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2024 Acuerdos transformativos CRUE-CSIC, Both EphB2- and EphB3-deficient mice exhibit profound histological alterations in the thymic epithelial network but few changes in T-cell differentiation, suggesting that this organization would be sufficient to produce functional T lymphocytes. Also, other antigen-presenting cells involved in immunological education could substitute the thymic epithelium. Accordingly, we found an increased frequency of plasmacytoid dendritic cells but not of conventional dendritic cells, medullary fibroblasts or intrathymic B lymphocytes. In addition, there are no lymphoid infiltrates in the organs of mutant mice nor do they contain circulating autoantibodies. Furthermore, attempts to induce arthritic lesions after chicken type II collagen administration fail totally in EphB2-deficient mice whereas all WT and half of the immunized EphB3−/− mice develop a typical collagen-induced arthritis. Our results point out that Th17 cells, IL4-producing Th2 cells and regulatory T cells are key for the induction of disease, but mutant mice appear to have deficits in T cell activation or cell migration properties. EphB2−/− T cells show reduced in vitro proliferative responses to anti-CD3/anti-CD28 antibodies, produce low levels of anti-type II collagen antibodies, and exhibit low proportions of T follicular helper cells. On the contrary, EphB3−/− lymph node cells respond accurately to the different immune stimuli although in lower levels than WT cells but show a significantly reduced migration in in vitro transwell assays, suggesting that no sufficient type II collagen-dependent activated lymphoid cells reached the joints, resulting in reduced arthritic lesions., Unión Europea. NextGenerationEU. Plan de Recuperación Transformación y Resiliencia, Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

CRUE-CSIC (Acuerdos Transformativos 2022), Eph receptors and their ligands, Ephrins, are involved in the thymocyte-thymic epithelial cell (TEC) interactions, key for the functional maturation of both thymocytes and thymic epithelium. Several years ago, we reported that the lack of EphA4, a Eph of the subfamily A, coursed with reduced proportions of double positive (DP) thymocytes apparently due to an altered thymic epithelial stroma [Munoz et al. in J Immunol 177:804–813, 2006]. In the present study, we reevaluate the lymphoid, epithelial, and extracellular matrix (ECM) phenotype of EphA4−/− mice grouped into three categories with respect to their proportions of DP thymocytes. Our results demonstrate a profound hypocellularity, specifc alterations of T cell diferentiation that afected not only DP thymocytes, but also double negative and single positive T cell subsets, as well as the proportions of positively and negatively selected thymocytes. In correlation, thymic histological organization changed markedly, especially in the cortex, as well as the proportions of both Ly51+UEA-1− cortical TECs and Ly51−UEA-1+ medullary TECs. The alterations observed in the expression of ECM components (Fibronectin, Laminin, Collagen IV), integrin receptors (VLA-4, VLA-6), chemokines (CXCL12, CCL25, CCL21) and their receptors (CXCR4, CCR7, CCR9) and in vitro transwell assays on the capacity of migration of WT and mutant thymocytes suggest that the lack of EphA4 alters T-cell diferentiation by presumably afecting cell adhesion between TECs and T-TEC interactions rather than by thymocyte migration., Ministerio de Ciencia e Innovación (MICINN), Instituto de Salud Carlos III (ISCIII), Comunidad de Madrid, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Intimate interactions between thymic epithelial cells (TECs) and thymocytes (T) have been repeatedly reported as essential for performing intrathymic T-cell education. Nevertheless, it has been described that animals exhibiting defects in these interactions were capable of a proper positive and negative T-cell selection. In the current review, we first examined distinct types of TECs and their possible role in the immune surveillance. However, EphB-deficient thymi that exhibit profound thymic epithelial (TE) alterations do not exhibit important immunological defects. Eph and their ligands, the ephrins, are implicated in cell attachment/detachment and govern, therefore, TEC–T interactions. On this basis, we hypothesized that a few normal TE areas could be enough for a proper phenotypical and functional maturation of T lymphocytes. Then, we evaluated in vivo how many TECs would be necessary for supporting a normal T-cell differentiation, concluding that a significantly low number of TEC are still capable of supporting normal T lymphocyte maturation, whereas with fewer numbers, T-cell maturation is not possible., Ministerio de Ciencia e Innovación (MICINN), Comunidad de Madrid, Instituto de Salud Carlos III (ISCIII), Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

