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Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells

Abstract

Innate lymphoid cells (ILCs) regulate stromal cells, epithelial cells and cells of the immune system, but their effect on B cells remains unclear. Here we identified RORγt+ ILCs near the marginal zone (MZ), a splenic compartment that contains innate-like B cells highly responsive to circulating T cell–independent (TI) antigens. Splenic ILCs established bidirectional crosstalk with MAdCAM-1+ marginal reticular cells by providing tumor-necrosis factor (TNF) and lymphotoxin, and they stimulated MZ B cells via B cell–activation factor (BAFF), the ligand of the costimulatory receptor CD40 (CD40L) and the Notch ligand Delta-like 1 (DLL1). Splenic ILCs further helped MZ B cells and their plasma-cell progeny by coopting neutrophils through release of the cytokine GM-CSF. Consequently, depletion of ILCs impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune system and circulatory system.

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Figure 1: Human splenic ILCs have a mucosa-like ILC3 phenotype and occupy MZ and perifollicular areas.
Figure 2: Human splenic ILCs establish bidirectional crosstalk with MRCs.
Figure 3: Human splenic ILCs express the MZ B cell–helper factors BAFF, CD40L and DLL1 and activate MZ B cells in cooperation with MRCs.
Figure 4: Human splenic ILCs help MZ B cells via BAFF, CD40L and DLL1.
Figure 5: Human splenic ILCs activate MZ B cell–helper neutrophils via GM-CSF.
Figure 6: Mouse splenic ILCs express the plasma cell–helper factors APRIL and DLL1 and enhance TI IgG3 responses.
Figure 7: Mouse splenic ILCs help plasmablasts and plasma cells emerging from TI IgG3 responses.
Figure 8: Mouse splenic neutrophils help plasma cells emerging from TI IgG3 responses and receive homeostatic signals from ILCs via GM-CSF.

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Acknowledgements

We thank A. Chorny, S. Casas, G.F. Sonnenberg and A. Bigas for technical assistance and help with discussions; A. Mensa for help with human samples; G. Dranoff (Dana-Farber Cancer Institute) for B16Csf2 melanoma cells; R. Gimeno (Institut Hospital del Mar d'Investigacions Mèdiques) for mouse OP9 and OP9-DLL1 stromal cell lines; M. López-Botet (Institut Hospital del Mar d'Investigacions Mèdiques) for antibody to human CD66; and E. Ramirez, E. Julià and O. Fornas for help with cell sorting. Supported by the European Research Council (ERC-2011-ADG-20110310 to A. Cerutti), Ministerio de Economia y Competitividad, Gobierno de España (M.G., S.B., C.M.B., I.P. and G.M.; SAF2011-25241 to A. Cerutti), the European Commission (PIRG-08-GA-2010-276928 to A. Cerutti), the US National Institutes of Health (R01 AI74378, R01 AI57653, U01 AI95613, U01 AI95776 IOF, P01 AI61093 and U19 096187 to A. Cerutti), the Ministry of Education, Culture, Sport, Science and Technology of Japan (Grant-in-Aid for Scientific Research 25293118 to S.F.) and Integrative Medical Sciences–Research Center for Allergy and Immunology (S.F.)

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G.M. and M. Miyajima designed and did research, discussed data and wrote the paper; S.B., A.M., I.P. and A. Chudnovskiy designed and did research; L. Cassis., C.M.B., L. Comerma., M.G., D.L. and M.C. did research; S.S., J.I.A., M.J. and J.Y. provided blood and tissue samples and discussed data; S.F. and M. Merad. designed research, provided reagents and discussed data; and A. Cerutti. designed research, discussed data and wrote the paper.

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Correspondence to Andrea Cerutti.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Human splenic ILCs include CD56+ and CD56 subsets of LinCD117+CD127+ cells that are distinct from NK cells and share ILC3 traits.

