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. Author manuscript; available in PMC: 2013 Jun 29.
Published in final edited form as: Immunity. 2012 May 17;36(6):921–932. doi: 10.1016/j.immuni.2012.04.006

Gene deregulation and chronic activation in natural killer cells deficient in the transcription factor ETS1

Kevin Ramirez 1, Katherine J Chandler 2, Christina Spaulding 4, Sasan Zandi 3, Mikael Sigvardsson 3, Barbara J Graves 2, Barbara L Kee 1,4
PMCID: PMC3389314  NIHMSID: NIHMS378665  PMID: 22608498

Abstract

Multiple transcription factors guide the development of mature functional natural killer (NK) cells yet little is known about their function. We used global gene expression and genome wide-binding analyses combined with developmental and functional studies to unveil three roles for the ETS1 transcription factor in NK cells. ETS1 functions at the earliest stages of NK cell development to promote expression of critical transcriptional regulators including T-BET and ID2, NK cell receptors (NKRs) including NKp46, Ly49H and Ly49D and signaling molecules essential for NKR function. As a consequence, Ets1−/− NK cells fail to degranulate after stimulation through activating NKRs. Nonetheless, these cells are hyper-responsive to cytokines and have characteristics of chronic stimulation including increased expression of inhibitory NKRs and multiple activation-associated genes. Therefore, ETS1 regulates a broad gene expression program in NK cells that promotes target cell recognition while limiting cytokine driven activation.

Keywords: ETS1, natural killer cells, natural cytotoxicity receptors, lymphocyte specification, cytokine

Introduction

Natural killer (NK) cells are lymphocytes that utilize germ line encoded activating and inhibitory receptors (NKRs) to recognize virus-infected, transformed and stressed cells. NK cells also contribute to adaptive immune responses through the production of inflammatory cytokines and by promoting the maturation or destruction of immature dendritic cells (Vivier et al., 2011). NK cells are activated when inhibitory NKRs recognizing classical or non-classical major histocompatibility complex (MHC) antigens fail to be engaged (“missing-self” recognition) and/or when activating NKRs detect their ligands thereby altering the balance between activating and inhibitory signals (Lanier, 2005). The mechanisms controlling the threshold for NK cell activation are not well understood but inhibitory receptor signaling appears to play a role in “licensing” or “arming or disarming” developing NK cells so that engagement of activating receptors results in a functional response (Joncker and Raulet, 2008; Yokoyama and Kim, 2006). NK cell immune deficiency results in susceptibility to infection and, while rare, NK cell malignancies are aggressive and difficult to treat. Therefore, understanding the mechanisms that control the development and function of NK cells has both basic biological and clinical significance.

NK cells develop in the bone marrow (BM) from common lymphoid progenitors (CLPs) through three major stages defined by expression of CD122 (interleukin 2 (IL-2) and IL-15 receptor-β chain), NK1.1 (an activating NKR expressed in only some strains of mice), and DX5 (integrin α2) (Kim et al., 2002; Rosmaraki et al., 2001). NK progenitors (NKPs) are CD122+ and lack NK1.1 and DX5 (CD49b). CD27, CD127 and CD244 mark a subset of NKPs (rNKP) enriched for NK cell potential as well as pre-NKP cells, a CD122− intermediate between CLP and rNKP (Fathman et al., 2011). Acquisition of NK1.1 occurs at the immature (i)NK cell stage where multiple NKRs initiate expression and the cells become dependent on IL-15 for survival (Vosshenrich et al., 2005). The mature (m)NK cell stage is defined by an increase in DX5, IL-15-driven expansion and licensing or arming of NK cells (Kim et al., 2002; Rosmaraki et al., 2001). Further maturation correlates with increased expression of CD11b and decreased expression of CD27 (Chiossone et al., 2009; Kim et al., 2002).

While stages in the NK cell program have been characterized little is known about the transcriptional networks that establish the NK cell gene program or promote NK cell function. A few transcription factors have been identified that play a major role in the generation of mNK cells including T-BET and EOMES (Gordon et al., 2012; Intlekofer et al., 2005), ETS1 (Barton et al., 1998), E4BP4 (Gascoyne et al., 2009; Kamizono et al., 2009), TOX1 (Aliahmad et al., 2010) and ID2 (Boos et al., 2007; Yokota et al., 1999). Although mNK cells largely fail to develop in these strains, the mechanisms underlying the observed phenotypes are not known. Moreover, the transcriptional programs controlling NKR expression, NK cell maturation or function remain to be determined, although a few factors such as TCF1, MEF1 and BLIMP1 play a role (Held et al., 1999; Kallies et al., 2011; Lacorazza et al., 2002).

ETS1, the founding member of the ETS family of transcription factors, has been known to be important for development of mNK cells for nearly 14 years and yet insight into how ETS1 functions is completely lacking (Barton et al., 1998). It is not known when ETS1 becomes essential and no target genes have been identified in the NK cell lineage. Here, we demonstrated that ETS1 functioned as early as the pre-NKP cell stage and that ETS1 regulated a broad spectrum of NK cell genes including transcription factors, NKRs and signaling molecules. We place ETS1 within a transcriptional network specifying the NK cell fate with direct targets including Tbx21 (T-BET) and Idb2 (ID2). Ets1−/− mNK cells failed to lyse NK cell targets and we demonstrated decreased expression or function of multiple activating NKRs. Unexpectedly however, Ets1−/− mNK cells had characteristics of chronic activation including increased expression of inhibitory NKRs Ly49G2 and Ly49E, increased expression of the IL-15 responsive gene Nfli3, encoding E4BP4, and increased Ikzf2, encoding HELIOS, a transcription factor associated with NK cell hyper-responsiveness (Narni-Mancinelli et al., 2012). Moreover, Ets1−/− mNK cells showed an augmented response to IL-15 in vitro. Our data provide insight into the molecular mechanisms underlying the requirement for ETS1 in NK cell development and function and provide a foundation for building the regulatory networks that control this important innate immune cell lineage.

