Background <p>B cells are key contributors to the pathogenesis of many autoimmune diseases (AID), including multiple sclerosis (MS), and appear to evade the peripheral tolerance checkpoints that normally maintain immune homeostasis. The fate of B cells at these checkpoints is believed to be regulated by intracellular Ca²⁺ signaling cascades triggered through engagement of B cell receptors (BCR), and by the suppressive effects of regulatory T cells (Tregs). However, most of the current knowledge about Treg–B cell interaction comes from animal studies, while data from human studies, particularly in the context of AID, are sparse. In contrast, impaired Treg-mediated inhibition of conventional T cells (Tcons) has already been described for several AID, including MS.</p> Objective <p>To assess the ability of Tregs to suppress activated B cells in healthy individuals and patients with MS.</p> Methods <p>B and T cell populations were isolated from 40 MS patients and 98 age- and sex-matched healthy donors (HD). Single-cell live Ca²⁺ imaging was used to assess early activation signals in B cells. In vitro proliferation assays and coculture experiments were employed to evaluate downstream responses, including proliferation, transcription factor activation (NFATc1, NF-ĸB), interleukin 6 (IL-6) release, and surface expression levels of antigen-presenting capacity (APC) markers both in anti-IgM/anti-CD40-stimulated B cells alone, and in the presence of Tregs.</p> Results <p>We demonstrate that Tregs exert a robust suppressive effect on B cell proliferation, IL-6 secretion and NFATc1 which is [<CitationRef CitationID="CR1">1</CitationRef>] independent of Ca²⁺ signaling [<CitationRef CitationID="CR2">2</CitationRef>], dependent on direct cell contact, and [<CitationRef CitationID="CR3">3</CitationRef>] impaired in MS. In contrast, early Ca²⁺ responses and downstream effects of anti-IgM/anti-CD40 stimulation, including activation of NFATc1 and NF-κB, as well as proliferation, did not differ between MS- and HD-derived B cells.</p> Conclusion <p>This study provides new data on Treg-mediated suppression of B cells in humans, including at single-cell level. Our findings show that the Treg dysfunction in MS previously described in the context of Tcon regulation extends to B cell regulation. Given the critical role of B cells in MS pathogenesis, this impaired Treg–B cell interaction may represent a previously underappreciated disease mechanism with potentially important therapeutic implications.</p>