Figure 2.

Ligand-dependent clustering of CD28 at TCR/CD3 MCs and at c-SMAC. (A, B) AND-Tg T cells expressing EGFP-CD28 were plated on a planar bilayer containing I-E^k and ICAM-1 (top) plus CD80 (bottom) (prepulsed with MCC_88–104). Cells were imaged at video rate (30 frames/s) using TIRFM (time shown above images). Images from 10 s to 5 min are depicted in (A) and at 10 min after initial contact in (B), interval between images is 10 s. Real-time images in (A, B) are available in Movie S1-S4. (C) Cells in (A, B) were fixed at 2 or 20 min after cell—bilayer contact, stained for CD3, and imaged by confocal microscopy. Histograms on right panels show fold fluorescence intensities of EGFP-CD28 (green) and CD3 (red) on the diagonal yellow lines in DIC images. (D) Fluorescence intensities of CD3 and EGFP-CD28 in CD28 (c-28) or CD3 clusters (c-3) in c-SMAC or p-SMAC (p) were compared to the intensities of entire interfaces in the cells in (C) (20 min, bottom two rows) (n=10). Asterisk, p-value <0.001. (E) AND-Tg T cells expressing EGFP-CD11a (top) or EGFP-CD28 (bottom) were stained with Alexa Fluor 546-labeled anti-TCRβ Abs Fab and were plated on a planar bilayer containing I-E^k, CD80, and Cy5-labeled ICAM-1 (prepulsed with MCC_88–104). Cells were imaged real-time by confocal microscopy 20 min after contacts. Histograms on the right panels are shown as in (C). Scale bars, 5μm.