CD3 organization at the resting and anergic human T-cell IS. (A) Organization of CD3ϵ at the T-cell-APC contact was categorized as follows: homogeneous indicates no clear polarization; arc/ring, ring- or arc-shaped CD3 accumulation; and single/multifocal, multiple foci or a single focus of CD3 accumulation. Examples of these categories, which are representative for both resting and anergic T cells, are given. (B) Charts show the percentage of each type of CD3 organization at synapses between APCs and resting or anergic 7P.73 T cells or Fel d 1 p4 T cells. P value is less than .005 for both 7P.73 and Fel d 1 p4, comparing the CD3 organization in resting versus anergic T cells. The amount of CD3 at the T-cell-APC contact was calculated as a percentage of total CD3 on the T cell (C), or as a fold increase in comparison with an area away from the intercellular contact (D). Plots show measurements for individual conjugates as dots and the mean for resting and anergic 7P.73 T-cell synapses. (E-H) Representative conjugates are shown that depict high or low levels of CD3 accumulation at the IS. The corresponding data points are marked on panel D. (E) Fel d 1 p4 resting T cell, (F) 7P.73 resting T cell, (G) Fel d 1 p4 anergic T cell, and (H) 7P.73 anergic T-cell-APC conjugates were stained with a pan class I MHC mAb to control for nonspecific membrane accumulation at the IS. The images from left to right are transmitted light; CD3ϵ; CD3ϵ intensity profile; class I MHC; class I MHC intensity profile. The intensity profiles have been color coded (confocal software; Leica) according to the scale given below the images. Scale bars represent 5 μm.