The mechanisms that determine the commitment of thymic epithelial precursors to the two major thymic epithelial cell lineages, cTECs and mTECs, remain unknown. Here we show that FoxN1 nu mutation, which abolishes thymic epithelium differentiation, results in the formation of a tubular branched structure according to a typical branching morphogenesis and tubulogenesis developmental pattern. In the presence of FoxN1, in alymphoid NSG and fetal Ikaros−/− thymi, there is no lumen formation and only partial apical differentiation. This initiates cortex–medulla differentiation inducing expression of medullary genes in the apically differentiating cells and of cortical genes in the non-apically differentiating cells, which will definitely differentiate in wt and postnatal Ikaros−/− mice. Therefore, the thymus development is based on a branching morphogenesis and tubulogenesis developmental pattern: FoxN1 expression in the thymic primordium inhibits tubulogenesis and induces the expression of genes involved in TEC differentiation, which culminates with the expression of functional cell markers, i.e., MHCII, CD80, Aire in both postnatal Ikaros−/− and WT thymi after arrival of lymphoid progenitor cells., Dos factores regulan el desarrollo del epitelio tímico, el factor de transcripción Foxn1 y las interacciones célula-célula entre timocitos y epitelio tímico, imprescindible a su vez para la maduración funcional de los timocitos. El trabajo re-evalúa el papel de ambos factores analizando comparativamente el desarrollo epitelial en timos sin Foxn1 ni timocitos (ratones nude), con Foxn1 pero sin timocitos (ratones NSG), con Foxn1 pero sin timocitos hasta después del nacimiento (ratones Ikaros-/-) y ratones WT con ambos factores durante todo el desarrollo. Los resultados confirman que el timo, como otras glándulas derivadas del endodermo, se desarrollan de acuerdo a un patrón de branching morphogenesis en el que la expresión de Foxn1 bloquea la formación del lumen de la glándula al tiempo que induce la diferenciación de genes epiteliales que culmina cuando la llegada de progenitores linfoides al primordio tímico induce la diferenciación de marcadores funcionales (MHC clase II, CD80, Aire)" Juan J. Muñoz, Esther Tobajas, Sonia Juara, Sara Montero, Agustín G. Zapata, Ministerio de Economía y Competitividad (MINECO), Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

This work was supported by the Spanish Ministry of Economy and Competitiveness under grants BFU2013-41112-R and Cell Therapy Network (RD12/0019/0007 and RD16/0011/0002)., EphB and their ligands ephrin-B are an important family of protein tyrosine kinase receptors involved in thymocyte-thymic epithelial cell interactions known to be key for the maturation of both thymic cell components. In the present study, we have analyzed the maturation of cortical thymic epithelium in EphB-deficient thymuses evaluating the relative relevance of EphB2 and EphB3 in the process. Results support a relationship between the epithelial hypocellularity of mutant thymuses and altered development of thymocytes, lower proportions of cycling thymic epithelial cells and increased epithelial cell apoptosis. Together, these factors induce delayed development of mutant cortical TECs, defined by the expression of different cell markers, i.e. Ly51, CD205, MHCII, CD40 and b5t. Furthermore, although both EphB2 and EphB3 are necessary for cortical thymic epithelial maturation, the relevance of EphB3 is greater since EphB3¡/¡ thymic cortex exhibits a more severe phenotype than that of EphB2-deficient thymuses., Ministerio de Economía y Competitividad, Red de Terapía Celular, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

El timo es un órgano linfoide primario responsable del desarrollo funcional de las células T inmunocompetentes. Está compuesto, principalmente, por dos elementos: el epitelio tímico (TEC) y las células linfoides cuyos progenitores colonizan el primordio del órgano alrededor del día 11.5F (Gordon y cols., 2004). Ambos componentes organizan en el timo adulto dos áreas histológica y funcionalmente diferenciadas, corteza y médula, donde acontece la selección positiva y negativa, respectivamente, de los receptores antigénicos de los timocitos en desarrollo, claves para la funcionalidad inmune (Blackburn y Manley, 2004). La maduración del epitelio y los timocitos conlleva interacciones célula a célula en la que estudios anteriores de nuestro grupo habían implicado a los receptores tirosina quinasa Eph y sus ligandos, las ephrinas, y más concretamente, a las EphB2 y EphB3. Nuestros estudios recientemente resumidos (Garcia‐Ceca y cols., 2015) relacionan estas moléculas con muchos de los procesos que suceden en el timo, pero no clarifica su origen durante la ontogenia del órgano ni los componentes celulares y moleculares específicamente implicados. Curiosamente, en ausencia de EphB2 o EphB3 los timos son hipoplásicos (Alfaro y cols., 2008) y muestran profundas alteraciones en la red epitelial que empiezan pronto en el desarrollo (Garcia‐Ceca y cols., 2009a), pero muchos menos defectos en el componente linfoide (Alfaro y cols., 2008)..., The thymus is a primary lymphoid organ concerned with the functional development of immunocompetent T cells. It consists, principally, of two cellular components: the thymic epithelium (TEC) and the lymphoid cells, whose progenitor cells colonize the thymic primordium around E11.5 (Gordon et al., 2004). Both components organize in the thymus two histological and functionally different areas, named cortex and medulla, in which positive and negative selection respectively, of the antigen receptors of developing thymocytes occurs. Both processes are key for reaching the immune functionality (Blackburn y Manley, 2004). The maturation of the two thymic components, epithelium and thymocytes, implies cell‐to‐cell interactions. Our own previous studies had demonstrated the relevance of tyrosine kinase receptors Eph and their ligands, the ephrins; concretely, of EphB2 and EphB3 in that process. Our studies associate these molecules with many of the functional events occurring in the thymus (Garcia‐Ceca et al., 2015), but they do not determine when this involvement appears during ontogeny neither the concrete cell types and molecules involved. Remarkably, the lack of EphB2 or EphB3 results in hypoplastic thymuses (Alfaro et al., 2008) exhibiting important alterations of the epithelial cell network that appear early in ontogeny (Garcia‐Ceca et al., 2009a) and a less severe lymphoid phenotype (Alfaro et al., 2008)...

