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Published in final edited form as: J Allergy Clin Immunol. 2014 Mar 31;134(1):195–203.e4. doi: 10.1016/j.jaci.2014.02.018

MicroRNA-150 regulates the cytotoxicity of natural killers by targeting perforin-1

Nayoung Kim a, Miju Kim c, Sohyun Yun a, Junsang Doh c,d, Philip D Greenberg e, Tae-Don Kim a,b,*, Inpyo Choi a,b,*
PMCID: PMC4125537  NIHMSID: NIHMS569926  PMID: 24698324

Abstract

Background

Perforin-1 is the predominant cytolytic protein secreted by natural killer cells. For a rapid immune response, resting NK cells contain high Prf1 mRNA concentrations while exhibiting minimal cytotoxicity due to a blockage of Prf1 protein synthesis, implying that an unknown post-transcriptional regulatory mechanism exists.

Objective

We sought to determine that microRNA-150 (miR-150) post-transcriptionally regulates Prf1 translation in both mouse and human NK cells at rest and at various time points after activation.

Methods

To investigate the role of miR-150 in NK cells, mouse NK cells with a targeted deletion of miR-150 (miR-150−/− NK cells), primary human NK cells, and NK92 MI cells were utilized. NK cell cytotoxicity assays and western blotting proved that activated miR-150−/− NK cells expressed upregulated Prf1, augmenting NK cell cytotoxicity. When immunodeficient mice were injected with miR-150−/− NK cells, there was a significant reduction in tumor growth and metastasis of B16F10 melanoma.

Results

We report that miR-150 binds to 3′ untranslated regions of mouse and human Prf1, post-transcriptionally downregulating its expression. Mouse wild-type NK cells displayed downregulated miR-150 expression in response to interleukin-15, which led to corresponding repression and induction of Prf1 during rest and after IL-15 activation, respectively.

Conclusion

Our results indicated that miR-150 is a common post-transcriptional regulator for Prf1 in mouse and human NK cells that represses NK cell lytic activity. Thus the therapeutic control of miR-150 in NK cells could enhance NK cell based immunotherapy against cancer, providing a better clinical outcome.

Keywords: miR-150, NK cells, Prf1, NK cell cytotoxicity, post-transcriptional regulation, immunotherapy, tumor growth and metastasis

NK cells kill target cells predominantly by secreting granule toxins including perforin-1 (Prf1) and granzymes (Gzms).1–3 Prf1 is a pore-forming protein, and Gzms are structurally-related serine proteases that lyse target cell protein at specific aspartate residues.4, 5. Prf1 disturbs the target cell membrane and facilitates the entry and/or trafficking of Gzms.6 Unlike multiple Gzms which can compensate each other, Prf1 is encoded by a single gene and does not have any functional redundancy.7

NK cells kill target cells “naturally” without prior antigen-specific recognition, allowing for rapid induction of lytic activity.8 For prompt immune responses, resting NK cells are in a “prearmed” state, containing high concentrations of Prf1 mRNA but they are minimally cytotoxic due to a blockage of Prf1 translation.9 Upon target recognition, activated NK cells immediately arm themselves with preformed Prf1 mRNA, correlating with an increase in Prf1 protein. Therefore, Prf1 is post-transcriptionally regulated by unknown regulators in resting NK cells.

MicroRNAs (miRNAs) are small noncoding RNAs of ~22 nucleotides that functions as post-transcriptional inhibitor by complementary to the 3′ UTR of their target mRNAs.10 Over the past few years, emerging data has implied that endogenously generated miRNAs are post-transcriptional regulators of immune cell development and function.11 miR-150 has been identified as a lymphocyte-specific miRNA because it is predominantly expressed in the lymph nodes, spleen, and thymus and is highly upregulated during lymphocyte maturation. miR-150 expression increases sharply in mature B and T lymphocytes as well as in mature NK and invariant NK T (iNKT) cells, but not in their progenitors.12–16

Although miR-150 expression is upregulated during lymphocyte maturation, it is downregulated again during the activation of mature B and T cells. Xiao et al. reported that miR-150 expression was downregulated in activated B cells, and miR-150-deficient mice exhibit enhanced humoral and T cell-dependent antibody responses with increased steady-state immunoglobulin (Ig) levels.14 Collectively, miR-150 appears to be inversely associated with immunological functions of activated B and T cells, but a relationship between miR-150 and the activation of NK cells has not yet been shown.

In this study, we report that miR-150 binds to 3′ UTR of mouse and human Prf1, post-transcriptionally downregulating its expression. Mouse WT NK cells exhibited biphasic up- and down-miR-150 expression in response to IL-15, which led to corresponding repression and induction of Prf1 during rest and after IL-15 activation, respectively. Primary human NK cells also display downregulated miR-150 and augmented Prf1 in response to IL-15. Our results suggest that miR-150−/− NK cells may provide clinical benefit to minimize spontaneous activation in resting NK cells while maximize cytotoxicity in activated NK cells. Therapeutic modulation of miR-150 may be a promising new approach for enhancing NK cell-mediated immunotherapy to treat various human pathologies.

