Effect of anti-KDR on the transendothelial migration of human CD4⁺ and CD8⁺ T cells. ECs were cultured to confluency on 3μM pore membranes in FluoroBlok transwells. CFSE-labeled CD4⁺ or CD8⁺ cells (5 × 10⁵ cells) were added into the upper chamber, and migration into the lower chamber was monitored in real time as described in “Lymphocyte migration assays.” Panel A illustrates the mean percent inhibition of transmigration of CD4⁺ or CD8⁺ cells (as indicated) by anti-KDR (R&D Systems) versus control IgG across unactivated (□) or TNFα–activated ECs (■, n = 5 experiments; *P < .01 vs contol IgG). Panel B illustrates a representative experiment showing patterns of inhibition of CD4⁺ or CD8⁺ T-cell transmigration by anti-KDR across TNFα-activated ECs (control IgG, solid dots; anti–human KDR, open squares). (C) Western blot analysis of control siRNA and KDR siRNA transfected ECs. Top and middle blots illustrate different exposure times of the same blot. (D) Representative experiments illustrating transmigration of CD4⁺ T cells across nontransfected, control siRNA-transfected ECs, and KDR siRNA-transfected endothelial cells. (E-F) Representative experiments illustrating the transmigration of CD4⁺ T cells across control siRNA-transfected ECs (E) or KDR siRNA-transfected ECs (F) in the absence (solid dots) or presence (open squares) of anti-KDR (2μg/mL). Panels G and H illustrate the mean percent inhibition (n = 3 experiments) of transmigration of CD4⁺ T cells (G) and CD8⁺ T cells (H) across control siRNA-transfected ECs compared with: nontransfected ECs (■) or KDR siRNA-transfected ECs () in the absence or presence of anti-KDR as indicated. Also shown is the inhibitory effect of anti-KDR on T-cell transmigration across control siRNA-transfected ECs (□, as in panel E) or KDR siRNA-transfected ECs (⊞, as in panel F). *P < .01 vs contol IgG.