Results

ETS1 functions at the earliest stages of NK cell development

Ets1−/− mice have a reduced number of mNK cells (Barton et al., 1998) but it is not known when or how ETS1 functions in the NK cell lineage. To begin to address this issue we rigorously analyzed NK cell development in Ets1−/− mice. As expected, in the BM and spleen of Ets1−/− mice mNK cell numbers were decreased by 90% and 80% respectively relative to wild-type (WT) mNK cells (Figure 1A, C, S1) (Barton et al., 1998). There was a decrease in the frequency of the most mature splenic mNK cells (CD27−CD11b+) but a similar frequency of these cells expressed KLRG1 (Figure S1) (Chiossone et al., 2009). ETS1 was required for development of approximately 50% of iNK cells but NKP numbers were similar to WT (Figure 1A, C). However, Ets1−/− rNKPs (Lin−CD27+CD244+Flt3−CD127+CD122+) were decreased by nearly 50% and their precursor pre-NKP (Lin−CD27+CD244+Flt3−CD127+CD122−) were decreased by 20% (Figure 1B, D). Ets1−/− mice also showed an approximate 50% decrease in pre-pro-NKb cells (Lin−CD117−Sca1+ CD127+Flt3−) (Figure S1) (Carotta et al., 2011). These data reveal a function for ETS1 at the earliest stages of NK cell development.

Figure 1.

Figure 1

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ETS1 functions at the earliest stages of NK cell development. Flow cytometry analysis of Ets1+/+ and Ets1−/− BM using (A) NK1.1 and DX5 to identify NKP (NK1.1− DX5−), iNK (NK1.1+DX5−) and mNK (NK1.1+DX5+) cells in CD122+ Lineage (CD19− CD3−TCRβ−CD4−CD8−Ter119−)− cells or (B) FLT3 and CD122 to identify CLP (Flt3+CD122−), pre-NKP (Flt3−CD122−) and rNKP (Flt3−CD122+) in Lin− CD27+CD244+CD127+ cells. The frequency of cells in the gated areas is indicated. The total number of (C) BM NKP, iNK and mNK and splenic mNK (sNK) or (D) CLP, pre-NKP and rNKP is presented. Each circle represents one mouse, horizontal bar = average. Closed circles = Ets1+/+ mice; open circles = Ets1−/− mice. n = 4-7 for each genotype. *= p<0.05, **= p<0.01, ***= p<0.001 using a students t-test, #= p<0.05 using a paired t-test but not significant with a standard t-test. (E) Relative contribution of Ets1+/+ and Ets1−/− Ly5.2+ cells in HSCs, MPPs, LMPPs, CLPs, NKP, iNK, mNK from mixed BM chimeras with WT Ly5.1+ competitors. Squares = Ets1+/+, Diamonds = Ets1−/−. n = 9 except CLPs where n = 7. See Figure S1 for analysis of splenic mNK cells, pre-pro-NK cells and flow cytometery analysis of BM chimeras.

To determine whether the requirements for ETS1 were cell-autonomous we created mixed BM chimeras where Ets1−/− cells developed in competition with WT cells. Both WT (CD45.2+) and Ets1−/− (CD45.2+) BM gave rise to hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), lymphoid primed MPPs (LMPPs) and CLPs that competed well with WT (CD45.1) cells (Figure 1E, S1). However, there was a >80% decline in NK lineage cells by the iNK cell stage (Figure 1E, S1). Therefore, there was a cell intrinsic requirement for ETS1 for NK cell development.

ETS1 regulates the NK cell transcriptome

To gain an understanding of how ETS1 functions in NK cells we performed a global analysis of gene expression in mNK cells isolated from Rag2−/−Ets1+/+ and Rag2−/− Ets1−/− mice. We used the Rag2−/− background to avoid contamination of NK cells with activated T lymphocytes (Stewart et al., 2007). We identified 216 genes that were decreased by 50% or increased by 2-fold by the absence of ETS1 (Figure 2A and Table S1). The distribution of these differentially expressed genes was examined across WT multipotent progenitor cell populations, proB cells and CD4+ T cells. Interestingly, nearly 50% of ETS1-dependent genes were expressed in CD4+ T cells but not in the other populations (Figure 2A). This finding suggested that ETS1 regulates a gene program shared with T cells, which also required ETS1 at multiple stages of development (Hollenhorst et al., 2009). However, slightly >50% of ETS1-dependent genes were unique to the NK cell lineage (Figure 2A).

Figure 2.

Figure 2

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Identification of ETS1-dependent genes in NK cells. (A) Microarray analysis of mRNA from Rag2−/−Ets1+/+ and Rag2−/−Ets1−/− mNK cells. Probe sets with differential expression in mNK cells are shown as is their mRNA expression across multiple hematopoietic cell populations. (B) Analysis of ETS1 binding to ETS1-dependent NK cell genes in a CD4+ T cell line by ChIP-seq. Genes associated with ETS1 bound regions were determined by the closest TSS. Pie graphs show the percent of ETS1-dependent NK genes that had human orthologs (upper chart) and the number of genes that were bound by ETS1 (lower chart). (C) MEME analysis of sequences bound by ETS1 in ETS1-dependent NK cell genes. (D) KEGG analysis for functional pathways regulated by NK cell genes using a 1.5X threshold of differential expression. See Table S1 for ETS1-dependent NK cell genes.

