Intravital imaging of T and B cell migration across high endothelial venules in lymph node

Abstract

Abstract High endothelial venules (HEVs) in lymph node (LN) facilitate an effective recruitment of circulating lymphocytes from the blood. HEVs have distinctive cuboidal-shaped endothelial cells and prominent perivascular sheaths consisting of fibroblastic reticular cells (FRCs). Yet, detailed dynamic behaviors of lymphocytes during trans-endothelial migration, intra-perivascular channel crawling and trans-FRC migration in HEV are still poorly understood. In this work, we adapted a custom-design confocal microscope to visualize T and B cell migration across HEV in real time in vivo. Actin-DsRed transgenic mice were used to visualize HEV-endothelial cells and T or B cells obtained from actin-GFP mice were adoptively transferred. At the same time, FRCs were labeled in vivo by injecting anti-ER-TR7 antibody conjugated with far-red fluorophore to the footpad. T and B cells squeezed in between endothelial cells (ECs) and then migrated along the perivascular channel, narrow space between ECs and FRCs, for searching a proper site to exit by trans-FRC migration. Interestingly, B cells spent twice longer time in the perivascular channel than T cells although their total moving distances in the perivascular channel were similar. In addition, we observed that there existed preferred exit sites (“exit ramps”) from the perivascular channel for both of T and B cell. To observe whether T and B cell exit through the same exit ramp, we simultaneously transferred DsRed+ T and GFP+ B cells into wild type mouse. Indeed, T and B cells followed each other though the same exit ramp from the perivascular channel. There also existed “entrance ramps” into the perivascular channel; preferred sites for trans-endothelial migration for both of T and B cells.