Phenotypic and Functional Plasticity of Murine Intestinal NKp46+ Group 3 Innate Lymphoid Cells

Thomas Verrier, Naoko Satoh-Takayama, Nicolas Serafini, Solenne Marie, James P. Di Santo and Christian A. J. Vosshenrich
J Immunol June 1, 2016, 196 (11) 4731-4738; DOI: https://doi.org/10.4049/jimmunol.1502673

Abstract

Group 3 innate lymphoid cells (ILC3) actively participate in mucosal defense and homeostasis through prompt secretion of IL-17A, IL-22, and IFN-γ. Reports identify two ILC3 lineages: a CCR6+T-bet− subset that appears early in embryonic development and promotes lymphoid organogenesis and a CCR6−T-bet+ subset that emerges after microbial colonization and harbors NKp46+ ILC3. We demonstrate that NKp46 expression in the ILC3 subset is highly unstable. Cell fate mapping using Ncr1CreGFP × Rosa26RFP mice revealed the existence of an intestinal RFP+ ILC3 subset (Ncr1FM) lacking NKp46 expression at the transcript and protein levels. Ncr1FM ILC3 produced more IL-22 and were distinguishable from NKp46+ ILC3 by differential CD117, CD49a, DNAX accessory molecule-1, and, surprisingly, CCR6 expression. Ncr1FM ILC3 emerged after birth and persisted in adult mice following broad-spectrum antibiotic treatment. These results identify an unexpected phenotypic instability within NKp46+ ILC3 that suggests a major role for environmental signals in tuning ILC3 functional plasticity.

Introduction

Innate lymphoid cells (ILCs) consist of an evolutionarily conserved and related set of innate immune effectors. Three ILC groups can be distinguished on the basis of their transcription factor profiles and cytokine production (1–3). T-bet–expressing group 1 ILCs (ILC1) are involved in responses against viruses, intracellular pathogens, and tumors and include eomesodermin (Eomes)-dependent cytotoxic ILC1 (NK cells) and Eomes-independent ILC1, both of which produce IFN-γ. Group 2 ILCs participate in protection against helminths and viruses via Gata-3–dependent production of IL-5, IL-13, and amphiregulin, but they may also aggravate allergic responses. Group 3 ILCs (ILC3) are enriched at mucosal sites, drive lymphoid tissue organogenesis during fetal life, and maintain intestinal homeostasis in adults through IL-22 production downstream of the transcription factor retinoic acid–related orphan receptor (ROR)γt. Two ILC3 sublineages have been proposed (4, 5): a CCR6+T-bet− subset that includes lymphoid tissue inducer (LTi) cells and a CCR6−T-bet+ subset that develops postnatally in response to microbial stimulation and gives rise to cells expressing the natural cytotoxic receptor (NCR) NKp46. These two ILC3 subsets share strong functional similarities (6) but occupy distinct niches (7–10). As such, their respective specific versus redundant roles in mucosal defense remain unclear.

Although NKp46 cross-linking triggers cytotoxicity in human and mouse NK cells, the functional role for NCR on ILC3 is poorly understood. Cytokine production in human ILC3 can be stimulated by anti-NKp44 Abs (11), although the ligands that can mimic this activity in vivo remain unknown. In the mouse, Ncr1 ablation (in Ncr1GFP/GFP mice) has little impact on ILC3 homeostasis, and cytokine responses to pathogenic Citrobacter rodentium are unaffected (12). Modulation of NKp46 expression on NK cells occurs during mouse CMV infection (13) and may “tune” ILC3 responses in a fashion similar to that proposed for “licensing” of cytokine and cytotoxic responses of NK cells via NKp46 (14).

To address the dynamics of NKp46 expression in mucosal ILC3, we performed in vivo fate-mapping studies using Ncr1CreGFP mice (15), which harbor a transgene containing the Ncr1 proximal promoter driving expression of a Cre GFP fusion protein (CreGFP). Analysis of Ncr1CreGFP × Rosa26RFP mice (in which NKp46-expressing cells are indelibly marked with RFP expression) revealed the existence of unusual NKp46−GFP−RFP+ cells uniquely within the intestinal mucosa. We report the phenotypic and functional characterization of these cells (referred to in this article as “Ncr1 fate-mapped” ILC3 or Ncr1FM ILC3) and discuss the potential implications for NKp46 plasticity in mucosal immunity.