Method

See supplemental materials and methods for further details.

Results

miR-150 expression is inversely proportional to Prf1 protein expression in both mouse and human NK cells during IL-15 activation

The expression of miR-150 in mouse WT NK cells exhibited a biphasic pattern during IL-15 stimulation. The level of miR-150 was sharply increased nearly two-fold at 4 h and maintained for a while. Then, its level was markedly declined to below the level of resting NK cells at 24 h and further downregulated at 72 h. miR-150 level was negatively associated with the expression of Prf1 protein (Fig 1, A). Primary human NK cells showed gradually decreased expression of miR-150 and elevated level of Prf1 protein in response to IL-15 (Fig 1, B). In both mouse and human NK cells, relatively high abundance of miR-150 and sustained expression of Prf1 and GzmB protein were observed during the first 6 h of IL-15 stimulation. Then, Prf1 and GzmB protein expression started to increase, accompanied by reduced expression of miR-150 at 24 h (Fig 1, C and D, top). In addition, GzmB mRNA was notably increased in a time-dependent manner correlating with an increase in GzmB protein, but abundant mRNA of Prf1 in resting NK cells remained largely unchanged during IL-15 activation in both mouse and human NK cells (Fig 1, C and D, bottom). It implies that Prf1 could be post-transcriptionally repressed by miR-150 in NK cells at rest and earlier time points after IL-15 activation.

FIG 1.

FIG 1

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miR-150 is involved in the posttranscriptional regulation of Prf1 in NK cells. miR-150 level and Prf1 protein were inversely plotted as means ± SD in primary mouse (A) and human NK cells (B). Protein and mRNA levels of Prf1 and GzmB were determined in mouse (C) and human NK cells (D) during IL-15 stimulation. Results are representative of three independent experiments.

miR-150−/− NK cells have augmented Prf1 protein expression and enhanced NK cell cytotoxicity

Resting WT and miR-150−/− NK cells minimally expressed Prf1 protein, but Prf1 protein was amplified in miR-150−/− NK cells at 48 h and 72 h of IL-15 stimulation. However, Prf1 mRNA abundance did not changed over time (Fig 2, A). After 48 h of IL-15 stimulation, miR-150−/− NK cells exhibited enhanced cytotoxicity by ~2-fold at an effector:target (E:T) ratio of 5:1 (Fig 2, B). Collectively, abundant Prf1 mRNA was post-transcriptionally suppressed in resting NK cells, but miR-150−/− NK cells augmented Prf1 protein expression and cytotoxicity upon IL-15 activation.

FIG 2.

FIG 2

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miR-150−/− NK cells exhibit upregulation of Prf1 and enhanced NK cell cytotoxicity. Protein and mRNA levels of Prf1 in WT and miR-150−/− NK cells after stimulation with IL-15 were presented (A). NK cell cytotoxicity was measured by standard 4 h 51Cr-release assays (B). Data represent means ± SDs of three independent experiments. (* P < 0.05, Student’s t-test).

WT and miR-150−/− NK cells have similar NK cell receptor profiles, degranulation, and death receptor/ligand interactions

NK cells express various receptors to recognize target cells. We, therefore, examined a broad range of activating NK cell receptors (NKG2D, NKp46, and Ly49D), inhibitory receptors (Ly49C/I, Ly49G2, and Ly49A), IL-15 receptors (CD122 and CD132), and chemokine receptors (CXCR5 and CCR6). IL-15 activated WT and miR-150−/− NK cells displayed similar frequencies in the receptor repertoire including IL-15 receptors and little or no chemokine receptor expressions (Fig 3, A). It led to similar NK cell degranulation evidenced by intensity of CD107a in WT and miR-150−/− NK cells after treatment with antibody against NKp46 in the presence or absence of target cells (Fig 3, B). NK cell cytotoxicity can also be mediated in the absence of Prf1 by the engagement of death receptors (e.g. Fas/CD95) on target cells via their cognate ligands (e.g. FasL) on NK cells.17 We investigated the level of two key effector ligands, FasL and TNF-related apoptosis-inducing ligand (TRAIL), and death receptor CD95 in IL-15 activated NK cells. miR-150 had no profound effects on the expression of these ligands and receptor (Fig 3, A and C). These data suggested that augmented cytotoxicity of miR-150−/− NK cells is caused predominantly by enhanced Prf1, and not by significant changes in NK cell receptor profiles, IL-15 receptor-mediated signaling pathways, or death receptor/ligand interactions.

