Andrew Freywald

Ephrin stimulation modulates T cell chemotaxis

Eph receptor tyrosine kinases and their ligands, the ephrins, are known to play an important role in regulating cell migration and targeting in neuronal and endothelial cells. Recently, it hasbeen shown that lymphoid cells also express Eph receptors, raising the possibility that Eph receptors may similarly regulate lymphocyte migration. Chemotaxis in response to soluble chemokine factors is an essential facet of T cell biology. We demonstrate here that T cell chemotaxis in response to both the stromal cell‐derived factor (SDF)‐1α and macrophage inflammatory protein 3β chemokines is modulated by costimulation with ephrins. Both ephrin‐A and ephrin‐B ligands were found to modify the chemotactic responses of a T cell line and primary T cells. Ephrin‐A1, in particular, strongly inhibited chemotaxis. In accordance with the tyrosine kinase activity of EphA receptors, ephrin‐A1 stimulation induced rapid intracellular tyrosine phosphorylation in T cells. Although strongly inhibiting chemotaxis, ephrin‐A1 costimulus did not affect many of the signaling events downstream of the SDF‐1α receptor CXCR4, including calcium flux and activation of MAPK. Rather, ephrin‐A1 altered the balance of small G protein activity in T cells. Ephrin‐A1 stimulation prevented SDF‐1α–induced activation of cdc42, while simultaneously inducing rho activation. Ultimately, ephrin‐A1 was found to inhibit chemokine‐induced actin polymerization, thereby blocking migration. Ubiquitous ephrin expression in vivo creates enormous potential for T cells to encounter these ligands, suggesting that Eph receptors and ephrins may be important regulators of T cell migration.

Ephrin-A1 induces c-Cbl phosphorylation and EphA receptor down-regulation in T cells.

Eph receptor tyrosine kinases are expressed by T lineage cells, and stimulation with their ligands, the ephrins, has recently been shown to modulate T cell behavior. We show that ephrin-A1 stimulation of Jurkat T cells induces tyrosine phosphorylation of EphA3 receptors and cytoplasmic proteins, including the c-cbl proto-oncogene. Cbl phosphorylation was also observed in peripheral blood T cells. In contrast, stimulation of Jurkat cells with the EphB receptor ligand ephrin-B1 does not cause Cbl phosphorylation. EphA activation also induced Cbl association with Crk-L and Crk-II adapters, but not the related Grb2 protein. Induction of Cbl phosphorylation upon EphA activation appeared to be dependent upon Src family kinase activity, as Cbl phosphorylation was selectively abrogated by the Src family inhibitor 4-amino-5(4-chlorophenyl-7-(tert-butyl)pyrazolo[3,4-d]pyrimidine, while EphA phosphorylation was unimpaired. Ephrin-A1 stimulation of Jurkat cells was also found to cause down-regulation of endogenous EphA3 receptors from the cell surface and their degradation. In accordance with the role of Cbl as a negative regulator of receptor tyrosine kinases, overexpression of wild-type Cbl, but not its 70-Z mutant, was found to down-regulate EphA receptor expression. Receptor down-regulation could also be inhibited by blockage of Src family kinase activity. Our findings show that EphA receptors can actively signal in T cells, and that Cbl performs multiple roles in this signaling pathway, functioning to transduce signals from the receptors as well as regulating activated EphA receptor expression.