Impaired cSMAC formation and pMHC recruitment by self-reactive T cells on planar lipid bilayers. (A) Representative images for HA-specific clones (HA:D7 and BA-8), MBP-specific clones (Ob.1A12, Ob.2F3, Hy.2E11, and Hy.1B11), and GAD65-specific clones (Agad303, Agad307, and Agad325) on lipid bilayers displaying ICAM-1 and pMHC. Images were selected from a 20–90-min time period. (B) Quantification of cSMAC formation. The percentage of cells with ICAM-1 accumulation that formed a cSMAC was determined. Mean of at least three different fields ± SEM over 90 min is shown. (C) Accumulation of pMHC at the interface. Integrated fluorescence of pMHC accumulation (in arbitrary units [AU]) was determined at 30 min for all cells that accumulated ICAM-1. Shown is the mean of 17–55 individual cells for each T cell clone ± SEM. (D and E) Representative images of Ob.1A12 or HA:D7 T cells incubated on lipid bilayers displaying ICAM-1, pMHC, and CD58 or CD80 (∼100 molec/µm²) at 30 min. (F and G) Representative images of T cells on lipid bilayers displaying anti-CD3ε Fab fragment (∼150 molec/µm²) and ICAM-1. T cells that accumulated ICAM-1 were categorized as follows: cSMAC formation, anti-CD3ε accumulation, or no visible anti-CD3ε clusters. Bar graph shows mean percentage ± SEM for 4–13 fields with 80–319 individual cells analyzed. Data are representative of at least two experiments. Bars, 5 µm.