Induction of T-cell activation during transmigration. (A) Additive effect of transendothelial migration and IL-15 on the expression of CD69 by T cells. PBTLs (>97% CD3⁺) were added to the upper compartment of Transwell chemotaxis chambers precoated with resting (HMEC-1) or TNF-–activated HMEC-1 cells (TNF–HMEC-1). In the lower chamber MIP1 (20 ng/mL) or IL-15 (15 ng/mL) were added as chemoattractants. As control, T cells were incubated with these cytokines in the absence of ECs. After 16 hours, control, upper chamber, and lower chamber T cells were separately collected and the expression of CD69 in CD3⁺ cells was measured by two-color flow cytometry. The thin lines and percentages correspond to the upper chamber T cells (NM, nonmigrated) and the thick lines and percentages to T cells migrated to the lower chamber (M, migrated).The proportion of migrated T cells for MIP1 is 25.1% (HMEC-1) and 30.6% (TNF–HMEC-1), and for IL-15 is 33.8% (HMEC-1) and 37.3% (TNF–HMEC-1). A representative experiment out of three is shown. (B) Additive effect of transmigration across ICAM-1–coated membranes and IL-15 on T-cell activation. PBTLs were plated in the upper compartment of FN (20 μg/mL) or rsICAM-1 (20 μg/mL) precoated Transwell chemotaxis chambers and MIP1 (20 ng/mL) or IL-15 (15 ng/mL) were added as chemoattractants in the lower chamber. Control cells were incubated as in (A). After 16 hours, T cells from both chambers were collected separately and the expression of CD69 in CD3⁺ cells was measured. The thin lines and percentages correspond to nonmigrated (NM) T cells from the upper chamber and the thick lines and percentages are referred to migrated (M) cells from the lower chamber. The proportion of migrated cells for MIP1 is 32.4% (FN) and 37.1% (ICAM-1), and for IL-15 is 42.8% (FN) and 47.6% (ICAM-1). A representative experiment out of three is shown.