Fig. 3: CD40 organisation.

a Cartoon of reconstituted SLBs. Left: Activated T-cell (blue) expresses CD40LG and interacts with the proteins on the SLB. Experimental molecular densities were: CD40-His-AF488 35-50, ICAM-1-His 200, and anti-CD3εFab-His 30 molecules/µm². Right: AlexaFluor647 (AF647) labelled antiCD40-Fab-His tethered to SLB. Primary lymphocytes expressing CD40 (B cells and monocytes) or lymphocytes lacking expression of CD40 (CD8⁺ T cells) were incubated with the bilayer allowing to follow CD40 organisation on cells. b Brightfield channel from confocal time-series for a stimulated (top) and quiescent (bottom) primary T-cell shortly after addition to SLB containing CD40-His-AF488. Scale bars are 5 µm. c, d Confocal and transmission images of a stimulated T cell (c) and quiescent T cell (d) on SLBs (labelled with CD40-AF488-His). Overview scale bar is 5 µm and zoom scale bar is 1 µm. Grey arrows indicate bright clusters of CD40-AF488-His. All confocal images are representative of at least 3 independent repeats. e, f sFCS raw correlation carpets for primary T-cells on locations exemplified by the purple lines in (c, d). g BTS histogram for CD40-His-AF488 on SLBs without T-cells (left), with quiescent cells (middle) and with stimulated T-cells (right). Control and quiescent and quiescent and stimulated are not significantly different (p = 0.15, p = 0.99 respectively; permutation test), control and stimulated are significant (p < 0.01). h Statistical analysis of sFCS transit time histograms indicating the relative likelihood (RL value) of free diffusion (value of 1 means free diffusion is the most likely cause of distribution shape). Every dot represents the assessment of one biological replicate (n = 4 for plain SLB, n = 6 for SLB with quiescent cells, and n = 7 for SLB with stimulated cells) for each pooling > 400 autocorrelation curves from various locations on >2 SLBs. i BTS diagram of antiCD40-Fab-His-AF647 as control (plain bilayer, left), incubated with T cells (middle), and B cells (right). Shifts in the BTS histograms are not significant p > 0.05 (permutation test). j Diffusion coefficients extracted from sFCS measurements in i reported as average per condition (ie., every dot represents >400 individual FCS curves pooled for each replicate, different donors for conditions with cells; n = 7 for antiCD40-Fab, n = 4 for T cells, n = 4 for B cells). Horizontal lines are mean values and error bars are standard deviation. P-values calculated using one-way ANOVA with Tukey’s test for multiple comparisons. All data were acquired at 37 °C.