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International Immunology Advance Access originally published online on November 13, 2007
International Immunology 2008 20(1):71-79; doi:10.1093/intimm/dxm120
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Runx proteins are involved in regulation of CD122, Ly49 family and IFN-{gamma} expression during NK cell differentiation

Shin-ichiro Ohno1, Takehito Sato1, Kazuyoshi Kohu2, Kazuyoshi Takeda3, Ko Okumura3, Masanobu Satake2 and Sonoko Habu1

1 Department of Immunology, Tokai University School of Medicine, Boseidai, Isehara, Kanagawa 259-1193, Japan
2 Department of Molecular Immunology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Aobaku, Sendai, Japan
3 Department of Immunology, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo, Japan

Correspondence to: T. Sato; E-mail: takehito{at}is.icc.u-tokai.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Runx family proteins play indispensable roles in the development of various hematopoietic lineage cells. However, their function in NK cells is still uncertain. We found that NK cells and CD8 T cells dominantly express Runx3 protein, whereas NKT cells and CD4 T cells express Runx1. Reverse transcription–PCR analysis revealed that Runx3 expression is initiated at the NK precursor stage and is maintained along the course of NK cell differentiation. In order to examine their role in the earlier stage of NK cell development, we introduced Runx dominant-negative (Runx dn) form into Lin–c-kit+Sca-1+ hematopoietic stem cells, which were applied to NK cell-inducing culture. Post-cultured cells showed a decreased expression of IL-2/IL-15 common receptor beta subunit (CD122), consistent with another finding that Runx binds to promoter region of CD122 gene. To examine the Runx function in the later developmental stage, we used transgenic mouse, in which Runx dn form is expressed in immature and mature NK cells. This mouse showed decreased expressions of NK maturation markers, such as Ly49 family, Mac-1 and CD43, whereas IFN-{gamma} production was greatly enhanced. These findings suggest that Runx proteins, especially Runx3, play multiple roles in NK cell differentiation.

Keywords: differentiation, NK cell, Runx


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
NK cells play important roles in innate immunity by eliminating virus-infected cells and tumor cells (1–3). NK cells develop from hematopoietic stem cells (HSCs) through multiple differentiation stages. In the presence of Fms-like tyrosine kinase 3 ligand (Flt3L) or stem cell factor (SCF), HSCs are induced to express IL-2/IL-15 common receptor beta subunit (CD122). Lin–CD122+ cells are regarded as NK precursors (NKPs) because the cells generate mature NK cells at high frequency in human and mouse (4, 5), but do not give rise to T, B, myeloid or erythroid cells under appropriate conditions (6). NKPs can differentiate into immature/mature NK stage in culture with bone marrow-derived stromal cells and IL-15. IL-15 signaling is known to be pivotal for NK cell development and peripheral survival (7–10). Although it is assumed that the proper expression of CD122 is important for lineage specification and differentiation of NK cells, the molecular basis for the expression control is not well understood.

Immature NK cells express NK1.1 (NKR-P1C), CD2 and CD94/NKG2, but they do not express high levels of several maturation markers such as Ly49, DX5, Mac-1 (CD11b) and CD43 (6, 11, 12). Then, during the maturation process, NK cells increase the expressions of these maturation markers. It is reported that the targeted deletion of GATA-3, IRF-2 or T-bet in NK cells resulted in decreased expression of some of the maturation markers (13–15), suggesting that these transcription factors are involved in the maturation process of NK cells. Using in vitro NK cell lines, Runx transcription factor was reported to control the expression of NK cell receptors such as the Ly49 family in mouse (16) and killer cell Ig-like receptors in human (17). However, it remains uncertain how Runx proteins play certain roles in the development of NK cells.

The Runx transcription factors Runx1 (AML1 and Cbfa2), Runx2 (AML3 and Cbfa1) and Runx3 (AML2 and Cbfa3) control the expression of various genes by recruiting co-activators or co-repressors to target sequences and by changing the chromatin structure in the vicinity of the sequences (18). All Runx family members have a well-conserved Runt domain, which binds to the target sequence (PuACCPuCA) in concert with common beta subunit (CBFβ) (19–21). Several investigators including our group have shown that Runx1 plays pivotal roles in the development of HSCs (22–25), megakaryocytes, B cells, T cells (26) and NKT cells (27) and also that Runx3 is important for CD8 T cell development (28–31).

