Intravital single-cell behavior profiling reveals disrupted germinal center B cell motility and interactions by EZH2 gain-of-function mutation

Germinal center (GC) B-cells give rise to the majority of non-Hodgkin lymphomas, underscoring the need to pinpoint critical processes that initiate and drive lymphomagenesis. Lymphoma driver mutations can alter GC B cell functions and B cell fate decisions. Here, we studied how EZH2 oncogenic mutation in GC B cells alters cellular motility and interactions with T follicular helper (Tfh) cells and follicular dendritic cells (FDCs) to determine B cell fate. By combining intravital imaging, single-cell behavior analyses, and RNA sequencing, we uncover how lymphoma-associated EZH2 mutations reprogram the behaviors of GC B cells in vivo. We found that EZH2 mutations increased single-cell motility speeds and morphological plasticity of GC B cells, redirecting migration toward the FDC–rich light zone subregions rather than to the dark zone. Although mutant EZH2 GC B cells exhibited normal engagement quality with FDCs, they showed shorter interaction times and reduced surface engagement with Tfh cells. Notably, EZH2 mutant B cells required prior contact with FDC before engaging with Tfh cells, thus impairing DZ recycling. This motility phenotype scaled with local mutant clone abundance, suggesting a behavioral strategy underlying how mutant cells outcompete WT cells. Lastly, we developed scMOTIPh, a computational framework that integrates single-cell behavioral features with transcriptomic profiles. Applying scMOTIPh to mutant GC B cells within the FDC-rich zone revealed enhanced ATP production, metabolic and antigen-presentation programs, and suppression of cell-death pathways, which is consistent with a tendency for malignant transformation and survival fitness. These findings provide an in vivo, single-cell view of how an epigenetic lesion rewires the local microenvironment by modulating single-cell behaviors within native GCs, revealing a dynamic mechanism for early lymphomagenesis.