To distinguish direct from possibly indirect targets of ETS1 we considered a genome wide analysis of ETS1 binding by ChIP-sequencing (ChIP-seq). However, such an approach was hampered by the low abundance of NK cells in vivo and because ETS1 was down-regulated in NK cell lines and primary NK cells cultured in vitro, limiting the use of in vitro expanded cells (Figure S2). Given that a set of ETS1-dependent NK cell genes was expressed in CD4+ T cells, we began by examining the overlap between these genes and ETS1 binding in a human CD4+ T cell line as determined by ChIP-seq (Hollenhorst et al., 2007). Of the 216 ETS1-dependent NK cell genes, 167 had human orthologs and 106 of these (63.5%) were associated with ETS1 binding in the T cell line (Figure 2B). Therefore, 106 (49%) of the differentially expressed genes we identified are high probability ETS1 target genes (Table S2).

We next determined whether any unique binding motifs were enriched among the sequences associated with ETS1 binding at ETS1-dependent NK cell genes using MEME (Bailey et al., 2009). An ETS binding motif was enriched that was nearly identical to the motif previously associated with ETS1 specific binding at distal (enhancer) sites (Figure 2C). These are sites that failed to be bound by ELF1 and GABPa in the CD4+ T cell line (Hollenhorst et al., 2009).

ETS1 is required for proper expression of multiple NKRs, signaling molecules and transcription factors

KEGG pathway analysis of the differentially expressed genes revealed their involvement in NK cell cytotoxicity, T cell receptor-, chemokine- and Janus kinase-signal transducer and activator of transcription (Jak-STAT)-signaling pathways (Figure 2D) (Huang da et al., 2009). A selected set of NK cell-associated genes is shown in Figure 3A and among these Ltb, Tbx21, Itk, Slamf6, Jak1, CD27, Lck, and Lair1 were bound by ETS1 in the CD4+ T cell line. We demonstrated that ETS1 binds directly to the Tbx21 and Cd122 genes in mNK cells by ChIP (Figure 3D), confirming that these are direct targets. We confirmed differential expression of Ncr1 (NKp46), Cd122, Idb2, Ltb, Lair1 and Tbx21 mRNA in Lin−CD122+DX5− proNK cells (NKP+iNK) and mNK cells by quantitative polymerase chain reaction (QPCR) (Figure 3B). Reduced expression of Ltb and Tbx21 mRNA was also confirmed in Ets1−/− NKPs (Figure 3C). Therefore, Ets1-deficiency results in decreased expression of critical regulators of NK cell development including transcription factors and NKRs.

Figure 3.

Figure 3

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Deregulation of multiple NK cell genes in the absence of ETS1. (A) Heat maps for a subset of ETS1-dependent genes in Rag2−/−Ets1+/+ and Rag2−/−Ets1−/− mNK cells (duplicates combined). * indicates that ETS1 was bound near this gene by ChIP-seq in CD4 T cells. (B) QPCR analysis Tbx21, Ncr1, Cd122, Idb2, Ltb and Lair1 mRNA in Ets1+/+ (black) and Ets1−/− (white) proNK cells (Lin−CD122+DX5-) and mNK cells. At least 3 independent experiments were performed. (C) QPCR analysis of Tbx21 and Ltb mRNA in NKPs. Error bars = s.d. of triplicate measurements. Two independent experiments were performed. (D) Anti-ETS1 ChIP for EBS in Tbx21 and Cd122. QPCR SP5, Hbb and Ebf1 are at regions that lack an EBS. See Table S2 for ETS1 binding sites in CD4+ T cells.

ETS1 regulates Idb2 in NK cells and their progenitors

The Idb2 gene, which encodes ID2, is required for proper development beyond the iNK cell stage (Boos et al., 2007) and its expression was dependent on ETS1 in mNK cells (Figure 3A). However, Idb2 was not bound by ETS1 in the CD4+ T cell line raising the possibility that Idb2 is not a direct target of ETS1. To gain insight into the mechanisms controlling Idb2 expression we performed mutational analysis of Idb2 promoter-luciferase reporters in an NKP cell line (Rodewald et al., 1992). We found that Idb2 reporters containing at least 225 bp of sequence upstream of the transcription start site (TSS) gave maximal luciferase activity (Figure 4A). In contrast, truncation to 130 bp, which removes a potential ETS binding site (EBS), decreased luciferase activity by 80% indicating that an important cis-regulatory element was deleted (Figure 4A). Mutation of this EBS in the context of the 670 bp or 225 bp promoter decreased luciferase activity by 45% and 68% respectively demonstrating that an ETS family protein was important for Idb2 transcription in this NKP line (Figure 4A).

Figure 4.

Figure 4

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Idb2 is regulated by ETS1 in NK cells and their precursors. (A) Promoter-luciferase reporter assay using regions 5′ of the Idb2 TSS, including the endogenous EBS site (GGA) or a mutated (m) EBS (GGT). One representative experiment from at least 4 is shown. (B) EMSA of proteins in PTL cell extracts binding to the Idb2 EBS in the presence (+) or absence (−) of cold competitor or α-ETS1, α-MEF1 or α-ELF1. (C) ChIP was performed on sorted splenic mNK cells using α-ETS1 followed by QPCR with primers flanking the EBS in Idb2 or for sequences lacking an EBS (SP5, Hbb and Ebf1). (D) QPCR analysis for Idb2, Ets1, Elf1 and Gabpa mRNA relative to Hprt mRNA in sorted NK cell and NK cell progenitor populations. Error bars represent standard error of triplicate measurements and one of at least 2-3 replicate experiments is shown. See Figure S2 for Ets1 mRNA expression in NK cell lines and activated NK cells.

