Distinct Requirements of Micrornas in NK Cell Activation, Survival, and Function

The Journal of Immunology

Distinct Requirements of MicroRNAs in NK Cell Activation, Survival, and Function

Natalie A. Bezman,*,† Elizabeth Cedars,*,†,‡ David F. Steiner,*,†,‡ Robert Blelloch,x,{ David G. T. Hesslein,*,† and Lewis L. Lanier*,†,‡

MicroRNAs (miRNAs) are small noncoding RNAs that have recently emerged as critical regulators of gene expression within the immune system. In this study, we used mice with conditional deletion of Dicer and DiGeorge syndrome critical region 8 (Dgcr8) to dissect the roles of miRNAs in NK cell activation, survival, and function during viral infection. We developed a system for deletion of either Dicer or Dgcr8 in peripheral NK cells via drug-induced Cre activity. We found that Dicer- and Dgcr8-deficient NK cells were significantly impaired in survival and turnover, and had impaired function of the ITAM-containing activating NK cell receptors. We further demonstrated that both Dicer- and Dgcr8-dependent pathways were indispensable for the expansion of Ly49H+ NK cells during mouse cytomegalovirus infection. Our data indicate similar phenotypes for Dicer- and Dgcr8-deficient NK cells, which strongly suggest that these processes are regulated by miRNAs. Thus, our findings indicate a critical role for miRNAs in controlling NK cell homeostasis and effector function, with implications for miRNAs regulating diverse aspects of NK cell biology. The Journal of Immunology, 2010, 185: 3835–3846.

