7). Collectively, these data suggest that EphB4 may contribute to the unique biphasic modulatory effect by ephrin-B1/B2 through

the recruitment of SHP1 (Fig. 6C). In contrast to ephrin-B1/B2, the phospho-EphB4 induced by ephrin-B3 could not couple with SHP1, which has the inhibitory effect of Lck phosphorylation. In this study, we elucidated that ephrin-B1 and ephrin-B2 belong to a novel class of costimulatory molecules with unique action, namely, a concentration-dependent switch from costimulation to inhibition; whereas, ephrin-B3 simply exerts a steadily increasing stimulatory effect in TCR-mediated regulation of primary T cells via Eph receptors other than EphB1/B2/B3/B6. The unique inhibitory effects Peptide 17 by the high concentrations of ephrin-B1/B2 occur buy GSK126 as a consequence of cross-talk of EphB4 signaling on TCR cascade, most likely targeting Lck. Although Eph receptors/ephrin ligands were initially recognized as mediators of repulsive signals in growing axons, it is now clear that their functions

are versatile, including attractive and adhesive property. In vivo, ephrin-Bs have been shown to act as both attractants and repellents for retinal axons during the developmental stage of the visual system [[24]]. However, it remains unclear whether the reciprocal effects in vivo are mediated by the same ephrin ligand in the same cell since these effects are dependent in time and space where the expression of Eph receptors/ephrin ligands would

be variable. Definitive demonstration of biphasic action of this system can be done in in vitro system. Recently, Hansen et al. elegantly demonstrated that ephrin-As induced the biphasic retinal axon growth [[21]]. Alfaro et al. [[7]] demonstrated that immobilized Eph-B2-Fc and ephrin-B1-Fc modulated the anti-CD3 antibody-induced apoptosis of CD4+CD8+ thymocytes in a concentration-dependent C-X-C chemokine receptor type 7 (CXCR-7) manner. Our present study has addressed the direct proof of biphasic effect of ephrin-Bs on the proliferation of the primary immune cells. In addition to the function in cell positioning including attraction, adhesion, and repulsion which have been mainly investigated in the nervous system, our study demonstrated for the first time that this biphasic regulation is functional in cell proliferation, as well. According to the studies for functional determination, Eph receptors can promote adhesion/attraction in a kinase-independent manner; whereas, repulsive function requires tyrosine kinase activity and receptor phosphorylation [[26, 38, 39]]. Eph receptors may possess two distinct functional sites, (i) adhesion by extracellular kinase-independent domain and (ii) repulsive/inhibitory signaling by intracellular kinase-dependent domain. The concentration of ephrin ligands would be one of the factors to determine the balance between these two functions.