Gene Deregulation and Chronic Activation in Natural Killer Cells Deficient in the Transcription Factor ETS1

Immunity

Article Gene Deregulation and Chronic Activation in Natural Killer Cells Deficient in the Transcription Factor ETS1 Kevin Ramirez,1 Katherine J. Chandler,3 Christina Spaulding,2 Sasan Zandi,4 Mikael Sigvardsson,4 Barbara J. Graves,3 and Barbara L. Kee1,2,* 1Committee

on Immunology of Pathology, Committee on Cancer Biology and The University of Chicago Comprehensive Cancer Center The University of Chicago, Chicago, IL 60615, USA 3Department of Oncological Sciences, University of Utah School of Medicine, Huntsman Cancer Center, University of Utah, Salt Lake City, UT 84112, USA 4Department of Clinical and Experimental Medicine, Experimental Hematopoiesis Unit, Faculty for Health Sciences, Linko ¨ ping University, 58183 Linkoping, Sweden *Correspondence: [email protected] DOI 10.1016/j.immuni.2012.04.006 2Department

SUMMARY

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 hyperresponsive 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.

INTRODUCTION Natural killer (NK) cells are lymphocytes that utilize germlineencoded 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 nonclassical 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, although 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-b chain), NK1.1 (an activating NKR expressed in only some strains of mice), and DX5 (integrin a2) (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 (rNKPs) enriched for NK cell potential as well as preNKP cells, a CD122
intermediate between CLP and rNKP (Fathman et al., 2011). Acquisition of NK1.1 occurs at the immature NK (iNK) cell stage where multiple NKRs initiate expression and the cells become dependent on IL-15 for survival (Vosshenrich et al., 2005). The mature NK (mNK) 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). Although 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 Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc. 921

Immunity ETS1 Targets in NK Cells

controlling NKR expression and 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. 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 hyperresponsiveness (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 (Figures 1A and 1C; Figure S1 available online; 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 (Figures 1A and 1C). However, Ets1
/
rNKPs (Lin
CD27+ CD244+FLT3
CD127+CD122+) were decreased by nearly 50% and their precursor pre-NKPs (Lin
CD27+CD244+FLT3
CD127+CD122
) were decreased by 20% (Figures 1B and 1D). 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. 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 922 Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc.

(CD45.1) cells (Figures 1E and S1). However, there was a >80% decline in NK lineage cells by the iNK cell stage (Figures 1E and 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; Table S1). The distribution of these differentially expressed genes was examined across WT multipotent progenitor cell populations, pro-B cells, and CD4+ T cells. 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). 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 downregulated 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 via 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 and in T cell receptor-, chemokine-, and Janus kinase-signal transducer and activator of transcription (Jak-STAT)-signaling pathways (Figure 2D; Huang et al., 2009). A selected set of NK cellassociated 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,

Immunity ETS1 Targets in NK Cells

C

0.10

85.1 9.0

NK1.1

102

Ets1+/+ Ets1-/-

101

NKP

D

Total cell number

10.3

CD27

ns

104

#

*

103 102

Ets1+/+ Ets1-/-

101 100

2.5

*** ***

1.25 1.00 0.75 0.50 0.25

K N m

K

0.00

H

CD122

Ets1+/+ Ets1-/-

1.75 1.50

iN

2.5

2.00

KP

10

rNKP

N

5

pre-NKP

LP

Flt3

88

Relative reconstitution

CD127 83

CLP

E

Flt3

2.8

mNK sNK

C

8.3

iNK

PP

CD244

Ets1-/-

*

103

105

Ets1+/+

13

ns

104

100 0.45

0.34

B

105

LM

0.75

**

**

106

PP

95.9

107

SC

DX5

Lin

Total cell number

CD122

0.91

Ets1-/-

M

Ets1+/+

A

Figure 1. ETS1 Functions at the Earliest Stages of NK Cell Development (A and B) 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
TCR-b
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. (C and D) 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 indicates average. Closed circles, Ets1+/+ mice; open circles, Ets1
/
mice. n = 4–7 for each genotype. *p < 0.05, **p < 0.01, ***p < 0.001 by a Student’s t test, #p < 0.05 by a paired t test but not significant with a standard t test. (E) Relative contribution of Ets1+/+ and Ets1
/
CD45.2+ cells in HSCs, MPPs, LMPPs, CLPs, NKP, iNK, and mNK from mixed BM chimeras with WT CD45.1+ 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.

