Editorial: Functional Relevance of Tetraspanins in the Immune System

AS is supported by a Netherlands Organization for Scientific Research Grant (NWO) Gravitation Programme 2013 grant (ICI-024.002.009), a Dutch Cancer Society grant (KUN2014-6845), and was awarded an European Research Council Consolidator Grant (Secret Surface, 724281). MY-M is supported by BIO2017-86500-R Research Grant from the Spanish Ministerio de Ciencia, Innovación y Universidades. CC is supported by SAF2016-77096-R Research Grant from the Spanish Ministerio de Ciencia, Innovación y Universidade

Embryonic trophectoderm secretomics reveals chemotactic migration and intercellular communication of endometrial and circulating MSCs in embryonic implantation

Embryonic implantation is a key step in the establishment of pregnancy. In the present work, we have carried out an in-depth proteomic analysis of the secretome (extracellular vesicles and soluble proteins) of two bovine blastocysts embryonic trophectoderm primary cultures (BBT), confirming different epithelial–mesenchymal transition stages in these cells. BBT-secretomes contain early pregnancy-related proteins and angiogenic proteins both as cargo in EVs and the soluble fraction. We have demonstrated the functional transfer of protein-containing secretome between embryonic trophectoderm and maternal MSC in vitro using two BBT primary cultures eight endometrial MSC (eMSC) and five peripheral blood MSC (pbMSC) lines. We observed that eMSC and pbMSC chemotax to both the soluble fraction and EVs of the BBT secretome. In addition, in a complementary direction, we found that the pattern of expression of implantation proteins in BBT-EVs changes depending on: (i) their epithelial–mesenchymal phenotype; (ii) as a result of the uptake of eMSC-or pbMSC-EV previously stimulated or not with embryonic signals (IFN-τ); (iii) because of the stimulation with the endometrial cytokines present in the uterine fluid in the peri-implantation perio

CD9 Controls Integrin α5β1-Mediated Cell Adhesion by Modulating Its Association With the Metalloproteinase ADAM17

Integrin α5β1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5β1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5β1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5β1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17

Bovine endometrial MSC: Mesenchymal to epithelial transition during luteolysis and tropism to implantation niche for immunomodulation

The uterus is a histologically dynamic organ, and the mechanisms coordinating its regeneration during the oestrous cycle and implantation are poorly understood. The aim of this study was to isolate, immortalize and characterize bovine endometrial mesenchymal stem cell (eMSC) lines from different oestrous cycle stages (embryo in the oviduct, embryo in the uterus or absence of embryo) and examine their migratory and immunomodulatory properties in an inflammatory or implantation-like environment, as well as possible changes in cell transdifferentiation. Methods: eMSCs were isolated and analysed in terms of morphological features, expression of cell surface and intracellular markers of pluripotency, inmunocytochemical analyses, alkaline phosphatase activity, proliferation and osteogenic or chondrogenic differentiation capacities, as well as their ability to migrate in response to inflammatory (TNF-α or IL-1β) or implantation (IFN-τ) cytokines and their immunomodulatory effect in the proliferation of T cells. Results: All eMSCs showed MSC properties such as adherence to plastic, high proliferative capacity, expression of CD44 and vimentin, undetectable expression of CD34 or MHCII, positivity for Pou5F1 and alkaline phosphatase activity. In the absence of an embryo, eMSC showed an apparent mesenchymal to epithelial transition state. eMSC during the entire oestrous cycle differentiated to osteogenic or chondrogenic lineages, showed the ability to suppress T cell proliferation and showed migratory capacity towards pro-inflammatory signal, while responded with a block in their migration to the embryo-derived pregnancy signal. Conclusion: This study describes for the first time the isolation, immortalization and characterization of bovine mesenchymal stem cell lines from different oestrous cycle stages, with a clear mesenchymal pattern and immunomodulatory properties. Our study also reports that the migratory capacity of the eMSC was increased towards an inflammatory niche but was reduced in response to the expression of implantation cytokine by the embryo. The combination of both signals (pro-inflammatory and implantation) would ensure the retention of eMSC in case of pregnancy, to ensure the immunomodulation necessary in the mother for embryo survival. In addition, in the absence of an embryo, eMSC showed an apparent mesenchymal to epithelial transition stateThis work was supported by grants from the Spanish Ministerio de Economía Industria y Competitividad to Ramírez M.A. (AGL2015-70140-R) and European Union’s Horizon 2020 Research and Innovation Programme under grant agreement N°731014 (Ramírez M.A.

CD81 controls sustained T cell activation signaling and defines the maturation stages of cognate immunological synapses

In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81-ICAM-1 and CD81- CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, contributing to sustained T cell receptor (TCR)-CD3-mediated signaling. Accordingly, we find that CD81 is required for proper T cell activation, regulating CD3ζ, ZAP-70, LAT, and extracellular signal-regulated kinase (ERK) phosphorylation; CD69 surface expression; and interleukin- 2 (IL-2) secretion. Our data demonstrate the important role of CD81 in the molecular organization and dynamics of the IS architecture that sets the signaling threshold in T cell activationThis work was supported by SAF2011-25834 from the Spanish Ministry of Science and Innovation, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid, Cardiovascular Network RD12-0042-0056 from the Instituto Salud Carlos III, and ERC-2011-AdG 294340-GENTRI

Dynamic interaction of VCAM-1 and ICAM-1 with moesin and ezrin in a novel endothelial docking structure for adherent leukocytes

