Impaired T cell functions after down modulation of focal adhesion molecules. (A) Representative images of the TCR (H57-Dy549) on shCtrl or shPxn + shPyk2 (dKD) cells stimulated with 1 µM Ag–loaded activated B cells are displayed (left). The frequencies of adhered and stopped cells upon interaction with APCs and the rate of the cells with cSMACs after 10 min in the adhered and stopped cells are plotted (right). Data are the mean from 59 or 65 cells ± SD. *, P < 0.05. DIC, differential interference contrast. (B) The shCtrl and dKD cells incubated with 1 µM Ag–loaded activated B cells for 10 min were fixed and analyzed for the phosphorylation levels of SLP76 and Erk by flow cytometry. Representative results from four experiments are shown. pSLP, phosphorylated SLP76. (C) The shCtrl, shPxn, shPyk2, and dKD cells were incubated with irradiated spleen cells at the indicated dose of Ag. IL-2 in the culture supernatant after 24 h was measured by ELISA. Representative results from three experiments are shown. Data are mean ± SD. *, P < 0.01. (D) T cell stimulation with lipid bilayer–coated beads. Three types of lipid bilayer–coated beads were used: I-E^k coated, both I-E^k and ICAM-1 coated, and a mixture of I-E^k and ICAM-1 coated. The shCtrl and dKD cells were stimulated with the glass beads with 1 µM Ag peptides for 10 min and analyzed for phosphorylation levels of Erk as in C. (E) The nontransfected cells were activated with ICAM-1, E^k, [E^k + ICAM], or [E^k] + [ICAM]. The proportions of pErk-positive cells from shRNA-treated cells in D and nontransfected cells are plotted. Data are the mean of four experiments ± SD. *, P < 0.05; **, P < 0.01. (F) Diagram of micro–adhesion rings and ISs (pSMAC, peripheral SMAC; left). When a T cell recognizes an Ag on an APC, TCR-MCs are generated at the interface and initiate activation signals. Then, TCR-MCs move to form cSMACs and generate a typical IS. The TCR-MCs in initial phase were surrounded by a micro–adhesion ring; this structure resembles mature ISs as the synapse-like structure in microscale. Our results revealed the following scenario. The integrin outside-in signal–induced micro–adhesion ring formation was composed of LFA-1, Pxn (Pax), Pyk2, vinculin, and MyoII. The micro–adhesion ring enhanced F-actin formation and TCR clustering, especially under weak TCR stimulation. (Middle) When F-actin was acutely broken by CytD treatment after TCR-MC generation, TCR-MCs and ZAP70 clusters remained, but the micro–adhesion ring and LAT and SLP76 clusters disappeared. However, when F-actin formation was inhibited before TCR-MC generation by CK666 treatment, the generation of both TCR-MCs and micro–adhesion rings was inhibited. Therefore, F-actin is important for both generation and maintenance of the TCR-MC signaling. (Right) In the dKD (shPxn + shPyk2) cells, the decrease of focal adhesion molecules caused defects in micro–adhesion ring formation. Consequently, small TCR-MCs and F-actin clusters were formed, and T cell activation such as phosphorylation of SLP76 and Erk and IL-2 production was impaired. (A and D) Bars, 5 µm.