Lair1, and Tbx21 mRNA in Lin
CD122+DX5
pro-NK 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. 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 Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc. 923

Immunity

A

mNK +/+ -/-

HSC MPP LMPP CLP B T G MP

ETS1 Targets in NK Cells

B

49 not annotated

167 human orthologs

61 no binding

106 bound

D KEGG Pathway Analysis of ETS1-dependent NK cell genes

2

p = 4.1e-125

b1 its

C

Category Natural killer cell mediated cytotoxicity T cell receptor signaling pathway Pathways in cancer Chemokine signaling pathway B cell receptor signaling pathway Jak-STAT signaling pathway

Count 28 24 46 30 15 22

Percent 1.9 1.7 3.2 2.1 1.0 1.5

p-value 5.6E-7 3.6E-5 9.2E-5 1.7E-4 3.5E-3 7.7E-3

0 1 2 3 4 5 6 7 8 9 10 1112 131415

Figure 2. 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 (left) and the number of genes that were bound by ETS1 (right). (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 with a 1.53 threshold of differential expression. See Table S1 for ETS1-dependent NK cell genes.

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). 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., 2007, 2009). 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. Although 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 924 Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc.

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 (Figures 5A and 5B). 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. 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 crosslinking to induce degranulation, as measured by surface CD107a. As expected, crosslinking of

Immunity

B 2.0

Tbx21

2.0

1.5

1.5

1.0

1.0

0.5

0.5

0.0

0.0

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Cd122

1.5

Ltb

Ncr1

Idb2

1.5

Lair1

1.0

0.5

0.5

1.25 1.00 0.75 0.50 0.25 0.00

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Ltb

1.00 0.75 0.50 0.25 0.00

NKP

3.0 2.5 2.0 1.5 1.0 0.5 0.0

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Relative mRNA expression

C

NKP

D Input (%)

Ets1* Idb2 Tbx21* Eomes Il2rβ Ltb* Jak1* Lck* Zap70* Itk* Slamf6* CD27* Hcst Ncr1 Klrk1 Klre1 Fcgr3* Klra16 Klra3 Klra17 Lair1* Crtam

Relative mRNA expression

Et

A

s1 +/ + Et s1 pro +/ B + Et m s1 N K -/m N K

ETS1 Targets in NK Cells

0.25 0.20 0.15 0.10 0.05 0.00 Tbx21 Cd122 SP5

Hbb

Ebf1

0.0

0.0

DX5-

DX5+

DX5-

DX5+

Figure 3. 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). Asterisk indicates that ETS1 was bound near this gene by ChIP-seq in CD4 T cells. (B) qPCR analysis of Tbx21, Ncr1, Cd122, Idb2, Ltb, and Lair1 mRNA in Ets1+/+ (black) and Ets1
/
(white) pro-NK cells (Lin
CD122+DX5
) and mNK cells. At least three independent experiments were performed. (C) qPCR analysis of Tbx21 and Ltb mRNA in NKPs. Error bars indicate SD 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.

NK1.1 or NKG2D resulted in increased CD107a compared to IgG on Ets1+/+ mNK cells (Figures 5D and 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 (Figures 5D and S3). Crosslinking 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 (Figures 5D and 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-g (IFN-g) production was not as severely affected, although crosslinking of NKG2D did not lead to a significant accumulation of IFN-g at this time point in Ets1+/+ or Ets1
/
mNK cells (Figures 5E and 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 GZMB (Granzyme B) and PRF (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 pro-NK cells (Figures 6A and 6C). Interestingly, Ikzf2 mRNA, which encodes HELIOS, whose increased expression contributes to hyperresponsiveness in Noe´ mice (NKp46-deficient) (Narni-Mancinelli et al., 2012), was increased in Ets1
/
mNK cells (Figures 6A and 6D). 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. 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 (Ly49E is normally not expressed on adult mNK cells) (Barao et al., 2011; Elpek et al., 2010; Fraser et al., 2002). We found an increased frequency of BM mNK cells expressing Ly49G2 and Ly49E but not Ly49A in Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc. 925

Immunity ETS1 Targets in NK Cells

B

A

C

D

Figure 4. Idb2 Is Regulated by ETS1 in NK Cells and Their Precursors

(A) Promoter-luciferase reporter assay via regions 50 of the Idb2 TSS, including the endogenous EBS site (GGA) or a mutated (m) EBS (GGT). One representative experiment from at least four is shown. (B) EMSA of proteins in PTL cell extracts binding to the Idb2 EBS in the presence (+) or absence (
) of cold competitor or a-ETS1, a-MEF1, or a-ELF1. (C) ChIP was performed on sorted splenic mNK cells via a-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 two to three replicate experiments is shown. See Figure S2 for Ets1 mRNA expression in NK cell lines and activated NK cells.

