IL-15 cooperates with TNF-–activated ECs and ICAM-1 in the induction of CD69 T-cell expression. (A) Activated ECs do not enhance the induction of CD25 promoted by IL-15 in T cells. PBLs incubated in the presence or absence of fixed TNF-–activated ECs (TNF-EC and control, respectively) were stimulated for 18 hours in the presence or absence of IL-15 (15 ng/mL). Cells were collected and CD69/CD25 expression of CD3⁺ cells was analyzed by three-color flow cytometry. Numbers correspond to the percent of positive cells. A representative experiment out of three is shown. (B) Additive effect of IL-15 and activated ECs on the induction of CD69 in CD45RO memory T cells. PBLs were incubated alone (control) or on fixed TNF-–activated EC monolayers in the presence or absence of IL-15. After 18 hours of culture, CD69/CD45RO expression of CD3⁺cells was analyzed by three-color flow cytometry. Numbers correspond to the percent of positive cells. A representative experiment out of three is shown. (C) Additive effect of IL-15 and ICAM-1 on the induction of expression of CD69 by T cells. PBLs were plated on BSA (1%) or ICAM-1 (20 μg/mL) precoated wells in the presence or absence of IL-15 (25 ng/mL). After 18 hours, cells were collected and CD69 expression in CD3⁺ T cells was measured and is shown as percentage of positive T cells. A representative experiment out of five is shown.