The Ets-1 Transcription Factor Is Required for the Development of Natural Killer Cells in Mice

Immunity, Vol. 9, 555–563, October, 1998, Copyright 1998 by Cell Press

The Ets-1 Transcription Factor Is Required for the Development of Natural Killer Cells in Mice Kevin Barton,*§ Natarajan Muthusamy,* Christopher Fischer,* Chao-Nan Ting,* Theresa L. Walunas,* Lewis L. Lanier,† and Jeffrey M. Leiden*‡ * Department of Medicine The University of Chicago Chicago, Illinois 60637 † Department of Immunobiology DNAX Research Institute of Molecular and Cellular Biology Palo Alto, California 94304

Summary In this report we have investigated the role of the Ets-1 transcription factor in the differentiation of the NK cell lineage in mice. Splenic NK cells express high levels of Ets-1. Ets-1-deficient mice produced by gene targeting developed mature erythrocytes, monocytes, neutrophils, and T and B lymphocytes. However, spleens from the Ets-1-deficient mice contained significantly reduced numbers of natural killer (NK) cells, and splenocytes from these mice lacked detectable cytolytic activity against NK cell targets in vitro. Moreover, unlike wild-type animals, Ets-1-deficient mice developed tumors following subcutaneous injection of NK-susceptible RMA-S cells. These NK cell defects could not be correlated with defects in the expression of IL-12, IL-15, and IL-18 or the IL-2 or IL-15 receptors. Thus, Ets-1 defines a novel transcriptional pathway that is required for the development of the NK cell lineage in mice. Introduction Natural killer (NK) cells are cytotoxic lymphocytes that play important roles in mammalian immune responses to tumor cells and cells infected with viruses, intracellular bacteria, and parasites (Trinchieri, 1989; Bancroft, 1993; Moore et al., 1996; Biron, 1997). Although NK cells and T cells appear to differentiate from a common T/NK progenitor (Rodewald et al., 1992; Sanchez et al., 1994; Carlyle et al., 1997), NK cell cytolytic activity, unlike cytotoxic T cell (CTL)-mediated lysis, does not require presensitization, is not MHC restricted, and is not dependent on rearranged antigen-specific receptor molecules such as the TCR (Gumperz and Parham, 1995). Instead, most NK cells appear to recognize class I MHCdeficient target cells via polymorphic but nonrearranging inhibitory receptors termed killer inhibitory receptors (KIR) and CD94/NKG2 in humans and Ly-49 in the mouse (Lanier, 1997, 1998). Thus, NK cells appear to function ‡ To whom correspendence should be addressed (e-mail: jleiden@

medicine.bsd.uchicago.edu). § Present address: Department of Medicine, Cardinal Bernadin Cancer Center, Loyola University Medical Center, Maywood, Illinois 60153.

as an important immunologic defense against tumor cells and pathogen-infected cells that have down-regulated endogenous class I MHC expression and are thereby invisible to class I MHC-restricted CTL (Karre et al., 1986; Liao et al., 1991). In addition to serving as mediators of innate immunity against malignant and virus-infected cells, NK cells have been implicated in bone marrow rejection (Ohlen et al., 1989; Yu et al., 1992; Murphy et al., 1993). During the last 5 years, a great deal has been learned about the signaling pathways and transcription factors that regulate T cell ontogeny (Zuniga-Pflucker and Lenardo, 1996). In contrast, relatively little is known about the molecular pathways that regulate NK cell development (Moore et al., 1996). The majority of NK cells develop in the bone marrow and NK cell differentiation has been shown to be dependent on the bone marrow microenvironment (Moore et al., 1996). Recent studies have demonstrated that IL-15 produced by radio-resistant cells in the bone marrow is required for NK cell development in vivo (Ogasawara et al., 1998). This bone marrow–derived IL-15 binds to the IL-15 receptor on NK cells, which is composed of a unique IL-15Ra chain and common IL-2Rb and -g chains. Several other cytokines including IL-2, IL-12, and IL-18 have also been implicated in the development of NK activity (Kobayashi et al., 1989; Chan et al., 1992; Magram et al., 1996; Puzanov et al., 1996; Carson et al., 1997; Suzuki et al., 1997; Takeda et al., 1998). Ets-1 is a member of the Ets family of winged helixturn-helix transcription factors. Ets proteins bind to purine-rich DNA motifs containing a conserved GGA core sequence and are important regulators of development in flies, worms, and mammals (Crepieux et al., 1994; Donaldson et al., 1996; Bassuk and Leiden, 1997). Multiple Ets family members are expressed in lymphoid cells and many lymphoid-specific genes contain functionally important Ets-binding sites (Bassuk and Leiden, 1997). However, until recently the function of individual Ets proteins in regulating lymphocyte development remained unknown. In previous studies, we and others used murine embryonic stem (ES) cells containing a targeted mutation of the Ets-1 gene in conjunction with the RAG2 complementation system to examine the role of Ets-1 in T and B cell development (Bories et al., 1995; Muthusamy et al., 1995). These studies demonstrated that Ets-1 is not required for the development of mature single-positive (CD41 or CD81) T cells or IgM1B2201 B cells. However Ets-1-deficient T cells displayed a severe defect in T cell antigen receptor (TCR)-mediated activation. In addition, the Ets-12/2RAG22/2 animals developed increased numbers of splenic and lymph node plasma cells and displayed 5- to 10-fold elevated serum IgM levels. Although useful for studying the role of Ets-1 in T and B cell development, the RAG2 complementation system did not allow us to assess the role of Ets-1 in the development and function of other hematopoietic cell lineages in vivo. Accordingly, in the studies described in this report, we have used gene targeting to produce

