A Tonsil Organoid Model for Epstein-Barr Virus-Host Immune Interactions in Germinal Centers

Introduction/Rationale: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that establishes lifelong persistence in B lymphocytes by adopting distinct latency programs within lymphoid tissues such as the tonsils. In vitro, EBV infection of primary B cells drives transformation into latency III lymphoblastoid cell lines (LCLs) with tumor-like phenotypes. In contrast, in vivo EBV-infected cells are predominantly found in germinal center (GC) and memory B cell compartments, exhibiting restricted latency states (IIa, I) with limited viral antigen expression. The developmental origin of these in vivo infected populations remains unknown, as existing models fail to recapitulate the tonsillar immune microenvironment necessary to study EBV-driven B cell differentiation pathways beyond LCL transformation.

Methods: We developed a tonsil organoid system preserving native immune cell composition and supporting GC formation. Organoids were infected with EBV and analyzed by flow cytometry and scRNAseq. Immune cell depletion experiments dissected cellular interactions driving EBV-induced B cell responses.

Results: We discovered that EBV directly drives naïve B cell differentiation into CD10+BCL6+ GC-like cells, a pathway distinct from canonical LCL transformation. A subset acquired latency IIa programming (LMP1+EBNA2-), recapitulating in vivo EBV+ GC cell phenotypes. This differentiation depends on germinal center immune microenvironment, and complete immune cell depletion abrogated GC-like B cell formation. Tfh cell depletion partially blocked differentiation, which was rescued by IL-21 supplementation, demonstrating Tfh-derived IL-21 as a critical driver. Conversely, CD8+ T cells from EBV-seropositive donors suppressed this differentiation, revealing adaptive immune control.

Conclusion: Our findings reveal how tonsillar immune cells orchestrate EBV-infected B cell fate and viral latency programming, providing a platform for developing immune-based therapies for EBV-associated lymphoproliferative diseases.