Regulation of NK cell development, maturation, and antitumor responses by the nuclear receptor NR2F6

Woelk, Johannes; Hornsteiner, Florian; Aschauer-Wallner, Stephanie; Stoitzner, Patrizia; Baier, Gottfried; Hermann-Kleiter, Natascha

doi:10.1038/s41419-025-07407-4

Download PDF

Article
Open access
Published: 07 February 2025

Regulation of NK cell development, maturation, and antitumor responses by the nuclear receptor NR2F6

Cell Death & Disease volume 16, Article number: 77 (2025) Cite this article

6765 Accesses
3 Citations
3 Altmetric
Metrics details

Subjects

Abstract

Natural killer (NK) cell development and functionality rely on precise regulation by specific transcription factors (TFs). Our study demonstrates that the nuclear orphan receptor NR2F6 represses the expression of the activating receptor NKp46, an established key player in NK cell-mediated cytotoxicity during infection and tumor rejection. Despite normal NK cell development in the bone marrow, germline Nr2f6-deficient mice exhibit impaired terminal maturation of NK cells in the periphery. Short-term NK cell responses to lipopolysaccharide (LPS) activation, independent of NKp46, are subsequently reduced in Nr2f6-deficient mice. Conventional type 1 dendritic cells (cDC1) and macrophage populations are decreased in spleens of Nr2f6-deficient mice, subsequently, IL-15-dependent NK cell priming is limited. Administration of exogenous IL-15 in vitro and as IL-15 complex in vivo can compensate for these deficits, promoting terminal maturation of NK cells in Nr2f6-deficient mice. Subsequent transcriptome analysis reveals significant changes in gene expression profiles of NK cells from IL-15 complex treated Nr2f6-deficient mice, with notable alterations in essential NK genes such as Klrg1, Prdm1, Stat5a, Zeb2, and Prf1. Consequently, Nr2f6-deficient IL-15 complex-treated NK cells raise enhanced effector responses of IFNγ, Perforin, and Granzyme B upon ex vivo activation. Of importance, Nr2f6-deficient mice are protected against MHC-I negative B16-F10 melanoma lung metastasis formation, especially with IL-15 complex treatment, indicating the potential of NR2F6 to affect NKp46-dependent NK cell-mediated tumor surveillance. The therapeutic targeting of NR2F6 may be a promising strategy for boosting NKp46-dependent NK-cell-mediated tumor surveillance and metastasis.

METTL3-mediated m⁶A RNA methylation promotes the anti-tumour immunity of natural killer cells

Article Open access 17 September 2021

Rapid functional impairment of natural killer cells following tumor entry limits anti-tumor immunity

Article Open access 24 January 2024

NKp46 enhances type 1 innate lymphoid cell proliferation and function and anti-acute myeloid leukemia activity

Article Open access 24 January 2025

Introduction

Natural killer (NK) cells are pivotal in regulating microbial infections, tumor surveillance, allograft rejection, pregnancy, and autoimmune responses [1,2,3]. Their inherent antitumor activity, independent of neo-antigen exposure, makes them crucial in preventing metastasis and a prime target in immuno-oncology therapies [4,5,6,7]. NK cell function is modulated by a diverse array of germline-encoded surface receptors, either activating or inhibiting, facilitating discrimination between “self” and “non-self” [8]. NK cells detect aberrantly expressed stress-induced ligands on unhealthy or major histocompatibility complex (MHC) class I-deficient cells [7, 9, 10].

The natural cytotoxicity receptors (NCRs) were the first identified for their ability to induce NK cell cytotoxicity against tumor cells. Functionally, genetic deficiency of the NCR member NKp46, encoded by Ncr1, leads to impaired clearance of subcutaneous melanoma and T cell lymphoma, whereas transgenic overexpression enhances rejection of metastasis in mice [11,12,13,14,15]. Recently, a fundamental role of NKp46 in the recognition and subsequent killing of ecto-calreticulin positive ER-stressed cells such as senescent or Zika virus (ZIKV)-infected cells, B16 melanoma, and Ras-driven lung carcinoma cells was detected [16]. Mechanistically, NKp46 recognition of the P domain of ecto-CRT triggers NK cell signaling and enhances tumor-infiltrating NK cell degranulation and cytokine secretion, but the upstream regulators of Ncr1 remain undefined [16].

NK cells are potent producers of pro-inflammatory cytokines and chemokines that enhance the recruitment and maturation of dendritic cells (DCs) [1, 17, 18]. In parallel, NK cells acquire their functionality through priming by accessory cells, such as DCs, macrophages, monocytes, or neutrophils, which stimulate the maturation and effector activity of NK cells [2, 19,20,21,22]. The trans-presentation of interleukin-15 (IL-15), critical for the survival and proliferation of NK cells, by the IL-15 receptor α (IL-15Rα) is important as IL-15 enhances NK cell cytotoxicity. Therefore, it is used already in clinical trials, either alone or in combination with other immunotherapies in human cancer patients (https://clinicaltrials.gov/search?cond=Cancer&intr=IL-15) [2, 19,20,21,22,23].

The orphan nuclear receptor subfamily 2, group F, member 6 (NR2F6, EAR2, COUP-TF III) belongs to the nuclear receptor (NR) family, which governs both pro-and anti-inflammatory processes [24, 25]. Network analysis representing TF downstream targets in human NK cells depicts NR2F6 at the core of the BCL11B and RUNX2 gene network [26]. We have been the first to establish an essential and non-redundant functional role of NR2F6 in T lymphocytes as an intracellular immune checkpoint during experimental autoimmunity, bacterial infection, and cancer immune surveillance and metastasis [27,28,29,30,31,32,33]. However, a functional investigation of NR2F6 in NK cells has not yet been performed.

