Intravital Two-Photon Imaging of Lymphocytes Crossing High Endothelial Venules and Cortical Lymphatics in the Inguinal Lymph Node.

Lymphocyte recirculation through lymph nodes (LNs) requires their crossing of endothelial barriers present in blood vessels and lymphatics by means of chemoattractant-triggered cell migration. The chemoattractant-chemoattractant receptor axes that predominately govern the trafficking of lymphocytes into, and out of, LNs are CCL19/CCR7 and sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1PR1), respectively. Blood-borne lymphocytes downregulate S1PR1 and use CCR7 signaling to adhere to high endothelial venules (HEVs) for transmigration. During their LN residency, recirculating lymphocytes reacquire S1PR1 and attenuate their sensitivity to chemokines. Eventually lymphocytes exit the LN by entering the cortical or medullary lymphatics, a process that depends upon S1PR1 signaling. Upon entering into the lymph, lymphocytes lose their polarity, downregulate their sensitivity to S1P due to the high concentration of S1P, and upregulate their sensitivity to chemokines. However, many of the details of lymphocyte transmigration across endothelial barriers remain poorly understood. Intravital two-photon imaging with advanced microscope technologies not only allows the real-time observation of immune cells in intact LN of a live mouse, but also provides a means to monitor the interactions between circulating lymphocytes and stromal barriers. Here, we describe procedures to visualize lymphocytes engaging and crossing HEVs, and approaching and crossing the cortical lymphatic endothelium to enter the efferent lymph in live mice.