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Figure 1. Ets-1 Expression in Splenic NK Cells and Generation of Ets-12/2 Mice (A) Immunoblot analysis of Ets-1 expression in wild-type thymocytes and purified DX51CD32 splenic NK cells. Murine Ets-1 migrates as a doublet in SDS-PAGE analysis (arrows). (B) Generation of Ets-1-deficient mice. Top panels, schematic representation of the wild-type Ets-1 locus (top), the Ets-1 targeting vector (middle), and the targeted Ets-1 allele (bottom). The targeting vector contains the neomycin resistance (neo) and the herpes simplex virus thymidine kinase (HSV-tk) genes, both under the control of the PGK promoter. The 6.4 and 2.7 kb wild-type and 4.5 and 5.9 kb targeted alleles are shown by arrows. Bottom left, Southern analysis of wild-type (1/1) and Ets-11/2 (1/2) ES cells. Bottom right, Southern analysis of offspring from an Ets-11/2Ets-11/2 mating. The positions of the wild-type (wt) and targeted (t) alleles are shown to the left of each blot. (C) Immunoblot analysis of Ets-1 expression in splenocytes from wild-type (1/1), heterozygous (1/2), and homozygous (2/2) Ets-1-deficient animals.

mice containing a null mutation of the Ets-1 gene (Ets-12/2 mice). Consistent with our previous results in the Ets-12/2RAG22/2 chimeras, we find that Ets-1 is not required for the development of mature T and B cells. Moreover, we show that Ets-1 is not required for the development of the erythroid, myelomonocytic, or megakaryocytic lineages. In contrast, Ets-1 appears to be essential for the development of mature NK cells in mice. Ets-1-deficient mice contained markedly decreased numbers of DX51CD32 splenic NK cells and displayed severely reduced or absent NK cell function both in vitro and in vivo. These defects in NK cell function could not be rescued by culturing Ets-12/2 bone marrow with exogenous IL-2, IL-12, or IL-15 and did not appear to result from the lack of expression of either IL-18 or the IL-2 or IL-15 receptors. Taken together, these results identify Ets-1 as an essential regulator of NK cell development and define a novel Ets-1-dependent transcriptional pathway that is required for NK cell differentiation and/or survival.

Results Expression of Ets-1 in Wild-Type NK Cells Previous studies have demonstrated that Ets-1 is expressed at high levels in mature B and T lymphocytes. To determine if Ets-1 is also expressed in the closely related NK cell lineage, we performed Western blot analyses on protein extracts derived from freshly isolated purified DX51CD32 splenic NK cells. As shown in Figure 1A, Ets-1 was expressed in NK cells at levels equivalent to those observed in freshly isolated thymocytes. Murine Ets-1 typically migrates as two bands in immunoblots. These bands may represent alternatively spliced and/ or differentially phosphorylated forms of the protein. In the experiment shown in Figure 1A, NK cells appear to contain relatively more slowly migrating Ets-1 than thymocytes. However, this was not a consistent finding in subsequent experiments (see, for example, Figure 1C; data not shown) and its significance therefore remains unclear.

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Figure 2. Lymphocyte Subsets in the Ets-1Deficient Animals (A) B and T lymphocyte subsets in spleens and thymi of wild-type (1/1) and Ets-1-deficient (2/2) mice. Lymphocyte subsets were quantitated by flow cytometry with antibodies specific for CD4, CD8, IgM, and B220. The data represents lymphocyte numbers from four to seven animals in each group. (B) Flow cytometric analysis of splenic T cells (top), B cells (middle), and NK cells (bottom). Note the significant reduction in DX51CD32 NK cells in the Ets-12/2 spleens. The percentage of cells in each quadrent is shown in each panel. (C) Numbers of DX51CD32 splenocytes in wild-type (1/1) and Ets-1-deficient (2/2) spleens. Data is shown as mean 6 SEM for at least seven animals in each group. (D) Serum IgM levels in age-matched wildtype (1/1) and Ets-1-deficient (2/2) mice.

Production of Ets-1-Deficient Mice To better define the role of Ets-1 in the development of non-B and -T cell hematopoietic lineages, we used homologous recombination in murine ES cells to produce a targeted deletion of exons 3 and 4 of the Ets-1 gene (Figure 1B). ES cells containing the Ets-1 mutation were injected into wild-type C57BL/6 mouse blastocysts to produce chimeric mice, two of which transmitted the targeted Ets-1 allele through the germ line. Mice heterozygous for the targeted allele (Ets1/2) were identified by Southern blot analysis (Figure 1B; data not shown). These mice were interbred to produce Ets-1-deficient animals (Ets-12/2) (Figure 1B). Immunoblot analyses demonstrated the lack of detectable full-length or truncated Ets-1 protein in splenocytes from the Ets-12/2 mice (Figure 1C), suggesting that this targeting construct had produced a null allele in these mice. The Ets-12/2 mice were viable and fertile. However, they displayed an increased perinatal mortality. Approximately 50% of the Ets-12/2 pups survived until weaning at 4 weeks of age. The cause of this premature mortality is unclear and remains under investigation. Adult Ets-12/2 mice contained normal numbers of peripheral blood erythrocytes, monocytes, granulocytes, and platelets (data not shown).