We have previously produced transgenic (Tg) mice expressing Runx1 Runt domain under CD2 promoter control (32). This truncated form lacks the trans-activating domain and binds to CBFβ and the DNA target sequence stronger than full-length Runx proteins. Runt domain can act as a dominant-negative form against all Runx family members. It is reported that Runx1 and 3 can substitute for each other (31, 33, 34) and that Runx3 down-regulates Runx1 expression (35). When one of the Runx genes is targeted, another Runx would be up-regulated to compensate for the compromised function (31). Therefore, Runx dominant-negative (Runx dn) form has been a powerful tool for investigating the function of Runx proteins. In this study, using Runx dn form Tg mouse and in vitro culture system, we showed that Runx proteins, especially Runx3, play important roles in NK cell development and functions through control of the expressions of CD122, Ly49 family, Mac-1, CD43 and IFN-{gamma}.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 Funding
 References
 
Mice
Runx dn-Tg mice (line 81) were previously reported (32). Runx dn form is expressed under the control of CD2 promoter/enhancer. Recombination-activating gene 2 (RAG2)-deficient mice were provided by the Central Institute of Experimental Animals (Kawasaki, Japan). Mice were maintained under specific pathogen-free conditions and were used at the age of 4–8 weeks. All mouse experiments were approved by the Animal Experimentation Committee, Isehara campus (Tokai University, Kanagawa, Japan).

Antibodies and flow cytometric analysis
Purified anti-mouse CD16/32 (93), APC-anti-mouse NK1.1 (PK136), APC-anti-mouse c-kit (2B8), PE-anti-mouse Sca1 (D7), FITC-anti-mouse TER119 (TER-119), FITC-anti-mouse NK1.1 (PK136), FITC-anti-mouse Mac1 (M1/70), FITC-anti-mouse Gr1 (RB6-8C5), PE-anti-mouse Ly49F (HBF-719), Biotin-anti-mouse CD94 (18d3) and Biotin-anti-mouse NK1.1 (PK136) were purchased from eBioscience (San Diego, CA, USA). FITC-anti-mouse Ly49C&I (5E6), D (4E5), G2 (4D11), PE-anti-mouse Ly49C&I (5E6), PerCPCy5.5-anti-mouse CD3 (145-2C11), PE-anti-mouse DX5, Biotin-anti-CD43 and PerCPCy5.5-streptavidin were purchased from BD Biosciences (San Diego, CA, USA). Multicolor flow cytometry was performed by FACScalibur (BD Biosciences). Data were analyzed by the CellQuest program (BD Biosciences).

Immunoblotting
NK, NKT, CD4 T and CD8 T cells were isolated from C57BL6/J mice by FACSvantage (BD Biosciences). The expression of Runx family protein was analyzed as described previously (29, 32). Briefly, cells (3 x 105 cells) were lysed for 30 min in ice-cold RIPA buffer. After centrifugation, cell lysate was subjected to SDS–10% (w/v) PAGE and transferred to an Immobilon-P membrane (Millipore, Billerica, MA, USA). The membrane was incubated with antiserum raised against the 15-amino acid carboxyl terminus of mouse Runx1, which reacts with all Runx family members (anti-pan-Runx antibody) (29, 32). Signals were detected by ECL advance blotting detection kit (Amersham Biosciences, Little Chalfont, UK).

Real-time reverse transcription–PCR analysis
Each lymphocyte subset was purified by a FACSvantage cell sorter (BD Biosciences). RNA was prepared with RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and first-strand cDNA was synthesized by Super-script 3 Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Real-time PCR was performed with ABI PRISM 7700 Sequence Detector and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster city, CA, USA) with primers and probes: for Runx1, Mm00486762_m1; for Runx3, Mm00490666_m1 and for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Rodent GAPDH Control Reagents (Applied Biosystems).

Reverse transcription–PCR
cDNA samples were normalized by the data of real-time PCR for GAPDH expression. PCR was carried out for 30 s at 94°C, 30 s at 60°C and 2 min at 74°C by TAKARA LA Taq with GC Buffer (TAKARA Bio Inc., Tokyo, Japan). The numbers of PCR cycles were 40, 38, 36 and 34. The primers used were as follows: for distal Runx1, 5'-AGAAGTGTAAGCCCAGCACA-3' and 5'-TTCTCAGTTCTGCCGAGTAG-3' and for distal Runx3, 5'-GGCTTCCAACAGCATCTTTG-3' and 5'-CGGAGTAGTTCTCATCATTG-3'. PCR samples were loaded onto the same gel and captured by Dual-intensity trans-illuminator AE-6911FXFD (ATTO, Tokyo, Japan). The intensity was measured by CS analyzer (ATTO).

Electrophonetic mobility shift assay (EMSA)
The NK1.1+CD3– cells were purified from wild-type (WT) and Runx dn-Tg splenocytes by FACSvantage. Whole-cell extracts were prepared as described previously (36). The DNA probe was the polyomavirus enhancer sequence (5'-GATCCAACTGACCGCAGCTG-3') end labeled with [32P] by Klenow enzyme. The underlined sequence represents a Runx-binding site. The mutated DNA probe sequence was 5'-GATCCAACTGAACGAAGCTG-3', where the underlined residues represent the mutated locations.

