EBI remains an orphan GPCR and the identity and
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 Sulfamethazine sale 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. For this study we have used the intermediate affinity mutant antigen HEL2× for immunization. However, we have observed a similar defect in the plasmablast differentiation of EBI2-deficient SWHEL B cells over a 10,000-fold affinity range by using WT HEL or the low-affinity mutant HEL3× (data not shown). This indicates that increasing or decreasing BCR signal strength, which is known to regulate the plasmablast response (Benson et al., 2007, Paus et al., 2006), does not correct or exacerbate the defective response of EBI2-deficient B cells. Consistent with this result, the in vitro activation and proliferation of B cells was not affected by absence of EBI2, and therefore a contribution of EBI2 to the signals required for efficient B cell stimulation is unlikely. Notably, expression of EBI2 was also not required for normal in vitro plasmablast differentiation. It is still possible, however, that signals delivered by EBI2 can directly trigger or regulate gene expression programs that drive plasmablast differentiation. On the other hand, the guidance of responding B cells to distinct microenvironments mediated by modulation of EBI2 expression may subject them to alternative extracellular milieus that could direct their subsequent lineage commitment. Thus, EBI2-deficient B cells may predominantly form GC B cells in vivo because of the fact that they remain in a microenvironment proximal to the FDC network, in which antigen deposits and follicular T helper cells drive their proliferation and selection into the long-term effectors of humoral immunity. At the same time, because the factors and myeloid cell populations supporting maturation and survival of plasmablasts are concentrated outside of B cell follicles, they are not encountered by most responding EBI2-deficient B cells (Garcia De Vinuesa et al., 1999, Mohr et al., 2009). The control of EBI2 expression, via NF-κB, Bcl-6, or other pathways, therefore plays an important role in determining the balance between plasmablast and GC differentiation. In vitro, cytokines such as interleukin (IL)-4, IL-6, and IL-10 have been shown to modulate Gpr183 expression (Lam et al., 2008, Shaffer et al., 2000). In contrast, we have only seen minimal differences in the expression of Gpr183 mRNA in SWHEL B cells stimulated with HEL antigen of different affinities (data not shown). Although regulation of EBI2 is an important component of the early commitment of responding B cells to the extrafollicular versus GC pathway of differentiation, it is clear that this fundamental decision in B cell fate is a complex interplay of many signals in vivo. However, it remains possible that abnormal regulation of EBI2 expression leads to autoimmune and inflammatory diseases in which the balance between plasmablast and GC B cell differentiation is lost.