Control of lymphocyte shape and the chemotactic response by the GTP exchange factor Vav

7 FiguresRho GTPases control many facets of cell polarity and migration; namely, the reorganization of the cellular cytoskeleton to extracellular stimuli. Rho GTPases are activated by GTP exchange factors (GEFs), which induce guanosine diphosphate (GDP) release and the stabilization of the nucleotide-free state. Thus, the role of GEFs in the regulation of the cellular response to extracellular cues during cell migration is a critical step of this process. In this report, we have analyzed the activation and subcellular localization of the hematopoietic GEF Vav in human peripheral blood lymphocytes stimulated with the chemokine stromal cell–derived factor-1 (SDF-1α). We show a robust activation of Vav and its redistribution to motility-associated subcellular structures, and we provide biochemical evidence of the recruitment of Vav to the membrane of SDF-1α–activated human lymphocytes, where it transiently interacts with the SDF-1α receptor CXCR4. Overexpression of a dominant negative form of Vav abolished lymphocyte polarization, actin polymerization, and migration. SDF-1α–mediated cell polarization and migration also were impaired by overexpression of an active, oncogenic Vav, although the mechanism appears to be different. Together, our data postulate a pivotal role for Vav in the transmission of the migratory signal through the chemokine receptor CXCR4.From the Servicio de Inmunología, Hospital Universitario de la Princesa,Madrid, Spain; Centro de Investigación del Cáncer (CIC), Campus Miguel deUnamuno, Salamanca, Spain; and Facultad de Medicina, Universidad Autónoma de San Luis Potosí (UASLP), San Luis Potosí, Mexico.Peer reviewe

Biocompatible Probes Based on Rare-Earth Doped Strontium Aluminates with Long-Lasting Phosphorescent Properties for In Vitro Optical IMAGING

In recent decades, the demand for biomedical imaging tools has grown very rapidly as a key feature for biomedical research and diagnostic applications. Particularly, fluorescence imaging has gained increased attention as a non-invasive, inexpensive technique that allows real-time imaging. However, tissue auto-fluorescence under external illumination, together with a weak tissue penetration of low wavelength excitation light, largely restricts the application of the technique. Accordingly, new types of fluorescent labels are currently being investigated and, in this search, phosphorescent nanoparticles promise great potential, as they combine the interesting size-dependent properties of nanoscale materials with a long-lasting phosphorescence-type emission that allows optical imaging well after excitation (so avoiding autofluorescence). In this work, core-shell structures consisting of SrAlO:Eu,Dy luminescent cores encapsulated within a biocompatible silica shell were prepared, showing a green persistent phosphorescence with an afterglow time of more than 1000 s. A high-energy ball milling procedure was used to reduce the size of the starting phosphors to a size suitable for cellular uptake, while the silica coating was produced by a reverse micelle methodology that eventually allows the excitation and emission light to pass efficiently through the shell. Confocal fluorescence microscopy using HeLa cancer cells confirmed the potential of the all-ceramic composites produced as feasible labels for in vitro optical imaging

The leukocyte activation receptor CD69 controls T cell differentiation through its interaction with galectin-1

CD69 is involved in immune cell homeostasis, regulating the T cell-mediated immune response through the control of Th17 cell differentiation. However, natural ligands for CD69 have not yet been described. Using recombinant fusion proteins containing the extracellular domain of CD69, we have detected the presence of a ligand(s) for CD69 on human dendritic cells (DCs). Pulldown followed by mass spectrometry analyses of CD69-binding moieties on DCs identified galectin-1 as a CD69 counterreceptor. Surface plasmon resonance and anti-CD69 blocking analyses demonstrated a direct and specific interaction between CD69 and galectin-1 that was carbohydrate dependent. Functional assays with both human and mouse T cells demonstrated the role of CD69 in the negative effect of galectin-1 on Th17 differentiation. Our findings identify CD69 and galectin-1 to be a novel regulatory receptor-ligand pair that modulates Th17 effector cell differentiation and functionThis work was funded by grants SAF2011-25834 and ERC-2011AdG 294340-GENTRIS to F.S.-M., RECAVA RD06/0014 from the Fondo de Investigaciones Sanitarias to J.V. and F.S.-M., and INDISNET 01592006 from the Comunidad de Madrid to F.S.-M. and P.M. and by grants from the Ministerio de Economia y Competitividad (PI11/01562 to P.N.) and the Generalitat de Catalunya-AGAUR (2009SGR1409 to P.N.). The Ministry of Science and Innovation and the Pro-CNIC Foundation support CNI

