MP-IVM investigations on T-cell interactions with HEVs. (A) A representative image of adoptively transferred WT T cells (blue) and α_L-I306A T cells (red) interacting with popliteal lymph node vessels (green) of GFP-chimeric recipient mice. (B) Gαi-independent arresting of KI cells. Firm adhesion of α_L-I306A T cells to HEV endothelial cells was independent of Gαi signals, whereas WT T-cell adhesion was reduced by pertussis toxin (PTX) treatment. (C-D) Motility parameters of T cells in the LN vessel compartment. Meandering index (C) and 3D-IV (D) are shown. (E) Overall transendothelial migration efficacy of arrested cells. Each migratory path of arrested WT and KI T cell on HEVs was traced and analyzed. The line in the box-plot indicates the median, the box-part represents the interquartile range, the whiskers depict the 5th and 95th percentiles, and the crosses represent the mean of 3 independent experiments. (F) A graphic representation of the crawling and TEM steps during adhesive interactions of T cells with HEVs. (G-H) Velocity (G) and traveling distance (H) during the crawling step. α_L-I306A T cells migrated slower and a shorter distance on HEVs than did WT T cells. (I) Time required for completing the TEM step. (J-K) Representative migration velocity profiles of WT (J) and KI (K) T cells during the crawling and TEM steps. (B-D,G) Data are expressed as the mean values ± SEM of 3 independent experiments. Two-tailed Student t test was used for statistical analyses. Statistical significance was defined as *P < .05 or ***P < .001. (C-D,H-I) A representative result from 3 independent experiments is shown. (C-D,I) Thick horizontal bars indicate mean values.