CCR7 signalosomes are preassembled on tips of lymphocyte microvilli in proximity to LFA-1

Abstract

Leukocyte microvilli are elastic actin-rich projections implicated in rapid sensing and penetration across glycocalyx barriers. Microvilli are critical for the capture and arrest of flowing lymphocytes by high endothelial venules, the main lymph node portal vessels. T lymphocyte arrest involves subsecond activation of the integrin LFA-1 by the G-protein-coupled receptor CCR7 and its endothelial-displayed ligands, the chemokines CCL21 and CCL19. The topographical distribution of CCR7 and of LFA-1 in relation to lymphocyte microvilli has never been elucidated. We applied the recently developed microvillar cartography imaging technique to determine the topographical distribution of CCR7 and LFA-1 with respect to microvilli on peripheral blood T lymphocytes. We found that CCR7 is clustered on the tips of T cell microvilli. The vast majority of LFA-1 molecules were found on the cell body, likely assembled in macroclusters, but a subset of LFA-1, 5% of the total, were found scattered within 20 nm from the CCR7 clusters, implicating these LFA-1 molecules as targets for inside-out activation signals transmitted within a fraction of a second by chemokine-bound CCR7. Indeed, RhoA, the key GTPase involved in rapid LFA-1 affinity triggering by CCR7, was also found to be clustered near CCR7. In addition, we observed that the tyrosine kinase JAK2 controls CCR7-mediated LFA-1 affinity triggering and is also highly enriched on tips of microvilli. We propose that tips of lymphocyte microvilli are novel signalosomes for subsecond CCR7-mediated inside-out signaling to neighboring LFA-1 molecules, a critical checkpoint in LFA-1-mediated lymphocyte arrest on high endothelial venules.