The putative EBS in the Idb2 promoter was identified previously as a target of the EWS-FLI1 and EWS-ERG fusion proteins found in Ewing’s sarcoma (Nishimori et al., 2002). FLI1 and ERG are members of a different clade of ETS family proteins and they have a DNA binding preference distinct from ETS1, therefore, it was not evident that ETS1 should regulate Idb2 through this EBS. However, the Idb2 EBS fits a consensus motif bound by multiple ETS family proteins including ETS1, ELF1 and GABPa (Hollenhorst et al., 2009; Hollenhorst et al., 2007). Electrophoretic mobility shift assays (EMSA) confirmed that both ETS1 and ELF1 were present in the NKP extract and were able to bind the Idb2 promoter EBS whereas MEF1 (Lacorazza et al., 2002), was not present in the bound complex (Figure 4B). Importantly, in mNK cells we detected binding of ETS1 at the Idb2 promoter indicating that ETS1 could directly regulate Idb2 (Figure 4C). Analysis of mRNA at defined stages of NK cell differentiation revealed an earlier onset of expression for Ets1 mRNA as compared to Idb2 and Ets1 expression peaks in rNKPs just prior to the peak of Idb2 at the iNK cell stage (Figure 4D). These data are consistent with the hypothesis that Idb2 mRNA is dependent on ETS1 at the initiation of NK cell lineage specification. While ID2 is not essential for early NK cell development its expression is one of the first indications of NK cell lineage specification and decreased expression of ID2 in Ets1−/− mNK cells is predicted to have an impact on the differentiation and function of these cells (Boos et al., 2007).

ETS1 is required for proper expression and function of multiple activating NKRs

The few mNK cells present in Ets1−/− mice are defective in their ability to kill cells lacking MHC Class 1 molecules (Barton et al., 1998). However, the mechanism underlying this loss of cytolytic function is not known. A failure to express activating receptors, or essential components of the signaling machinery activated by these receptors, would explain this defect. Our microarray analysis revealed decreased expression of genes encoding the activating receptor NKp46 and multiple Ly49 receptors (encoded by the Klra genes) as well as proteins involved in signal transduction by these receptors. We confirmed the decreased expression of NKp46, Ly49D and Ly49H on BM and splenic mNK cells from Ets1−/− mice by flow cytometry (Figure 5A, B). These activating NKRs were also reduced on Ets1−/− mNK cells isolated from mixed BM chimeras indicating that this alteration was cell intrinsic (Figure S3). Therefore, the failure of Ets1−/− mNK cells to kill NK cell targets may be, in part, a consequence of decreased expression of multiple activating NKRs.

Figure 5.

Figure 5

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Ets1−/− mNK cells had decreased activating NKR expression and activating NKR triggered degranulation. (A) Flow cytometry analysis for the activating NKRs NKp46, Ly49D and Ly49H in BM and spleen of Ets1+/+ (dark line) and Ets1−/− (grey line) mice. Shaded histogram = isotype control. (B) Summary of the percent of mNK cells expressing NKp46, Ly49D and Ly49H. Ets1+/+ (filled) Ets1−/− (open), horizontal bar = average. * = p<0.05, ** = p<0.01, *** = p<0.001 n = 4, ns = p > 0.05 (C) Flow cytometry analysis for the activating NKRs NK1.1 and NKG2D on Ets1+/+ and Ets1−/− mNK cells. Histograms are as indicated in (A). Summary of percent (D) CD107a+ or (E) IFN-γ+ after a 5 hour stimulation with PMA plus ionomycin, α-NK1.1, α-NKG2D or IgG. See Figure S2 for activating NKR expression in mixed BM chimeras and representative flow cytometery staining for CD107a and IFN-γ.

In contrast to NKp46, Ly49D and Ly49H the activating receptors NK1.1 and NKG2D were expressed appropriately on BM and splenic mNK cells (Figure 5C) suggesting that these receptors are available for NK cell target recognition. To determine whether these receptors were functional, we tested the ability of NK1.1 or NKG2D cross-linking to induce degranulation, as measured by surface CD107a. As expected, cross-linking of NK1.1 or NKG2D resulted in increased CD107a compared to IgG on Ets1+/+ mNK cells (Figure 5D, S3). In contrast, NKG2D stimulation of Ets1−/− mNK cells did not induce CD107a above that observed with IgG, although CD107a was higher on IgG stimulated Ets1−/− mNK cells compared to Ets+/+ mNK cells (Figure 5D, S3). Cross-linking of NK1.1 on Ets1−/− mNK cells increased surface CD107a but the frequency of CD107a+ cells was lower than observed on Ets1+/+ cells. In contrast, CD107a was efficiently induced by phorbol myristate acetate (PMA) + ionomycin in both Ets1+/+ and Ets1−/− mNK cells (Figure 5D, S3). These observations indicated that Ets1−/− mNK cells were intrinsically defective in their ability to degranulate in response to activating NKR stimulation. In contrast, interferon-γ (IFN-γ) production was not as severely affected, although cross-linking of NKG2D did not lead to a significant accumulation of IFN-γ at this time point in Ets1+/+ or Ets1−/− mNK cells (Figure 5E, S3). The reduced expression of many activating NKRs and the impaired exocytosis function in Ets1−/− mNK cells is sufficient to explain the decreased cytolytic function of these cells.

Ets1−/− NK cells have characteristics of chronic cytokine stimulation

In addition to the ETS1-dependent genes we noted that multiple genes associated with NK cell activation were increased in Ets1−/− mNK cells. Gzmb and Prf1 mRNA, encoding the cytolytic proteins Granzyme B and Perforin, were increased as were mRNAs encoding the serine protease inhibitors Serpinb6a and Serpinb9b (Figure 6A). We confirmed that Gzmb mRNA was increased in Ets1−/− mNK cells by qPCR (Figure 6B). Nfil3 mRNA, encoding a cytokine responsive transcription factor (Gascoyne et al., 2009; Kamizono et al., 2009), was also increased in Ets1−/− mNK cells as well as in proNK cells (Figure 6A, C). Interestingly, Ikzf2 mRNA, which encodes HELIOS, whose increased expression contributes to hyper-responsiveness in Noé mice (NKp46-deficient) (Narni-Mancinelli et al., 2012), was increased in Ets1−/− mNK cells (Figure 6A, D). Ets1−/− mNK cells isolated from mixed BM chimeras also showed increased granularity and increased expression of the activation marker CD69 as measured by flow cytometry (Figure 6E). Taken together these data indicate that Ets1−/− mNK cells are in an activated state.

