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La regulación del citoesqueleto de actina es muy importante en la respuesta inmunitaria. En los linfocitos T CD4+, esta red de actina es esencial para el establecimiento de la sinapsis inmunitaria (SI), una estructura especializada que se forma entre una célula presentadora de antígeno y un linfocito T. En el contexto de la SI, el reconocimiento por parte del receptor de la célula T (TCR) de un péptido antigénico específico presentado en el complejo mayor de histocompatibilidad conlleva el inicio de la señalización mediada por el TCR y reordenamientos del citoesqueleto que estabilizan el contacto. La dinámica del citoesqueleto de actina en los linfocitos T está controlada por un amplio número de proteínas, como cofilina, un factor que promueve el corte y despolimerización de la actina. La interacción y función de cofilina sobre los filamentos de actina están reguladas por varios factores entre los que se encuentran las proteínas WDR1 y coronina, cuyas mutaciones en humanos resultan en cuadros de inmunodeficiencia. Un correcto establecimiento de la SI es esencial para conseguir una completa activación y función efectora del linfocito T llevada a cabo por la señalización del TCR, el cual inicia una cascada de fosforilaciones y desfosforilaciones de proteínas regulada por quinasas y fosfatasas. Las fosfatasas de regeneración hepática (PRLs: PRL-1, PRL-2 y PRL-3) se expresan en las células T, pero su papel en la activación del linfocito T está aún por explorar. Nuestro grupo ha demostrado que la activación T causa una bajada de expresión de PRL-2, que resulta en un incremento del ratio PRL-1/PRL-2 en la célula, y que la PRL-1 sobreexpresada se polariza a la SI en la línea celular T CD4+ Jurkat (JK). Además, el uso de fármacos inhibidores de la actividad catalítica de las PRLs reduce la polarización de la actina filamentosa (F-actina) a la SI y la secreción de interleuquina-2 (IL-2) tras la estimulación del linfocito T. Estos resultados sugieren que las PRLs poseen un ro, Regulation of the actin cytoskeleton is essential for the immune response. In CD4+ T cells, the actin networks are important during the assembly of the immune synapse (IS), a specialized structure formed between an antigen presenting cell and a T lymphocyte. In the IS context, the T cell receptor (TCR) engagement with a specific antigenic peptide presented on the major histocompatibility complex results in TCR downstream signalling and cytoskeletal rearrangements that stabilize the contact. Actin cytoskeleton dynamics in T cells is controlled by a wide range of proteins, including the severing and depolymerization factor cofilin. Cofilin interaction and function on actin filaments is controlled by several factors such as WDR1 and coronin proteins, whose mutations result in human immunodeficiencies.Proper IS formation is essential to achieve full T cell activation and effector function driven by TCR signalling, which starts a cascade of phosphorylation and dephosphorylation of proteins regulated by kinases and phosphatases. The phosphatases of regenerating liver (PRLs: PRL-1, PRL-2 and PRL-3) are expressed in T lymphocytes and their regulatory role during T cell activation is still to explore. Our group have showed that T cell activation downregulates the expression of PRL-2, resulting in an increased PRL-1/PRL-2 ratio in the cell, and that overexpressed PRL-1 is delivered to the IS formed by the Jurkat CD4+ T cell line (JK). Moreover, pharmacological inhibition of the catalytic activity of the PRLs reduces actin polarization at the IS and secretion of interleukin-2 (IL-2). These results suggest a regulatory role of PRLs during T cell activation, but the exact molecular mechanism is unknown...