Results

NKp46 expression within peripheral Nr2f6-deficient NK cells is highly enhanced

To explore the NR2F6-regulated transcriptional landscape in NK cells under steady-state conditions, we sorted splenic CD3^-NK1.1⁺NKp46⁺CD49b⁺ NK cells from healthy wild-type and germline Nr2f6-deficient mice and performed RNA-Seq (Fig. 1A, S1A-C). Notably, the expression of several genes within the cluster of splenic NK cell-defining genes [34] in mice displayed enhanced expression in Nr2f6-deficient NK cells. This cluster included the activating receptor Ncr1 (NKp46); the IL-18 coreceptor (Il18rap); the cell membrane proteins, lymphocyte antigen 6 family memberC2 (Ly6c2) and the killer cell lectin-like receptor subfamily A, member 9 (Klra9); as well as the inhibitory receptor, killer cell lectin-like receptor subfamily B, member 1B (Klrb1b) (Fig. 1A, B). Additionally, the expression of two chemokine receptor genes, C-C motif chemokine receptor (Ccr) 2 and Ccr5, were significantly enhanced in Nr2f6-deficient NK cells, along with DENN domain containing 2B (St5) (Fig. 1A, B).

**Fig. 1: Characterization of *Nr2f6*-deficient NK cells via RNA-Seq analysis.**

Simultaneously, we investigated downregulated genes. In parallel to Nr2f6, the expression of various family members of the tripartite motif-containing Trim family (Trim 12a, Trim5, Trim 30 d&b, and Trim34a), as well as Annexin A8 (Anxa8); Sideroflexin 2 (Sfxn2) and galactosidase, beta 1 (Glb1) was reduced (Fig. 1A, B). Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted, yet no upregulated gene sets were identified.

We investigated the protein levels of RNA-Seq candidates, including NKp46 and CCR5, along with other essential NK cell markers such as NK1.1, NKG2D, or DNAM-1 (CD226) via flow cytometry (Fig. S1D). Of note, splenic cell numbers of Nr2f6-deficient mice were generally decreased therefore, a substantial reduction in total cell numbers of NK1.1⁺NKp46⁺, NKG2D⁺, and DNAM-1⁺ NK cells was observed in Nr2f6-deficient mice (Fig. 1C, D, S1E, F). Consistent with our RNA-Seq dataset, the expression levels of NKp46 (determined as mean fluorescent intensity (MFI)) and CCR5 were enhanced, as were the frequencies of CCR5 expressing splenic Nr2f6-deficient NK cells when compared to wild-type controls (Fig. 1E–G).

The frequencies of DNAM-1 expressing splenic Nr2f6-deficient NK cells were slightly enhanced, whereas NKG2D frequencies were marginally lower, but the MFI of NK1.1, DNAM-1, and NKG2D were not altered when compared to wild-type controls (Fig. 1H–J).

Along this line, in the blood, the expression of NKp46 but also the frequencies of DNAM-1⁺ Nr2f6-deficient NK cells were significantly elevated compared to wild-type controls (Fig. 2A, B). To investigate if NKp46 expression is cell-autonomously regulated by NR2F6, wild-type or Nr2f6-deficient splenic NK cells were isolated and cultured in vitro for 7 days in the presence of IL-15. Subsequently, the NK cells were stimulated for 5 hours under different conditions (resting, IL-12 + IL-18, IL-15, B16-melanoma cells). Regardless of the stimuli, Nr2f6-deficient NK cells consistently exhibited elevated NKp46 levels compared to wild-type controls (Fig. 2C).

**Fig. 2: NKp46 expression within *Nr2f6*-deficient NK cells in the blood is highly enhanced.**

To decipher potential NR2F (COUP) DNA binding sites (TGACCT) within the murine Ncr1 promoter, we employed the TRANSFAC database, which identified two putative binding sites at 912 and 154 base pairs upstream of the Ncr1 transcription start site (Fig. 2D) [35]. We performed chromatin immunoprecipitation (ChIP) assays in isolated splenic NK cells, utilizing either anti-NR2F6 or IgG-control antibodies and confirmed functional relevance at the −912 site within the promoter locus (Fig. 2E).

In summary, loss of NR2F6 significantly enhances NKp46 expression at the transcriptional and surface receptor levels in peripheral splenic and blood-derived NK cells. ChIP results provide evidence supporting the regulatory function of NR2F6 in suppressing murine Ncr1 gene expression.

NKp46 expression is enhanced in bone marrow-derived Nr2f6-deficient NK cells

To investigate the onset of NKp46 expression during NK cell development in the bone marrow, together with NK cell precursors, we analyzed progenitor populations in healthy wild-type and Nr2f6-deficient mice. The frequencies and total cell numbers of common lymphoid progenitors (CLP), pre-NK precursor (NKPs), and refined NK precursor (rNKPs) were similar between genotypes (Fig. 3A-C; S2A-C). During NK cell development, immature NK (iNK) cells pass through three developmental stages [36]. Percentages and cell numbers of stages A, B, and C were similar between the genotypes, however, upon reaching stage C, characterized by the upregulation of NKp46, Nr2f6-deficient CD3^-NK1.1⁺ cells exhibited significantly elevated levels of NKp46 expression (Fig. 3B, D, E, S2D). Along this line, it is worth noting that Nr2f6 expression is barely detectable in CLP; but gets upregulated during NK cell development (Fig. 3F) [37]. Further characterization of the maturation of NK (mNK) cells in the BM from immature CD27⁺CD11b^-, CD27⁺CD11b⁺, to mature CD27^-CD11b⁺ did not reveal differences between genotypes (Fig. 3G). Taken together, in the BM, loss of NR2F6 does not alter NK cell progenitor populations or maturation but enhances NKp46 expression upon induction.

**Fig. 3: *Nr2f6*-deficient mice have normal NK cell development in the bone marrow, NKp46 expression is increased.**

The loss of NR2F6 blocks peripheral NK cell maturation and short-term effector responses

Nr2f6 expression is increased in splenic NK cell subsets compared to naïve, effector, and memory CD8 T cell subsets in the spleen, in which the role of Nr2f6 has already been well characterized (Fig. 4A) [27, 29, 32, 37]. Upon analyzing the maturation status of Nr2f6-deficient NK cells in both the spleen and blood, unexpectedly, and in contrast to the bone marrow, we observed an over-representation of immature NK cells, coupled with a significant decrease in the frequencies of mature and terminally matured (CD27^-CD11b⁺KLRG1⁺) NK cell populations compared to wild-type controls (Figs. 4B–D; S3A-C). Along this line the MFI of CD27 was enhanced, whereas the MFI of CD11b was reduced when compared to wild-type controls (Fig. S3D).