Transfection of hematopoietic precursors
Gene segments for hemagglutinin (HA), Runx dn form (Runt domain) and nuclear localization signal (NLS) were fused as described previously (32). HA-NLS-Runx dn form was inserted into EcoR1 site of MIGR1 (kindly donated by W. Pear, University of Pennsylvania Medical Center, Philadelphia, PA, USA). HA-NLS-Runx dn/MIGR1 or empty control/MIGR1 was transfected transiently into PLAT-E (37), and the culture supernatants were harvested and used as the source of the retrovirus. Purified lineage markers Lin(Ter119, Gr-1, Mac-1 and NK1.1), c-kit+ and Sca-1+ cells from RAG2–/– bone marrow cells were infected with the retrovirus through centrifugation (7500 r.p.m., 1.5 h) in the presence of polybrene (8 µg ml–1) and SCF (100 ng ml–1). After incubation at 37°C for 3 h, the cells were used as gene-transfected hematopoietic precursors. Infected cells could be distinguished by their expression of green fluorescence protein.

Stromal cells
OP9 cells were purchased from RIKEN (Yokohama, Japan). The cells were cultured with MEM (Invitrogen) containing 20% FCS, 200 nM glutamine, 100 µg ml–1 streptomycin sulfate and 100 U ml–1 penicillin.

In vitro generation of NK cells from marrow cells
In vitro NK cell differentiation from HSCs was performed according to Williams et al. (38). Briefly, HSCs (Lin–, c-kit+ and Sca-1+ marrow cells) were isolated from RAG2–/– C57BL6/J mice by FACSvantage (BD Biosciences). Sorted HSCs were cultured in 96-well U-bottom plates at 1 x 104 to 2 x 104 cells per well in 0.2 ml of RPMI medium supplemented with 10% FCS and a mixture of 0.5 ng ml–1 murine IL-7, 30 ng ml–1 murine SCF and 100 U ml–1 murine Flt3L (PeproTech, Rocky Hill, NJ, USA). The cells were replaced with the same medium on day 3 and on day 5 were replated at 1 x 104 to 2 x 104 per well in RPMI medium containing 150 ng ml–1 human IL-15 (PeproTech) and a confluent monolayer of OP9 stromal cells. On day 8, cultured cells were refed with the same medium, and on day 12, cells were harvested for analysis.

Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assay using anti-Runx antibody was performed as previously reported (29). Chromatin of NK cells was fixed and immunoprecipitated with anti-pan-Runx antibody or normal rabbit IgG by using the ChIP assay kit (Upstate Cell Signaling Solution, Charlottesville, VA, USA) as described. After DNA purification, the presence of selected DNA sequences was assessed by PCR. The primers used were as follows: for CD122 promoter, 5'-AGAAGTCGATGGTGTTAGGGT-3' and 5'-TCCTGTATAGAGGATGAGGG-3'. PCR was carried out for 1 min at 94°C, 1 min at 59°C and 1 min at 72°C. The numbers of PCR cycles for immunoprecipitants were 30, 32, 34, 36 and 38 and for input DNA were 24, 26, 28, 30 and 32.

Cytotoxicity assay
Spleen NK cells were enriched from Runx dn-Tg mice and littermate control mice by MACS beads separation system (Miltenyi Biotec, Bergisch Gladbach, Germany). After depleting CD4+ and CD8+ cells by anti-CD4 and anti-CD8 MACS beads (Miltenyi Biotec), NK1.1+ cells were collected using APC-anti-NK1.1 (PK136) and anti-APC-MACS beads (Miltenyi Biotec). Target YAC-1 cells were labeled with 51Cr for 45 min at 37°C and extensively washed before use. 51Cr-labeled target cells and effector cells were mixed in a 96-well plate at the indicated effector:target cell ratios in triplicate. After incubation for 4 h at 37°C in 5% CO2, a half volume of supernatant was collected from each well to count the released radioactivity. Percent-specific lysis was calculated as 100 x (experimental release – spontaneous release)/(maximal release – spontaneous release).

