In an effort to discover additional GPCRs directing the migr
In an effort to discover additional GPCRs directing the migratory events of responding B cells, we identified Epstein-Barr virus (EBV)-induced gene 2 (EBI2) as a promising candidate. The gene encoding EBI2 (Ebi2, also known as Gpr183) was originally identified together with Ebi1 (Ccr7) as the most highly upregulated gene in EBV-infected Burkitt\'s lymphoma cells (Birkenbach et al., 1993). EBI2 was subsequently reported to be most homologous to members of the lipid and purine GPCR family (Rosenkilde et al., 2006, Surgand et al., 2006). Although lacking any close homology partner in the chemokine receptor family, EBI2 signals through the pertussis-sensitive Gαi protein, similarly to many chemokine receptors (Rosenkilde et al., 2006). This orphan GPCR is predominantly expressed in lymphoid tissues and high mRNA expression has been observed in naive ABT-199 (Birkenbach et al., 1993, Rosenkilde et al., 2006). The already high constitutive expression of Gpr183 present in naive B cells is further increased by B cell receptor (BCR)-triggered NF-κB activation, although this upregulation is only transient (Glynne et al., 2000). In contrast, GC B cell differentiation is associated with the shut down of Gpr183 expression, which is controlled by the transcriptional repressor Bcl-6 (Shaffer et al., 2000). Despite this notable pattern of expression linked to B cell differentiation, the biological function of EBI2 remains undefined.
Discussion The early control of extrafollicular versus GC localization of responding B cells is controlled to a large extent by the activity of the chemokine receptors CXCR4, CXCR5, and CCR7. However, CXCR4-deficient plasma cells are not impacted in their access to extrafollicular sites, and GC clusters form, albeit with disorganized structure or orientation, in absence of CXCR4- or CXR5-mediated chemotaxis (Allen et al., 2004, Hargreaves et al., 2001). Thus, the existing experimental evidence points toward the involvement of an additional signal in the organization of early migratory events in T cell-dependent B cell responses. Our findings demonstrate that EBI2-driven positioning of B cells is a critical component of the mechanism that segregates activated B cells into the extrafollicular versus GC compartments. First, EBI2-dependent migration of responding B cells toward the bridging channels was required for the generation of the early extrafollicular plasmablast response, a process that was greatly enhanced by enforced expression of EBI2. Furthermore, the downregulation of EBI2 expression associated with GC B cell differentiation was sufficient to drive the accumulation of B cells in the deep FDC-rich regions of the follicle where GCs originate and enhanced the formation of GC responses. Although it is clear from the migration pattern of naive EBI2-deficient B cells that other activation-induced changes are typically required for B cells to enter GCs, our data indicate that the downregulation of Gpr183 by Bcl-6 is one of the critical activities of this transcription factor in mediating GC B cell differentiation (Shaffer et al., 2000). EBI2 remains an orphan GPCR and the identity and source of its ligand are yet to be described. Molecular studies of EBI2 have suggested that this receptor has constitutive activity, similar to that observed for many herpesvirus-encoded 7TM receptors (Benned-Jensen and Rosenkilde, 2008, Rosenkilde et al., 2006). It is conceivable that heterodimerization of EBI2 with other chemokine receptors could positively or negatively regulate their activity (Levoye et al., 2006). Accordingly, EBI2 deficiency would be expected to affect the in vitro chemotactic responses of B cells to chemokines. Such an outcome was not the case as far as tested. Thus, although our results do not exclude a constitutive receptor activity, they indicate that the potential agonist-independent signaling of EBI2 has no detectable impact on the migration of B cells to chemokines. Nevertheless, it remains possible that EBI2 modulates B cell responsiveness to chemokines when bound to its ligand. This orphan GPCR therefore is most likely to regulate B cell localization through a ligand that exhibits a spatially defined pattern of production. The migratory behavior of both naive and activated EBI2-deficient B cells indicated that these B cells failed to be attracted to the periphery of B cell follicles and extrafollicular regions, suggesting the presence of an agonist in these areas. Accumulation of B cells and plasma cells in the marginal-zone-bridging channels has been observed as a result of unbalanced chemokine responsiveness (Hargreaves et al., 2001, Reif et al., 2002). Similarly, autoreactive B cell blasts tend to localize to the red pulp-T zone border (Phan et al., 2003, Seo et al., 2002, William et al., 2002). The reasons for this homing pattern are unclear and suggest the existence of an unknown factor driving the lodgment of cells to this splenic subcompartment. Although our findings reveal EBI2 as a good candidate for mediating such localization, the extent of its contribution remains to be defined. Nevertheless, EBV-infected B cells, which are induced to express high amounts of EBI2 (Birkenbach et al., 1993), have been reported to migrate to extrafollicular regions and to avoid GCs during infectious mononucleosis (Niedobitek et al., 1992), which is in accordance with our results on EBI2 function.