Abstract
Lymph nodes (LN) are major checkpoints for circulating T lymphocytes to recognize foreign antigens collected from peripheral tissue. High endothelial venule (HEV) in LN facilitates the effective transmigration of circulating T lymphocytes from the blood into LN. There have been many efforts to visualize T lymphocytes trafficking across HEV to understand the underlying mechanism. However, due to insufficient spatiotemporal resolution and the lack of an in vivo labeling method, clear visualization of dynamic behaviors of rapidly flowing T lymphocytes in HEV and their transmigration have been difficult. In this work, we adapted a custom-designed video-rate laser scanning confocal microscopy system to track individual flowing T lymphocytes in the HEV in real time in vivo. We demonstrate that the HEVs in LN can be clearly identified in vivo with its distinctive cuboidal morphology of endothelial cells fluorescently labeled by intravenously injected anti-CD31 antibody conjugated with Alexa fluorophore. By visualizing the adaptively transferred T lymphocytes, we successfully analyzed dynamic flowing behaviors of T lymphocytes and their transendothelial migration while interacting with the endothelial cells in HEV.
Highlights
To achieve an effective immune surveillance over wide area of human body, the immune system is operated by highly mobile lymphocytes
3.1 In Vivo Visualization of T Lymphocytes Interacting with Endothelial Cells in high endothelial venule (HEV) of Lymph Nodes
Some T lymphocytes firmly adhered in endothelial cells started to transmigrate across the endothelial cells from the lumen of HEV [Fig. 2(d) and Video 3]
Summary
To achieve an effective immune surveillance over wide area of human body, the immune system is operated by highly mobile lymphocytes. Lymph nodes (LN) are small nodular tissues encapsulating numerous lymphocytes, and which are distributed throughout the body. Due to the low frequency of naïve T lymphocytes for any specific antigen, a huge population of T lymphocytes constantly recirculates LN over the body via the blood and lymph network to ensure prompt induction of immune response. To facilitate the effective recruitment of T lymphocytes from the blood to LN, a specialized blood vessel called high endothelial venule (HEV)[1,2,3] exists in LN. The endothelial cells constituting an innermost layer of the HEV are directly exposed to the rapid blood flow while interacting with flowing lymphocyte to recruit them. Several intravital imaging studies have documented the contribution of various adhesion molecules involved in lymphocyte trafficking across HEV.[4,5,6,7] It has been shown that naïve T lymphocytes express a surface receptor L-selectin, which can bind
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