Induction of IFN-{gamma} production
Either total splenocytes or purified NK1.1+CD3– splenocytes were cultured in the presence of human IL-2 (1000 U ml–1) and murine IL-12 (10 ng ml–1) for 2 h and then further incubated in the presence of brefeldin A (10 µg ml–1) for 4 h. The cells were stained and fixed with 4% (w/v) PFA in PBS and permeabilized in a solution containing 50 mM NaCl, 5 mM EDTA, 0.02% (w/v) NaN3, pH 7.5 and 0.5% (w/v) Triton X-100. After blocking with PBS containing 3% (w/v) BSA, the cells were stained with APC-anti-IFN-{gamma} (XMG1.2).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 Funding
 References
 
NK cells dominantly express Runx3
The Runx transcription factor family consists of three homologous members (Runx1, Runx2 and Runx3). Runx1 is expressed in almost all hematopoietic lineage cells, but Runx3 in relatively limited populations. In the T cell lineage, Runx1 is preferentially expressed on thymocytes at the immature CD4–CD8– (double negative, DN), pre-mature CD4+CD8+ (double positive, DP) and mature CD4 single-positive stages and CD4+ peripheral T cells, but Runx3 is detectable only in CD8 cell lineage of thymocytes and peripheral T cells (32; Fig. 1). Furthermore, we and several groups (26–32) have reported that Runx1 is pivotal for the development of megakaryocytes, B cells, T cells and NKT cells and that Runx3 plays important roles for CD8 T cell differentiation in the thymus through reactivating CD8 and silencing CD4 of DP cells. It was recently reported that Runx3 plays pivotal roles for activating IFN-{gamma} and silencing IL-4 in Th1 cells (39). However, the role of Runx family proteins in NK cells was not elucidated. To address this issue, we first examined whether NK cell lineage expresses Runx family members. For protein level analysis, we used an antibody raised against the 15-amino acid carboxyl terminus of mouse Runx1, which reacts with all Runx family proteins (32). Immunoblotting showed that isolated NK cells of spleen expressed Runx3 dominantly but Runx1 at a low level, as CD8+ cells do. CD4 T cells preferentially expressed Runx1, and NKT cells expressed Runx1 and Runx3 at a similar level (Fig. 1).


Figure 1
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  		Fig. 1. Expression of Runx family protein in CD4+ T, CD8+ T, NKT and NK cells. The protein extracts corresponding to equivalent cell numbers (3 x 105 per lane) were processed for immunoblot analysis using anti-pan-Runx antibody (29, 32) and anti-β-actin antibody (loading control). The expression levels of Runx1 and Runx3 proteins were determined by densitometry and the index was calculated. Data are representative of three separate experiments.

 
To explore how Runx1/3 transcription is controlled in NK cell lineage in the developing pathway, its expression was examined in Lin–c-kit+Sca-1+ HSCs and NK lineage cells of developing stages: NKPs (CD122+NK1.1–), immature NK cells (CD122+NK1.1+DX5–) and mature NK cells (CD122+NK1.1+DX5+) (38; Fig. 2, top). Using the real-time reverse transcription (RT)–PCR system, Runx3 transcription was detected at a similar level in immature and mature NK cells, but it was barely detected in HSCs and NKPs (black bars in Fig. 2A). Runx1 transcription was not prominent in any of the populations (white bars in Fig. 2A). However, it should be noted that the amplified products could be derived from distinct transcripts. Transcription of Runx genes is driven by two promoters, proximal and distal. It is reported that proximal promoter-driven transcripts, but not distal promoter-driven ones, are poorly translated, as their 5'-untranslated region forms some secondary structure that inhibits ribosome scanning (40).


Figure 2
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  		Fig. 2. Expressions of Runx1 and Runx3 mRNAs in NK cell differentiation pathway. (A) Total Runx1 and Runx3 transcripts in NK cell differentiation pathway were quantitated by real-time PCR. Sorted RAG2–/– marrow HSCs (Lin–, c-kit+ and Sca-1+ cells) were cultured in SCF, Flt3L and IL-7 for 7 days. On day 7, CD122+NK1.1– cells were isolated as NK progenitors (NKP) using a cell sorter. CD3–NK1.1+DX5– cells and CD3–NK1.1+DX5+ cells in C57BL/6 mouse were prepared as immature NK cells and mature NK cells, respectively. mRNAs were extracted from these cells and real-time PCR analysis was performed. Data represent mean ± SD (n = 3 – 7). (B) Distal-type Runx1 and Runx3 transcripts in NK cell differentiation pathway were determined by RT–PCR. cDNA samples used in (A) were normalized by real-time PCR data for GAPDH transcripts. Data are representative of three separate experiments.

