Fig. 2

BTK inhibition limits B-cell activation and differentiation in NMOSD. A Experimental design to examine the effect of BTK inhibition on B-cell subtypes, production of AQP4 antibody and intracellular signaling pathway under different stimulants. B, C Representative graph and quantitative analysis of intracellular BTK expression from a NMOSD patient. (B: sorted B cells after 2 day culture of either untreated or activated with F(ab′)₂ anti-human IgM; C: PBMC after 7 day culture of either untreated or activated). D–I Quantitative analysis of the proportions of naive B cells (D) switched memory B cells (E) and ASCs (F) and the expression of CD86 (G), CD69 (H), HLA-DR (I) in magnetic-activated cell-sorted B cells from healthy controls or NMOSD patients after 2 days of stimulation with F(ab’)₂ anti-human IgM (10 μg/ml) in the absence or presence of BTKi (zanubrutinib, 10 nM) (n = 6 per group). J Binding of patient IgG from culture supernatants to live HEK-293 T cells co-expressing AQP4 and green fluorescent protein (GFP) from a single plasmid. Quantitative titres defined as the difference in mean fluorescence intensity (MFI) between the GFP-expressing and GFP-negative cell populations (expressed as △MFI). K NF-κB nuclear translocation staining showed B cells failed to translocate NF-κB from cytoplasm to the nucleus upon stimulation after administration of zanubrutinib. L Analyses of similarity scores showed NF-κB translocation to the nucleus of B cells. Representative of 3 experiments. The spatial relationship between the NF-κB and nuclear images was measured using the ‘Similarity’ feature in the IDEAS® software. Data are displayed as means ± SEM. D–I Statistical analysis was conducted by Mix-effects two-way ANOVA, L Repeated measure two-way ANOVA