c-Maf restrains T-bet-driven programming of CCR6-negative group 3 innate lymphoid cells

RORγt+ group 3 innate lymphoid cells (ILC3s) maintain intestinal homeostasis through secretion of type 3 cytokines such as interleukin (IL)−17 and IL-22. However, CCR6- ILC3s additionally co-express T-bet allowing for the acquisition of type 1 effector functions. While T-bet controls the type 1 programming of ILC3s, the molecular mechanisms governing T-bet are undefined. Here, we identify c-Maf as a crucial negative regulator of murine T-bet+ CCR6- ILC3s. Phenotypic and transcriptomic profiling of c-Maf-deficient CCR6- ILC3s revealed a hyper type 1 differentiation status, characterized by overexpression of ILC1/NK cell-related genes and downregulation of type 3 signature genes. On the molecular level, c-Maf directly restrained T-bet expression. Conversely, c-Maf expression was dependent on T-bet and regulated by IL-1β, IL-18 and Notch signals. Thus, we define c-Maf as a crucial cell-intrinsic brake in the type 1 effector acquisition which forms a negative feedback loop with T-bet to preserve the identity of CCR6- ILC3s.

The transcription factor (TF) RORgt is strictly required for the development of all ILC3s, as mice deficient for RORgt lack all ILC3 subsets Sanos et al., 2009). RORgt also controls the functionality of ILC3s by regulating the production of effector cytokines, such as IL-17 and IL-22 (Ivanov et al., 2006;Rutz et al., 2013). Interestingly, CCR6 -ILC3 co-express RORgt and the master regulator of type 1 immunity, T-bet. T-bet is key to the differentiation of NKp46 + CCR6 -ILC3s, as those cells fail to develop in mice lacking the gene encoding T-bet, Tbx21 (Klose et al., 2013;Rankin et al., 2013). Importantly, T-bet not only contributes to NKp46 + CCR6 -ILC3 development, but an increasing T-bet gradient enables functional plasticity of NKp46 + CCR6 -ILC3s by instructing a type 1 effector program in ILC3s (Klose et al., 2013;Sciumé et al., 2012;Klose et al., 2014;Cella et al., 2019). Tunable T-bet expression in NKp46 + CCR6 -ILC3s serves as a dynamic molecular switch from a type 3 to a type 1 phenotype (Klose et al., 2013). Once T-bet expression reaches a sufficient level, it can also act as a repressor of RORgt, resulting eventually in a full conversion of ILC3s to ILC1-like cells (referred to as ILC3-to-1 plasticity) (Vonarbourg et al., 2010;Cella et al., 2019;Bernink et al., 2015). Thus, the balance between RORgt versus T-bet expression dictates the fate and function of CCR6 -ILC3s (Fang and Zhu, 2017).
Here, we demonstrate that c-Maf was essential for CCR6 -ILC3s to establish a physiological equilibrium between type 1 and type 3 effector states. c-Maf directly restrained T-bet expression, thereby preventing CCR6 -ILC3s from acquiring excessive type 1 effector functions. c-Maf expression itself was dependent on T-bet and tightly correlated with its expression level. Upstream, we identified IL-1ß-and IL-18-mediated NF-kB, as well as Notch signals, as potent extrinsic enhancers of c-Maf expression in CCR6 -ILC3s. Thus, our data define c-Maf as an integral regulator within the type 3-to-1 conversion program that acts as a cell-intrinsic gatekeeper of T-bet expression to maintain the function and lineage-stability of CCR6 -ILC3s.
Results and discussion c-Maf specifically preserves the type 3 identity of CCR6 -ILC3s Given the pivotal role of c-Maf in CD4 + T cells, we aimed to define its function in ILCs, which share a similar transcriptional program with T cells (Vivier et al., 2018). We first investigated the expression pattern of c-Maf in different ILC subsets of the small intestinal lamina propria (siLP) by staining for c-Maf. This analysis showed that ILC3s expressed higher levels of c-Maf when compared to ILC1s or ILC2s ( Figure 1A, gating strategy see Figure 1-figure supplement 1). Among the ILC3 subsets, c-Maf was particularly highly expressed by NKp46 + CCR6 -ILC3s at levels comparable to RORgt + CD4 + T cells ( Figure 1B). Collectively, these data suggested a potential function of c-Maf in these cells.
In order to directly study the role of c-Maf in ILC3s, we crossed mice carrying floxed Maf alleles (Maf fl/fl ) to mice expressing Cre recombinase driven by the regulatory elements of the Rorc(gt) gene locus (Rorc-Cre Tg ), thereby generating mice with a specific deletion of c-Maf in RORgt + ILC3s and T cells (Rorc Cre Maf fl/fl ). Phenotypic analysis of siLP ILCs revealed decreased frequencies of ILC3s in the absence of c-Maf, whereas frequencies and total numbers of ILC1s and ILC2s were increased in Rorc-Cre Maf fl/fl mice when compared to littermate controls ( Figure 1C). Among ILC3s, we detected a selective loss of NKp46 -CCR6 -ILC3s in Rorc Cre Maf fl/fl mice, while total numbers of NKp46 + CCR6 -ILC3s and CCR6 + ILC3s were not significantly changed ( Figure 1D).
