Defect in entering and leaving lymph node. (A-D) Imaging of events at the endothelial interface in lymph node. HEVs were stained by a low concentration of fluorescence-labeled MECA-79 (visualized as gray). T-lymphocytes from WT mice were stained red (CMTMR) and from ezrin T567E transgenics were stained green (CMFDA), mixed and injected intravenously while inguinal lymph node was being imaged at frame rate of 5 seconds. (A) A representative still image of HEVs showing lymphocytes at the endothelial surface and in the immediate vicinity of the vessel 40 minutes after cell injection. (B) Four sequential still images with corresponding time stamp. Dashed line indicates the edge of vessel delineated by MECA-79 stain, which is better seen on lower magnification view. Sequence illustrates an ezrin T567E transgenic T-cell initiating transmigration (maximal at 8:55), then withdrawing extension (11:05) and de-attaching (11:45). (C) Scoring of the success or failure of WT or T567E transgenic T-lymphocytes to transmigrate from endothelium into interstitium. (D) Average time taken for lymphocytes to transmigrate (P = .0185). Data are mean ± SEM: WT (53.9 ± 8.2; N = 9), Tg (100.6 ± 14.1; N = 16). P values were calculated with Mann-Whitney test by Prism Version 5 (GraphPad). *P < .05. (E) Thirty minutes of cumulative image of cell movement in representative lymph node in efferent lymph. Arrow indicates direction of lymph flow. (F) Comparison of the relative number of lymphocytes of ezrin T567E versus WT-lymphocytes from panel E in cortical sinus compared with cortex. ***P < .01.