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

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.

INTRODUCTION

The spleen is a highly perfused organ specialized in host defense against blood-borne pathogens. Interposed between the follicles of the splenic white pulp and the circulation, the marginal zone (MZ) contains B cells enmeshed with macrophages and dendritic cells (DCs) in a stromal reticular cell network[1-3]. All of these cells provide an efficient immunosurveillance of the circulatory system by readily interacting with circulating antigens from commensal or pathogenic microbes owing to the slow flow rate of the blood passing through the MZ[4]. Following antigen capture, macrophages, DCs and possibly neutrophils of the innate immune system expose antigen to MZ B cells, a unique subset of antibody-producing lymphocytes that develop from transitional B cells in response to NOTCH2 signals[5].

Lymphoid sites positioned between the host and the environment contain innate-like B and T cells that belong to the adaptive immune system, but share several properties with effector cells of the innate immune system. Mucosal and serosal membranes include innate-like B-1 cells that generate a first line of protection through early production of low-affinity immunoglobulin M (IgM) to bacteria[6]. When microbes breach the mucosal barrier and enter the general circulation, innate-like MZ B cells provide a second line of protection via low-affinity IgM and IgG that bridge the temporal gap required for the slower production of high-affinity IgG by follicular (FO) B cells[4].

Similar to B-1 cells, MZ B cells express clonally distributed and somatically recombined but rather unspecific B cell receptor (BCR) molecules encoded by poorly diversified immunoglobulin (Ig) genes[4, 6]. MZ B cells also express non-clonally distributed and germline-encoded Toll-like receptors (TLRs)[7], a subfamily of nonspecific microbial sensors generally known as pattern recognition receptors. Typically expressed by effector cells of the innate immune system, TLRs activate MZ B cells after recognizing conserved microbial molecular signatures in cooperation with BCRs[8]. The activation of MZ B cells is further enhanced by B cell-stimulating cytokines released by DCs, macrophages and neutrophils[9, 10].

Besides innate-like lymphocytes, mucosal surfaces include innate lymphoid cells (ILCs) that express neither somatically recombined antigen receptors nor conventional surface lineage molecules[11]. These ILCs require the transcriptional repressor inhibitor of DNA 2 (Id2) and the cytokine interleukin-7 (IL-7) for their development and generate cytokine secretion patterns that mirror those of T helper (TH) cells of the adaptive immune system[12, 13]. Similar to pro-inflammatory TH1 cells, group 1 ILCs (ILC1) release interferon-γ (IFN-γ) and require the transcription factor T-bet for their development as do natural killer (NK) cells of the innate immune system[14]. ILC2, which include natural helper cells and nuocytes, secrete IL-5 and IL-13 and require the transcription factor GATA-3, thus resembling pro-inflammatory TH2 cells[15-17]. Finally, ILC3 require the transcription factors retinoic acid receptor-related orphan receptor-γt (RORγt) and aryl hydrocarbon receptor (AhR) and include mucosal NK-22 cells, which secrete IL-22 and thus mimic non-inflammatory TH22 cells[18-21], as well as fetal and mucosal lymphoid tissue inducer (LTi) cells, which produce IL-22 and IL-17 and thus resemble pro-inflammatory TH17 cells[22-24]. While NK-22 cells express natural cytotoxicity receptors (NCRs) usually associated with NK cells and mediate mucosal homeostasis by targeting epithelial cells via IL-22 (refs. 25-27), LTi cells lack NCRs and promote fetal lymphoid organogenesis and post-natal mucosal immunity by targeting stromal cells via lymphotoxin (LT) and tumor necrosis factor (TNF)[28-30].

Mucosal NK-22 cells, also defined as NCR+ ILC3 to distinguish them from inflammatory NCR– ILC3 characterized by constitutive IL-17, IL-22 and activation-induced IFN-γ production[31, 32], express B cell-activating factor of the TNF family (BAFF)[20], a cytokine used by DCs, macrophages and neutrophils to help MZ B cells and plasma cells in a T cell-independent (TI) manner[1, 9, 10]. BAFF and its homologue a proliferation-inducing ligand (APRIL) are related to CD40 ligand (CD40L), a TNF family member used by T follicular helper (TFH) cells to activate FO B cells[33]. Given their involvement in mucosal TI antibody production[29, 34], ILCs could regulate humoral immunity also in the MZ, a lymphoid area that is continually exposed to antigen as are mucosal membranes.

Here we identified ILCs with mucosa-like properties in the MZ and perifollicular zone of the spleen. These ILCs required survival signals from marginal reticular cells (MRCs), a MZ subset of stromal cells that responded to TNF and LT from ILCs. In addition to stimulating MZ B cells and plasma cells via BAFF, APRIL, CD40L and the NOTCH2 ligand Delta-like 1 (DLL1), splenic ILCs co-opted MZ B cell-helper neutrophils via granulocyte monocyte-colony stimulating factor (GM-CSF). Consequently, ILC depletion hampered MZ B cell production of antibodies to TI antigens.

