Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system and a leading cause of neurologic disability in young adults. Relapsing‑remitting disease courses are marked by episodic symptomatic flares separated by remission, but the molecular events that immediately precede clinical relapse are poorly resolved because relevant immune perturbations can be transient and presymptomatic. Existing biomarkers such as serum neurofilament light chain reflect downstream tissue injury rather than upstream triggers. Given the central role of B cells in MS pathogenesis and the established association between EBV infection and MS risk, this study sought to define time‑resolved peripheral immune changes that occur before clinical relapse.
The investigation was an observational study of longitudinal blood samples from patients with relapsing‑remitting MS. The authors combined multiple immune profiling modalities to capture cellular and molecular dynamics surrounding relapse: single‑cell RNA sequencing, bulk transcriptomics, multiparameter flow cytometry and targeted viral reverse transcription quantitative PCR (RT‑qPCR). These complementary approaches were used to generate a temporally resolved atlas of peripheral immune perturbations that emerge before, during and after relapse.
Analyses identified a reproducible pre‑relapse transcriptional program detectable in both monocytes and B cells, with components of this signature emerging up to roughly 3 months before clinical symptom onset. The signature included modules of host genes that are responsive to factors associated with EBV lytic reactivation, indicating that the peripheral immune compartment shows coordinated transcriptional changes well before overt relapse.
Targeted viral RT‑qPCR confirmed the presence of elevated EBV transcripts—specifically increased LMP‑1 RNA—in B cells sampled during the pre‑relapse period. This molecular evidence supports the transcriptomic enrichment for EBV‑responsive host genes and suggests active EBV gene expression in peripheral B cells prior to clinical relapse.
Multiparameter flow cytometry demonstrated expansion of CD11c+ atypical memory B cells (ABCs) in the pre‑relapse window. These expanded B cell populations displayed the EBV surface glycoprotein gp350, linking the phenotypic expansion of ABCs to detectable viral protein expression on B cells during the pre‑relapse period. The findings align with prior descriptions of ABC expansion in autoimmune diseases and chronic viral infections and with observations that ABCs in MS express interferon‑stimulated genes and antigen‑presentation machinery.
Pre‑relapse transcriptional modules substantially overlapped with genetic risk regions identified by MS genome‑wide association studies and with enhancers bound by the EBV nuclear antigen EBNA‑2. This overlap suggests that inherited susceptibility and EBV‑responsive transcriptional programs may act through shared regulatory elements to shape peripheral immune responsiveness in the months preceding relapse.
The study establishes a temporal association between peripheral EBV reactivation signatures and subsequent clinical relapse, but causal relationships and the pathway linking peripheral events to CNS lesion formation were not established. How peripheral B cell activation and EBV expression translate into CNS immune activation, lesion accrual or blood–brain barrier perturbation remains to be determined. Details of sample sizes, cohort demographics, exact timing windows for all patients, and other protocol specifics were reported in the primary article but are not restated here. The generalizability across MS subtypes and treatment backgrounds likewise requires further study.
These findings support a model in which EBV reactivation within a genetically predisposed peripheral immune environment is a proximal precursor of MS relapse. The results reinforce interest in EBV‑targeted strategies and in monitoring EBV activity or ABC expansions as potential early indicators of imminent relapse. Future work should address mechanistic links between peripheral EBV‑related activation and CNS pathology, evaluate whether these peripheral signatures predict radiographic activity, and determine if interventions that suppress EBV reactivation or modulate ABCs alter relapse risk. Until such causal and interventional data are available, the clinical application of these biomarkers remains investigational.