EZH2 oncogenic mutation disrupts germinal center B cell dynamics and promotes early lymphomagenic behaviors

Abstract A major clinical challenge in germinal center (GC)-derived lymphomas, such as follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL), is early relapse and acquired resistance to targeted therapies, including EZH2 inhibitors. These outcomes likely reflect early, mutation-driven changes in B cell behavior and microenvironmental interactions that are not captured in patient biopsies or ex vivo assays. Mechanistic in vivo studies are necessary to define how oncogenic mutations reprogram cell dynamics and immune interactions in native GC niches, providing a foundation for improved therapeutic strategies. GCs are essential sites for B cell clonal expansion, affinity maturation, and the origin of most B cell lymphomas. These functions depend on precise B cell migration between the dark zone (DZ) and light zone (LZ), and coordinated interactions with T follicular helper (Tfh) cells and follicular dendritic cells (FDCs). Somatic gain-of-function mutations in the histone methyltransferase EZH2, most commonly at Y641 residue, are early genetic lesions found in ~25-30% of FL and DLBCL, yet how these mutations alter B cell behavior within the GC microenvironment to initiate lymphomagenesis remains poorly defined. To address this, we used intravital two-photon microscopy combined with high-dimensional single-cell behavioral and transcriptomic analyses to define, in real time, how mutant EZH2 reshapes B cell dynamics in vivo. We generated mice in which mutant EZH2 GC B cells expressed YFP, WT GC B cells expressed CFP (with reciprocal fluorophore switching to control for potential artifacts), Tfh cells expressed tdTomato, and FDCs were visualized via anti-CD35 fluorophore-labeled antibody (AlexaFluor594). Mutant EZH2 GC B cells displayed increased motility (p<0.05), morphological deformability (p<0.001), and spatiotemporal heterogeneity (p<0.05) within the LZ. These behavioral alterations were particularly evident across spatially distinct LZ subregions, defined by FDC density, and were coupled with defective transitions to the DZ, resulting in reduced interzonal migration. Although mutant GC B cells retained normal engagement with FDCs, they displayed reduced interaction stability (p<0.001) and contact volume (p<0.005) with Tfh cells, aberrant pre-contact motility (p=0.03), and failed to initiate canonical DZ re-entry following T cell engagement. Notably, successful Tfh engagement in mutant EZH2 B cells required prior FDC interaction, indicating increased reliance on FDC signaling. Transcriptional profiling of behaviorally defined single cells revealed enrichment of pathways linked to ATP production, migration, proliferation, and antigen presentation despite reduced Tfh engagement, reflecting a reprogrammed B cell state poised for malignant transformation. Together, these findings establish EZH2 mutation as a driver of aberrant GC B cell dynamics that disrupts interzonal migration and cellular interactions essential for normal selection. The resulting impaired selection, LZ retention and reduced dependence on T cell help, explain how early genetic lesions rewire B cell behaviors and establish a cellular framework for malignant transformation in GC-derived lymphomas. More broadly, this work represents the first in vivo demonstration of how an epigenetic driver mutation alters GC B cell behavior at single-cell resolution, directly linking altered cell dynamics to disrupted spatial organization and immune communication in early lymphomagenesis.