RESULTS

Splenic ILCs have type-3 mucosal features

The MZ is continually exposed to blood-borne antigens and may thus require homeostatic signals from ILCs as mucosal membranes do[4]. Flow cytometry showed that histologically normal adult human spleens contained cells that lacked common lymphoid and myeloid lineage (Lin)-associated molecules, but expressed the IL-7 receptor CD127 and the stem cell growth factor receptor CD117 ( and ). These ILCs resembled mucosal NCR+ ILC3s[19, 20], because they expressed the NK cell molecules CD56, CD96, CD161, NKp44 and NKp46, the CCL20 chemokine receptor CCR6, the activation molecules CD25 and CD69, and NOTCH2 ( and ). Splenic ILCs expressed neither the CD8 co-receptor, which is associated with cytotoxic T cells, nor the integrin CD103, which is expressed by some mucosal lymphocytes, nor the CD4 co-receptor and the prostaglandin D2 receptor CRTH2 ( and ), which are expressed by mouse LTi cells and human ILC2, respectively[16, 22]. Albeit expressing as much CD161 as NK cells, splenic ILCs showed more NKp44, CCR6 and CD96 but less NKp46 than NK cells did ().

Gene expression studies performed through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) showed that splenic ILCs expressed the transcription factors RORγt, AhR and Id2, the cytokines IL-22, IL-26, LT-α, LT-β and TNF and the IL-23 receptor, whereas splenic NK cells, T cells and macrophages did not, except B cells that expressed abundant LT-β ( and ). Compared to mucosal ILCs from tonsils, splenic ILCs expressed comparable RORγt and AhR, but less IL-22 and more LT-α, LT-β and TNF (). These latter cytokines are also hallmarks of LTi cells together with IL-17 (refs. 23, 30). However, splenic ILCs lacked IL-17, which was instead detected in T cells (). Of note, splenic ILCs included CD56+ and CD56– subsets with an overlapping ILC3 gene expression profile that did not include IFN-γ and Perforin-1, which were instead abundant in NK cells ( and ). Flow cytometry showed that splenic RORγt+ ILCs lacked T-bet, a transcription factor that was instead expressed by RORγt– NK cells ().

Flow cytometry and enzyme-linked immunosorbent assay (ELISA) demonstrated that splenic ILCs were more abundant than mucosal ILCs () and displayed NCR+ ILC3 properties[19, 20]. Indeed, splenic ILCs received anti-apoptotic signals from the stromal cytokine IL-7 and the innate cytokines IL-1β and IL-23 and secreted IL-22 in response to IL-1β and IL-23 as mucosal ILCs did (). Finally, tissue immunofluorescence analysis (IFA) and immunohistochemistry (IHC) showed that RORγt+NKp44+CD117+tryptase– ILCs occupied MZ and perifollicular zone (PFZ) areas and were distinct from CD117+tryptase+ mast cells and CD3+RORγt–NKp44– T cells ( and ). Some ILCs were also found in the periarteriolar lymphoid sheath (not shown). Thus, the human spleen contains mucosa-like ILCs different from NK cells and positioned nearby MZ B cells.

Splenic ILCs receive survival signals from MRCs

Given that LTi cells interact with stromal cells via LT and TNF[22, 29, 30], we sought to identify stromal cells in the human MZ by tissue IFA, IHC and flow cytometry. Fibroblast-like cells expressing mucosal addressing cell adhesion molecule-1 (MAdCAM-1) were detected in the MZ nearby IgD+ B cells and some RORγt+ ILCs ( and ). These MAdCAM-1+ cells did not express the coagulation protein von Willebrand factor (vWF), which characterizes endothelial cells, whereas MAdCAM-1– sinusoid-lining cells (SLCs) from the red pulp did (). MAdCAM-1+ cells also lacked the endothelial molecules platelet endothelial cell adhesion molecule-1 (PECAM-1) and CD34 as well as the leukocyte molecule CD45, but expressed the stromal molecules Thy-1 (or CD90), CD141 (thrombomodulin), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and α-smooth muscle actin ( and ), and were thus considered equivalent to mouse MRCs[3].

Human MRCs expressed TLR3, which binds double-stranded RNA, TLR4, which binds lipopolysaccharide (LPS), and TLR9, which binds unmethylated CpG-rich DNA (), pointing to a role of microbial products in the regulation of MRCs. Laser capture microdissection followed by qRTPCR demonstrated that MRC-enriched MAdCAM-1+ tissue contained more ILC survival factors such as IL-1β, IL-7 and IL-23 and more ILC-recruiting factors such as CCL20 (a CCR6 ligand) than SLC-enriched mannose receptor (MR, an endocytic receptor)+ tissue or control MR– tissue ().

As shown by flow cytometry, splenic ILCs up-regulated the MAdCAM-1-binding integrin α4β7 in response to IL-7 and IL-1β, pointing to a role of MAdCAM-1-α4β7 interaction in the perifollicular positioning of ILCs. Conversely, MRCs up-regulated ICAM-1 and VCAM-1 in response to splenic ILCs or a combination of LT and TNF or LPS ( and ). Blocking experiments and qRT-PCR indicated that ILCs activated MRCs via LT and TNF, whereas MRCs up-regulated IL-7, CCL20 and MAdCAM-1 in response to LT and TNF ( and ). Of note, transwell assays showed that MRCs supported ILC survival via contact-dependent and contact-independent signals that included IL-7 (). Finally, splenic macrophages and DCs expressed more IL-1β and IL-23 than MRCs (), suggesting that ILCs establish a bi-directional interplay with MRCs in splenic niches that may also entail innate immune cells.