This work was supported by funds of the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2012-33218 and SAF2016-75656-P) and the European Union (FP7-PEOPLE-2012-CIG) to P. R.-N. These projects support P. C.-S. and R. R.-M. The PEARL study is supported by PI14/00442 to IG-A and the work of A. L. was supported by PIE13/00041, both grants from the MINECO (‘Instituto de Salud Carlos III’), co-funded by ‘Fondo Europeo de Desarrollo Regional’ (FEDER). A. L. was co-funded by SER (Sociedad Espanola de Reumatolog ~ ıa) to A. O., Phosphotyrosine phosphatases (PTPs) control phosphorylation levels and, consequently, regulate the output of intracellular signalling networks in health and disease. Despite the high number of PTPs expressed in CD4 T cells and their involvement in autoimmunity, information about the expression profile of PTPs in these cells has not been obtained in patients diagnosed with autoimmune diseases. Here, we compare the expression profile of PTPs in CD4 T cells of healthy volunteers and patients submitted to an early arthritis clinic, due to suspicion of rheumatoid arthritis, an autoimmune disease mediated by CD4 T cells. We found lower transcript levels of the mitogen-activated protein kinase (MAPK) phosphatase dual-specific phosphatase-7 (DUSP7) and the cell division cycle-25B (CDC25B) in T cells of patients. While the low expression level of DUSP7 was restricted to patients with positive rheumatoid factor and anti-citrullinated protein antibodies, the altered expression of CDC25B correlated with the activity of the disease. Low levels of CDC25B might contribute to the progression of the autoimmune arthritis and/or might be consequence of the inflammatory environment in the active disease. The possible role of DUSP7 and CDC25B as biomarkers of the disease in clinical protocols is discussed., Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Nuclear deformability plays a critical role in cell migration. During this process, the remodeling of internal components of the nucleus has a direct impact on DNA damage and cell behavior; however, how persistent migration promotes nuclear changes leading to phenotypical and functional consequences remains poorly understood. Here, we described that the persistent migration through physical barriers was sufficient to promote permanent modifications in migratory-altered cells. We found that derived cells from confined migration showed changes in lamin B1 localization, cell morphology and transcription. Further analysis confirmed that migratory-altered cells showed functional differences in DNA repair, cell response to chemotherapy and cell migration in vivo homing experiments. Experimental modulation of actin polymerization affected the redistribution of lamin B1, and the basal levels of DNA damage in migratory-altered cells. Finally, since major nuclear changes were present in migratory-altered cells, we applied a multidisciplinary biochemical and biophysical approach to identify that confined conditions promoted a different biomechanical response of the nucleus in migratory-altered cells. Our observations suggest that mechanical compression during persistent cell migration has a role in stable nuclear and genomic alterations that might handle the genetic instability and cellular heterogeneity in aging diseases and cancer.

Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR−EpCAM−) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity.

La respuesta immunitaria adaptativa depende de la correcta regulación del citoesqueleto de actina. En los linfocitos T, el citoesqueleto es importante durante el reconocimiento antigénico, en la formación de la sinapsis inmunitaria (IS) y en la migración celular. Como parte de la IS, se produce la reorganización del citoesqueleto de actina por la actividad de proteínas reguladores. Estas proteínas reguladoras son también importantes para mantener la direccionalidad celular durante la migración. El citoesqueleto de actina también es importante en las células dendríticas (DCs), tanto en su estado inmaduro para facilitar su movimiento en los tejidos, gracias a unas estructuras denominadas podosomas, como en células maduras, facilitando la migración hacia los nódulos linfáticos y favoreciendo la presentación de antígenos. El citoesqueleto de actina está formado por monómeros que polimerizan formando filamentos. Estos filamentos se encuentran continuamente polimerizando en un extremo y depolimerizando en el otro, en una dinámica denominada “actin turnover”, regulado por proteínas que se encargan de controlar la polimerización, nucleación, organización y depolimerización del citoesqueleto. Una de estas proteínas es cofilina, un factor desestabilizante de actina que fragmenta filamentos y favorece la depolimerización del citoesqueleto. Cofilina es una proteína de especial interés en la regulación de procesos inmunitarios, como la IS o la migración celular. La actividad de cofilina está a su vez regulada por una serie de proteínas, entre las que se incluye la fosfatasa Slingshot-1 (SSH1), responsable de la defosforilación y activación de cofilina. A pesar de su importancia como regulador de cofilina, el papel de SSH1 en el sistema inmunitario apenas ha sido estudiado..., Regulation of the actin cytoskeleton is essential for the adaptive immune response. In CD4 T cells, the actin cytoskeleton is important during antigen recognition, in the assembly of the immune synapse (IS) and during cell migration. During the IS, the actin cytoskeleton is rearranged, due to the activation of many actin regulating proteins. These proteins are also important to regulate the directionality of movement during cell migration. Actin cytoskeleton is also important in dendritic cells (DCs), in both its immature state, facilitating their movement in the tissues, aided by specialized actin structures termed podosomes, and in mature cells, increasing their migration speed towards the lymph nodes and favouring antigen presentation.The actin cytoskeleton is formed mainly by actin monomers that polymerise forming actin filaments. These filaments are continuously polymerising in an end and depolymerising in the other, in a process termed “actin turnover”, tightly regulated by proteins that are specialized in controlling polymerization, nucleation, organization and depolymerisation. One of these is cofilin, an actin destabilising factor that severs actin filaments and increase the depolymerisation rate, and has been identified as an actin regulatory protein essential for immune processes such as the IS or cell migration. Activity of cofilin is regulated by a number or proteins, including the phosphatase Slingshot-1 (SSH1), responsible for the dephosphorylation and activation of cofilin. Despite being a major activator of cofilin, the role of SSH1 in the immune system has been poorly studied...