Thus, c-Maf was selectively required for the maintenance of intestinal NKp46 -CCR6 -ILC3s. Its absence in ILC3s resulted in significant changes in the proportions of individual ILC subsets in the gut. Importantly, flow cytometric intracellular staining also revealed a significant downregulation of  RORgt protein levels in c-Maf-deficient NKp46 + and NKp46 -CCR6 -ILC3s, which was not observed in CCR6 + ILC3s ( Figure 1E). Next, we tested the functionality of c-Maf-deficient ILC3s by assessing their capacity to produce the type 3 signature cytokines IL-17A and IL-22. In line with the decrease in RORgt expression, NKp46 + and NKp46 -CCR6 -ILC3s from Rorc Cre Maf fl/fl mice exhibited significantly reduced frequencies of IL-17A and IL-22 producers after ex vivo restimulation as compared to control cells ( Figure 1F and G). Again, no differences were detected in CCR6 + ILC3s, corroborating the selective role of c-Maf for the homeostasis and function of CCR6 -ILC3s. Notably, c-Maf did not act as a repressor of IL-22 production in ILC3s, as it was shown for Th17 cells (Rutz et al., 2011), indicating essential differences in c-Maf function between ILCs and T cells.
In summary, these findings demonstrated a crucial requirement of c-Maf in maintaining the type 3 identity of CCR6 -ILC3s, including their expression of RORgt, IL-17A and IL-22.
Interestingly, c-Maf expression strongly correlated with T-bet expression in both NKp46 + and NKp46 -CCR6 -ILC3s ( Figure 2A). More importantly, c-Maf deficiency resulted in strong upregulation of T-bet and NKp46 expression, both on the population (frequencies) and at single cell level (gMFI), in CCR6 -ILC3s ( Figure 2B). These data, together with the selective loss of NKp46 -CCR6 -ILC3s, which are considered to contain precursors of NKp46 + CCR6 -ILC3s, in Rorc Cre Maf fl/fl mice ( Figure 1D), suggested an amplified type 1 conversion of CCR6 -ILC3s in the absence of c-Maf.
In accordance with the increase in T-bet expression, we also detected increased frequencies of IFN-g producing cells within c-Maf-deficient CCR6 -ILC3s as compared to c-Maf-competent control cells ( Figure 2C). Of note, Ki67 staining of c-Maf-deficient CCR6 -ILC3s was not altered, ruling out that proliferative differences facilitated the skewing towards a type 1 phenotype in the absence of c-Maf (Figure 2-figure supplement 1A).
In addition to the lack of c-Maf expression in ILC3s, Rorc Cre Maf fl/fl mice also harbour a c-Maf-deficient T cell compartment, due to the expression of RORgt during T cell development (Sun et al., 2000). Moreover, conditional deletion of c-Maf in T cells (Cd4 Cre Maf fl/fl or Foxp3 Cre Maf fl/fl ) was reported to cause disturbances in intestinal homeostasis (Neumann et al., 2019;Imbratta et al., 2019), raising the possibility that changes in the gut microenvironment contributed to the 'hyper type 1' phenotype of CCR6 -ILC3s in Rorc Cre Maf fl/fl mice. To interrogate this scenario, we analysed intestinal CCR6 -ILC3s from Cd4 Cre Maf fl/fl mice. The expression of T-bet by CCR6 -ILC3s was not altered when c-Maf was selectively deleted in T cells, excluding that T cell-dependent alterations affected the type 1 conversion of ILC3s in Rorc Cre Maf fl/fl mice ( Figure 2-figure supplement 1B).
To better understand the global c-Maf-dependent changes in gene expression programs, we performed gene set enrichment analysis (GSEA). Given the strong upregulation of type 1 and NK cell features in c-Maf-deficient CCR6 -ILC3s, we made use of published RNA-seq data comparing ILC1s and NK cells with CCR6 -ILC3s (Pokrovskii et al., 2019). In detail, we created sets of genes that were most highly overexpressed in ILC1s or NK cells, thereby defining ILC1 and NK cell gene signatures that distinctly separated those lineages from CCR6 -ILC3s. Importantly, when applied to GSEA, both gene signatures were significantly enriched in c-Maf-deficient NKp46 + CCR6 -ILC3s ( Figure 2F), indicating a global shift in gene expression towards an ILC1/NK cell phenotype.
In summary, these data demonstrated that c-Maf was essential to globally balance type 1 and type 3 gene programs within CCR6 -ILC3s. This function of c-Maf was specific to NKp46 + CCR6 -ILC3s as opposed to CCR6 + ILC3s, most likely due to the particularly high c-Maf expression and the selective accessible of type 1 gene loci in these cells (Pokrovskii et al., 2019;Shih et al., 2016). In the absence of c-Maf, NKp46 + CCR6 -ILC3s downregulated type 3 effector genes, while overexpressing numerous genes encoding for type 1 and cytotoxic effector molecules. The latter finding is particularly interesting, since NKp46 + CCR6 -ILC3s are largely considered to be non-toxic cells (Melo-Gonzalez and Hepworth, 2017). Nevertheless, NKp46 + CCR6 -ILC3s share a considerable transcriptional overlap with ILC1s, which also exhibit a degree of cytotoxic capacity Cortez and Colonna, 2016). Thus, c-Maf-deficiency may result in marked cytotoxicity of NKp46 + CCR6 -ILC3s. More work is needed to precisely define the role of c-Maf for ILC3 functionality during homeostasis and in the context of intestinal inflammation.