FIG 3.

FIG 3

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miR-150 has no significant effects on NK cell receptor profiles (A), degranulation (B), and death receptor/ligand interactions (C). WT and miR-150−/− NK cells were cultured in IL-15 for 48 h, and then cell surfaces were stained with indicated antibodies. Anti-NKp46 antibody was used to activate NKp46 receptors on NK cells. Data represent means ± SDs of three independent experiments.

miR-150−/− NK cells show potent lytic granules hit at the immunological synapse

NK cells are functionally heterogenous, and thus, only a small portion of NK cells kill target cells.18 To assess the dynamics of individual NK cell cytotoxicity, lytic granules of NK cells were labeled with LysoSensor Green and target cells were labeled with DDAO-SE and then co-cultured in propidium iodide (PI)-containing media. Larger amounts of lytic granules containing Prf1 and GzmB were found in miR-150−/− NK cells than WT NK cells indicated by microscopic granule intensity showing about 2-fold increase (Fig 4, A). In accordance, mean fluorescence intensity (MFI) of intracytoplasmic Prf1 by flow cytometry showed increased level of Prf1 protein in miR-150−/− NK cells (Fig 4, B). Additionally, WT and miR-150−/− NK cells were stained with CD107a, which is a sensitive marker of NK cell degranulation,19 in the absence or presence of target cells. WT and miR-150−/− NK cells were minimally degranulated without targets, but substantially degranulated following simulation with target cells. However, miR-150 had no significant effects on the extent of degranulation, and thus, the overall degree of degranulation were similar in WT and miR-150−/− NK cells (Fig 4, C).

FIG 4.

FIG 4

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miR-150−/− NK cells induce a potent lytic hit at the immunological synapse. NK cells were stained with LysoSensor (A), Prf1 (B), or CD107a (C). Dynamic interface formed between NK cells and target cells were photographed (D) and illustrated (E). Percentage of NK cells remaining at each step (F), kinetics of steps C, D, and E (G) and cytotoxicity (H) indicated. MFI; mean fluorescence intensity.

In order to understand NK -target interactions, we examined sequential stages of lytic synapses: the initiation stage, the effector stage, and the termination stage (Orange, 2008). In initiation stage, NK cells transiently contact with target cells either with diffused granules (Step A) or with polarized granules (Step B). In the effector stage, NK cells formed stable contacts with their targets and polarized lytic granules to distal poles (Step C). Then, lytic granules were transported to the immunological synapse (IS) (Step D). In the termination stage, NK cells eventually killed target cells determined either by the formation of membrane blebs (Step E) or increase in PI fluorescence (Fig 4, D, right panel). These sequential steps are illustrated in Fig 4, E. Among NK cells encountered target cells, the percentages of NK cells remaining at each step at the end of 2 h time-lapse imaging were measured and plotted in Fig 4, F (See Supplementary Movies E1-5 for each case). The kinetics of Steps C, D and E in WT and miR-150−/− NK cells were further assessed at various time points after co-culture. Throughout the experiments, the higher percentage of WT NK cells remained at Step D, but miR-150−/− NK cells exhibited higher cytotoxicity than WT NK cells when the duration of NK-target contact became longer than 60 min (Fig 4, G and H). It suggests that polarized granules in WT NK synapses may not be as effective as those in miR-150−/− NK cells to kill target cells. Since the levels of degranulation were comparable between WT and miR-150−/− NK cells (Fig 4, C), it is likely that miR-150−/− NK cells exhibit more potent lytic hits to targets than WT NK cells potentially due to higher contents of lytic granules.

miR-150 directly targets Prf1 in both mouse and human NK cells

To test whether miR-150 directly targets Prf1, aralkylamine N-acetyltransferase (AANAT) reporter assays were performed.20 AANAT reporter plasmids contain a neomycin resistant gene (NeoR) and AANAT coding region attached to GzmB 3′ UTR or Prf1 3′ UTR (Fig 5, A). According to the miRanda algorithm (www.microrna.org), miR-150 has one putative binding site in the 3′ UTR of mouse Prf1 (Fig 5, B). Moreover, it has a predicted binding site in the 3′ UTR of both human GzmB and Prf1 which have less complementary base pairing compared to mouse Prf1 (Fig 5, B and C). miR-150 mimic did not change the expression of AANAT containing the 3′ UTR of mouse or human GzmB suggesting GzmB was not the real target of miR-150. In contrast, miR-150 mimic significantly reduced the expression of AANAT containing mouse Prf1 3′ UTR in a dose-dependent manner (Fig 5, D, top) and human Prf1 3′ UTR to a lesser extent as predicted by less complementarity (Fig 5, E, top). miR-150 mutant, which had mutations in the 5′ end of miRNA referred to as seed region (Fig 5, B and C), lost binding capacity to the target, and thus attenuated its inhibitory effects on mouse or human Prf1 3′ UTR (Fig 5, D and E, top). To confirm that these phenomena dominantly occurred by miR-150-mediated AANAT reduction and were not caused by exogenous AANAT mRNA, the levels of AANAT mRNA were normalized to the amount of NeoR mRNA. Minimal AANAT mRNA changes were observed among transfected cells (Fig 5, D and E, bottom). AANAT mRNA level was relatively higher in human Prf1 (> 1.6 fold), but the AANAT protein expression was rather downregulated by miR-150 mimic (Fig 5, E, bottom, lane 2). Taken together, these data suggest that Prf1 is a conserved functional target of miR-150 in both mouse and human NK cells.