Ets1
/
as compared to WT mice (Figures 6F and 6G). 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 as shown by the fact that they were observed on Ets1
/
mNK cells in mixed BM chimeras (Figure S4). Taken together with the increased expression of Nfil3, Gzmb, and Prf1 mRNA and CD69, our findings indicate that Ets1
/
mNK cells resembled NK cells chronically stimulated by IL-15. Ets1–/– mNK Cells Are Hyperresponsive 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 GZMB and proliferation by means of BrdU incorporation. Within 24 hr, Ets1
/
mNK cells showed an increase in GZMB 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 (Figures 7C and 7D). In addition, whereas Ets1+/+ mNK cells showed little induction of GZMB or BrdU incorporation when cultured in 926 Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc.

1 ng/ml IL-15, Ets1
/
mNK cells showed a 4-fold higher response (Figures 7C and 7D). 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. 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-g 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

Immunity ETS1 Targets in NK Cells

44 6

95 47

B

50 21

Max (%)

98 72

56 30

Spleen

BM

98 99

Ly49H

Spleen

100 100

NK1.1

75

50

40

40

25

20

20

0

0

BM spleen

*

50

*

*

25

**

80

**

60

**

*

60

0

BM spleen

BM spleen

100 75 50 25 0

0

G

Ig

2D

.1

KG

N

2D

I A/

K1

N

PM

G

Ig

.1

K1

I A/

Max (%)

80

Ly49H

100

75

KG N

97 90

100

Ly49D

100

E

N

96.5

NKp46 ***

***

100

PM

87 86

D

CD107a+ (%)

Ly49D

NKp46

C

53 14

Positive (%)

BM

IFN-γ + (%)

A

NKG2D

Figure 5. 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
/
(gray line) mice. Shaded histogram shows isotype control. (B) Summary of the percent of mNK cells expressing NKp46, Ly49D, and Ly49H. Ets1+/+ (filled), Ets1
/
(open); horizontal bar indicates 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). (D and E) Summary of percent (D) CD107a+ or (E) IFN-g+ after a 5 hr stimulation with PMA plus ionomycin, a-NK1.1, a-NKG2D, or IgG. See Figure S2 for activating NKR expression in mixed BM chimeras and representative flow cytometery staining for CD107a and IFN-g.

probably other external stimuli, which may prevent expansion and activation in nonpathogenic conditions. In the absence of ETS1, mNK cells had hallmarks of chronic IL-15 stimulation and they had a heightened response to a suboptimal 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 although NKP cell 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 prepro-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-7Ra) is critical for identification of these cells and was reported to be an ETS1 target in peripheral CD8+ T cells (Grenningloh et al., 2011). Importantly, we did not find decreased CD127 on Ets1
/
CLPs via 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 Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc. 927

Immunity

F

D 6

10

E

3 16 36

2 1

+/+ -/CD11bBM

G

Spleen

SSC 44 77

Nfil3

Max (%)

7 6 5 4 3 2 1 0

41 55

7 38

spleen Ly49A

4

Ets1+/+ Ets1-/Relative mRNA expression

+/+ -/CD11b+

6

0

C

BM

8

2

11 71

4

0

Gzmb

45 77

Ikzf2

5

100

Positive (%)

Relative mRNA expression

B

Relative mRNA expression

Gzma Serpinb9b Gzmb* Klra5 Serpinb6a Klrg1 GzmK Klra20 Nfil3 Prf1* Ikzf2

25 42

Max (%)

Et

A

s1 Et +/+ s1 p Et +/+ roB s1 m -/- N m K N K

ETS1 Targets in NK Cells

8 26

Ly49A

80

40

Ly49G2 *

60 NS

Ly49E

Ly49G2

Ly49E *

NS

NS

**

20 0

BM spleen

BM spleen

BM spleen

CD69

+/+ -/DX5-

+/+ -/DX5+

Figure 6. 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. Asterisk indicates that ETS1 was bound near this gene in CD4+ T cells as determined by ChIP-seq. (B–D) qPCR analysis for (B) Gzmb, (C) Nfil3, and (D) Ikzf2 mRNA in Ets1+/+ (black) and Ets1
/
(white) mNK cells. Expression is plotted relative to transcripts from Hprt. Error bars represent standard error of triplicate measurements and one of at least two to three replicate experiments are shown. (E and F) 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
/
(gray). (G) Summary of the percent of mNK cells expressing Ly49A, Ly49G2, and Ly49E. Ets1+/+ (filled), Ets1
/
(open), horizontal bar indicates average. *p < 0.05, **p < 0.01, ***p < 0.001; n = 4; ns, p > 0.05. Cells in (E) were isolated from CD45.1 (WT):CD45.2 (WT or Ets1
/
) chimeric mice. See Figure S3 for Ly49A, Ly49G2, and Ly49E staining on mNK cells from mixed BM chimeras.