atural killer cells are a key component of the innate im- complexes results in recruitment and activation of protein tyrosine mune system, providing early cellular defense against kinases, which initiate the signaling cascade, resulting in secretion N viruses and intracellular pathogens, and contributing to of effector cytokines, such as IFN-g, and target cell cytotoxicity. the early detection and destruction of transformed cells (1). NK cells Lack of NK cells renders both human and mice susceptible to develop from a common lymphoid precursor in the bone marrow certain infections, particularly the herpesviruses, including human and undergo terminal maturation in the periphery, acquiring opti- cytomegalovirus and MCMV (5). Therefore, experimental in- mal cytolytic and effector functions (2). Mature NK cells are rela- fection of mice with MCMV provides a useful model for studying tively static in terms of proliferative capacity until transferred into factors that are important for NK cell activation in vivo. Although a lymphopenic environment (3, 4) or challenged with pathogens (5). much has been learned about the signaling molecules and tran- NK cell activation and function are determined by a balance of scription factors that control activation of NK cells, less is known signals transmitted by inhibitory and activating NK cell receptors about the posttranscriptional mechanisms that regulate NK cell (NKRs) (1). Many of the inhibitory NKRs, including Ly49 recep- function. tors in mice and inhibitory killer cell Ig-like receptors in humans, MicroRNAs (miRNAs) are short (∼22 nt) noncoding RNAs, recognize self-ligands, such as MHC class I molecules. Activating expressed from endogenous genes, that act on protein-encoding NKRs recognize host-encoded molecules induced by transfor- mRNAs, targeting them for translational repression or degrada- mation or viral infection (e.g., NKG2D ligands) (6), or nonself- tion (9). The biogenesis of miRNAs involves two processing steps. ligands, including the mouse cytomegalovirus (MCMV) m157 gp, Primary miRNA transcripts are first cleaved by the nuclear “micro- which is recognized by Ly49H (7, 8). Some activating receptors processor” complex containing the RNAse III enzyme Drosha and associate with adapter proteins that contain ITAMs, including its dsRNA-binding partner DiGeorge syndrome critical region 8 DAP12, FcεRIg, and CD3z. Ligation of ITAM-containing receptor (Dgcr8), resulting in short hairpin structures (precursor miRNAs) (10, 11). These precursor miRNAs are then exported to the cytoplasm, and are further processed by another RNAse III enzyme, *Department of Microbiology and Immunology, †Cancer Research Institute, ‡Bio- medical Sciences Graduate Program, xEli and Edythe Broad Center of Regeneration Dicer, to generate the mature duplex miRNAs (12). Finally, a single Medicine and Stem Cell Research, and {Department of Urology, University of Cali- strand of the mature miRNA duplex, loaded into the RNA-induced fornia San Francisco, San Francisco, CA 94143 silencing complex, targets specific sequences within mRNA 39 Received for publication March 26, 2010. Accepted for publication July 19, 2010. untranslated regions resulting in mRNA degradation and/or trans- This work was supported by National Institutes of Health Grant AI068129. L.L.L. is lational repression. an American Cancer Society Professor. N.A.B. is an American Cancer Society Post- Genetic studies showed a critical requirement for Dicer in vivo. doctoral Fellow and was supported by National Institutes of Health Training Grant T32. D.G.T.H. is a special fellow of the Leukemia and Lymphoma Society. R.H.B. Ablation of Dicer in the mouse germline produces a lethal pheno- was supported by National Institutes of Health/National Institute of Neurological type (13), and conditional deletion of Dicer in various hemato- Disorders and Stroke Grants K08 NS048118 and R01 NS057221. poietic lineages has been shown to have detrimental effects, such as Address correspondence and reprint requests to Dr. Lewis L. Lanier, University of impaired cell differentiation, proliferation, and survival (14–21). California, San Francisco, 513 Parnassus Avenue, HSE 1001G, Box 0414, San Fran- cisco, CA 94143. E-mail address: [email protected] The function of Dicer, however, is not limited to miRNA biogenesis. The online version of this article contains supplemental material. Dicer is also required for the processing of small inhibitory RNAs Abbreviations used in this paper: Dgcr8, DiGeorge syndrome critical region 8; LNA, (siRNAs) derived from endogenous dsRNA transcripts or exoge- locked nucleotide acid; MCMV, mouse cytomegalovirus; MFI, mean fluorescence nous sources, including viral dsRNAs (22). Endogenous siRNAs intensity; miRNA, microRNA; NKR, NK cell receptor; PI, postinfection; siRNA, were discovered in mammalian oocytes and embryonic stem cells small inhibitory RNA; YFP, yellow fluorescent protein. (23–25) and were shown to be essential for oocyte maturation Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 (26). Thus, although the loss of miRNA-dependent regulation has www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000980 3836 DICER- AND DGCR8-DEPENDENT PATHWAYS REGULATE NK CELL FUNCTION been implicated in the phenotypes caused by Dicer deficiency, at layer were isolated from PBMCs (Stanford Blood Center, Palo Alto, CA), + 2 least in some cases they are the consequence of the loss of other stained with anti-CD56 and anti-CD3 mAbs, and CD56 CD3 lympho- . small RNA classes. Unlike Dicer, Dgcr8 is essential for the bio- cytes were sorted. The purity of the recovered NK cells was typically 98%. genesis of canonical miRNAs, but not in the generation of siRNAs In vivo BrdU labeling (23, 27). In this study, we developed a system for the deletion of Mice were injected i.p. with 200 mg BrdU (Sigma-Aldrich) every 24 h for either Dicer or Dgcr8 in NK cells via drug-induced Cre activity to 3 consecutive days (Fig. 3B) or once for 2 h (Fig. 3E) and then sacrificed. dissect the roles of miRNAs in NK cell activation, survival, and For the detection of incorporated BrdU, cells were first stained for surface function during infection. Ags, then fixed, permeabilized, treated with DNase I, and stained with allophycocyanin-conjugated anti-BrdU mAb (BD Pharmingen). Materials and Methods CFSE labeling and adoptive transfer Mice and infections A 1:1 splenocyte mixture of CD45.1+ (from wild-type C57BL/6 mice) and + D/+ D/D The Cre-ERT2 C57BL/6 transgenic mice were kindly provided by Dr. CD45.2 (from either Dgcr8 or Dgcr8 C57BL/6 mice) cells was M. Matloubian (University of California San Francisco, San Francisco, treated with ACK lysis buffer to remove RBCs, labeled for 8 min with 3 7 CA) (28), R26R-yellow fluorescent protein (YFP) knockin C57BL/6 mice 0.5 mM CFSE, and washed twice in PBS. A total of 1 10 labeled cells were provided by Dr. N. Killeen (University of California San Francisco) were transferred i.v. into Ly49H-deficient C57BL/6 mice. Twenty-four (29), and Ly49H-deficient (Klra82/2) mice were from Dr. S. Vidal (McGill hours later, recipient mice were infected with MCMV and sacrificed University, Montreal, Quebec, Canada). Mice carrying the conditional 4 d later. Adoptively transferred NK cells were analyzed for Ly49H ex- floxed alleles of Dicer (DicerF/F) (14) and Dgcr8 (Dgcr8F/F) (30) were pression and CFSE dilution by flow cytometry. backcrossed onto a C57BL/6 background for eight generations. To gen- MiRNA array profiling erate bone marrow chimeras, 6- to 8-wk-old CD45.1-congenic C57BL/6 mice (National Cancer Institute, Frederick, MD) were lethally irradiated Total RNA was extracted from sorted mouse splenic and human peripheral with 1000 rad. Donor bone marrow was harvested from DicerF/+, DicerF/F, blood NK cells by using TRIzol (Invitrogen). RNA quality was verified by an Dgcr8F/+, or Dgcr8F/F mice carrying Cre-ERT2 transgene and mixed 1:1 Agilent 2100 Bioanalyzer profile. A mixture of equal amounts of RNA from with bone marrow harvested from CD45.1 C57BL/6 mice. Cells were then mouse and human NK cells were made as reference. Total RNA (5 mg) from injected into irradiated recipients by the intraocular vein in the retro-orbital the samples (mouse or human NK cells) and a reference pool were labeled plexus. The chimeras were treated with tamoxifen 7 wk after reconstitution with Hy5 and Hy3 fluorescent label, respectively, using the miRCURY and infected i.p. with 5 3 104 PFU of a salivary stock of MCMV (Smith locked nucleotide acid (LNA) miRNA array labeling kit (Exiqon, Den- strain) 2 wk later (31). All experiments were conducted in accordance with mark). The Hy5-labeled samples and Hy3-labeled reference pool RNA sam- University of California San Francisco Institutional Animal Care and Use ples were mixed pairwise and hybridized to the LNA array (Gladstone Geno- Committee guidelines. mics Core, San Francisco, CA), which contains capture probes targeting human, mouse, and rat miRNAs. The hybridization was performed on Tamoxifen treatment GeneTAC hybridization station (Genomic Solutions, Boston, MA). The Mice were administered tamoxifen (Sigma-Aldrich, St. Louis, MO) dis- slides were scanned by Axon GenePix 4000B scanner (Molecular Devices, solved in corn oil via oral gavage for 5 consecutive days. Unless otherwise Sunnyvale, CA). GPR files containing fluorescent ratios (sample/control) stated, animals were sacrificed for analysis 10–20 d posttreatment. were generated using GenePix Pro 6.0 software. Flow cytometry and functional assays Quantitative RT-PCR + Single-cell suspensions were used for flow cytometry. Fc receptors were Total RNA was isolated from sorted YFP NK cells by using the mirVana blocked with anti-CD16 and CD32 mAb (clone 2.4G2) at 10 mg/ml prior to miRNA Isolation kit (Ambion, Austin, TX). The expression levels of spe- surface staining with the indicated Abs (all purchased from BD Biosciences, cific miRNAs were examined by using the TaqMan MicroRNA Assay kit San Jose, CA, eBioscience, or BioLegend, San Diego, CA). Rae-1 expres- (Applied Biosystems, Foster City, CA) with gene-specific stem-loop PCR sion was measured using anti–pan-Rae-1 mAb (clone 186107). Splenocytes primers and TaqMan probes (IDT, Coralville, IA) to detect mature miRNA were stimulated in tissue culture plates treated with N-(1-(2,3-dioleoyloxyl) transcripts. Quantitative RT-PCR reaction was performed on an Applied propyl)-N,N,N-trimethylammonium methylsulfate (Sigma-Aldrich) and Biosystems 7500 Fast Real-Time PCR System. snoRNA202 was used as endogenous control. Relative