(a) Flow cytometry of viable (DAPI) splenocytes stained for CD19, CD3, CD14, CD117, CD127 and CD56. FSC-A, forward scatter area; SSC-A, side scatter area. (b) Flow cytometry of CD8, CRTH2, NOTCH2 and CD103 on splenic LinCD127+CD117+ ILCs. Gray shading, isotype-matched control antibody. (c) Flow cytometry of NKp44, NKp46, CCR6, CD96, CD103 and CD161 on splenic LinCD127+CD117+ ILCs (red lines) and splenic CD56+CD3CD127CD117 NK cells (blue lines). (d) qRT-PCR of mRNAs for ID2 (Id2), IL17A (IL-17A), IL23R (IL-23 receptor) and IL26 (IL-26) in splenic ILCs, NK cells, macrophages (Mφ), B cells and T cells. Results are normalized to ACTB mRNA (β-actin) and presented as relative expression (RE) compared with that of fresh NK cells. (e) Gating strategy adopted to FACSort splenic CD56+ ILCs (red gate), CD56 ILCs (orange gate) and NK cells (blue gate). (f) qRT-PCR analysis of RORC (RORγt), IL22 (IL-22), TNF (TNF), LTB (LT-β), and PRF1 (Perforin-1) mRNAs from splenic CD56+ ILCs, CD56 ILCs and NK cells. Results are normalized to ACTB mRNA and presented as RE compared with that of fresh NK cells. Error bars, s.e.m.; *P <0.05 (two-tailed unpaired Student's t test). Data summarize three measurements from three pooled experiments with one donor in each (d,f) or show one of fifteen (a) or four (b,c,e) experiments with similar results.

Supplementary Figure 2 Human splenic ILCs express NKp44 and CD117 and occupy MZ, perifollicular zone and red pulp areas.

(a) IHC of spleen stained for NKp44 and counterstained with hematoxylin. Original magnification, ×20 (left), ×40 (right), zoom ×2 (inset). Solid and dashed lines demarcate the follicle (FO) and MZ, respectively. (b) IHC of spleen (top) and tonsil (bottom) tissue sections stained for CD117 (red) and tryptase (brown) and counterstained with hematoxylin. Red CD117+tryptase ILCs can be clearly distinguished from brown CD117+tryptase+ mast cells, which are abundant in tonsils but not spleen. MC, mast cell. Original magnification ×20 (left) and ×40 (right). (c) Spleen sections immunohistochemically stained for CD117 (red) and tryptase (brown) scanned with ScanScope and visualized with ImageScope viewer to quantify CD117+tryptase ILCs in MZ-PFZ (blue stars) or red pulp (black stars) from nine microscopic ×20 fields of two spleens. PFZ: perifollicular zone. Original magnification, ×20; zoom ×2 (rightmost-bottom image). (d) IFA of spleens stained for RORγt (red), NKp44 (green) and CD3 (blue). Nuclear DNA was counterstained with DAPI. Dashed line demarcates the follicle. Original magnification, ×20. Data show one of two-four experiments with similar results.

Supplementary Figure 3 Human MRCs express a stromal phenotype and respond to lymphotoxin and TNF from ILCs.

(a) IFA of spleen stained for MAdCAM-1 (red or green), RORγt (green), Thy-1 (red), IgD (blue), and/or CD141 (purple). Original magnification, ×20 (leftmost larger image) or ×40 (rightmost smaller images). (b) IHC of spleen stained for CD117 (red) and α-SMA (brown). Arrowheads point to ILCs. Original magnification, ×40 and zoom ×2 (inset). (c) Left images: IFA of MAdCAM-1 (red) and DAPI-stained nuclear DNA (blue) in purified splenic MRCs. Original magnification, ×63. Right profiles: intracellular flow cytometry of MAdCAM-1 in splenic MRCs. Gray shading, isotype-matched control antibody. (d,e) Flow cytometry of ICAM-1 and VCAM-1 on splenic MRCs cultured with or without TNF and LT (d, left) and in the presence or absence of anti-LT and anti-TNF antibodies (d, right) or with or without LPS (e) for 72 hours. (f) Flow cytometry of VCAM-1 on MRCs cultured for 72 h with or without ILCs in the presence or absence of control Ig, anti-TNF antibody, anti-LTαβ antibody or a combination of anti-TNF plus anti-LTαβ antibodies. (g) qRT-PCR of MADCAM1 (MAdCAM-1), IL7 (IL-7) and CCL20 (CCL20) mRNAs from splenic MRCs cultured with or without LT plus TNF for 72 h. Results are normalized to ACTB mRNA (β-actin) and presented as relative expression (RE) compared with that of unstimulated MRCs. (h) qRT-PCR of IL7 (IL-7), IL1b (IL-1β) and IL23 (IL-23) mRNAs from splenic MRCs, macrophages (Mφ) and DCs. Results are normalized to ACTB mRNA and presented as RE compared with that of MRCs. Error bars, s.e.m.; *P <0.05 (two-tailed unpaired Student's t test). Data show one of four experiments with similar results (a-f) or summarize three experiments (g,h) with one donor in each.

Supplementary Figure 4 Mouse splenic LinCD117+CD127+ ILCs include CD4+ and CD4 fractions and their loss does not cause gross splenic anatomical defects in Rorc–/– mice.

