EBI2 mediates B cell segregation between the outer and centre follicle - PubMed (original) (raw)
. 2009 Aug 27;460(7259):1122-6.
doi: 10.1038/nature08226. Epub 2009 Jul 13.
Affiliations
- PMID: 19597478
- PMCID: PMC2809436
- DOI: 10.1038/nature08226
EBI2 mediates B cell segregation between the outer and centre follicle
João P Pereira et al. Nature. 2009.
Abstract
B cell follicles are specialized microenvironments that support events necessary for humoral immunity. After 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. Subsequently, some cells move to the follicle centre, become germinal centre B cells and undergo antibody affinity maturation. Although germinal centres within follicles were described in 1885 (ref. 12), the molecular cues mediating segregation of B cells between the outer and centre follicle have remained undefined. Here we present a role for the orphan G-protein-coupled receptor, Epstein-Barr virus induced molecule-2 (EBI2, also known as GPR183), in this process. EBI2 is expressed in mature B cells and increases in expression early after activation, before being downregulated in germinal centre B cells. EBI2 deficiency in mice 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 centre, whereas EBI2 overexpression was sufficient to promote B cell localization to the outer follicle. In mixed bone marrow chimaeras, EBI2-deficient B cells phenocopied germinal centre B cells in preferentially localizing to the follicle centre. When downregulation of EBI2 in wild-type B cells was antagonized, participation in the germinal centre 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.
Figures
Figure 1. Ebi2 upregulation in activated B cells and down-regulation in GCs
a, Flow cytometric detection of GFP fluorescence in the indicated BM, spleen and lymph node (LN) cell subsets from Ebi2GFP/+ mice. PC, plasma cells. b, Q-PCR analysis of Ebi2 transcript abundance in the indicated cell populations. Expression is shown relative to Hprt1. c and d, Flow cytometric detection of GFP fluorescence in B cells stimulated for one day with anti-IgM or anti-IgM and anti-CD40 (c) or that were stimulated for 1 h with anti-IgM and exposed in vivo for 2 days to T cell help (red) or not provided with T cell help (gray) (d). Numbers in a indicate percent of cells in gate. PC, plasma cells; FO, follicular B cells. Gray histograms in c indicate unstimulated cells. Bar graph in c shows geometric mean Ebi2GFP/+ fluorescence for one and two day cultures and summarizes 3 experiments. e, Immunofluorescence microscopy of fixed spleen tissue from an Ebi2GFP/+ mouse, stained to detect GFP+ cells (green) and CD169+ marginal zone macrophages (blue, left panel) or GL7+ GC B cells and CD4 T cells (red and blue, respectively, right panel).
Figure 2. EBI2 promotes localization of activated B cells in the outer follicle
a-c, Immunohistochemical staining of spleen cryosections. a, Distribution of wild-type and EBI2-deficient B cells that had been stimulated with anti-IgM in vitro for 1h, analyzed 6 h after being transferred to wild-type hosts. Ebi2−/− B cells were CFSE labeled prior to transfer. Sections were stained with an antibody to detect CFSE (Ebi2-/-) or co-transferred wild-type Igha (IgMa, Ebi2+/+) B cells (blue) and endogenous B cells (IgD, brown). b, Distribution of anti-IgM treated wild-type and EBI2-deficient B cells (CFSE, blue), and internal control Igha B cells (IgMa, blue), after 2 days exposure to T cell help in bm12 hosts. Upper and lower panels are serial sections. Endogenous B cells were detected with anti-IgD (brown). c, Distribution of B cells transduced with control or _Ebi2_-expressing retrovirus (hCD4, blue), one day after transfer. Endogenous B cells were detected with anti-IgD (brown). d, Anti-NP IgG1 and IgM serum titers in wild-type and EBI2-deficient mice on day 7 following immunization with NP-CGG in alum. R.U., relative units.
Figure 3. EBI2-deficient B cells localize to the follicle center in a LTα1β2- and CXCL13-dependent manner
a, Distribution of wild-type and EBI2-deficient B cells in spleen, lymph nodes (LN) and Peyer's patches (PP) of 20:80 mixed BM chimeras (20% Ighb Ebi2+/+ or Ebi2−/− and 80% Igha wild-type). Sections were stained to detect Ebi2+/+ or Ebi2−/− B cells (IgDb, green), Igha control B cells (IgDa, red), and T cells (CD4+CD8, blue). b, Spleen sections from 90:10 mixed BM chimeras (90% Ighb Ebi2+/+ or Ebi2−/− and 10% Igha wild-type) stained to detect B cells as in a and for CD35 to highlight FDC networks (blue). c, Similar analysis to b in control or 3 week LTβR-Fc treated 90:10 Ebi2-/- mixed BM chimeras. d, Distribution of wild-type and EBI2-deficient B cells in CXCL13−/− hosts reconstituted with 70:30 BM mixtures (70% Ighb Ebi2+/+ or Ebi2−/− and 30% Igha wild-type). Spleen sections stained to detect EBI2-deficient B cells (IgDb, green), wild-type B cells (IgDa, red) and marginal zone macrophages (CD169, blue).
Figure 4. Maintained Ebi2 expression impairs participation in GC response
a, Flow cytometric analysis of spleen cells from an immunized mouse receiving _Ebi2_-transduced Ig-transgenic (IgMa) B cells, 4 days after transfer, showing gating scheme to identify representation of transduced (hCD4+) cells amongst GC B cells (B220+IgMa+IgDloFashi) and plasma cells (PC, B220loIgMa+). Numbers indicate frequency of cells in the indicated gate. b, Number of transduced (hCD4+) B cells having a GC or plasma cell phenotype. c, Distribution of transduced B cells (hCD4+, blue) in sections of spleen from mice receiving control vector or _Ebi2_-transduced B cells. Endogenous naïve B cells are stained brown (IgD) and GCs are detected in serial sections using GL7 (blue, lower panels).
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