Fig 2: Characterization of S1P- and cenerimod-mediated S1P1 receptor internalization, and reciprocal S1P1 receptor and CD69 expression kinetics upon activation of primary human T cells. (A) Concentration–response curves of min/max normalized S1P1 receptor surface expression after 30 min ligand (S1P or cenerimod) incubation with the half maximal effective concentration (EC50) values given. (B) Detection of T cell proliferation and concurrent expression of S1P1 receptor and CD69. T cells were left non-activated (top) or were activated for 48 h (anti-CD3 and anti-CD28), followed by two days of proliferation, in the constant presence of S1P (0.5 µM), cenerimod (0.5 µM) or DMSO (control). (C) Detection of CD69 and S1P1 receptor expression kinetics in CD4+ T cells following activation by anti-CD3 and anti-CD28. CTV (CellTrace™ Violet, proliferation staining). One representative experiment from one healthy donor (n = 3) is shown.

Fig 3: Antagonism of S1P-mediated migration of primary human B cells by cenerimod is not affected by prednisolone. 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. As indicated prednisolone (100 nM) or solvent control was added 30 min before cenerimod. 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 < 0.0419, ** p < 0.0029 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 4: 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 5: Migration of activated primary human T cells towards S1P is effectively antagonized by cenerimod. (A) Experimental setup of real-time migration assay towards the chemoattractant (S1P or cenerimod) with or without pretreatment of different cenerimod concentrations to test the effectiveness of migration prevention. Vertical-directed cell migration towards the chemoattractant (applied to the reservoir) was quantified by the decrease of cell counts on the top of the insert membrane over time. (B) Representative pictures taken of the insert surface to detect decrease of cell count due to migration. The experimental procedure was performed as described in (A). (C) Migration of activated (anti-CD3 and anti-CD28, 48 h) T cells towards different concentrations of S1P or cenerimod. (D) Migration of activated (anti-CD3 and anti-CD28, 48 h) T cells towards S1P (80 nM) in the presence of different cenerimod concentrations applied to cells 30 min before S1P addition as chemoattractant. (C/D) Cell migration is shown by decrease of cell count on the top of the insert membrane after 90 h of chemoattractant application, normalized to solvent control. Data are means ± SEM (technical quadruplicate). ** p < 0.0023 by Kruskal–Wallis nonparametric test and Dunn’s post-hoc test to compare each group to either solvent control (C) or S1P (80 nM only, no cenerimod) (D). One representative experiment from one healthy donor (n = 3) is shown.