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-dayold fetal mice decreases the numbers of both double-positive (CD4 + CD8 + ) and singlepositive (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.

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, EphB2(LacZ) 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 EphB2(LacZ) 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.