Splenic ILCs and MRCs deliver B cell-helper signals

Given their MZ and perifollicular location, human splenic ILCs may regulate MZ B cells. As shown by tissue IFA and qRT-PCR, splenic RORγt+ ILCs were positioned nearby CD20+ B cells and, compared to other splenic leukocyte subsets or mucosal ILCs, expressed more BAFF, CD40L and DLL1 (), three major B cell and plasma cell helper factors[5, 9, 10, 35]. Unlike splenic macrophages, splenic ILCs expressed little or no APRIL (), a BAFF-related plasma cell survival factor[36].

As shown by flow cytometry and ELISA, splenic ILCs expressed surface BAFF, CD40L and DLL1 and secreted BAFF, whereas APRIL was mainly secreted by macrophages (). Accordingly, splenic ILCs stimulated the survival, proliferation, IgM secretion and plasmablast differentiation of IgDloCD27+ MZ but not IgDhiCD27– FO B cells (). Having shown that MRCs provide survival and activation signals to ILCs, we verified whether MRCs augmented the B cell-helper function of ILCs. Compared to MZ B cells exposed to ILCs or MRCs alone, MZ B cells exposed to both ILCs and MRCs not only underwent more IgM secretion and plasmablast differentiation, but also acquired the ability to release IgG and IgA, particularly in the presence of CpG DNA (). Thus, human splenic ILCs may integrate stromal and innate immune signals to orchestrate antibody production.

Splenic ILCs help MZ B cells via BAFF, CD40L and DLL1

Next, we used flow cytometry and ELISA to determine the mechanism by which human splenic ILCs help MZ B cells. BAFF-R-Ig, a soluble decoy receptor that blocks the binding of BAFF to B cells, attenuated the survival of MZ B cells exposed to ILCs (). Of note, MZ B cells underwent plasmablast differentiation and IgM secretion in response to ILCs but not ILC-conditioned medium (), suggesting the involvement of contact-dependent signals from membrane-bound BAFF, CD40L and/or DLL1. Accordingly, BAFF-R-Ig, CD40-Ig or a NOTCH signaling inhibitor mitigated ILC-induced IgM secretion, particularly when these inhibitors were used in combination ().

Although required for the early development of MZ B cells, NOTCH2 remains expressed on mature MZ B cells and may thus modulate their function[5]. Indeed, when combined with BAFF, DLL1-expressing OP9 stromal cells stimulated plasma cell differentiation and IgM secretion through NOTCH signals in MZ B cells (). In contrast, BAFF alone or combined with DLL1-negative OP9 cells had little or no stimulatory effect (). Thus, besides eliciting MZ B cell survival and activation, human splenic ILCs promote plasmablast differentiation and survival via BAFF, CD40L and DLL1.

Splenic ILCs activate perifollicular neutrophils via GM-CSF

In human spleens, MZ B cells interact with neutrophils (termed NBH cells) possibly originating from circulating precursors (termed NC cells)[10]. Given the involvement of RORγt in GM-CSF expression by TH17 cells and the important role of GM-CSF in neutrophil activation and survival[37], we wondered whether splenic ILCs expressed GM-CSF. Tissue IHC showed CD117+ ILCs nearby CD66 (CEACAM-1)+ NBH cells positioned around the MZ (). qRT-PCR, ELISA and flow cytometry demonstrated the expression of abundant GM-CSF in splenic ILCs, but not other leukocyte subsets (). Splenic ILCs further expressed the neutrophil-recruiting chemokine IL-8 (or CXCL8), which was also produced by macrophages and NK cells, albeit in lesser amounts ().

Given that GM-CSF enhances the B cell-helper function of neutrophils[10], we used flow cytometry, ELISA, IFA and qRT-PCR to establish whether GM-CSF-producing splenic ILCs induce NBH cell-like properties to NC cells. In the presence of splenic ILCs, NC cells not only acquired phenotypic traits typical of NBH cells[10], such as up-regulation of the activation molecules CD69, CD11b and CD24 and down-regulation of the adhesion molecule CD62 ligand (CD62L), but also received survival signals from GMCSF (). When incubated with splenic ILCs or their conditioned medium, NC cells up-regulated APRIL and thus stimulated more IgA production in MZ B cells (). ILC-conditioned medium also augmented the capacity of NBH cells to induce IgM, IgG and IgA in MZ B cells (). Similar to GMCSF or LPS, ILC-conditioned medium stimulated NC cells to form neutrophil extracellular traps (NETs) (), which are antigen-trapping projections often emanating from NBH cells[10]. Thus, human splenic ILCs may amplify MZ B cell responses by stimulating perifollicular neutrophils via GM-CSF.

Splenic ILCs enhance TI antibody production

To elucidate the in vivo contribution of splenic ILCs to humoral immunity, we initially took advantage of Rorc–/– mice, which lack the RORγt transcription factor required for the development of ILC3 (refs. 22, 28). Rorc–/– mice also lack TH17 cells, a subset of CD4+ T cells that regulate neutrophils via IL-17 and GM-CSF[37-39]. Flow cytometry showed that Rorc–/– mice had fewer splenic Lin–CD117+CD127+ ILCs than Rorc+/+ mice (. In these wild type mice, flow cytometry and qRT-PCR demonstrated that splenic ILCs included both CD4+ and CD4– subsets that expressed more RORγt, IL-22, LT-α and TNF than did other splenic leukocytes ( and ). In contrast to human splenic ILCs, mouse splenic ILCs lacked BAFF and CD40L, but expressed APRIL and DLL1 ().