 
Therefore, the expression level of the distal promoter-driven mRNA may be correlated with the protein expression level. Thus, we established a novel RT–PCR to specifically amplify distal promoter-driven transcripts and verified the correlation between the expression of protein and distal-type transcripts in CD4 and CD8 T cells (Supplementary Data 1, available at International Immunology Online). In CD4 T cells, distal-type Runx1 was highly detected, but the detection of distal-type Runx3 was only slight. In CD8 T cells, the expression of distal-type Runx3 was prominent, while that of Runx1 was less than that in CD4 T cells (Supplementary Data 1B, available at International Immunology Online). The ratio of the expression index of distal-type transcripts was well correlated with the protein expression (Supplementary Data 1C, available at International Immunology Online), although the results obtained by conventional real-time PCR did not show any correlation (Supplementary Data 1A and C, available at International Immunology Online).

Then, we examined the expression level of distal-type mRNAs of Runx1 and Runx3 (Fig. 2B). While distal-type Runx1 transcripts were detected in HSCs but not in other cells, distal-type Runx3 transcripts were evident in immature and mature NK cells. In both stages, Runx1 expression was decreased to a very low level. These results are consistent with the previous report that Runx1 mRNA was decreased when common lymphoid progenitor cells differentiated to NK cells (41). Interestingly, NKPs expressed significant amounts of distal-type Runx3, suggesting that Runx3 expression is initiated at the NKP stage. In consideration of the recent report that Runx3 expression is associated with the commitment process to CD8 T cells and {gamma}{delta} T cells (29, 42), our findings led us to the speculation that Runx3 plays an important role around the commitment process into NK cell lineage.

Runx controls CD122 expression in early stage of NK cell development
In the developmental pathway of NK cell lineage, Runx3 was first detected in the NKP stage, where CD122 expression is initiated. We therefore examined whether Runx3 is involved in the control of CD122 gene transcription. In the database analysis, we found that several Runx-binding sites were located in the promoter region of murine (Fig. 3A) and human (data not shown) CD122 gene. Then, we confirmed whether Runx protein substantially binds to the CD122 promoter by ChIP assay. As shown in Fig. 3(B), ChIP assay using anti-Pan-Runx antibody (29) revealed that Runx protein binds to murine CD122 promoter region in NK cells.


Figure 3
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  		Fig. 3. Runx is involved in CD122 expression in early NK cell differentiation stage. (A) The promoter region of IL-2/15 receptor beta contains Runx-binding sites. Downward arrows indicate putative Runx-binding sites. Upward arrows indicate binding sites for transcription factors that regulate CD122 transcription in human or mouse [references for transcription factors: EWS-WT1 (55), Egr-1 (56, 57) and Ets-1/GABP (57, 58)]. Horizontal arrows indicate the sites of PCR primers used in (B). Numbers indicate nucleotide positions relative to the transcription start site. (B) ChIP assay. Sorted NK cells were analyzed with anti-Pan-Runx antibody and Rabbit IgG (negative control). PCR of immunoprecipitated samples is compared with DNA before immunoprecipitation (input). Data are representative of three separate experiments. (C) Sorted RAG2–/– marrow Lin–, c-kit+ and Sca-1+ cells were transfected with Runx dn form by retrovirus vector and cultured in SCF, Flt3L and IL-7. On day 5, after washing, cells were cultured in a 24-well plate with a confluent monolayer of OP9 stromal cells in the presence of IL-15. On day 12, total cultured cells were harvested, stained with antibodies and analyzed by flowcytometry. The numbers indicated in each quadrant represent the percentage of the cells. Data are representative of five separate experiments.

 
In order to clarify whether Runx is involved in CD122 expression in the NK cell developmental pathway, we examined the effect of decreased Runx function in NK induction culture by inducing Runx dn form to express in bone marrow HSCs of RAG2-deficient mice by retrovirus transfer system. The DNA-binding domain of Runx, called Runt domain, lacks a trans-activation domain but binds to DNA target sequence and β subunit stronger than full-length Runx protein does. Since Runt domain is well conserved among the family members, enforced expression of Runt domain inhibits the activity of all Runx family members (29, 32). Runt domain-transfected HSCs, which were distinguished by enhanced green fluorescent protein (EGFP) expression, were cultured with Flt3L, SCF and IL-7 for 5 days and with IL-15 for further 7 days as described in Fig. 2. CD122-expressing cells appeared on day 5 (data not shown), after which CD122+ NK1.1+ cells appeared in control vector-transfected cells on day 12 (Fig. 3C, ‘Empty’). In Runx dn-transfected cells, however, approximately half of the NK1.1+ cells did not express a detectable level of CD122, and the proportion of NK1.1+CD122+ immature NK cells was smaller than that of control vector-transfected cells (2.1 versus 14.2%, Fig. 3C). Therefore, CD122 expression seemed to be positively regulated by Runx protein in NK1.1+ cells.