El proyecto tiene como objetivo proporcionar a los alumnos del máster de Investigación en Inmunología de la UCM una formación teórica y práctica para la presentación de trabajos científicos en congresos en formatos de presentación oral y en póster.

T cell-redirecting strategies have emerged as effective cancer immunotherapy approaches. Bispecific antibodies (bsAbs) are designed to specifically recruit T cells to the tumor microenvironment and induce the assembly of the immunological synapse (IS) between T cells and cancer cells or antigen-presenting cells. The way that the quality of the IS might predict the effectiveness of T cell-redirecting strategies, including those mediated by bsAbs or by chimeric antigen receptors (CAR)-T cells, is currently under discussion. Here we review the organization of the canonical IS assembled during natural antigenic stimulation through the T cell receptor (TCR) and to what extent different bsAbs induce T cell activation, canonical IS organization, and effector function. Then, we discuss how the biochemical parameters of different formats of bsAbs affect the effectivity of generating an antigen-induced canonical IS. Finally, the quality of the IS assembled by bsAbs and monoclonal antibodies or CAR-T cells are compared, and strategies to improve bsAb-mediated T cell-redirecting strategies are discussed., Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Fundación CRIS del Cáncer, Asociación Española Contra el Cáncer, Fundación “La Caixa”, Fundación de Investigación Biomédica 12 de Octubre, Depto. de Inmunología, Oftalmología y ORL, Fac. de Medicina, TRUE, pub

Este proyecto se basa en diseñar una estrategia para la internalización de las asignaturas de grado y posgrado de la Facultad de Medicina, especialmente los impartidos por el Departamento de Inmunología, Oftalmología y ORL., This project is based on the design of a strategy to internalize the main courses for the BSc and MSc at the School of Medicine, especially by the subjects taught at the Department of Immunology, Ophthalmology and ORL.

Ministerio de Economía y Competitividad (MINECO), Depto. de Inmunología, Oftalmología y ORL, Fac. de Medicina, TRUE, pub