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 ETS1dependent NK cell genes were bound by ETS1 in CD4+ T cells, probably 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., 2007, 2009), 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., 2007, 2009). However, although 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 928 Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc.

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 probably 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, 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., 2007, 2009), posttranslational modification (Cowley and Graves, 2000; Lee et al., 2008), DNA methylation (Gaston and Fried, 1995; Yokomori et al., 1995), and cooperative interactions with

Immunity ETS1 Targets in NK Cells

100

Ets1+/+

Ets1-/-

(A) Percent of single Ets1+/+ (filled) and Ets1
/
(open) pro-NK 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 GZMB and BrdU incorporation in splenic mNK cells 24 hr after initiation of culture in varying concentrations of IL-15. The percent of Granzyme B+ BrdU+ cells is indicated. One of three experiments is shown. (D) Average ± SD of percent GZMB+ BrdU+ cells from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. The splenic mNK cells were isolated from BM chimeras.

75 50

NS

NS

25

FSC

0

DX51 ng/ml 0.216

C

Figure 7. Ets1–/– mNK Cells Are Hyperresponsive to IL-15

B

SSC

IL-2 responsive (%)

A

DX5+ 5 ng/ml 2.78

10 ng/ml 6.3

50 ng/ml 21

100 ng/ml 41.8

Granzyme B

Ets1+/+

2.6

5.84

16.2

48.3

Granzyme B+ BrdU+ (%)

BrdU D

60

Ets1+/+ Ets1-/-

50

***

*

40 30

**

20

**

10 0 1

10

et al., 2002). Moreover, in in vitro cytokine-dependent cultures, Ets1
/
NK cells cloned well with larger colony Ets1-/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 GZMB more rapidly than WT mNK cells at all concentrations of IL-15. The mechanism underlying the heightened activation in Ets1
/
NK cells probably involves the deregulation of multiple genes encoding signaling proteins and transcription factors (for example, Itk, Jak1, Lck, Ppp1r3b, Ptpn3, Egr3, 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 hyperresponsive 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 probably contributes to the hyperresponsive phenotype. However, in Ets1
/
mNK cells the compounded defects in activating receptor expression and degranulation probably 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 because Ets1
/
T cells fail to become activated after 50

100

IL-15 (ng/mL)

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 lineagespecific 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 upregulated by chronic cytokine stimulation (Barao et al., 2011; Elpek et al., 2010; Fraser

Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc. 929

Immunity ETS1 Targets in NK Cells

stimulation (Muthusamy et al., 1995). Importantly, although 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.

(ebio244F4). Propidium idodide was used to exclude dead cells. Cells were analyzed on a FACS Canto, LSRII, or Fortessa or sorted with a FACS ARIAII (Becton Dickenson). Lineage cocktail for HSC, MPP, LMPP, CLPs: B220, CD3ε, CD4, CD8, NK1.1, Ter119, CD11b, Ly-6G; for NK cells: CD19, CD3ε, CD4, CD8, Ter119; and for CLP, pre-NKP, rNKP populations: CD19, CD11b, CD3ε, Ly6d, and NK1.1.

EXPERIMENTAL PROCEDURES

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 hr. At t = 48 hr, NK cells were isolated by flow cytometry. Alternatively, 100 mg of poly(I:C) was injected intraperitoneally (i.p.) followed by isolation of NK cells at t = 24 hr. For a-NK1.1, a-NKG2D, or IgG stimulation of NK cells, mNK cells were cultured on antibodycoated plates in 1,000 IU/ml IL-2 plus Golgi Plug (Becton Dickenson) for 5 hr. Fluorochrome-labeled a-CD107a (1D4B) or isotype control antibodies (5 mg/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).

