Fig. 3: The four stages of close-contact formation.

a Key stages of close-contact formation. Images show a J8-GECI cell interacting with an SLB2 presenting pMHC^null plus pMHC^9V-hi (i.e., ~100 molecules of pMHC^9V/μm²). The different stages were identified by simultaneously imaging close contacts (black holes in the SLB glycocalyx fluorescence), cell footprint (cell membrane area), and triggering state (calcium signal) of a cell. A cartoon of each stage is indicated below. See main text for stage descriptions. b–e Image-based analysis of J8-GECI cells on an SLB2 presenting pMHC^null ± pMHC^9V-lo or pMHC^9V-hi. The baseline response is given by non-signaling cells on pMHC^null. See Supplementary Movies 1, 4, and 5. b Cumulative distribution of the searching to scanning stage transition for J8-GECI cells on SLB2s. The analysis uses both signaling and non-signaling cells; n = 26 (pMHC^9V-hi), 26 (pMHC^9V-lo), and 34 (pMHC^null) cells. Plotted is the cumulative distribution function of the Kaplan–Meier estimator with the exponential Greenwood confidence interval. A pairwise log-rank test indicated that there were no significant differences. c Cell footprint versus time, plotted relative to the first appearance of a close contact (timepoint 0 s). Plotted is the mean (±S.D.); n = 18 (pMHC^9V-hi, 5 SLBs), 17 (pMHC^9V-lo, 5 SLBs), and 11 (pMHC^null, 6 SLBs) signaling cells, and 23 (pMHC^null, 6 SLBs) non-signaling cells. d Number of close contacts versus time. Plotted is the mean (±S.D.). The analysis uses the same cells as in (c). e Area and diameter of individual close contacts within the first 10 s after their formation. Plotted is the mean (±S.D.). The analysis uses the same cells as in (c). f–i Same analysis as in (b–e) for primary CD8⁺ T cells. UCHT-1 Fab-HaloTag was used as an agonist. See Supplementary Movies 6 and 7. f Cumulative distribution of the searching to scanning stage transition for primary cells on SLB2s. The analysis uses data for both signaling and non-signaling cells [n = 19 (UCHT-1 Fab-HaloTag) and 28 (pMHC^null) cells]. g Cell footprint versus time, plotted relative to the first appearance of a close contact (timepoint 0 s). Plotted is the mean (±S.D.); n = 8 signaling cells (UCHT-1 Fab-HaloTag, 3 SLBs) and 14 non-signaling cells (pMHC^null, 6 SLBs). h Number of close contacts versus time. Plotted is the mean (±S.D.). The analysis uses the same cells as in (g). i Area and diameter of individual close contacts within the first 10 s after their formation. Plotted is the mean (±S.D.). The analysis uses the same cells as in (g). j, k J8-GECI cells interacting with SLB2s presenting pMHC^null plus pMHC^9V-hi. j Detection of pMHC from a single close contact at calcium release. See Supplementary Movie 8. Images are representative of J8-GECI cells for n = 3 independent SLBs. k Accumulation of ZAP70 at close contacts prior to calcium release. See Supplementary Movie 9. Images are representative of J8-GECI cells for n = 3 independent SLBs. l Total area of close contacts versus total cell membrane area at calcium release. ‘UCHT-1’ refers to UCHT-1 Fab-HaloTag. m Number of close contacts at calcium release. In l, m, boxplots indicate the quartiles with a line at the median. Whiskers extend to points that lie within 1.5 IQRs of the lower and upper quartile. Conditions were compared using the Kruskal–Wallis H test, and, if p < 0.05, further compared using the pairwise Mann–Whitney U test. The p values are shown. The analysis uses the same cells as in (c) for J8-GECI cells and (g) for primary cells. n Detection of single bound pMHC^9V at close contacts prior to (t = −4 s) and at calcium release (t = 0 s). Images show a J8-GECI cell interacting with an SLB2 presenting pMHC^null plus pMHC^9V-lo. The arrow indicates a single pMHC^9V molecule at a close contact. o Maximum number of pMHC^9V bound per close contact per cell for signaling and non-signaling cells; n = 13 FOVs, with 8 calcium signaling cells. Source data are provided in the Source data file.