Fig. 4

2D2 CD4⁺Vα3.2⁺c-Met⁺ polarized Th1 T cells showed greater adhesion and transmigration properties in vitro. A 2D2 CD4⁺Vα3.2⁺c-Met⁻ and c-Met⁺ T cells labeled with CFSE were seeded on the surface of confluent bEnd.3 cells. After 1 h of incubation and several washes, the attached 2D2 CD4⁺ T cells were analyzed. Representative immunofluorescent images (left panels) and quantification of the average numbers (right panel) of CFSE labeled T cells per well are shown. The values obtained in each group were calculated relative to the control condition (inactivated bEnd.3). Scale bar = 100 μm. B Transwell migration assays were performed by precoating the upper chambers with confluent monolayer of bEnd.3. A total of 10⁵ 2D2 CD4⁺Vα3.2⁺c-Met⁻ or c-Met⁺ T cells were blocked with 10ug/ml of indicated blocking antibodies, washed and seeded on the layer of activated bEnd.3. The transwell migration units were then incubated in presence of CXCL12 (100 ng/ml) in the lower chamber. After 6 h, all the cells contained in the lower chamber were harvested and the relative number of transmigrated cells was shown. C Schematic presentation of the flow assay system: T cell were captured from free flow and firmly adhered to HUVEC luminal surfaces (captured; step 1). T cell moves into the abluminal side by transmigrating between junctions of adjacent endothelial cells (transmigrating; step 2), which is followed by total transmigration (transmigrated T cells; step 3). Representative videomicrographs of CD4⁺Vα3.2⁺ Th1 cells (bottom panels) of the same area at indicated timepoint. Orange arrow indicates the same Th1 cell during the 3 phases; scale bar = 10 μm. D Adherent polarized 2D2 CD4⁺Vα3.2⁺c-Met⁻ and c-Met⁺ Th1 T cells on activated HUVECs were individually tracked and monitored for transmigration between different compartments for 50 min. Note that HUVEC cells were activated with CXCL12 and washed prior the adding of polarized Th1 cells. Captured, transmigrating and transmigrated T cells were analyzed separately. Data are presented as the means ± SD of 3 fields. Data are representative of three independent experiments and mean values ± SEM are shown (A, B); **p ≤ 0.01 ***p ≤ 0.001, ****p ≤ 0.0001, two-way ANOVA followed by Tukey’s post hoc test for multiple groups