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 RNA was purified with 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 DCT 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 with SacI and XhoI and cloned into pGL3. PTL cells were transfected with DEAE-dextran with 8 mg of pGL3 constructs and 0.5 mg of pRL-CMV as an internal control (Kee and Murre, 1998). Lysates were prepared 48 hr after transfection and assayed with the Dual-Glo Luciferase kit (Promega). Electrophoretic Mobility Shift Analysis Nuclear extracts were prepared and EMSA performed as described (Kee and Murre, 1998). The Idb2 EBS sequence was 50 -GGTATTGGCTGCGAACGCG GAAGAACC-30 and the Idb2 EBS mutant sequence was 50 -GGTATTGGCT GCGAACGCGGTAGAACC-30 . 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 mM 2-mercaptoethanol, 100 units/ml penicillin, 100 mg/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 (1,000 IU/ml, NIH Reagents program), CD117 (1:1,000 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 H.-R. Rodewald by in vitro culture of fetal thymus-derived FcRgII or FcRgIII+ NK and T cell progenitors (Rodewald et al., 1992) and was adapted for growth in Opti-MEM. The KY1, KY2, and NKCRq cell lines were provided by W. Yokoyama and C. Roth (Caraux et al., 2006; Karlhofer et al., 1995). IL-15 responsiveness was determined by culturing 1,500–3,000 flow cytometry-sorted splenic mNK cells, isolated from chimeric mice, in multiple concentrations of recombinant mIL-15. At t = 24 hr, 1 mM BrdU was added for 45 min prior to intracellular staining for BZMB and BrdU. Flow Cytometry Cells were stained with fluorochrome- or biotin-labeled antibodies for 20 min 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: CD45.2 (104), CD45.1 (A20), CD19 (1D3), B220 (Ra3-6B2), CD3ε (145-2C11), CD4 (RM4-5), CD8a (Ly-2), TCR-b (H57-597), TCR-gd (UC7-13D5), CD11b (M1/ 70), Ter-119 (Ly-76), Gr-1 (RB6-8C5), IL-7Ra (A7R34), CD117 (2B8), Sca1 (E13-161.7), FLT3 (A2F10), CD122 (TM-b1), 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), IFN-g (XMG1.2), GZMB (NGZB or 16G6), and 2B4

930 Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc.

BM Chimeras Chimeric mice were established by retrorbital injection of 2.5 3 106 bone marrow cells from CD45.1 WT mice and 2.5 3 106 bone marrow cells from CD45.2 Ets1+/+ or CD45.2 Ets1
/
mice into 8-week-old lethally irradiated (1,000 rad) CD45.1 and CD45.2 or CD45.1 recipients. Recipients were maintained on Bactrim and analyzed 8 weeks posttransplantation. 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 were normalized with RMAexpress (http://rmaexpress. bmbolstad.com/) and analyzed by dChip (http://www.biostat.harvard.edu/ complab/dchip/). Probe set annotation was obtained from Affymetrix. 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 that the minimum motif length was set to 8 and maximum to 15. Only the motifs with the lowest E-value are reported. Chromatin Immunoprecipitation 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 mg 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 with PCR spin columns (QIAGEN) and amplified by qPCR with 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). ACCESSION NUMBERS The microarray data are available in the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/gds) under the accession number GSE37301. SUPPLEMENTAL INFORMATION Supplemental Information includes four figures and two tables and can be found with this article online at doi:10.1016/j.immuni.2012.04.006. ACKNOWLEDGMENTS We thank E. Svensson and K. Barton for providing Ets1
/
mice. We are grateful to V. Kumar for helpful discussions and comments on this manuscript. This

Immunity ETS1 Targets in NK Cells

work was supported by the National Institutes of Health (CA099978 to B.L.K., CA099978-S to 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). Received: October 26, 2011 Revised: March 2, 2012 Accepted: April 19, 2012 Published online: May 17, 2012 REFERENCES Aliahmad, P., de la Torre, B., and Kaye, J. (2010). Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages. Nat. Immunol. 11, 945–952. Bailey, T.L., Boden, M., Buske, F.A., Frith, M., Grant, C.E., Clementi, L., Ren, J., Li, W.W., and Noble, W.S. (2009). MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37 (Web Server issue), W202–W208. Barao, I., Alvarez, M., Ames, E., Orr, M.T., Stefanski, H.E., Blazar, B.R., Lanier, L.L., Anderson, S.K., Redelman, D., and Murphy, W.J. (2011). Mouse Ly49G2+ NK cells dominate early responses during both immune reconstitution and activation independently of MHC. Blood 117, 7032–7041. Barton, K., Muthusamy, N., Fischer, C., Ting, C.N., Walunas, T.L., Lanier, L.L., and Leiden, J.M. (1998). The Ets-1 transcription factor is required for the development of natural killer cells in mice. Immunity 9, 555–563. Boos, M.D., Yokota, Y., Eberl, G., and Kee, B.L. (2007). Mature natural killer cell and lymphoid tissue-inducing cell development requires Id2-mediated suppression of E protein activity. J. Exp. Med. 204, 1119–1130. Caraux, A., Lu, Q., Fernandez, N., Riou, S., Di Santo, J.P., Raulet, D.H., Lemke, G., and Roth, C. (2006). Natural killer cell differentiation driven by Tyro3 receptor tyrosine kinases. Nat. Immunol. 7, 747–754.

