Loss of β2–kindlin-3 interactions in CD4 T cells results in impaired adhesion strengthening and impaired migration on ICAM-1 in vitro. (A) AFM measurements of the forces required to detach WT and KI effector CD4 T cells from an ICAM-1–coated surface, presented as maximum unbinding force. The control readings (Ctr) indicate WT cell adhesion to bare plastic, and WT cell adhesion to ICAM-1 in the presence of EDTA. At all contact times, adhesion of both WT and knock-in cells to ICAM-1 was significantly above binding to bare plastic or ICAM-1 in the presence of EDTA. N = 50 individual T cells, with mean ± SEM. (B) Still images of WT and KI effector CD4 T cells plated onto ICAM-1–coated surfaces. Scale bars represent 50 μm. Images are representative of cells from N = 8 mice. (C) WT and KI effector CD4 T-cell spreading on ICAM-1 was quantified in terms of area, longest axis, and perimeter. Cells are from N = 3 mice. (D) Effector CD4 T-cell 2-dimensional migration on ICAM-1 was viewed using time-lapse microscopy over a 15-minute period and videos analyzed to quantify the parameters indicated. Cells are from N = 8 mice, with mean ± SEM. Student t test was used to calculate significance values. NS, not significant.