Metodología experimental aplicada a la Inmunología Molecular

El objetivo general del proyecto es aplicar un modelo pedagógico en el que participen los alumnos de manera activa y apliquen el método científico en base a los conocimientos que han adquirido, resolviendo y realizando un caso práctico en el laboratorio. Integra una estrategia didáctica que va a fomentar la participación activa del alumnado provocando un aprendizaje significativo, ya que el alumno tiene que resolver mediante el razonamiento un caso práctico y luego integrarlo en el laboratorio con el uso de una técnica ampliamente utilizada en Inmunología, como es la citometría de flujo.Depto. de Arquitectura de Computadores y AutomáticaFac. de InformáticaFALSEInnovasubmitte

La molécula de activación leucocitaria CD69 como regulador de los procesos inflamatorios ; director Francisco Sánchez-Madrid

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 21 de Mayo de 2010

Close Encounters of Lymphoid Cells and Bacteria

During infections, the first reaction of the host against microbial pathogens is carried out by innate immune cells, which recognize conserved structures on pathogens, called pathogen-associated molecular patterns. Afterward, some of these innate cells can phagocytose and destroy the pathogens, secreting cytokines that would modulate the immune response to the challenge. This rapid response is normally followed by the adaptive immunity, more specific and essential for a complete pathogen clearance in many cases. Some innate immune cells, usually named antigen-presenting cells, such as macrophages or dendritic cells, are able to process internalized invaders and present their antigens to lymphocytes, triggering the adaptive immune response. Nevertheless, the traditional boundary of separated roles between innate and adaptive immunity has been blurred by several studies, showing that very specialized populations of lymphocytes (cells of the adaptive immunity) behave similarly to cells of the innate immunity. These “innate-like” lymphocytes include γδ T cells, invariant NKT cells, B-1 cells, mucosal-associated invariant T cells, marginal zone B cells, and innate response activator cells, and together with the newly described innate lymphoid cells are able to rapidly respond to bacterial infections. Strikingly, our recent data suggest that conventional CD4+ T cells, the paradigm of cells of the adaptive immunity, also present innate-like behavior, capturing bacteria in a process called transinfection. Transinfected CD4+ T cells digest internalized bacteria like professional phagocytes and secrete large amounts of proinflammatory cytokines, protecting for further bacterial challenges. In the present review, we will focus on the data showing such innate-like behavior of lymphocytes following bacteria encounter.This work was supported by the grants from the Spanish Ministry of Science and Technology (MICINN; BFU2011-29450) to EV and Ministry of Economy and Competitiveness (MINECO; SAF2014-58895-JIN to AC-A and SAF2014-56716-REDT and BFU2014-59585-R to EV).Peer reviewedPeer Reviewe

Close encounters of lymphoid cells and bacteria

During infections, the first reaction of the host against microbial pathogens is carried out by innate immune cells, which recognize conserved structures on pathogens, called pathogen-associated molecular patterns (PAMPs). Afterwards, some of these innate cells can phagocytose and destroy the pathogens, secreting cytokines that would modulate the immune response to the challenge. This rapid response is normally followed by the adaptive immunity, more specific, and essential for a complete pathogen clearance in many cases. Some innate immune cells, usually named antigen presenting cells (APCs), like macrophages or dendritic cells, are able to process internalized invaders and present their antigens to lymphocytes, triggering the adaptive immune response. Nevertheless, the traditional boundary of separated roles between innate and adaptive immunity has been blurred by several studies, showing that very specialized populations of lymphocytes (cells of the adaptive immunity), behave similarly to cells of the innate immunity. These innate-like lymphocytes include γδ T cells, invariant NKT cells, B-1 cells, mucosal associated invariant T (MAIT), marginal zone B cells (MZ B cells), innate response activator (IRA) cells and together with the newly described innate lymphoid cells (ILCs), are able to rapidly respond to bacterial infections. Strikingly, our recent data suggest that conventional CD4+ T cells, the paradigm of cells of the adaptive immunity, also present innate-like behavior, capturing bacteria in a process called transinfection. Transinfected CD4+ T cells digest internalized bacteria like professional phagocytes and secrete large amounts of pro-inflammatory cytokines, protecting for further bacterial challenges. In the present review, we will focus on the data showing such innate-like behavior of lymphocytes following bacteria encounter