Figure 2 continued
Recipient mice were CD90.1 positive. Representative flow cytometric profiles of T-bet vs. CCR6 expression are shown left; quantification on the right (n = 7, mean ± SEM, **p<0.01). All statistical differences were tested using an unpaired Students' t-test (two-tailed). (E) NKp46 + CCR6 -ILC3s were sorted from siLP of Rorc Cre Maf fl/fl and control mice and subjected to RNA sequencing. Vulcano plot showing comparison of gene expression between c-Maf-deficient and control NKp46 + CCR6 -ILC3s. Data represent the combined analysis of three biologically independent samples. Genes considered significant (FC > 1.5, FDR < 0.05) fall into the grey background, while selected genes are highlighted in red. (F) Gene set enrichment plots showing enrichment of ILC1 and NK cell signature genes in c-Maf-deficient vs. control NKp46 + CCR6 -ILC3s (FDR < 0.01). Normalized enrichment score (NES). The online version of this article includes the following figure supplement(s) for figure 2:  c-Maf restrains T-bet expression by directly repressing the Tbx21 promoter The strong upregulation of T-bet expression in c-Maf-deficient CCR6 -ILC3s raised the possibility that c-Maf acted as a direct repressor of T-bet, thereby restraining the type 1 differentiation program. Indeed, in silico analysis identified Maf response elements (MARE) within the Tbx21 promoter, as well as in a conserved distant Tbx21 enhancer (Kataoka et al., 1994;Yang et al., 2007), both regions accessible in NKp46 + CCR6 -ILC3s as evidenced by ATAC-sequencing (Shih et al., 2016; Figure 3A and B).
In order to study the transcriptional activity of c-Maf at these sites, we cloned the Tbx21 promoter alone or in conjunction with the Tbx21 enhancer upstream of a luciferase reporter ( Figure 3C). Indeed, the Tbx21 promoter showed strong transcriptional activity when compared to a promoterless control vector ( Figure 3D). The Tbx21 enhancer further increased this activity when cloned upstream of the Tbx21 promoter ( Figure 3D). Importantly, upon exogenous overexpression of c-Maf, we detected a strong de-repression of the reporter signal when we mutated the MARE sites within the Tbx21 promoter ( Figure 3B and E). Notably, mutating the Tbx21 enhancer did not result in further increase of reporter activity, indicating that c-Maf facilitated its suppressive function mainly by acting on the Tbx21 promoter ( Figure 3B and E).
Thus, these data identified the Tbx21 promoter as a c-Maf-responsive region through which c-Maf directly controls T-bet expression.

