Early signaling events in self-reactive T cells with altered synapse formation. (A) Detection of microclusters of pCD3ζ in T cells conjugated for 15 min with peptide-pulsed B cells. Images on the right show an en face view of the contact area. Bars, 5 µm. (B) Kinetics of CD3ζ phosphorylation upon antigen stimulation. T cells were stimulated with peptide-pulsed B cells; after stimulation, CD3ζ was immunoprecipitated. Blots were probed for phosphorylated tyrosine (pTyr) and total CD3ζ (loading control). (C) Calcium flux. Fura-2–labeled T cells were stimulated with peptide-loaded B cells. The profiles represent a mean of 20–45 individual cells, with time 0 being the initial contact of the T cell with the B cell. See Videos 4–7 for examples. (D) Calcium mobilization in mouse 5C.C7 and mOb.1A12 TCR transgenic T cell blasts on lipid bilayers displaying ICAM-1 and pMHC. The profiles represent a mean of 33 individual cells, with time 0 being the initial contact with the lipid bilayer. Data are representative of at least two independent experiments.