IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches

Abstract

Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer's patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-β receptor (LTβR)-dependent maintenance of DCs. PP DCs augment IgA production by integrin αvβ8-mediated activation of transforming growth factor-β (TGFβ). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC-B cell interactions needed for TGFβ activation and induction of mucosal IgA responses.

Figure 1. B cell access to the PP subepithelial dome (SED) is CCR6-dependent

(A) Representative images of Peyer’s patch (PP) dome stained with anti-CD11c (blue) and anti-IgD (brown) (left panel) or with anti-GFP (green) and anti-IgD (blue) (right panel). Dashed white line demarcates the follicle-SED boundary. Scale bar is 20μm. (B) Representative flow cytometric analysis of CD19⁺ B cells in PPs for IgD and CCR6 expression. (C, D) Representative FACS staining of transferred CellTrace Violet-labeled polyclonal B cells (red) in MD4 hosts (endogenous B cells, black) for IgD and GL7 at day 7 (C) and IgD and CCR6 at days 3 and 4 after transfer. (E) Migration of PP follicular and pre-GC B cells from Ccr6^+/+ and Ccr6^−/− mice to the indicated chemokines. (F) Representative CCR6 expression on transferred wild-type (WT) and Cd40^−/−B cells in MD4 hosts (upper panels) or wild-type B cells in MD4 hosts treated with either isotype control or anti-CD40L antibody (lower panels) after 7 days. (G) Representative images of distribution of transferred B cells (Thy1.1, brown) in sections of PP from mice receiving control vector or CCR6-transduced B cells. Slides were counterstained with hematoxylin. (H) Representative images of distribution of B cells in PPs of chimaeras reconstituted with 50% Igh^b Ccr6^+/+ or Ccr6^−/− and 50% Igh^a wild-type BM. Sections were stained to detect Ccr6^+/+ or Ccr6^−/− B cells (IgD^b, blue) and control B cells (IgD^a, brown). (I) Distribution of polyclonal Ccr6^+/+ and Ccr6^−/− B cells in PPs of mixed transfer MD4 recipients (75% CD45.2 Ccr6^+/+ or Ccr6^−/− and 25% CFSE CD45.1 wild-type) 4 days after transfer. Sections were stained to detect Ccr6^+/+ or Ccr6^−/− B cells (CD45.2, blue) and control B cells (CFSE, brown). Each symbol in (E) represents an individual mouse, and data are pooled from at least three independent experiments. Data in (A), (B), (C), (D), (F), (G), (H) and (I) are representative of at least three independent experiments. *, p<0.05; **, p<0.01, ***, p<0.005 (unpaired Student’s T-test).

Figure 2. B cell isotype switching to IgA is CCR6-dependent

(A) Frequency of WT and Ccr6^+/+ or Ccr6^−/− GC B cells expressing IgA or IgG1 in PPs from mixed BM chimeras, as determined by FACS. (B) GC and memory contribution to IgA⁺ B cell pool in PPs. (C) Representative FACS of frequency of WT and Ccr6^+/+ or Ccr6^−/− B cells expressing IgA or IgG1 in PPs from mixed BM chimeras. (D) Representative FACS staining (left panel) and frequency (right panel) of IgA⁺ B cells amongst transferred B cells in MD4 recipient PPs after 7 days. (E) Fecal IgA from WT (Igh^a) and Ccr6^+/+ or Ccr6^−/− (Igh^b) mixed BM chimeras as measured by allotypic ELISA. (F) Percentage of fecal bacteria coated with IgA^a or IgA^b measured by FACS from mixed BM chimeras as in A. (G) Fecal CT-specific IgA from WT (Igh^a) and Ccr6^+/+ or Ccr6^−/− (Igh^b) mixed BM chimeras orally treated with CT, measured by allotypic ELISA. (H) Fecal IgA from WT (Igh^a) and Ccr6^+/+ or Ccr6^−/− (Igh^b) B cells co-transferred in to either Rag1^−/− or μMT, or from mixed BM chimeras as in A, measured by allotypic ELISA Each symbol in (A), (B), (E), (F), (G) and (H) represents an individual mouse, and data are pooled from at least three independent experiments. Data in (C) and (D) are representative of at least three independent experiments. *, p<0.05; **, p<0.01, ***, p<0.005 (unpaired Student’s T-test).

Figure 3. Pre-GC B cells in PPs initiate IgA class switch

(A, B) Representative semi-quantitative (A) or quantitative (B) RT-PCR on B cells from PPs sorted according to IgD and CCR6 expression, for the indicated transcripts. (C) Representative FACS staining (left panel) and frequency (right panel) of AID-GFP⁺ PP B cells from reporter mice, according to IgD and CCR6 expression. Each symbol in (B) and (C) represents an individual mouse and data are pooled from three independent experiments. Data in (A) are representative of three independent experiments.

