(A) Baseline F-actin content of CD4⁺CD25⁺ Tregs from Dock8^–/– and WT mice, and effect of CD3 crosslinking on the F-actin content of Tregs from Dock8^–/– and WT mice. Results are expressed as the change in the MFI of F-actin from the baseline (time 0). (B) Representative images of CD4⁺CD25⁺ Tregs from Dock8^–/– mice and WT controls plated on anti-CD3– and ICAM-1–coated glass chambers and stained for F-actin by phalloidin at 5 and 45 minutes (original magnification, ×100). (C) Relative number of adherent cells per unit area at 5 minutes. (D) Quantitative analysis of F-actin staining of Tregs after 5 and 45 minutes of stimulation. (E) Localization of pTYR, F-actin, and DOCK8 in Dock8^–/– and WT Tregs at 10 minutes (original magnification, ×100). (F) A representative TIRF image of DOCK8 and actin distribution across the synapse after 10 minutes of synapse formation. The graph shows line scan profiles of DOCK8 and ACTIN across the middle of the cell; the green trace represents ACTIN intensity distribution, and the red trace represents DOCK8 intensity distribution. Note that DOCK8 and ACTIN intensities coincide in the cell periphery, represented by the peaks at the beginning and end of the line scan profiles. (G) Side view of 2 representative T cells, showing the whole cell ACTIN, pTYR, and DOCK8 distribution. Cells stained with DOCK8, pTYR, and ACTIN were imaged using confocal microscopy, and the images show a side view of maximum intensity projection of the confocal images. (H) Quantification of cell edge roughness determined by “shape descriptors” utility of ImageJ at 5 and 45 minutes. (I) Measurement of immune synapse instability, as denoted by the kinapse index (transient interactions index > 1) at 5 and 45 minutes. (J) Relative staining intensity of pTYR, phospho-ZAP70 (pZAP70), and TALIN at 10 minutes. (K) Transendocytosis of CD86-GFP by Tregs from Dock8^–/– and WT mice cocultured with CD86-GFP–expressing CHO cells. The percentage of GFP⁺ Tregs of total Tregs is shown. Results in A and K are representative of 3 independent experiments. Results in B–J are representative of 2 independent experiments. Symbols represent individual measurements, and error bars represent mean and SEM. Significance was determined by unpaired t test. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.