B-cell depletion is an efficacious treatment in relapsing and progressive multiple sclerosis (MS). A phase II trial (NCT02975349) showed promising results for Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of MS. Previously, we found that T-bet+ B cells preferentially infiltrate the central nervous system and are induced under IFN-γ- and TLR9-stimulating, germinal center-like conditions in MS.
We aimed to elucidate how BTK is expressed and activated in distinct ex vivo B-cell subsets during the course of MS. Moreover, the relation between BTK activity and T-bet+ B-cell differentiation was assessed both ex vivo and in vitro.
We determined BTK and phosphorylated BTK (pBTK) levels in transitional, naive mature, class-switched and non class-switched B cells in blood from both treatment-naive patients with CIS, RRMS, SPMS and PPMS and healthy controls (HC; n=30 per group), as well as clinical MS responders and non-responders to natalizumab (pre- vs 1y post-treatment) using flow cytometry. Purified naive mature and memory B cells were cultured under several IL-21/CD40L-inducing conditions with and without evobrutinib.
BTK was mainly expressed in non-class-switched memory B cells, while pBTK levels were high in both class-switched and unswitched memory B cells. In contrast to BTK, pBTK was significantly higher in ex vivo memory B cells of RRMS and SPMS compared to CIS, PPMS and HC groups. In both RRMS and SPMS, pBTK was also less induced after a-IgM stimulation. BTK and pBTK levels were elevated in blood B cells from clinical responders, but not in non-responders to natalizumab. These levels correlated positively with CXCR3 and VLA-4 expression. No correlation was seen for CXCR4, CXCR5, CD40 and HLA-DR. In vitro experiments revealed that pBTK in B cells was particularly triggered by IFN-γ and TLR9 induction. Evobrutinib attenuated class-switching during in vitro cultures of naive B cells, while it interfered with plasmablast formation in memory B-cell cultures. T-bet and T-bet-related markers (CD21, CD11c) were only affected by evobrutinib in IFN-γ- and TLR9-stimulating naive B-cell cultures.
These data demonstrate that BTK is more activated in memory B cells from RRMS and SPMS patients and functionally related to pathogenic T-bet+ B-cell development. This study provides new mechanistic insights into how evobrutinib intervenes in human B-cell differentiation and can modulate the clinical course of MS.