Ezrin, radixin, and moesin (ERM) regulate cortical morphogenesis and cell adhesion by connecting membrane adhesion receptors to the actin-based cytoskeleton. We have studied the interaction of moesin and ezrin with the vascular cell adhesion molecule (VCAM)-1 during leukocyte adhesion and transendothelial migration (TEM). VCAM-1 interacted directly with moesin and ezrin in vitro, and all of these molecules colocalized at the apical surface of endothelium. Dynamic assessment of this interaction in living cells showed that both VCAM-1 and moesin were involved in lymphoblast adhesion and spreading on the endothelium, whereas only moesin participated in TEM, following the same distribution pattern as ICAM-1. During leukocyte adhesion in static or under flow conditions, VCAM-1, ICAM-1, and activated moesin and ezrin clustered in an endothelial actin-rich docking structure that anchored and partially embraced the leukocyte containing other cytoskeletal components such as α-actinin, vinculin, and VASP. Phosphoinositides and the Rho/p160 ROCK pathway, which participate in the activation of ERM proteins, were involved in the generation and maintenance of the anchoring structure. These results provide the first characterization of an endothelial docking structure that plays a key role in the firm adhesion of leukocytes to the endothelium during inflammation

Immunoassays for scarce tumour-antigens in exosomes: Detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma

Abstract Background Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. Methods Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. Results Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. Conclusions These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques.This work has been supported by Grants from Madrid Regional Government [IMMUNOTHERCAN-CM (S2010/BMD-2326)] and the Spanish Ministry of Economy [SAF2015-69169-R (MINEICO/FEDER) and MAT2017-84959-C2-1-R.; the Network of Excellence for Research in Exosomes, Rediex (MINEICO/FEDER); the Consejería de Economía y Empleo del Principado de Asturias (Plan de Ciencia, Tecnología e Innovación 2013-2017), under the Grant GRUPIN14-022. SL-C was a recipient of a FPU fellowship (MECD), and a travel fellowship from the Spanish Group of Extracellular Vesicles (GEIVEX); CC-S was a recipient of a master’s fellowship from “Postgrado de la Fundación Ramón Areces-UAM

Endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms

VCAM-1 and ICAM-1, receptors for leukocyte integrins, are recruited to cell–cell contact sites on the apical membrane of activated endothelial cells. In this study, we show that this recruitment is independent of ligand engagement, actin cytoskeleton anchorage, and heterodimer formation. Instead, VCAM-1 and ICAM-1 are recruited by inclusion within specialized preformed tetraspanin-enriched microdomains, which act as endothelial adhesive platforms (EAPs). Using advanced analytical fluorescence techniques, we have characterized the diffusion properties at the single-molecule level, nanoscale organization, and specific intradomain molecular interactions of EAPs in living primary endothelial cells. This study provides compelling evidence for the existence of EAPs as physical entities at the plasma membrane, distinct from lipid rafts. Scanning electron microscopy of immunogold-labeled samples treated with a specific tetraspanin-blocking peptide identify nanoclustering of VCAM-1 and ICAM-1 within EAPs as a novel mechanism for supramolecular organization that regulates the leukocyte integrin–binding capacity of both endothelial receptors during extravasation

CD9 controls integrin α5β1-mediated cell adhesion by modulating its association with the metalloproteinase ADAM17

Integrin α5β1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5β1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5β1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5β1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17.This work has been supported by the grant SAF2016-77096-R from Ministerio Español de Economía y Competitividad (MINECO) awarded to CC, by a grant from Fundación Ramón Areces Ayudas a la Investigación en Ciencias de la Vida y de la Materia, 2014 awarded to MY-M, and by the Deutsche Forschungsgemeinschaft (SFB 877, A6, Z3 and SPP1710) to JG and I

RIAM-VASP module relays integrin complement receptors in outside-in signaling driving particle engulfment

The phagocytic integrins and complement receptors M 2/CR3 and X 2/CR4 are classically associated with the phagocytosis of iC3b-opsonized particles. The activation of this receptor is dependent on signals derived from other receptors (inside-out signaling) with the crucial involvement of the Rap1-RIAM-Talin-1 pathway. Here, we analyze the implication of RIAM and its binding partner VASP in the signaling events occurring downstream of 2 integrins (outside-in) during complement-mediated phagocytosis. To this end, we used HL-60 promyelocytic cell lines deficient in RIAM or VASP or overexpressing EGFP-tagged VASP to determine VASP dynamics at phagocytic cups. Our results indicate that RIAM-deficient HL-60 cells presented impaired particle internalization and altered integrin downstream signaling during complement-dependent phagocytosis. Similarly, VASP deficiency completely blocked phagocytosis, while VASP overexpression increased the random movement of phagocytic particles at the cell surface, with reduced internalization. Moreover, the recruitment of VASP to particle contact sites, amount of pSer157-VASP and formation of actin-rich phagocytic cups were dependent on RIAM expression. Our results suggested that RIAM worked as a relay for integrin complement receptors in outside-in signaling, coordinating integrin activation and cytoskeletal rearrangements via its interaction with VASPThis work has been supported by Ministerio Español de Economía y Competitividad (MINECO) grants: SAF2016-77096-R (ELD and CC), BIO2014:54164-R (PAR), BIO2017-86500-R (MY-M), EI/MICIU EXPLORA BIO2017-91272-EXP & FEDER (PI17/02303) (S.R.-P). AT-G is supported by a predoctoral fellowship from MINECO, JLS-T is supported by a predoctoral fellowship from Universidad Complutense de Madrid, RT-R is supported by a postdoctoral fellowship from Asociación Española Contra el Cáncer (AECC