**Fig. 4: Terminal NK cell maturation block in *Nr2f6*-deficient mice and reduced NKp46 independent short-term effector responses.**

Next, we aimed to assess Nr2f6-deficient splenic NK cell effector responses in a model independently of NKp46 activation. We employed the in vivo short-term LPS-driven inflammation model established by the group of Granucci (Fig. 4E) [2]. Following LPS injection, DCs are activated and secrete IL-2, IL-18, and IFN-β, which induces NK cell activation. Although IFNγ production in NK cells was robustly induced in both genotypes, the frequencies, cell numbers, and IFNγ expression (MFI) were significantly reduced in Nr2f6-deficient NK1.1⁺NKp46⁺ NK cells when compared to wild-type (Fig. 4E-H; S3E). Notably, the MFI of NKp46 remained elevated in Nr2f6-deficient NK cells (Fig. 4F, H).

Taken together, in contrast to the bone marrow, Nr2f6-deficient peripheral NK cell maturation in the spleen and the blood and short-term effector response are decreased.

A comprised myeloid compartment blocks peripheral NK cell maturation in Nr2f6-deficient mice

NK cells acquire full maturation potential and functionality through priming by accessory myeloid cells such as DCs, macrophages, or monocytes [19,20,21,22]. We characterized the splenic myeloid compartment to pinpoint the eventual NK cell-extrinsic factor missing for priming Nr2f6-deficient NK cells in vivo (Fig. S4A). Our analysis revealed significantly reduced frequencies of the cross-presenting XCR1⁺cDC1s both on a percentage of parent and within living CD45⁺ cells compared to wild-type controls (Fig. 5A–C). Given that NK cell maturation relies on IL-15 cross-presentation by accessory myeloid cells, we further explored the expression (MFI) of IL-15Rα [21, 22]. Our analysis revealed lower IL-15Rα on Nr2f6-deficient DCs than wild-type controls (Fig. 5D).

**Fig. 5: Loss of NR2F6 reduces the splenic cDC1 and macrophage compartment.**

The Nr2f6-deficient CD11b^lo-midF4/80⁺ macrophage compartment was also strongly reduced both in terms of frequencies of the parent population and when calculated per live CD45⁺ cells, but the expression of IL-15Rα on Nr2f6-deficient macrophages was similar to wild-type (Fig. 5E–H).

To investigate the role of IL-15 during NK cell priming in vitro, we cultured isolated splenic NK cells with IL-15 for one week (Fig. 5I). In vitro expanded Nr2f6-deficient NK cells decreased IFNγ and TNFα responses upon stimulation with IL12 + IL-18, but increased following NKp46 activation compared to wild-type controls. Furthermore, when co-cultured with B16 tumor cells, Nr2f6-deficient NK cells exhibited significantly enhanced IFNγ production in percentages and MFI (Fig. 5J–L). Therefore, a comprised splenic myeloid compartment reduces IL-15 priming of NK cells and, subsequently, maturation and effector responses in Nr2f6-deficient mice.

Overcoming IL-15 limitation in vivo enables Nr2f6-deficient NK cells to develop their full maturation potential

We next assess the potential of IL-15-complex (IL-15C) treatment, which is more potent than IL-15 alone, in rescuing Nr2f6-deficient NK cell maturation and effector functions in vivo (Fig. 6A; S5A) [38,39,40]. Following three times IL-15C injections, total splenic and NK cell numbers increased in Nr2f6-deficient mice and were similar to wild-type (Fig. S5B). The frequencies of IL-15C-treated NK cells were especially high in the spleen and blood of Nr2f6-deficient mice and strongly enhanced when compared to IL-15C-treated wild-type controls (Figs. 6B; S5C-D). Of importance, the MFI of NKp46 remained significantly elevated in splenic and blood-derived Nr2f6-deficient NK cells when compared to wild-type (Figs. 6C, D; S5C, E). IL-15C treatment normalized the maturation of Nr2f6-deficient CD11b⁺NK cells in the spleen and blood and significantly increased frequencies of terminally mature CD11b⁺KLRG1⁺ NK cells even surpassing wild-type levels (Fig. 6C, E, F; S5C, F, G).

**Fig. 6: IL-15 complex treatment restores NK maturation.**

We sorted NK cells following IL-15C treatment and performed RNA-Seq analysis (Fig. 7A, S1A, B; S6A). In parallel to the steady state conditions, expression of Ncr1, Ly6c2, Ccr2, St5, and Il18ra were significantly upregulated, whereas Ccr5 expression was reduced in Nr2f6-deficient IL-15C treated NK cells compared to wild-type controls (Fig. 7B). Following IL-15C treatment expression of Fcgr3, Itga2a, Prdm1, and in line with the flow cytometric data, Klrg1 mRNA expression was significantly enhanced in Nr2f6-deficient IL-15C treated NK cells (Fig. 7B). Furthermore, expression of the TFs Stat5a, Zeb2, and the effector molecule perforin (Prf1) was significantly enhanced. In contrast, the TF Eomes and the expression of the inhibitory receptors Lag3 and Tigit were downregulated considerably in Nr2f6-deficient NK cells following IL-15C treatment (Fig. 7B).

**Fig. 7: Characterization of IL-15C treated *Nr2f6*-deficient NK cells via RNA-Seq analysis.**

Furthermore, ex vivo culture of IL-15C treated Nr2f6-deficient splenocytes under resting or cytokine stimulatory conditions (IL-12 + IL-18) significantly increased Nr2f6-deficient NK cell effector functions measured by the percentage of IFNγ, Perforin, and Granzyme B-producing NK cells, compared to wild-type controls (Fig. 7C–E).

Loss of NR2F6 protects against B16-F10-B2m ^−/− melanoma lung metastasis formation

In healthy mice, only a minor fraction of blood-derived soluble IL-15 is complexed, whereas, in inflammatory settings, most of the circulating IL-15 converts to the soluble complex isoform, thus acting as a rapid alert response [40]. Therefore, we suggested that elevated NKp46 expression could still enhance antitumor NK cell responses in Nr2f6-deficient mice during cancer progression. We chose the B16-F10-B2m^−/− (melanoma) tumor metastasis model, which is CD8 T cell-independent, established by the group of Raulet and others [7, 41]. Upon intravenous injection of 2.5×10⁵ B16-F10-B2m^−/− cells, metastasis formation on day 16 was significantly reduced in Nr2f6-deficient mice (Fig. 8A). To rule out that the observed maturation defect in Nr2f6-deficient mice was related to the reduced number of metastasis, we pre-treated Nr2f6-deficient mice with IL-15C one day prior to the injection of B16-F10-B2m^−/− cells and administered IL-15C again on days 2 and 5 post-injection. After 16 days, metastasis formation was significantly reduced in IL-15C-treated Nr2f6-deficient mice compared to Nr2f6-deficient untreated mice, with 8 out of 10 treated mice showing no metastatic foci at all (Fig. 8A).

**Fig. 8: Loss of NR2F6 protects against B16-F10-*B2m*^−/− lung metastasis formation.**

Tumor-infiltrating NK cell frequencies were similar between genotypes, as were frequencies in the blood and spleens of Nr2f6-deficient mice (Fig. 8B). NKp46 levels were consistently enhanced within Nr2f6-deficient NK cells in the tumor, blood and spleen (Fig. 8C). Of importance, whereas frequencies of terminal mature CD27^-CD11b⁺KLRG1⁺ NK cells in the spleen were still reduced in Nr2f6-deficient mice, they were rescued to wild-type controls in the blood and the tumor (Fig. 8D).

Hence, Nr2f6-deficient NK cells exhibit superior functionality compared to their wild-type counterparts in the context of MHC-I deficient lung metastasis rejection.

Discussion

NK cells have emerged as pivotal players in the development of immuno-oncologic treatments. Despite their potential, the full extent of their immunotherapeutic capability has not yet been fully established in the clinic [5, 6, 42,43,44]. In parallel to transcription factors that have been shown recently to regulate antitumor NK cell responses, such as HIF-1α, BACH2, or Zhx2, germline Nr2f6-deficient mice can reject B16-F10-B2m^-/- lung metastasis [43, 45,46,47,48,49]. Mechanistically, we identified NR2F6 as a negative regulator of Ncr1 gene expression. In the absence of NR2F6, bone marrow-derived, peripheral, tumor-infiltrating, and in vitro expanded NK cells express significantly enhanced levels of the activating receptor NKp46. Along this line, blockage of NKp46 leads to the inhibition of NK cell-mediated killing of diverse cancer targets, and in the absence of Ncr1, mice displayed impaired tumor immunosurveillance [15, 50, 51]. In a clinical setting, tumor patients with elevated levels of NKp46 expression demonstrate a significantly improved prognosis compared to their counterparts characterized by low NKp46 levels [52]. Enhanced NK cell-specific NKp46 expression has also been observed in patients diagnosed with the autoimmune disease systemic lupus erythematosus (SLE) [53]. Our previous research has shown that deletion of NR2F6 in mice leads to a SLE-like immunopathological condition, especially in aged animals or following diverse immunization protocols [27, 31]. These findings raise the question of whether the augmented expression of NKp46 on NK cells, in addition to the primarily investigated CD4 follicular T helper and Th17 cell responses, might play a role in the progression of autoimmune diseases such as SLE, in germline Nr2f6-deficient mice.

A comprehensive understanding of how NRs regulate antitumor NK cell-specific responses has only recently begun. Retinoic acid and its receptor (RARα, NR1B1) regulate Granzyme B and NKp46 expression in the human NK-92 cell line [54, 55]. In the context of mouse tumor models, NR4A1 (Nuclear Receptor 4A1) has emerged as a significant player, mediating NK-cell dysfunction in hepatocellular carcinoma through the IFNγ/p-STAT1/IRF1 pathway [56]. Conversely, ROR (RAR-related orphan receptor) α has been identified as an essential factor for the maintenance and antitumor immunity of liver-resident NK cells [57].

In Nr2f6-deficient mice, the numbers of cDC1s and splenic macrophages are reduced. This results in a reduced IL-15 trans-presentation responsible for the maturation defect in NK cells observed under steady-state conditions. NK cell development and maturation in the bone marrow is independent of IL-15 trans-presentation by DCs or macrophages and, therefore, unaltered in Nr2f6-deficient mice [58]. As IL-15C treatment in vivo upregulates the expression of genes known to promote antitumor NK cell responses such as Perf1, Stat5a, Zeb2, Fcgr3, and Itga2a [59,60,61,62,63], while downregulating checkpoint inhibitors like Lag3 and Tigit [64], it is plausible that Nr2f6-deficient NK cells exhibit enhanced efficacy during NK cell-mediated tumor immunosurveillance, due to their intrinsic elevation in Ncr1 expression.

Considering that loss of NR2F6 enhances antitumor responses in T cells, especially during immune-checkpoint inhibition [29, 30], loss of NR2F6 in NK cells may be significant for tumors that lack potent T cell epitopes or have downregulated MHC-I expression as an escape mechanism, offering a potential avenue for a combinatorial therapeutic intervention.

Material and methods

Mice

Nr2f6-deficient mice on the C57BL/6 background have been described previously [29, 31, 32],. Mice were age (8-12 weeks) and sex-matched for individual experiments in a non-randomized manner. Animal procedures were approved by the Austrian Federal Ministry of Education, Science and Research (BMWFW-66.011/0064-WF/V/3b/2016; BMWFW-66.011/0112-WF/V/3b/2017, GZ: 2023-0.315.075, GZ: 2021-0.406.862, GZ: 2023-0.623.434, and GZ: 2024-0.885.804).

B16-F10-B2m^−/− melanoma lung metastasis was performed as described previously [29].

Tissue sampling

Spleens were homogenized through a 100 μm cell strainer in PBS (Sigma Aldrich-Aldrich, P5493-1L) supplemented with 3% FBS, 1% Penicillin/Streptomycin (Sigma-Aldrich, A2213). Blood was collected via the femoral artery during sacrifice or through the mandibular vein. Bone marrow (BM) was extracted from the femur and tibia via centrifugation (3000 g, 30 sec). If necessary, red blood cell lysis was performed using erythrocyte lysis buffer, as described previously [31, 32].

Flow cytometry

Flow cytometric staining and analysis were performed as described previously [31, 32]. The complete list of used antibodies can be found under supplementary methods Table 1.

NK Isolation and in vitro culture

According to the manufacturer’s instructions, NK cells were negatively isolated from total splenocytes using the mouse NK cell isolation kit (Miltenyi Biotec, 130 115 818). 1–2 × 10⁵ NK cells were cultured in 96-well plates in IMDM (Sigma-Aldrich, I3390) supplemented with 10% FBS, 1x non-essential amino-acids, and 50 µM β-mercaptoethanol (Sigma-Aldrich, M3148-25ML) in the presence of 50 ng/ml IL-15 (Biolegend, 566302), or 50 ng/ml IL-15 and 25 ng/ml IL-18 (BioLegend, 767002), or 25 ng/ml IL-2 (BioLegend, 575402).

NK cell stimulation

Total splenocytes or isolated NK cells were stimulated for 5 hours under the following conditions: 25 ng/ml IL-12 (BioLegend, 577002) and 25 ng/ml IL-18 (BioLegend, 767002), 5 µg/ml αNKp46, or co-cultured with 1×10⁴ B16 cells for 24 h. For intracellular cytokine measurement, GolgiPlug™ was added 4 h before the harvest.

ChIP

Chromatin immunoprecipitation assays were performed as previously described [31].

In vivo NK cell activation and IFN-γ secretion

To activate NK cells in vivo, age-matched mice were injected i.v. with 2 μg LPS (LPS-SM Ultrapure (LPS from S. minnesota R595), InvivoGen). Mice were euthanized 2.5 hr before the 5 hr time points, to culture RBC-lysed splenocytes in the presence of brefeldin A (BFA; 10 μg/ml; Sigma-Aldrich) for the remaining time. This allowed measuring direct IFN-γ release by NK cells in the absence of artificial restimulation [2, 65].

IL-15 complex treatment

Wild-type or Nr2f6-deficient mice were i.v. injected with 2 µg IL-15 complex (2:9 ratio: 0,36 µg recombinant mIL-15 (BioLegend, 566302) and 1,63 µg recombinant mouse IL-15R alpha (R&D Systems, 551-MR-100) complexed for 30 minutes at 37°C) or PBS as described previously every third day and analyzed after 9 days [39]. During tumor formation IL-15C was injected on day -1 and then on day 2 and 5 after B16-F10-B2m^-/- melanoma lung metastasis induction.

Cell sorting and RNA preparation

Total splenic NK cells were sorted as live/Lin^-/CD45⁺/NK1.1⁺/NKp46⁺/CD49b⁺ on a FACS Aria III (BD Biosciences). RNA from sorted cells was extracted using the RNeasy mini Kit (Qiagen, 74104). RNA quantity and integrity were determined by Agilent 5400, and only samples with > 100 ng RNA and RNA integrity number > 4 were used. PolyA enrichment, non-directional library preparation, and Illumina sequencing were performed by Novogene.

Gene counts were derived based on the number of reads mapped to each gene using featureCounts (v1.5.0-p3) [66]. Differentially expression (DE) analysis was performed using the DESeq2 R package (1.20.0) [67]. The resulting p values were adjusted using the Benjamini and Hochberg’s approach to control the false discovery rate. Genes with an adjusted p value <=0.05 were considered as differentially expressed [68]. DESeq2 normalized gene expression values were used to perform KEGG WebGestalt [69] and GSEA [70, 71].

Statistical analysis

Statistical analysis of experimental data was performed using Prism 10.2.3. The Gaussian distribution of the data was assessed using the Shapiro-Wilk test, and variance discrepancies were evaluated with the F-test. For gaussian distributed samples, differences were analyzed using an unpaired Student’s t-test, while non-gaussian distributed samples were compared using the Mann-Whitney U test. Randomization, blinding, or sample size estimation tests were not applied to our animal studies.

Data availability

The raw data supporting the conclusions of this article will be made available by the authors without undue reservation. RNAseq primary data deposition is found under GEO accession GSE263734.

References

Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol. 2008;9:503–10.
Article CAS PubMed Google Scholar
Zanoni I, Spreafico R, Bodio C, Di Gioia M, Cigni C, Broggi A, et al. IL-15 cis presentation is required for optimal NK cell activation in lipopolysaccharide-mediated inflammatory conditions. Cell Rep. 2013;4:1235–49.
Article CAS PubMed Google Scholar
Gianchecchi E, Delfino DV, Fierabracci A. NK cells in autoimmune diseases: Linking innate and adaptive immune responses. Autoimmun Rev. 2018;17:142–54.
Article CAS PubMed Google Scholar
Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, et al. Innate or adaptive immunity? The example of natural killer cells. Science. 2011;331:44–9.
Article CAS PubMed PubMed Central Google Scholar
Glasner A, Levi A, Enk J, Isaacson B, Viukov S, Orlanski S, et al. NKp46 Receptor-mediated interferon-gamma production by natural killer cells increases fibronectin 1 to alter tumor architecture and control metastasis. Immunity. 2018;48:396–8.
Article CAS PubMed PubMed Central Google Scholar
Cozar B, Greppi M, Carpentier S, Narni-Mancinelli E, Chiossone L, Vivier E. Tumor-infiltrating natural killer cells. Cancer Discov. 2020;11:34–44.
Nicolai CJ, Wolf N, Chang IC, Kirn G, Marcus A, Ndubaku CO, et al. NK cells mediate clearance of CD8 T cell-resistant tumors in response to STING agonists. Sci Immunol. 2020;5:eaaz2738.
Long EO, Kim HS, Liu DF, Peterson ME, Rajagopalan S. Controlling natural killer cell responses: integration of signals for activation and inhibition. Annual Rev Immunol. 2013;31:227–58.
Article CAS Google Scholar
Wu Y, Tian Z, Wei H. Developmental and functional control of natural killer cells by cytokines. Front Immunol. 2017;8:930.
Article PubMed PubMed Central Google Scholar
Zhang X, Feng J, Chen S, Yang H, Dong Z. Synergized regulation of NK cell education by NKG2A and specific Ly49 family members. Nat Commun. 2019;10:5010.
Article PubMed PubMed Central Google Scholar
Halfteck GG, Elboim M, Gur C, Achdout H, Ghadially H, Mandelboim O. Enhanced in vivo growth of lymphoma tumors in the absence of the NK-activating receptor NKp46/NCR1. J Immunol. 2009;182:2221–30.
Article CAS PubMed Google Scholar
Lakshmikanth T, Burke S, Ali TH, Kimpfler S, Ursini F, Ruggeri L, et al. NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo. J Clin Invest. 2009;119:1251–63.
Article CAS PubMed PubMed Central Google Scholar
Glasner A, Ghadially H, Gur C, Stanietsky N, Tsukerman P, Enk J, et al. Recognition and prevention of tumor metastasis by the NK receptor NKp46/NCR1. J Immunol. 2012;188:2509–15.
Article CAS PubMed Google Scholar
Glasner A, Isaacson B, Viukov S, Neuman T, Friedman N, Mandelboim M, et al. Increased NK cell immunity in a transgenic mouse model of NKp46 overexpression. Sci Rep-Uk. 2017;7:13090.
Barrow AD, Martin CJ, Colonna M. The natural cytotoxicity receptors in health and disease. Front Immunol. 2019;10:909.
Article CAS PubMed PubMed Central Google Scholar
Sen Santara S, Lee DJ, Crespo A, Hu JJ, Walker C, Ma X, et al. The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells. Nature. 2023;616:348–56.
Article PubMed PubMed Central Google Scholar
Böttcher JP, Bonavita E, Chakravarty P, Blees H, Cabeza-Cabrerizo M, Sammicheli S, et al. NK cells stimulate recruitment of cDC1 into the tumor microenvironment promoting cancer immune control. Cell. 2018;172:1022.
Article PubMed PubMed Central Google Scholar
Barry KC, Broz ML, Cueto FJ, Binnewies M, Combes AJ, Nelson AE, et al. A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments. Nat Med. 2018;24:1178–1191.
Article CAS PubMed PubMed Central Google Scholar
Welte S, Kuttruff S, Waldhauer I, Steinle A. Mutual activation of natural killer cells and monocytes mediated by NKp80-AICL interaction. Nat Immunol. 2006;7:1334–42.
Article CAS PubMed Google Scholar
Lucas M, Schachterle W, Oberle K, Aichele P, Diefenbach A. Dendritic cells prime natural killer cells by trans-presenting interleukin 15. Immunity. 2007;26:503–17.
Article CAS PubMed PubMed Central Google Scholar
Soderquest K, Powell N, Luci C, van Rooijen N, Hidalgo A, Geissmann F, et al. Monocytes control natural killer cell differentiation to effector phenotypes. Blood. 2011;117:4511–8.
Article CAS PubMed Google Scholar
Jaeger BN, Donadieu J, Cognet C, Bernat C, Ordonez-Rueda D, Barlogis V, et al. Neutrophil depletion impairs natural killer cell maturation, function, and homeostasis. J Exp Med. 2012;209:565–80.
Article CAS PubMed PubMed Central Google Scholar
Waldmann TA, Dubois S, Miljkovic MD, Conlon KC. IL-15 in the combination immunotherapy of cancer. Front Immunol. 2020;11:868.
Article CAS PubMed PubMed Central Google Scholar
Glass CK, Saijo K. Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells. Nat Rev Immunol. 2010;10:365–76.
Article CAS PubMed Google Scholar
Leopold Wager CM, Arnett E, Schlesinger LS. Macrophage nuclear receptors: Emerging key players in infectious diseases. PLoS Pathog. 2019;15:e1007585.
Article PubMed PubMed Central Google Scholar
Holmes TD, Pandey RV, Helm EY, Schlums H, Han H, Campbell TM, et al. The transcription factor Bcl11b promotes both canonical and adaptive NK cell differentiation. Sci Immunol. 2021;6:1–16.
Article Google Scholar
Hermann-Kleiter N, Gruber T, Lutz-Nicoladoni C, Thuille N, Fresser F, Labi V, et al. The nuclear orphan receptor NR2F6 suppresses lymphocyte activation and T helper 17-dependent autoimmunity. Immunity. 2008;29:205–16.
Article CAS PubMed PubMed Central Google Scholar
Hermann-Kleiter N, Meisel M, Fresser F, Thuille N, Muller M, Roth L, et al. Nuclear orphan receptor NR2F6 directly antagonizes NFAT and RORgammat binding to the Il17a promoter. J Autoimmun. 2012;39:428–40.
Article CAS PubMed PubMed Central Google Scholar
Hermann-Kleiter N, Klepsch V, Wallner S, Siegmund K, Klepsch S, Tuzlak S, et al. The nuclear orphan receptor NR2F6 is a central checkpoint for cancer immune surveillance. Cell Rep. 2015;12:2072–85.
Article CAS PubMed PubMed Central Google Scholar
Klepsch V, Gerner RR, Klepsch S, Olson WJ, Tilg H, Moschen AR, et al. Nuclear orphan receptor NR2F6 as a safeguard against experimental murine colitis. Gut. 2018;67:1434–44.
Article CAS PubMed Google Scholar
Olson WJ, Jakic B, Labi V, Schoeler K, Kind M, Klepsch V, et al. Orphan nuclear receptor NR2F6 suppresses T follicular helper cell accumulation through regulation of IL-21. Cell Reports. 2019;28:2878.
Article CAS PubMed Google Scholar
Jakic B, Olson WJ, Siegmund K, Klepsch V, Kimpel J, Labi V, et al. Loss of the orphan nuclear receptor NR2F6 enhances CD8(+) T-cell memory via IFN-gamma. Cell Death Dis. 2021;12:187.
Article CAS PubMed PubMed Central Google Scholar
Vyas M, Requesens M, Nguyen TH, Peigney D, Azin M, Demehri S. Natural killer cells suppress cancer metastasis by eliminating circulating cancer cells. Front Immunol. 2022;13:1098445.
Article CAS PubMed Google Scholar
Crinier A, Milpied P, Escaliere B, Piperoglou C, Galluso J, Balsamo A, et al. High-dimensional single-cell analysis identifies organ-specific signatures and conserved NK cell subsets in humans and mice. Immunity. 2018;49:971–86.e5.
Article CAS PubMed PubMed Central Google Scholar
Matys V, Kel-Margoulis OV, Fricke E, Liebich I, Land S, Barre-Dirrie A, et al. TRANSFAC and its module TRANSCompel: transcriptional gene regulation in eukaryotes. Nucleic Acids Res. 2006;34:D108–10.
Article CAS PubMed Google Scholar
Abel AM, Yang C, Thakar MS, Malarkannan S. Natural killer cells: development, maturation, and clinical utilization. Front Immunol. 2018;9:1869.
Article PubMed PubMed Central Google Scholar
Heng TS, Painter MW. Immunological Genome Project C. The Immunological Genome Project: networks of gene expression in immune cells. Nat Immunol. 2008;9:1091–4.
Article CAS PubMed Google Scholar
Elpek KG, Rubinstein MP, Bellemare-Pelletier A, Goldrath AW, Turley SJ. Mature natural killer cells with phenotypic and functional alterations accumulate upon sustained stimulation with IL-15/IL-15R alpha complexes. P Natl Acad Sci USA. 2010;107:21647–52.
Article CAS Google Scholar
White JT, Cross EW, Burchill MA, Danhorn T, McCarter MD, Rosen HR, et al. Virtual memory T cells develop and mediate bystander protective immunity in an IL-15-dependent manner. Nat Commun. 2016;7:11291.
Article PubMed PubMed Central Google Scholar
Fiore PF, Di Matteo S, Tumino N, Mariotti FR, Pietra G, Ottonello S, et al. Interleukin-15 and cancer: some solved and many unsolved questions. J Immunother Cancer. 2020;8.
Torrejon DY, Abril-Rodriguez G, Champhekar AS, Tsoi J, Campbell KM, Kalbasi A, et al. Overcoming genetically based resistance mechanisms to PD-1 Blockade. Cancer Discov. 2020;10:1140–57.
Article CAS PubMed PubMed Central Google Scholar
Baginska J, Viry E, Paggetti J, Medves S, Berchem G, Moussay E, et al. The critical role of the tumor microenvironment in shaping natural killer cell-mediated anti-tumor immunity. Front Immunol. 2013;4:490.
Article PubMed PubMed Central Google Scholar
Ni J, Wang X, Stojanovic A, Zhang Q, Wincher M, Buhler L, et al. Single-Cell RNA sequencing of tumor-infiltrating NK cells reveals that inhibition of transcription factor HIF-1 alpha unleashes NK cell activity. Immunity. 2020;52:1075.
Article CAS PubMed Google Scholar
Bald T, Krummel MF, Smyth MJ, Barry KC. The NK cell-cancer cycle: advances and new challenges in NK cell-based immunotherapies. Nat Immunol. 2020;21:835–47.
Article CAS PubMed PubMed Central Google Scholar
Stojanovic A, Fiegler N, Brunner-Weinzierl M, Cerwenka A. CTLA-4 is expressed by activated mouse NK Cells and Inhibits NK Cell IFN-gamma production in response to mature dendritic cells. J Immunol. 2014;192:4184–91.
Article CAS PubMed Google Scholar
Hsu J, Hodgins JJ, Marathe M, Nicolai CJ, Bourgeois-Daigneault MC, Trevino TN, et al. Contribution of NK cells to immunotherapy mediated by PD-1/PD-L1 blockade. J Clin Invest. 2018;128:4654–68.
Article PubMed PubMed Central Google Scholar
Kohlhapp FJ, Broucek JR, Hughes T, Huelsmann EJ, Lusciks J, Zayas JP, et al. NK cells and CD8+ T cells cooperate to improve therapeutic responses in melanoma treated with interleukin-2 (IL-2) and CTLA-4 blockade. J Immunother Cancer. 2015;3:18.
Article PubMed PubMed Central Google Scholar
Tan SY, Guo XW, Li MZ, Wang TX, Wang ZH, Li CY. et al. Transcription factor Zhx2 restricts NK cell maturation and suppresses their antitumor immunity. J Exp Med. 2021;218:e20210009.
Article CAS PubMed PubMed Central Google Scholar
Imianowski CJ, Whiteside SK, Lozano T, Evans AC, Benson JD, Courreges CJF, et al. BACH2 restricts NK cell maturation and function, limiting immunity to cancer metastasis. J Exp Med. 2022;219:e20211476.
Article CAS PubMed PubMed Central Google Scholar
Sivori S, Vitale M, Morelli L, Sanseverino L, Augugliaro R, Bottino C, et al. p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J Exp Med. 1997;186:1129–36.
Article CAS PubMed PubMed Central Google Scholar
Sivori S, Pende D, Bottino C, Marcenaro E, Pessino A, Biassoni R, et al. NKp46 is the major triggering receptor involved in the natural cytotoxicity of fresh or cultured human NK cells. Correlation between surface density of NKp46 and natural cytotoxicity against autologous, allogeneic or xenogeneic target cells. Eur J Immunol. 1999;29:1656–66.
Article CAS PubMed Google Scholar
Chretien AS, Devillier R, Fauriat C, Orlanducci F, Harbi S, Le Roy A, et al. NKp46 expression on NK cells as a prognostic and predictive biomarker for response to allo-SCT in patients with AML. Oncoimmunol. 2017;6:e1307491.
Article Google Scholar
Lin SJ, Kuo ML, Hsiao HS, Lee PT, Chen JY, Huang JL. Activating and inhibitory receptors on natural killer cells in patients with systemic lupus erythematosis-regulation with interleukin-15. PLoS One. 2017;12:e0186223.
Article PubMed PubMed Central Google Scholar
Li A, He ML, Wang H, Qiao B, Chen P, Gu H, et al. All-trans retinoic acid negatively regulates cytotoxic activities of nature killer cell line 92. Biochem Bioph Res Co. 2007;352:42–7.
Article CAS Google Scholar
Kim CH. Control of Innate and Adaptive Lymphocytes by the RAR-Retinoic Acid Axis. Immune Netw. 2018;18:e1.
Article PubMed PubMed Central Google Scholar
Yu W, He J, Wang F, He Q, Shi Y, Tao X, et al. NR4A1 mediates NK-cell dysfunction in hepatocellular carcinoma via the IFN-γ/p-STAT1/IRF1 pathway. Immunology. 2023;169:69–82.
Article CAS PubMed Google Scholar
Song JX, Song H, Wei HM, Sun R, Tian ZG, Peng H. Requirement of ROR alpha for maintenance and antitumor immunity of liver-resident natural killer cells/ILC1s. Hepatology. 2022;75:1181–93.
Article CAS PubMed Google Scholar
Mortier E, Advincula R, Kim L, Chmura S, Barrera J, Reizis B, et al. Macrophage- and dendritic-cell-derived interleukin-15 receptor alpha supports homeostasis of distinct CD8(+) T cell subsets. Immunity. 2009;31:811–22.
Article CAS PubMed Google Scholar
vandenBroek HF, Kagi D, Zinkernagel RM, Hengartner H. Perforin dependence of natural killer cell-mediated tumor control in vivo. Eur J Immunol. 1995;25:3514–6.
Article CAS Google Scholar
Gotthardt D, Putz EM, Grundschober E, Prchal-Murphy M, Straka E, Kudweis P, et al. STAT5 is a key regulator in NK cells and acts as a molecular switch from tumor surveillance to tumor promotion. Cancer Discovery. 2016;6:414–29.
Article CAS PubMed Google Scholar
van Helden MJ, Goossens S, Daussy C, Mathieu AL, Faure F, Marcais A, et al. Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection. J Exp Med. 2015;212:2015–25.
Article PubMed PubMed Central Google Scholar
Mei J, Zhou WJ, Zhu XY, Lu H, Wu K, Yang HL, et al. Suppression of autophagy and HCK signaling promotes PTGS2(high) FCGR3(-) NK cell differentiation triggered by ectopic endometrial stromal cells. Autophagy. 2018;14:1376–97.
Article CAS PubMed PubMed Central Google Scholar
Shannon MJ, Mace EM. Natural killer cell integrins and their functions in tissue residency. Front Immunol. 2021;12:1–23.
Article Google Scholar
Cai LT, Li YC, Tan JX, Xu L, Li YQ. Targeting LAG-3, TIM-3, and TIGIT for cancer immunotherapy. J Hematol Oncol. 2023;16:101.
Bär E, Whitney PG, Moor K, Sousa CRE, LeibundGut-Landmann S. IL-17 regulates systemic fungal immunity by controlling the functional competence of NK Cells. Immunity. 2014;40:117–27.
Article PubMed Google Scholar
Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–30.
Article CAS PubMed Google Scholar
Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15:1–21.
Article Google Scholar
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008;5:621–8.
Article CAS PubMed Google Scholar
Liao Y, Wang J, Jaehnig EJ, Shi Z, Zhang B. WebGestalt 2019: gene set analysis toolkit with revamped UIs and APIs. Nucleic Acids Res. 2019;47:W199–W205.
Article CAS PubMed PubMed Central Google Scholar
Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34:267–73.
Article CAS PubMed Google Scholar
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005;102:15545–50.
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We want to thank Paul Hoeller for his assistance and support with mouse care. The authors thank Veronika Sexl, Dagmar Gotthardt, and Agnes Dadak (University of Veterinary Medicine Vienna, Austria) for their technical advice. Thanks to Verena Labi and Julia Heppke for their help in cell sorting (Medical University Innsbruck, Austria). We thank Chris Nicolai and David H. Raulet for providing the B16-F10-B2m^-/- cell line (University of California, Berkeley, CA 94720, USA).

Funding

This research was funded in part by the Austrian Science Fund (FWF) [Grant-DOI: 10.55776/P28694; 10.55776/DOC82] awarded to N-HK, [DOIs10.55776/P33855] awarded to PS, the Tirolean Science Fund (F.30904/7-2021) awarded to N-HK, the Austrian Society of Gastroenterology and Hepatology (ÖGGH) awarded to N-HK, the Austrian Academy of Sciences with a PhD-fellowship awarded to FH (OAW DOC/26015) and the ERC_ADG #786462 awarded to GB. For open access purposes, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising from this submission.

Author information

Stephanie Aschauer-Wallner
Present address: Regenerative Medicine, Spinal Cord Injury and Tissue Regeneration Center Salzburg, Paracelsus Medical University Salzburg, Salzburg, Austria

Authors and Affiliations

Institute of Cell Genetics, Department for Genetics and Pharmacology, Medical University of Innsbruck, Innsbruck, Austria
Johannes Woelk, Gottfried Baier & Natascha Hermann-Kleiter
Department of Dermatology, Venereology & Allergology, Medical University of Innsbruck, Innsbruck, Austria
Florian Hornsteiner & Patrizia Stoitzner
Laboratory of Tumor Immunology, Tyrolean Cancer Institute & Internal Medicine V, Medical University of Innsbruck, 6020, Innsbruck, Austria
Stephanie Aschauer-Wallner

Authors

Johannes Woelk
View author publications
Search author on:PubMed Google Scholar
Florian Hornsteiner
View author publications
Search author on:PubMed Google Scholar
Stephanie Aschauer-Wallner
View author publications
Search author on:PubMed Google Scholar
Patrizia Stoitzner
View author publications
Search author on:PubMed Google Scholar
Gottfried Baier
View author publications
Search author on:PubMed Google Scholar
Natascha Hermann-Kleiter
View author publications
Search author on:PubMed Google Scholar

Contributions

N-HK. developed the concept and design for the study. J.W. and N-HK. performed most experiments with the help of F.H., P.S., and S.A-W. FH. and P.S. performed analysis of the myeloid compartment, S.A-W helped with first LPS assay. J.W., F.H., P.S., S-AW. and N-HK., analyzed the data. J.W. and N-HK. prepared and designed the figures. N-HK. supervised the work. G.B. gave intellectual input. N.-H.K. wrote, and all authors read, edited, and approved the final version of the manuscript.

Corresponding author

Correspondence to Natascha Hermann-Kleiter.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics

Animal procedures were approved by the Austrian Federal Ministry of Education, Science and Research (BMWFW-66.011/0064-WF/V/3b/2016; BMWFW-66.011/0112-WF/V/3b/2017, GZ: 2021-0.406.862, GZ: 2023-0.315.075, GZ: 2023-0.623.434, and GZ: 2024-0.885.804.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by Massimiliano Agostini

Supplementary information

Supplementary Files

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Woelk, J., Hornsteiner, F., Aschauer-Wallner, S. et al. Regulation of NK cell development, maturation, and antitumor responses by the nuclear receptor NR2F6. Cell Death Dis 16, 77 (2025). https://doi.org/10.1038/s41419-025-07407-4

Download citation

Received: 17 May 2024
Revised: 12 January 2025
Accepted: 29 January 2025
Published: 07 February 2025
Version of record: 07 February 2025
DOI: https://doi.org/10.1038/s41419-025-07407-4