Materials and Methods

Mice

Tg(Ncr1-cre/EGFP)#Cajv mice (Ncr1CreGFP) were generated and crossed to Rosa26RFP mice, as described (15). Ncr1GFP/+ mice (16) were the kind gift from O. Mandelboim (Hebrew University, Jerusalem, Israel). Unless specified otherwise, adult mice were used between 7 and 13 wk of age. For embryonic day (E)18.5 embryos, mice were cohoused overnight, and female mice with a vaginal plug were separated the next day, with embryos considered as E0.5.

Antibiotic treatment

An antibiotic mixture was used to deplete intestinal commensal microbiota, as previously described (4). Ampicillin (1 g/l), colistin (1 g/l), streptomycin (5 g/l), and sucrose (20%; Sigma-Aldrich) were diluted in drinking water and filter sterilized (0.22 μm). Plugged female mice were treated with antibiotics; the drinking water was changed each week. Four weeks after birth, pups were weaned, and treatment continued until sacrifice 3 wk later.

Isolation of lamina propria lymphocytes

Small intestine lamina propria (SI-LP) were extracted after perfusion of intestines with cold RPMI 1640 medium to remove feces. Peyer’s patches (PPs) were removed, and the intestine was cut open longitudinally and washed again. Intestines were cut into 1-cm pieces and agitated in prewarmed RPMI 1640 medium (Life Technologies) containing 10 mM EDTA (Sigma-Aldrich) at 37°C for 15 min, followed by a 15-min wash with RPMI 1640 alone at 37°C. Pieces of intestine were harvested, cut into small pieces, and digested in RPMI 1640 (Life Technologies) containing 5% FCS and Collagenase VII (0.75 mg/ml; Sigma-Aldrich) (twice for 20 min each). After each extraction, SI-LP were collected, filtered (100 μm), pooled, and centrifuged (300 × g; 10 min). Lymphocytes were enriched by Percoll 80/40% gradient centrifugation. For stimulation, 1–2 × 106 cells were incubated at 37°C with IL-1β (R&D Systems; 100 ng/ml), IL-2 (PeproTech; 100 ng/ml), IL-6 (R&D Systems; 20 ng/ml), and IL-23 (R&D Systems; 50 ng/ml) in RPMI 1640 medium containing 10% FCS, penicillin (Life Technologies), and streptomycin (Life Technologies) for 1 h. PMA (50 ng/ml) and ionomycin (500 ng/ml) were added together with BD GolgiPlug (BD Pharmingen; 555029) for the last 3 h before analysis of intracellular cytokines by FACS.

Flow cytometry and transcriptional analyses

Cell suspensions were incubated for 40 min with cold PBS (Life Technologies) containing 10% FCS, FcR block (2.4G2), a viability dye (eFluor 506; eBioscience #65-0866-14) and fluorochrome-bound Abs. To detect intracellular Ags, cells were fixed for 40 min with 2% paraformaldehyde, followed by a permeabilization/staining step for transcription factors and/or cytokines with the Foxp3 permeabilization kit (eBioscience). The following Abs were used: CD3 (17A2), CD4 (RM4-5), NK1.1 (PK136), CD127 (A7R34), CD117 (2B8), ROR, IL-22 (IL22JOP), NKp46 (29A1.4), CCR6 (29-2L17), T-bet (ebio4B10), MHC class II (M5/114.15.2), CD49a (Ha31/8), Ki67 (SoIA15), DNAX accessory molecule (DNAM; 10E5). All of the samples were acquired on an LSR FORTESSA (BD), and data were analyzed with FlowJo software (TreeStar; version 9.8.5 & 10.0.8). Quantitative RT-PCR was performed, as previously described (7, 10).