FIG 5.

FIG 5

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miR-150 directly targets Prf1 in NK cells. A scheme of reporter vector (A) and sequence alignments of miR-150 to targets (B and C) are shown. 3′ UTR AANAT reporter containing mouse GzmB/Prf1 (D) or human Prf1/GzmB (E) co-transfected with indicated miRNAs and then AANAT proteins and mRNAs were analyzed. Data represent means ± SDs of three independent experiments. (+) 50nM, (++) 100nM.

Lentivirus-mediated overexpression of miR-150 shows a significant decrease in Prf1 and diminished NK cell mediated cytotoxicity

To test whether miR-150 overexpression and miR-150 deficiency would cause opposite effects on mouse or human NK cells, miR-150−/− NK cells were transduced by lentivirus containing miR-150 precursor (LV-miR150) and cultured in high concentration of IL-15 (100 ng/ml) for 2 d. LV-mediated upregulation of miR-150 markedly repressed Prf-1 protein expression compared with cells transduced with a control vector (Fig 6, A), which led to diminished NK cell cytotoxicity (Fig 6, B). In this case, higher NK cell cytotoxicity was observed in a high concentration of IL-15 (100 ng/ml) (Fig 6, B) compared with a modest concentration of IL-15 (25ng/ml) (Fig 2, B) at the same E:T ratio.

FIG 6.

FIG 6

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Lentivirus-mediated overexpression of miR-150 suppresses Prf1 and cytotoxicity. After LV-transduction, miR-150−/− NK cells and NK92 MI showed decreased Prf1 protein (A and E) and NK cell cytotoxicity (B and F), respectively. Low endogenous miR-150 in NK92 MI (C) was increased after LV-transduction (D). Data represent the means ± SDs from three independent experiments. * P < 0.05, ** P < 0.01.

Compared with in vitro differentiated-human mature NK cells (mNK), NK92 MI cells expressed a significantly low level of endogenous miR-150 (Fig 6, C), and thus were selected for applying LV-mediated overexpression. During LV-transduction, the level of miR-150 in NK92 MI was increased in a time-dependent manner (Fig 6, D). Modest upregulation of miR-150 after 2 d of LV transduction did not change human Prf1 protein, but robust expression of miR-150 significantly inhibited Prf1 production at 4 d after LV-150 transduction (Fig 6, D and E), which contributed to reduced NK cell cytotoxicity (Fig 6, F). Collectively, these data imply that overexpression of miR-150 can act as a negative regulator of NK cell lytic activity by repressing Prf1 in both mouse and human NK cells.

miR-150−/− NK cells significantly reduce tumor growth and metastasis in immunocompromised mice

To investigate the role for miR-150 in immune surveillance against tumor cells in vivo, activated NK cells were injected intravenously, and then B16F10 melanoma were transplanted subcutaneously into Rag2−/−γC−/− mice, which lack B, T, and NK cells.21 The adoptive transfer of miR-150−/− NK cells significantly reduced tumor volumes to an average of 71.23 ± 20.31 mm3 (mean ± SD, n = 5), but WT NK cells less substantially reduced tumor volumes with an average of 146.62 ± 109.34 mm3 (mean ± SD, n = 5) on day 11 after B16F10 implantation (Fig 7, A).

FIG 7.

FIG 7

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miR-150−/− NK cells exhibit enhanced tumor surveillance in immunocompromised mice. Adoptive transfer of miR-150−/− NK cells effectively reduced tumor growth (A) and lung metastasis of B16F10 melanoma (B and C) compared to WT NK cells in Rag2−/−γC−/− mice. Data represent the means ± SDs of two independent experiments. * P < 0.05.

