<p>Lymphocytes play a central role in adaptive immunity and represent the primary source of RANKL in periodontitis. Understanding their organization and activation within gingiva is therefore essential. This study investigates the role of tertiary lymphoid structures (TLSs), ectopic lymphoid aggregates that orchestrate local immune responses. Using multiplex immunohistochemistry staining, we found that TLSs form and mature with increasing inflammatory severity. These structures function as hubs for B cell activation and were associated with poorer surgical outcomes. Given the known role of fibroblasts in initiating TLS assembly, we explored the underlying mechanism. Using single-cell RNA sequencing and in vitro assays, we identified that oral pathogens activate the IFN-I–IRF7 axis in gingival fibroblasts, leading to overproduction of IFNβ and driving their differentiation into a CXCL13-producing subtype that facilitates TLS organization. Furthermore, in a mouse periodontitis model, we demonstrated that activated IgD⁺CD80⁺ B cells within TLSs promote osteoclastogenesis and bone resorption via RANKL secretion. Therapeutically, inhibition of IFN-I signaling disrupted this pathogenic cascade by suppressing TLS formation and subsequent RANKL production, thereby mitigating periodontitis bone loss. In conclusion, our findings establish TLSs as pathological hubs that coordinate B cell-mediated bone destruction in periodontitis and highlight the IFN-I signaling pathway as a promising target for therapeutic intervention.</p>