Epstein-Barr virus (EBV)-induced gene 2 (EBI2, aka GPR183) is a G protein-coupled receptor that is required for humoral immune responses and polymorphisms in the receptor have been associated with inflammatory autoimmune diseases1-3. The natural ligand for EBI2 has been unknown. Here we describe identification of 7α, 25-dihydroxycholesterol (5-cholesten-3β, 7α, 25-triol; 7α, 25-OHC) as a potent and selective agonist of EBI2. Functional activation of EBI2 by 7α, 25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high affinity radioligand binding. Furthermore we find that 7α, 25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A key enzyme required for the generation of 7α, 25-OHC is cholesterol 25-hydroxylase (Ch25h)4. Similar to EBI2 receptor knockout mice, mice deficient in Ch25h fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that Ch25h generates EBI2 bioactivity in vivo and suggests that the EBI2 − oxysterol signaling pathway plays an important role in the adaptive immune response.

B cell follicles are specialized microenvironments that support events necessary for humoral immunity 1, 2, 3. Following antigen encounter, activated B cells initially seek T cell help at the follicle-T zone boundary and then move to interfollicular and T-zone distal (outer) regions of the follicle 4, 5, 6, 7, 8, 9, 10. Subsequently, some cells move to the follicle center, become germinal center (GC) B cells and undergo antibody affinity maturation 1, 2, 11. Although germinal ‘centers’ within follicles were described in 1885 12, the molecular cues mediating segregation of B cells between outer and center follicle have remained undefined. Here we establish a role for the orphan G-protein coupled receptor, Epstein Barr Virus-induced molecule-2 (EBI2) 13, in this process. EBI2 is expressed in mature B cells and increases in expression early after activation before being down-regulated in GC B cells. EBI2 deficiency led to a reduction in the early antibody response to a T-dependent antigen. EBI2-deficient B cells failed to move to the outer follicle at day 2 of activation and instead were found in the follicle center, whereas EBI2 over-expression was sufficient to promote B cell localization to the outer follicle. In mixed bone marrow chimeras, EBI2-deficient B cells phenocopied GC B cells in preferentially localizing to the follicle center. When down-regulation of EBI2 in wild-type B cells was antagonized, participation in the GC reaction was impaired. These studies identify an important role for EBI2 in promoting B cell localization in the outer follicle, and show that differential expression of this receptor helps position B cells appropriately for mounting T-dependent antibody responses.

Summary 7α,25-dihydroxycholesterol (7α,25-OHC) is a ligand for the G-protein coupled receptor EBI2 (GPR183); however, the cellular sources of this oxysterol are undefined. 7α,25-OHC is synthesized from cholesterol by the stepwise actions of two enzymes, CH25H and CYP7B1, and is metabolized to a 3-oxo derivative by HSD3B7. We show that all three enzymes control EBI2-ligand concentration in lymphoid tissues. Lymphoid stromal cells are the main CH25H and CYP7B1-expressing cells required for positioning of B cells and they also mediate 7α,25-OHC inactivation. CH25H and CYP7B1 are abundant at the follicle perimeter whereas CH25H expression by follicular dendritic cells is repressed. CYP7B1-, CH25H- and HSD3B7-deficiencies each result in defective T-cell dependent plasma cell responses. These findings establish that CYP7B1 and HSD3B7, as well as CH25H, have essential roles in controlling oxysterol production in lymphoid tissues and they suggest that differential enzyme expression in stromal cell subsets establishes 7α,25-OHC gradients required for B cell responses.