The influence of anti-CD20-directed B cell depletion across different agents on Epstein-Barr virus-specific humoral and cellular immunity in patients with multiple sclerosis

Abstract Background Epidemiological evidence has established a central role of Epstein-Barr virus (EBV) in the pathogenesis of multiple sclerosis (MS). While EBV-specific T-cell responses may influence disease progression, the impact of anti-CD20 B-cell depletion therapy (BCDT) on EBV-specific cellular and humoral immunity remains insufficiently characterized, particularly in comparison with healthy controls (HC), untreated people with MS (pwMS), and pwMS on different BCDT agents. Main body Methods In this cross-sectional study, pwMS receiving BCDT, untreated pwMS at initial diagnosis (MS-ID), and age- and sex-matched HC were enrolled. EBV-specific and polyclonal CD8 + and CD4⁺ T-cell responses were quantified and functionally characterized following stimulation with a EBV-derived peptide-mix predominately containing MHC class I binding peptides or Staphylococcus aureus enterotoxin B (SEB) using multiparametric flow cytometry. Anti-EBNA1- and anti-VCA-IgG levels were measured by ELISA. Results A total of 183 participants were included: 54 pwMS receiving BCDT (mean age 47.0±15.6 years; 31 females), 107 controls (47.2±18.3 years, 54 females), and 22 MS-ID (33.4±11.1 years, 20 females). EBV-specific CD8⁺ and CD4⁺ T-cell levels were similar in controls and MS-ID. In contrast, BCD-treated pwMS showed significantly lower frequencies of EBV-specific CD8⁺ ( p <0.0001) and CD4⁺ T cells ( p =0.0004) than controls, and lower EBV-specific CD8⁺ T-cell levels than MS-ID ( p =0.0023). In contrast, polyclonally stimulated CD8⁺ T-cell responses were largely comparable across groups, indicating preserved polyclonal T-cell function. EBV-specific CD8⁺ T cells in both patients and controls were predominantly polyfunctional, coexpressing IFNγ, IL-2, and TNF. Longer BCDT duration was associated with lower frequencies of EBV-specific CD8⁺ T cells ( p =0.0003). Ofatumumab-treated patients exhibited higher EBV-specific CD8⁺ T-cell frequencies than ocrelizumab-treated patients ( p <0.0001). Despite BCDT, anti-EBNA1- and anti-VCA-IgG levels were significantly higher in pwMS than in MS-ID or controls, and were unrelated to treatment duration, disease duration, disability, or BCDT agents. Conclusions Unlike in treatment-naïve pwMS, BCDT is associated with selectively lower EBV-specific T-cell frequencies while EBV-specific and polyclonal T-cell functionality was largely preserved. In contrast, EBV-specific antibody levels were persistently higher despite treatment. Given the remarkably high clinical efficacy of BCDT in this cohort, the findings support a prominent role of EBV-specific cellular immunity in ongoing MS pathophysiology and suggest that reduced EBV-reactive T-cell responses may contribute to this potency. Differences between BCDT agents warrant further investigation.