Regulation of germinal center B cell fate choice by Hedgehog signaling

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Project Summary Effective humoral immunity critically depends on the formation of germinal centers (GC) that generate high- affinity, long-lived memory and plasma cells. Within mature GCs, histologically distinct dark (DZ) and light zones (LZ) can be observed, the latter defined by the presence of a stromal cell type, follicular dendritic cells (FDCs). FDCs express high levels of complement and Fc receptors that retain immune complexed antigen. In addition to a depot of antigen, the LZ also harbors the majority of T follicular helper cells (Tfh). GC B cells undergo many rounds of cell division and profound shifts in gene expression during cycles of selective clonal expansion. The selection of LZ GC B cells with B cell receptors (BCR) of higher affinity involves antigen acquisition, BCR signaling and competition for Tfh cell contacts. After engagement and instruction by Tfh cells, LZ B cells undergo transcriptional rewiring that propels their movement to the DZ where they complete one or more cell divisions. Through a poorly understood process, DZ B cells eventually lose their propensity to remain within that zone and migrate back to the LZ as centrocytes. This iterative cyclic process ultimately results in the selective expansion of higher affinity BCR variants. In addition to their clonal self-renewal, GC B cells also give rise to long-term memory B cell and plasma cell lineages that exit the GC during maturation. The factors that regulate the cell fate choice of reactivated GC B cells are incompletely understood. Recent studies are consistent with the idea that stronger BCR signaling within the LZ promotes the plasma cell lineage while the formation of memory B cells predominates among GC B cells with BCRs of lower affinity for antigen, presumed to be the result of insufficient engagement of Tfh cells due to poor antigen acquisition and presentation. However, the role that FDC-derived factors might play in GC B cell fate choices have not been examined. The factors that regulate the cell fate choice of reactivated LZ B cells is poorly understood. We questioned whether stromal cell-derived factors influencing cell behavior in those niches could also play a role in GCs. We hypothesize that Hedgehog signaling plays a role in critical transitions during GC B cell re-activation that influence cell fate. We propose to define the Hedgehog dependent and independent events and their impact on GC B cell lineage divergence. These questions will be addressed through the inhibition or ablation of Hedgehog signaling and an examination of the effect on GC B cell self-renewal or the initiation of differentiation to alternative cell lineages within the GCs. Proposed experiments to assess the molecular impact of Hedgehog signaling include scRNAseq of GC B cells.