Cell culture of ILC3

ILC3 from the SI-LP of Ncr1GFP/+ mice were isolated as CD45intCD90hiNK1.1− and dissected according to GFP, CD49a, and CCR6 expression. Cells were sorted using a FACSAria II (BD) and cultured on OP9 or OP9-DL1 stromal cells (kind gift of A. Cumano, Institut Pasteur) in IMDM (Lonza) containing 10% FCS, 1× penicillin/streptomycin (Life Technologies), and 60 μM 2-ME (Sigma-Aldrich), rIL-7 (20 ng/ml), and rSCF (20 ng/ml). Two and four days later, cells were harvested and analyzed (as stated above) for CD45, CD90, CD49a, CCR6, NKp46, and NK1.1 expression by FACS.

Statistical analysis

Data were compiled and analyzed with GraphPad Prism software (version 6). Paired and unpaired Student t tests were used to determine statistical differences between groups. All graphs show mean + SEM. The p values < 0.05 were considered significant.

Results

In vivo fate mapping of Ncr1+ ILC subsets

Ncr1CreGFP mice express CreGFP under the control of a minimal Ncr1 promoter and were shown to faithfully report Ncr1-directed GFP expression and Cre recombinase activity in known NKp46+ cells, including NK cells, ILC1, and NKp46+ ILC3 (15). We crossed Ncr1CreGFP mice with Rosa26RFP Cre reporter mice to embellish NKp46+ cells with a stable genetic mark (RFP) that would allow us to investigate the stability of NKp46 expression (Supplemental Fig. 1A). In agreement with our previous results (15), RFP+ cells isolated from bone marrow, spleen, lymph nodes, salivary glands, liver, and lung of Ncr1CreGFP × Rosa26RFP mice uniformly expressed cell surface NKp46 and NK1.1+ (Fig. 1A, Supplemental Fig. 1B), thus identifying these RFP+ cells as ILC1.

FIGURE 1.
FIGURE 1.

An unusual “ex-NKp46” ILC3 subset in the small intestine. (A) Analysis of NK1.1, RORγt, and NKp46 expression in RFP+ cells from the indicated organs in Ncr1CreGFP × Rosa26RFP mice. Gating on live CD45+RFP+ cells (left panels). (B) Gating strategy used to identify RFP+NKp46−GFP− ILC3 in SI-LP lymphocytes from Ncr1CreGFP × Rosa26RFP mice. CD4+ LTi cells (CD3−CD127+RORγt+CD4+) were used as a control to set the negative gate (NKp46 versus GFP). (C) Ncr1 mRNA expression levels by sorted intestinal populations: NK1.1+ cells, CD3−CD45.2+NK1.1+NKp46+GFP+RFP+; Ncr1+ ILC3, CD3−NK1.1−NKp46+GFP+RFP+; Ncr1FM ILC3, CD3−NK1.1−NKp46−GFP−RFP+; CD4+ LTi cells, CD3−NK1.1−NKp46−RFP−CD117+CD4+. (D) Representative FACS plot of CD127 versus CD117 in RFP+NK1.1− cells. (E) Expression levels (MFI) of CD127 (left panel) and CD117 (right panel) by the indicated populations, as measured by FACS. (F) Representative FACS plot of NKp46, GFP, RFP, and CCR6 expression in ILC3 (gated on live CD45+CD3−CD127+RORγt+). (G) Distribution of RFP+ among intestinal ILC3 (gated on CD3−CD127+RORγt+) shown as percentages (left panel) and absolute numbers (right panel) (mean + SEM; n = 15). (H) Overlays of the indicated populations. Ncr1+ and Ncr1FM were gated as described in (C). T cells (gray line and shaded) were used as a negative and positive control for intracellular staining of NKp46 and CD3, respectively. ***p < 0.0001, **p < 0.005.

In contrast, RFP+ cells isolated from gut-associated tissues, including SI-LP and PPs, were clearly different, with a fraction of cells lacking cell surface NKp46 and GFP expression (Fig. 1A, Supplemental Fig. 1C). These NKp46−GFP− cells represented ∼30% of the total RFP+ cells within the SI-LPL or PP, were uniformly NK1.1−, and highly expressed RORγt (Fig. 1A, Supplemental Fig. 1C). Because RORγt and Eomes expression in CD3−CD19− intestinal cells are mutually exclusive (Supplemental Fig. 1D), NKp46−GFP−RFP+ cells are not related to Eomes+ ILC1. As such, RFP+NKp46−GFP− cells appeared as “ex-NKp46-ILC3” that had lost NKp46 expression. We purified NKp46−GFP−RFP+ cells and confirmed that they were significantly depleted in mRNA for Ncr1 that encodes NKp46 (Fig. 1C). We refer to the NKp46−GFP−RFP+ cells in Ncr1CreGFP × Rosa26RFP mice as “Ncr1 fate-mapped” ILC3 or Ncr1FM ILC3. Ncr1FM ILC3 were not detected in the large intestine under steady-state conditions (Supplemental Fig. 1C).