Figure 6.

Figure 6

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Ets1−/− mNK cells have characteristics of chronic cytokine stimulation. (A) Heat maps for a subset of ETS1-repressed genes in Rag2−/−Ets1+/+ and Rag2−/−Ets1−/− mNK cells. * indicates that ETS1 was bound near this gene in CD4+ T cells as determined by ChIP-seq. QPCR analysis for (B) Gzmb (C) Nfi13 and (D) Ikzf2 mRNA in Ets1+/+ (black) and Ets1−/− (white) mNK cells. Expression is plotted relative to transcripts from Hprt. Flow cytometry analysis for (E) SSC and CD69 or (F) the inhibitory NKRs Ly49A, Ly49G2 and Ly49E on BM and splenic mNK cells from Ets1+/+ (black) and Ets1−/− (grey). (G) Summary of the percent of mNK cells expressing Ly49A, Ly49G2 and Ly49E. Ets1+/+ (filled) Ets1−/− (open), horizontal bar = average. * = p<0.05, ** = p<0.01, *** = p<0.001. n = 4, ns = p>0.05. Cells in E were isolated from Ly5.1 (WT):Ly5.2 (WT or Ets1−/−) chimeric mice. See Figure S3 for Ly49A, Ly49G2 and Ly49E staining on mNK cells from mixed BM chimeras.

Our observation that at least two IL-15-regulated genes are increased in Ets1−/− mNK cells led us to question whether these cells have other characteristics of chronic cytokine stimulation. Chronic IL-15 stimulation leads to increased expression of the inhibitory NKRs Ly49G2 and Ly49E, which is normally not expressed on adult mNK cells (Barao et al., 2011; Elpek et al., 2011; Fraser et al., 2002). We found an increased frequency of BM mNK cells expressing Ly49G2 and Ly49E but not Ly49A in Ets1−/− as compared to WT mice (Figure 6F, G). The intensity of Ly49G2 and Ly49E staining was also increased on Ets1−/− mNK cells in both the BM and spleen. (Figure 6F). These alterations in inhibitory NKR expression were cell intrinsic since they were observed on Ets1−/− mNK cells in mixed BM chimeras (Figure S4). Taken together with the increased expression of Nfil3, Gzmb, Prf1 mRNA and CD69, our findings indicate that Ets1−/− mNK cells resembled NK cells chronically stimulated by IL-15.

Ets1−/− mNK cells are hyper-responsive to IL-15

Given the phenotype of Ets1−/− mNK cells we questioned how Ets1−/− NK cells would respond to cytokines. Single cell analysis of Ets1−/− and Ets1+/+ DX5− and DX5+ NK cells cultured in vitro revealed that a comparable frequency of cells could form colonies in response to IL-2 (Figure 7A). However, Ets1−/− colonies were larger and the cells were more granular than their WT counterparts (Figure 7B). To determine whether Ets1−/− mNK cells were more responsive to IL-15 than WT mNK cells, we titrated IL-15 in cultures of Ets1−/− and Ets1+/+ mNK cells and measured induction of GRANZYME B and proliferation, using BrdU incorporation. Within 24 hours, Ets1−/− mNK cells showed an increase in Granzyme B and BrdU incorporation compared to Ets1+/+ mNK cells at all concentrations of IL-15 (Figure 7C). The augmented response of Ets1−/− mNK cells was particularly evident when IL-15 was present at 50 ng/ml, the concentration commonly used for expansion of NK cells in vitro (Figure 7C, D). In addition, whereas Ets1+/+ mNK cells showed little induction of Granzyme B or BrdU incorporation when cultured in 1 ng/ml IL-15, Ets1−/− mNK cells showed a 4-fold higher response (Figure 7C, D). These experiments were performed with mNK cells isolated from mixed BM chimeras allowing us to exclude in vivo homeostatic proliferation as a factor predisposing Ets1−/− mNK cells to an increased cytokine response. Taken together with the data in Figure S2, showing that Ets1 mRNA decreased when NK cells were stimulated in vivo for 2 days with IL-2 or 1 day with poly I:C, our findings suggest a role for ETS1 in limiting NK cell activation in response to cytokines. Our data support a model in which ETS1 controls expression of a broad spectrum of NK cell genes including transcription factors, NKRs, and signaling molecules at the earliest stages of NK cell development allowing for appropriate NK cell activation in pathogenic conditions.

Figure 7.

Figure 7

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Ets1−/− mNK cells are hyper-responsive to IL-15. (A) Percent of single Ets1+/+ (filled) and Ets1−/− (open) proNK and mNK cells giving rise to visible colonies after 10 days in cultures supplemented with IL-2. (B) FSC versus SSC on typical mNK cell progeny from A. (C) Flow cytometry analysis for Granzyme B and BrdU incorporation in splenic mNK cells 24 hours after initiation of culture in varying concentrations of IL-15. The percent of Granzyme B+ BrdU+ cells is indicated. One of 3 experiments is shown. (D) Average +/− S.D. of percent Granzyme B+ BrdU+ cells from 3 independent experiments. * = p<0.05, ** = p<0.01, *** = p<0.001. The splenic mNK cells were isolated from BM chimeras.

Discussion

In this study, we have revealed at least three major functions for ETS1 in NK cells. First, ETS1 directly regulates expression of Idb2 and Tbx21, whose protein products ID2 and T-BET comprise a part of the transcriptional circuitry necessary for NK cell differentiation. Second, ETS1 is required for expression and function of multiple activating NKRs that are necessary for induction of NK cell-mediated cytolysis. This functional deficit was revealed primarily as a failure of degranulation rather than IFN-γ production. Thus, the inability of Ets1−/− NK cells to kill NK cell targets can be explained by their decreased ability to degranulate in response to activating NKR ligands. Third, ETS1 sets the threshold for responsiveness to cytokine, and likely other external stimuli, which may prevent expansion and activation in non-pathogenic conditions. In the absence of ETS1, mNK cells had hallmarks of chronic IL-15 stimulation and they had a heightened response to a sub-optimal dose of IL-15. Taken together, our data provide insight into the functions of this critical transcriptional regulator in NK cells and provide a foundation on which to build the regulatory circuits driving NK cell development and function.

The absence of ETS1 resulted in alterations in NK cell progenitors at the earliest stages of development, placing ETS1, along with ID2, TOX1 and E4BP4 (Aliahmad et al., 2010; Kamizono et al., 2009), as the earliest acting transcriptional regulators identified in NK cells. We showed that Ets1 mRNA expression precedes Idb2 mRNA, which was previously the earliest known marker of NK cell differentiation. Therefore, ETS1 is positioned to play a key role in NK cell lineage specification. In order for ETS1 to function in NK cell specification its expression should precede NK cell lineage restriction. We previously found that Ets1 was among the genes primed by E2A in LMPPs (Dias et al., 2008). During specification of the NK cell lineage E2A function is antagonized by ID2 and ID3 (Boos et al., 2007) and yet Ets1 mRNA increases. Therefore, the transcription factors controlling Ets1 must evolve as the NK cell fate is specified. This shift in transcriptional control could occur as a consequence of the induction of NK cell-associated transcription factors such as T-BET, or alternatively, ETS1 may autoregulate its own expression (Hollenhorst et al., 2009; Seth and Papas, 1990). There are multiple ETS1 binding events near the Ets1 gene in CD4+ T cells indicating that ETS1 may control its own expression (Hollenhorst et al., 2009). Based on these considerations, and our current knowledge of transcriptional networks in B and T cell development (Nutt and Kee, 2007), we hypothesize that ETS1 functions in a transcriptional network with re-enforcing feedback loops to control NK cell lineage specification.

It is important to note that while NKP numbers in Ets1−/− mice were indistinguishable from Ets1+/+ mice, more highly enriched progenitor populations revealed a requirement for ETS1. However, identification of pre-NKP, rNKP and pre- pro-NK cells relies on expression of surface proteins reported to be ETS1 targets raising the possibility that altered gene expression rather than altered development is responsible for this decrease. CD127 (IL-7Rα) is critical for identification of these cells and was reported to be an ETS1 target in peripheral CD8+ T cells (Grenningloh et al., 2010). Importantly, we did not find decreased CD127 on Ets1−/− CLPs using multiple different staining strategies or within the larger Lin−CD244+CD27+ population containing NK cell progenitors. Therefore, ETS1 is not essential for CD127 expression in multipotent progenitors of NK cells. Nonetheless, if CD127 were an ETS1 target in the earliest NK cell progenitors this would further support our conclusion that ETS1 controls gene expression at this early stage of NK cell development.

We have defined a minimal set of high probability ETS1 target genes by correlating ETS1-dependent gene expression with ETS1 DNA binding events in a CD4+ T cell line. This is a minimal set because not all ETS1-dependent NK cell genes are expressed in CD4+ T cells, therefore, ETS1 binding could not be assessed at all NK cell targets. Nonetheless, 49% of ETS1-dependent NK cell genes were bound by ETS1 in CD4+ T cells likely reflecting overlapping functions for ETS1 in these cell types. Indeed, ETS1 regulates genes involved in T cell activation in CD4+ T cells (Hollenhorst et al., 2009; Hollenhorst et al., 2007) and we identified “T cell receptor signaling”, in addition to “NK cell cytotoxicity”, as pathways associated with ETS1-dependent genes in NK cells. Consistent with this common function, we found that ETS1-regulated NK genes had ETS binding motifs nearly identical to an ETS1-specific motif reported in T cell studies (Hollenhorst et al., 2009; Hollenhorst et al., 2007). However, while this site was not associated with ELF1 binding in CD4+ T cells, it shows similarities to ETS binding motifs reported for other ETS family proteins (Hollenhorst et al., 2009; Wei et al., 2010). We speculate that other ETS factors may occupy some of these sites in the absence of ETS1, providing an explanation for the only partial decrease in many putative ETS1 target genes in Ets1−/− mNK cells. In addition, some genes, such as Idb2, have an ETS binding motif that can be bound by ELF1 and other ETS family proteins (Hollenhorst et al., 2009) and we found ELF1 binding to this site in an NKP line. Therefore, ETS family proteins likely play a more crucial role in NK cell development than revealed by the Ets1−/− mouse.

An emerging question is why ETS1 induces some genes specifically in NK cells but not in T cells and B cells where it is also expressed (Eyquem et al., 2004a; Eyquem et al., 2004b). The unique chromatin landscape present in each of these cells undoubtedly plays an important role. However, there are also factors that may influence ETS1 target gene selection such as ETS1 concentration and DNA sequence affinity (Hollenhorst et al., 2009; Hollenhorst et al., 2007), post-translational modification (Cowley and Graves, 2000; Lee et al., 2008), DNA methylation (Gaston and Fried, 1995; Yokomori et al., 1995) and cooperative interactions with neighboring transcription factors (Cowley and Graves, 2000; Pufall et al., 2005). In T cells, ETS1 and RUNX1 bind cooperatively at the Tcra enhancer and in B cells ETS1 is recruited to the Cd79b promoter via association with PAX5 (Fitzsimmons et al., 2009; Hollenhorst et al., 2009). Future studies analyzing cis-regulatory elements at shared and NK cell specific ETS1 targets could provide insight into the mechanisms of lineage specific gene expression by ETS proteins. Our study provides a critical first step in this analysis by identifying potential shared and NK cell specific ETS1 target genes.

Multiple observations lead us to conclude that ETS1 limits the NK cell response to cytokines. In addition to having an activated phenotype Ets1−/− NK cells showed elevated Nfil3 mRNA and Nfil3 is regulated downstream of IL-15 and is sufficient to rescue NK cell differentiation in Il15ra−/− NKPs cultured in vitro (Gascoyne et al., 2009; Kamizono et al., 2009). Ly49G2 and Ly49E were both highly expressed on Ets1−/− compared to Ets1+/+ mNK cells and their expression is up-regulated by chronic cytokine stimulation (Barao et al., 2011; Elpek et al., 2011; Fraser et al., 2002). Moreover, in in vitro cytokine-dependent cultures, Ets1−/− NK cells cloned well with larger colony sizes compared to Ets1+/+ NK cells and both in vitro and in vivo, and under competitive reconstitution conditions, Ets1−/− NK cells had an activated phenotype. Most importantly however, Ets1−/− mNK cells incorporated more BrdU and induced Granzyme B more rapidly than WT mNK cells at all concentrations of IL-15. The mechanism underlying the heightened activation in Ets1−/− NK cells likely involves the deregulation of multiple genes encoding signaling proteins and transcription factors (for example, Itk, Jak1, Lck, Ppp1r3b, Ptpn3, Erg3, Tbx21, Ikzf2 or Nfat1c). In addition, the decreased expression and function of activating NKRs may change the “tuning” of the intracellular signaling milieu resulting in an altered response to multiple cell surface receptors (Joncker et al., 2009; Joncker and Raulet, 2008). Indeed, mice lacking NKp46 are hyper-responsive to MCMV and the NK cell target YAC-1 and their increased responsiveness requires HELIOS (Narni-Mancinelli et al., 2012), which is increased in Ets1−/− NK cells and therefore likely contributes to the hyper-responsive phenotype. However, in Ets1−/− mNK cells the compounded defects in activating receptor expression and degranulation likely limited NK cell mediated lysis. The hypothesis that ETS1 influences lymphocyte activation potential is consistent with a previously reported role for ETS1 in the B cell response to TLR9 (John et al., 2008; Wang et al., 2005). Moreover, ETS1 influences cytokine responsiveness and activation in T lymphocytes (Clements et al., 2006; Grenningloh et al., 2005; Higuchi et al., 2007; Moisan et al., 2007; Russell and Garrett-Sinha, 2010) indicating that targets of ETS1 contribute to the signaling milieu in adaptive lymphocytes. The barrier to NK cell activation imposed by ETS1 may reflect involvement of ETS1 targets in the unique mechanisms controlling NK cell activation since Ets1−/− T cells fail to become activated after stimulation (Muthusamy et al., 1995). Importantly, while ETS1 deficiency phenocopies many aspects of chronic cytokine stimulation, Ets1−/− mice do not develop leukemia as was observed in IL-15 transgenic mice (Fehniger et al., 2001). Leukemogenesis may be limited by the arrested differentiation that accompanies ETS1 deficiency at the earliest stages of NK cell development.

Methods

Mice

C57BL/6 or 129/SvJ Ets1−/− mice (Barton et al., 1998) were housed at the University of Chicago Animal Resources Center in accordance with the guidelines of the University of Chicago Institutional Animal Care and Use Committee. 129/SvJ Rag2−/− mice were purchased from Jackson labs.

Quantitative real-time PCR (QPCR)

RNA was purified using the RNeasy micro kit (Qiagen), reverse-transcribed with SuperScriptIII (Invitrogen) and primed with random hexamers as described (Boos et al., 2007). Expression is reported as ΔCT relative to Hprt mRNA. QPCR primer sequences are available upon request.

Luciferase reporter assays

The 670 bp and 225 bp Idb2 promoter fragments were PCR amplified from genomic DNA and cloned into pGL3. The 130 bp Idb2 fragment was digested from pGL3-225-Idb2p using SacI and XhoI and cloned into pGL3. PTL cells were transfected using DEAE-dextran with 8 ug of pGL3 constructs and 0.5 ug of pRL-CMV as an internal control (Kee and Murre, 1998). Lysates were prepared 48 hours after transfection and assayed using the Dual-Glo Luciferase kit (Promega).

Electrophoretic Mobility Shift Analysis (EMSA)

Nuclear extracts were prepared and EMSA performed as decscribed (Kee and Murre, 1998). The Idb2 EBS sequence was 5′-GGTATTGGCTGCGAACGCGGAAGAACC-3′ and the Idb2 EBS mutant sequence was 5′-GGTATTGGCTGCGAACGCGGTAGAACC-3′. Antibodies to ETS1, ELF1, and MEF1 were purchased from Santa Cruz Biotechnology.

Cell culture

Cells lines were maintained in Opti-MEM or RPMI-1640 supplemented with 10% FBS, 80 μM 2-mercaptoethanol, 100 units/ml penicillin, 100ug/ml streptomycin, and 29.2 mg/ml glutamine. Primary NKPs were grown on OP9 stromal cells (10,000 OP9 cells/well of a 96 well plate) supplemented with IL-2 (1000 IU/ml, NIH Reagents program), c-Kit (1:1000 dilution from CHO-MGF cells), and Flt3 (10 ng/ml). Primary mNK cells and NK cell lines were cultured in media supplemented with IL-2. The PTL line was generated by Dr. Hans-Reimer Rodewald by in vitro culture of fetal thymus derived FcRγII or III+ NK and T cell progenitors (Rodewald et al., 1992) and was adapted for growth in Opti-MEM. The KY1, KY2 and NKCRθ cell lines were provided by Wayne Yokoyama and Claude Roth (Caraux et al., 2006; Karlhofer et al., 1995).

