Fig. 6.

BTK inducibility depends on B-cell maturation and activation, evobrutinib reduces cytokine production. PBMCs from healthy controls (HC) or MS patients were isolated freshly for B-cell purification or thawed from − 80 °C storage. a Purified B cells were stained using Fluo-3 and Fura Red for calcium mobilization assay. After 25 s baseline recording, cells were stimulated with 40 µg/ml anti-IgM/anti-IgG. PBMCs stained for surface markers and stimulated for 30 s using anti-IgM/anti-IgG. After immediate fixation and permeabilization, intracellular antibodies for BTK and pBTK (Y223) were incubated for 1 h. b Frequency of B cells/B-cell subsets on single cells. c BTK expression by MFI d BTK phosphorylation inducibility. e Isolated B cells were stimulated using 10 µg/ml anti-IgM for 22 h. Cytokine production of IL-6, IFN-γ and IL-10 was analyzed by ELISA and normalized to vehicle control. Median, n = 6–18, pooled from at least 3 independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001