Assembly of the resting and anergic T-cell IS after 10 and 30 minutes. (A) Organization of CD3ζ at the HA308 T-cell-APC contact was categorized as follows: homogeneous indicates no clear polarization; ring/arc, ring- or arc-shaped CD3 accumulation; and single/multifocal, multiple foci or single focus of CD3 accumulation. (B) Charts show the percentage of resting T-cell-APC or anergic T-cell-APC conjugates exhibiting each type of CD3ζ organization at the IS. Cells were fixed and stained 10 or 30 minutes after coincubation. Comparing CD3ζ organization at the IS formed by anergic or resting T cells, P was less than .005 for both 10 minutes and 30 minutes, even though at 30 minutes the difference in distribution of patterns is more striking. □ indicates homogeneous; ▦, arc/ring; ▪, single/multifocal. (C) The percentage of resting and anergic HA308 T cells conjugated to APCs was assessed by flow cytometry at 0 and 60 minutes after coincubation with APCs in the presence or absence of agonist peptide.