c-Maf expression in CCR6 -ILC3s is dependent on T-bet
Despite the fact that c-Maf acted as a direct repressor of T-bet, we observed strong correlation of c-Maf expression with T-bet expression in CCR6 -ILC3s ( Figure 2A). This finding let us hypothesize that c-Maf expression was co-regulated with T-bet expression as part of the type one conversion program. To explore this hypothesis, we studied c-Maf expression in CCR6 -ILC3s from T-bet-deficient mice. Indeed, in the absence of T-bet, c-Maf expression was strongly reduced when compared to T-bet-sufficient cells ( Figure 3F), indicating that T-bet positively regulated its own repressor to establish an equilibrated state within the ILC3-to-ILC1 continuum.
In silico analysis of the Maf locus using ATAC-Seq (Shih et al., 2016) and
Since both IL-1ß and IL-18 signal via NF-kb and a conserved NF-kb binding site is present within CNS-0.5 upstream of Maf (Figure 3-figure supplement 1C and D), we hypothesized that NF-kb is involved in regulation of c-Maf expression downstream of IL-1ß/IL-18. Indeed, pharmacological inhibition of NF-kb signalling completely abrogated the cytokine-mediated induction of c-Maf expression ( Figure 3H). In addition to IL-1ß/IL-18, we found that IL-12 suppressed Maf expression ( Figure 3G). However, IL-12 did not interfere with the IL-1ß/IL-18-mediated c-Maf induction nor was c-Maf expression altered in NKp46 + CCR6 -ILC3s from Il12a -/mice as compared to controls ( (Shih et al., 2016). The Tbx21 promoter and enhancer regions are highlighted in red (Yang et al., 2007). ATAC sequencing tracks were visualized using the WashU browser from the Cistrome project (Mei et al., 2017). Of note, the cytokine-mediated induction of Maf expression was accompanied by a concomitant decrease in Tbx21 expression (Figure 3-figure supplement 2D). Yet, this suppression was independent of NF-kb and c-Maf, since Tbx21 expression was similarly suppressed by IL-1ß/IL-18 upon NF-kb inhibiton and in c-Maf-deficient NKp46 + CCR6 -ILC3s (Figure 3-figure supplement 2E and F). Taken together, these data demonstrate that, besides their reciprocal regulation, c-Maf and T-bet expression level are further critically controlled by cytokine signals.
In addition to cytokines, we also tested Notch signals as a potential extrinsic cue controlling both type 1 conversion of and c-Maf expression by ILC3s. Indeed, Notch was shown to be necessary for the induction and maintenance of T-bet and NKp46 expression in CCR6 -ILC3s (Rankin et al., 2013;Viant et al., 2016). Similarly, we could recently identify Notch signalling as a potent inducer of c-Maf expression in T cells (Neumann et al., 2019;Neumann et al., 2014). To test the role of Notch we cultured purified NKp46 + CCR6 -ILC3s on OP9 or OP9-DLL1 stromal cells, the latter ectopically express the Notch ligand Delta-like 1 (DLL1). As reported earlier, Notch signals were essential to drive type 1 conversion of NKp46 + CCR6 -ILC3s, as evidenced by reduction and loss of NKp46 expression in the absence of Notch ( Figure 3I). Importantly, c-Maf showed a similar expression pattern, being significantly reduced in NKp46 + CCR6 -ILC3s cultured on OP9 cells as compared to cells cultured on OP9-DLL1 cells ( Figure 3J). This downregulation of NKp46 and c-Maf expression in the absence of continuous Notch signalling was independent of potential survival promoting effects of Notch (Figure 3-figure supplement 3), establishing a molecular link between Notch signalling and type 1 conversion of NKp46 + CCR6 -ILC3s.