NK cells also play critical roles in reducing lung metastasis in various murine cancer models, and Prf1 is highly involved in the inhibition of tumor metastasis.22 To define the potential role for miR-150 in limiting lung metastasis of melanoma in vivo, activated WT and miR-150−/− NK cells were injected intravenously and then highly metastatic murine B16F10 cells were injected into the tail veins of Rag2−/−γC−/− mice at 2 d after NK cell transplantation. The numbers of pulmonary metastatic colonies were quantitated and photographed at 14 d after B16F10 implantation (Fig 7, B and C). miR-150−/− NK cells effectively inhibited lung metastasis of B16F10 melanoma to an average of 170.71 ± 25.23 (mean ± SD, n = 7) compared with WT NK cells with an average of 65.67 ± 21.42 (mean ± SD, n = 6) (Fig 7, B). Taken together, these results demonstrate that miR-150−/− NK cells exhibit augmented NK cell cytotoxicity against tumor growth and lung metastasis of B16F10 melanoma in immunodeficient mice.

Discussion

Prf1 was first isolated and characterized from cytotoxic lymphocytes in the mid-1980s, and significant progress has been made in understanding how NK cells use Prf1 to eliminate target cells. However, our knowledge of how preformed Prf1 mRNA is post-transcriptionally regulated in resting NK cells is still limited. Recent achievements have highlighted the importance of miRNA-mediated Prf1 regulation in human NK cells and two studies have been summarized.23, 24 A study from our laboratory demonstrated that miR-27a* targeted the 3′ UTRs of Prf1 and GzmB in in-vitro differentiated human NK cells at rest or after IL-15 activation. 20 However, freshly isolated resting human NK cells expressed very low levels of miR-27a*. 25 It suggests that in-vitro differentiated human NK cells that result from long-term culture with high levels of cytokines have significantly different miRNA expression than freshly isolated primary human NK cells. Wang et al. reported that miR-30e repressed Prf1 in human NK cells upon IFN-α stimulation.25 The level of miR-30e was shown to be inversely associated with Prf1 protein expression in vitro, but its role for immune surveillance against tumor cells in vivo still need to be elucidated. Although these studies address the role for miRNAs in human NK cells, extensive research on miRNA-mediated Prf1 regulation in both mouse and human NK cells at rest and at various time points after activation has not been investigated.

Here, we report that miR-150 post-transcriptionally regulates Prf1 in freshly isolated primary mouse and human NK cells at rest and after IL-15 activation. Unlike exogenous Prf1 gene transfer to NK cells, which constitutively expressing Prf1 without stimuli, an inverse correlation between miR-150 level and Prf1 expression during IL-15 activation serves two purposes. First, mature resting or insufficiently activated NK cells contain substantial amounts of endogenous miR-150, which prevents spontaneous activation of the newly-generated NK cells without adequate stimuli through repression of pre-existing Prf1 mRNA translation. Second, reduced miR-150 expression in fully activated NK cells allows enhanced production of Prf1 from preformed Prf1 mRNA for prompt and potent immune responses.

Even though miR-150−/− NK cells exhibited amplified Prf1 translation at 48 and 72 h of IL-15 stimulation, resting miR-150−/− NK cells still minimally express Prf1 protein similarly to resting WT NK cells (Fig 2, A, top). Xiao et al. also reported that activated miR-150−/− B cells highly expressed c-Myb protein, a known target of miR-150, compared to WT B cells at 48 and 72 h of anti-IgM stimulation, but resting miR-150−/− B cells minimally expressed c-Myb protein 14. How resting miR-150−/− NK cells are minimally cytotoxic still need to be clarified, but this phenomenon makes miR-150−/− NK cells more attractive for adoptive NK cell therapy because of their unique ability to maintain minimal cytotoxicity at rest while exhibiting maximal lytic activity upon activation.

miR-150 is highly upregulated in mature B, T, and NK cells, but not in their progenitors.12–16 Ectopic expression of miR-150 in the mouse B cell precursors leads to severe defects in B cell development at the transition from the pro-B to pre-B cells stage by targeting c-Myb, but impairment of T cell development was less severe.14, 26 miR-150 deficient mice have significant reductions in NK cell development and maturation.12 Here, we revealed that same number of miR-150−/− NK cells showed enhanced cytotoxicity than WT NK cells by augmented Prf1 production which led to more powerful lytic hits to target cells. Collectively, it implies that miR-150−/− mice might compensate for reduced numbers of mature NK cells by alternatively improving NK cell effector function.

miR-150 also plays a critical role as a tumor suppressor by targeting the proto-oncogene c-Myb and Notch3. miR-150 is significantly downregulated in severe sepsis,27 a broad range of acute leukemias,28 chronic myeloid leukemias,29 malignant NK/T-cell lymphomas,30 colorectal cancers,31 and hepatocellular carcinomas.32 Therefore, miR-150 was highly expressed in normal mature NK cells, but significantly downregulated in NK92 MI cells derived from a patient with non-Hodgkin’s lymphoma (Fig 6, C). Lentivirus-mediated overexpression of miR-150 in NK/T-cell lymphoma cells resulted in increased expression of tumor suppressors, such as p53 and Bim, and decreased levels of AKT2 becoming more susceptible to the anticancer drug, etoposide.30 On the contrary, the transcription levels of proto-oncogenes c-Myb, c-kit and Bcl-2 were increased in sorted miR-150−/− NK cells,12 which raises the interesting possibility that miR-150−/− NK cells could be more resistant to anticancer drugs.