We then verified whether Rorc–/– mice had an altered MZ. As shown by tissue IFA, Rorc–/– mice had no perturbations of either MOMA1+ metallophilic macrophages, ER-TR9+ MZ macrophages and ICAM-1+ marginal sinus cells from the MZ or IgM+ B cells from the MZ and white pulp or F4/80+ macrophages and VCAM-1+ endothelial cells from the red pulp (). Flow cytometry, tissue IFA and ELISA demonstrated that spleens from non-immunized Rorc–/– mice contained conserved B cells, but fewer plasmablasts and plasma cells expressing IgG3 ( and ), an antibody produced by MZ B cells through a TI pathway under pre-immune conditions or after immunization[40, 41]. Besides less total serum IgG3, Rorc–/– mice had less serum IgG3 to the bacterial TI antigen phosphorylcholine (PC) (). To ascertain the relative contribution of ILCs and T cells to pre-immune IgG3 responses, Rorc+/– and Rorc–/– mice were crossed with T cell-deficient Cd3e–/– mice. In agreement with the TI nature of pre-immune IgG3 production[40, 41], non-immunized Rorc+/–Cd3e–/– mice had normal pools of splenic B cells, plasma cells and plasmablasts expressing IgG3 (). Compared to these mice, Rorc–/–Cd3e–/– mice showed conserved B cells, but fewer plasmablasts and plasma cells expressing IgG3 and less pre-immune serum IgG3 ().

Unlike pre-immune serum IgG3, pre-immune serum IgM was augmented in Rorc–/– mice together with splenic IgMhiB220int/loCD5int B-1 cells but not B220+CD21+CD23+ FO B cells and B220+CD21hiCD23– MZ B cells (). The expansion of IgM-producing B-1 cells in Rorc–/– mice may result from enhanced migration of B-1 cells from the peritoneal cavity to the spleen due to the lack of gut-associated lymphoid tissue and increased systemic translocation of gut bacteria[22, 28, 34, 42]. Nonetheless, Rorc–/– mice had less pre-immune serum IgM to PC (), suggesting a role of ILCs in at least some TI IgM responses.

To dissect the MZ B cell-helper function of ILCs in a more physiological model, an anti-Thy-1.2 antibody was used to deplete ILCs in Thy-1-disparate Rag1–/– chimeric mice generated as described in a recently published study[43]. Compared to mice treated with a control antibody, mice treated with anti-Thy-1.2 showed fewer splenic ILCs, fewer IgG3-expressing plasmablasts and plasma cells, and reduced pre-immune serum IgG3 ( and ). Furthermore, anti-Thy-1.2-treated mice had impaired post-immune IgG3 responses to 2,4,6-trinitrophenyl (TNP)-Ficoll (), a TI antigen that induces antibody production in innate-like B cells, including MZ B cells[40].

The involvement of splenic ILCs in TI antibody production was also demonstrated by reconstituting lethally irradiated Rorc–/– mice with a mix of bone marrow cells from Rag1–/– mice – which generate ILCs but not T and B cells – and Rorc–/– mice – which generate T and B cells but not ILCs. ILC-sufficient (ILC+) chimeric mice developed splenic ILCs, whereas control ILC– mice generated by reconstituting irradiated Rorc–/– mice with bone marrow cells from Rorc–/– mice did not (). Compared to ILC+ mice, ILC– mice showed fewer splenic plasmablasts and plasma cells expressing IgG3 and less pre-immune serum IgG3 (). In all mouse models, IgM responses were only marginally affected (not shown), indicating that splenic ILCs predominantly help IgG3-expressing plasmablasts and plasma cells emerging from MZ B cell responses to TI antigens.

Splenic ILCs regulate neutrophil homeostasis

We next determined the role of mouse splenic ILCs in the homeostasis of NBH cells. Tissue IFA and flow cytometry showed that ILC– bone marrow chimeric mice had fewer splenic Ly6G+CD11b+ neutrophils and extrafollicular IgG3-producing plasmacytes than control ILC+ mice (). Splenic neutrophils were also decreased in Rorc–/– mice with either a T cell-sufficient or T cell-deficient background (), indicating that splenic ILCs provide helper signals to neutrophils independently of T cells. To ascertain the contribution of splenic neutrophils to pre-immune IgG3 production, ILC-reconstituted (ILC+) bone marrow chimeric mice were depleted of splenic neutrophils with an anti-Ly6G antibody (). These mice had normal splenic ILCs and IgG3-expressing B cells and plasmablasts, but fewer splenic IgG3-expressing plasma cells and decreased pre-immune serum IgG3 compared to ILC+ mice treated with a control antibody (). Thus, unlike ILCs, neutrophils may predominantly control terminally differentiated IgG3-secreting cells.

