Figure 3.

Adhesion and transmigration of CD28SA-activated CD4⁺ effector memory T cells. Human CD4⁺ T_EMs were stimulated with plate-bound anti-CD3 mAb (CD3, 5 μg/ml); NIB1412 (NIB1412, 10 μg/ml); anti-CD3 mAb and NIB1412 (CD3 and NIB1412); control category included cells without any treatment (Control). Cells were harvested at 48 h and labeled with CellTracker™ Green CMFDA. (A) Immunofluorescent microscopy of HDMEC monolayer with adherent CMFDA-labeled T_EMs initially stimulated with the indicated antibodies and stained for F-actin and DNA using fluorescently labeled phalloidin (red) and DAPI (blue), respectively. (Scale bar = 20 microns). Phase optics was adjusted to emphasize adherent cells. (B) CMFDA-labeled T_EMs were added to confluent HDMECs and incubated for 30 min. Cells were washed and the remaining adherent cells visualized under fluorescence microscopy. Vertical axis represents the number of adherent T_EMs per field (**p < 0.01; ***p < 0.001, unpaired t test). Results from four independent donors are represented as means ± SD (C) Transmigration of stimulated CD4⁺ T_EMs across HDMEC layer on transwell inserts. Transmigrated cells were quantified by fluorescence and the results are presented as the mean (± SD) number of transmigrated cells from 3 replicate wells per condition. Results are representative of three independent experiments (***p < 0.001; unpaired t test).