IL-15 responsiveness was determined by culturing 1500-3000 flow cytometry-sorted splenic mNK cells, isolated from chimeric mice, in multiple concentrations of recombinant mIL-15. At T=24 hours, 1 mM BrdU was added for 45 minutes prior to intracellular staining for Granzyme B and BrdU.

Flow cytometry

Cells were stained with fluorochrome- or biotin-labeled antibodies for 20 minutes on ice. The following antibodies conjugated to FITC, PE, PerCP-Cy5.5, PerCP-ef710, PeCy7, APC, APC-ef780, Pacific Blue, or Brilliant Violet 421 were purchased from eBioscience, BD Pharmingen, or Biolegend: Ly5.2 (104), Ly5.1 (A20), CD19 (1D3), B220 (Ra3-6B2), CD3ε (145-2C11), CD4 (RM4-5), CD8α (Ly-2), TCRβ (H57-597), TCRγδ (UC7-13D5), CD11b (M1/70), Ter-119 (Ly-76), Gr-1 (RB6-8C5), IL-7Rα (A7R34), c-Kit (2B8), Sca1 (E13-161.7), Flt3 (A2F10), CD122 (TM-β1), NK1.1 (PK136), CD49b (DX5), CD94 (18d3), NKG2ACE (20d5), NKp46 (29A1.4), Klrg1 (CF1), CD69 (H1.2F3), Ly49D (4E5), Ly49H (3D10), Ly49G2 (ebio4D11), Ly49A (A1), LyEF (CM4), Ly49CIFH (14B11). ). Ly-6d (49-H4), CD27 (LG.7F9), Klrg1 (2F1), BrdU (Bu20a or PRB-1), IFNγ (XMG1.2), Granzyme B (NGZB or 16G6), and 2B4 (ebio244F4). Propidium idodide was used to exclude dead cells. Cells were analyzed on a FACS Canto, LSRII or Fortessa or sorted using a FACS ARIAII (Becton Dickenson).

Lineage cocktail for HSC, MPP, LMPP, CLPs: B220, CD3ε, CD4, CD8, NK1.1, Ter119, CD11b, Ly-6G and for NK cells: CD19, CD3ε, CD4, CD8, Ter119 and for CLP, pre-NKP, rNKP populations-CD19, CD11b, CD3ε, Ly6d, and NK1.1.

NK cell activation

In vivo activation of NK cells was accomplished by an intravenous (i.v.) injection of 1,000,000 IU IL-2 at t=0 and t=24 hours. At t=48 hours NK cells were isolated by flow cytometry. Alternatively, 100 ug of poly I:C was injected intraperitoneally (i.p.) followed by isolation of NK cells at t= 24 hours. For α-NK1.1, α-NKG2D or IgG stimulation of NK cells mNK cells were cultured on antibody coated plates in 1000 IU/ml IL-2 plus Golgi Plug (Becton Dickenson) for 5 hours. Fluorochrome labeled α-CD107a (1D4B) or isotype control antibodies (5 ug/ml) were added at t=0. Cells were stained with DX5 and NKp46 prior to flow cytometry analysis. Alternatively, cells were cultured with PMA (100 ng/ml) and ionomycin (2 mM).

BM chimeras

Chimeric mice were established by retrorbital injection of 2.5 × 106 bone marrow cells from Ly5.1 WT mice and 2.5 × 106 bone marrow cells from Ly5.2 Ets1+/+ or Ly5.2 Ets1−/− mice into 8 week old lethally irradiated (1,000 rad) Ly5.1 and Ly5.2 or Ly5.1 recipients. Recipients were maintained on Bactrim and analyzed 8 weeks post transplantation.

Microarray analysis

cDNA prepared from 10,000 Lin−CD122+DX5+ cells was used to probe Affymetrix MOE 430_2 arrays as previously described (Dias et al., 2008). Raw array data was normalized using RMAexpress (http://rmaexpress.bmbolstad.com/) and analyzed by dChip (http://www.biostat.harvard.edu/complab/dchip/). Probe set annotation was obtained from Affymetrix. GEO Accession (GSE37301).

MEME

Multiple Em for Motif Elicitation was used to identify repeated motifs in ETS1 ChIP-Seq sequences from the CD4+ T cell line Jurkat. MEME was run with default setting, except the minimum motif length was set to 8 and maximum to 15. Only the motifs with the lowest E-value are reported.

Chromatin Immunoprecipitation (ChIP)

Primary mouse mNK cells were crosslinked in 1% formaldahyde and sheared on a Branson sonicator. Protein-DNA complexes were immunoprecipitated with polyclonal anti-ETS1 (C-20) or IgG (Santa Cruz). For each sample, 1,000,000 cell equivalents of chromatin were incubated with 5 ug of antibody. Protein G coupled magnetic beads were used to isolate immune complexes. Crosslinks were reversed by heating at 65°C followed by proteinase K treatment. DNA was purified using PCR spin columns (Qiagen) and amplified by QPCR using primers specific for the Tbx21, Cd122 or Idb2 EBS or irrelevant genomic regions (SP5, Hbb, Ebf1). ChIP-sequencing was described in (Hollenhorst et al., 2009).

Supplementary Material

01
02
03

Acknowledgements

We thank Eric Svensson and Kevin Barton for providing Ets1−/− mice. We are grateful to Vinay Kumar for helpful discussions and comments on this manuscript. This work was supported by the National Institutes of Health (CA099978 to B.L.K., CA099978-S for K.R., GM38663 to B.J.G. and CA42014 to the Huntsman Cancer Institute for support of core facilities). B.L.K. is supported by a scholar award from the Leukemia and Lymphoma Society. K.R. is supported by the Interdisciplinary Training Program in Immunology (T32AI007090).

Footnotes

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01
NIHMS378665-supplement-01.xls (74.5KB, xls)
02
NIHMS378665-supplement-02.xlsx (22.8KB, xlsx)
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NIHMS378665-supplement-03.pdf (534.9KB, pdf)

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