Because NKp46 expression levels are lower in ILC3 compared with NK/ILC1, a clear separation of these subsets by Ab staining alone is not possible. Therefore, we examined NKp46 cell surface versus GFP expression in RORγt+ ILC3 within total RFP+ cells (Fig. 1B). This approach allowed us to unambiguously identify Ncr1FM ILC3 as NKp46−GFP−RFP+ cells within the RORγt+ gate. ILC3 subsets coexpress CD117 (c-kit) and CD127 (IL-7Rα), albeit at varying levels (Fig. 1D). For example, previous studies showed that CCR6+ ILC3 were CD117hiCD127hi as opposed to NKp46+CCR6− ILC3, which express lower CD117 and CD127 levels (4). We found that Ncr1FM ILC3 expressed significantly higher levels of CD127 and CD117 compared with NKp46+ ILC3 (Fig. 1E). Further analyses revealed that a fraction of Ncr1FM ILC3 expressed CCR6 (Fig. 1F). On average, we found 4 × 104 Ncr1FM ILC3 in the SI-LP from Ncr1CreGFP × Rosa26RFP mice, of which 33.5 ± 5.7% expressed CCR6 (Fig. 1G). Thus, CCR6+ Ncr1FM ILC3 represented ∼10% of the total CCR6+ ILC3 pool (Fig. 1G). Some of these CCR6+ Ncr1FM ILC3 expressed MHC class II molecules at the cell surface (Supplemental Fig. 2A), but these represented a minute fraction of the total MHC class II+ ILC3 pool. The remaining Ncr1FM ILC3 lacked CCR6 and CD4 expression (Supplemental Fig. 2B) and would be contained within the NKp46−CCR6− ILC3 gate. Within this ILC3 subpopulation, Ncr1FM ILC3 accounted for ∼17% of cells. Thus, a substantial fraction of previously identified NKp46−CCR6− ILC3 are, in fact, Ncr1FM ILC3 that lost NKp46 expression.

Because some T cells express NKp46 (17, 18) or RORγt (19, 20), we assessed whether Ncr1FM ILC3 could represent a peculiar T cell subset that lost surface expression of CD3 and NKp46. However, neither NKp46+RFP+ nor NKp46−RFP+ ILC3 expressed cell surface CD5 or intracellular CD3 protein (Fig. 1H). Importantly, intracellular NKp46 protein was not detected in Ncr1FM ILC3, ruling out that the loss of cell surface NKp46 was due to protein internalization (Fig. 1H).

Taken together, analyses of Ncr1CreGFP × Rosa26RFP mice provide clear evidence for an intestinal ILC3 subset that has modulated NKp46 expression at the protein and transcriptional levels.

Functional capacities of Ncr1FM ILC3

Ncr1FM ILC3 did not stain for Ki67, suggesting that they were primarily resting cells (Fig. 2A, 2B). Given that one major ILC3 function is the production of IL-22, we assessed whether Ncr1FM ILC3 were able to produce this cytokine upon stimulation in vitro. Of note, IL-22 protein was readily detectable in Ncr1+ ILC3 and Ncr1FM ILC3, even without IL-23 stimulation (Fig. 2C); however, steady-state IL-22 production by Ncr1FM ILC3 was significantly greater than that of Ncr1+ ILC3 on a population level, as well as on a per-cell basis (Fig. 2D, 2E). Nevertheless, upon stimulation with IL-23, Ncr1+ ILC3 and Ncr1FM ILC3 produced similar levels of IL-22 (Fig. 2C–E). These results suggest that Ncr1FM ILC3 may have received activating signals in situ; such pathways might also be involved in NKp46 downmodulation, although this remains speculative.

FIGURE 2.
FIGURE 2.

