To uncover how BTK activity is regulated in B cells during the MS course and whether it contributes to the development of brain-homing T-bet⁺ B cells using evobrutinib.

A phase II trial (NCT02975349) showed promising results for Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of MS. Recently, we found that CXCR3⁺ B cells preferentially infiltrate the CSF, meninges and brain tissues from MS patients and are selectively triggered under IFN-γ- and TLR9-inducing, germinal center-like conditions. CXCR3 is the surrogate marker of T-bet, which is enriched in age-associated B cells.

BTK and pBTK levels in transitional, naive mature, class-switched and unswitched memory B cells were compared between CIS, RRMS, SPMS, PPMS and matched healthy control blood (n=30 per group). The impact of evobrutinib on in vitro differentiation and transmigration of B cells was studied under T-bet-, IL-21/CD40L-inducing conditions and using monolayers of human brain endothelial cells.

BTK was mainly expressed in unswitched memory B cells, while pBTK levels were highest in both class-switched and unswitched memory B cells. In contrast to BTK, pBTK was significantly higher in memory B cells of RRMS and SPMS versus CIS, PPMS and healthy control groups. In RRMS and SPMS, pBTK was less induced after a-IgM stimulation. These levels correlated with CXCR3 and VLA-4 expression. In vitro experiments demonstrated that pBTK was upregulated after IFN-γ and TLR9 induction and that CXCR3, T-bet and CD21 were downregulated by evobrutinib irrespective of anti-IgM triggering. Evobrutinib inhibited class-switching, the formation of IgG-producing plasmablasts and transmigration of CXCR3⁺ memory B cells.

BTK is more activated in memory B cells of both RRMS and SPMS patients and functionally related to T-bet⁺ memory B cells. This provides key insights into how evobrutinib intervenes in pathogenic B-cell differentiation and can modulate the clinical course of MS.