Concluding remarks
Collectively, our study adds c-Maf as a novel key factor to the complex transcriptional network that governs the differentiation and function of ILC3s. In line with the emerging concept that co-expression and cross-regulation of multiple master regulators determines the fate and function of ILCs (Fang and Zhu, 2017), we have uncovered an essential negative feedback loop between c-Maf and T-bet, which restrains the type 1 conversion of ILC3s. Given the antagonism between T-bet and RORgt, the c-Maf-dependent suppression of T-bet also indirectly stabilizes RORgt expression, thus preserving the type 3 identity of ILC3s. In addition, c-Maf may also directly contribute to RORgt expression in ILC3s, as it has been reported for T cells (Tanaka et al., 2014;Zuberbuehler et al., 2019). Our data supports and extends the findings of a very recent report that was released after completion of this manuscript (Parker et al., 2020). sequences are shown in lower lines in red. (C) Schematic representation of plasmids containing the Tbx21 promoter/enhancer linked to the firefly luciferase reporter gene (Luc). Blue dots indicate MARE sites within the Tbx21 promoter and enhancer region. (D) Relative luciferase activity (RLU) of different reporter constructs driven by the Tbx21 promoter alone or in combination with an enhancer sequence compared to a promoterless (w/o) control vector (pGL3 basic) (n = 3, mean ± SEM, *p<0.05, ***p<0.001). (E) Analysis of c-Maf-dependent suppression of luciferase activity. The MARE sites within the Tbx21 promoter or the Tbx21 promoter and enhancer were mutated in the Tbx21 enhancer/promoter construct. Comparison of RLU between unmutated and mutated Tbx21 enhancer/promoter constructs upon c-Maf overexpression (n = 3, mean ± SEM, ***p<0.001). All reporter assay data are pooled from three independent experiments. (F) Expression of c-Maf by siLP CCR6 -ILC3s from T-bet-deficient (T-bet -/-) and -sufficient (T-bet +/ + ) mice. Representative flow cytometric profiles are shown on the left; graph on the right shows quantification of c-Maf gMFI (n = 4, mean ± SEM, ***p<0.001). (G-H) Sort-purified siLP NKp46 + CCR6 -ILC3s from Rorc Cre R26 EYFP mice were cultured in vitro for 36 hr in the presence of IL-7/SCF (w/o) or IL-7/SCF plus indicated cytokines. Subsequently, Maf expression was measured by qPCR (n = 4, mean ± SEM, *p<0.05, **p<0.01, ***p<0.001). In one condition the NF-kb inhibitor BMS-345541 (BMS) was added at 1 mM to the culture. Data are pooled from two independent experiments each with two replicate wells. (I) Sort-purified siLP NKp46 + CCR6 -ILC3s from Rorc Cre R26 EYFP mice were cultured in the presence of IL-7/SCF on OP9 or OP9-DLL1 stromal cells as indicated. After 12 days, cells were analysed by flow cytometry for the cell surface expression of NKp46. Representative contour plots are shown on the left (pregated on CCR6 -ILC3s); graph on the right shows quantification of the frequency of NKp46 + cell among CCR6 -ILC3s (n = 10, mean ± SEM, ***p<0.001). Data are pooled from two independent experiments with 4 to 6 replicate wells. (J) Expression of c-Maf by NKp46 + CCR6 -ILC3s cultured on OP9 or OP9-DLL1 cells. Representative histogram is shown left; graph on the right shows quantification of c-Maf gMFI (n = 10, mean ± SEM, ***p<0.001). All statistical differences were tested using an unpaired Students' t-test (two-tailed). The online version of this article includes the following figure supplement(s) for figure 3:

