Antiviral and inflammatory immune signatures in anti-CD20-treated multiple sclerosis: a cross-sectional study

CD20-targeting monoclonal antibodies are highly effective therapies for relapsing multiple sclerosis (MS). We examined how anti-CD20 therapy reshapes antiviral T-cell immunity, innate inflammatory tone, and blood markers of neuroaxonal injury. In this cross-sectional study, adults with relapsing MS were enrolled at two neurological centers in Rome. An immunophenotyping cohort (45 untreated, 25 anti-CD20-treated, all receiving ocrelizumab) was profiled by high-parameter flow cytometry across more than 40 immune populations. A separate antiviral cohort (24 untreated, 13 treated, 19 healthy donors) was assessed for T-cell responses to Epstein-Barr virus (EBV), cytomegalovirus (CMV) and SARS-CoV-2 by activation-induced marker assay, for cytokine and chemokine release by multiplex assay, and for serum neurofilament light chain (sNfL) by single-molecule array. Groups were compared by Kruskal-Wallis with Dunn's correction; correlations used Spearman's rank test. Anti-CD20 therapy depleted total and switched-memory B cells while sparing antibody-secreting cells and reduced absolute counts of follicular helper T cells and mucosal-associated invariant T cells. Untreated patients with MS (pwMS) showed elevated monocyte interleukin-1β and monocyte chemotactic protein-3 (MCP-3) and impaired pDC cell interferon-α production, whereas pDC IFN-α responses in ocrelizumab-treated pwMS were comparable to those in HD. EBV-specific CD4+ T-cell counts and stimulation index were selectively lower ocrelizumab-treated pwMS, whereas CMV- and SARS-CoV-2-specific responses were comparable across groups. sNfL was elevated in untreated pwMS and correlated with MCP-3 at baseline and after EBV stimulation. Anti-CD20 therapy acts on immune circuits beyond B cells, selectively contracting EBV-directed helper T-cell immunity while sparing other antiviral responses, and identifies MCP-3 as a candidate blood marker associated with neuroaxonal injury in pwMS.