Ncr1FM ILC3 exhibit elevated IL-22 production and reduced T-bet expression. (A) Representative FACS plot of Ki67 versus NKp46 in RFP+NK1.1− cells and CD3+ T cells (control). (B) Statistical evaluation of Ki67 data obtained from analyses as in (A). (C) Representative FACS plot of IL-22 expression by the indicated subsets (among RFP+NK1.1−) after 4 h of stimulation or not with IL-23 (50 ng/ml). (D) Percentages of IL-22+ cells in RFP+NKp46+ (open bar) and RFP+NKp46− (filled bar) (mean + SEM; n = 9). (E) Expression levels (MFI) of intracellular IL-22 protein in unstimulated or IL-23–stimulated cells from the indicated populations. (F) Representative FACS plot of IFN-γ and IL-22 expression after stimulation with various cytokine mixes, as indicated. Cells were incubated for 1 h with IL-1β, IL-2, IL-6, and IL-23, and PMA and ionomycin were added for the last 3 h of stimulation. (G) Overlays of T-bet expression in RFP+NK1.1+ (red), RFP+NKp46+ (gray), RFP+NKp46− (blue), and CD4+ LTi (dark gray). (H) MFI were normalized to RFP+NK1.1+ (mean + SEM; n = 4). **p < 0.01, ***p < 0.001. NS, nonstimulated.

We explored the cytokine-production capacities of Ncr1FM ILC3 using various stimuli. Only NKp46+GFP+RORγt− cells produced IFN-γ upon stimulation with IL-2, IL-12, and IL-18 (Fig. 2F), whereas Ncr1+ ILC3 and Ncr1FM ILC3 produced IL-22 under these conditions (Fig. 2F). Interestingly, approximately 25% of the IL-22–producing Ncr1+ ILC3 and Ncr1FM ILC3 also produced GM-CSF (Supplemental Fig. 3A). The frequencies of IL-22–producing cells and IL-22/GM-CSF–producing cells increased when stimulated with a mixture consisting of IL-1β, IL-2, IL-6, IL-23, and PMA/ionomycin (Fig. 2F, Supplemental Fig. 3A). Under these conditions, approximately half of the IL-22+ Ncr1+ ILC3 produced IFN-γ, whereas Ncr1FM ILC3 did not (Supplemental Fig. 3A). RFP+ cells failed to produce IL-17A after stimulation (Supplemental Fig. 3B). Consistent with their ability to produce IFN-γ, Ncr1+ ILC3 expressed T-bet (Fig. 2G) and require this transcription factor for development (5, 21). In contrast, Ncr1FM ILC3 expressed significantly less T-bet protein compared with NK1.1+ ILC1 or with Ncr1+ ILC3 (Fig. 2G, 2H).

Ontogeny of Ncr1FM ILC3 and the role of microbiota

We next explored at what stage Ncr1FM ILC3 emerged during ontogeny. CCR6+ ILC3 were detected as early as E13.5 (22). However, only few RFP+ cells were found at E18.5 (Fig. 3A), and these cells uniformly expressed NK1.1 (Supplemental Fig. 4A). One week after birth, RFP+NK1.1− cells were apparent; the majority were Ncr1FM ILC3 (Fig. 3B), which indicates that NKp46 expression was initiated, but not maintained, in these RFP+ cells. Interestingly, Ncr1FM ILC3 exhibited higher IL-22 production capacity at this stage, under steady-state conditions and after IL-23 stimulation (Fig. 3C), the latter contrasting with the results obtained in adult mice (Fig. 2C, 2D). We also noted an age-dependent decrease in the frequency of Ncr1FM among total ILC3 (13.2 ± 2.9% and 9.4 ± 2.7% in 1- and 7-wk-old mice, respectively). This might point to a specific function of these cells during the early postnatal period.

FIGURE 3.
FIGURE 3.

