Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin α4β1

The 41 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migra- tion and activation. We show here that 41 and CCR9 are coexpressed mainly on double- and single-positive thymocytes and that CCL25 strongly stimulates CD4CD8 and CD4CD8 - adhesion to CS-1/FN and VCAM-1. CCL25 rap- idly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9/41 human T cell line Molt-4. To study the role of CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTP- interacting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and adapter protein that binds selec- tively to active Rap1 (RIAM), we knocked down their expression and tested transfectant attach- ment to 41 ligands. We found that WAVE2 and RAPL but not RIAM were required for effi- cient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strength- ening but not during initial 41-ligand interac- tions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by 41 could contribute to control their trafficking in the thymus during maturation and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in inside- out signaling, leading to stimulated adhesion. J. Leukoc. Biol. 82: 000-000; 2007.