sICAM-5 down-regulates the anti-CD3-induced T-cell activation. Ligation of immobilized anti-CD3 mAb induces TCR signaling and leads to up-regulation of CD69, CD40L, and CD25 on human T cells. The expression level of each marker was analyzed by flow cytometry and presented as the percentage of expression level in panels A and B, or as the mean fluorescent value in panels C-F. CD69 and CD40L were significantly decreased in sICAM-5 D1–2-Fc (50 μg/mL) treated T cells within 24 hours. CD40L remained suppressed until the late activation stage (24-48 hours), when CD25 was also decreased (A). The overlaid histograms of CD69 expression by nonactivated T cells (dot line), anti-CD3-activated T cells (solid dark line), and anti-CD3 plus sICAM-5-treated T cells (solid gray line) at 5 hours, 24 hours, and 48 hours are inserted in panel A. Furthermore, T cells were double stained for CD45RO and CD40L after each treatment for 20 hours and represented as dot plots. The percentage of positive cells is marked in each quadrant. sICAM-1-Fc (75 μg/mL) increased, whereas sICAM-5-Fc reduced, the proportion of CD40L⁺ T cells (panel B left). Moreover, sICAM-1-Fc significantly enhanced, whereas sICAM-5-Fc decreased, CD40L expression in activated CD45RO^Low naive, but not in CD45RO^High memory T cells (panel B right). The level of integrin CD11a polypeptide was not significantly changed by ICAM-1 or ICAM-5 (panel B right). The presence of higher than 30 μg/mL sICAM-1-Fc further promoted the up-regulation of CD25 (C,D). By contrast, sICAM-5 D1-2-Fc decreased CD25 expression at concentrations greater than 10 μg/mL (C,D). Moreover, CD25 expression on activated CD4⁺ and CD8⁺ T cells was decreased by 50 μg/mL sICAM-5 D1-2-Fc (E). Neither sICAM-1 nor sICAM-5 induced apoptosis of the treated T cells, as illustrated by annexin V staining (F). Data are shown as means plus or minus SD of triplicates (*P < .05, **P < .01).