Redistribution of intracellular LFA-1 during T cell activation. (A) Representative flow cytometry analysis of T cell activation (CD69 and CD25) and proliferation (CFSE dilution) after stimulation of naive T cells with cognate ligand (N4) or altered peptide ligand (D7)–loaded irradiated splenocytes; n = 4 mice. (B) Representative images from real-time T cell contacts with APCs loaded with N4 or D7 peptide on plates coated with ICAM-1 and CCL21. Bars, 5 µm. In the graph, each bar represents the percentage of total cells scored after 45 min of co-culture. The gray portion of each bar is the fraction of cells exhibiting dominant YFP signal at the immunological synapse (IS) region, and the white portion is the fraction of the cells that showed YFP signal at the posterior region. Data represent mean ± SEM; n = 3 mice/group (30–42 cells per mouse). *, P < 0.05. (C) Representative fluorescence intensity of CD11a-mYFP cell surface from B. YFP fluorescence intensity is shown in a pseudocolor scale (from low [black] to high [red]). +/− 180°, rear of cell; 0°, leading edge; white lines depict the T cell–APC interface; arrowheads indicate the beginning of the T cell–APC contact. (D) Representative Western blot analysis of YFP expression in cell cytosol and plasma membrane (PM) fractions from naive CD11a-mYFP CD8⁺ cells or cells stimulated with CD3/CD28 antibodies for 30 min. Note that CD11a-mYFP protein level was increased in PM after T cell activation. n = 3 mice. (E) Flow cytometry analysis of surface LFA-1, VLA-4, and TCR levels after indicated times of naive CD11a-mYFP/OT-I CD8⁺ T cell and peptide-pulsed or PBS-treated BMDC co-culture. YFP⁺ T cells were fixed at indicated times and stained for surface expression. Total LFA-1 levels measured by mYFP intensity (yellow line). Data normalized to PBS control. Data are expressed as mean ± SEM of six separate experiments. *, P < 0.001. (F) Representative image of naive CD11a-mYFP CD8⁺ T cells stained with ER Tracker (MTOC; red) during N4-loaded APC interaction on the ICAM-1+CCL21–coated surface. Note that CD11a-mYFP and the MTOC are not colocalized during the LFA-1 redistribution. Bar, 5 µm. (G) The MTOC and CD11a-mYFP are colocalized during migration and mature APC contact, but not during early LFA-1 translocation to the APC contact (“early contact”) when stimulated by N4. The Pearson’s correlation coefficient was generated as YFP (LFA-1)/red (MTOC). Circles represent individual cells from three independent experiments with mean shown as a line. Data represent mean ± SEM. *, P < 0.01.