Finally, we determined whether splenic ILCs regulate neutrophils via GM-CSF. As shown by qRTPCR, splenic ILCs from wild-type mice expressed more GM-CSF than other leukocyte subsets did, including macrophages and NK cells (). Compared to controls, GM-CSF (also known as colony-stimulating factor 2)-deficient Csf2–/– mice had reduced pre-immune serum antibodies to bacterial PC and fewer splenic neutrophils, which increased after inoculation of GM-CSF-expressing B16 melanoma cells ( and ). Also Rag1–/–Il2rg–/– mice, which lack the IL-2 receptor common γ chain mandatory for IL-7-dependent ILC development[13], had fewer splenic neutrophils compared to Rag1–/–Il2rg+/+ controls (). Moreover, adoptive transfer of ILCs from Csf2+/+ but not Csf2–/– mice increased splenic neutrophils in Rag1–/–Il2rg–/– mice (). Thus, in addition to helping MZ B cells and plasma cells through BAFF (or APRIL), CD40L and DLL1, splenic ILCs co-opt neutrophils via GM-CSF to enhance TI antibody production ().

DISCUSSION

We have shown that the MZ and PFZ of the spleen contained group-3 mucosa-like ILCs that released LT and TNF to establish a bi-directional crosstalk with stromal MRCs. In addition to stimulating MZ B cells and plasma cells through BAFF, APRIL, CD40L and DLL1, splenic ILCs co-opted neutrophils with MZ B cell- and plasma cell-helper functions through GM-CSF, thereby sustaining TI antibody production.

Mucosal and serosal membranes contain evolutionarily primitive lymphocyte subsets that include B-1 cells, γδ T cells, invariant NKT cells and mucosa-associated invariant T cells[44]. By sensing conserved microbial signatures through somatically recombined and germline-encoded receptors, these innate-like lymphocytes rapidly activate protective programs that crossover the conventional boundaries between the innate and adaptive immune systems[44]. Mucosal membranes also contain ILCs that lack somatically recombined receptors and yet mount prompt T cell-like responses[13]. Here, we found that splenic ILCs enhanced antibody production to TI antigens by activating innate-like B cells positioned at the interface between the immune and circulatory systems.

Similar to mucosal ILC3 (refs. 19, 20), human splenic ILCs contained both RORγt and AhR transcription factors, expressed NCRs along with various activation molecules, and secreted IL-22 in response to IL-23. In addition, human splenic ILCs activated MZ-based stromal MRCs via LT and TNF and thus also resembled fetal or mucosal LTi cells[28-30, 45]. However, unlike LTi cells[23, 30], human splenic ILCs did not express IL-17, raising questions as to the ontogenetic relationship between these ILC subsets. Mouse splenic ILCs had a phenotype and gene expression profile similar to those of human splenic ILCs, but expressed CD4, thus showing a closer affiliation with LTi cells.

Human splenic ILCs not only expressed the gut homing receptor α4β7, but also interacted with MRCs equipped with MAdCAM-1, an α4β7 ligand usually associated with gut endothelial cells[34]. Given their additional expression of CCL20 and the presence of the CCL20 receptor CCR6 on splenic ILCs, MRCs might be involved in the positioning of ILCs within and around the MZ. Consistent with their stroma phenotype and reticular morphology, MRCs lacked endothelial molecules and exhibited strong responsiveness to LT and TNF from ILCs, thus resembling MAdCAM-1+ stromal cells lining the mouse marginal sinus[46]. Conversely, MRCs delivered both contact-dependent and contact-independent survival signals to ILCs, including IL-7. Remarkably, MRCs increased IL-7 expression in response to LT and TNF, pointing to the presence a bi-directional functional crosstalk between MRCs and ILCs in the MZ and PFZ of the spleen. Additional ILC survival factors such as IL-1β and IL-23 were predominantly expressed by splenic DCs and macrophages, suggesting that ILCs inhabit a splenic niche that entails survival signals from both stromal and immune cells. Of note, MRCs received further activation signals from TLR ligands such as LPS, lending support to the possibility that commensal antigens from the intestinal mucosa help the post-natal development and function of the splenic MZ[4, 10, 47, 48].

In humans, splenic ILCs enhanced the survival of MZ B cells through a contact-independent mechanism involving soluble BAFF, but triggered the differentiation of MZ B cells into plasmablasts through contact-dependent signals provided by membrane-bound BAFF, DLL1 and CD40L. Of note, DLL1 synergized with soluble BAFF to induce IgM secretion, possibly owing to the pro-survival effect of soluble BAFF on plasmablasts[35]. Given that DLL1 also cooperates with BAFF to stimulate the early development of MZ B cells[5], there is the possibility that DLL1 on ILCs operates at both early and late stages of MZ B cell differentiation with distinct signaling programs. Of note, splenic ILCs further helped MZ B cells by co-opting NBH cells through GM-CSF. This cytokine may also enhance the MZ B cell-stimulating function of DCs and possibly macrophages[1, 4], which may explain why a relative small number of ILCs is sufficient to enhance TI antibody responses.

Despite expressing CD40L, a class switch-inducing factor typically detected on TFH cells[33], human splenic ILCs did not trigger IgG or IgA production in MZ B cells. Nonetheless, MZ B cells induced IgA and IgG when exposed to ILCs in the presence of MRCs and microbial TLR ligands. Accordingly, small amounts of these bacterial components can be detected in splenic perifollicular areas together with plasma cells releasing IgM, IgG or IgA[10]. Thus, similar to gut LTi cells[29], splenic ILCs may integrate stromal and innate immune signals to rapidly generate antibodies for antimicrobial protection.

