Figure 1.

All Major Resting T Cell Subsets, Except Human CD8 Memory Cells, Spend More Time in the Kinapse Mode during Interaction with Uniformly Coated Stimulatory Surfaces

(A) Four representative tracks of human naive CD8 T cells. Periods of positional stability, presumably corresponding to synapse mode of interaction, are highlighted using red arrows and numbered for reference. Additional periods of positional stability that one may visually infer are either of too short a duration or represent drastic turns during motility.

(B) The speed of the cells shown in (A) over the duration of the tracks. The speed plotted here is smoothed by averaging the instantaneous speed over the two frames before and after the frame in question. However, the profile shows abrupt changes and fails to demarcate the periods of positional stability.

(C) The confinement score (originally termed probability level, L) identifies the periods of relative confinement (Simson et al., 1995). L > 3 represents a probability of < 0.017 that the confinement is due to random chance, which is the threshold value used to demarcate periods of relative confinement.

(D) The positional spread within each period of relative confinement is considered to determine whether the cell was in the stable state (value of 1) that is synonymous with synapse mode of interaction. Positional spread is defined by R²/t, where t is the period of relative confinement, and R is the diameter of the confined zone. A value of < 0.666 μm²/frame was found to represent positional stability. The fraction of time the cell spends in the stable state is called the positional stability index. Thus, a positional stability index of > 0.5 means that the cell has spent more time in stable synapse mode than in motile kinapse mode and vice versa.

(E–L) The positional stability index of resting T cell subsets on coverglass uniformly coated with anti-CD3 and ICAM1. The type of T cell subset examined is denoted at the bottom or in the panels and in the category names of dot plots (for example, naïve and memory cells from human CD8 T cells in E and human CD4 T cells in I). Inhibitors of PKCθ shift the balance from kinapses toward synapses in human naive CD8 T cells (G and H). The data points in (F) and (J) represent population means from separate blood donors, whereas, in rest of the panels, they represent individual tracks of cells from a particular donor. Mean values are given as red horizontal bars. The data shown in (G), (H), (K), and (L) are representative of two independent experiments.