Fig. 1

Single-cell RNA-sequencing and flow cytometry characterization of BTK expression in NMOSD. A Visualization of clustering by t-distributed stochastic neighbor embedding (t-SNE) plot of all B cells obtained from blood, CSF and bone marrow. B Heat map of scaled gene expression for top ten differentially expressed genes identifying each cluster, with selected genes listed. C Average proportion of each cell type derived from healthy controls (HCs, blood, n = 3; bone marrow, n = 2) and NMOSD (blood, n = 6; bone marrow, n = 2; CSF, n = 4) samples. D Volcano plot of differential expression genes of B cells between blood of patients with NMOSD and HCs or CSF/bone marrow from patients with NMOSD. Green, downregulated; red, upregulated. E Heatmap of differential expression genes (DEGs) involving BTK-related genes in B cells from different sources. F Expression of BCR signaling-related genes mRNA in peripheral blood B cells from HCs and patients with NMOSD was analyzed using real-time PCR. BTK (HCs, n = 9 and NMOSD, n = 18); LYN (HCs, n = 8 and NMOSD, n = 15); BCL2, PIK3CA, CD40, JAK1 (HCs, n = 6 and NMOSD, n = 15), STAT1 (HCs, n = 5 and NMOSD, n = 15) and BLK1 (HCs, n = 7 and NMOSD, n = 15). G Representative flow plots show BTK protein expression in a NMOSD patient, a healthy control, and a fluorescence minus one (FMO). H BTK protein expression was measured by intracellular flow cytometry in total B cells and B-cell subpopulations in HCs and NMOSD patients, normalized to BTK expression in total B cells of HCs, which was set to 1.0. (n = 16 per group). Phospho-BTK (pBTK) expression in total B cells and the indicated B-cell subpopulations that were either left unstimulated (pBTK_unstim) or were stimulated (pBTK_stim) for 30 s with anti-IgM (20 μg/ml) in blood of NMOSD patients and HCs (I) (n = 16 per group) or in blood and CSF from NMOSD patients (J) (blood, n = 16; CSF, n = 6). Data are displayed as means ± SEM. Statistical analysis was conducted by Student’s t test