Graduate School of Nanoscience and Technology-WCU, Korea Advanced Institute of Science and Technology-KAIST, 291 Deahak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
J Biomed Opt. 2013 Mar;18(3):036005. doi: 10.1117/1.JBO.18.3.036005.
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.
淋巴结 (LN) 是循环 T 淋巴细胞识别从外周组织收集的外来抗原的主要检查点。LN 中的高内皮小静脉 (HEV) 促进了循环 T 淋巴细胞从血液有效迁移到 LN。人们已经做出了许多努力来可视化 T 淋巴细胞穿过 HEV 的迁移，以了解其潜在机制。然而，由于时空分辨率不足以及缺乏体内标记方法，难以清晰地可视化 HEV 中快速流动的 T 淋巴细胞的动态行为及其迁移。在这项工作中，我们采用了定制设计的视频速率激光扫描共聚焦显微镜系统，实时在体内跟踪单个流过的 HEV 中的 T 淋巴细胞。我们证明，用静脉内注射的抗 CD31 抗体与 Alexa 荧光染料缀合标记的内皮细胞，LN 中的 HEV 可以在体内被清晰地识别，其具有独特的立方体形貌。通过可视化适应性转移的 T 淋巴细胞，我们成功地分析了 T 淋巴细胞的动态流动行为及其与 HEV 内皮细胞相互作用时的跨内皮迁移。