Analyses of B and T cell development in these mice demonstrated the phenotypic characteristics observed previously in the Ets-12/2RAG22/2 chimeras (Bories et al., 1995; Muthusamy et al., 1995). Thymocyte numbers in 4- to 8-week-old Ets-12/2 mice were reduced by approximately 65% as compared to those of wild-type littermates (Figure 2A). However, the relative proportions of double-negative (DN) (CD42CD82), double-positive (DP) (CD41CD81), and single-positive (SP) (CD41 and CD81) thymocytes were not significantly different in the wild-type and Ets-1-deficient animals (Figure 2A; data not shown). Unlike wild-type CD81 thymocytes, many of the Ets-12/2 CD81 thymocytes also expressed low levels of CD4. The significance of this finding remains unclear. Numbers of Ets-12/2 SP splenic T cells were slightly reduced and this reduction was more pronounced in the CD81 than in the CD41 population (Figure 2A). In addition, Ets-12/2 splenic T cells displayed a marked defect in activation following cross-linking of the T cell antigen receptor (data not shown). Numbers of IgM1B2201 splenic B cells were normal in the Ets-12/2 mice (Figure 2A). However, these animals had increased numbers of splenic and lymph node plasma cells and serum IgM levels were elevated by approximately 5-fold (Figure 2D). Serum levels of the other Ig isotypes were

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Figure 3. NK Cytolytic Activity of Ets-1-Deficient Splenocytes In vitro cytotoxicity against YAC-1 (A) and b2m2/2 lymphoid blasts (B) using unfractionated poly (I:C)-stimulated splenocytes. Cytotoxicity assays were performed at effector to target cell ratios (E:T) of 6.25:1 to 100:1. Data are presented as mean 6 SEM for cells from three different animals in each group.

normal (data not shown). Thus, Ets-1 does not appear to be required for the development of mature erythrocytes, neutrophils, monocytes, platelets, or B and T cells. However, in the absence of Ets-1 there is a modest reduction in thymocyte numbers, mature T cells cannot be activated normally through the TCR, and there appears to be increased differentiation of B cells into IgM-secreting plasma cells. Ets-1 Is Required for NK Cell Development To assess directly the role of Ets-1 in NK cell development, freshly isolated splenocytes from wild-type or Ets-12/2 mice were analyzed by two-color flow cytometry using antibodies against CD3 and DX5. The majority of mature NK cells in the spleen and bone marrow are contained within the DX51CD32 population (DiSanto et al., 1995). Although the epitope recognized by the DX5 MAb remains unknown, it was used in these studies because it detects most or all NK cells in both C57BL/6 and 129 mice (the Ets-12/2 mice are in a mixed C57BL/ 6-129 background). As shown in Figures 2B and 2C, the numbers of splenic DX51CD32 NK cells were markedly reduced in the Ets-12/2 animals as compared to their wild-type littermates (p # 0.00001). Similar reductions in NK cell numbers were observed when splenocytes were stained with antibodies against NK1.1 and CD3, demonstrating that the defect in NK cell numbers did not simply reflect a lack of expression of the DX5 epitope on the Ets-1-deficient splenocytes (data not shown). However, the NK1.1 analysis was complicated by the fact that only some of the C57BL/6-129 mixed background mice inherited the NK1.1 allele, which is expressed on C57BL/6 but not on 129 NK cells. Finally,

the lymph nodes and bone marrow of the Ets-12/2 mice also did not contain detectable DX51CD32 cells (data not shown). Ets-1-Deficient Splenocytes Display Reduced NK Cell Activity In Vitro The severe reduction in the numbers of splenic NK cells in the Ets-12/2 mice suggested that these mice might display defects in NK cell activity. Indeed, unlike wildtype splenocytes, Ets-12/2 splenocytes were unable to lyse NK-susceptible YAC-1 tumor cells in vitro (Figure 3A). Consistent with previous reports, conconavalin A–stimulated lymphoid blasts from b2-microglobulin-deficient (b2m2/2) mice, which lack cell-surface expression of class I MHC molecules, were lysed efficiently by wildtype NK cells (Figure 3B) (Liao et al., 1991). In contrast, Ets-12/2 splenocytes also failed to lyse these b2m2/2 lymphoid blasts in vitro (Figure 3B). The lack of NKmediated target cell lysis by unfractionated Ets-12/2 splenocytes might have reflected the absence of functional splenic NK cells or, alternatively, a reduction in the numbers of NK cells in the spleens of these animals. To distinguish these alternatives, we repeated the in vitro NK cell cytolytic assays using purified DX51CD32 splenocytes from wild-type and Ets-1-deficient mice. DX51CD32 splenocytes from the Ets-1-deficient animals were purified at least 100-fold by flow cytometry (data not shown). However, the cytolytic activity of these purified cells against YAC-1 and RMA-S NK target cells increased only minimally (Figures 4A and 4B). This result suggested either that the residual DX51CD32 Ets-1-deficient splenocytes were not bona fide NK cells (and instead reflected low-level background staining with the Figure 4. NK Cytolytic Activity of Purified DX51CD32 Ets-1-Deficient NK Cells In vitro cytotoxicity against YAC-1 (A) and RMA-S (B) targets of purified poly (I:C)-stimulated DX51CD32 splenic NK cells. Cytotoxicity assays were performed at effector to target cell ratios (E:T) of 0.15:1 to 2.5:1. Data in (A) is presented as mean 6 SEM for cells from three different animals in each group. Data in (B) represents values from a single animal in each group.

