Fig. 2.

Central TCR accumulation is rapid and requires high concentrations of agonist peptide. (A) The percent of T cell/APC couples with central TCR accumulation in at least one of the four time points analyzed (1′, 3′, 5′, and 7′–15′; central, black bars); any other accumulation phenotype (other, gray bars) or no accumulation (none, unfilled bars) are given for different TCR transgenic T cells as indicated in the first column. Fig. 9 provides a kinetic analysis of the same data. TCR accumulation was determined by using EL-4 target cell transfection with H2-D^b-GFP or T cell transduction with TCR-ζ/GFP or ZAP-70 double SH2-GFP (2SH2) as indicated in the second column at the given concentration of peptide (third column). The ZAP-70 double SH2 domain-GFP protein is further characterized in Supporting Text and Fig. 11, which is published as supporting information on the PNAS web site. Imaging probes are discussed in Supporting Text. Agonist peptides were used throughout with the exception of the A4Y partial P14 agonist and the addition of 10 μg/ml blocking B7-1/B7-2 antibodies (B7) to one set of DO11.10 samples. n, cells from at least three independent experiments were analyzed per condition. (B) An interaction of a P14 T cell that has been transduced with ZAP-70 double SH2–GFP (Lower) with an EL4 APC incubated with 10 μM p33 peptide (Upper) is shown at the indicated time points relative to tight cell couple formation (t = 0:00) in still images derived from Movie 1, which is published as supporting information on the PNAS web site. (Upper) Bright field images are displayed. (Lower) Matching projections of the three-dimensional ZAP-70 double SH2 domain–GFP fluorescence data in a false color scale (increasing from purple to red and white).