Phenotypic and functional characteristics of randomly moving of CD8⁺ T cells. (A) In transwell migration experiments, migration of CD8⁺ T cells through fibronectin-coated 5-μM pore membranes (left panel) and through HUVECs grown on 5-μM pore membranes (right panel) was assessed in absence of a chemotactic gradient. Ex vivo expression of CCR7 and CD45RA, CD44 and CD62L, and the activation marker CD69 was compared with expression on migrating cells (16 hours migration). Migrating CD8⁺ T cells were skewed toward a CCR7⁻ and CD62L⁻ phenotype. (B) As assessed in transwell migration experiments, the frequency of EBV-specific CD8⁺ T cells producing IFNγ was higher amid migrating than nonmigrating cells. *P < .05. (C) Using time-lapse microscopy, the movement pattern of sorted CCR7⁻ and CCR7⁺CD8⁺ T cells on fibronectin-coated cell-culture plates and on HUVECs was visualized. The velocity of crawling and noncrawling cells was analyzed assessing 1-minute–spaced pictures. The average speed of crawling cells was similar among subsets on both fibronectin and HUVECs, whereas noncrawling cells moved slightly more rapid on fibronectin (top panel). Crawling and noncrawling cells were enumerated on 5-minute–spaced pictures throughout a 1-hour observation period, and the mean percentage of crawling cells compared among CCR7⁻ and CCR7⁺CD8⁺ T cells. Crawling was a prominent feature of CCR7⁻CD8⁺ T cells on fibronectin (∼10% of all cells). On HUVECs the frequency of crawling CD8⁺ T cells was higher among the CCR7⁺ subset, but always remained less than 5% (bottom panel). ***P < .001 (D) On representative time-lapse video pictures crawling CD8⁺ T cells on fibronectin and HUVECs are marked with a circle. The insert shows the typical features of a crawling cell at higher magnification. Scale bar represents 0.1 mm.