Resolving the cellular profile of germinal center-analogous microstructures in zebrafish

Teleost fish lack canonical germinal centers (GCs); however, they can mount antigen-specific immune responses, raising longstanding questions about how antibody affinity maturation is organized in cold-blooded vertebrates. Melanomacrophage centers (MMCs) have been proposed as GC analogues, yet their cellular composition and immune organization remain poorly defined. To address this, we isolated autofluorescent MMCs from zebrafish spleens and performed single-cell RNA sequencing to characterize their immune and stromal cell populations at high resolution. Our analysis identified centroblast-like and memory-like B cells, helper-like CD4+ T cells, cytotoxic and memory CD8+ T cells, and melanomacrophages with features associated with marginal zone and tingible body macrophages. We also identified a progenitor fibroblastic reticular cell-like population enriched for follicular dendritic cell-associated markers, suggesting a potential role in antigen retention and local immune organization. This was supported by integrating our dataset with publicly available single-cell gene expression data from sharks and frogs. Chemokine expression patterns, including putative CXCR4-CXCL12 interactions, further supported coordinated stromal-lymphocyte organization within MMCs. Together, these findings indicate that zebrafish MMCs contain conserved cellular features associated with affinity maturation, support their role as germinal center-analogues, and provide insight into how germinal centers may have evolved in vertebrates.