Poster CC10 - Dynamic behaviors of Ezh2 mutant B cell and interaction with follicular dendritic cell and T follicular helper cell within germinal centers

Introduction: Germinal centers (GC) are transient structures within secondary lymphoid organs where mature B cells undergo proliferation, somatic hypermutation of their immunoglobulin loci, selection and eventually differentiation into high-affinity memory B cells and long-lived plasma cells. To achieve this, GC B cells undergo iterative cycles of dynamic trafficking between dark zone (DZ) and light zone (LZ) of the GC, that encompass complex lymphocyte behavior patterns as well as interaction with follicular dendritic cells (FDC), which expose the antigen, and T follicular helper (Tfh) cells, which positively or negatively select the GC B cells based on the affinity of their B cell receptor for antigen. Ezh2, which is an epigenetic regulator that represses transcription, is required for GC formation and maintenance, allowing proliferation and inhibiting differentiation of GC B cell. The point mutation of Ezh2 at Y641 residue is a gain of function mutation associated with lymphomagenesis by attenuating GC B cell requirement for T cell help through epigenetic silencing of genes involved in immune synapse signaling and rendering GC B cells more dependent for their survival on their interactions with FDCs. However, it is unclear how Ezh2 mutation affects emergent cellular behaviors and interaction patterns with FDCs and Tfhs to determine B cell fate. We hypothesized that GC B cellular dynamic behaviors determine the quantity and quality of their interactions with Tfhs and FDC. By comprehensively mapping lymphocyte behavior patterns within GCs, we provide new insights on GC dynamics and determine how dynamic interactions can lead to dysfunctions.

Materials and

Methods: Three-dimensional movies of cell movements were recorded within the LZ of GCs of popliteal lymph nodes of previously immunized animals using intravital two-photon microscopy. Using Imaris, we generated 3D segmentation of cells and tracked the positions and morphologies of fluorescently tagged CD4 Tfh cells, wt (wilde-type) GC B cells, and EZH2-mutant (mt) GC B cells for a duration of 10 minutes at 30 second-intervals. We then extracted ~100 behavior features that encode information about the spatiotemporal dynamics and ~50 colocalization features that describe interaction patterns with Tfhs and FDCs, identified by fluorochrome-conjugated anti CD35 antibody injected into live mice. Unsupervised K-means clustering were performed to identify distinct migration modes that exist within the GC. (Fig A).

Results, Conclusions, and Discussions: The behaviors of the wt and Ezh2 mutant (mt) GC B cell were mapped onto the UMAP space and we identified a unique distribution of each cell types (Fig B). We defined 12 distinct patterns of behavioral clusters that share similar movement patterns (Fig C) and found that mt GC B cells display high heterogeneity across behavior patterns (Fig D). In contrast, wt GC B cells mostly populate within cluster 8, typically representing low displacement and low tortuosity (Fig D). The Ezh2 mutation also increased speed but did not change the directionality of movement (Fig E, F). We then defined 3 distinct zones within GC: the dark zone (DZ), the sparse light zone (sLZ), and the dense light zone (dLZ). DZ is where FDCs are largely absent, sLZ is where FDC exist but with small density of the FDC and dLZ is where the FDC is dense. GC B cells exhibited susceptibility to changes in migration mode that depended on the location within FDC meshwork (Fig I). Specifically, the lowest correlation between wt and mt in behavior patterns was within the DZ (Fig I). As mt GC B is located in the dense FDC region, they show enrichment in the cluster 8 pattern and thus behave similar to wt cells (Fig H). This interzonal rapid transition of behavior patterns by mt GC B cells was a critical characteristic, potentially leading to increased dependency on FDCs. We further observed that these distinct behavior patterns led to high frequent contact but decreased long-term engagement with Tfh (Fig J).

We identified significant differences between wt and mt GC B cell behavior dynamics as a function of the location defined by the FDC density. EZH2 mutant GC B cells displayed a significant behavioral shift from mt, especially in the DZ and sLZ regions, and confirmed that pre-malignant lymphoma B cells undergo rapid behavioral reprogramming that influences interaction dynamics with FDCs and Tfhs. This analysis gives a full interpretation of the behavior dynamics of GC lymphocytes and novel insights into other physiological settings where the spatiotemporal dynamics in single-cell resolution are a key component.

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