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EBI2 Mediates B Cell Segregation Between the Outer and Centre Follicle

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EBI2 Mediates B Cell Segregation Between the Outer and Centre Follicle

João P Pereira et al. Nature.

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
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
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
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
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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