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Figure 5. IFNg Production by Ets-1-Deficient Splenocytes Wild-type (1/1) or Ets-1-deficient (2/2) splenocytes were stimulated in vitro with poly (I:C) for 18 hr, and IFNg was measured in culture supernatants by ELISA. Data is presented as mean 6 SEM for cells from three different animals in each group.

DX5 antibody) or that these cells were not functional for NK-mediated lysis. NK cells are an important early source of IFNg following pathogen invasion (Biron, 1997). In keeping with the finding of markedly reduced numbers of splenic NK cells in the Ets-1-deficient mice, Ets-12/2 splenocytes stimulated with poly (I:C) in vitro produced significantly reduced amounts of IFNg as compared to wild-type control cells (Figure 5). Thus, two important in vitro NK cell functions, cytolysis of NK cell targets and IFNg production, were defective in the Ets-1-deficient animals. Ets-1-Deficient Mice Display Defective NK Cell Activity In Vivo The rejection of many class I MHC-deficient tumors is dependent on intact NK cell function in vivo. One example of such an NK cell–dependent class I MHC-deficient tumor is RMA-S (van den Broek et al., 1995). To determine if the defects in in vitro NK cell function observed in the Ets-12/2 mice were reflected in abnormal NK cell function in vivo, wild-type and Ets-12/2 mice were injected subcutaneously with 105 RMA-S cells and followed for 40 days for tumor progression (Figure 6). None of the wild-type mice developed detectable tumors during the 40 day time course of the experiment. In contrast, 6 of 7 Ets-12/2 animals developed subcutaneous tumors $ 1 cm between 17 and 26 days after injection (Figure 6). Thus, Ets-1 appears to be necessary for the development of normal NK activity as assayed both in vitro and in vivo. Cytokines and Cytokine Receptors and the NK Cell Defect of Ets-1-Deficient Mice A number of cytokines have been implicated in the development and function of NK cells. These include IL-2, IL-12, IL-15, and IL-18. Accordingly, we studied the expression of these cytokines and their receptors in the Ets-12/2 animals. Mice with a targeted mutation of the b subunit of the IL-2 receptor (IL-2Rb) display a reduction in NK cell numbers and deficient NK cell–mediated cytolytic activity (Suzuki et al., 1997). However, expression of both the IL-2Rb and common g chains were normal in Ets-12/2 splenocytes as assessed by RNase protection assays (Figure 7A). Consistent with these

Figure 6. In Vivo NK Cell Function in Ets-1-Deficient Mice (A) Photographs of representative wild-type (1/1) and Ets-1-deficient (2/2) mice following subcutaneous injection with 105 RMA-S tumor cells. Note the tumor in the right hindlimb of the Ets-1-deficient mouse, which is not seen in the wild-type mouse injected with the same cells. (B) Incidence of tumors following injection of wild-type (n 5 9) and Ets-1-deficient (n 5 7) mice with 105 RMA-S cells.

findings, flow cytometric analyses of splenocytes from the Ets-1-deficient mice demonstrated high level expression of the IL-2Rb chain on CD81 T cells (Figure 7B). Activated T cells from the Ets-1-deficient mice also demonstrated normal expression of IL-2Ra and -g chains by flow cytometry (data not shown). Furthermore, addition of exogenous IL-2 did not rescue the cytolytic activity of Ets-12/2 bone marrow against YAC-1 targets (Figure 7C). IL-15 is produced by many cells including bone marrow stromal cells and activated monocytes and is thought to be important for NK cell development (Puzanov et al., 1996; Carson et al., 1997). The IL-15 receptor (IL-15R) is composed of a unique IL-15a chain in combination with the IL-2Rb chain and the common g chain. As described above, expression of the IL-2Rb and -g chains were not significantly reduced in Ets-1-deficient splenocytes, nor was expression of the IL-15Ra chain (Figure 7A). Moreover, addition of exogenous IL-15 did not rescue the cytolytic activity of Ets-12/2 bone marrow cells against YAC-1 targets (Figure 7C). Similarly, addition of both IL-2 and IL-15 failed to restore the cytolytic activity of Ets-12/2 bone marrow cells against YAC-1 targets (Figure 7C). IL-12 enhances NK cytolytic activity, induces secretion of IFNg, and is mitogenic for T and NK cells (Kobayashi et al., 1989; Chan et al., 1992). IL-12 expression, as measured by RNase protection assay, was moderately

