Dynamic insight into naive lymphocyte extravasation in vivo

Authors

• SL Yan
• O Kamenyeva
• IY Hwang
• JH Kehrl

Abstract

The trafficking of immune cells in lymph nodes plays a critical role in immunity. Lymphocytes recirculate from the blood, through lymph nodes (LNs), into lymphatics and back to the blood in search of foreign antigens. However, the mechanisms regulating lymphocyte morphology, movement, and routes of lymphocyte transmigration are still undefined. Here we established a novel four-dimensional imaging platform to precisely determine the profile and dynamics of naïve lymphocyte transmigration in vivo that allows live-imaging by laser scanning microscopy over prolonged periods time (>4 h). This 4D imaging system allows for advanced spatial and temporal resolution. Actin dynamics are essential for various cellular processes, including the control of cell shape and movement. By utilizing this high-resolution confocal intravital imaging technique with LifeAct-GFP bone-marrow reconstituted mice, we observed that actin accumulation at the leading edge of the lymphocytes are required for lymphocyte extravasation. Various reports suggest that lymphocyte transmigration can occur via junctions between adjacent endothelial cells (paracellular), or through the body of endothelial cells (transcellular). Here we labeled the LN vasculature with fluorescently-labeled antibody against PECAM-1 and found that naïve lymphocytes preferentially cross the HEVs via the paracellular route. In conclusion, this novel imaging technique allowed us to confirm the importance of actin polymerization during lymphocyte extravasation in vivo, and determine the routes of lymphocyte transmigration. The confocal intravital imaging technique established here can be adapted to address many peripheral LN-related biological questions, including the dynamics of cell migration, cell-cell interactions, and changes in HEV morphology.

Scientific Focus Area: Immunology

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