Although lacking both CD40L and BAFF, mouse splenic ILCs expressed APRIL, a BAFF-related molecule that enhances plasma cell survival[36]. By also expressing DLL1, mouse splenic ILCs would sustain the differentiation and/or survival of plasmablasts and plasma cells emerging during IgG3 responses to TI antigens. In mice, ILC depletion did not impair IgM production, suggesting that ILCs predominantly enhance MZ B cell-derived IgG3 rather than B-1 cell-derived IgM responses. Nonetheless, splenic ILCs sustained pre-immune production of IgM to bacterial PC, which points to the involvement of ILCs in IgM responses to some but not all TI antigens.

In ILC-deficient mice, impaired TI IgG3 production was associated with a reduction of NBH cells, which help MZ B cells via BAFF and APRIL[10]. Accordingly, human splenic ILCs enhanced the survival and B cell-helper activity of NBH cells through GM-CSF. This cytokine also induced NET-like structures, which might enable NBH cells to capture blood-borne antigens transiting through the MZ[4, 10]. Human splenic ILCs also released IL-8, which may contribute to the recruitment of NBH cells. Mice do not express IL-8, but their splenic ILCs may attract NBH cells via alternative factors, including GM-CSF. Remarkably also splenic innate response activator (IRA) B cells express GM-CSF[49], which could explain the persistence of some NBH cells in mice lacking ILCs.

GM-CSF further derives from TH17 cells[37], but the analysis of T cell-deficient mice suggested that ILCs can regulate the homeostasis of NBH cells independently of T cells. In mice, splenic ILCs sustained both IgG3+ plasmablasts and IgG3+ plasma cells, whereas mouse NBH cells predominantly helped IgG3+ plasma cells, suggesting that multiple subsets of innate immune cells orchestrate TI IgG3 responses in a hierarchical manner. Of note, IgG3 may provide protection against certain pathogens following its interaction with ficolins, a family of soluble pattern recognition receptors released by hepatocytes and various cells of the innate immune system[41]. In summary, our studies indicate that splenic ILCs orchestrate a stromal-innate cell network that fosters TI antibody production by MZ B cells. Harnessing this network with adjuvants may enhance protective humoral responses to poorly immunogenic TI antigens[4, 50], including viral glycoproteins and bacterial carbohydrates.

ONLINE METHODS

Human samples

Mononuclear cells and neutrophils were isolated from the peripheral blood of healthy volunteers or histologically normal spleens from deceased organ donors or splenectomized trauma patients without clinical signs of infection or inflammation[10]. Tonsils were from individuals with follicular hyperplasia[10]. The use of blood and tissues was approved by the Ethical Committee for clinical investigation of Institut Hospital del Mar d'Investigacions Mèdiques (CEIC-IMIM 2011/4494/I). Tissue samples were collected by the department of Pathology of Hospital del Mar. Prior to collection, signed informed consent was obtained from the patient or his/her parent or guardians. All the blood and tissue samples were coded and relevant clinical information remained anonymous.

Mice

Rorc–/– (Rorcgfp/gfp), Cd3e–/–, Rag1–/–, Rag1–/–Il2rg–/– and Csf2–/– mice on the C57BL/6 background have been described previously[29,51-54]. C57BL/6 CD90.1 mice were purchased from the Jackson Laboratory. All mice were housed in specific pathogen-free conditions. Male and female mice were used at age 8-12 weeks unless specified in the text. All the experiments involving mice were performed in accordance with approved protocols from the Institutional Animal Care at RIKEN and Icahn School of Medicine at Mount Sinai.

Mouse chimeras and neutrophil depletion

4 × 107 bone marrow cells from Rag1–/– mice were mixed with 1 × 107 bone marrow cells from Rorc–/– mice and injected intravenously into 10-Gy irradiated 2-month old Rorc–/– mice. After transplantation, mice were given 500 mg/L ampicillin (Sigma) and 1 g/L neomycin (Sigma) in drinking water for 2 weeks. To deplete neutrophils, control RTK2758 monoclonal antibody (mAb) or neutrophil-depleting 1A8 mAb to Ly6G (BioXCell) were administrated intraperitoneally every 5 days at a dose of 250 μg/mouse for a month. Thy-1 disparate Rag1–/– chimeras were generated and depleted of ILCs with a 30-H12 mAb to Thy-1.2 (BioXCell) injected intraperitoneally every 3 days at a dose of 500 μg/mouse for a month as previously published[43]. LTF-2 mAb was used as control.

Adoptive transfers

2–3 × 104 ILCs from the small intestine of either Csf2+/+ or Csf2–/– mice were adoptively transferred into Rag1–/–Il2rg–/– mice via retro orbital injection as previously described and splenic neutrophils were analyzed 21 days later. 1 × 105 B16 melanoma cells overexpressing GM-CSF (from G. Dranoff) were injected into Csf2–/– mice and splenic neutrophils were analyzed 12–14 days later.

Immunization

Serum TNP-specific antibody titers were determined by ELISA 7 days after intraperitoneal injection of 50 μg TNP-Ficoll.