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Figure 7. Cytokines and Cytokine Receptors and the NK Defect of Ets-1-Deficient Mice (A) Expression of cytokine and cytokine receptor mRNAs in Ets-1 deficient poly (I:C)-stimulated splenocytes as determined by ribonuclease protection assay. Values were normalized first to an internal GAPDH control in each sample and then to wild-type mRNA levels as described in Experimental Procedures. (B) Flow cytometric analysis of IL-2Rb expression on CD81 splenocytes from wild-type (1/1) and Ets-1-deficient (2/2) mice. (C) Effect of exogenous cytokines on the in vitro cytotoxicity of wild-type (1/1) or Ets-1-deficient (2/2) bone marrow. All assays utilized YAC-1 targets. Effector to target (E:T) ratios are shown. Data from two animals of each genotype are shown. (D) Effect of exogenous IL-12 on the in vitro cytotoxicity of wild-type (1/1) and Ets-1-deficient (2/2) splenocytes. Data from one animal of each genotype is shown.

reduced in poly (I:C)-stimulated Ets-12/2 splenocytes (Figure 7A). However, this reduction in IL-12 production appears unlikely to account for the NK cell abnormalities seen in the Ets-12/2 mice for two reasons. First, unlike the Ets-1-deficient mice, IL-12 (p40)-deficient mice display only a modest reduction in NK cytolytic activity (Magram et al., 1996). Moreover, addition of exogenous IL-12 did not restore the cytolytic activity of Ets-1-deficient splenocytes against YAC-1 targets (Figure 7D). Like IL-12, IL-18, which is secreted from activated macrophages, induces secretion of IFNg and enhances NK cell activity. Recently, IL-18-deficient mice have been reported to display moderately reduced NK cell activity in vitro (Takeda et al., 1998). However, it appears unlikely that deficiencies in the expression of IL-18 or IL-18R were responsible for the NK cell phenotype seen in the Ets-12/2 animals. Unlike the Ets-1-deficient mice, which displayed markedly reduced numbers of splenic NK cells, the IL-182/2 mice contained normal numbers of NK cells in the spleen (Takeda et al., 1998). Second, as shown in Figure 7A, expression of IL-18 by Ets-1deficient splenocytes was normal. Discussion In the studies described in this report, we have demonstrated that the Ets-1 transcription factor is not required

for the development of mature erythrocytes, neutrophils, monocytes, megakaryocytes, or B and T lymphocytes, but it is necessary for the development and/or survival of the NK cell lineage in mice. Ets-1-deficient mice contained significantly reduced splenic NK cells and displayed markedly reduced NK cell cytolytic activity against both tumor cell and class I MHC-deficient targets in vitro and in vivo. These defects in NK cell function did not appear to reflect defective expression of either IL-18 or the IL-2 or IL-15 receptors and could not be rescued by culturing Ets-12/2 bone marrow with exogenous cytokines IL-2, IL-12, or IL-15. Thus, Ets-1 appears to define a novel transcriptional pathway that distinguishes the development of the NK cell lineage from that of closely related B and T lymphocytes. This pathway appears to be different from those regulated by transcription factors such as Ikaros, which is required for the development of all (B, T, and NK) lymphoid lineages (Georgopoulos et al., 1994). The mechanism by which Ets-1 deficiency causes defects in NK cell activity remains unclear. Our finding of markedly reduced NK cells in the spleens of the Ets1-deficient mice strongly suggests that the functional defect in NK activity seen in these animals reflects a defect in the production and/or survival of mature NK cells. However, the lack of lineage-specific markers that allow the identification of specific populations of NK cell

