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

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. 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. 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. 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.