Materials and methods Animals
To generate conditional c-Maf-deficient mice, Rorc Cre mice or Cd4 Cre mice were crossed to Maf flox mice (provided by C. Birchmaier, MDC, Berlin, Germany) (Wende et al., 2012). Cd4 Cre (Lee et al., 2001), Rorc Cre , R26 EYFP (Srinivas et al., 2001) and T-bet-deficient mice (Szabo et al., 2002) were described before. Il12a -/mice were kindly provided by U. Schleicher, Erlangen. All mice were on a C57BL/6 background and bred and maintained under specific pathogen-free conditions at our animal facilities (FEM Charité Berlin, Germany). All animal experiments were in accordance with the ethical standards of the institution or practice at which the studies were conducted and were reviewed and approved by the responsible ethics committees of Germany (LAGeSo Berlin, I C 113 -G0172/14) and Russia.

Antibodies
A list of antibodies used in this study is provided in Supplementary file 3.

Cell isolation from small intestine and flow cytometry
Small intestinal tissue was treated with HBSS buffer (without calcium and magnesium) containing 5 mM EDTA and 10 mM HEPES (pH 7.5) at 37˚C for 30 min to remove epithelial cells, minced and digested in HBSS buffer (with calcium and magnesium) containing 10 mM HEPES, 4% FCS, 0.5 mg/ ml Collagenase D, 0.5 mg/ml DNaseI (Sigma), 0.5 U/ml Dispase (BD) with constantly stirring at 37˚C for 30 min. The supernatant was filtered and the remaining tissue was mashed through a 70 mm mesh. siLP cells were separated using a 40%/80% step-gradient (Percoll solution, GE Healthcare). Flow cytometry was performed according to previously defined guidelines (Cossarizza et al., 2019). In detail, single-cell suspensions were stained with different antibodies (Supplementary file 3). For cytokine analysis, cells were restimulated with PMA (Sigma, 10 ng/ml), ionomycin (Sigma, 1 mg/ml) and IL-23 (50 ng/ml) for 5 hr in TexMACS medium (Miltenyi Biotec) containing 10% FCS. After 1 hr of stimulation, Brefeldin A (Sigma, 5 mg/ml) was added to block cytokine secretion. For intracellular staining of cytokines and transcription factors, cells were first stained for surface markers and dead cells were labeled with Fixable Viability Dye eFluor780 (eBioscience). After that, cells were fixed in Fix/Perm buffer (eBioscience) at 4˚C for 1 hr, followed by permeabilization (eBioscience) at 4˚C for 2 hr in the presence of antibodies. Cells were acquired with a BD LSRFortessa X-20 and analysis was performed with FlowJo (Tree Star) software.

RNA-seq analysis
NKp46 + CCR6 -ILC3s and CCR6 + ILC3s were sorted from the siLP of 8-12 weeks old Rorc Cre Maf fl/fl or littermate Maf fl/fl control mice using a BD FACSAria sorter (sorting strategy Figure 2-figure supplement 2A). RNA was isolated with the RNeasy Micro kit from Qiagen according to the manufacturer's protocol. RNA libraries were prepared using the Smart-Seq V4 Ultra low Input RNA kit (Takara Clontech). Sequencing was performed on an Illumina Nextseq 500 generating 75 bp pairedend reads. Three biological replicates of each subset were sequenced. Raw sequence reads were mapped to the mouse GRCm38/mm10 genome with TopHat2 (Kim et al., 2013) in very-sensitive settings for Bowtie2 (Langmead and Salzberg, 2012). Gene expression was quantified either by HTSeq (Anders et al., 2015) for total RNA or featureCounts (Liao et al., 2014) for mRNA and analyzed using DESeq2 (Love et al., 2014). A cut-off (FC > 1.5 and p-value < 0.05) was applied for calling differentially expressed genes. Furthermore, differentially expressed genes were filtered for 'gene_type = protein_coding' before further analysis.

Gene set enrichment analysis (GSEA)
GSEA was performed using the GSEA tool from the Broad Institute. Gene sets used in this study were generated by taking the top upregulated genes (log 2 FC > 2) from published differential gene expression analysis of RNA-seq data comparing ILC1s or NK cells with CCR6 -ILC3s (Pokrovskii et al., 2019).

