Altered synapse formation of self-reactive Ob.1A12 T cells on planar lipid bilayers displaying pMHC and ICAM-1. (A and B) Titration of pMHC complexes. HA:D7 and Ob.1A12 T cells were incubated on glass-supported planar lipid bilayers displaying ICAM-1 (∼200 molec/µm²) and pMHC (DR4-HA or DR15-MBP) at ∼1, 10, and 100 molec/µm². Representative images at 30 min are shown. (C and D) Kinetics of IS formation by HA:D7 and Ob.1A12 T cells incubated on planar lipid bilayers containing pMHC (∼100 molec/µm²) and ICAM-1. Each cell with ICAM-1 accumulation was categorized as showing cSMAC formation, pMHC accumulation without cSMAC formation, or no visible pMHC clusters. Graphs show mean percentage ± SEM for three to four fields with 48–65 individual cells analyzed. (E) Specificity of pMHC accumulation. HA:D7 or Ob.1A12 T cells were incubated on lipid bilayers displaying control pMHC (DR4-CLIP or DR15-CLIP at ∼100 molec/µm²) and ICAM-1. CLIP (class II–associated invariant chain peptide) was used as a control peptide. Images of ICAM-1 and pMHC at 30 min are shown. (F) Mouse 5C.C7 and mOb.1A12 T cell blasts were incubated on lipid bilayers displaying pMHC (∼100 molec/µm²) and ICAM-1. Representative images at 30 min are shown. T cells with ICAM-1 accumulation were categorized as in C and D. Bar graph shows mean percentage ± SEM for 7–11 fields with 130–161 individual cells analyzed. Data are representative of at least two experiments. Bars, 5 µm.