Innate lymphoid cell recovery and occurrence of GvHD after hematopoietic stem cell transplantation

Lymphocytes are essential for microbial immunity, tumor surveillance, and tissue homeostasis. However, the in vivo development and function of helper-like innate lymphoid cells (ILCs) in humans remain much less well understood than those of T, B, and NK cells. We monitored hematopoietic stem cell transplantation (HSCT) to determine the kinetics of ILC development in both children and adults. It was found that, unlike NK cells, helper-like ILCs recovered slowly, mirroring the pattern observed for T cells, with normalization achieved at 1 year. The type of graft and the proportion of CD34+ cells in the graft did not significantly affect ILC reconstitution. As HSCT is often complicated by acute or chronic graft-versus-host disease (GVHD), the potential role of ILC subsets in maintaining tissue integrity in these conditions was also analyzed. It was found that GVHD was associated with lower levels of activated and gut-homing NKp44+ ILCP, consistent with a non-redundant role of this ILC subset in preventing this life-threatening disorder in lymphopenic conditions.Depto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Type 3 Innate Lymphoid Cells as Regulators of the Host-Pathogen Interaction

Type 3 Innate lymphoid cells (ILC3s) have been described as tissue-resident cells and characterized throughout the body, especially in mucosal sites and classical first barrier organs such as skin, gut and lungs, among others. A significant part of the research has focused on their role in combating pathogens, mainly extracellular pathogens, with the gut as the principal organ. However, some recent discoveries in the field have unveiled their activity in other organs, combating intracellular pathogens and as part of the response to viruses. In this review we have compiled the latest studies on the role of ILC3s and the molecular mechanisms involved in defending against different microbes at the mucosal surface, most of these studies have made use of conditional transgenic mice. The present review therefore attempts to provide an overview of the function of ILC3s in infections throughout the body, focusing on their specific activity in different organs.Programa Ramón y CajalMinisterio de Ciencia, Innovación e UniversidadesFondo Europeo de Desarrollo RegionalDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu

Optimized Protocol for Characterization of Mouse Gut Innate Lymphoid Cells

Since their discovery, innate lymphoid cells (ILCs) have gradually been gaining greater relevance in the field of immunology due to their multiple functions in the innate immune response. They can mainly be found in mucosal and barrier organs like skin, gut, and lungs, and have been classified into five main types (NKs, ILC1s, ILC2s, ILC3s, and Lti cells) according to their function and development. They all play major roles in functions such as tissue homeostasis, early pathogen defense, regulation of inflammation, or tissue remodeling. ILCs are mostly tissue-resident cells tightly bound to the tissue structure, a fact that requires long and complex protocols that do not always provide sufficient yield for analysis. This suggests the need for optimized approaches aimed at ensuring that enriched and viable ILC samples are obtained, in order to furnish quality results. Herein a detailed protocol is established for obtaining a single-cell suspension highly enriched in lymphoid cells from mouse gut in order to identify the different subsets of ILCs by means of flow cytometry. The cell marker panel and flow cytometry gating strategies for identification and quantification of all the different ILC populations are provided for simultaneous analysis. Moreover, the protocol described includes a procedure for studying the different cytokines produced by ILC3s involved in maintaining the integrity of the gut barrier and defending against extracellular pathogens. As a result, herein an efficient method is presented for studying mouse ILCs within the lamina propria of the small intestine and colon; this can constitute a useful tool for future investigations in the field.Ministerio de Ciencia, Innovación e UniversidadesAgenda Estatal de InvestigaciónFondo Europeo de Desarrollo RegionalDepto. de Inmunología, Oftalmología y ORLFac. de MedicinaTRUEpu