A Novel Feedback Mechanism by Ephrin-B1/B2 In T Cell Activation: Concentration-Dependent Switch From Costimulation to Inhibition

Abstract 277

Eph is the largest known family of receptor tyrosine kinases, and bind to a cell surface-associated ligand, ephrin on neighboring cells upon direct cell-cell contact. The ensuing bidirectional signals have been recognized as a major form of contact-dependent cell communications, such as cell attraction and repulsion to control accurate spatial and temporal patterning in the development of the central nervous system. EphBs, EphB6 in particular, are expressed in T cells and its specific ligand, ephrin-B2 has been shown to act as a costimulatory molecule for the T cell receptor (TCR)-mediated cell proliferation. Recently, another remarkable feature of ephrins, a concentration-dependent transition from promotion to inhibition in axon growth has emerged in ephrin-As. Thus, we postulated that this type of ligand concentration dependent functional transition would be suitable for the delicate tuning of immune responses to avoid reckless drive. To figure this out, we carefully evaluated the costimulatory effects of ephrin-Bs by using murine primary T cells. Interestingly, low doses of solid phase ephrin-B1 as well as ephrin-B2 (at up to 5μ g/ml) costimulated, to the comparable level with anti-CD28, T cell proliferation induced by suboptimal concentration of immobilized anti-CD3 antibody, but high concentrations of ephrin-B1/B2 inhibited the TCR-mediated proliferation significantly (by approximately 70% reduction from the baseline at 20μ g/ml). The similar concentration-dependent transition from coactivation to inhibition was also observed under the optimal CD3 stimulation. The concentration-dependent biphasic effects, positively at low concentration and negatively at high concentration, by ephrin-B1/B2 in T cell activation were confirmed in the cytokine production such as TNF-α, IL-2, and IFN-γ. In contrast, ephrin-B3 showed steadily increasing stimulatory effect even in higher concentrations in proliferation and cytokine production. We speculated that these unique modulations were partly mediated by EphB6 because EphB6 transfected in HEK293T cells has been shown to exert biphasic effects in cell adhesion and migration in response to different concentrations of ephrin-B2. T cell derived from Ephb6 -/- mice showed decreased CD3-stimulated cell proliferation as reported previously. However, the unique comodulatory pattern by each ephrin-B was virtually preserved in Ephb6 -/- T cells. Since the functions of Eph family could be redundant, we further investigated by generating multiple EphB knockout mice lacking four genes, Ephb1, Ephb2, Ephb3 and Ephb6. Surprisingly, no further alteration was observed in T cells from the quadruple knockout mice compared to the Ephb6 single deficiency. We also confirmed that EphA4, an exception in EphA receptor family which binds ephrin-Bs, was not expressed in T cells by RT-PCR. Taken together with the fact that EphB5 does not exist in mammals, the unique comodification by ephrin-Bs might be regulated by EphB4. Next, we examined the cross-talk of EphB forward signaling with TCR pathway. The inhibitor of p38^MAPK and p44/42^MAPK significantly reduced the TCR-mediated proliferation, but did conserve the concentration-dependent effects of ephrin-B1/B2, suggesting the interference with EphB signaling in TCR signal transduction at the upstream of MAPKs which are important for cell growth and survival. Immuno-blot analyses revealed that high concentrations of ephrin-B1/B2, but not ephrin-B3, clearly inhibited the anti-CD3 induced phosphorylation of Lck and its downstream signaling molecules such as ZAP70, c-Raf, MEK1/2, Erk, and Akt, although the phosphorylation of CD3ζ was not inhibited by high concentrations of any ephrin-Bs. These data suggest that Eph signaling upon stimulation by high concentrations of ephrin-B1/B2 may engage in negative feedback to TCR signals via Lck. The present studies demonstrate that TCR-mediated primary T cell activation may be highly governed by EphB/ephrin-B axis with a complexity determined by the combination as well as the concentration of different ephrin-Bs expressed in immunological microenvironments. EphB-involved in negative feedback of T cell activation could be a novel therapeutic target to inhibit the most proximal TCR signaling molecule, Lck. The generation of strong signaling molecule which mimics ephrin-B1/B2 would be an effective strategy to control T cell mediated immune disorders.