Fig 2: Migration of primary human B cells towards S1P is effectively antagonized by cenerimod. (A) B cell migration towards different concentrations of S1P and cenerimod. B lymphocytes were activated with CD40L (2 μg/mL), IL-21 (20 ng/mL) and anti-IgM/IgG (10 μg/mL) for 48 h or left untreated. (B) Non-activated B cell migration towards S1P (80 nM) in the presence of different cenerimod concentrations applied to cells 30 min before the addition of S1P as chemoattractant. (A/B) Cell migration is shown by decrease of cell area on the top of the insert membrane after 30 h of chemoattractant application, normalized to solvent control. Data are means ± SEM (technical triplicate). * p < 0.0184, ** p < 0.0066 by Kruskal–Wallis nonparametric test and Dunn’s post-hoc test to compare each group to solvent control. One representative experiment from one healthy donor (n = 3) is shown.

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.

Fig 4: 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 5: Characterization of lymphoid tissue-specific B cell populations(A) Experimental workflow (n = 11 donors).(B) Violin plots of molecules significantly differentially expressed by at least two tissues (p < 0.005).(C) IgH isotype usage by tissue. ND denotes “not determined”; IgMD denotes co-expression of IgM and IgD.(D) Subset composition by tissue.(E) Manhattan distance between each tissue based on subset composition.(F) Biaxial of B cells colored as GC or other.(G) Biaxial of B cells colored as CD39+ tonsillar or other.(H) UMAP plot generated from an equal subsampling by tissue, then an equal subsampling by subset.