Early Combined B-Cell Depletion and BTK Inhibition Reduced TLS-like Structures and Relapse in PLP139–151-Induced EAE

B-cell-depleting therapies have revolutionized multiple sclerosis (MS) treatment, yet relapses persist in some patients—suggesting additional pathogenic drivers beyond peripheral B cells. Tertiary lymphoid structures (TLS) are extensively documented in progressive MS at autopsy, but whether their formation begins during the relapsing-remitting phase and how they evolve during the transition to progression remain undefined. Here, using the relapsing-remitting PLP139–151-induced EAE model, we uncover that TLS-like structures form in the subventricular zone during relapse, once established, persist through remission as niches containing both B cells and persistently activated microglia. Neither B-cell depletion alone nor BTK inhibition alone fully prevents relapse. Strikingly, early combined B-cell depletion and BTK inhibition virtually abolishes TLS-like structure formation and may effectively prevent complete disease relapse in this model. By contrast, late initiation of the same combination fails to resolve existing TLS-like structures or prevent relapse, although it attenuates disease severity. These data indicate that established TLS-like structures may represent treatment-resistant compartments, and that both B cells and microglia may be crucial during early formation for sustaining their disease relapse-driving activity. Our study confirms that TLS-like structures may be a key factor driving the compartmentalization of central nervous system inflammation, points to a potentially narrow therapeutic window for intervention, and proposes that early combined B-cell depletion and BTK inhibition may represent a promising strategy worthy of further investigation.