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precursors makes it difficult or impossible to precisely determine the stage at which NK cell development is defective in the Ets-1-deficient mice. It appears unlikely that the observed NK cell defect is a result of the abnormalities in T cell activation or B cell development seen in the Ets-12/2 mice, because normal NK cells develop in the absence of T and B cells in both SCID and RAG2deficient animals (Trinchieri, 1989; Moore et al., 1996). Because Ets-1 is expressed at high levels in mature NK cells, the simplest model to explain our findings is that Ets-1 regulates the expression of NK cell genes that are required for the normal development or survival of the lineage; i.e., the defect seen in the Ets-12/2 mice is intrinsic to the NK cells or their precursors. However, we cannot at present rule out the possibility that Ets-1 instead regulates the expression of non-NK cell cytokines or cell surface receptors, which in turn modulate the differentiation of NK cells in a paracrine fashion. A more definitive answer to this question will await the results of experiments in which Ets-1-deficient bone marrow is transplanted into lethally irradiated wild-type hosts (and vice versa). Our preliminary studies have suggested that the defect in TCR-mediated activation observed in the Ets-1deficient T cells reflects abnormalities in membrane-proximal signaling events. Thus, for example, the Ets-12/2 T cells display markedly reduced activation following TCR cross-linking. This defect is associated with decreased tryrosine phosphorylation and activation of LAT and ZAP70, two proximal T cell signal transduction molecules (T. W. et al., unpublished data). These results have suggested a working model in which Ets-1 regulates the expression of one or more proximal signal transduction molecules in wild-type resting T cells. Recent studies have suggested that the development and activation of NK cells and T cells may be mediated at least in part by common proximal signal transduction pathways (Leibson, 1997). For example, cross-linking of the a/b TCR is known to result in phosphorylation of ITAM-containing CD3 molecules, which in turn recruit and activate the ZAP70 protein tyrosine kinase, leading to the tyrosine phosphorylation of a set of downstream signaling molecules (Qian and Weiss, 1997). Similarly, in NK cells, KIR molecules lacking ITIM motifs have recently been reported to interact with the ITAM-containing adapter DAP12, which in turn appears to activate ZAP70 and Syk protein tyrosine kinases leading to the phosphorylation of downstream signaling molecules and NK cell activation. Given these similarities, it is tempting to speculate that Ets-1 may regulate the expression of one or more proximal signal transduction molecules that are important for both thymocyte and NK cell development. In such a model, the decreased expression of such signal transduction molecules in the Ets-1-deficient mice might only partially inhibit thymocyte development (thereby accounting for the modest reductions in the numbers of thymocytes and splenic T cells seen in these mice) but might be more essential for NK cell development (thereby accounting for the more severe defect in NK cell development seen in these mice [Lanier et al., 1998]). Ongoing studies designed to identify the target genes regulated by Ets-1 in both T and NK cells should shed light on this question and thereby help to elucidate

the molecular pathways underlying the development of natural immunity in mammals. Experimental Procedures Generation of Ets-1-Deficient Mice The targeting vector used to produce the deletion of exons 3 and 4 of the murine Ets-1 gene has been described previously (Muthusamy et al., 1995). RW ES cells were electroporated and selected by growth in G418 and ganciclovir. ES cell clones containing the targeted deletion of the Ets-1 gene were identified by Southern blot analysis with a 0.9 kb XbaI/BglII probe (probe A, Figure 1B) and were injected into C57BL/6 blastocysts to produce chimeras. Chimeric males were mated with C57BL/6 females, and the resulting agouti pups were genotyped by Southern blot analysis with a 1.0 kb XbaI probe (probe B, Figure 1B). Mice heterozygous for the Ets-1 deletion were interbred to produce Ets-1-deficient mice. Agematched Ets-12/2 and wild-type 6- to 12-week-old mice were used for all NK studies. All animal experimentation was carried out in the FMI animal facility of the University of Chicago in accordance with NIH guidelines. Immunoblotting Whole-cell extracts from 0.5 3 106 thymocytes; 0.5 3 106 flow-sorted DX51/CD32 splenocytes; or 4 3 106 Ets-11/1, Ets-12/1, or Ets-12/2 splenocytes were subjected to immunoblot analysis with the Ets1-specific rabbit polyclonal antiserum C-20 (Santa Cruz) (2 mg/ml). Flow Cytometry Single-cell suspensions were obtained by mechanical disruption. Cells (0.5 3 106 to 1.0 3 106) were blocked with anti-Fc receptor and stained on ice for 30 min with phycoerythrin (PE)- and fluorescein isothiocyanate (FITC)-conjugated antibodies. The antibodies used in these experiments included anti-Fc-g III/II (2.4 G2), anti-CD3 (2C11), anti-CD4 (RM4–5), anti-CD8 (53–6.7), anti-B220 (RA3–6B2), anti-IgM (II/41), anti-DX5 (DX5) and anti-NK-1.1 (PK136), and anti-IL-2Rb (TMb1) (PharMingen). The DX5 antibody recognizes an unknown epitope on most or all NK cells from all known mouse strains and is therefore a useful reagent for identifying NK cells in gene-targeted mice produced from C57BL/6-129 chimeras. This epitope appears to be distinct from NKR-P1, CD56, or CD94. Cells were washed and twocolor flow cytometric analyses were performed on a FACScan (Becton-Dickinson). Gating for viable cells was performed using propidium iodide exclusion. Each plot represents analysis of more than 104 events using Lysis II software. Measurement of Serum IgM Serum IgM levels were measured by ELISA (Su et al., 1997) using the Clonotyping System/AP (Southern Biotechnology Associates). The enzymatic reaction was developed with p-NPP phosphatase Substrate System (Kirkegaard & Perry Laboratories) according to the manufacturer’s recommendations. Measurement of IFNg Splenocytes were cultured in vitro for 18 hr with poly (I:C) (100 mg/ ml). Supernatants were removed and IFNg measured by ELISA using the murine IFNg Endogen MiniKit according to the manufacturer’s instructions. In Vitro Cytotoxicity Effector cells consisted of fresh splenocytes that had been stimulated in vivo for 18–24 hr by IP injection of 100 mg poly (I:C) (Sigma). In some experiments, DX51CD32 splenocytes were purified by sorting on a FACstar flow cytometer (Becton Dickinson) prior to use in cytotoxicity assays. Target cells included YAC-1, a Moloney leukemia virus–induced lymphoma of the H-2a haplotype, RMA-S, the class I MHC-deficient variant of the Rauscher virus–induced T cell lymphoma of C57BL/6 origin, and concanavalin A-stimulated blasts from 48 hr cultures of splenocytes of b2m2/2 mice. For the IL-12 add-back experiments, splenocytes were cultured in vitro for 20 hr at 1 3 107 cells/ml with recombinant murine IL-12 (20 ng/ml) (RandD

