We propose the following model for
We propose the following model for how B cell-guiding EBI2 ligand gradients are established in lymphoid tissues. CH25H and CYP7B1 are needed in radiation-resistant stromal cells, are abundantly expressed and active in CXCL13+ and CCL21+ FRCs, but not lymphocytes, and are poorly expressed in the inner follicle compared to surrounding regions. This enzyme distribution results in more ligand production at the follicle perimeter than at the follicle center, accounting for the propensity of EBI2hi cells to be attracted to the follicle perimeter (Gatto et al., 2011; Kelly et al., 2011; Pereira et al., 2009). HSD3B7, by contrast, is present in similar amounts in the inner follicle and at the perimeter, and thus we propose that by shortening the 7α,25-OHC half-life, this enzyme ensures that the concentration of ligand closely mirrors the distribution of the biosynthetic enzymes. CYP7B1 and CH25H are also abundant in the T zone, but in this compartment HSD3B7 is highly expressed, causing T zone 7α,25-OHC to be relatively low. As a result, ligand concentrations are most likely higher along the follicle-T zone interface than within the T zone proper. In the first hours after B cell activation, CCR7 and EBI2 are both upregulated, and Indirubin move to the T zone in a CCR7-dependent manner (Gatto et al., 2009; Kelly et al., 2011; Pereira et al., 2009; Reif et al., 2002); however, their propensity to remain at and distribute along the length of the B-T zone interface is promoted by the abundance of EBI2 ligand in this region. Later, after activation (∼day 2), when B cells have received T cell help, they maintain high EBI2 function (Figure 1) and CXCR5 expression but downregulate CCR7 function (Chan et al., 2009; Coffey et al., 2009; Kelly et al., 2011). As a result, these cells are less strongly attracted to the B-T zone interface and relocate to the outer follicle in an EBI2-dependent manner. A recent study suggested EBI2 transmits proproliferative signals to B cells (Benned-Jensen et al., 2011). We have not found 7α,25-OHC to have mitogenic effects on B cells (T.Y., L.M.K., and J.G.C., unpublished data). Our findings of similar B cell positioning and antibody response defects in mice that are unable to make 7α,25-OHC (CH25H- and CYP7B1-deficient) and in mice that have an elevated abundance of 7α,25-OHC (HSD3B7-deficient) are most consistent with B cell EBI2 functioning principally as a guidance receptor. More abundant CH25H and CYP7B1 expression in the outer versus inner follicle highlights the specialization of follicular stromal cells in these regions. One subset of stromal cells situated immediately beneath the marginal and subcapsular sinuses is the MRC (Katakai et al., 2008). Although there is currently no method available for isolating these cells, the abundance of the MRC marker TRANCE in our FRC preparations confirms the presence of these cells and is consistent with the possibility that they are a source of CH25H and CYP7B1. We attempted to further test whether CH25H was enriched in TRANCE+ stromal cells using single-cell PCR but found the detection of TRANCE via this procedure to be variable. We had similar difficulties with reproducible detection of CYP7B1 in the single-cell analysis. However, the single-cell approach did allow us to demonstrate lower expression of CH25H by CR1-CR2hi cells than by the majority of CXCL13+ FRCs, in agreement with the findings for flow-cytometry-purified FDCs. These data are in accord with the laser-capture microscopy analysis results, suggesting that the stromal cells at the follicle center manifest only low amounts of CH25H. We propose that the loss of EBI2 gradient information following FDC ablation occurs because of the role of FDCs in repressing FRCs within follicles. The rapid invasion or induction of follicular FRCs following FDC ablation leads to a more uniform expression of CH25H and CYP7B1 throughout the B cell area, and this probably causes the loss of a gradient in 7α,25-OHC biosynthetic activity. FDCs are lymphotoxin dependent, and the loss of EBI2-guided B cell segregation in follicles following lymphotoxin blockade (Pereira et al., 2009) is also consistent with this model of EBI2 ligand maintenance by FDCs. Although FDCs express CXCL13, a finding confirmed here in the single-cell PCR analysis, their ablation does not cause a reduction in CXCL13 abundance in the follicle (Wang et al., 2011). Moreover, EBI2-dependent segregation remains in Cxcl13–/– recipients (Pereira et al., 2009), and we have not observed marked differences in Cxcl13 mRNA or protein between the inner and outer follicle (Cyster et al., 2000; Wang et al., 2011), making it unlikely that the loss of EBI2-dependent B cell segregation following FDC-ablation is due to altered CXCL13 distribution.