These data suggest that the downregulation of miR-150 confers a growth advantage to malignant cells, as well as premature and activated lymphocytes, possibly by upregulating c-Myb and Bcl-2. Upon activation, miR-150 was downregulated in mature B, T, NK, and iNKT cells, potentially releasing mature lymphocytes from growth arrest and allowing rapid proliferation to occur for proper immune responses. In summary, the downregulation of miR-150 can be a promising approach to NK cell therapies by enhancing NK cell cytotoxicity while possibly reducing NK cell apoptosis to anticancer agents. The ultimate goal of immunotherapy in many settings would not only include boosting the tumor-killing ability of immune effector cells but also making the effector cells more resistant to the anticancer drugs being concurrently administered to patients.33 Additionally, one study reported that allergen-specific cytotoxic T cells require sufficient Prf1 expression to suppress allergic airway inflammation.34 This implies that NK cells may contribute to Prf1-mediated allergic responses. Actually, NK cells have been reported to be involved in allergic diseases.35–37 Patients with atopic dermatitis showed defects in NK cell cytotoxicity and IFN-γ production.38

Thus, therapeutic control of miR-150 in NK cells potentially opens new avenues for enhanced miRNA-mediated NK cell immunotherapy against various human pathologies including cancer and allergy, providing a better clinical outcome.

Supplementary Material

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Key Messages.

  • miR-150 post-transcriptionally downregulated Prf1 expression in both mouse and human NK cells at rest and after IL-15 activation.

  • Activated miR-150−/− NK cells expressed upregulated Prf1, augmenting cytotoxicity.

  • The adoptive transfer of miR-150−/− NK cells significantly reduced tumor growth and metastasis of B16F10 melanoma in immunocompromised mice.

Acknowledgments

We thank Youngju Kang, Yeonkyung Kim, DongO Kim, Sungjin Yoon, Mijeong Kim, Wonsam Kim, Soojin Kim, Sooyoung Jun, and Hyangran Yoon for expert technical assistance. We also thank Haiyoung Jung, Youngjun Park, Sukran Yoon, David Kim and Gouyoung Koh for fruitful discussions. This work was supported in part by grants from the GRL project (FGM1401223), the Ministry of Education, Science & Technology, the Korean Health Technology R&D Project (A121934), Ministry of Health and Welfare, KRIBB Research Initiative Program, and Basic Science Research Program through the National Research Foundation of Korea (RBM0261312). P.D.G. was supported in part by grants from the KRIBB and NIH CA33084.

Abbreviations used

WT

wild-type

NK

natural killer

miRNAs

MicroRNAs

Prf1

perforin 1

Gzms

granzymes

UTR

untranslated regions

Ig

immunoglobulin

IL-15

interleukin-15

E:T

effector:target

PI

propidium iodide

LV

lentivirus

IS

immunological synapse

AANAT

aralkylamine N-acetyltransferase

pA

polyadenylation

Footnotes

Disclosure of conflict of interest: The authors declare no competing financial interests.

Authorship

Contribution: N.Y.K. designed and performed experiments, analyzed results, and wrote the manuscript; M.J.K. conducted experiments and analyzed data; S.H.Y. performed revision experiments and analyzed data; J.S.D. interpreted results and wrote part of manuscript; P.D.G. provided critical comments; and T.D.K and I.P.C. assisted in experimental design and supervised the project.