Figure 4. LTβR-dependent PP DC support IgA class switching

(A) Quantitative PCR analysis of Ltbr transcript abundance in the indicated DC subsets from PPs (left panel) and median fluorescence intensity (MFI) ratio for LTβR on the DC subsets from PPs of BM chimeras reconstituted with WT or Ltbr^−/− BM (right panel). (B) Representative FACS staining (left panel) and absolute cell number (right panel) of the indicated DC subset from PPs of BM chimeras reconstituted with WT or Ltbr^−/− BM, (C) Frequency of IgA⁺ and IgG1⁺ GC B cells in PPs from BM chimeras as in B, determined by FACS. (D) Frequency of IgA⁺ and IgG1⁺ GC B cells in PP from mixed chimeras reconstituted with CD11c-DTR and either Ltbr^+/+ or Ltbr^−/− BM, treated with DT for 3 weeks. (E) Absolute number of the indicated DC subset from PPs of WT or LT-transgenic (tg) mice. (F) Frequency of IgA⁺ and IgG1⁺ GC B cells in PPs from WT or LT-tg mice, as determined by FACS. (G) Absolute number of the indicated DC subset from PPs of mice treated with LTβR-Fc or hIgG-Fc for 7 days. (H) Representative FACS staining of polyclonal B cells in PPs 7 days after transfer to MD4 recipients that were treated with LTβR-Fc or hIgG-Fc (left panel) and frequency of IgA⁺ B cells and GC B cells in PPs amongst transferred B cells (right panels). Each symbol in (A), (B), (C), (D), (E), (F), (G) and (H) represents an individual mouse, and data are pooled from at least three independent experiments. In (B), (C), (D), (E), (F), (G) and (H) *, p<0.05; **, p<0.01, ***, p<0.005 (unpaired Student’s T-test); in A, ***, p<0.005 (one-way ANOVA with Bonferroni’s post-hoc test).

Figure 5. LT- and ILC3-dependence of PP IgA response

(A) Representative FACS staining showing gating strategy and LTα1β2 on ILC3s in PP of WT mouse using LTβR-Fc. (B) Frequency of IgA⁺ and IgG1⁺ GC B cells in PPs from chimeras reconstituted with WT or Rorc^−/− BM. (C) Frequency of IgA⁺ and IgG1⁺ GC B cells in PPs as determined by FACS in BM chimeras reconstituted with WT, Rorc^−/− or a mix of Rorc^−/− and Rag1^−/− BM. (D) Frequency of IgA⁺ and IgG1⁺ GC B cells in PPs as determined by FACS in BM chimeras reconstituted with a mix of Lta^−/− BM and either WT or Rorc^−/− BM. (E) Representative immunofluorescence of PP SED from Rorc(γt)-EGFP mouse stained for the indicated markers. Yellow arrowheads indicate ILC3s proximal to DCs. Scale bar is 20μm. Each symbol in (B), (C) and (D), represents an individual mouse, and data are pooled from at least three independent experiments. Data in (A) and (E) are representative of at least three independent experiments. In B and D,***, p<0.005 (unpaired Student’s T-test); in C,, ***, p<0.005 (one-way ANOVA with Bonferroni’s post-hoc test).

Figure 6. PP B cell migration dynamics and interaction with SED DCs

(A) Representative TPLSM of PP for transferred CFP⁺ B cells (green) in CD11c-YFP mice. YFP⁺ cells (red) are shown using volume rendering. White dotted line indicates location of the SED. (B) Median velocity and (C) displacement versus square root of time of B cells in follicle (black) and in the SED (blue). (D) Percentage of B cells in the SED pausing (Pause), scanning (Scan) or not contacting (No contact) CD11c-YFP⁺ cells. (E) Representative time-lapse images of CFP⁺ B cell interaction with CD11c-YFP⁺ cell in the SED. Arrowhead highlights a single B cell-DC interaction. (Yellow color is due to bleed-through between channels). Time is shown in min:sec. (F) Representative z-projection view of PP follicle and SED of the type in A, showing only the CFP⁺ B cells. White dotted line, SED-follicle border; yellow lines, tracks of B cells in the follicle; blue lines, track of B cells in the SED; pink lines, tracks of B cells moving from the SED to the follicle; white lines, tracks of B cells moving from the follicle to the SED. Each symbol in (B) and (D), represents an individual mouse, and data are pooled from at least three independent experiments. Data in (A), (C), (E) and (F) are representative of at least three independent experiments.

Figure 7. DC integrin-β8 promotes TGFβ -dependent IgA switching

(A) Quantitative PCR analysis of Itgb8 (left panel) and Itgav (right panel) transcript abundance in the indicated DC subsets from PPs. (B) Median fluorescence intensity (MFI) ratio for β8 on the indicated DC subsets from PPs of either CD11c-Cre⁻ (WT) or CD11c-Cre⁺ Itgb8^fl/fl mice. (C) Frequency of IgA⁺ and IgG1⁺ GC B cells in PPs of either CD11c-Cre⁻ or CD11c-Cre⁺ Itgb8^fl/fl mice as determined by FACS. (D) Frequency of IgA⁺ and IgG1⁺ GC B cells in PPs 7 days after adoptive transfer in MD4 mice and treatment with either isotype control or anti-β8. (E) Frequency of IgA⁺ splenic B cells upon 5 days of culture with DCs sorted from PPs or mLNs of CD11c-Cre⁻ or CD11c-Cre⁺ Itgb8^fl/fl mice. (F) Frequency of IgA⁺ splenic B cells upon 5 days of culture with DCs sorted from PPs and incubated with the indicated antibodies. (G) Frequency of IgA⁺ splenic B cells upon 5 days of culture with the indicated DC subset sorted from PPs. (H) Median fluorescence intensity (MFI) for β8 on the BMDCs generated from CD11c-Cre⁻ or CD11c-Cre⁺ Itgb8^fl/fl BM upon 7 days of culture and incubation with the indicated reagents. Each symbol in (B), (C), (D), (E), (F), (G) and (H) represents an individual mouse, and data are pooled from at least three independent experiments. Data in (A) are pooled from three independent experiments In B, F and G *, p<0.05; **, p<0.01, ***, p<0.005 (one-way ANOVA with Bonferroni’s post-hoc test); in C, D and E **, p<0.01, ***, p<0.005 (unpaired Sudent’s T-test).