quantification using the DDct method in coated with anti-NK1.1, anti-NKp46, anti-Ly49H, or control mAb for 4 h at D/D D/D 37˚C in the presence of GolgiStop (BD Pharmingen, San Diego, CA), fol- tamoxifen-treated Dicer or Dgcr8 versus control mice was carried out lowed by staining for lysosome-associated membrane protein-1 (CD107a, and relative changes were calculated for each miRNA. BD Pharmingen) and intracellular IFN-g (BD Pharmingen). For control PCR analysis of DicerF/F and Dgcr8F/F mice experiments, splenocytes were stimulated with IL-12 (20 ng/ml) and IL-18 (10 ng/ml). For apoptosis analysis, freshly isolated splenocytes were first The floxed Dicer allele was genotyped as previously described (18). The stained with anti- NK1.1, anti–TCR-b, and Live/Dead fixable near-IR stain same primers were used to quantitate the floxed Dicer alleles by real-time (Invitrogen, Carlsbad, CA), washed, and then stained with annexin V-PE PCR with the SYBR green PCR Master Mix (Applied Biosystems). The PU.1 (BD Pharmingen), according to manufacturer’s protocol. For expansion of sequences 59-CTTCACTGCCCATTCATTGGCTCATCA-39 (forward) and NK cells in vitro, splenocytes were stained with anti-CD4, anti-CD8, anti- 59-GCTGGGGACAAGGTTTGATAAGGGAA-39 (reverse) were used for Ter119, and anti–GR-1 mAbs and then incubated with goat anti-rat IgG- normalization. The floxed Dgcr8 allele was genotyped using primers 59- coated and goat anti-mouse IgG-coated magnetic beads (Qiagen, Valencia, CTGGAGTAGGCATGTTGATTTC-39 (forward) and 59-CCTGATTCAC- CA) to deplete T cells, B cells, and GR-1+ cells, respectively. NK cells were TTACAACACAACC-39 (reverse). stained with PE-conjugated DX5 mAb and positively selected by using an anti-PE mAb-conjugated magnetic bead system (Miltenyi Biotec, Auburn, Statistical analysis CA). The resulting NK cells were cultured with 1333 U/ml recombinant All data shown are the mean 6 SEM unless stated otherwise. Comparisons human IL-2 (National Institutes of Health Biological Resources Branch between samples were performed by using a two-tailed Student t test. Preclinical Repository, Frederick, MD). NK cells were counted and stained Statistics were determined using Prism software (GraphPad Software, La with PE-conjugated anti-NKp46, PerCP-Cy5.5–conjugated-CD3, and Jolla, CA).The p values were denoted in the following manner: pp , 0.05; annexin V-conjugated A647 after culture for 4 d. Flow cytometry was per- ppp , 0.01; pppp , 0.001. formed by using a LSRII flow cytometer and analyzed with FlowJo software (TreeStar, Ashland, OR). Results Cell sorting Inducible deletion of either Dicer or Dgcr8 leads to a global NK cells from spleen were enriched with mAbs against CD5, CD4, CD8, miRNA deficiency in NK cells Ter119, Gr-1, and CD19 (University of California San Francisco Ab Core), We determined the expression profile of miRNAs in mouse and hu- and anti-rat IgG-coated magnetic beads (Miltenyi Biotec). NK cells were + 2 + 2 man NK cells. The miRNAs obtained from sorted NK1.1 TCR-b then stained with anti-NK1.1 and anti-TCRb mAbs, and NK1.1 TCRb + 2 cells were sorted using a FACSAria (Becton Dickinson, San Jose, CA). For mouse NK cells and from CD56 CD3 human NK cells were human NK cells, the low-buoyant density cells from the Percoll interface profiled with a LNA-based microarray (Supplemental Table I). The The Journal of Immunology 3837 preliminary analysis indicated that 80% of all miRNAs present in and Dgcr8D/D mice. Flow cytometric analyses of YFP+ lympho- human NK cells were also expressed in mouse NK cells, and 59% of cytes in the spleen, liver, and blood showed a marked reduction of mouse miRNAs were present in human NK cells (Supplemental Fig. the overall percentage of NK1.1+ TCR-b2 cells (2-fold reduction 1A). Quantitative RT-PCR confirmed expression of the top 20 com- on average) in DicerD/D and Dgcr8D/D mice compared with litter- mon miRNAs in mouse NK cells (Supplemental Fig. 1B). mate controls (Fig. 2A,2B). Using the same R26R-YFP reporter To understand the role of miRNAs in NK cell biology, we induced mice, it was previously demonstrated that in the absence of Dicer, ablation of the conditional DicerF (14) and Dgcr8F (30) alleles there is a strong deletion of CD4+ and CD8+ T cells that have using a drug-inducible Cre recombinase. This inducible system is expressed Cre (16). We also found a smaller proportion of YFP+ dependent upon three sets of genes. The first are the genes encoding cells in the splenic and liver NK populations in DicerD/D and Dicer and Dgcr8 in which both alleles are flanked by two loxP sites Dgcr8D/D mice compared with controls (Fig. 2C). Taken together (DicerF/F and Dgcr8F/F, respectively). The second gene is a Cre with the reduced frequency of NK cells within the YFP+ lympho- recombinase-human estrogen receptor (Cre-ERT2) chimeric mole- cytes, the absolute numbers of YFP+ NK cells were significantly cule under the control of the ubiquitin promoter. This fusion protein decreased in the spleen of DicerD/D and Dgcr8D/D mice (2.4-fold, is sequestered within the cytoplasm in the absence of the estrogen p , 0.0001, and 3.3-fold, p = 0.0024, respectively). Similar reduc- analog, tamoxifen (28, 32). In the presence of tamoxifen, the Cre- tion in numbers of YFP+ NK cells were seen in liver of DicerD/D and ERT2 protein shuttles into the nucleus and gains access to loxP Dgcr8D/D mice (6.3-fold, p = 0.0066, and 3.3-fold, p = 0.033, re- sites. This approach allows for the constitutive expression of a Cre spectively) (Fig. 2D). This decrease in peripheral NK cell numbers recombinase yet prevents it from acting on loxP sites until ta- in DicerD/D and Dgcr8D/D mice occurs in the context of reduced, but moxifen is administered. The third component is an enhanced YFP not abolished, miRNA function. As such, it is possible that the gene inserted into the Rosa-26 locus (29). The YFP cassette is reason that we observed only 50–75% reduction in miRNA levels preceded by a loxP-flanked tpA transcriptional stop signal. This (Fig. 1D) is because these are the only cells that are viable. construct permits expression of YFP and identification of cells that In contrast to the NK cell compartment, the frequencies of B cells currently or previously expressed an activated Cre. When tamox- and CD4+ T cells were unchanged in the spleens of DicerD/D and ifen is administered to mice, Cre becomes active and deletes the Dgcr8D/D mice compared with controls. CD8+ T cells were con- floxed Dicer or Dgcr8 alleles and the R26R stop cassette, resulting sistently moderately reduced in DicerD/D and Dgcr8D/D mice, al- in DicerD/DYFP+ and Dgcr8D/DYFP+ phenotypes, respectively (Fig. though this difference did not reach statistical significance (Fig. 2E, 1A). To serve as a control for these mice, we generated mice in 2F). Spleen cellularity in Dicer- and Dgcr8-deficient mice, how- which loxP sites flank only one allele, the other allele is wild-type ever, was significantly reduced, as compared with controls (67.6 3 (DicerF/+ and Dgcr8F/+ mice). Following tamoxifen treatment, these 106 6 8.5 3 106 [n = 9 DicerD/+] versus 39.8 3 106 6 4.9 3 106 mice would be referred to as DicerD/+ and Dgcr8D/+, respectively. [n = 14 DicerD/D]; p = 0.006 and 59.4 3 106 6 12.8 3 106 [n =8 We determined the kinetics of YFP expression and concomitant Dgcr8D/+] versus 30.0 3 106 6 2.8 3 106 [n = 8 Dgcr8D/D]; p = reduction of miRNA expression in NK cells from DicerD/D and 0.04). As a result, the numbers of B cells, CD4+ T cells, and CD8+ Dgcr8D/D mice after administration of tamoxifen. YFP expression T cells, were lower (∼2-fold, 2.3 fold, and 5-fold, respectively) in appeared on day 5 after tamoxifen treatment was initiated (data DicerD/D and Dgcr8D/D mice compared with that in controls. This is not shown) and was maximal by day 14 posttreatment (Fig. 1B). consistent with prior reports showing that Dicer deletion resulted in Only ∼25% of NK cells in DicerD/+ and Dgcr8D/+ control mice a dramatic reduction of B cells and T cells (15–17). Although produced the YFP protein, suggesting that this system under- iNKT cell development was recently reported to be severely im- reports Dicer (and perhaps Dgcr8) deletion, as has been reported paired in the absence of Dicer (19, 20), we detected only minor for other genes (33). In fact, Dicer was also excised in YFP2 reduction in the number of NK1.1+ TCR-b+ cells in our DicerD/D NK cells in DicerD/D mice, albeit not as efficiently as in YFP+ and Dgcr8D/D mice (∼1.3-fold and ∼1.2-fold reduction com- NK cells (Supplemental Fig. 2A). To confirm that deletion occurred pared with controls, respectively) (data not shown). Taken together, in the studied cells, all experiments were performed on cells gated these data show that miRNAs are required for the maintenance of as YFP+. normal lymphocyte numbers, particularly those of the NK cell By day 14 posttreatment, miRNA expression in YFP+ NK cells lineage. from Dgcr8D/D mice decreased to 7–22% of the miRNA expression level in NK cells from untreated Dgcr8F/F mice (Fig. 1C). MiRNAs regulate survival and turnover of NK cells Because the half-life of naive NK cells is ∼17 d (4) [and potential To determine whether the reduction in NK cell numbers in DicerD/D global effects of Dicer and Dgcr8 deficiency on the mouse im- and Dgcr8D/D mice was the result of increased cell death, impaired mune system may exist (18, 21, 34)], we limited our analysis of expansion of immature NK cells, or both, we stained freshly NK cells from these mice to days 10–20 posttamoxifen treatment. isolated cells with annexin Vand a viability dye to assess apoptosis Approximately 50–75% reduction of select miRNAs was con- (live, annexin V+) and cell death (dead, annexin V+). There was a firmed in YFP+ NK cells from several DicerD/D and Dgcr8D/D mice consistent increase of both apoptotic and dead NK cells from (Fig.1D, Supplemental Fig. 2B). Importantly, miRNAs expressed DicerD/D and Dgcr8D/D versus control mice (Fig. 3A,3B). The rate at high (miR21 and miR16), medium (miR29a and miR30b), and of apoptosis and death of DicerD/D NK cells was further increased low levels (miR222) were similarly affected by Dicer and Dgcr8 and significantly higher than that of DicerD/+ NK cells if cells were deletion. These studies demonstrate that inducible Cre-ERT2– cultured in the presence of IL-2 in vitro (Fig. 3C,3D). In addition, mediated deletion of either Dicer or Dgcr8 leads to a significant we examined NK cell turnover using BrdU incorporation. Ap- depletion of miRNAs in YFP+ NK cells. proximately 10% of wild-type NK1.1+ TCRb2 NK cells incor-