Impact of ontogeny and microbiota on Ncr1FM ILC3 generation. (A) Representative FACS plot of CD4, RFP, NKp46, and GFP expression in ILC3 (gated on live CD45+CD3−CD127+RORγt+) from the SI-LP of Ncr1CreGFP × Rosa26RFP mice at the indicated time points. (B) Percentages of Ncr1+ and Ncr1FM ILC3 among NK1.1− cells (mean + SEM; n ≥ 9). (C) Percentages of IL-22+ cells in Ncr1+ (open bar) and Ncr1FM (filled bar) in SI-LP lymphocytes from 1-wk-old mice (mean + SEM; n = 9). (D) Percentages of RORγt+ among CD3+ T cells with or without antibiotic treatment in control (open bars) or antibiotic-treated (filled bars) mice (mean + SD; n = 3). Antibiotics were administered in drinking water to pregnant mice and mice after birth until sacrifice (6–7 wk old). (E) Representative FACS plot of RFP+NK1.1− ILC3, with or without antibiotic treatment. (F) Percentages of Ncr1+ and Ncr1FM ILC3 among NK1.1−RFP+ (E) in control (empty bars) or antibiotic-treated (black bars) mice (mean + SD; n = 3). **p < 0.01, ****p < 0.0001.

IL-23–driven T-bet expression in CCR6− ILC3 was shown to be induced by microbiota, resulting in the generation of NKp46+ ILC3 (5). Because Ncr1+ ILC3 emerged after birth and increased with age (4), we hypothesized that microbiota might also regulate Ncr1FM ILC3 homeostasis. To address this point, we treated Ncr1CreGFP × Rosa26RFP mice (in utero and throughout life until adulthood) with broad-spectrum antibiotics that were shown to largely ablate commensal microbial communities (9). As expected (9), CD3+RORγt+ T cells were decreased after antibiotic treatment (Fig. 3D). In contrast, we did not observe any changes in the proportions of Ncr1+ ILC3 or Ncr1FM ILC3 (Fig. 3E, 3F). These results suggest that antibiotic-sensitive microbiota are not a major driving force in shaping Ncr1FM ILC3 homeostasis in adult mice. Nevertheless, future studies using germ-free Ncr1CreGFP × Rosa26RFP mice should be performed to confirm whether homeostasis of the Ncr1FM ILC3 subset is microbiota independent.

CD49a and CD226 expression by ILC3 subsets

CD49a (encoded by Itga1) is highly expressed by Eomes-independent hepatic ILC1 (DX5−NK1.1+ cells) but is largely absent on Eomes+DX5+ NK cells (23, 24). Subsequently, CD49a was proposed as a surface marker to identify ILC1 in various organs (25). Because ILC1 and NKp46+ ILC3 require T-bet for their development (5, 21, 24, 25), we assessed whether CD49a might also be differentially expressed by intestinal ILC3 subsets. We found that CD49a was uniformly expressed by NKp46+ and a subset of NKp46−CCR6− ILC3 (Fig. 4A). Importantly, CD49a was coexpressed with T-bet in both populations, whereas CCR6 and CD49a expression were mutually exclusive (Fig. 4A), consistent with previous transcriptomic analysis of ILC3 subsets (6). Consequently, all Ncr1+ ILC3 expressed CD49a (Fig. 4B). In contrast, only a fraction of Ncr1FM ILC3 stained positive for this marker (Fig. 4B).

FIGURE 4.
FIGURE 4.

Phenotype and plasticity of Ncr1FM ILC3. (A) Representative FACS plot of CD49a and T-bet expression in the indicated ILC3 subsets from the SI-LP of C57BL/6 mice. (B) Representative FACS plot of CD49a versus CCR6 expression in Ncr1+ (left panel) and Ncr1FM (right panel) from SI-LP lymphocytes of Ncr1CreGFP × Rosa26RFP mice. (C) Representative FACS plot of DNAM-1 versus CD49a and CCR6 expression by cells from the indicated subsets from Ncr1CreGFP × Rosa26RFP mice. (D) CD45intCD90hiNK1.1−CD49a+GFP+ were sorted from the SI-LP of Rag2−/− Ncr1GFP/+ mice and cultured on OP9- or OP9-DL1–expressing stromal cells in the presence (white bar) or absence (black bar) of TGF-β (100 ng/ml) for up to 4 d. MFI of GFP in sorted CD49a+GFP+ cells was assessed by flow cytometry at the indicated time points (mean + SD; n = 3). *p < 0.05, **p < 0.01.

