Recent clinical trials have shown promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti–VLA-4 antibody) treatment. Under in vitro T follicular helper–like conditions , BTK phosphorylation was enhanced by T-bet–inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet–associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3 ⁺ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.

The effector programs of CD8⁺ memory T cells are influenced by the transcription factors RUNX3, EOMES and T-bet. How these factors define brain-homing CD8⁺ memory T cells in multiple sclerosis (MS) remains unknown. To address this, we analyzed blood, CSF and brain tissues from MS patients for the impact of differential RUNX3, EOMES and T-bet expression on CD8⁺ T cell effector phenotypes. The frequencies of RUNX3- and EOMES-, but not T-bet-expressing CD8⁺ memory T cells were reduced in the blood of treatment-naïve MS patients as compared to healthy controls. Such reductions were not seen in MS patients treated with natalizumab (anti-VLA-4 Ab). We found an additional loss of T-bet in RUNX3-expressing cells, which was associated with the presence of MS risk SNP rs6672420 (RUNX3). RUNX3⁺EOMES⁺T-bet⁻ CD8⁺ memory T cells were enriched for the brain residency-associated markers CCR5, granzyme K, CD20 and CD69 and selectively dominated the MS CSF. In MS brain tissues, T-bet coexpression was recovered in CD20^dim and CD69⁺ CD8⁺ T cells, and was accompanied by increased coproduction of granzyme K and B. These results indicate that coexpression of RUNX3 and EOMES, but not T-bet, defines CD8⁺ memory T cells with a pre-existing brain residency-associated phenotype such that they are prone to enter the CNS in MS.