D/D D/D porated BrdU over 3 d of labeling, consistent with previous reports Reduction of the NK cell compartment in Dicer and Dgcr8 (4). In contrast, DicerD/D and Dgcr8D/D NK cells incorporated BrdU mice at a lower rate, 6.3 6 0.3% and 5.8 6 2.0% of NK cells, respectively To investigate the role for Dicer- and Dgcr8-controlled miRNAs in (Fig. 3E). Consistent with the decreased turnover rate, DicerD/D and the regulation of NK cell homeostasis, we ﬁrst determined the Dgcr8D/D NK cells did not exhibit activated phenotypes, shown by frequency and number of NK cells in peripheral organs of DicerD/D the similar expression levels of CD69 and KLRG1 (Fig. 4A). These 3838 DICER- AND DGCR8-DEPENDENT PATHWAYS REGULATE NK CELL FUNCTION

FIGURE 1. Inducible deletion of Dicer and Dgcr8 leads to miRNA deficiency in NK cells. DicerF/F or Dgcr8F/F mice were crossed with Cre-ERT2 transgenic and R26R-YFP knockin mice to delete one (DicerD/+ or Dgcr8D/+) or both (DicerD/D or Dgcr8D/D) alleles, expressing YFP upon deletion of a floxed stop cassette inserted into the Rosa-26 locus. A, Diagram of the floxed loci before and after tamoxifen-induced activation of the Cre-ERT2-fusion protein. B, NK1.1 and YFP expression in lymphocytes isolated from DicerD/+, DicerD/D, Dgcr8D/+, and Dgcr8D/D mice on day 14 posttreatment. C and D, Quantification of miRNA levels using quantitative RT-PCR. Levels of mature miRNAs were normalized to sno202 and relative expression of poorly (miR222), moderately (miR29a and miR30b), and highly (miR16 and miR21) expressed miRNAs are shown. C, RNA was isolated from YFP+ NK1.1+ TCR-b2 cells sorted from Dgcr8D/D mice on treatment day 1 and day 5, and day 7 and day 14 posttreatment. Data are representative of two experiments. D, RNAwas isolated from YFP+ NK1.1+ TCR-b2 cells sorted from mice of the indicated genotypes on days 10–20 after treatment. Mean 6 SEM is shown for DicerD/D (n = 4) relative to DicerD/+ (n = 2), and for Dgcr8D/D (n = 4) relative to DicerD/+ (n = 2).

