Actin cloud is formed at the center of the T-cell–APC interface. (A) An actin cloud is formed at the T-cell–APC interface. Jurkat cells (left) or IL-2–cultured human PBLs (right) were stimulated with SDF-1 and conjugated with Raji cells for 30 minutes in the absence of Ag. Stimulated cells were fixed and stained with phalloidin–Alexa 488. Images in both xy-axes (top) and xz-axes (bottom) axes are shown. Those in the z-axis are reconstituted with a TCS SP2 confocal laser microscope equipped with a galvo stage. The images after staining B-cell surface with anti-CD19 to identify the border between T and B cells are shown in Figure S2. (B) The actin cloud accumulated LFA-1 and phosphoproteins but not TCR. SDF-1–stimulated Jurkat cells were conjugated with Raji cells in the absence of Ag (top panels), whereas Jurkat cells were mixed with Raji cells that had been pulsed with SEE (200 ng/mL; bottom panels). After incubation for 30 minutes, the cells were fixed and stained with phalloidin–Alexa 488, anti-CD3, and anti–mouse LFA-1β, followed by avidin-Cy3 or anti–mouse IgG–APC. The white arrow indicates representative actin cloud. All analyses were performed on more than 100 cells.