Fig. 6. BTK expression in microglia/macrophages within lesion tissue from PMS patients.

A Immunohistochemistry analysis for myelin PLP and BTK in post-mortem brain tissue from a PMS patient. BTK-positive cells colocalized with an IBA1-positive rim around white matter lesions. Human BTK histology was the result of many experiments to determine optimal antibodies and concentrations to selectively stain for BTK+ cells. B BTK positivity in demyelinated regions compared with NAWM (n = 7 patients). BTK mRNA expression (C) and protein levels (D; assessed by Western blot) in post-mortem bulk brain tissue samples from controls and MS patients (either grouped by individual MS subtype or as all MS combined). P-values were calculated using one-way ANOVA with post hoc Dunnett test for (D). The p-values shown in (C) from left to right are 0.021, 0.023, and 0.008 and in (D) is 0.0019. (N = 5–18 and 5–6 patients for (C) and (D), respectively). Whiskers in (C) denote minimum and maximum values. E Co-immunostaining for BTK and microglia/macrophages, using anti-IBA antibody (for visualization) or anti-CD68 antibody (for quantification in lesion tissue compared with NAWM, where n = 3 patients). F Hierarchical clustering of PMS patient lesion samples from NAWM samples using the BTK-reversed signature identified in human iPSC-derived microglia³². Heatmap displays log2(FPKM + 1) data scaled using a Z-score. Differential expression analysis was performed in Array Studio using a general linear model. The tolebrutinib-reversed transcriptional signature consists of 144 differentially expressed genes (absolute(fold-change) ≥1.2 and p-value ≤ 0.05) in IgG + tolebrutinib vs IgG + Vehicle that were reversed in IgG + Vehicle vs Vehicle. G, H Cellular enrichment of BTK mRNA expression in brain samples from PMS patients, including t-SNE analysis (H)³². I Expression of BTK-regulated gene targets in brain samples from controls and SPMS patients. P-values are indicated by * ≤ 0.05, ** ≤ 0.01, *** ≤0 .001, and **** ≤ 0.0001. Data are presented as mean values ± SEM. BTK Bruton’s tyrosine kinase, CD68 cluster of differentiation 68, DAPI = 4′,6-diamidino-2-phenylindole, IBA1 ionized calcium-binding adapter molecule 1, MS multiple sclerosis, NAWM normal-appearing white matter, Oligo oligodendrocyte, OPC oligodendrocyte progenitor cell, PLP proteolipid protein, PMS progressive MS, PPMS primary PMS, RRMS relapsing-remitting MS, SPMS secondary PMS. Source data are provided as a Source Data file.