Effect of anti-KDR on the transendothelial migration of mitogen-activated human CD4⁺ and CD8⁺ T cells. ECs were cultured to confluency on FluoroBlok 3μM pore transwells. Subsequently, 72-hour mitogen-activated (anti-CD3/CD28) CD4⁺ or CD8⁺ T cells were CFSE labeled and added into the upper chamber of transwells, and migration into the lower chamber was monitored in real time. (A) Mitogen-activated T cells were pretreated with control IgG or anti-KDR for 3 hours and were washed in culture medium prior to the migration assay. Representative experiments showing transmigration patterns across untreated or TNFα–activated ECs are illustrated. The bar graphs represent mean percent inhibition of transmigration by anti-KDR pretreatment across unactivated ECs (□) or TNFα-activated ECs (■) in 5 experiments at each time point. **P < .05 comparing anti-KDR pretreated cells versus cells pretreated with control IgG. (B) Transmigration of mitogen-activated CD4⁺ or CD8⁺ T cells across unactivated ECs or TNFα-activated ECs in the presence of control IgG (solid dots) or anti-KDR (open squares) for the entire period of the assay. The bar graphs illustrate mean percent inhibition of transmigration by anti-KDR across unactivated ECs (□) or TNFα–activated ECs (■) in n = 5 experiments. *P < .01 comparing the effect of anti-KDR versus control IgG. (C) Unactivated (▨) or 72-hour mitogen-activated CD4⁺ T cells (■) were placed in the upper chamber of a microchemotaxis Boyden chamber, and migration into the lower chamber was assessed after 4 hours, as described in “Lymphocyte migration assays.” The chemotaxis response to VEGF or IP-10 (as a positive control) is illustrated. As indicated, the T cells were pretreated with SU5416, a pharmacologic KDR signaling inhibitor, prior to and during the chemotaxis assay. The illustrated experiment is representative of at least 3 performed in triplicate wells. P values were calculated using the Student t test (*P < .01).