NK cells in Runx dn form Tg mice have immature surface phenotype
To examine the role of Runx in the process from immature to mature NK cells, we used Tg mice expressing Runx dn form under the control of CD2 promoter (32) because CD2 expression begins at the immature stage of NK cells (6; Fig. 4A). We crossed the mice to obtain offspring possessing two chromosomes harboring the transgene. As shown in Fig. 4(B), EMSA assay displayed the dosage effect of Runx dn form in isolated NK1.1+ CD3– cells. In Runx dn-Tg mice, the absolute number of NK cells was not significantly different between WT and Runx dn-Tg mice (Fig. 4C). CD122 expression was only slightly reduced in NK cells of Runx dn-Tg mouse (Supplementary Data 3, available at International Immunology Online), suggesting that Runx is required for the acquisition of CD122 expression in the earlier developmental stage of NK cells but is not essential for the maintenance of CD122 expression in peripheral NK cells at the later developmental stage.


Figure 4
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  		Fig. 4. Surface phenotype of NK cells in Runx dn-Tg mice. (A) Expression of the transgene is initiated at immature NK cell stage since Runx dn-Tg mice express Runx dn form under the control of CD2 promoter (6, 32). (B) Endogenous Runx1 and Runx3 DNA-binding activities in NK cells from Runx dn-Tg were measured by EMSA. CD3–NK1.1+ cells were isolated from the Runx dn-Tg mice (+/–, +/+) and a non-Tg litter. Whole-cell extracts were prepared and subjected to EMSA. The DNA probe used was the polyomavirus enhancer sequence. The bands indicated represent the Runx1/DNA and Runx3/DNA complexes. (C) The number of NK cells in spleen (left) and liver (right) from mice with indicated genotypes are as follows: WT, n = 9; Runx dn-Tg+/–, n = 12 and Runx dn-Tg+/+, n = 5. Data are shown as mean ± SD from 5 to 12 mice in each group. (D) Expressions of CD94, Ly49 family (Ly49C&I, G2 and D), DX5, Mac-1 and CD43 on CD3–NK1.1+ spleen cells of WT, Runx dn heterozygous (Runx dn-Tg+/–) and homozygous mice (Runx dn-Tg+/+). Representative data are indicated by histogram plots (top). Percentages of positive cells are shown as mean ± SD from four to nine mice in each group (bottom). *Significant decreases in percentages of positive cells as compared with those in littermate control mice (P < 0.01). (E) The expression of Ly49C&I, Ly49F, Ly49G2 and Ly49D on CD3–NK1.1+ spleen cells from Runx dn-Tg mice. Percentages of positive cells are indicated. (F) The expression of Ly49 family and Mac-1 on lymphocytes in liver of littermate control and Runx dn-Tg mice. Percentages of positive cells are indicated. (G) Mac-1 and CD43 expression in spleen CD3–NK1.1+ cells of Runx dn-Tg mice (left). Numbers given in the individual quadrants indicate the percentages of cells in each gate. Data represent three different mice with control littermates. Number of Mac-1highCD43high mature NK cells in spleen from Runx dn-Tg mice (right). The number of mature NK cells was determined by calculating the absolute number of total cells from the FACS profiles. Data are shown as mean ± SD from three to six mice in each group. *Significant decreases in absolute cell number as compared with those in littermate control mice (P < 0.01).

 
Next, we examined the expression status of NK cell maturation markers in NK1.1+CD3– spleen cells among Runx dn-hetero mice (Runx dn-Tg+/–), -homo mice (Runx dn-Tg+/+) and WT littermates (Fig. 4D). In Runx dn-Tg mice, expressions of Ly49 family (Ly49C, I, G2 and D), Mac-1 and CD43 were significantly decreased, while expressions of CD94 and DX5 had not changed (Fig. 4D). It is to be noted that the extent of decrease was dose dependent on Runx dn form. The proportions of Ly49high cells in NK cells were 73.1, 56.3 and 39.6% in WT, Runx dn+/– and Runx dn+/+ mice, respectively. A decreased expression was not restricted to certain particular Ly49 family members. Expressions of both inhibitory type (Ly49C&I, Ly49F and Ly49G2) and activating type (Ly49D) receptors were significantly decreased (Fig. 4E). Similarly, Mac-1high cells were 48.8, 41.8 and 23.3%. CD43high cells were 54.5, 43.4 and 17.0% in WT, Runx dn+/– and Runx dn+/+ mice, respectively. Similar results were obtained from liver NK cells in Runx dn-homo mice (Fig. 4F).

We also examined the absolute numbers of immature and mature NK cells in Runx dn-Tg mice. The cell number of mature type NK cells (CD43highMac-1high) was reduced in Runx dn-Tg mice, whereas total NK cells did not change significantly (Fig. 4C and G). These findings indicated that Runx is involved in the expression control of several NK cell maturation markers.

