Wt and DOCK2^–/–T lymphocytes arrest normally on TNFα-activated murine EC and integrin ligands under shear flow in response to different chemokine signals. (A) CD3⁺ splenocytes from wt and DOCK2–/– mice were double stained for CD4 and CD8, and the fractions of single-positive subsets are indicated. (B) FACS assay showing expression of α₄ integrins (probed with an anti–α₄ subunit mAb) and of LFA-1 (probed with an anti–α_L subunit mAb) on wt (gray lines) and DOCK2^–/– (black lines) CD3⁺ purified T splenocytes. Functional CCR7 expression was assayed by CCL19-Fc binding probed with antihuman Fc. CXCL12 receptors were assayed by similar staining using CXCL12-Fc. Background staining is depicted by dotted lines. (C) Arrest of CD3⁺ splenocytes (T lymphocytes) perfused for 1 minute over TNFα-activated bEnd.3 EC monolayers in the absence or presence of overlaid CCL21. The percentages of rapidly arrested cells are expressed as the fractions of the lymphocyte flux in close contact with the endothelial monolayer. Results presented are mean values ± range of 4 independent fields. (Inset) Effect of pretreatment with combined anti-α₄ (VLA-4; PS/2) and anti-α_L (LFA-1; M17/4) mAbs on CCL21-triggered arrest of either wt or DOCK2^–/– T lymphocytes. The experiments are representative of 3 independent runs with different animals and batches of endothelial cells. (D-E) Frequency and strength of tethering of wt and DOCK2^–/– T lymphocytes to VCAM-1–Fc coated at 0.5 μg/mL alone or with the indicated immobilized (imm.) chemokines at 2 μg/mL (D) or to ICAM-1–Fc coated alone at 2 μg/mL or with the indicated immobilized chemokines at 2 μg/mL (E). Tethers were determined at a shear stress of 0.5 dyne/cm². More than 90% of tethers to either VCAM-1 or ICAM-1 were blocked, respectively, by treating lymphocytes with either 20 μg/mL α₄-or α_L-blocking mAbs as in the inset of panel C (data not shown). Mean values ± range determined in 2 fields are presented.