In this study we generated mice in which
In this study we generated mice in which the native Bcl6 locus was engineered to produce a form of Bcl6 containing a mutant RD2 domain that disrupts its repressor function. We found that RD2 domain is essential for pre-GC B cell differentiation and clustering into nascent GC within follicles, in part through repressing key trafficking receptors S1pr1 and Gpr183 by recruiting Hdac2. Thus, these findings implicate the Bcl6 RD2 domain as defining the fate of activated Levodopa synthesis toward the GC phenotype, which is mechanistically distinct from its role in enabling proliferation and survival upon induction of the proliferative burst within GCs. The activities of Bcl6 can thus be parsed out into unique biochemical elements, each contributing different actions to the functionality of immune system.
Discussion The development of GCs is a sequential and complex process, during which BCL6 serves as master regulator in multiple cell types (Goodnow et al., 2010, Klein and Dalla-Favera, 2008, McHeyzer-Williams et al., 2012), There are many unanswered questions regarding the mechanisms and stages at which Bcl6 controls this process. Dissection of these events is further complicated by the diverse functions of Bcl6 in B cells, T cells, etc., as well as the multifaceted biochemical mechanisms of action of Bcl6 (Bunting and Melnick, 2013). After T cell-dependent antigen stimulation, Bcl6 protein begins to accumulate in pre-GC B cells in the outer follicle regions and is maintained at high levels within GC B cells (Kerfoot et al., 2011, Kitano et al., 2011). Its role in mature, established GCs is fairly well defined and is linked to its silencing of cell cycle, DNA damage sensing, and apoptosis checkpoint genes as well as repression of genes involved in terminal differentiation (Bunting and Melnick, 2013, Klein and Dalla-Favera, 2008). These well-known GC B cell functions are mediated through Bcl6 BTB domain-dependent recruitment of the SMRT, NCOR, and BCOR corepressors (Hatzi et al., 2013, Huang et al., 2013, Polo et al., 2004). In contrast, the significance of Bcl6 in B cells at the pre-GC stage has not been confirmed, and the molecular basis for Bcl6-mediated pre-GC B cell differentiation remains unknown. Our current data showing that the Bcl6 RD2 domain plays an essential role in pre-GC B cell differentiation and nascent GC formation thus significantly extends knowledge of how B cells adopt the GC fate under the control of a specific BCL6 biochemical mechanism distinct from its known canonical function (Figure 4). The profound defect in early pre-GC differentiation in RD2-deficent B cells best explains the complete abrogation of GC formation in Bcl6 knockout mice and emphasizes the importance of Bcl6 in early GC B cell commitment. The canonical BCL6 repressive mechanism of action in GC B cells is mediated by recruitment of the SMRT, NCOR, and BCOR corepressors to the BCL6 BTB domain (Hatzi and Melnick, 2014). However, we find that Bcl6 BTB mutant mice are still mostly capable of forming early GC clusters. This phenotype is in contrast to the complete abrogation of GC formation observed in BCL6 RD2 mutant mice. Hence, whereby BCL6 mediates early steps in commitment to the GC fate and clustering into nascent GCs through the RD2 domain, once GCs are established, Bcl6 is then required to maintain the proliferation and survival by repressing genes through its BTB domain. These findings suggest a model of sequential and biochemically distinct biological functions of BCL6 at different GC B cell developmental stages (Figure S6). It is also possible that the RD2 domain might also contribute to later events in established GCs such as regulation of terminal differentiation (Fujita et al., 2004, Parekh et al., 2007), although such functions cannot be assessed in the RD2 animal model developed here. This pre-GC B cell differentiation deficiency caused by RD2 loss of function was explained at least in part by the finding that the BCL6 RD2 domain is required to repress expression of the key migration factors GRP183 and S1PR1. Downregulation of GRP183 is critically important for B cell migration into the follicle center (Gatto et al., 2009, Pereira et al., 2009). S1PR1 plays a key role in enabling B cell trafficking out of follicles (Cinamon et al., 2004). Our finding that BCL6 directly represses these genes in GC cells suggests a role for BCL6 at an early stage of the GC response, whereby BCL6 can enable “capture” of B cells within follicles to enable their clustering. At the same time, S1PR2 upregulation in GC B cells confines them to an S1P-low niche within follicles (Green et al., 2011). We observed that S1PR1 overexpression promoted GC-type B cell migration and antagonized S1PR2 (Figure S5G). Taken together, the data suggest that dynamic regulation and equilibrium of S1PR1 and S1PR2 is a critical event in the early pre-GC phase of the humoral immune response, controlled at least in part through BCL6 direct binding and repression of S1PR1.