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Systems) prior to use in cytotoxicity assays. Target cells were labeled with 100 mCi sodium (51Cr) chromate for 1–2 hr at 378C. Labeled target cells were washed at least three times and plated at 104 cells per round-bottom well with appropriately diluted effector cells. Plates were incubated at 378C for 4–5 hr, after which they were spun down and supernatants counted in a scintillation counter. Percent specific lysis was calculated as ((experimental release2spontaneous release)/(maximal release2spontaneous release)) 3 100. In Vivo Cytotoxicity Mice were injected with 105 NK-susceptible RMA-S tumor cells subcutaneously in the right flank (Karre et al., 1986; van den Broek et al., 1995) and followed for tumor development for 40 days. Animals were sacrificed following development of a tumor $ 1 cm in diameter. In Vitro Bone Marrow Culture Bone marrow obtained from the femur was cultured in RPMI 1640, 10% FCS, 2 mM l-glutamine, and 5 3 1025 M 2-mercaptoethanol at 3 3 106 cells per well in 24-well plates (Kalland, 1986). The cells were either unstimulated or stimulated with recombinant murine IL-2 (50 U/ml), recombinant human IL-15 (10 ng/ml), or both (RandD Systems). Cytotoxicity against YAC-1 targets was measured after 4 days in culture. Ribonuclease Protection Assays Splenocytes were cultured in vitro for 18 hr with poly (I:C) (100 mg/ ml). The cells were harvested and total RNA prepared using Trizol (GIBCO BRL). Cytokine and cytokine receptor mRNA quantitation was carried out using the commercially available RiboQuant kit (PharMingen) according to the manufacturer’s instructions. A PhosphorImager (Molecular Dynamics) and ImageQuant software were used to quantitate RNAase-protected bands. Values were normalized first to an internal GAPDH control in each sample and then to wild-type mRNA levels that were assayed with the same probe on the same gel. Acknowledgments

Chan, S.H., Kobayashi, M., Santoli, D., Perussia, B., and Trinchieri, G. (1992). Mechanisms of IFNg induction by natural killer cell stimulatory factor (NKSF/IL-12). Role of transcription and mRNA stability in the synergistic interaction between NKSF and IL-2. J. Immunol. 148, 92–98. Crepieux, P., Coll, J., and Stehelin, D. (1994). The Ets family of proteins: weak modulators of gene expression in quest for transcriptional partners. Crit. Rev. Oncog. 5, 615–638. DiSanto, J.P., Muller, W., Guy-Grand, D., Fischer, A., and Rajewsky, K. (1995). Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain. Proc. Natl. Acad. Sci. USA 92, 377–381. Donaldson, L.W., Peterson, J.M., Graves, B.J., and McIntosh, L.P. (1996). Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif. EMBO J. 15, 125–143. Georgopoulos, K., Bigby, M., Wang, J.H., Molnar, A., Wu, P., Winandy, S., and Sharpe, A. (1994). The Ikaros gene is required for the development of all lymphoid lineages. Cell 79, 143–156. Gumperz, J.E., and Parham, P. (1995). The enigma of the natural killer cell. Nature 378, 245–248. Kalland, T. (1986). Generation of natural killer cells from bone marrow precursors in vitro. Immunology 57, 493–498. Karre, K., Ljunggren, H.J., Piontek, G., and Kiessling, R. (1986). Selective rejection of H-2-deficient lymphoma variants suggests alternative immune defence strategy. Nature 319, 675–678. Kobayashi, M., Fitz, L., Ryan, M., Hewick, R.M., Clark, S.C., Chan, S., Loudon, R., Sherman, F., Perussia, B., and Trinchieri, G. (1989). Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biological effects on human lymphocytes. J. Exp. Med. 170, 827–845. Lanier, L.L. (1997). Natural killer cells: from no receptors to too many. Immunity 6, 371–378. Lanier, L.L. (1998). Follow the leader: NK cell receptors for classical and nonclassical MHC class I. Cell 92, 705–707. Lanier, L.L., Corliss, B.C., Wu, J., Clement, L., and Phillips, J.H. (1998). Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature 391, 703–707.

We thank T. Ley for the generous gift of the RW ES cells, P. Cresswell for the RMA-S cells, and C. Clendenin and K. Sigrist for help with the generation of the Ets-1-deficient mice. L. Gottschalk helped with the preparation of illustrations. P. Lawrey assisted with the preparation of the manuscript. This work was supported in part by a grant from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (to J. L.) and by the Cancer Center of the University of Chicago. DNAX is supported by Schering-Plough Corporation.

Leibson, P.J. (1997). Signal transduction during natural killer cell activation: inside the mind of a killer. Immunity 6, 655–661.

Received June 15, 1998; revised August 26, 1998. References

Moore, T.A., Bennett, M., and Kumar, V. (1996). Murine natural killer cell differentiation: past, present and future. Immunol. Res. 15, 151–162.

