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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. [ABSTRACT FROM AUTHOR]

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, specific alterations of T cell differentiation that affected 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 differentiation by presumably affecting cell adhesion between TECs and T-TEC interactions rather than by thymocyte migration. [ABSTRACT FROM AUTHOR]

Epithelial-free areas, present in both thymic cortex and medulla, have been studied in WT and EphB-deficient mice that have important alterations in the development of thymic epithelium due to the lack of proper thymocyte-thymic epithelial cell interactions. In both WT and mutant thymuses, the number and size of epithelial-free areas are significantly larger in the medulla than in the cortex. The two parameters show a reverse correlation: low numbers of these areas course with large epithelial-free areas and vice versa. However, their structure and cell content are similar in mutant and WT thymuses. Cortical epithelial-free areas just contain DP thymocytes, while the medullary ones consist of SP cells, blood vessels, mesenchyme-derived ER-TR7 cells and components of the extracellular matrix (i.e., collagen IV, fibronectin, laminin). Other components, such as desmin, αSMA, PDGFRβ and Ng2, frequently associated with blood vessel walls, also appear. Vimentin, although present in medullary epithelial-free areas, does not co-express with epithelial cells. Other markers related to epithelial-mesenchymal transitions, such as Snail, Slug or FSP1, are not expressed. These results suggest that alterations in the cell interactions between distinct thymic cell components that induce both increased proportions of apoptotic thymic epithelial cells and altered behavior of the mesenchyme associated with the medullary vasculature could explain the appearance of these areas and their differences in the cortex and medulla. [ABSTRACT FROM AUTHOR]

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 β5t. 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. [ABSTRACT FROM AUTHOR]

Thymus development is a complex process in which cell-to-cell interactions between thymocytes and thymic epithelial cells (TECs) are essential to allow a proper maturation of both thymic cell components. Although signals that control thymocyte development are well known, mechanisms governing TEC maturation are poorly understood, especially those that regulate the maturation of immature TEC populations during early fetal thymus development. In this study, we show that EphB2-deficient, EphB2LacZ and EphB3-deficient fetal thymuses present a lower number of cells and delayed maturation of DN cell subsets compared to WT values. Moreover, deficits in the production of chemokines, known to be involved in the lymphoid seeding into the thymus, contribute in decreased proportions of intrathymic T cell progenitors (PIRA/B) in the mutant thymuses from early stages of development. These features correlate with increased proportions of MTS20 cells but fewer MTS20 cells from E13.5 onward in the deficient thymuses, suggesting a delayed development of the first epithelial cells. In addition, in vitro the lack of thymocytes or the blockade of Eph/ephrin-B-mediated cell-to-cell interactions between either thymocytes-TECs or TECs-TECs in E13.5 fetal thymic lobes coursed with increased proportions of MTS20 TECs. This confirms, for the first time, that the presence of CD45 cells, corresponding at these stages to DN1 and DN2 cells, and Eph/ephrin-B-mediated heterotypic or homotypic cell interactions between thymocytes and TECs, or between TECs and themselves, contribute to the early maturation of MTS20 TECs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Eph receptors and their ligands, ephrins, are molecules involved in the morphogenesis of numerous tissues, including the central nervous system in which they play a key role in determining cell positioning and tissue domains containing or excluding nerve fibers. Because common features have been suggested to occur in the microenvironmental organization of brain and thymus, a highly compartmentalized organ central for T cell differentiation, we examined the expression and possible role of Eph/ephrins in the biology of the thymus gland. We reviewed numerous in vivo and in vitro results that confirm a role for Eph and ephrins in the maturation of the thymic epithelial cell (TEC) network and T cell differentiation. Their possible involvement in different steps of early thymus organogenesis, including thymus primordium branching, lymphoid colonization, and thymocyte–TEC interactions, that determine the organization of a mature three-dimensional thymic epithelial network is also analyzed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In order to carry out an in-depth study of the roles of EphB receptors in T-cell development and to determine the specific relevance of forward and reverse signals in the process, we established severe combined immunodeficient (SCID) mice chimeras with wild-type (WT) or EphB-deficient bone marrow cells. The obtained results demonstrate that EphB2 contributes more significantly than EphB3 in the control of CD4−CD8− (DN)-CD4+CD8+ (DP) progression, and that reverse signals generated in SCID mice receiving EphB2LacZ precursors, which express the EphB2 extracellular domain, partially rescue the blockade of DN cell maturation observed in EphB2-null chimeras. In addition, increased apoptotic DP thymocytes occurring in EphB2 and/or EphB3 SCID chimeras also contribute to the reduced proportions of DP cells. However, EphB2LacZ chimeras do not show any changes in the proportions of apoptotic DP cells, thus suggesting that there is a role for ephrinB reverse signaling in thymocyte survival. The maturation of DP to CD4+CD8− or CD4−CD8+ seems to need EphB2 forward signaling and EphB3; a fact that was confirmed in reaggregates formed with either EphB2- or EphB3-deficient DP thymocytes and WT thymic epithelial cells (TECs). The DP thymocyte-TEC conjugate formation was also affected by the absence of EphB receptors. Finally, EphB-deficient SCID chimeras show profoundly altered thymic epithelial organization that confirms a significant role for EphB2 and EphB3 receptors in the thymocyte-TEC crosstalk. [ABSTRACT FROM AUTHOR]

