Fig 2: Characterization of S1P- and cenerimod-mediated S1P1 receptor internalization, and S1P1 receptor and CD69 expression kinetics upon cell activation in primary human B cells. (A) Concentration–response curves of min/max normalized S1P1 receptor surface expression after 30 min ligand (S1P or cenerimod) incubation, with EC50 values given. (B) Detection of B cell proliferation and concurrent expression of S1P1 receptor and CD69 after four days of B cell activation by CD40L (2 μg/mL) alone or combined activation using CD40L (2 μg/mL), IL-21 (20 ng/mL) and anti-IgM/IgG (10 μg/mL) compared to non-activated control (solvent). (C) Expression kinetics of CD69 and S1P1 receptor on B cells from Day 0 to Day 4 post activation treatment as described in (B). (D) Total S1P1 receptor expression level after four days of B cell activation compared to non-activated control with treatments as described in (B) analyzed by Western Blot. CTV (CellTrace™ Violet, proliferation staining). One representative experiment from one healthy donor (n = 3) is shown.

Fig 3: Oligomerisation in vitro on reconstituted supported lipid bilayers.a Cartoon depicting the experimental set-up of a supported lipid bilayer with tethered His-EGFP (visualised as green beta barrel inspired by PDB 1f0b102). Oligomerisation was induced by use of primary (orange) and secondary antibodies (magenta). b Confocal images of SLBs composed of DOPC and DGS-Ni-NTA (4%) labelled with EGFP-His (left), treated with primary (mouse) anti-GFP antibody (middle), and addition of secondary antibody (anti-mouse, right). Scale bar is 5 µm. Representative images from >3 independent experiments. c BTS diagram for EGFP (left, control), EGFP and anti-GFP antibody (+Primary, middle), and EGFP, primary, and secondary antibody (+Primary/Secondary, right). All BTS histograms are significantly different from each other with p < 0.01 (permutation test). d Changes in numbers of particles per area as determined by sFCS. e Experimental fluctuation data quality measured by nRMSD and SNR as violin plots (dashed grey lines represent median and dotted lines represent quartile values). f Cartoon depicting the interaction of a membrane-anchored EGFP with a fluorescently tagged nanobody undergoing energy transfer (FRET) g BTS histograms for EGFP (control, left) and with anti-GFP nanobody (labelled with AbberiorSTAR635P, right), p < 0.01 (permutation test) h Cartoon of the reconstituted system for investigation of CD40 organisation. AlexaFluor488 conjugated to a His6-tag (AF488-His) was used as a monomeric control (left). Recombinant CD40-His was labelled with AF488 (orange with green star). Perturbations performed with anti-CD40 antibodies (black) or recombinant CD40LG (red). i Confocal images of SLBs with varying amounts (measured by sFCS) of CD40-AF488-His. Scale bar is 5 µm. j BTS histogram for AF488-His on a SLB as monomeric control k sFCS BTS diagrams for different surface concentrations of CD40-AF488-His. 25 molecules/µm2 is significantly different from 100 molecules/µm2 (p < 0.01) whereas 175 molecules/µm2 is not significantly different from 100 molecules/um2 (p > 0.05, permutation test). l, m sFCS BTS diagram for CD40-AF488-His (monomeric control, l) and under perturbation (m) with anti-CD40 antibodies (5C3 and HB14) or recombinant CD40LG-His (all unlabelled). Conditions in m are significantly different from the control in n with p < 0.01 (permutation test). 5C3 is not significantly different from HB14. CD40L vs 5C3 p = 0.1 and CD40L vs HB14 p = 0.01 (permutation test). The experimental histograms are representative data from one repeat and integrate more than 10 sFCS acquisitions on various bilayer locations (>500 autocorrelation curves) acquired at ambient temperature (24 °C).

Fig 4: Segregating B cells into phenotypically and isotypically distinct subsets(A) Experimental workflow (n = 3 donors).(B) Median expression by subset.(C) Percent of B cells for all subsets, colored as in (B).(D) UMAP plot generated from an equal subsampling by subset.(E) IgH isotype usage by subset. ND denotes “not determined”; IgMD denotes co-expression of IgM and IgD.(F) Euclidean distance between each B cell subset based on median expression profile. White boxes denote column minimum.(G) Subset composition by isotype as determined by canonical gating or meta-clustering.(H) Contour plots by IgH isotype. Dots and error bars indicate mean and SEM of individual donors.(I) Relative contribution of phenotype and isotype to the variance explained by linear models created to predict single-cell expression of CD79b or surface Ig.

Fig 5: 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.