An EBV-associated atypical B cell signature in clinically isolated syndrome is implicated in progression of multiple sclerosis

Expansion and pathogenicity of CD19+/CD20+/CD11c+/T-bet+ atypical B cells (ABCs) are hallmarks of numerous autoimmune disorders and chronic infections. In many such cases Epstein-Barr virus (EBV) is another associated or etiologic factor, though EBV involvement in these diseases remains poorly understood. Notably, the expansion of pro-inflammatory ABCs and a putative causal role for EBV have been identified independently in multiple sclerosis (MS). A common precipitating event in MS onset is Clinically Isolated Syndrome (CIS), a neuroinflammatory demyelinating condition of which 60-80% of cases progress to relapsing-remitting MS (RRMS). Here we report single-cell gene and surface protein expression (scRNA/CITE-seq) in peripheral B cells collected longitudinally from patients with CIS during the Immune Tolerance Network STAyCIS Trial. We focus on the transcriptomic signatures of ABCs from this cohort, publicly available scRNA-seq datasets from six other autoimmune and chronic infectious diseases, and in vitro EBV infection. Conservation of an expanded ABC expression profile across diseases establishes ABC dysregulation as a feature of CIS. Critically, we also observed transcriptomic features that distinguished CIS and de novo EBV-infected ABCs from those found in healthy controls and other disease contexts. Outcome stratification of CIS samples revealed a rare yet distinctive pro-inflammatory ABC subset that was significantly underrepresented in long-term non-progressor (LTNP) versus cases with RRMS activity (∼5-fold difference). Collectively, this study provides evidence for altered ABC regulation – possibly arising from niche-specific responses to EBV infection – preceding MS onset. SUMMARY Single-cell transcriptomics establishes an EBV-associated signature in T-bet+ atypical B cells in CIS and a pro-inflammatory phenotype underrepresented in patients with no disease progression. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT00094172 ### Funding Statement Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH NIAID award #UM1AI109565) and an ITN award to S.G.G.. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. E.D.S. wishes to acknowledge funding from the American Cancer Society (ACS; award PF-21-084-01-DMC). M.A.L. acknowledges support from the NIDCR (R01DE025994). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: An Internal Review Board (IRB) of Duke University gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors