NK cells are critical innate immune cells that target the tumor cells and cancer-initiating cells and clear viruses by producing cytokines and cytotoxic granules. However, the role of the purinergic receptor P2Y6 in the NK cells remains largely unknown. In this study, we discovered that the expression of P2Y6 was decreased upon the activation of the NK cells. Moreover, in the P2Y6-deficient mice, we found that the deficiency of P2Y6 promoted the development of the NK precursor cells into immature NK and mature NK cells. We also found that the P2Y6 deficiency increased, but the P2Y6 receptor agonist UDP or UDP analog 5-OMe-UDP decreased the production of IFN-γ in the activated NK cells. Furthermore, we demonstrated that the P2Y6-deficient NK cells exhibited stronger cytotoxicity in vitro and antimetastatic effects in vivo. Mechanistically, P2Y6 deletion promoted the expression of T-bet (encoded by Tbx21), with or without the stimulation of IL-15. In the absence of P2Y6, the levels of phospho-serine/threonine kinase and pS6 in the NK cells were significantly increased upon the stimulation of IL-15. Collectively, we demonstrated that the P2Y6 receptor acted as a negative regulator of the NK cell function and inhibited the maturation and antitumor activities of the NK cells. Therefore, inhibition of the P2Y6 receptor increases the antitumor activities of the NK cells, which may aid in the design of innovative strategies to improve NK cell–based cancer therapy.

Natural killer cells are important innate immune cells that play a key role in mediating antitumor and antiviral responses by secreting proinflammatory cytokines and cytotoxic granules (1). NK cells are known to originate from the bone marrow; however, recent findings have shown that they can also develop from the lymph nodes (2, 3). Based on the expression of surface markers on NK cells, the development of murine NK cells is divided into three stages: NK precursor (NKp) cells (CD122+NK1.1CD11b), immature NK (iNK) cells (CD122+NK1.1+CD11b), and mature NK (mNK) cells (CD122+NK1.1+CD11b+). Upon acquisition of NK1.1 surface expression, murine NK cells are further classified into three stages based on the surface expression of CD11b and CD27: CD11bCD27+ NK cells, CD11b+CD27+ NK cells, and CD11b+CD27 NK cells (46). Transcription factors are important for the development of NK cells (e.g., IKAROS, PU.1, ETS1, and VDUP-1) and modulate the early stages of NK cell development by promoting the production of NKp cells. However, E4BP4, ID2, and MEF promote early maturation of NK cells during the iNK cell phase, whereas GATA-3, TOX, TBX21, EOMES, FOXO1, and IRF2 mainly regulate the terminal maturation of NK cells (713).

G protein–coupled receptors (GPCRs) are not only closely related to cell development and differentiation, but also play an important role in the immune system (14, 15). Recent studies have demonstrated that many extracellular nucleotides are released during pathogen infection and cell injury. These extracellular nucleotides can act as mediators of inflammatory responses by binding to extracellular nucleotide receptors. These receptors are called purine receptors, which are widely expressed in cells of the immune system (16). The purine receptor includes P1 and P2 receptors. The P2 receptor is divided into two subtypes: P2X (ligand-gated ion channels) and P2Y (GPCR) receptors (17, 18).

As a GPCR, the P2Y6 receptor is highly expressed on stromal cells, and its ligand is UDP (19). Recent in vivo studies have shown that activation of the P2Y6 receptor inhibits bacterial and viral infection (20, 21). In addition, P2Y6 receptor activation increases the secretion of chemokines from monocytes, dendritic cells, epithelial cells, and endothelial cells, thus promoting the migration of inflammatory cells to the site of inflammation and infection (2227).

It has been reported that the damaged nerve cells release UDP and UTP, increasing the expression of the P2Y6 receptor in microglia and promoting the ability of microglia to engulf dead cells. Thus, UDP activates the P2Y6 signal that can be used as an “eat-me” signal of microglia, promoting the removal of dead cells or fragments in the nervous system (19, 22). In addition, the expression of the P2Y6 receptor is increased in intestinal epithelial cells of inflammatory bowel disease (28). The incidence of inflammatory bowel disease is more severe in P2Y6 knockout mice than in wild-type (WT) mice (24). P2Y6 receptor deficiency promotes the occurrence of chronic lung diseases and aggravates experimental autoimmune encephalomyelitis (25, 29). In atherosclerotic mouse models, UDP promotes chronic inflammatory disorders by activating the P2Y6 receptor (30). In summary, these studies indicate that activation of the P2Y6 receptor enhances the innate immune response. However, the function of GPCRs in regulating the development and function of NK cells, especially the P2Y6 receptor, remains largely unknown. In a previous study, two articles reported that the function of GPCRs on NK cells, which is ATP, inhibits the chemotaxis and toxicity of NK cells by activating the P2Y11 receptor (31), and the GPR56 receptor negatively regulates human NK cell function (32).

In this study, we demonstrated that the P2Y6 receptor was downregulated in both mouse and human activated NK cells. Meanwhile, deletion of the P2Y6 receptor promoted the NK cell maturation and activation by upregulating the expression of T-bet. We also found that the recombinant cytokine IL-15 induced the expression of T-bet in the NK cells by activating the mammalian target of rapamycin (mTOR) signaling pathway, whereas the P2Y6 receptor inhibited the expression of T-bet by suppressing the levels of phospho-serine/threonine kinase and pS6 in the NK cells upon IL-15 stimulation, thereby leading to a decrease in the activation of NK cells and eventually inhibiting the effector and antitumor functions of NK cells in vitro and in vivo. In this study, we demonstrated that the P2Y6 receptor is an important negative regulatory checkpoint during the maturation and actions of the NK cells and also elucidated its molecular mechanism, which further highlights its potential to be used for cancer therapy.

The following Abs were used for flow cytometry: anti-NK1.1 (PK136; 1:200), anti-CD122 (5H4; 1:200), anti-CD11b (M1/70; 1:200), anti-CD3 (17A; 1:300), anti-CD27 (LG.7F9; 1:200), anti-CD132 (TuGm2; 1:200), anti-CD4 (GK1.5; 1:300), anti-CD8 (53-6.7; 1:300), anti-CD71 (C2; 1:200), anti-CD98 (RL388; 1:200), anti–T-bet (eBIO4B10; 1:100), and anti-Eomes (Dan11m9g; 1:100). They were purchased from eBioscience (San Diego, CA), BioLegend (San Diego, CA), or BD Biosciences (San Jose, CA).

Abs against FOXO1 (catalog no. 2880T; 1:500), p-FOXO1 (Ser319, catalog no. 9461T; 1:500), phospho-serine/threonine kinase (Ser473; catalog no. 5315; 1:500), p-4EBP1 (catalog no. 7547; 1:50), and p-S6 (catalog no. 4851; 1:50) were purchased from Cell Signaling Technology (Danvers, MA). The anti-P2Y6 Ab was purchased from Alomone Labs (catalog no. APR-011; Jerusalem, Israel). UDP was obtained from Sigma-Aldrich (catalog no. U4125; St. Louis, MO). Poly(I:C) was obtained from InvivoGen (catalog no. tlrl-pic; San Diego, CA). The RPMI 1640 medium and bovine serum were obtained from Life Technologies (Carlsbad, CA). Recombinant murine cytokines IL-12 and IL-15 were obtained from PeproTech (East Windsor, NJ). The Leukocyte Activation Cocktail with BD GolgiPlug (catalog no. 550583) and BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit (catalog no. 555028) were purchased from BD Pharmingen (San Jose, CA). Magnetic beads coated with the Ab to mouse NK1.1 were purchased from EasySep (STEMCELL Technologies, Cambridge, MA). The PrimeScript RT-PCR Kit and SYBR Premix Ex Taq kit were obtained from Takara Bio (Shiga, Japan).

Six- to 12-wk-old C57BL/6 (CD45.2) mice and B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mice were obtained from the Laboratory Animal Center (East China Normal University, Shanghai, China). P2Y6 knockout mice on a C57BL/6 background were prepared as previously described (20). In the bone marrow chimera experiment, the recipient (CD45.1) mice were irradiated and engrafted with the WT or P2Y6 knockout (P2Y6−/−) donor bone marrow cells from C57BL/6 (CD45.2) via tail vein injections. All mice were maintained under specific pathogen-free conditions. All animal experiments were performed with the approval of the Animal Ethics Committee of East China Normal University.

The surface markers were: NK1.1, CD3, CD122, CD11b, CD27, CD71, CD98, and CD132. Cells (2 × 106) were stained with Abs in PBS containing 2% FBS. After 30 min, the cells were washed three times, followed by flow cytometry analysis. Intracellular staining was used to detect the NK cell transcription factors, cytokines, and phosphorylated proteins. Spleen cells (2 × 106) were cultured in RPMI 1640 medium and stimulated with 1 μg/ml Poly(I:C), 100 ng/ml IL-15, and 10 ng/ml IL-12 in the presence of 2 μl/ml Leukocyte Activation Cocktail with BD GolgiPlug for 4 h. Cells were harvested and washed three times. The surface markers of NK cells were stained with anti-CD3 and anti-NK1.1. After 30 min, the cells were washed three times and resuspended in 200 μl BD Cytofix/Cytoperm Plus fixation/permeabilization buffer for 30 min. The cells were washed three times and stained with anti–IFN-γ. For T-BET, EOMES, and FOXO1 staining, 2 × 106 spleen cells were stimulated with or without 100 ng/ml IL-15 for 12 h. Cells were harvested and washed three times. The surface markers of NK cells were stained with anti-CD3 and anti-NK1.1. After 30 min, the cells were washed three times and resuspended in 200 μl FIX & PERM Cell Permeabilization buffer (Thermo Fisher Scientific, Waltham, MA) for 30 min. Cells were washed three times and stained with anti–T-BET, anti-EOMES, and anti-FOXO1 Abs. For detection of phosphorylated signaling proteins, 2 × 106 spleen cells were stimulated with or without 100 ng/ml IL-15 for 1 h or 2 h. Cells were harvested and stained with anti-CD3 and anti-NK1.1. After 30 min, the cells were washed three times and fixed with Phosflow Lyse/Fix buffer, followed by permeabilization with Phosflow Perm Buffer III (BD Pharmingen) and staining with Abs to detect AKT phosphorylated at Ser473, S6 phosphorylated at Ser235/236, and 4EBP1 phosphorylated at Thr37/46. Flow cytometry data were acquired using LSR Fortessa (BD Biosciences) and analyzed using FlowJo software.

Total RNA was extracted from cultured cells using TRIzol reagent (Takara Bio) according to the manufacturer’s instructions. cDNA was synthesized with 1000 ng RNA using a reverse transcription kit (PrimeScript First Strand cDNA Synthesis kit, catalog no. R047A; Takara Bio), according to the manufacturer’s protocols. The expression of mRNA encoding P2Y6 was quantified by quantitative PCR (qPCR) with SYBR Premix Ex Taq kit (Takara Bio) and was normalized to the expression of GAPDH. cDNA was amplified using a Light Cycle (Agilent Technologies). The human P2Y6 primers for real-time PCR in this study were as follows: P2Y6 sense, 5′-GTGTCTACCGCG-3′; P2Y6 antisense, 5′-CCAGAGCAAGGTTTAGGGTGTA-3′. The mouse P2Y6 primers for real-time PCR were as follows: P2Y6 sense, 5′-CCTTCGCTGCTGCCTACAA-3′; P2Y6 antisense: 5′-TCTCTGCCTCTGCCACTTG-3′. All real-time qPCR (RT-qPCR) data were analyzed using the 2−ΔΔCt change-in-cycling-threshold method.

Cells were washed with ice-cold PBS and suspended in lysis buffer (Cell Signaling Technology) on ice for 30 min. Equal amounts of protein were resolved by 12.5% SDS-PAGE gels and then transferred onto a PVDF membrane (Millipore, Billerica, MA). The membrane was incubated in 5% skim milk in TBS for 1 h at room temperature and then incubated with a primary Ab at 4°C for 16 h and an HRP-conjugated secondary Ab for 2 h at room temperature. The immune-reactive proteins were detected using the Odyssey laser digital imaging system (Gene Company).

In cytotoxicity assays, NK cells were isolated from the spleens of mice using the NK cell isolation kit (STEMCELL Technologies) according to the manufacturer’s protocols. YAC-1-GFP cells were cocultured at 1 × 104 cells/well in 96-well microtiter plates with NK cells or 100 ng/ml IL-15–activated NK cells in triplicate in 96-well microtiter plates at the indicated effector/target ratios. After 4 h of incubation at 37°C, the cells were harvested and incubated with 7-aminoactinomycin D for 5 min. Cells were analyzed using an FACSCalibur flow cytometer (BD Biosciences).

NK cells were isolated from 8–12-wk-old WT or P2Y6−/− mice. The 6–8-wk-old WT mice were used as recipient mice for lung experimental metastasis assays. A total of 2 × 106 WT or P2Y6−/− NK cells and 1 × 105 B16F10 melanoma cells were coinjected into WT mice by i.v. injection. Two weeks after the injection, the mice were sacrificed to analyze the lung weight and tumor nodules.

Splenocytes were collected from 6–8-wk-old WT mice and stained with Abs against CD3, NK1.1, CD11b, and CD27. After 30 min, the cells were harvested and washed three times. CD11bCD27 NK cells, CD11bCD27+ NK cells, CD11b+CD27+ NK cells, and CD11b+CD27 NK cells (gated on CD3NK1.1+) were sorted using a flow cytometer (BD Biosciences).

Bone marrow chimeras were prepared using 8-wk-old mice as donors and recipients. C57BL/6 CD45.2+ donor bone marrow cells (1 × 106) collected from WT or congenic age- and sex-matched P2Y6 knockout mice were i.v. injected into C57BL/6 CD45.1 congenic recipient mice, which were lethally irradiated (4 cGy twice on the same day) using an x-ray irradiator. The recipient mice were maintained in antibiotic water containing enrofloxacin. Bone marrow–transplanted mice were sacrificed and analyzed 3–4 wk posttransplantation.

All experiments were analyzed using Microsoft Excel or GraphPad Prism v.7.0 (https://www.graphpad.com/scientific-software/prism/) software. Results are presented as the mean ± SD, and the difference between groups was calculated using the two-tailed Student t test for unpaired data. Multiple-group comparisons were performed using one-way ANOVA. Differences were considered significant when *p < 0.05, **p < 0.01, or ***p < 0.001.

It has been reported that the P2Y6 receptor was expressed in human NK cells (31). But how the P2Y6 receptor regulates NK cell function remains unknown. To investigate the function of the P2Y6 receptor in NK cells, we analyzed the expression of the P2Y6 receptor in activated mouse NK cells upon exposure to different stimuli. Murine splenocytes were treated with or without Poly(I:C) for 6 h or 12 h. NK cells were sorted by flow cytometry, and mRNA was extracted immediately. The expression of the P2Y6 receptor was analyzed using RT-qPCR. Our results showed that P2Y6 expression was decreased in mouse NK cells after Poly(I:C) treatment for 6 and 12 h in vitro (Fig. (1A). Consistently, the in vivo results showed that the expression of P2Y6 receptor was significantly decreased in splenic NK cells and splenocytes isolated from the mice that were i.p. injected with Poly(I:C) for 6 h compared with the control mice without i.p. injection of Poly(I:C) (Fig. (1B, 1C). Therefore, both in vitro and in vivo experiments showed that the expression of the P2Y6 receptor in NK cells was significantly decreased upon Poly(I:C) treatment. To investigate whether other treatments that activated NK cells can also affect the expression of the P2Y6 receptor, we treated mouse NK cells with IL-15 or Poly(I:C) and analyzed the protein expression of the P2Y6 receptor in NK cells with or without Poly(I:C) or IL-15 treatment. P2Y6 receptor protein expression was detected in the WT NK cells, but not in the P2Y6−/− NK cells using immunoblotting (Fig. (1D) and flow cytometry (Fig. (1E). As expected, the expression of the P2Y6 receptor in mouse NK cells was also significantly decreased in the protein level upon Poly(I:C) or IL-15 stimulations (Fig. (1F) compared with the control cells. To investigate whether the expression of P2Y6 was similarly decreased in human NK cells, we treated human NK cell line NK-92 cells with or without Poly(I:C) or IL-15. Similarly, the expression of P2Y6 receptors in NK-92 cells also decreased (Fig. (1G). In addition, similar results were found in NK cells isolated from human PBMCs treated with IL-15 (Fig. (1H). Altogether, these data suggest that the P2Y6 receptor is significantly decreased in activated NK cells.

FIGURE 1.

Expression of P2Y6 receptor was decreased after NK cell activation. (A) Murine splenocytes were stimulated with Poly(I:C) for 6 h or 12 h. NK cells were sorted by flow cytometry, and mRNA was extracted immediately. RT-qPCR analyzed the expression of P2Y6 receptor. (B and C) Splenic NK cells or splenocytes were purified from WT mice i.p. injected with Poly(I:C) (125 μg/mouse) for 6 h. The expression of P2Y6 receptor was analyzed using RT-qPCR. (D) The protein expression of P2Y6 in WT and P2Y6−/− NK cells was analyzed using by immunoblotting. (E) The protein expression of P2Y6 in WT and P2Y6−/− NK cells was analyzed using flow cytometry. (F) Splenic NK cells were stimulated with or without Poly(I:C) or IL-15 for 24 h, and the mean fluorescence intensity (MFI) of P2Y6 receptor in NK cells (gated CD3NK1.1+ cells) was analyzed by flow cytometry (right). (G) NK-92 cells were stimulated by Poly(I:C) or IL-15 for different time, and the expression of the P2Y6 receptor was analyzed using RT-qPCR. (H) Human NK cells purified from PBMCs were stimulated with IL-15 (100 ng/ml) for 6 h, and the expression of the P2Y6 receptor was analyzed using RT-qPCR. Graphs show mean ± SD (n = 4–6). Data are representative of three independent experiments. Statistical significance was assessed using Student t test (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 1.

Expression of P2Y6 receptor was decreased after NK cell activation. (A) Murine splenocytes were stimulated with Poly(I:C) for 6 h or 12 h. NK cells were sorted by flow cytometry, and mRNA was extracted immediately. RT-qPCR analyzed the expression of P2Y6 receptor. (B and C) Splenic NK cells or splenocytes were purified from WT mice i.p. injected with Poly(I:C) (125 μg/mouse) for 6 h. The expression of P2Y6 receptor was analyzed using RT-qPCR. (D) The protein expression of P2Y6 in WT and P2Y6−/− NK cells was analyzed using by immunoblotting. (E) The protein expression of P2Y6 in WT and P2Y6−/− NK cells was analyzed using flow cytometry. (F) Splenic NK cells were stimulated with or without Poly(I:C) or IL-15 for 24 h, and the mean fluorescence intensity (MFI) of P2Y6 receptor in NK cells (gated CD3NK1.1+ cells) was analyzed by flow cytometry (right). (G) NK-92 cells were stimulated by Poly(I:C) or IL-15 for different time, and the expression of the P2Y6 receptor was analyzed using RT-qPCR. (H) Human NK cells purified from PBMCs were stimulated with IL-15 (100 ng/ml) for 6 h, and the expression of the P2Y6 receptor was analyzed using RT-qPCR. Graphs show mean ± SD (n = 4–6). Data are representative of three independent experiments. Statistical significance was assessed using Student t test (*p < 0.05, **p < 0.01, ***p < 0.001).

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To determine whether the P2Y6 receptor plays a crucial role in regulating NK cells, we used P2Y6-deficient mice to study the development and function of NK cells in vivo. We analyzed the proportion of immune cells in P2Y6−/− mice and found that there was no significant difference in T, B, and CD11b+ monocyte cells in the spleen of P2Y6−/− mice compared with WT mice (Supplemental Fig. 1). P2Y6 receptor deletion did not alter the percentage and absolute number of CD3NK1.1+ NK cells in the bone marrow, spleen, lung, and liver (Fig. (2A, 2B), suggesting that the P2Y6 receptor did not affect the percentage and absolute number of CD3NK1.1+ NK cells in mice. However, we observed that the percentage of CD122+NK1.1CD11b cells was decreased and CD122+NK1.1+CD11b cells increased significantly in P2Y6−/− mice compared with WT mice (Fig. (2C, 2D), indicating that NKp cells were decreased and iNK cells were increased. Taken together, these results indicate that the P2Y6 receptor inhibits the development of NKp cells into iNK cells.

FIGURE 2.

P2Y6 receptor inhibits the development of NKp cells into iNK cells. (A) FACS analysis the CD3NK1.1+ NK cells in the bone marrow, spleen, liver, and lung from WT and P2Y6−/− mice. (B) The number of CD3NK1.1+ NK cells in the bone marrow, spleen, liver, and lung in WT and P2Y6−/− mice. (C and D) FACS analysis of CD11bCD3CD122+NK1.1+ NK cells from WT and P2Y6−/− mice. Graphs show mean ± SD (n = 4–6). Data are representative of three independent experiments. Statistical significance was assessed using Student t test (*p < 0.05, ***p < 0.001).

FIGURE 2.

P2Y6 receptor inhibits the development of NKp cells into iNK cells. (A) FACS analysis the CD3NK1.1+ NK cells in the bone marrow, spleen, liver, and lung from WT and P2Y6−/− mice. (B) The number of CD3NK1.1+ NK cells in the bone marrow, spleen, liver, and lung in WT and P2Y6−/− mice. (C and D) FACS analysis of CD11bCD3CD122+NK1.1+ NK cells from WT and P2Y6−/− mice. Graphs show mean ± SD (n = 4–6). Data are representative of three independent experiments. Statistical significance was assessed using Student t test (*p < 0.05, ***p < 0.001).

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To further investigate the effect of the P2Y6 receptor on NK cell maturation, we examined the expression of P2Y6 at different maturation stages of NK cells isolated from the spleen. The data showed that the P2Y6 receptor was highly expressed in iNK cells; however, the expression level gradually decreased, along with NK cell maturation (Fig. (3A), suggesting that the expression level of the P2Y6 receptor was downregulated with the maturation of NK cells.

FIGURE 3.

P2Y6 receptor inhibits the maturation of NK cells. (A) Splenic NK cell subsets were purified using FACS. The expression of P2Y6 receptor was analyzed using RT-qPCR. (BF) FACS analysis of the percentages and numbers of CD27 and CD11b (gated NK1.1+CD3) NK cell subsets in the bone marrow, lymph node, spleen, liver, and lung of WT and P2Y6−/− mice. Graphs show mean ± SD (n = 5). (G) Percentages of CD27 and CD11b (gated CD45.2+NK1.1+CD3) NK cell subsets were analyzed by flow cytometric analysis in the liver, spleen, and bone marrow of CD45.1 recipients, which were engrafted with bone marrow cells of WT or P2Y6−/− mice littermates. Data are representative of three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 3.

P2Y6 receptor inhibits the maturation of NK cells. (A) Splenic NK cell subsets were purified using FACS. The expression of P2Y6 receptor was analyzed using RT-qPCR. (BF) FACS analysis of the percentages and numbers of CD27 and CD11b (gated NK1.1+CD3) NK cell subsets in the bone marrow, lymph node, spleen, liver, and lung of WT and P2Y6−/− mice. Graphs show mean ± SD (n = 5). (G) Percentages of CD27 and CD11b (gated CD45.2+NK1.1+CD3) NK cell subsets were analyzed by flow cytometric analysis in the liver, spleen, and bone marrow of CD45.1 recipients, which were engrafted with bone marrow cells of WT or P2Y6−/− mice littermates. Data are representative of three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001).

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Next, we analyzed NK cell subsets in the bone marrow, lymph node, spleen, liver, and lung from WT and P2Y6−/− mice. We observed that the proportion of CD11bCD27+ NK cells, CD11b+CD27+ NK cells, and CD11b+CD27 NK cells (gated on CD3NK1.1+) in the bone marrow and lymph nodes was similar in WT and P2Y6−/− mice (Fig. (3B, 3C). However, the proportion of CD11b+CD27 NK cells in the spleen, liver, and lung was significantly higher in P2Y6−/− mice than in WT mice (Fig. (3D–F). Collectively, the above data strongly support the idea that the P2Y6 receptor suppresses the maturation of NK cells.

To further determine whether the effect of P2Y6 on the maturation of NK cells is cell intrinsic, a bone marrow chimera experiment was performed. Three to four weeks after engraftment, the liver, splenic, and bone marrow NK cells were analyzed by flow cytometry. The data showed that there were more mNK cells in the liver and spleen of recipient CD45.1 after the injection of P2Y6−/− bone marrow cells compared with the injection of WT bone marrow cells, whereas there was no significant difference in mNK cells in the bone marrow of recipient CD45.1 mice after the injection of P2Y6−/− bone marrow cells and WT bone marrow cells (Fig. (3G). These data indicate that the effect of P2Y6 on NK cell maturation is cell intrinsic.

To investigate whether the deletion of the P2Y6 receptor affects the activation of NK cells in a steady state, we used an intracellular staining assay to detect the levels of IFN-γ and granzyme B. We observed that there was no significant difference in the production of IFN-γ and granzyme B in splenic NK cells from WT and P2Y6−/− mice (Supplemental Fig. 2A). Furthermore, P2Y6 receptor deletion did not alter the expression of the activating receptor NKG2D and inhibitory receptors NKG2A and KLRG1 on the surface of NK cells (Supplemental Fig. 2B). These data suggest that the P2Y6 receptor does not affect the biological characteristics of NK cells in the steady state.

To further investigate whether the P2Y6 receptor affects IFN-γ secretion in activated NK cells, mouse splenocytes were treated with Poly(I:C), IL-15, IL-12, IL-15, or PMA. We observed the level of IFN-γ production in NK cells from P2Y6−/− mice was higher than that in NK cells from WT mice (Fig. (4A). We also treated mouse NK cells, human peripheral blood NK cells, or NK-92 cells with the P2Y6 receptor agonist UDP or UDP analog 5-OMe-UDP in the presence or absence of Poly(I:C) or recombinant cytokine IL-15. Our results showed that UDP or 5-OMe-UDP inhibited the production of IFN-γ in NK cells (Fig. (4B–D). Collectively, these data indicate that the P2Y6 receptor suppresses the activation of NK cells.

FIGURE 4.

P2Y6 receptor inhibits the activation of NK cells. (A) Intracellular flow cytometric analysis of IFN-γ production by WT and P2Y6−/− splenic NK cells stimulated with Poly(I:C) (100 μg/ml), IL-15 (100 ng/ml), IL-12 (10 ng/ml) plus IL-15 (100 ng/ml), or PMA and ionomycin for 4 h in the presence of GolgiPlug. (B) Intracellular flow cytometric analysis of IFN-γ production by splenic NK cells treated with Poly(I:C) or IL-15 for 12 h in the presence of 10 μM or 100 μM UDP. The percentage of IFN-γ+ NK cells has been statistically analyzed. (C) Intracellular flow cytometric analysis of IFN-γ production by human PBMC NK cells treated with IL-15 for 12 h in the presence of 100 μM UDP or 10 μM 5-OMe-UDP. (D) Intracellular flow cytometric analysis of IFN-γ production by NK-92 cells treated with or without IL-15 (right) or PBS (left) for 12 h in the presence of 100 μM UDP or 10 μM 5-OMe-UDP. Graphs show mean ± SD (n = 3–4). Data are representative of three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (*p < 0.05, **p < 0.01).

FIGURE 4.

P2Y6 receptor inhibits the activation of NK cells. (A) Intracellular flow cytometric analysis of IFN-γ production by WT and P2Y6−/− splenic NK cells stimulated with Poly(I:C) (100 μg/ml), IL-15 (100 ng/ml), IL-12 (10 ng/ml) plus IL-15 (100 ng/ml), or PMA and ionomycin for 4 h in the presence of GolgiPlug. (B) Intracellular flow cytometric analysis of IFN-γ production by splenic NK cells treated with Poly(I:C) or IL-15 for 12 h in the presence of 10 μM or 100 μM UDP. The percentage of IFN-γ+ NK cells has been statistically analyzed. (C) Intracellular flow cytometric analysis of IFN-γ production by human PBMC NK cells treated with IL-15 for 12 h in the presence of 100 μM UDP or 10 μM 5-OMe-UDP. (D) Intracellular flow cytometric analysis of IFN-γ production by NK-92 cells treated with or without IL-15 (right) or PBS (left) for 12 h in the presence of 100 μM UDP or 10 μM 5-OMe-UDP. Graphs show mean ± SD (n = 3–4). Data are representative of three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (*p < 0.05, **p < 0.01).

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As NK cells are innate effector lymphocytes, their cytotoxicity may be affected by their activation. We performed both in vitro and in vivo experiments to investigate the role of the P2Y6 receptor in the cytotoxicity of NK cells. The results showed that P2Y6−/− NK cell cytotoxicity against YAC-1 target cells was significantly increased in the presence or absence of IL-15 compared with WT NK cells (Fig. (5A, 5B). To further study the role of P2Y6 deletion in the function of NK cells in vivo, we established a B16F10 melanoma metastasis model by i.v. injection to examine NK cell–mediated tumor surveillance. Our results showed that there was lower lung weight and fewer numbers of lung tumor nodules in the mice injected with P2Y6−/− NK cells compared with those injected with WT NK cells (Fig. (5C–E). In summary, these data demonstrate that the P2Y6 receptor suppresses the effector function of NK cells and antitumor activity in vitro and in vivo.

FIGURE 5.

P2Y6 receptor suppressed cytotoxic and antitumor metastatic potential of NK cells. (A) Assessment of natural cytotoxicity of WT and P2Y6−/− splenic NK cells against YAC-1 target cells using FACS. (B) Assessment of natural cytotoxicity of WT and P2Y6−/− splenic NK cells on YAC-1 target cells after stimulation with IL-15. (CE) B16/F10 metastasis assay; the indicated mice were injected i.v. with 1 × 105 B16/F10 or 2 × 106 FACS WT and P2Y6−/− splenic NK cells. The mice were sacrificed 14 d later, and the lung weights and numbers of tumor nodules were counted. Graphs show mean ± SD (n = 6). Data are representative of two or three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (*p < 0.05, **p < 0.01).

FIGURE 5.

P2Y6 receptor suppressed cytotoxic and antitumor metastatic potential of NK cells. (A) Assessment of natural cytotoxicity of WT and P2Y6−/− splenic NK cells against YAC-1 target cells using FACS. (B) Assessment of natural cytotoxicity of WT and P2Y6−/− splenic NK cells on YAC-1 target cells after stimulation with IL-15. (CE) B16/F10 metastasis assay; the indicated mice were injected i.v. with 1 × 105 B16/F10 or 2 × 106 FACS WT and P2Y6−/− splenic NK cells. The mice were sacrificed 14 d later, and the lung weights and numbers of tumor nodules were counted. Graphs show mean ± SD (n = 6). Data are representative of two or three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (*p < 0.05, **p < 0.01).

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T-bet and Eomes have been reported to play key roles in the maturation and activation of NK cells (33, 34). To further investigate the mechanism by which the P2Y6 receptor affects the maturation and activation of NK cells, we detected the expression of transcription factors T-bet and Eomes in NK cells and their subsets in the spleen of WT and P2Y6−/− mice. We found that the expression of T-bet in P2Y6−/− NK cells was significantly higher than that in WT NK cells, but there was no obvious change in the expression of Eomes (Fig. (6A, 6B). Similar results were found in splenic NK cell subsets CD11b-CD27+ NK cells, CD11b+CD27+ NK cells, and CD11b+CD27 NK cells from P2Y6−/− mice compared with WT mice (Fig. (6C, 6D), indicating that P2Y6 suppressed T-bet expression in NK cells.

FIGURE 6.

P2Y6 receptor inhibits the expression of T-bet in NK cells with or without IL-15 stimulation. (A and B) Representative overlaid histograms showing T-bet and Eomes expression by the indicated NK cells. Red lines, WT NK cells stained with T-bet or Eomes Ab; blue lines, P2Y6−/− NK cells stained with anti–T-bet or Eomes Ab. Quantifications of mean fluorescence intensity (MFI) are shown (right panel). (C and D) Representative overlaid histograms showing T-bet and Eomes expression by the indicated NK cell subsets. Red lines, WT NK cells stained with T-bet or Eomes Ab; blue lines, P2Y6−/− NK cells stained with T-bet or Eomes Ab. Quantifications of MFI are shown (right panel). Representative overlaid histograms showing T-bet (E) and Eomes (F) expression by the indicated NK cells after treatment with IL-15. Blue lines, WT NK cells stained with T-bet or Eomes Ab; red lines, P2Y6−/− NK cells stained with T-bet or Eomes Ab. Quantifications of MFI are shown (right panel). (G) Representative overlaid histograms showing T-bet expression by NK-92 cells after the stimulation with IL-15 in the presence or absence of UDP or 5-OMe-UDP. (H) Representative histogram plot showing T-bet expression in human PBMC NK cells after the stimulation with IL-15 in the presence of 5-OMe-UDP. Graphs show mean ± SD (n = 6). Data are representative of three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (*p < 0.05, **p < 0.01).

FIGURE 6.

P2Y6 receptor inhibits the expression of T-bet in NK cells with or without IL-15 stimulation. (A and B) Representative overlaid histograms showing T-bet and Eomes expression by the indicated NK cells. Red lines, WT NK cells stained with T-bet or Eomes Ab; blue lines, P2Y6−/− NK cells stained with anti–T-bet or Eomes Ab. Quantifications of mean fluorescence intensity (MFI) are shown (right panel). (C and D) Representative overlaid histograms showing T-bet and Eomes expression by the indicated NK cell subsets. Red lines, WT NK cells stained with T-bet or Eomes Ab; blue lines, P2Y6−/− NK cells stained with T-bet or Eomes Ab. Quantifications of MFI are shown (right panel). Representative overlaid histograms showing T-bet (E) and Eomes (F) expression by the indicated NK cells after treatment with IL-15. Blue lines, WT NK cells stained with T-bet or Eomes Ab; red lines, P2Y6−/− NK cells stained with T-bet or Eomes Ab. Quantifications of MFI are shown (right panel). (G) Representative overlaid histograms showing T-bet expression by NK-92 cells after the stimulation with IL-15 in the presence or absence of UDP or 5-OMe-UDP. (H) Representative histogram plot showing T-bet expression in human PBMC NK cells after the stimulation with IL-15 in the presence of 5-OMe-UDP. Graphs show mean ± SD (n = 6). Data are representative of three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (*p < 0.05, **p < 0.01).

Close modal

We also found that the expression of T-bet in P2Y6−/− NK cells was significantly higher than that in WT NK cells after IL-15 stimulation, but the expression of Eomes remained unaltered (Fig. (6E, 6F). To further verify this result, we also used the P2Y6 receptor agonist UDP or 5-OMe-UDP to treat NK-92 cells or human peripheral blood NK cells. The results showed that the expression of T-bet was significantly decreased after UDP or 5-OMe-UDP treatment in the presence of Poly(I:C) or IL-15 compared with the control group (Fig. (6G, 6H). Taken together, these data indicate that the P2Y6 receptor represses the expression of T-bet and suppresses the maturation and activation of NK cells.

To investigate the mechanism by which P2Y6 receptor deficiency increases the expression of T-bet in NK cells, leading to the maturation and activation of NK cells, we detected the downstream signaling pathways of IL-15. The results showed that there was no significant difference in the level of p-FOXO1, which is one of the downstream proteins of IL-15 signaling pathways that control the maturation and activation of NK cells (10), between WT and P2Y6−/− NK cells (Fig. (7A). Furthermore, compared with WT NK cells, the expression level of FOXO1 did not change in the different mature stages of NK cells in P2Y6−/− NK cells (Fig. (7B), suggesting that the P2Y6 receptor does not affect FOXO1 expression and phosphorylation in NK cells. We next detected the expression of IL-15 receptor CD122 and CD132 in WT and P2Y6−/− NK cells and found that there was no significant difference between them (Supplemental Fig. 3). We then detected the expression of nutrition proteins CD71 and CD98, which are associated with the mTOR signaling pathway. We found that after IL-15 stimulation, the expression of CD71 and CD98 in P2Y6−/− NK cells was significantly higher than that in WT NK cells (Fig. (7C, 7D), suggesting that the P2Y6 receptor may inhibit mTOR signaling. Thus, we investigated the activation of the mTOR signaling pathway in WT and P2Y6−/− NK cells upon IL-15 stimulation and found that the phosphorylation level of Akt and S6 protein after IL-15 stimulation in WT NK cells was significantly lower than that in P2Y6−/− NK cells, but the level of 4EBP1 phosphorylation was not significantly altered (Fig. (7E–G). Taken together, our results demonstrate that the P2Y6 receptor inhibits the phosphorylation of Akt and S6 proteins upon IL-15 stimulation in NK cells.

FIGURE 7.

P2Y6 receptor suppresses the activation of the mTOR signaling pathway in NK cells. (A) Splenic NK cells were stimulated with IL-15 (100 ng/ml). Cells were harvested at the indicated time points, followed by FACS analysis using a p-FOXO1Ser256 Ab (n = 3). (B) FACS analysis of the expression of FOXO1 in splenic NK cell subsets (n = 5). (C and D) Splenocytes from WT or P2Y6−/− mice were treated with IL-15 overnight. Expression of CD71 (C) and CD98 (D) was detected by flow cytometry, and the mean fluorescence intensity (MFI) was calculated (n = 3). (EG) WT and P2Y6−/− splenic NK cells were treated for 0, 1, or 2 h. Intracellular p-AktS473, p-S6, and p-4EBP1 were detected by flow cytometry (top), and the MFI was calculated (bottom) (n = 3). Graphs show mean ± SD. Data are representative of three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (***p < 0.001).

FIGURE 7.

P2Y6 receptor suppresses the activation of the mTOR signaling pathway in NK cells. (A) Splenic NK cells were stimulated with IL-15 (100 ng/ml). Cells were harvested at the indicated time points, followed by FACS analysis using a p-FOXO1Ser256 Ab (n = 3). (B) FACS analysis of the expression of FOXO1 in splenic NK cell subsets (n = 5). (C and D) Splenocytes from WT or P2Y6−/− mice were treated with IL-15 overnight. Expression of CD71 (C) and CD98 (D) was detected by flow cytometry, and the mean fluorescence intensity (MFI) was calculated (n = 3). (EG) WT and P2Y6−/− splenic NK cells were treated for 0, 1, or 2 h. Intracellular p-AktS473, p-S6, and p-4EBP1 were detected by flow cytometry (top), and the MFI was calculated (bottom) (n = 3). Graphs show mean ± SD. Data are representative of three independent experiments. Statistical significance was assessed using Student t test or one-way ANOVA test (***p < 0.001).

Close modal

As an important immune cell, NK cells kill viral-infected cells and tumors by releasing proinflammatory cytokines and cytotoxic granules and play a very important role in resisting infections and killing tumors (5, 3538). NK cells secrete more inflammatory cytokines and cytotoxic granules after maturation, resulting in stronger cytotoxicity (39, 40). We first found that the expression of the P2Y6 receptor is reduced after NK cell activation, which suggests that the P2Y6 receptor may regulate the function of NK cells. Therefore, we performed a series of studies on how the P2Y6 receptor regulates the development, maturation, and function of NK cells. We found that the proportion and absolute number of T cells, B cells, and CD11b+ cells did not change in P2Y6−/− mice compared with WT mice. Similarly, the proportion and absolute number of CD3-NK1.1+ NK cells did not change in the different organs and tissues of WT and P2Y6−/− mice, suggesting that the P2Y6 receptor does not affect NK cell numbers. However, our results showed that the proportion of NKp cells was significantly decreased, and the proportion of iNK was significantly increased in P2Y6−/− mice, indicating that the P2Y6 receptor inhibits the development of NK cells. As the proportion of iNK cells is increased significantly in P2Y6-deficient mice, they can develop into more mNK cells. Thus, we hypothesized that the P2Y6 receptor might affect the maturation of NK cells. We also observed that the proportion of CD11b+CD27 NK cells in the spleen, liver, and lung was significantly increased, but the proportion of CD11b+CD27 NK cells in the bone marrow and lymph nodes from WT and P2Y6−/− mice was similar, indicating that the P2Y6 receptor inhibits the development of NK cells from NKp to iNK cells, and eventually suppresses the terminal maturation of NK cells.

Mature NK cells produce more cytokines, especially IFN-γ, and show stronger cytotoxicity (5, 41). We show that P2Y6−/− NK cells produce more IFN-γ after treatment with Poly(I:C), IL-15, IL-12 plus IL-15, or PMA. We also discovered that the production of IFN-γ was significantly decreased in murine NK cells, NK-92, and human NK cells isolated from human PBMC upon treatment with P2Y6 receptor agonist UDP or analog 5-OMe-UDP. Therefore, we hypothesize that P2Y6 receptor deficiency can promote the cytotoxicity of NK cells by increasing the production of IFN-γ. Thus, we designed a cytotoxic experiment in vitro and a tumor metastasis model in vivo and found that the cytotoxicity of P2Y6−/− NK cells against YAC-1 cells was stronger than that of the WT NK cells. The P2Y6−/− NK cell cytotoxicity was also stronger than that of WT NK cells after IL-15 treatment. To verify the cytotoxicity of NK cells in vivo, we established a tumor metastasis model. We found that mice injected with P2Y6−/− NK cells had fewer lung tumor nodules and lower lung weight than mice injected with WT NK cells. These results indicate that the P2Y6 receptor suppresses the maturation and activation of NK cells and eventually inhibits the cytotoxicity of NK cells.

Recent findings suggest that many transcription factors, especially T-bet and Eomes, regulate the development, maturation, and function of NK cells (810, 33, 34). Our data showed that the expression of T-bet, but not Eomes, in P2Y6−/− NK cells was significantly higher than that in WT NK cells. Further analysis of the expression of T-bet in the three stages of NK cell maturation showed that the expression of T-bet in CD11bCD27+ NK cells, CD11b+CD27+ NK cells, and CD11b+CD27 NK cells was significantly higher in P2Y6−/− mice than in WT mice, but there was no significant difference in the expression of Eomes. We also used the P2Y6 receptor agonist UDP or UDP analog 5-OMe-UDP to treat mouse NK cells, NK-92, or human peripheral blood NK cells. The results showed that the expression of T-bet was significantly decreased compared with that in the control group. These results indicate that the P2Y6 receptor inhibits the expression of T-bet in NK cells.

It has been reported that a decrease in transcription factor FOXO1 expression in NK cells leads to an increase in T-bet expression (10, 42). Our results showed that there was no significant difference in the expression of FOXO1 in WT and P2Y6−/− NK cells, and there was no significant difference in the level of p-FOXO1 in WT and P2Y6−/− NK cells after IL-15 treatment (13, 4345). Therefore, we believe that the high expression of T-bet in P2Y6−/− NK cells was not related to p-FOXO1. NK cells can activate the PI3K-Akt-mTOR signaling pathway after IL-15 treatment. We first explored whether P2Y6 receptor knockout affects the expression of IL-15 receptors on NK cells. The results showed that IL-15 receptor expression in WT and P2Y6−/− NK cells was not altered. We further hypothesized that the P2Y6 receptor may affect downstream signaling pathways related to IL-15 receptors. The nutrient proteins CD71 and CD98 are related to the metabolism of NK cells and activation of the mTOR signaling pathway (40). We found that the expression of CD71 and CD98 in P2Y6−/− NK cells was significantly increased after IL-15 treatment overnight. Therefore, we speculated that P2Y6 may affect the activation of the mTOR signaling pathway.

mTOR protein is an atypical serine/threonine kinase that exists in two different complexes, mTORC1 and mTORC2. It is a major growth regulator that can be felt and combined with different nutritional and environmental factors, including growth factors, energy levels, cell stress, and amino acids. It combines these signals by phosphorylation to enhance anabolism (such as mRNA translation and lipid synthesis), or limit catabolism (such as autophagy), or promote cell growth. It has been reported that NK cells activate PI3K and induce PIP3 production after IL-15 stimulation in NK cells. PIP3 induces Akt phosphorylation and mTORC2 activation (46, 47). We found that upon IL-15 stimulation for 2 h, the level of p-Akt in P2Y6−/− NK cells was increased significantly compared with that in WT NK cells. The phosphorylation of Akt inhibits the expression of TSC1/TSC2 and the activity of the GTP enzyme Rheb activation while inducing the activation of mTORC1, which leads to the phosphorylation of S6 and 4EBP1. Our results showed that the level of pS6 in P2Y6−/− NK cells was significantly increased after IL-15 treatment for 1 h and 2 h, but the level of p-4EBP1 did not significantly change. These results indicate that the P2Y6 receptor inhibits the activation of the mTOR signaling pathway induced by IL-15, thereby inhibiting the activation of NK cells. It has been reported that Gαq inhibits the expression of Akt (48, 49), whereas the P2Y6 receptor can activate Gαq. Therefore, we speculate that the P2Y6 receptor may inhibit the expression of Akt by activating Gαq, thus suppressing the level of p-Akt, which eventually represses the activation of the mTOR signaling pathway and eventually inhibits the maturation and function of NK cells. Based on the advantages of the P2Y6 receptor, engineering P2Y6 knockout chimeric Ag receptor NK cells (CAR-NK) or combining chimeric Ag receptor NK cells with a P2Y6 inhibitor may enhance antitumor effects.

In this study, we identified the novel functions of the P2Y6 receptor in the NK cells. We found that as a negative regulator, P2Y6 inhibited the maturation and activation of the NK cells and suppressed the cytotoxicity of the NK cells via the mTOR signaling pathway. Therefore, P2Y6 may act as a potential target for the immunoregulation of NK cells.

We thank Prof. Min Qian for providing P2Y6-knockout mice.

This work was supported by the National Natural Science Foundation of China (81771306 and 81072459), the Science and Technology Commission of Shanghai Municipality (21S11906200, 201409002900, and 14140904200), the National Key Research and Development Program of China (2016YFC1200400), the Program for New Century Excellent Talents in University (NCET-12-0179), and the ECNU Public Platform for Innovation (011).

The online version of this article contains supplemental material.

Abbreviations used in this article

     
  • GPCR

    G protein–coupled receptor

  •  
  • iNK

    immature NK

  •  
  • MFI

    mean fluorescence intensity

  •  
  • mNK

    mature NK

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • NKp

    NK precursor

  •  
  • qPCR

    quantitative PCR

  •  
  • RT-qPCR

    real-time qPCR

  •  
  • WT

    wild-type.

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The authors have no financial conflicts of interest.

Supplementary data