Fitzsimmons, D., Lukin, K., Lutz, R., Garvie, C.W., Wolberger, C., and Hagman, J. (2009). Highly cooperative recruitment of Ets-1 and release of autoinhibition by Pax5. J. Mol. Biol. 392, 452–464. Fraser, K.P., Gays, F., Robinson, J.H., van Beneden, K., Leclercq, G., Vance, R.E., Raulet, D.H., and Brooks, C.G. (2002). NK cells developing in vitro from fetal mouse progenitors express at least one member of the Ly49 family that is acquired in a time-dependent and stochastic manner independently of CD94 and NKG2. Eur. J. Immunol. 32, 868–878. Gascoyne, D.M., Long, E., Veiga-Fernandes, H., de Boer, J., Williams, O., Seddon, B., Coles, M., Kioussis, D., and Brady, H.J. (2009). The basic leucine zipper transcription factor E4BP4 is essential for natural killer cell development. Nat. Immunol. 10, 1118–1124. Gaston, K., and Fried, M. (1995). CpG methylation has differential effects on the binding of YY1 and ETS proteins to the bi-directional promoter of the Surf-1 and Surf-2 genes. Nucleic Acids Res. 23, 901–909. Gordon, S.M., Chaix, J., Rupp, L.J., Wu, J., Madera, S., Sun, J.C., Lindsten, T., and Reiner, S.L. (2012). The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation. Immunity 36, 55–67. Grenningloh, R., Kang, B.Y., and Ho, I.C. (2005). Ets-1, a functional cofactor of T-bet, is essential for Th1 inflammatory responses. J. Exp. Med. 201, 615–626. Grenningloh, R., Tai, T.S., Frahm, N., Hongo, T.C., Chicoine, A.T., Brander, C., Kaufmann, D.E., and Ho, I.C. (2011). Ets-1 maintains IL-7 receptor expression in peripheral T cells. J. Immunol. 186, 969–976. Held, W., Kunz, B., Lowin-Kropf, B., van de Wetering, M., and Clevers, H. (1999). Clonal acquisition of the Ly49A NK cell receptor is dependent on the trans-acting factor TCF-1. Immunity 11, 433–442. Higuchi, T., Bartel, F.O., Masuya, M., Deguchi, T., Henderson, K.W., Li, R., Muise-Helmericks, R.C., Kern, M.J., Watson, D.K., and Spyropoulos, D.D. (2007). Thymomegaly, microsplenia, and defective homeostatic proliferation of peripheral lymphocytes in p51-Ets1 isoform-specific null mice. Mol. Cell. Biol. 27, 3353–3366.

Carotta, S., Pang, S.H., Nutt, S.L., and Belz, G.T. (2011). Identification of the earliest NK-cell precursor in the mouse BM. Blood 117, 5449–5452.

Hollenhorst, P.C., Shah, A.A., Hopkins, C., and Graves, B.J. (2007). Genomewide analyses reveal properties of redundant and specific promoter occupancy within the ETS gene family. Genes Dev. 21, 1882–1894.

Chiossone, L., Chaix, J., Fuseri, N., Roth, C., Vivier, E., and Walzer, T. (2009). Maturation of mouse NK cells is a 4-stage developmental program. Blood 113, 5488–5496.

Hollenhorst, P.C., Chandler, K.J., Poulsen, R.L., Johnson, W.E., Speck, N.A., and Graves, B.J. (2009). DNA specificity determinants associate with distinct transcription factor functions. PLoS Genet. 5, e1000778.

Clements, J.L., John, S.A., and Garrett-Sinha, L.A. (2006). Impaired generation of CD8+ thymocytes in Ets-1-deficient mice. J. Immunol. 177, 905–912.

Huang, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57.