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References

  • 1.Grossman WJ, Revell PA, Lu ZH, Johnson H, Bredemeyer AJ, Ley TJ. The orphan granzymes of humans and mice. Curr Opin Immunol. 2003;15:544–52. doi: 10.1016/s0952-7915(03)00099-2. [DOI] [PubMed] [Google Scholar]
  • 2.Henkart PA. Mechanism of lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 1985;3:31–58. doi: 10.1146/annurev.iy.03.040185.000335. [DOI] [PubMed] [Google Scholar]
  • 3.Russell JH, Ley TJ. Lymphocyte-mediated cytotoxicity. Annu Rev Immunol. 2002;20:323–70. doi: 10.1146/annurev.immunol.20.100201.131730. [DOI] [PubMed] [Google Scholar]
  • 4.Baran K, Dunstone M, Chia J, Ciccone A, Browne KA, Clarke CJ, et al. The molecular basis for perforin oligomerization and transmembrane pore assembly. Immunity. 2009;30:684–95. doi: 10.1016/j.immuni.2009.03.016. [DOI] [PubMed] [Google Scholar]
  • 5.Voskoboinik I, Thia MC, Fletcher J, Ciccone A, Browne K, Smyth MJ, et al. Calcium-dependent plasma membrane binding and cell lysis by perforin are mediated through its C2 domain: A critical role for aspartate residues 429, 435, 483, and 485 but not 491. J Biol Chem. 2005;280:8426–34. doi: 10.1074/jbc.M413303200. [DOI] [PubMed] [Google Scholar]
  • 6.Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, et al. Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell. 2000;103:491–500. doi: 10.1016/s0092-8674(00)00140-9. [DOI] [PubMed] [Google Scholar]
  • 7.Lichtenheld MG, Podack ER. Structure of the human perforin gene. A simple gene organization with interesting potential regulatory sequences. J Immunol. 1989;143:4267–74. [PubMed] [Google Scholar]
  • 8.Kiessling R, Klein E, Wigzell H. “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol. 1975;5:112–7. doi: 10.1002/eji.1830050208. [DOI] [PubMed] [Google Scholar]
  • 9.Fehniger TA, Cai SF, Cao X, Bredemeyer AJ, Presti RM, French AR, et al. Acquisition of murine NK cell cytotoxicity requires the translation of a pre-existing pool of granzyme B and perforin mRNAs. Immunity. 2007;26:798–811. doi: 10.1016/j.immuni.2007.04.010. [DOI] [PubMed] [Google Scholar]
  • 10.Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136:215–33. doi: 10.1016/j.cell.2009.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Baltimore D, Boldin MP, O’Connell RM, Rao DS, Taganov KD. MicroRNAs: new regulators of immune cell development and function. Nat Immunol. 2008;9:839–45. doi: 10.1038/ni.f.209. [DOI] [PubMed] [Google Scholar]
  • 12.Bezman NA, Chakraborty T, Bender T, Lanier LL. miR-150 regulates the development of NK and iNKT cells. J Exp Med. 2011;208:2717–31. doi: 10.1084/jem.20111386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Fedeli M, Napolitano A, Wong MP, Marcais A, de Lalla C, Colucci F, et al. Dicer-dependent microRNA pathway controls invariant NKT cell development. J Immunol. 2009;183:2506–12. doi: 10.4049/jimmunol.0901361. [DOI] [PubMed] [Google Scholar]
  • 14.Xiao C, Calado DP, Galler G, Thai TH, Patterson HC, Wang J, et al. MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb. Cell. 2007;131:146–59. doi: 10.1016/j.cell.2007.07.021. [DOI] [PubMed] [Google Scholar]
  • 15.Zheng Q, Zhou L, Mi QS. MicroRNA miR-150 is involved in Valpha14 invariant NKT cell development and function. J Immunol. 2012;188:2118–26. doi: 10.4049/jimmunol.1103342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Zhou B, Wang S, Mayr C, Bartel DP, Lodish HF. miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely. Proc Natl Acad Sci U S A. 2007;104:7080–5. doi: 10.1073/pnas.0702409104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Oshimi Y, Oda S, Honda Y, Nagata S, Miyazaki S. Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells. J Immunol. 1996;157:2909–15. [PubMed] [Google Scholar]
  • 18.Vanherberghen B, Olofsson PE, Forslund E, Sternberg-Simon M, Khorshidi MA, Pacouret S, et al. Classification of human natural killer cells based on migration behavior and cytotoxic response. Blood. 2013;121:1326–34. doi: 10.1182/blood-2012-06-439851. [DOI] [PubMed] [Google Scholar]
  • 19.Peters PJ, Borst J, Oorschot V, Fukuda M, Krahenbuhl O, Tschopp J, et al. Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med. 1991;173:1099–109. doi: 10.1084/jem.173.5.1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Kim TD, Lee SU, Yun S, Sun HN, Lee SH, Kim JW, et al. Human microRNA-27a* targets Prf1 and GzmB expression to regulate NK-cell cytotoxicity. Blood. 2011;118:5476–86. doi: 10.1182/blood-2011-04-347526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Colucci F, Soudais C, Rosmaraki E, Vanes L, Tybulewicz VL, Di Santo JP. Dissecting NK cell development using a novel alymphoid mouse model: investigating the role of the c-abl proto-oncogene in murine NK cell differentiation. J Immunol. 1999;162:2761–5. [PubMed] [Google Scholar]