results indicate that Dicer- and Dgcr8-deficiency affects both the isolated YFP+ NK cells. The maturation status of peripheral NK turnover rate, as well as survival, of NK cells. cells is classified based on their expression of CD11b and CD27, lo hi D/D with CD27 CD11b NK cells being the most mature population Phenotype and receptor repertoire of NK cells in Dicer and D/D D/D (35). The percentage of this mature NK cell subset in Dicer Dgcr8 mice and Dgcr8D/D mice was decreased compared with control mice Dicer-deficiency has been shown to affect the development of Treg (Fig. 4B). Expression levels of NK1.1, NKp46, Ly49H, Ly49D, and iNKT lineages, and the differentiation of CD4+ T cell into Th1 CD94, Ly49G2, Ly49C/I, and Ly49A receptors were unaffected in and Th2 cells (15, 16, 18–21). To determine whether miRNAs could DicerD/D and Dgcr8D/D mice (Fig. 4C, Supplemental Table II). The affect the maturation of peripheral NK cells, we analyzed the ex- only exception was NKG2D whose level was often diminished on pression of maturation markers and surface receptors on freshly NK cells from DicerD/D and Dgcr8D/D mice. NKG2D recognizes The Journal of Immunology 3839

FIGURE 2. Preferential reduction of NK cells in DicerD/D and Dgcr8D/D mice. Flow cytometric analysis of lymphocytes from DicerD/D (n = 16), Dgcr8D/D (n = 9), and control DicerD/+ (n = 13) and Dgcr8D/+ (n = 11) mice. A, Contour plots depict frequency of NK (NK1.1+ TCR-b2) cells in spleen (top panels), liver (middle panels), and blood (bottom panels). Gated on YFP+ lymphocytes. B, Summary (mean 6 SEM) of data shown in A. C, Selective loss of YFP+ DicerD/D and YFP+ Dgcr8D/D NK cells. Histograms depict relative cell number versus Cre-induced YFP fluorescence within NK cells in spleen (top row) and liver (bottom row). Gated on NK1.1+ TCR-b2 cells and percentages of YFP-expressing cells are indicated. D, Bar graph shows the number of YFP+ NK cells from spleen (left panel) and liver (right panel) of DicerD/D, Dgcr8D/D, and control DicerD/+, Dgcr8D/+ mice. E and F, Comparison of B (CD19+), CD4+ T (CD4+ TCR-b+), and CD8+ T (CD8+ TCR-b+) cell frequencies from spleen of DicerD/D, Dgcr8D/D and control DicerD/+, Dgcr8D/+ mice. E, Flow cytometric analysis of populations in total YFP+ splenocytes (top panel) and YFP+ TCR-b+ splenocytes (bottom panel). Percentages are shown. F, Summary (mean values 6 SEM) of data shown in E from four to six mice per group. Data are representative of at least five experiments. several cellular ligands, which are expressed at low levels in NKG2D correlated with higher expression of Rae-1 ligands on healthy cells, but are often upregulated in stressed, virally infected, DicerD/D (Fig. 4D), and Dgcr8D/D (data not shown) NK cells. To- or tumor cells (36). We found that the reduced expression of gether, our results indicate that Dicer- and Dgcr8-deficiency effects 3840 DICER- AND DGCR8-DEPENDENT PATHWAYS REGULATE NK CELL FUNCTION

FIGURE 3. Increased apoptosis and reduced basal turnover of Dicer- and Dgcr8-deﬁcient NK cells. A and B, Annexin V staining of freshly isolated splenic NK cells. A, Per- centages of apoptotic (live annexin V+) and dead cells in control versus DicerD/D and Dgcr8D/D mice are indicated within the quadrants. B, Summary (mean values 6 SEM) of data shown in (A) from three to ﬁve mice per group. C, Enumeration of NK cells after 4 d culture in medium supplemented with IL-2. Values shown represent the mean 6 SEM of NK cell numbers (triplicate wells). D, Histogram shows annexin V expression by DicerD/+ (black line) and DicerD/D (gray line) NK cells after 4 d culture in medium supplemented with IL-2. E, Intracellular staining for the incorporation of BrdU in control, DicerD/D,andDgcr8D/D mice. Per- centage of NK cells that incorporated BrdU is shown. A–E, Gated on YFP+ NK cells. Data are representative of two to three independent experiments.

NK cell maturation and elicits upregulation of NKG2D ligand and Dgcr8D/D mice compared with controls (Fig. 5A). Further- expression, but does not globally affect the NKR repertoire. more, fewer DicerD/D and Dgcr8D/D NK cells produced IFN-g when activated by Abs to NK1.1, NKp46, or Ly49H (Fig. 5B). DicerD/D and Dgcr8D/D NK cells produce less CD107a and To determine whether this is a generalized hyporesponsiveness IFN-g when stimulated through ITAM-containing receptors or an exclusive defect associated with ITAM-containing receptors, we stimulated NK cells with IL-12 and IL-18. Interestingly, Many of the activating NKRs, such as NK1.1, NKp46, and Ly49H, DicerD/D, Dgcr8D/D, and control NK cells produced comparable ε associate with the ITAM-containing Fc RIg or DAP12 adapter amounts of IFN-g in the presence of IL-12 and IL-18 (Fig. 5C). proteins (1). Ligation of an ITAM-associated NKR results in de- This demonstrates that Dicer- and Dgcr8-deﬁcient NK cells are granulation, cytokine production, and killing of the target cell. fully capable of responding through their cytokine receptors; Several studies have recently reported that miRNAs regulate ac- however, function of their ITAM-containing receptors is impaired. tivation of signaling pathways downstream of ITAM receptors (37, + 38). We asked whether Dicer- or Dgcr8-deﬁcient NK cells show Dicer and Dgcr8 are critical for Ly49H NK cell expansion impaired function of their ITAM-containing receptors. Freshly during MCMV infection isolated NK cells were activated by plate-bound anti-NK1.1, anti- To investigate whether miRNAs regulate NK cell function in vivo, NKp46, and anti-Ly49H Abs, and surface CD107a (LAMP-1) and we asked whether Dicer and Dgcr8 are necessary for NK cell intracellular IFN-g were measured. There were fewer CD107a- cytokine production and expansion in response to viral infection. positive cells among gated YFP+ NK populations from DicerD/D During infection with MCMV, NK cells bearing the activating The Journal of Immunology 3841

FIGURE 4. Dicer- and Dgcr8-deficeint NK cells do not posses an activated phenotype and have normal receptor expression with the exception of NKG2D. Splenocytes from DicerD/D, Dgcr8D/D, and control DicerD/+ and Dgcr8D/+ mice were stained with mAbs against activation markers CD69 and KLRG1 (A), maturation markers CD11b and CD27 (B), and activating NKRs NK1.1, NKp46, Ly49H, and NKG2D (C). Gated on YFP+ NK1.1+ TCRb2 cells. Data are representative of at least five experiments. D, Expression levels of Rae-1 on YFP+ NK1.1+ TCR-b+ cells in the spleens of DicerD/+ and DicerD/D mice. Mean fluorescence intensity (MFI) values are shown as mean 6 SD using two mice per group. Data are representative of three experiments.

Ly49H receptor recognize the viral protein m157 (7, 8), and re- that in the DicerD/+ and Dgcr8D/+ chimeric mice, consistent with our spond to inflammatory cytokines, such as IL-12 produced by den- earlier findings (Supplemental Fig. 3A,3B). Importantly, NK cell dritic cells (39). Signaling primarily via ITAM-containing DAP12, frequency in the CD45.1+ compartment was similar in DicerD/D, and augmented through DAP10, Ly49H+ NK cells mount a rapid Dgcr8D/D, and DicerD/+, Dgcr8D/+ chimeric mice, thus providing an antiviral response, secrete cytokines, including IFN-g, and un- internal control as a standard for each sample. dergo expansion (40). Following MCMV infection, we stained for intracellular IFN-g To measure MCMV-specific immune responses by Dicer- and immediately ex vivo on day 1.5 postinfection (PI). The 45.5 6 Dgcr8-deficient NK cells, we generated chimeric mice by recon- 6.5% and 47.9 6 3.6 of CD45.2+ NK cells from DicerD/D and stituting lethally irradiated mice with 1:1 mixed bone marrow from DicerD/+ chimeric mice, respectively, produced IFN-g (Fig. 6B, wild-type (CD45.1+) and experimental (DicerF/F, Dgcr8F/F, and 6C). No differences in intracellular IFN-g production were ob- control DicerF/+, Dgcr8F/+ [CD45.2+]) mice (Fig. 6A). Prior to ta- served between CD45.2+ NK cells from Dgcr8D/D and Dgcr8D/+ moxifen treatment, wild-type and experimental bone marrow chimeric mice (54.9 6 6.7% and 52.3 6 3.5%, respectively). reconstituted the NK cell compartment equally well (Supplemental Together, these results indicate that IFN-g production by NK cells Fig. 3A). Posttamoxifen treatment, however, the frequency of during MCMV infection proceeds independently of miRNAs. NK cells was significantly decreased in the CD45.2+ DicerD/D and During the NK cell response against MCMV in wild-type mice, CD45.2+ Dgcr8D/D compartments of chimeric mice, compared with the Ly49H+ NK cells preferentially expand during the first several 3842 DICER- AND DGCR8-DEPENDENT PATHWAYS REGULATE NK CELL FUNCTION

FIGURE 5. Defective degranulation and IFN-g production after stimulation of the activating NKRs in Dicer- and Dgcr8-deﬁcient NK cells. A, NK cells were incubated in wells either coated with IgG or mAbs NK1.1, NKp46, or Ly49H or with no mAb in the presence of anti-CD107a. Only cells that had degranulated displayed CD107a on the cell surface. Percentages of NK cells (gated on YFP+ DX5+ TCR-b2 cells) expressing CD107a. Right, Percentages of NK cells expressing CD107a (mean 6 SEM of four to nine mice/group). B and C, NK cells were stimulated as above or incubated with IL-12 and IL-18 in the presence of brefeldin A. Intracellular staining for IFN-g in YFP+ DX5+ TCR-b2 cells is shown. Right, Percentages of NK cells producing IFN-g (mean 6 SEM of three to seven mice/group). C, Data are mean 6 SEM of three mice/group. Data are representative of at least ﬁve experiments. days of infection (41, 42). When we infected DicerD/+ and Dgcr8D/+ number of Ly49H+ NK cells at day 7 PI expanded ∼10.6- and chimeric mice with MCMV, both mice showed an increase in ∼13.8-fold, respectively (Fig. 6E). In contrast, fewer Ly49H+ Ly49H+ NK cell frequency (Fig. 6D), and numbers (Fig. 6E) at day 7 NK cells were found in both DicerD/D and Dgcr8D/D chimeric mice at PI. With precursor numbers of ∼0.8 3 105 (DicerD/+) and ∼0.66 3 day 7 PI. The absolute number of Ly49H+ NK cells in DicerD/D and 105 (Dgcr8D/+) total Ly49H+ NK cells in the spleen, the absolute Dgcr8D/D chimeric mice expanded only ∼3- and ∼2.1-fold, The Journal of Immunology 3843

FIGURE 6. Dicer and Dgcr8 are necessary for Ly4H+ NK cell expansion during MCMV infection. A–F, Mixed bone marrow chimeric mice (1:1 mixture of wild-type [CD45.1+] and either DicerF/+, DicerF/F, Dgcr8F/+, or Dgcr8F/F [CD45.2+] cells) were treated with tamoxifen and then given PBS (uninfected) or infected with MCMV. B and C, Intracellular staining for IFN-g in NK cells immediately ex vivo at day 1.5 PI. B,Representa- tive scatter plots gated on CD45.2+ NK1.1+ TCR-b2 cells from DicerD/+, DicerD/D,Dgcr8D/+, and Dgcr8D/D chimeric mice are shown, and the numbers represent percentages of cells in the indicated quadrants. C,Cumu- lative results (n = 4–8 mice/group) are quantiﬁed. Percentages (D) and absolute numbers (E)ofCD45.2+ Ly49H+ NK cells in DicerD/+, DicerD/D, Dgcr8D/+,orDgcr8D/D chimeric mice at day 7 PI. F, Absolute number of wild-type CD45.1+ Ly49H+ NK cells in the same DicerD/+, DicerD/D, Dgcr8D/+,orDgcr8D/D chimeric mice at day 7 PI. E and F, The number of Ly49H+ NK cells from uninfected chimeric mice (gray bars) and from MCMV-infected mice (black bars) is shown. Data are mean 6 SEM of three mice per group and are representative of three independent experiments.

respectively. Importantly, wild-type CD45.1+ Ly49H+ NK cells possibilities, we monitored proliferation in vivo by analyzing from every set of chimeric mice (DicerD/D and Dgcr8D/D, and control BrdU incorporation. Following a 2-h pulse at day 5 PI, similar DicerD/+ and Dgcr8D/+) expanded equally well, indicating that all percentages of DicerD/D, Dgcr8D/D, and DicerD/+, Dgcr8D/+ NK groups of mice were able to mount an effective antiviral response cells from mixed bone marrow chimeric mice incorporated (Fig. 6F). Altogether, these experiments demonstrate that miRNAs BrdU (Fig. 7A). Adoptive transfer of CFSE-labeled NK cells into are required for robust NK cell expansion during MCMV infection. Ly49H-deficient recipients further confirmed that both Dgcr8D/D D/+ + + and Dgcr8 Ly49H cells were dividing to a similar extent, fully MiRNAs regulate survival of Ly49H NK cells during MCMV diluting their CFSE during the 4-d following infection (Fig. 7B). infection We also monitored apoptosis by annexin V staining at the same Defective expansion of DicerD/D and Dgcr8D/D Ly49H+ NK cells time point PI. Notably, a higher percentage of apoptotic cells was could result from decreased NK cell proliferation and/or impaired detected in Dgcr8D/D mice compared with that in Dgcr8D/+ control survival during MCMV infection. To discriminate between these mice (Fig. 7C). Together these data indicate that miRNAs are not 3844 DICER- AND DGCR8-DEPENDENT PATHWAYS REGULATE NK CELL FUNCTION required for the proliferative responsiveness of Ly49H+ NK cells, recently showed that inhibition of miR21 in the human NK cell line but regulate the survival of NK cells during MCMV infection. led to increased apoptosis associated with the upregulation of pro- apoptotic miR21 targets PTEN, PDCD4, and Bim. Although it is Discussion very likely that multiple miRNAs take part in the coordinate regu- In this study, we have shown identical phenotypes when either Dicer lation of NK cell survival, it would be informative to test whether or Dgcr8 gene was inactivated in NK cells. These results strongly miR21 is a key regulator of NK cell survival. suggest that these two molecules function in the same biological Maturation of NK cells is characterized by a decrease in the ex- pathway in miRNA biogenesis, and demonstrate that the deficiency pression of CD27, active proliferation, and a concomitant increase in miRNAs is the primary cause underlying the observed pheno- in the levels of CD11b and effector functions (35, 45). Analysis of types. Although it is possible that Dicer-deficient cells also exhibit DicerD/D and Dgcr8D/D mice revealed a relative accumulation of other more subtle phenotypes, such as the derepression of retro- more immature CD27hi CD11blo NK cells. This could be due to transposons (24, 25), it appears that it is miRNAs, rather than other a selective loss of more mature CD27lo CD11bhi NK cells or Dgcr8-independent, Dicer-dependent small RNAs, that are critical a maturation defect. We did not detect any preferential difference in for NK cells. the death or turnover in the CD27hi CD11blo or CD27lo CD11bhi Ablation of the miRNA biogenesis pathway, through deletion of NK cell subsets (N. Bezman, unpublished observations). Further- Dicer or Dgcr8, led to increased apoptosis of peripheral NK cells. more, we found a similar accumulation of CD27hi CD11blo Similarly, Dicer deletion in developing B cells (17), thymocytes (15, NK cells in CD45.2+ subsets of DicerD/D and Dgcr8D/D mixed bone 16), or iNKT cells (19) resulted in increased cell death. These marrow chimeras (data not shown). These data indicate that im- results suggest that the miRNA pathway plays an important role paired transition from CD27hi CD11blo to CD27lo CD11bhi of in controlling cell survival. Potential mechanisms include mitotic Dicer- and Dgcr8-deficient NK cells is hematopoietic cell intrinsic defects due to centromere dysfunctions (43), defects in heterochro- and cell autonomous, suggesting that miRNAs might regulate matin maintenance (44), and aberrant over expression of proapo- NK cell maturation. Interestingly, miR150 has been shown to ptotic protein Bim (17). In addition, our preliminary studies indicate regulate B cell development and this regulation occurs through the that Bcl-2 mRNA level was consistently decreased in DicerD/D inhibition of Myb expression (46, 47). MiR150 is dynamically and Dgcr8D/D NK cells (Supplemental Fig. 4). Yamanaka et al. (38) regulated during NK cell maturation (N. Bezman, unpublished

FIGURE 7. Robust proliferation, but decreased survival, of Dicer- and Dgcr8-deficient NK cells during MCMV infection. A, BrdU incorporation (day 5 PI) by NK1.1+ TCR-b2 cells from mixed bone marrow chimeric mice (Fig. 6). Percentages of BrdU incorporation by wild- type (CD45.1+) cells (lower panel) and either DicerD/+, DicerD/D, Dgcr8D/+, or Dgcr8D/D (CD45.2+) cells (top panel) are shown. Data are representative of two experiments. B, CFSE- labeled 1:1 mix of wild-type (CD45.1+) and either Dgcr8D/+ or Dgcr8D/D (CD45.2+) NK cells were transferred into Ly49H-deficient recipients. Ly49H+ NK cells were analyzed at day 4 PI. Data are representative of three independent experiments. C, NK cells from Dgcr8D/+ or Dgcr8D/D mice were transferred into Ly49H- deficient recipients and were stained for annexin Vat day 4 PI. Bar graph indicates the percentage of NK1.1+ TCR-b2 cells (mean 6 SD) that are live and are staining positive for annexin V (indicating apoptotic). Data are representative of three independent experiments using two mice per group. The Journal of Immunology 3845 observations), and Chiossone et al. (45) reported that Myb is dif- this study, we determined the miRNA expression profile of mouse ferentially upregulated in CD27hi CD11blo NK cells. Future studies and human NK cells. Naive NK cells and CD8+ T cells share will determine whether NK cell development or maturation is a large part of their miRNA profile: miR142-3p, miR142-5p, regulated by miR150. miR150, miR16, miR23a, miR15b, miR29a, miR29b, miR30b, In contrast to having no significant effect on many of the ac- and miR26a are highly expressed in both naive NK cells and CD8+ tivating and inhibitory NKRs analyzed, Dicer and Dgcr8-deficiency T cells [Supplemental Fig. 1 (55, 56)]. Similar to NK cells, the had an impact on the level of NKG2D on the surface of NK cells. In frequency and number of naive CD8+ T cells were preferentially addition, we found elevated surface levels of Rae-1 on NK cells reduced in Dicer- and Dgcr8-deficient mice. Thus, miRNAs from Dicer-deficient mice (Fig. 4D). NKG2D is an activating re- shared by naive NK cells and CD8+ T cells might be implicated in ceptor that binds to a diverse family of ligands that are distant the regulation of common molecular pathways, such as regulation relatives of MHC class I molecules, including MICA and MICB in of homeostasis. humans and Rae-1a-ε, MULT1, and H60 in mice (36). Engagement NK cells also share a miRNA profile with effector and memory of NKG2D by its ligands leads to the direct activation of killing and CD8+ T cells. MiR21, miR221, and miR222 are expressed in both cytokine secretion by NK cells. Because of this potent killing NK and effector CD8+ T cells, whereas miR146a is found in both ability, surface expression of NKG2D ligands is tightly regulated so NK cells and memory CD8+ T cells [Supplemental Fig. 1 (56)]. It that the immune response is not triggered inappropriately. Our is intriguing to speculate that the miRNAs shared by NK cells, findings suggest that miRNAs may regulate the expression of the effector CD8+ T cells, and memory CD8+ T cells might be im- mouse NKG2D ligand Rae-1. One possible mechanism would be plicated in the regulation of common pathways that lead to the a specific miRNA targeting Rae-1 mRNA. Several human miRNAs acquisition of the effector phenotype and survival. Our identifi- were recently identified to bind the 39 untranslated regions of cation of a requirement of Dicer and Dgcr8 for Ly49H+ NK cell MICA and MICB and to repress their translation (48, 49). Alter- expansion during MCMV infection predicts miRNAs might target natively, Dicer or Dgcr8 deficiency in NK cells might elicit a DNA factors that are important for maintaining the NK cell population damage response, which in turn results in the induction of Rae-1 during viral infection. In conclusion, our results present evidence expression (50). Consistent with this hypothesis, silencing of Dicer for miRNAs regulating diverse aspects of NK cell biology, in- in HEK293T and human hepatoma cells led to the upregulation of cluding basic processes, such as turnover and survival, as well as MICA and MICB (51). In either of these scenarios, the increased the function of activating NKRs during MCMV infection. levels of Rae-1 would likely lead to down modulation of NKG2D on NK cells, as has previously been demonstrated (36). Acknowledgments We have shown that degranulation and IFN-g production were We thank Drs. Michael McManus and Eric Brown for mice. We also thank impaired in Dicer- and Dgcr8-deficient NK cells following stim- Jessica Jarjoura for assistance with cell sorting, Hernan Consengco for as- ulation via NK1.1, NKp46, or Ly49H. This impairment in IFN-g sistance with genotyping, and members of the Lanier laboratory for critical production might be caused by the reduction in the CD27lo reading of the manuscript. CD11bhi NK cell subset in DicerD/D and Dgcr8D/D mice, because this subset has been demonstrated to play a dominant role in cy- Disclosures tokine production (45). To this end, we costained NK cells with The authors have no financial conflicts of interest. anti-CD27 and anti-CD11b Abs and measured intracellular IFN-g levels. Both CD27hi CD11blo and CD27lo CD11bhi NK cell sub- References sets from DicerD/D and Dgcr8D/D mice produced less IFN-g com- 1. Lanier, L. L. 2008. Up on the tightrope: natural killer cell activation and inhibition. Nat. 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