Cowley, D.O., and Graves, B.J. (2000). Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition. Genes Dev. 14, 366–376. Dias, S., Ma˚nsson, R., Gurbuxani, S., Sigvardsson, M., and Kee, B.L. (2008). E2A proteins promote development of lymphoid-primed multipotent progenitors. Immunity 29, 217–227. Elpek, K.G., Rubinstein, M.P., Bellemare-Pelletier, A., Goldrath, A.W., and Turley, S.J. (2010). Mature natural killer cells with phenotypic and functional alterations accumulate upon sustained stimulation with IL-15/IL-15Ralpha complexes. Proc. Natl. Acad. Sci. USA 107, 21647–21652. Eyquem, S., Chemin, K., Fasseu, M., and Bories, J.C. (2004a). The Ets-1 transcription factor is required for complete pre-T cell receptor function and allelic exclusion at the T cell receptor beta locus. Proc. Natl. Acad. Sci. USA 101, 15712–15717. Eyquem, S., Chemin, K., Fasseu, M., Chopin, M., Sigaux, F., Cumano, A., and Bories, J.C. (2004b). The development of early and mature B cells is impaired in mice deficient for the Ets-1 transcription factor. Eur. J. Immunol. 34, 3187–3196. Fathman, J.W., Bhattacharya, D., Inlay, M.A., Seita, J., Karsunky, H., and Weissman, I.L. (2011). Identification of the earliest natural killer cell-committed progenitor in murine bone marrow. Blood 118, 5439–5447. Fehniger, T.A., Suzuki, K., Ponnappan, A., VanDeusen, J.B., Cooper, M.A., Florea, S.M., Freud, A.G., Robinson, M.L., Durbin, J., and Caligiuri, M.A. (2001). Fatal leukemia in interleukin 15 transgenic mice follows early expansions in natural killer and memory phenotype CD8+ T cells. J. Exp. Med. 193, 219–231.

Intlekofer, A.M., Takemoto, N., Wherry, E.J., Longworth, S.A., Northrup, J.T., Palanivel, V.R., Mullen, A.C., Gasink, C.R., Kaech, S.M., Miller, J.D., et al. (2005). Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat. Immunol. 6, 1236–1244. John, S.A., Clements, J.L., Russell, L.M., and Garrett-Sinha, L.A. (2008). Ets-1 regulates plasma cell differentiation by interfering with the activity of the transcription factor Blimp-1. J. Biol. Chem. 283, 951–962. Joncker, N.T., and Raulet, D.H. (2008). Regulation of NK cell responsiveness to achieve self-tolerance and maximal responses to diseased target cells. Immunol. Rev. 224, 85–97. Joncker, N.T., Fernandez, N.C., Treiner, E., Vivier, E., and Raulet, D.H. (2009). NK cell responsiveness is tuned commensurate with the number of inhibitory receptors for self-MHC class I: the rheostat model. J. Immunol. 182, 4572–4580. Kallies, A., Carotta, S., Huntington, N.D., Bernard, N.J., Tarlinton, D.M., Smyth, M.J., and Nutt, S.L. (2011). A role for Blimp1 in the transcriptional network controlling natural killer cell maturation. Blood 117, 1869–1879. Kamizono, S., Duncan, G.S., Seidel, M.G., Morimoto, A., Hamada, K., Grosveld, G., Akashi, K., Lind, E.F., Haight, J.P., Ohashi, P.S., et al. (2009). Nfil3/E4bp4 is required for the development and maturation of NK cells in vivo. J. Exp. Med. 206, 2977–2986. Karlhofer, F.M., Orihuela, M.M., and Yokoyama, W.M. (1995). Ly-49-independent natural killer (NK) cell specificity revealed by NK cell clones derived from p53-deficient mice. J. Exp. Med. 181, 1785–1795.