We next examined whether DNAM-1 (or CD226) was expressed by Ncr1FM ILC3. CD226 identifies NK/ILC1 subsets with enhanced IFN-γ production (26). DNAM ligands, CD112 and CD155, are expressed by dendritic cells and other cells (27), and CD226–CD155 interactions were shown to be involved in NK cell–dendritic cell cross-talk (28). CD226 expression varied on ILC3 subsets: it was expressed at high levels by all CCR6− ILC3 (Fig. 4C), whereas the majority of CCR6+ ILC3 expressed intermediate levels, and a small fraction of CCR6+ ILC3 were CD226− (Fig. 4C). We found that Ncr1+ ILC3 expressed high levels of CD226, whereas Ncr1FM ILC3 contained CD226hi and CD226int cells (Fig. 4C). A high level of CD226 expression by Ncr1FM ILC3 was associated with CD49a expression (Fig. 4C), whereas CD49a− Ncr1FM ILC3 expressed intermediate levels of CD226 and harbored the CCR6+ subset (Fig. 4C). Together, these data reveal a cluster of markers (NKp46, CD49a, CD226) that is highly expressed by T-bet+ ILC3 and is downregulated in Ncr1FM ILC3.

Signals that modulate NKp46 expression on intestinal ILC3

Membrane-bound and soluble factors regulate NK cell receptor expression. With regard to NCRs, Notch was proposed as a key positive regulator for mucosal ILC3 in mice (21), whereas TGF-β can modulate human NKp30 expression in vitro (29). NKp46 expression is reduced in cancer patients (30) and during influenza and HIV infection (30–32). To begin to investigate the signals that might be involved in the regulation of NKp46 expression in Ncr1FM ILC3, we used Ncr1GFP/+ (16) mice in which one Ncr1 allele harbors a fluorescent reporter generating high GFP expression by all NKp46+ cells. We then isolated CD49a+GFP+, CD49a+GFP−, and CCR6+ ILC3 subsets and monitored GFP expression after cell culture on OP9 stromal cells that differentially expressed Notch ligands (21). We detected a significant decrease in the GFP expression level (mean fluorescence intensity [MFI]) between days 2 and 4 by CD49a+GFP+ cells cultured on OP9 cells, whereas GFP expression remained constant on OP9-DL1 cells (Fig. 4D). The addition of TGF-β accelerated GFP downregulation in CD49a+GFP+ cells cultured on OP9, but not on OP9-DL1, cells (Fig. 4D). In contrast, GFP expression was not induced in cultured CCR6+ or CD49a+ GFP− ILC3, and no changes in CD49a or CCR6 expression were observed under any conditions (Supplemental Fig. 4B). These results suggest that TGF-β– and Notch-derived signals regulate NKp46 expression in cultured Ncr1+ ILC3 and may be involved in the maintenance of NKp46 expression on Ncr1+ ILC3 or the generation of Ncr1FM ILC3.

Discussion

Using in vivo fate mapping, we identified an unusual subset of intestinal ILC3 that lost previous NKp46 expression; these “ex-NKp46 ILC3” are denoted Ncr1FM ILC3. As a result, a fraction of CD4−NKp46− ILC3 are derived from Ncr1+ ILC3 and demonstrate that NKp46+ ILC3 is not a stable or determinant phenotype. Our results also indicate that the CD4−NKp46− ILC3 subset is heterogeneous and show that at least two pathways are involved in the generation of this population.

Interestingly, a small subset of Ncr1FM ILC3 expresses CCR6, thus resembling adult CD4+ ILC3 (LTi-like cells). It is a not clear whether CCR6+ ILC3 represent a stable lineage (33). Constantinides et al. (33) identified early ILC precursors using Zbtb16CreGFP mice and, through fate mapping, showed that, although most NKp46+ ILC3 were derived from Zbtb16-expressing precursors, a minor fraction of CCR6+ ILC3 was also fate-mapped. Conceivably, the small subset of CCR6+ Ncr1FM ILC3 that we identified might correspond to (or be contained within) the CCR6+ Zbtb16-derived ILC3 population (33). Because NKp46+ ILC3 are CCR6−, this receptor appears to be induced on a fraction of Ncr1FM ILC3, possibly in response to environmental cues. As such, the generation of CCR6+ Ncr1FM ILC3 may allow for a specific postnatal positioning of these IL-22–producing ILC3 cells within the intestine.