Los linfocitos CD4 naíf se diferencian a distintos subtipos de células efectoras Th (Th1, Th2, Th17 y Treg) tras ser estimulados por células presentadoras de antígeno (APCs) en el ganglio linfático. Las células Th generadas migran a la periferia, donde un segundo encuentro con el mismo antígeno presentado por APCs conducirá a su activación y función efectora. Tanto la activación inicial de la célula naíf en el ganglio como la reactivación de la célula efectora en la periferia tienen lugar en el contexto de la sinapsis inmunológica (IS), un contacto dinámico y especializado entre una APC y un linfocito. El contacto inicial entre el linfocito T y la APC ocurre en el lamelipodio, donde la estimulación del receptor de la célula T (TCR) y la señalización a través de moléculas de adhesión y de coestimulación induce reordenamientos en el citoesqueleto que estabilizan el contacto, conduciendo a la formación de una IS madura. La estimulación por TCR y moléculas coestimuladoras desencadena redes de señalización que conducen a la activación del linfocito T y su función efectora. La dinámica de actina juega un papel fundamental tanto en el establecimiento de la IS como en la progresión de la señalización necesaria para alcanzar la completa activación del linfocito T., Stimulation of naïve CD4 T cells in the lymph node by antigen presenting cells (APCs) induces their differentiation into effector T helper (Th) cells. Effector Th cells then migrate to peripheral sites, where a secondary encounter with the same antigen presented by APCs will lead to their activation and effector function, including secretion of appropriate cytokines. Both the initial activation of naïve cells in the lymph node and the reactivation of effector cells in the periphery take place in the context of the immunological synapse (IS). The IS is a specialized dynamic contact between an APC and a lymphocyte. Initial contact between a migrating T cell and an APC takes place at the leading lamellipodium, where T cell receptor (TCR) engagement and signaling through adhesion and costimulatory molecules induces cytoskeletal rearrangements that stabilize the contact, leading to the formation of a mature IS. Recognition of the antigen by the TCR, together with costimulation, triggers the activation of signaling networks that lead to full T cell activation and effector function. Actin dynamics play a central role in both the establishment of the IS and the progression of sustained signaling required for full T cell activation...

Durante la activación del linfocito T, se establece una interacción estrecha con la célula presentadora de antígeno (APC). Esta unión se denomina sinapsis inmunológica (SI) y es necesaria para la correcta activación del linfocito. La formación de la SI implica la polarización de orgánulos celulares y múltiples moléculas de señalización hacia la zona de contacto, donde se reorganizan diferencialmente en anillos concéntricos o SMACs (SupraMolecular Activation Clusters). En la zona más periférica de la SI (dSMAC) se acumula la actina, cuya dinámica es esencial no sólo para el ensamblaje de la SI sino también para las funciones efectoras de los linfocitos. La necesidad de una correcta dinámica de actina hace muy interesante el estudio de las proteínas que la regulan. Una de las más importantes es cofilina, que a su vez está activada por la fosfatasa SSH1, cuyo papel durante la activación linfocitaria se desconoce actualmente..., During T-cell activation, a stable interaction with the antigen-presenting cell (APC) is established. This junction is called the immunological synapse (IS) and is necessary for the proper activation of T cells. The formation of the IS involves the polarization of cell organelles and multiple signaling molecules towards the contact area, where they are differentially reorganized in concentric rings or SMACS (SupraMolecular Activation Clusters). In the outermost part of the IS (dSMAC), the actin is accumulated, and its dynamics are essential not only for the assembly of the IS, but also for the effector functions of the lymphocytes. The need for proper actin dynamics makes the study of its regulatory proteins very interesting. One of the most important proteins is cofilin, which is in turn activated by the phosphatase SSH1, whose role during T-cell activation is currently unknown...

Durante la activación del linfocito T, se establece una interacción estrecha con la célula presentadora de antígeno (APC). Esta unión se denomina sinapsis inmunológica (SI) y es necesaria para la correcta activación del linfocito. La formación de la SI implica la polarización de orgánulos celulares y múltiples moléculas de señalización hacia la zona de contacto, donde se reorganizan diferencialmente en anillos concéntricos o SMACs (SupraMolecular Activation Clusters). En la zona más periférica de la SI (dSMAC) se acumula la actina, cuya dinámica es esencial no sólo para el ensamblaje de la SI sino también para las funciones efectoras de los linfocitos. La necesidad de una correcta dinámica de actina hace muy interesante el estudio de las proteínas que la regulan. Una de las más importantes es cofilina, que a su vez está activada por la fosfatasa SSH1, cuyo papel durante la activación linfocitaria se desconoce actualmente...