Immunity 36, 921–932, June 29, 2012 ª2012 Elsevier Inc. 931

Immunity ETS1 Targets in NK Cells

Kee, B.L., and Murre, C. (1998). Induction of early B cell factor (EBF) and multiple B lineage genes by the basic helix-loop-helix transcription factor E12. J. Exp. Med. 188, 699–713. Kim, S., Iizuka, K., Kang, H.S., Dokun, A., French, A.R., Greco, S., and Yokoyama, W.M. (2002). In vivo developmental stages in murine natural killer cell maturation. Nat. Immunol. 3, 523–528. Lacorazza, H.D., Miyazaki, Y., Di Cristofano, A., Deblasio, A., Hedvat, C., Zhang, J., Cordon-Cardo, C., Mao, S., Pandolfi, P.P., and Nimer, S.D. (2002). The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells. Immunity 17, 437–449. Lanier, L.L. (2005). NK cell recognition. Annu. Rev. Immunol. 23, 225–274. Lee, G.M., Pufall, M.A., Meeker, C.A., Kang, H.S., Graves, B.J., and McIntosh, L.P. (2008). The affinity of Ets-1 for DNA is modulated by phosphorylation through transient interactions of an unstructured region. J. Mol. Biol. 382, 1014–1030. Moisan, J., Grenningloh, R., Bettelli, E., Oukka, M., and Ho, I.C. (2007). Ets-1 is a negative regulator of Th17 differentiation. J. Exp. Med. 204, 2825–2835. Muthusamy, N., Barton, K., and Leiden, J.M. (1995). Defective activation and survival of T cells lacking the Ets-1 transcription factor. Nature 377, 639–642. Narni-Mancinelli, E., Jaeger, B.N., Bernat, C., Fenis, A., Kung, S., De Gassart, A., Mahmood, S., Gut, M., Heath, S.C., Estelle´, J., et al. (2012). Tuning of natural killer cell reactivity by NKp46 and Helios calibrates T cell responses. Science 335, 344–348. Nishimori, H., Sasaki, Y., Yoshida, K., Irifune, H., Zembutsu, H., Tanaka, T., Aoyama, T., Hosaka, T., Kawaguchi, S., Wada, T., et al. (2002). The Id2 gene is a novel target of transcriptional activation by EWS-ETS fusion proteins in Ewing family tumors. Oncogene 21, 8302–8309.

Fc gamma RII/III contains precursors of T lymphocytes and natural killer cells. Cell 69, 139–150. Rosmaraki, E.E., Douagi, I., Roth, C., Colucci, F., Cumano, A., and Di Santo, J.P. (2001). Identification of committed NK cell progenitors in adult murine bone marrow. Eur. J. Immunol. 31, 1900–1909. Russell, L., and Garrett-Sinha, L.A. (2010). Transcription factor Ets-1 in cytokine and chemokine gene regulation. Cytokine 51, 217–226. Seth, A., and Papas, T.S. (1990). The c-ets-1 proto-oncogene has oncogenic activity and is positively autoregulated. Oncogene 5, 1761–1767. Stewart, C.A., Walzer, T., Robbins, S.H., Malissen, B., Vivier, E., and Prinz, I. (2007). Germ-line and rearranged Tcrd transcription distinguish bona fide NK cells and NK-like gammadelta T cells. Eur. J. Immunol. 37, 1442–1452. Vivier, E., Raulet, D.H., Moretta, A., Caligiuri, M.A., Zitvogel, L., Lanier, L.L., Yokoyama, W.M., and Ugolini, S. (2011). Innate or adaptive immunity? The example of natural killer cells. Science 331, 44–49. Vosshenrich, C.A., Ranson, T., Samson, S.I., Corcuff, E., Colucci, F., Rosmaraki, E.E., and Di Santo, J.P. (2005). Roles for common cytokine receptor gamma-chain-dependent cytokines in the generation, differentiation, and maturation of NK cell precursors and peripheral NK cells in vivo. J. Immunol. 174, 1213–1221. Wang, D., John, S.A., Clements, J.L., Percy, D.H., Barton, K.P., and GarrettSinha, L.A. (2005). Ets-1 deficiency leads to altered B cell differentiation, hyperresponsiveness to TLR9 and autoimmune disease. Int. Immunol. 17, 1179–1191. Wei, G.H., Badis, G., Berger, M.F., Kivioja, T., Palin, K., Enge, M., Bonke, M., Jolma, A., Varjosalo, M., Gehrke, A.R., et al. (2010). Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo. EMBO J. 29, 2147–2160.

Nutt, S.L., and Kee, B.L. (2007). The transcriptional regulation of B cell lineage commitment. Immunity 26, 715–725.

Yokomori, N., Kobayashi, R., Moore, R., Sueyoshi, T., and Negishi, M. (1995). A DNA methylation site in the male-specific P450 (Cyp 2d-9) promoter and binding of the heteromeric transcription factor GABP. Mol. Cell. Biol. 15, 5355–5362.

Pufall, M.A., Lee, G.M., Nelson, M.L., Kang, H.S., Velyvis, A., Kay, L.E., McIntosh, L.P., and Graves, B.J. (2005). Variable control of Ets-1 DNA binding by multiple phosphates in an unstructured region. Science 309, 142–145.

Yokota, Y., Mansouri, A., Mori, S., Sugawara, S., Adachi, S., Nishikawa, S., and Gruss, P. (1999). Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 397, 702–706.

Rodewald, H.R., Moingeon, P., Lucich, J.L., Dosiou, C., Lopez, P., and Reinherz, E.L. (1992). A population of early fetal thymocytes expressing

Yokoyama, W.M., and Kim, S. (2006). Licensing of natural killer cells by selfmajor histocompatibility complex class I. Immunol. Rev. 214, 143–154.

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