Cells

Human splenocytes or tonsillar mononuclear cells were obtained from fresh tissue samples as reported in published studies[10]. Human ILCs, NK cells, naïve B cells, MZ B cells, macrophages, T cells, NC cells and NBH cells were sorted by flow cytometry as described previously[10] or reported below. To isolate human MRCs, splenic tissues were enzymatically digested in Hank's balanced salt solution (Lonza) containing 1 mg/ml collagenase-IV (Invitrogen) and 50 ng/ml DNAse-I (50 ng/ml) at 37 °C for 45 min, followed by separation on Ficoll-Hypaque gradient. Splenocytes were resuspended in EGM medium (Lonza) and cultured in 0.1% gelatin (Sigma) pre-coated plate. 72 h later, adherent cells were obtained with Accutase cell detachment solution as instructed by the manufacturer (Millipore). CD45– CD31– adherent cells were sorted by flow cytometry and expanded up to three passages in EGM medium. Murine OP9 and OP9-DLL1 stromal cell lines (from R. Gimeno) were tested for mycoplasma and grown in essential medium-α (Invitrogen) supplemented with 20% Fetalclone I (Hyclone).

Cultures and reagents

Human splenic ILCs (5 × 104/well) were plated in 96 U-bottom plates and cultured in complete RPMI medium with or without 50 ng/mL IL-7, 50 ng/mL IL-1β and/or 50 ng/mL IL-23 (Peprotech). Conditioned medium was obtained by culturing splenic ILCs in complete medium for 24 h. In co-culture experiments, splenic ILCs were first expanded with IL-7 and IL-1β for 5-8 days as previously published. ILCs were co-cultured with confluent human MRCs in flat-bottom plates for 72 h. To avoid cell-to-cell contact, some co-cultures were performed in a 24-transwell system (Corning). Human ILCs were also co-cultured with human MZ B cells, NC cells and/or NBH cells at a 1:10 ratio. Some co-cultures were supplemented with 500 ng/ml BAFF (Alexis), 0.25 μg/ml CpG ODN-2006 (InvivoGen), 30 μg/ml BAFF-R-Ig, 5 μg/ml CD40-Ig, 5 μg/ml, 10 μg/ml neutralizing MAB215 mAb to human GM-CSF, or 10 μg/ml isotype control IgG1 mAb (R&D system). To inhibit NOTCH2, human MZ B cells were incubated with 25, 5 or 2.5 μM N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) (Sigma), which interferes with the cleavage of NOTCH proteins by γ-secretase. In some experiments, human MRCs were stimulated with 100 ng/ml LTα1β2 and 10 ng/ml TNF (R&D Systems) or with ILCs in the presence or absence of 10 μg/ml mAbs MAB2101 to TNF or MAB1370 to LTα2β1/α1β2 (R&D Systems). Human NC cells were stimulated with 50 ng/ml GM-CSF (Peprotech) or 100 ng/ml LPS (InvivoGen).

Flow cytometry

Cells were incubated with Fc blocking reagent (Miltenyi Biotec) at 4 °C before adding appropriate cocktails of fluorochrome-labeled mAbs (). Dead cells were excluded with 4’-6-diamidine-2’-phenylindole (DAPI) as indicated by the manufacturer (Boehringer Mannheim). To stain intracellular transcription factors, human ILCs or NK cells were stained with mAbs to cell-specific surface molecules, permeabilized, fixed with a Transcription Factor Buffer Set as indicated by the manufacturer (eBioscience), and labeled with mAbs to human RORγt or T-bet (). For intracellular cytokine staining, human ILCs were cultured for 4 h in complete RPMI medium with GolgiStop (BD Pharmingen) in the presence or absence of 10−7 M phorbol myristate acetate and 0.5 μg/ml ionomycin (Sigma). These cells were stained with mAbs to specific surface molecules, fixed, permeabilized using the Cytofix/Cytoperm kit (BD Pharmingen), and finally incubated with mAbs to human GM-CSF or IL-8 (). To stain intracellular IgG3, mouse splenocytes were stained with mAbs to specific surface molecules, permeabilized, fixed with the Cytofix/Cytoperm kit (BD Pharmingen), and labeled with R40-82 mAb to mouse IgG3 (). Cells were acquired using FACS calibur, FACS Aria II, LSRII or LSRFortessa (BD Biosciences) and further analyzed using the FlowJo software (Tree Star).

FACSorting

Human CD3–CD14–CD19–CD117+CD127+ ILCs, CD3–CD14–CD19–CD117–CD127– CD56+ NK cells, CD19+IgDhiCD27– naïve B cells, CD19+IgDloCD27+ MZ B cells, CD14+ macrophages and CD3+ T cells as well as mouse CD3+ T cells, B220+ B cells, CD11c+ DCs, CD11b+ macrophages, CD49b+ NK cells, Ly6G+ neutrophils, CD4+CD45+CD117+CD127+Lin– (CD3–B220–CD11b–CD11c– Ly6G–) ILCs and CD4–CD45+CD117+CD127+Lin– ILCs were stained with appropriate cocktails of fluorochrome-labeled mAbs () and sorted using FACSAria II (BD Biosciences) after exclusion of dead cells through DAPI staining. The purity of FACSorted cells was consistently > 95%.