Adult thymuses lacking either ephrin type B receptor 2 (EphB2) or EphB3, or expressing a truncated form of EphB2, the forward signal-deficient EphB2LacZ, have low numbers of early thymic progenitors (ETPs) and are colonized in vivo by reduced numbers of injected bone marrow (BM) lineage-negative (Lin2) cells. Hematopoietic progenitors from these EphB mutants showed decreased capacities to colonize wild type (WT) thymuses compared with WT precursors, with EphB22/2 cells exhibiting the greatest reduction. WT BM Lin2 cells also showed decreased colonizing capacity into mutant thymuses. The reduction was also more severe in EphB22/2 host thymuses, with a less severe phenotype in the EphB2LacZ thymus. These results suggest a major function for forward signaling through EphB2 and, to a lesser extent, EphB3, in either colonizing progenitor cells or thymic stromal cells, for in vivo adult thymus recruitment. Furthermore, the altered expression of the molecules involved in thymic colonization that occurs in the mutant thymus correlates with the observed colonizing capacities of different mutant mice. Reduced production of CCL21 and CCL25 occurred in the thymus of the 3 EphB-deficient mice, but their expression, similar to that of P-selectin, on blood vessels, the method of entry of progenitor cells into the vascular thymus, only showed a significant reduction in EphB22/2 and EphB32/2 thymuses. Decreased migration into the EphB22/2 thymuses correlated also with reduced expression of both ephrinB1 and ephrinB2, without changes in the EphB2LacZ thymuses. In the EphB32/2 thymuses, only ephrinB1 expression appeared significantly diminished, confirming the relevance of forward signals mediated by the EphB2-ephrinB1 pair in cell recruitment into the adult thymus. J. Leukoc. Biol. 98: 883–896; 2015., Ministerio de Economía y Competitividad (MINECO), Ministerio de Sanidad, Servicios Sociales e Igualdad (España), Ministerio de Educación, Cultura y Deporte y CAPES-DGU, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

Numerous studies emphasize the relevance of thymocyte–thymic epithelial cell (TECs) interactions for the functional maturation of intrathymic T lymphocytes. The tyrosine kinase receptors, Ephs (erythropoietin-producing hepatocyte kinases) and their ligands, ephrins (Eph receptor interaction proteins), are molecules known to be involved in the regulation of numerous biological systems in which cell-to-cell interactions are particularly relevant. In the last years, we and other authors have demonstrated the importance of these molecules in the thymic functions and the T-cell development. In the present report, we review data on the effects of Ephs and ephrins in the functional maturation of both thymic epithelial microenvironment and thymocyte maturation as well as on their role in the lymphoid progenitor recruitment into the thymus., Ministerio de Economía y Competitividad (MINECO), Ministerio de Salud (España), Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub

The signals that determine the survival/death of the thymic epithelial cells (TECs) component during embryonic development of the thymus are largely unknown. In this study, we combine different in vivo and in vitro experimental approaches to define the role played by the tyrosine kinase receptors EphB2 and EphB3 and their ligands, ephrinsB, in the survival of embryonic and newborn (NB) TECs. Our results conclude that EphB2 and EphB3 are involved in the control of TEC survival and that the absence of these molecules causes increased apoptotic TEC proportions that result in decreased numbers of thymic cells and a smaller-sized gland. Furthermore, in vitro studies using either EphB2-Fc or ephrinB1-Fc fusion proteins demonstrate that the blockade of Eph/ephrinB signalling increases TEC apoptosis, whereas its activation rescues TECs from cell death. In these assays, both heterotypic thymocyte–TEC and homotypic TEC–TEC interactions are important for Eph/ephrinB-mediated TEC survival., Ministerio de Educación y Ciencia, Ministerio de Salud y Consumo, Comunidad de Madrid/Universidad Complutense de Madrid, Depto. de Biología Celular, Fac. de Ciencias Biológicas, TRUE, pub