Runx protein controls IFN-{gamma} production but not cytolytic activity of NK cells
Since NK cells play important roles in innate immunity by killing some target cells directly and producing cytokines such as IFN-{gamma}, we examined whether Runx protein is involved in the cytotoxicity and IFN-{gamma} production of NK cells. We collected NK1.1+CD3– cells from the spleen and performed 51Cr-release assay against YAC-1 as target cells. We found that the percentage lysis after 4-h incubation with target cells was almost equal in WT and Runx dn-Tg mice (Fig. 5A). The expression level of NKG2D, known to be a major receptor for YAC-1 recognition (43, 44), was not significantly different between WT and Runx dn-Tg mouse NK cells (data not shown). Therefore, Runx is not crucial for the cytolytic activity of NK cells. As for the IFN-{gamma} production in NK cells, NK1.1+CD3– cells of WT and Runx dn-Tg mice were stimulated with IL-2 and IL-12. As shown in Fig. 5, the production of IFN-{gamma} was greatly enhanced in Runx dn-Tg mice (Fig. 5B), suggesting that Runx protein negatively controls IFN-{gamma} production in NK cells. Collectively, NK1.1+CD3– cells in Runx dn-Tg mice possessed enhanced potential for IFN-{gamma} production and equivalent cytolytic activity, but showed relatively immature surface phenotype.


Figure 5
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  		Fig. 5. Functional analysis of NK cells in Runx dn-Tg mice. (A) NK activity in Runx dn-Tg mice. Cytolytic activity of sorted CD3–NK1.1+ splenocytes from Runx dn-Tg mice and littermate control was examined by 51Cr-release assay against YAC-1 target cells at the indicated effector:target (E/T) ratios. Data are expressed as percentage of specific release. Three independent experiments were performed with similar results, and data represent the mean of triplicate determinations from one representative experiment. (B) IFN-{gamma} production of NK cells in Runx dn-Tg mice. Splenocytes from Runx dn-Tg mice were stimulated with IL-2 + IL-12 or only IL-2 (negative control) and analyzed for intracellular IFN-{gamma}. Gated CD3– NK1.1+ cells are shown, and the numbers represent the percentages of cells in each gate. One representative experiment of three is shown. In addition, stimulation of isolated CD3–NK1.1+ cells showed similar results (Supplementary data 4, available at International Immunology Online).

 

   Discussion
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 Methods
 Results
 Discussion
Supplementary data
 Funding
 References
 
In this study, we demonstrated that Runx proteins are involved in NK cell development and functions. We showed that Runx3 is induced to express in NK cell lineage from the NKP stage to mature NK cells. In the developmental pathway, Runx controlled the expression of NKP marker, CD122, and NK cell maturation markers such as Ly49 family, Mac-1 and CD43. At the same time, we also demonstrated that Runx negatively controls IFN-{gamma} production in NK cells.

In order to examine whether Runx plays a role in the early NK cell development, Runx dn form-transfected HSCs were applied to NK cell-inducing culture. Among NK1.1+ cells, the CD122– cell proportion was larger in Runx dn form-transfected cells than in control vector-transfected cells after the culture (Fig. 3C, day 12). When EGFP-positive cells within Runx dn form-transfected cells were subdivided into EGFP-high (EGFP-High) and -middle (EGFP-Mid)-positive cells, the proportion of CD122+ cells in NK1.1+ cells was smaller in EGFP-High cells than in -Mid cells (Supplementary Data 2, available at International Immunology Online). This suggests that Runx dn form inhibits CD122 expression in a dose-dependent manner. However, the number of EGFP-High cells appearing in control vector-transfected cells was significantly smaller than that in Runx dn form-transfected cells for unknown reason. Therefore, we cannot absolutely deny the possibility that the detection of CD122 expression in EGFP-High cells was a result of an extremely high expression of retrovirus vector. In addition, it is to be confirmed whether Runx controls CD122 gene expression directly or indirectly, although the ChIP assay showed that Runx protein binds to the promoter region of CD122 gene (Fig. 3B).

As shown in Fig. 2, HSCs express Runx1 but not Runx3 and vice versa in NK cell lineage from the NKP stage. Similarly, a Runx1 to Runx3 transcription switch has been observed in the CD8 thymocyte differentiation pathway (29) and in the activation process of B cells (35). Recently, it was reported that a Runx1 and Runx3 switch was observed in Th1 differentiation from naive CD4 T cells (39). It was also reported that Runx3 can repress Runx1 expression through binding to the distal promoter region of Runx1 gene (35). Considering that CD122 expression on HSCs is correlated to the initiation of NK cell lineage (12), we can hypothesize that the switch from Runx1 to Runx3 expression is correlated with the commitment to NK lineage cells, resulting in the acquisition of CD122 expression. Although Runx1 and Runx3 share a redundant function at least in part, each protein may have a certain unique function. The physiological significance of the switch from Runx1 to Runx3 is sure to be elucidated in the near future.