In the present study, we have analysed the phenotype of EphB2 and/or EphB3 deficient thymocytes confirming and extending previous studies on the role of this family of molecules in T-cell differentiation. In all mutant thymuses statistically significant reduced cell contents were observed. This reduction of thymic cellularity correlated with increased proportions of apoptotic cells, largely both double negative (DN; CD4− CD8−) and double positive (CD4+ CD8+) cells, and decreased proportions of DN cycling cells. Adult deficient thymuses also showed increased proportions of DN cells but not significant variations in the percentages of other thymocyte subsets. In absolute terms, the thymocyte number decreased significantly in all thymocyte compartments from the DN3 (CD44− CD25+) cell stage onward, without variations in the numbers of both DN1 (CD44+ CD25−) and DN2 (CD44+ CD25+) cells. Remarkably, all these changes also occurred from the 15-day fetal EphB2 and/or EphB3 deficient mice, suggesting that adult phenotype results from the gradual accumulations of defects appearing early in the thymus ontogeny. As a reflection of thymus condition, a reduction in the number of T lymphocytes occurred in the peripheral blood and mesenteric lymph nodes, but not in spleen, maintaining the proportions of T-cell subsets defined by CD4/CD8 marker expression, in all cases. [ABSTRACT FROM AUTHOR]

Our understanding about medullary compartment, its niches composition and formation is still limited. Previous studies using EphB2 and/or EphB3 knockout mice showed an abnormal thymic development that affects mainly to the epithelial component, including the cortex/medulla distribution, thymic epithelial cell (TEC) morphology and different epithelial-specific marker expression. We have already demonstrated that the lack of ephrinB1 and/or ephrinB2, either on thymocytes or on TECs, alters the cell intermingling processes necessary for thymus organization and affect cortical TEC subpopulations. In the present work, we have used the Cre-LoxP model to selectively delete ephrinB1 and/or ephrinB2 in thymocytes (EfnB1, EfnB2, EfnB1EfnB2 mice) or TECs (EfnB1, EfnB2, EfnB1EfnB2 mice) and have analyzed their role on the medullary compartment. In all the studied mutants, medullary areas are smaller and more compact than in the wt thymuses. In most of them, we observe abundant big cysts and a higher proportion of UEAMTS10 cells than in wt mice, which are often forming small cysts. On EfnB1EfnB2, changes affecting organ size and medullary compartment start at perinatal stage. Our data shed some light on knowledge about wt medulla histological structure and cysts meaning and formation process and on the role played by ephrinB in them. [ABSTRACT FROM AUTHOR]

Previous analysis on the thymus of erythropoietin-producing hepatocyte kinases (Eph) B knockout mice and chimeras revealed that Eph-Eph receptor-interacting proteins (ephrins) are expressed both on T cells and thymic epithelial cells (TECs) and play a role in defining the thymus microenvironments. In the current study, we have used the Cre-LoxP system to selectively delete ephrin-B1 and/or ephrin-B2 in either thymocytes (EfnB1thy/thy, EfnB2thy/thy, and EfnB1thy/thyEfnB2thy/thy mice) or TECs (EfnB1tec/tec, EfnB2tec/tec, and EfnB1tec/tecEfnB2tec/tec mice) and determine the relevance of these Eph ligands in T cell differentiation and thymus histology. Our results indicate that ephrin-B1 and ephrin-B2 expressed on thymocytes play an autonomous role in T cell development and, expressed on TECs, their nonautonomous roles are partially overlapping. The effects of the lack of ephrin-B1 and/or ephrin-B2 on either thymocytes or TECs are more severe and specific on thymic epithelium, contribute to the cell intermingling necessary for thymus organization, and affect cortical TEC subpopulation phenotype and location. Moreover, ephrin-B1 and ephrin-B2 seem to be involved in the temporal appearance of distinct cortical TECs subsets defined by different Ly51 levels of expression on the ontogeny. [ABSTRACT FROM AUTHOR]