at the IS is linked to multivesicular bodies that polarize to the cell–cell contact zone, but not to plasma membrane or Golgi complex. These data underscore the role of clathrin as a platform for the recruitment of proteins that promote actin polymerization at the interface of T cells and APCs.

Immunomodulatory role of microRNAs transferred by extracellular vesicles.

The immune system is composed of different cell types localised throughout the organism to sense and respond to pathological situations while maintaining homeostasis under physiological conditions. Intercellular communication between immune cells is essential to coordinate an effective immune response and involves both cell contact dependent and independent processes that ensure the transfer of information between bystander and distant cells. There is a rapidly growing body of evidence on the pivotal role of extracellular vesicles (EVs) in cell communication and these structures are emerging as important mediators for immune modulation upon delivery of their molecular cargo. In the last decade, EVs have been shown to be efficient carriers of genetic information, including microRNAs (miRNAs), that can be transferred between cells and regulate gene expression and function on the recipient cell. Here, we review the current knowledge of intercellular functional transfer of EV‐delivered miRNAs and their putative role in immune regulation.

The leukocyte activation antigen CD69 limits allergic asthma and skin contact hypersensitivity

BACKGROUND Allergic diseases have a major health care impact in industrialized countries. The development of these diseases is influenced by exposure to allergen and to immunological and genetic factors. However, the molecular mechanisms underlying the inflammatory response that triggers allergy are not well defined. OBJECTIVE We have investigated the role of the leukocyte activation antigen CD69 in the regulation of two allergic diseases, asthma and contact dermatitis. METHODS Analysis of two models of allergic diseases in CD69 knockout and wild-type mice: ovalbumin-induced allergic airway inflammation (BALB/c genetic background) and contact hypersensitivity to oxazolone (C57BL/6J genetic background). RESULTS CD69 deficiency dramatically enhanced the inflammatory response in the ovalbumin-induced asthma model of antigen-induced airway allergy. CD69 knockout mice showed exacerbated pulmonary eosinophil recruitment, high vascular cell adhesion molecule 1 expression levels in lung vasculature, and enhanced T(H)2 and T(H)17 cytokines in the bronchoalveolar space and lung tissue. In the hapten-induced cutaneous contact hypersensitivity model, both CD69 deficiency and treatment with anti-CD69 mAb increased inflammation. Treatment with contact allergens induced enhanced T(H)1 and T(H)17 responses in CD69 deficient mice, and neutralizing anti-IL-17 antibodies reduced skin inflammation. In both experimental systems, adoptive transfer of lymph node cells from CD69 knockout mice increased the inflammatory response in recipient mice. CONCLUSION These results demonstrate that the early activation receptor CD69 is an intrinsic modulator of immune allergic processes through the negative regulation of allergen-induced T-cell effector responses.

Analysis of MicroRNA and Protein Transfer by Exosomes During an Immune Synapse

Immune cells release microRNA-containing exosomes that can be taken up by recipient cells. Exosomes can thus act as mediators of cell-cell communication through direct exchange of genetic material between cells. Exosome-mediated transfer of miRNAs between T cells and antigen-presenting cells (APCs) can take place over long distances. Our work has shown that this transfer is enhanced by the formation of a functional immune synapse. Here we give a detailed description of the isolation of exosomes produced by immune cells by ultracentrifugation, their quantification by flow cytometry, and the analysis of miRNA and protein exchange between T cells and APCs, both at a distance and after the formation of an immune synapse.

Extracellular vesicles as a source for non-invasive biomarkers in bladder cancer progression.

&NA; Bladder cancer is the second most frequent malignancy of the urinary tract after prostate cancer. Current diagnostic techniques, such as cystoscopy and biopsies are highly invasive and accompanied of undesirable side effects. Moreover, there are no suitable biomarkers for relapse or progression prognosis. We analysed whether the specific composition of microRNAs (miRNAs) and proteins of extracellular vesicles (EVs) that urothelial tumour cells of bladder mucosa release into the urine, could reflect their pathologic condition. For this purpose, urinary EVs were isolated and their protein and miRNA composition evaluated in healthy donors and low or high‐grade bladder cancer patients. Using a microarray platform containing probes for 851 human miRNAs we found 26 deregulated miRNAs in high‐grade bladder cancer urine EVs, from which 23 were downregulated and 3 upregulated. Real‐time PCR analysis pointed to miR‐375 as a biomarker for high‐grade bladder cancer while miR‐146a could identify low‐grade patients. Finally, several protein markers were also deregulated in EVs from tumour patients. Our data suggest that the presence of ApoB in the 100,000 pellet is a clear marker for malignancy. Graphical abstract Figure. No caption available.