Saleh et al. (16) reported that Runx binds to the immature NK stage-specific promoter (Pro1) element of some Ly49 family genes, such as Ly49j, Ly49i, Ly49g, Ly49c and Ly49e. They argued the possibility that the proper activation of Pro1 at the immature NK stage leads to the activation of downstream, mature NK stage-specific promoter (Pro2). In Runx dn-Tg mice, we found decreased expression of the Ly49 family and assumed that Runx protein, presumably Runx3, is involved in the initial decision of whether each Ly49 family member is to be expressed. In the same mice, Ly49D, which lacks Runx-binding motif in the Pro1 element (data not shown), also showed the decreased expression, pointing to the possibility that Runx can control Ly49 expression not only through Pro1 elements.

In addition to the Ly49 family expression, we found decreased expression of NK cell maturation markers such as Mac-1 and CD43 in Runx dn-Tg mice (Fig. 4). We cannot completely rule out the possibility that the changes of NK cell maturation marker expression in Runx dn-Tg mice had resulted from the defect of T cells since T cell development was impaired in these mice (32). Still, this possibility seems unlikely because NK cell maturation is intact in RAG-deficient mice that completely lack T cells (45–47). In contrast to the decreased expression of NK cell maturation markers, IFN-{gamma} production was enhanced in Runx dn-Tg mice (Fig. 5B). Vosshenrich et al. (48) reported that thymic NK cells also showed enhanced IFN-{gamma} production with decreased expression of CD43 and Mac-1. It can be assumed that thymic NK cells develop under a condition of Runx being down-regulated. On the other hand, it has been reported that NK cells deficient in any of the transcription factors GATA-3 (13), IRF-2 (14) or T-bet (15) showed decreased expressions of Mac-1 and CD43, while IFN-{gamma} production was reduced in these knockout mice. Collectively, the expression of maturation markers and IFN-{gamma} production are independently regulated. Brenner et al. (49) showed that the expressions of IFN-{gamma}, GATA-3 and T-bet were increased in the colonic mucosa of Runx3-deficient mice. They claimed that Runx3 negatively regulates IFN-{gamma} production via the inhibition of GATA-3 and T-bet expressions. Alternatively, it may be speculated that Runx regulates IFN-{gamma} production through transforming growth factor-β (TGF-β) signaling pathway because it is known that TGF-β represses IFN-{gamma} production in NK cells (50, 51) and that Runx plays important roles in TGF-β signaling through the binding to Smad proteins (52). Recently, it was reported that Runx3 binds to the promoter of IFN-{gamma} gene and positively controls its expression (39). It is known that Runx proteins can control the expression of target genes either positively or negatively. For example, we have recently reported that Runx3 has the dual functions of reactivating CD8 as well as silencing CD4 in DP thymocytes, resulting in the generation of CD8 T lineage-committed thymocytes (29). Similarly, {alpha}E/CD103 expression is controlled both positively and negatively by Runx3 (53). Such capability for both directions is based on the fact that Runx proteins can recruit both co-activators such as p300 and co-repressors such as Groucho/TLE (53, 54). We can assume that Runx3 recruits some co-repressors in NK cells but recruits some co-activators in Th1 cells. The molecular basis for the control of IFN-{gamma} via Runx proteins is to be further investigated.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
Funding
 References
 
Supplementary data are available at International Immunology Online.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
References
 
Ministry of Education, Culture, Sports, Science and Technology, Japan (18590477).


   Acknowledgements
 
We thank Yoshinori Okada and Chiharu Sato for their technical assistance.


   Abbreviations
 
CBFβ, common beta subunit
ChIP, chromatin immunoprecipitation
EGFP, enhanced green fluorescent protein
EMSA, electrophonetic mobility shift assay
Flt3L, Fms-like tyrosine kinase 3 ligand
GAPDH, glyceraldehyde-3-phosphate dehydrogenase
HA, hemagglutinin
HSC, hematopoietic stem cell
NKP, NK precursor
NLS, nuclear localization signal
RAG2, recombination-activating gene 2
RT, reverse transcription
Runx dn, Runx dominant negative
SCF, stem cell factor
Tg, transgenic
TGF-β, transforming growth factor-β
WT, wild type

   Notes
 
Transmitting editor: T. Watanabe

Received 21 February 2007, accepted 17 October 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 

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