1. Microenvironmental cues that promote lymphomagenesis in mLN Germinal centers within mucosal lymphoid tissues such as mLN and Peyer's Patches (PPs) are thought to form in response to chronic stimulation by microbial products and other stimuli derived from the gut. We find that Ga13-deficiency in B cells promotes GC B cell survival most robustly in the mLN and to a lesser degree in PPs. Surprisingly, Ga13-deficiency does not promote increased GC B cell survival within peripheral LNs or the spleen following immunization with model antigens or viral infection. In aged Ga13-deficient mice, lymphomas initially develop in the mLN and then spread to distant sites. These data suggest that there are unique cues within the mLN that support the development of GC-derived lymphoma. In the mouse, each lobe of the mLN drains a distinct segment of the gut. Aged Ga13-deficient animals initially develop lymphomas in mLN lobes draining the distal portions of the small intestine and cecum but not the proximal small intestine. Additionally, lobes of the mLN draining distal portions of the small intestine and cecum most strongly promote survival of Ga13-deficient GC B cells. These data suggest that there are unique cues derived from lymph draining these areas that promote survival or expansion of Ga13-deficient GC B cells and subsequent lymphomagenesis. One potential factor accounting for these regional differences is the gut microbiota. The diversity and load of microbiota is increased in distal portions of the small intestine compared to more proximal portions of the gut. In preliminary data, we have found that the outgrowth of Ga13-deficient GC B cells in mLN can be abrogated in animals treated with certain combinations of broad spectrum antibiotics but not others. In future experiments, we will treat animals with narrow spectrum antibiotic regimens and assess whether the presence or absence of certain species of microbiota correlates with outgrowth of Ga13-deficient GC B cells. In preliminary data, we have also found that dendritic cells migrating from the gut to the mesenteric lymph node are required for the outgrowth of Ga13-deficient GC B cells. In future experiments, we will attempt to determine whether a specific dendritic cell subset can be identified that promotes Ga13-deficient GC outgrowths. 2. Control of germinal center polarity by Tgf-b signaling Iterative cycling of GC B cells between the light zone (LZ) and dark zone (DZ) is required for antibody affinity maturation. Recent work has demonstrated that the transcription factor forkhead box protein O1 (Foxo1) is required for GC B cells to maintain the dark zone state. Foxo1 was shown to be more active in DZ GC B cells. In the LZ, Foxo1is phosphorylated preventing it from entering the nucleus and targeting it for degradation. A fraction of LZ GC B cells show active nuclear Foxo1 and these cells are thought to be in the process of transitioning to the DZ. The cues in the GC microenvironment that might induce nuclear translocation of Foxo1 in LZ cells and allow for transition to the DZ state have not been defined. Peyer's patches (PP) are a key site for the induction of IgA, the most abundant immunoglobulin in the body. The role of Tgfbin supporting the induction of IgA in B cells both in vitro and in vivo has been well described. In the absence of Tgf-breceptor on B cells, IgA induction is lost and there is hyperplasia of PP germinal center (GC) B cells. Recent work has demonstrated that induction of IgA occurs in activated B cells in a specialized area of the PP called the subepithelial dome (SED) where B cells interact with dendritic cells that are thought to present active Tgfb. However, it has not been directly demonstrated that Tgfbsignaling occurs in activated B cells in situ. It has also been proposed that other cells in the PP, such as follicular dendritic cells (FDCs), a specialized population of stromal cells present in the LZ of the GC, may provide active Tgfbto GC B cells. Whether Tgfbsignaling occurs in PP GC B cells or GC B cells in non-mucosal sites has not been demonstrated in situ nor is it clear what role Tgfb signaling in GC B cells might play in IgA induction or GC homeostasis. We developed a staining protocol to determine with high resolution the sites of Tgfb signaling in situ. We found that Tgfbsignaling occurs in rare activated B cells in the SED in PP, however we also found that GC B cells in mucosal and, surprisingly, non-mucosal sites showed evidence of strong Tgfbsignaling. To determine what the consequences of Tgfbsignaling were in activated B cells versus GC B cells, we crossed Tgfbr1-floxed animals to animals expressing cre in all mature B cells and animals expressing cre only in GC B cells. We found that in the absence of Tgfbr1 in all mature B cells there was a loss of IgA, while when Tgfbr1 was lost in GC B cells, class switch recombination to IgA could still occur. In both models, there was a cell-intrinsic expansion of mucosal GC B cells, most prominently in PP GCs, and an increase in LZ phenotype cells in mucosal and, importantly, in non-mucosal GCs. The accumulation of LZ GC B cells in the absence of Tgfb signaling occurred likely as a result of reduced activation of Foxo1. Additionally, we found that Tgfb signaling in GCs promoted antibody affinity maturation. Finally, we demonstrated that FDCs are required to promote Tgfb signaling in GC B cells. This work identified Tgfb signaling in GC B cells as an important microenvironmental cue that supports GC polarity in both mucosal and nonmucosal sites that is distinct from its role in supporting IgA induction. 3. Molecular mechanism of Ga13 signaling in GC B cells Ga13-signaling in GC B cells suppresses cell survival and the development of lymphoma and represents an important tumor suppressive pathway in human GC-derived lymphomas. Ga13 triggers guanine nucleotide exchange on the small GTPase Rho by activating the guanine nucleotide exchange factor (GEF) ARHGEF1 (also known as P115 RhoGEF and Lsc). In previous work we and others have found that Ga13 stimulation can suppress cellular migration induced by Gai-coupled stimuli and pAkt in GC B cells ex vivo. We speculated that inhibition of pAkt was the primary mechanism by which Ga13 inhibits GC B cell survival in vivo. To more rigorously test this assumption and to discover novel effectors of Ga13 signaling, in collaboration with the laboratory of Louis Staudt, we developed two GCB-DLBCL cell line models expressing Cas9 where we could stimulate Ga13 and inhibit cell survival. In these two cell lines, we performed a whole genome CRISPR screen to identify unknown components of this signaling pathway. Importantly in both cell lines GNA13 and ARHGEF1were among the top hits in our screen. ARHGEF1 mutations have been reported in GCB-DLBCL, however whether these mutations disrupt its function is unknown. We developed a reconstitution system to functionally characterize most mutations of ARHGEF1 that have been published in publicly available data sets. We found that approximately one third of these mutations disrupt ARHGEF1 function. We are currently trying to assess whether loss of Arhgef1 is sufficient to promote lymphomagenesis in vivo. Finally, there were a number of hits from our screen in both cell lines that were required to suppress cell survival downstream Ga13 signaling but were not required for inhibition of Akt signaling. Several of these hits were required to inhibit cell cycle progression downstream of Ga13 in vitro. We are currently trying to determine how Ga13 signaling might suppress cell cycle progression and whether Ga13 signaling can suppress cell cycle progression in GC B cells in vivo.