  • 22.Smyth MJ, Thia KY, Cretney E, Kelly JM, Snook MB, Forbes CA, et al. Perforin is a major contributor to NK cell control of tumor metastasis. J Immunol. 1999;162:6658–62. [PubMed] [Google Scholar]
  • 23.Sullivan RP, Leong JW, Fehniger TA. MicroRNA regulation of natural killer cells. Front Immunol. 2013;4:44. doi: 10.3389/fimmu.2013.00044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Leong JW, Sullivan RP, Fehniger TA. Natural killer cell regulation by microRNAs in health and disease. J Biomed Biotechnol. 2012;2012:632329. doi: 10.1155/2012/632329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Liu X, Wang Y, Sun Q, Yan J, Huang J, Zhu S, et al. Identification of microRNA transcriptome involved in human natural killer cell activation. Immunol Lett. 2012;143:208–17. doi: 10.1016/j.imlet.2012.02.014. [DOI] [PubMed] [Google Scholar]
  • 26.Lu J, Guo S, Ebert BL, Zhang H, Peng X, Bosco J, et al. MicroRNA-mediated control of cell fate in megakaryocyte-erythrocyte progenitors. Dev Cell. 2008;14:843–53. doi: 10.1016/j.devcel.2008.03.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Vasilescu C, Rossi S, Shimizu M, Tudor S, Veronese A, Ferracin M, et al. MicroRNA fingerprints identify miR-150 as a plasma prognostic marker in patients with sepsis. PLoS One. 2009;4:e7405. doi: 10.1371/journal.pone.0007405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Jiang X, Huang H, Li Z, Li Y, Wang X, Gurbuxani S, et al. Blockade of miR-150 maturation by MLL-fusion/MYC/LIN-28 is required for MLL-associated leukemia. Cancer Cell. 2012;22:524–35. doi: 10.1016/j.ccr.2012.08.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Agirre X, Jimenez-Velasco A, San Jose-Eneriz E, Garate L, Bandres E, Cordeu L, et al. Down-regulation of hsa-miR-10a in chronic myeloid leukemia CD34+ cells increases USF2-mediated cell growth. Mol Cancer Res. 2008;6:1830–40. doi: 10.1158/1541-7786.MCR-08-0167. [DOI] [PubMed] [Google Scholar]
  • 30.Watanabe A, Tagawa H, Yamashita J, Teshima K, Nara M, Iwamoto K, et al. The role of microRNA-150 as a tumor suppressor in malignant lymphoma. Leukemia. 2011;25:1324–34. doi: 10.1038/leu.2011.81. [DOI] [PubMed] [Google Scholar]
  • 31.Ma Y, Zhang P, Wang F, Zhang H, Yang J, Peng J, et al. miR-150 as a potential biomarker associated with prognosis and therapeutic outcome in colorectal cancer. Gut. 2012;61:1447–53. doi: 10.1136/gutjnl-2011-301122. [DOI] [PubMed] [Google Scholar]
  • 32.Jiang J, Gusev Y, Aderca I, Mettler TA, Nagorney DM, Brackett DJ, et al. Association of MicroRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival. Clin Cancer Res. 2008;14:419–27. doi: 10.1158/1078-0432.CCR-07-0523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Markasz L, Stuber G, Vanherberghen B, Flaberg E, Olah E, Carbone E, et al. Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells. Mol Cancer Ther. 2007;6:644–54. doi: 10.1158/1535-7163.MCT-06-0358. [DOI] [PubMed] [Google Scholar]
  • 34.Enomoto N, Hyde E, Ma JZ, Yang J, Forbes-Blom E, Delahunt B, et al. Allergen-specific CTL require perforin expression to suppress allergic airway inflammation. J Immunol. 2012;188:1734–41. doi: 10.4049/jimmunol.1102699. [DOI] [PubMed] [Google Scholar]
  • 35.Wei H, Zhang J, Xiao W, Feng J, Sun R, Tian Z. Involvement of human natural killer cells in asthma pathogenesis: natural killer 2 cells in type 2 cytokine predominance. J Allergy Clin Immunol. 2005;115:841–7. doi: 10.1016/j.jaci.2004.11.026. [DOI] [PubMed] [Google Scholar]
  • 36.Farhadi N, Lambert L, Triulzi C, Openshaw PJ, Guerra N, Culley FJ. Natural killer cell NKG2D and granzyme B are critical for allergic pulmonary inflammation. J Allergy Clin Immunol. 2013 doi: 10.1016/j.jaci.2013.09.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Deniz G, van de Veen W, Akdis M. Natural killer cells in patients with allergic diseases. J Allergy Clin Immunol. 2013;132:527–35. doi: 10.1016/j.jaci.2013.07.030. [DOI] [PubMed] [Google Scholar]
  • 38.Luci C, Gaudy-Marqueste C, Rouzaire P, Audonnet S, Cognet C, Hennino A, et al. Peripheral natural killer cells exhibit qualitative and quantitative changes in patients with psoriasis and atopic dermatitis. Br J Dermatol. 2012;166:789–96. doi: 10.1111/j.1365-2133.2012.10814.x. [DOI] [PubMed] [Google Scholar]

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