Role of Rap1 GTPase in LFA-1– and VLA-4–mediated T-cell adhesion. (A) Adhesion of GFP-control– or GFP-SPA1–transfected T cells to immobilized ICAM-1 (2.5 μg/mL) or VCAM-1 (2 μg/mL) after treatment with 1 μg/mL PMA or 50 nM Mn²⁺. Percentage of adherent GFP-positive cells from initial GFP-positive cell input is expressed as mean plus or minus standard error of the mean (SEM). SPA1-GFP-positive cells failed to adhere to ICAM-1 after stimulation with PMA, whereas adhesion to VCAM-1 and Mn²⁺-induced adhesion to ICAM-1 were unaffected. *P < .001. (B) Effect of VCAM-1 concentration on cell adhesion. T cells were transfected as in panel A and assayed for PMA-induced adhesion to the indicated concentrations of immobilized VCAM-1. Adhesion of cells overexpressing GFP-SPA1 was comparable with GFP-control cells at all doses of VCAM-1 analyzed. (C) T cells transfected with Rap1GAP failed to adhere to immobilized ICAM-1 after PMA stimulation compared with cells transfected with GFP-control alone but were capable of adhering to immobilized VCAM-1 after similar PMA stimulation. *P < .05. (D) Adhesion to VCAM-1 is α4-dependent. T cells were incubated with a functional blocking antibody to VLA-4 (mAb HP2/1), and PMA-induced adhesion to ICAM-1 and VCAM-1 was performed as described in panel A. Blocking α4 had no effect on ICAM-1 binding but completely abolished adhesion to VCAM-1. *P < .001. (E) Effects of inactivation of Rap1 by SPA1 on SDF1-stimulated adhesion to ICAM-1 or VCAM-1 under shear flow conditions. GFP-control or GFP-SPA1-positive T cells were perfused over ICAM-1 or VCAM-1 coimmobilized with SDF-1α at a shear stress rate of 0.75 dyne/cm². Percentage of adherent GFP-positive cells from total GFP-positive cell input is expressed as the mean plus or minus SEM; *P < .01. T cells overexpressing GFP-SPA1 failed to adhere to ICAM-1 but were able to adhere to VCAM-1 in response to SDF-1α compared with GFP-control cells. (F) Flow cytometry of GFP-control or GFP-SPA1-positive T cells stained with the high-affinity extension reporter mAb KIM127 in the absence (Basal) or presence (top left) of PMA or SDF-1α (top right) or Mn²⁺ (bottom). GFP-SPA1 cells failed to expose the neoepitope KIM127 in response to both PMA and SDF-1α compared with a substantial induction in GFP-control cells. In contrast, SPA1 cells expressed the KIM127 neoepitope in response to Mn²⁺. (G) sVCAM-1/Fc binding was determined by flow cytometry after 30 s stimulation with vehicle control, PMA, SDF-1α, or Mn²⁺. Data are expressed as percentage of events of GFP-positive cells that were positive for APC. Agonist-induced up-regulation of α₄ integrin affinity as measured by sVCAM-1/Fc binding was similar in SPA-1 and GFP-control cells. Three to 5 independent experiments were done for each panel.