Lymphoid and Nonlymphoid Tissues B Cells and Ligand Activity Is Detectable in EBI2 Guides Serial Movements of Activated

EBV-induced gene 2 (EBI2) was recently shown to direct the delayed movement of activated B cells to interfollicular and outer follicular regions of secondary lymphoid organs and to be required for mounting a normal T-dependent Ab response. In this study, we show that EBI2 promotes an early wave of Ag-activated B cell migration to the outer follicle in mice. Later, when B cells have moved to the T zone in a CCR7-dependent manner, EBI2 helps distribute the cells along the B zone–T zone boundary. Subsequent EBI2-dependent movement to the outer follicle coincides with CCR7 downregulation and is promoted by CD40 engagement. Using a bioassay, we identify a proteinase K-resistant, hydrophobic EBI2 ligand activity in lymphoid and nonlymphoid tissues. Production of EBI2 ligand activity by a cell line is sensitive to statins, suggesting production in a 3-hydroxy-3-methyl-glutaryl-CoA reductase–dependent manner. CD40-activated B cells show sustained EBI2-dependent responsiveness to the bioactivity. These findings establish a role for EBI2 in helping control B cell position at multiple stages during the Ab response and they suggest that EBI2 responds to a broadly distributed lipid ligand. B cells migrate into lymphoid follicles in a CXCR5-dependent manner, responding to the CXCR5 ligand CXCL13, which is abundantly displayed on follicular stromal cells (1–3). Within the follicle B cells migrate at an average velocity of 6 mm/min in a " random walk, " surveying for Ags displayed by sinus-associated macrophages, follicular dendritic cells, or conduits or that have diffused into the follicle (3). Within 6 h after Ag encounter, Ag-engaged B cells move to the B zone–T zone (B–T) boundary in a CCR7-dependent manner, responding to CCL21 and CCL19 made by T zone stromal cells, to interact with Th cells (4). CXCR5 remains expressed by activated B cells and helps distribute cells along the B–T boundary. By day 2 of T-dependent responses, some activated B cells relocalize to the outer follicular and interfollicular regions (5, 6). Plasmablasts then emerge, particularly in interfollicular regions, and germinal center (GC) B cells soon accumulate at the follicle center (6, 7). EBV-induced gene 2 (EBI2) is an orphan GPCR that was identified during a screen for EBV-induced genes (8). Transcript analysis and studies in an EBI2-GFP reporter mouse line showed that EBI2 is abundantly expressed in B cells and it is further upregulated following activation; expression was also found in some T cells and myeloid cells (9–14). Studies in two EBI2-knockout (KO) mouse …