In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4⁺CD8⁺) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 ^−/− phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44⁻CD25⁺ triple-negative cells and, sometimes, of CD44⁺CD25⁻ triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 ^−/− epithelial cells affects their development and finally results in altered T cell development.

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2‐Fc or ephrinB1‐Fc fusion proteins to fetal thymus organ cultures established from 17‐day‐old fetal mice decreases the numbers of both double‐positive (CD4⁺CD8⁺) and single‐positive (both CD4⁺CD8^– and CD4^–CD8⁺) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4⁺CD8⁺ thymocytes, we have also demonstrated that ephrinB1‐Fc proteins are able to disorganize the three‐dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB‐Fc treatment also decreases the formation of cell conjugates by CD4⁺CD8⁺ thymocytes and TEC as well as the TCR‐dependent signaling between both cell types. Finally, immobilized EphB2‐Fc and ephrinB1‐Fc modulate the anti‐CD3 antibody‐induced apoptosis of CD4⁺CD8⁺ thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three‐dimensional organization of TEC.

Recruitment of lymphoid progenitors to the thymus is compromised by the lack of Eph/ephrin signaling in both T-cell progenitors and thymic microenvironmental cells The ephrin-Eph ligand receptor pair is known to control the repulsion/adhesion process in different tissues, including the immune system. Herein, we evaluated the role of EphB2 receptors in T cell progenitor migration during in vitro thymus colonization and to ECM or chemokine stimuli. EphB2 and their ligands, ephrin-B1 and ephrin-B2, are expressed in BM-derived progenitors, and EphB2−/− cells had diminished thymus colonization capacity. Conversely, EphB2LacZ cells, which maintain a preserved ephrin-binding domain, were capable of colonizing WT thymuses similarly to WT progenitors, highlighting the importance of reverse signals transmitted to normal fetal thymus. However, the EphB2 receptor expressed by microenvironmental cells also drives progenitor immigration, as recolonization of EphB2-deficient fetal thymuses was compromised profoundly. Additionally, we observed lower depositions of ECM and chemokines on EphB2-deficient thymuses but no changes in their receptor expression on BM-derived progenitors and developing thymocytes. Migration of EphB2-deficient progenitors and thymocytes was also reduced through ECM or chemokine stimuli. Furthermore, ephrin-B1 costimulation also inhibited haptotaxis and chemotaxis of WT but not EphB2LacZ cells, demonstrating the specific involvement of EphB2 signaling on T cell progenitor migration. Our data suggest the relevance of a nonactivated EphB2 for regulating T cell progenitor migration and its modulation upon ephrin-B engagement.

Summary: 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.

In the current study, we extend our own previous results on the thymocyte phenotype of EphB2 and/or EphB3 deficient mice by analyzing the phenotype and the histological organization of their thymic epithelial stroma. All studied adult EphB-deficient thymi showed profound alterations with respect to the wild-type (WT) ones. Each mutant exhibited a specific phenotype, but also showed common features including occurrence of K5+K8+MTS10+ immature medullary epithelial cells, numerous K5-K8-MTS20+ cells and K5+K8+ cells in the thymic cortex and cortical and medullary K5-K8- areas devoid of epithelial cell markers. In addition, comparative analysis of WT and EphB-deficient embryonic and newborn thymi demonstrated that the observed adult phenotype was a consequence of the gradual accumulation of early phenotypic and morphological defects, becoming more severe at the end of embryonic life and in newborn animals. Together, these results confirm a role for EphB2 and EphB3 in thymus morphogenesis. The obtained data are discussed from the point of view of the recognized role played by these two Ephs in the homeostasis of other epithelia and their possible relationships with molecules known to be involved in thymic epithelial cell development.