(a) Flow cytometry of mouse viable propidium (PI) splenocytes stained for CD45, Lin molecules (CD3, B220, CD11b, CD11c, Ly6C/G, Ter119), CD4, CD117 and CD127. FSC, forward scatter; FSC-W, forward scatter width; SSC, side scatter. (b) IFA of spleens from Rorc+/+ and Rorc–/– mice stained for ER-TR9 or VCAM-1 (green), F4/80 or IgM (red) and MOMA-1 or ICAM-1 (blue). Original magnification, ×10. Data show one of three experiments with similar results.

Supplementary Figure 5 Mouse splenic ILCs enhance preimmune IgG3 production but not IgM production, except phosphorylcholine-specific IgM production.

(a) IFA of splenic IgM (green) and IgG3 (red) from Rorc+/+ and Rorc–/– mice. Original magnification, ×40. (b) ELISA of total serum IgM in Rorc+/+ (n = 7) and Rorc–/– (n = 7) mice. (c) Absolute numbers of splenic FO B cells, MZ B cells and B-1 cells from Rorc+/+ (n = 3) and Rorc–/– (n = 3) mice determined by flow cytometric analysis of B220+CD21+CD23+, B220+CD21hiCD23 and IgMhiB220int/loCD5int B-1 cells, respectively. (d) ELISA of serum phosphorylcholine-reactive (PC-R) IgM in Rorc+/+ (n = 6) and Rorc–/– mice (n = 6). (e) Flow cytometric analysis of frequency of splenic B220+CD21+CD23+ FO B cells (blue gate), B220+CD21hiCD23 MZ B cells (red gate), B220+CD21CD23 transitional B cells (green gate) and T cell receptor β (TCRb)+CD4+ T cells from disparate chimeric mice treated with control (ctrl) (n = 7) or anti-Thy.1.2 antibodies (n = 7). Error bars, s.d.; *P <0.05 (unpaired Student's t test). Data show one of three experiments with similar results (a,e) or summarize three experiments containing 3-7 animals per group (b-d).

Supplementary Figure 6 Mouse splenic ILCs control the homeostasis of NBH cells.

(a) Flow cytometric analysis of the frequency (left panels) and absolute number (right bars) of splenic Ly6G+CD11b+ neutrophils (orange gate or bars) and splenic Ly6GCD11b+ macrophages (pink gate or bars) in Rorc+/+ (n =5) and Rorc–/– mice (n = 5). (b) IFA of IgG3 (green), Ly6G (red) and IgM (blue) in spleens from Rorc+/+ and Rorc–/– mice. Original magnification, ×5. (c) Flow cytometric analysis of the frequency (left panels) and absolute number (right bars) of splenic Ly6G+CD11b+ neutrophils in Rorc+/–Cd3e–/– mice (n =4), Rorc–/–Cd3e–/– mice (n = 3). (d) ELISA of serum Phosphorylcholine-reactive (PC-R) IgM in Cfs2+/+ (n =7) and Cfs2–/– mice (n =7). Error bars, s.d.; *P <0.05 (one-tailed unpaired Student's t test). Data summarize results from at least 3 mice in each group (a,c: bars; d) or show one of at least three experiments with similar results (b)

Supplementary Figure 7 Splenic ILCs integrate stromal and immunological signals to enhance TI antibody production by MZ B cells.

(a) Location of splenic ILCs, MZ B cells and NBH cells in the human spleen. PFZ, perifollicular zone. (b) Splenic ILCs release LT and TNF, which induce MRC production of splenic ILC survival factors such as IL-1β, IL-7 and IL-23. Some of these cytokines are also produced by splenic DCs and macrophages. In addition to promoting splenic ILC survival, IL-1β, IL-7 and IL-23 enhance splenic ILC expression of B cell-helper factors. In humans, splenic ILCs express BAFF, CD40L and DLL1, which cooperate with MRCs and microbial TLR ligands such as CpG DNA to promote MZ B cell survival, activation, plasmablast differentiation as well as IgM, IgG and IgA secretion. In mice, ILCs lack BAFF and CD40L, but express APRIL and DLL1 and enhance pre-immune and post-immune IgG3 responses to TI antigens by promoting the survival and possibly the differentiation of IgG3-expressing plasmablasts and plasma cells emerging from B cells, including MZ B cells. ILCs further enhance antibody production by co-opting NBH cells through GM-CSF. Besides promoting the survival of NBH cells, GM-CSF enhances the MZ B cell-helper function of NBH cells, which largely depends on APRIL and (not shown in this study) BAFF. In addition, GM-CSF helps NBH cells to form DNA-containing NET-like structures, which may have an antigen-trapping function.

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Magri, G., Miyajima, M., Bascones, S. et al. Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells. Nat Immunol 15, 354–364 (2014). https://doi.org/10.1038/ni.2830

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