Viability and proliferation assays

Cell survival was measured using the Annexin-V Apoptosis Detection Kit II (BD Pharmingen). Gates and quadrants were drawn to give ≤ 1% total positive cells in samples incubated with isotype control mAbs. Cell proliferation was assessed by CFSE staining using the CellTrace CFSE Cell Proliferation Kit (Invitrogen).

Laser capture microdissection

Microdissections were perfomed on fresh human splenic tissues frozen in optimal cutting temperature (OCT) compound using the ArcturusXT Laser Capture Microdissection System following the manufacturer's instructions (Life Technologies). In brief, 10-μm tissue slices were isolated on membrane slides (Life Technologies) and fixed in cold acetone for 10 s. Immunohistochemistry with specific mAbs was followed by tissue dehydration and fixation with xylene. Groups of cells from completely dried tissues were acquired by microdissection and RNA was extracted using the Arcturus PicoPure RNA Extraction Kit (Life Technologies).

Immunofluorescence analysis

Frozen tissues and cells from humans or mice were fixed as previously published[10] and stained with various combinations of Abs (). Biotinylated Abs were detected using horseradish peroxydase (HRP)-conjugated streptavidin followed by tetramethylrhodamine from a Tyramide Signal Amplification Kit (PerkinElmer Life Sciences) or streptavidin-Alexa Fluor conjugates. Nuclear DNA was stained with DAPI. Coverslips were applied with FluorSave reagent (Calbiochem). Images were obtained using Axioplan2 (Carl Zeiss) microscopy and further analyzed using Axiovision (Carl Zeiss) software. To perform NET formation assays, human circulating neutrophils were seeded on poly D-lysin coated glass cover slips and stimulated for 3 h with either human GM-CSF or human splenic ILC-conditioned medium. Cells were fixed with 4% paraformaldehyde and stained with mAbs NP57 or ab21595 to human elastase and DAPI.

Immunohistochemistry

5-μm thick formalin-fixed and paraffin-embedded tissue sections were stained with various mAbs () using EnVision™ + Dual Link System-HRP (DAB+) for single stainings and EnVision G/2 Doublestain System Rabbit/Mouse (DAB+/Permanent Red) for double stainings (Dako). Sections were counterstained with hematoxylin.

ELISA

Total human IgM, IgG and IgA were measured as previously published[10]. Soluble human BAFF ELISA Kit (Adipogen), LEGEND MAX Human APRIL/TNFSF13 ELISA Kit (Biolegend), Human GMCSF ELISA Development Kit, and Human IL-8 ELISA Development Kit (Peprotech) were used to measure human BAFF, APRIL, GM-CSF and IL-8. Human Th1/Th2/Th9/Th17/Th22 13plex FlowCytomix Multiplex (eBioscience) was used to measure human IL-17 and IL-22. The concentration of mouse serum IgM, IgG3 and IgA were determined with Mouse ELISA Quantification Set (Bethyl Laboratories). To detect mouse TNP-specific IgG3, ELISA plates coated with 5 μg/ml bovine serum albumin (BSA)-conjugated TNP (BioSearch Technologies) were sequentially incubated with mouse serum and HRP-conjugated goat polyclonal antibody A90-111P to mouse IgG3 (Bethyl Laboratories). To detect mouse phosphorylcholine-specific IgM, ELISA plates coated with 5 μg/ml BSA-conjugated phosphorylcholine were sequentially incubated with serum and an HRP-conjugated goat polyclonal antibody A90-101P to IgM (Bethyl Laboratories). BSA-conjugated phosphorylcholine was synthesized as follows. 1 mg of BSA was coupled to 1 mg p-aminophenylphosphorylcholine in 0.1 M 2-(N-morpholino)ethanesulfonic buffer (Sigma) at pH 4.5 supplemented with 2 mg 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride in a 1.6 ml final volume. This mixture was incubated for 2 h at 20 °C. Conjugated BSA was dialyzed against PBS at pH 7.4 and 4 °C.

qRT-PCR

To quantify human gene products, total RNA was extracted and reverse transcribed into cDNA as reported in previously published studies[10]. qRT-PCRs were performed as reported previously[10] using specific primer pairs (). To quantify mouse gene products, total RNA was extracted with TRIzol reagent (Gibco BRL). After DNase I (Invitrogen) treatment, random hexamers (Invitrogen) were used for first-strand cDNA synthesis. qRT-PCRs were performed in 96-well plates with a LightCycler 480 real-time PCR instrument (Roche Diagnostics) using the LightCycler 480 SYBR Green I Master kit (Roche Diagnostics) and specific primer pairs (). Gene expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase in each sample.

Statistical analysis

Values were expressed as mean ± standard error of the mean (s.e.m.) or standard deviation (s.d.). Statistical significance was assessed with two-tailed or one-tailed unpaired Student's t-test unless specified otherwise. Mann-Whitney U-test or Wilcoxon matched-pairs signed rank test were performed on non-parametric data. Results were analyzed with Prism software (Graph Pad) and P values less than 0.05 were considered significant. In animal experiments, littermates (minimum 3 mice/group) were randomly distributed to the treatment groups so that all groups were age-matched and sex-matched. No specific randomization or blinding protocol was used and no animals were excluded from the analysis.