We found that CD49a and CD226 are differentially expressed on intestinal ILC3. Ncr1+ ILC3 express both markers, whereas Ncr1FM ILC3 have reduced levels. Because CCR6 was exclusively expressed by Ncr1FM ILC3, we propose the following differentiation sequence: RFP+NKp46+CD49a+CD226hiCCR6− → RFP+NKp46−CD49a+CD226hiCCR6− → RFP+NKp46−CD49a−CD226intCCR6+. The change in CD49a and CCR6 expression might allow for a differential localization of Ncr1+ and Ncr1FM ILC3 within the tissue, enabling them to exert their function in the correct cellular context. Indeed, Ncr1FM ILC3 displayed spontaneous production of IL-22, suggesting that they may have received stimulatory signals in situ. Further studies on the localization of Ncr1+ and Ncr1FM ILC3 should provide important insights into the specific roles for these ILC subsets in immune homeostasis.

Previous work revealed that, under the influence of environmental cues, ILC3 acquire the expression of T-bet and the concomitant ability to produce IFN-γ (34). T-bet upregulation is associated with the loss of RORγt expression and IL-22–production capabilities, allowing ILC3 to acquire an ILC1-like phenotype (“ex-ILC3” ILC1). In contrast, Ncr1FM ILC3 do not show signs of ILC1-like plasticity. They retain characteristics of ILC3 (RORγt+, IL-22 production) in naive mice and when stimulated in vitro. Furthermore, Ncr1FM ILC3 expressed lower levels of T-bet than did Ncr1+ ILC3 and were unable to produce IFN-γ. However, similar to Ncr1+ ILC3, they can produce GM-CSF. Whether Ncr1FM ILC3 show additional plastic features with resultant loss of RORγt expression (similar to this ILC3 to ILC1 differentiation) is not known. Of note, our analyses demonstrated that all RFP+ ILC1 in Ncr1CreGFP × Rosa26RFP mice coexpress NK1.1 and NKp46. Thus, if such a plasticity (Ncr1FM ILC3 to ILC1) occurs, it would involve concomitant upregulation of NKp46 and NK1.1 by those cells, rendering them indistinguishable from “normal” ILC1.

Taken together, NKp46 modulation in ILC3 likely reflects an intrinsic program influenced by environmental cues. AhR activation by dietary ligands may explain higher CD117 and IL-22 expression in Ncr1FM (35), whereas cellular partners that emerge after birth, such as monocyte-derived CX3CR1+ macrophages, may stabilize NKp46 expression by providing Notch ligands (36). To balance their function, TGF-β might dampen an ILC3 proinflammatory program that is dependent on NKp46 expression. Remarkably, NKp46+ ILC3 are the only immune cell to express Gata-3, RORγt, and T-bet simultaneously. The interplay among these transcription factors in ILC3 differentiation is complex, and NKp46 expression appears to be tightly controlled by Gata-3 and T-bet (37). As such, relative amounts of these transcription factors, balanced by extrinsic signals, might finely tune the emergence and the function of Ncr1+ ILC3 and their transition to Ncr1FM ILC3.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Ana Cumano for the gift of OP9 stromal cells, Ofer Mandelboim for Ncr1GFP mice, and Hans-Jorg Fehling for Rosa26RFP mice.

Footnotes

  • This work was supported by grants from the Institut Pasteur, INSERM, and the Ligue Nationale contre le Cancer as an Equipe Labelisée. T.V. was supported by a Ph.D. training grant from the French government and the Ligue Nationale contre le Cancer.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    CreGFP
    Cre GFP fusion protein
    DNAM
    DNAX accessory molecule
    E
    embryonic day
    Eomes
    eomesodermin
    ILC
    innate lymphoid cell
    ILC1
    group 1 ILC
    ILC3
    group 3 ILC
    LTi
    lymphoid tissue inducer
    MFI
    mean fluorescence intensity
    NCR
    natural cytotoxicity receptor
    PP
    Peyer’s patch
    ROR
    retinoic acid–related orphan receptor
    SI-LP
    small intestine lamina propria.

  • Received January 4, 2016.
  • Accepted March 21, 2016.

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