Luciferase reporter assay
HEK293T cells were transfected with the pGL3 basic luciferase plasmid (Promega) containing the T-bet promoter alone or the T-bet promoter in combination with an upstream enhancer region (Yang et al., 2007), or the empty pGL3 basic in combination with an internal control pRL-TK Renilla plasmid (Promega). The T-bet enhancer/promoter plasmid was described before (Hosokawa et al., 2013) and kindly provided by H. Hosokawa (Tokai University, Japan). In order to assess gene regulation by c-Maf, putative Maf responsive elements in the promoter and enhancer were mutated using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). In addition to mutated reporter plasmids, cells were co-transfected with c-Maf coding sequence in pMSCV. Luciferase activity was measured on a SpectraMax i33 microplate reader (Molecular Devices) after 24 hr using dual luciferase assay system (Promega). Luciferase activity was determined relative to Renilla.
In vitro culture of NKp46 + CCR6 -ILC3s on OP9-DLL1 cells CD45 + Lineage -(Lineage: anti-CD19, anti-Gr-1, anti-CD3, anti-CD5) RORgt fm+ CD127 + NKp46 + CCR6cells were sort-purified from the siLP of 11-14 week old Rorc Cre R26 EYFP mice. Sorted cells were transferred in complete RPMI medium to OP9 or OP9-DLL1 cells at a density of 10.000 cells/ well and cultured in the presence of IL-7 (20 ng/ml) and SCF (20 ng/ml) for 12 days before flow cytometric analysis. OP9 cells are murine stromal cells derived from OP/OP mice used as feeder cells in lymphocyte differentiation assays. OP9-DLL1 cells are transfected with Notch ligand delta-like-1 (Schmitt and Zúñiga-Pflücker, 2002). Prior to adding isolated lymphocytes, confluent feeder cells were treated with 5 mg/ml Mitomycin C (Sigma) for 3 hr at 37˚C and subsequently seeded on a 96 flat bottom well plate at a density of 50.000 cells/well.

Bone marrow chimeras
Bone marrow cells from wild-type CD45.1 + CD90.2 + C57BL/6 and CD45.2 + CD90.2 + Rorc Cre Maf fl/fl mice were mixed in a 1:1 ratio and intravenously injected into sub-lethally irradiated CD90.1 + wildtype recipient mice. Small and colonic lamina propria of reconstituted mice were analysed 6 weeks after cell transfer. qPCR mRNA for real-time qPCR was isolated with the RNeasy Plus Micro Kit according to the manual of the manufacturer (QIAGEN). Reverse transcription was done with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) as it is described in the manufacturer's protocol. qPCR was performed using a Quant Studio five system (Applied Biosystems) and the SYBR Green PCR Master Mix Kit (Applied Biosystems). The mRNA expression is presented relative to the expression of the housekeeping gene hypoxanthine-guanine phosphoribosyl-transferase (HPRT). Real-time qPCR primer can be found in Supplementary file 4.

Statistical analysis
Data are the mean with SEM and summarize or are representative of independent experiments as specified in the text. Statistical analyses were performed using Prism software (GraphPad) with twotailed unpaired Student's t test (except RNA-seq data). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics
All animal experiments were in accordance with the ethical standards of the institution or practice at which the studies were conducted and were reviewed and approved by the responsible ethics committees of Germany (LAGeSo Berlin, I C 113 -G0172/14) and Russia.

Supplementary files
. Supplementary file 1. Differentially expressed genes between c-Maf-deficient and -sufficient NKp46 + CCR6 -ILC3s. NKp46 + CCR6 -ILC3s were sorted from siLP of Rorc Cre Maf fl/fl and control mice and subjected to RNA sequencing. 941 genes were identified as differentially expressed (FC >1.5, p-value<0.05). Data represent the combined analysis of three biologically independent samples.
. Supplementary file 3. List of antibodies used in this study.
. Supplementary file 4. qPCR Primer used in this study.
. Transparent reporting form

Data availability
Sequencing data supporting the findings of this study have been deposited in the Gene Expression Omnibus (GEO) database under the GEO accession number: RNA-Seq: GSE143867.
The following dataset was generated: Author (