Bancroft, G.J. (1993). The role of natural killer cells in the innate resistance to infection. Curr. Opin. Immunol. 5, 503–510.

Murphy, W.J., Reynolds, C.W., Tiberghien, P., and Longo, D.L. (1993). Natural killer cells and bone marrow transplantation. J. Natl. Cancer Inst. 85, 1475–1482.

Bassuk, A.G., and Leiden, J.M. (1997). The role of Ets transcription factors in the development and function of the mammalian immune system. Adv. Immunol. 64, 65–104.

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.

Biron, C.A. (1997). Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9, 24–34.

Ogasawara, K., Hida, S., Azimi, N., Tagaya, Y., Sato, T., YokochiFukuda, T., Waldmann, T.A., Taniguchi, T., and Taki, S. (1998). Requirement for IRF-1 in the microenvironment supporting development of natural killer cells. Nature 391, 700–703.

Bories, J.-C., Willerford, D.M., Grevin, D., Davidson, L., Camus, A., Martin, P., Stehelin, D., and Alt, F.W. (1995). Increased T-cell apoptosis and terminal B-cell differentiation induced by inactivation of the Ets-1 proto-oncogene. Nature 377, 635–638. Carlyle, J.R., Michie, A.M., Furlonger, C., Nakano, T., Lenardo, M.J., Paige, C.J., and Zuniga-Pflucker, J.C. (1997). Identification of a novel developmental stage marking lineage commitment of progenitor thymocytes. J. Exp. Med. 186, 173–182. Carson, W.E., Fehniger, T.A., Haldar, S., Eckhert, K., Lindemann, M.J., Lai, C.F., Croce, C.M., Baumann, H., and Caligiuri, M.A. (1997). A potential role for interleukin-15 in the regulation of human natural killer cell survival. J. Clin. Invest. 99, 937–943.

Liao, N.-S., Bix, M., Kijlstra, M., Jaenisch, R., and Raulet, D. (1991). MHC class I deficiency: susceptibility to natural killer (NK) cells and impaired NK activity. Science 253, 199–202. Magram, J., Connaughton, S.E., Warrier, R.R., Carvajal, D.M., Wu, C.Y., Ferrante, J., Stewart, C., Sarmiento, U., Faherty, D.A., and Gately, M.K. (1996). IL-12-deficient mice are defective in IFNg production and type 1 cytokine responses. Immunity 4, 471–481.

Ohlen, C., Kling, G., Hoglund, P., Hansson, M., Scangos, G., Bieberich, C., Jay, G., and Karre, K. (1989). Prevention of allogeneic bone marrow graft rejection by H-2 transgene in donor mice. Science 246, 666–668. Puzanov, I.J., Bennett, M., and Kumar, V. (1996). IL-15 can substitute for the marrow microenviroment in the differentiation of natural killer cells. J. Immunol. 157, 4282–4285. Qian, D., and Weiss, A. (1997). T cell antigen receptor signal transduction. Curr. Opin. Cell Biol. 9, 205–212. Rodewald, H.-R., Moingeon, P., Lucich, J.L., Dosoiu, C., Lopez, P.,

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and Reinherz, E.L. (1992). A population of early fetal thymocytes expressing FcgRII/III precursors of T lymphocytes and natural killer cells. Cell 69, 139–150. Sanchez, M.J., Muench, M.O., Roncarolo, M.G., Lanier, L.L., and Phillips, J.H. (1994). Identification of a common T/natural killer cell progenitor in human fetal thymus. J. Exp. Med. 180, 569–576. Su, G.H., Chen, H.-M., Muthusamy, N., Garrett-Sinha, L.A., Tenen, D.G, and Simon, M.C. (1997). Disruption of the gene encoding Spi-B, an Ets protein causes defective B cell receptor mediated responses. EMBO J. 16, 7118–7129. Suzuki, H., Duncan, G.S., Takimoto, H., and Mak, T.W. (1997). Abnormal development of intestinal intraepithelial lymphocytes and peripheral natural killer cells in mice lacking the IL-2 receptor b chain. J. Exp. Med. 185, 499–505. Takeda, K., Tsutsui, H., Yoshimoto, T., Adachi, O., Yoshida, N., Kishimoto, T., Okamura, H., Nakanishi, K., and Akira, S. (1998). Defective NK cell activity and TH1 response in IL-18-deficient mice. Immunity 8, 383–390. Trinchieri, G. (1989). Biology of natural killer cells. Adv. Immunol. 47, 187–376. van den Broek, M.F., Kagi, D., Zinkernagel, R.M., and Hengartner, H. (1995). Perforin dependence of natural killer cell-mediated tumor control in vivo. Eur. J. Immunol. 25, 3514–3516. Yu, Y.Y.L., Kumar, V., and Bennett, M. (1992). Murine natural killer cells and marrow graft rejection. Annu. Rev. Immunol. 10, 189–213. Zuniga-Pflucker, J.C., and Lenardo, M.J. (1996). Regulation of thymocyte development from immature progenitors. Curr. Opin. Immunol. 8, 215–224.