ECs induce the expression of CD69 on PBTLs. (A) ECs induce the expression of CD69 but not CD25 on PBTLs. PBLs were incubated alone (control) or on fixed TNF-–activated EC monolayers (TNF-EC). After 18 hours of culture, CD69 and CD25 expression on CD3⁺ cells was analyzed. Dotted lines represent negative control corresponding to P3X63. Numbers correspond to the percent of positive cells. A representative experiment out of three is shown. (B) Induction of CD69 T-cell expression by ECs activated with different stimuli. ECs were incubated with medium alone (untreated) or with IFN-γ (250 U/mL), TNF- (20 ng/mL), LPS (50 ng/mL), and IL-1β (1 ng/mL) for 20 hours at 37°C, fixed, and extensively washed. Then, PBLs pretreated or not with the blocking anti–ICAM-1 RR1/1 MoAb were added to wells containing medium or fixed HUVEC. After 18 hours, CD69 expression in CD3⁺ T cells was measured. The percentage of induction over the control level is shown as arithmetic mean ± 1 standard deviation (SD). The results correspond to three independent experiments. (▨), EC; (▪), EC + anti–ICAM-1. (C) Effect of adhesion receptor blockade on EC-mediated activation of T cells. PBLs preincubated 30 minutes at 4°C with blocking anti-CD18 Lia3/2 and anti-CD49d HP2/1 MoAbs were added to confluent monolayers of resting and TNF-–activated ECs and incubated for 18 hours. Then, the expression of CD69 on CD3⁺ cells was analyzed by two-color flow cytometry. Control cells corresponded to PBLs incubated in the absence of ECs. The percentage of induction over the control level is shown as arithmetic mean ± 1 SD, and the results correspond to three independent experiments. (□), no MoAb; (▨), anti-CD18; (▩), anti-CD49d; (▪), anti-CD18 + anti-CD49d.