br EBI structure and expression The gene encoding EBI
EBI2 structure and expression The gene encoding EBI2 was originally identified together with EBI1 (CCR7) as the most highly upregulated gene in Epstein–Barr virus-infected Burkitt\'s lymphoma cells . EBI2 belongs to the rhodopsin-like subfamily of seven transmembrane GPCRs . Although it is not highly homologous to any other known GPCRs, sequence alignments have clustered EBI2 with lipid receptors and indicated highest sequence similarity with GPR18 19, 20. Studies of the activity of EBI2 have shown that it signals through the pertussis-sensitive Gαi protein . Expression of EBI2 is most abundant in lymphoid tissues, with B cells, T cells, and various myeloid cells displaying high levels of EBI2 mRNA 15, 18, 19. EBI2 is expressed constitutively by mature biotin-LC-LC-tyramide  and displays a characteristic expression pattern linked to B cell differentiation in both humans and mice. The already high constitutive EBI2 expression of naïve B cells is transiently increased after activation 9, 14, 17, 21, 22. By contrast, GC B cell differentiation is associated with the shutdown of EBI2 expression 14, 17, 21, 22. Plasma cells maintain expression of EBI2, albeit at lower levels than do naïve B cells 14, 17. Little is known about the transcriptional and molecular regulation of EBI2 expression. In vitro, cytokines such as interleukin (IL)-4, IL-6, and IL-10 have been shown to modulate EBI2 expression, whereas lipopolysaccharide (LPS) stimulation upregulates EBI2 transcription in most leukocyte populations 16, 22, 23. Similar to chemokine receptors, cell surface EBI2 expression is downregulated by ligand binding through β-arrestin-mediated receptor internalization 15, 16, 24.
Oxysterols: the endogenous ligands for EBI2 Although EBI2 was initially suggested to have constitutive activity , it was subsequently deorphanized through the screening of tissue extracts with classic analytical methods and was shown to be the first GPCR activated by oxysterols 15, 16. The oxysterol 7α,25-dihydroxycholesterol (7α,25-OHC) was found to be a potent and selective agonist of EBI2 and its most likely endogenous ligand 15, 16. 7α,25-OHC is an oxygenated derivative of cholesterol and was previously identified as an intermediate product in the synthesis of bile acids . Conversion of cholesterol into 7α,25-OHC is carried out by the biosynthetic enzymes cholesterol 25-hydroxylase (CH25H) and oxysterol 7α-hydroxylase (CYP7B1) , which are highly expressed by lymphoid stromal cells 15, 24. Although 7α,25-OHC appears as the major endogenous ligand for EBI2, inactivation of 7α,25-OHC synthesis via deletion of the Ch25h gene does not completely remove EBI2-ligand activity . Thus natural oxysterols other than 7α,25-OHC may trigger EBI2 in vivo, consistent with the capacity of the receptor to bind and respond to several cholesterol derivates with high structural homology to 7α,25-OHC 15, 16. Expression of both CH25H and CYP7B1 by lymphoid stromal cells is required for the synthesis of 7α,25-OHC in lymphoid tissues and the correct positioning of B cells. Production of EBI2 ligand by hematopoietically derived cells appears limited  but may occur under some circumstances, given that both Ch25h and Cyp7b1 can be expressed by hematopoietic cells . Degradation of 7α,25-OHC is mediated by the enzyme 3β-hydroxy-Δ5-C27 steroid oxidoreductase (HSD3B7) , which is also expressed in lymphoid stromal cells and in T cell zone DCs . Differential expression of CH25H, CYP7B1, and HSD3B7 in stromal cells present in distinct compartments of secondary lymphoid organs appears to establish a gradient of 7α,25-OHC. The biosynthetic enzymes are more highly expressed in stromal cells at the perimeter of the follicle compared to stromal cells and FDCs in the center of the follicles, therefore, EBI2 ligand is higher at the follicle perimeter than in the inner follicle (Figure 1) . By contrast, the degradation of 7α,25-OHC is increased in T cell zones, due to high levels of HSD3B7 expression in stromal cells within these areas, keeping levels of EBI2 ligand low . HSD3B7 expression by splenic DCs, in particular of the CD8+ subset, helps maintain low EBI2 ligand concentrations in the T cell zone. Levels of 7α,25-OHC have been shown to be increased in mouse lymphoid tissues upon LPS injection in a CH25H-dependent manner 15, 16, suggesting that EBI2 ligand production may be regulated during infection, as has been observed for lymphoid chemokines .