TBX21+CD21loCXCR3+B cell expansion with an EBV-associated phenotype in clinically isolated syndrome conversion to multiple sclerosis

Epstein-Barr virus (EBV) infection precedes multiple sclerosis (MS) onset and plays an etiologic role in the disease, although the nature of viral involvement is poorly understood. A common early event in the course of MS is Clinically Isolated Syndrome (CIS), a neuroinflammatory demyelinating condition for which 60-80% of cases progress to relapsing-remitting MS (RRMS). To investigate possible viral pathogenesis during early disease stages, we analyzed single-cell gene and surface protein expression of CIS peripheral B cells collected longitudinally during the Immune Tolerance Network (ITN) STAyCIS Trial. CIS B cell profiles were compared to publicly available scRNA-seq data from in vitro EBV infection, other autoimmune disorders, chronic infectious diseases, and healthy controls. Analyses focused on CD19+/CD20+/CD21lo/CD11c+/T-bet+ atypical B cells (ABCs), which are expanded pathogenic effectors in MS and other autoimmune and chronic infectious diseases. CIS PBMCs contained significantly higher frequencies of ABCs than healthy adult controls by scRNA-seq and flow cytometry, establishing ABC compartment expansion as a clinical feature of CIS. An EBV-associated ABC transcriptome, including expression of CXCR3 and immune checkpoint molecules (PD-L1, PD-L2), was enriched in CIS versus controls; however, evidence of direct EBV infection within the ABC niche was infrequently observed. CIS ABCs exhibited significant enrichment of inflammatory cytokine mRNAs including CXCL8 (IL8), IL18, and VEGFA. Several of these cytokines (IL-8, VEGF) were secreted by B cells upon de novo EBV exposure. CIS outcome stratification revealed a rare yet distinctive inflammatory ABC signature significantly underrepresented in long-term non-progressors (LTNP) versus people with primary endpoint RRMS activity. Moreover, CXCR3+ ABCs increased after baseline CIS diagnosis and were significantly enriched in MS vs LTNP outcomes after controlling for age and sex. Thus, we find evidence for ABC expansion, inflammatory responses, and checkpoint that precede MS onset, possibly as a bystander response to EBV infection.