Fig. 1: Requirement of CD99 for IS formation and actin reorganization.

a Fluorescence microscopy images of wild type (WT) or Cd99^−/− H60-CTLs (green) coincubated with cognate H60-DCs or control OVA-DCs (red). Numbers of CTLs in contact with one DC (n = 263, 369, 386, and 192 conjugates, from left to right) and the proportion of DCs in contact with more than three CTLs (n = 5 fields per group) are plotted. b Confocal images of phalloidin-stained WT or Cd99^−/− H60-CTLs, and control OVA-DC or cognate H60-DCs (cognate DC) after 15 min of coincubation. Polarized CTLs were quantified by identifying cells with higher F-actin intensity at the IS compared to the rear of the CTL (n = 6, 12, 5, and 12 fields, from left to right). Representative single Z-section images of the synapse are shown. Each data point in the graph indicates percentage of polarized cells per field. c–e Longitudinal analysis of actin reorganization in anti-CD3-stimulated WT- and Cd99^−/−-cells (see Supplementary Movie 1). Time-lapse images of F-actin (Lifeact-mCherry) against black or differential interference contrast (DIC) image background (c). Time in minutes:seconds. Plots of cellular fluorescence intensity expressed in arbitrary units (arb.units) (n = 6 cells per group) (d). Plots of the synapse area (a, white dotted line on cell image) and lamellipodia width (b, black line) at different time points, and distance of the actin-microcluster (MC) from the plasma membrane (c, yellow double-headed arrow) measured at 15–20 min (n = 9 cells per group) (e). f Quantification of nuclear translocation of NFAT (n = 9, 35, 28, 40, 8, 40, 33, and 35 cells, from left to right) and NFκB (n = 23, 24, 35, 38, 24, 20, 37, and 35 cells, from left to right), and phosphorylation of ERK (n = 28, 48, 46, 43, 38, 43, 39, and 34 cells, from left to right) at the indicated time points (see Supplementary Fig. 1e–g). All representative images were obtained from at least 5 randomly selected fields from each group. Data (a, b, d–f) are presented as mean ± SEM from at least three independent experiments. Statistical significance was assessed using unpaired two-tailed t-test (a, b, d, f), and two-way repeated measures ANOVA with Sidak post-hoc test (d, e). Exact p-values are presented in the figure. Source data are provided as a Source Data file.