Recently, it was reported that the expression of Runt-related transcription factor 3 (Runx3) is up-regulated in CD4+ helper T cells during Th1 cell differentiation, and that Runx3 functions in a positive feed-forward manner with the T-box family transcription factor, T-bet, which is a master regulator of Th1 cell differentiation. The relative expression levels of IFN-γ and IL-4 are also regulated by the Th2-associated transcription factor, GATA3. Here, we demonstrate that Runx3 was induced in Th2 as well as Th1 cells and that Runx3 interacted with GATA3 and attenuated GATA3 transcriptional activity. Ectopic expression of Runx3 in vitro in cultured cells or transgenic expression of Runx3 in mice accelerated CD4+ cells to a Th1-biased population or down-modulated Th2 responses, in part by neutralizing GATA3. Our results suggest that the balance of Runx3 and GATA3 is one factor that influences the manifestation of CD4+ cells as the Th1 or Th2 phenotypes.

The CD4+ helper T cells play a central role in acquired immunity. Naive CD4+ T cells in peripheral lymphoid tissues are activated by Ag and induced to differentiate into effector Th cells, which secrete large amounts of effector cytokines. Distinct subsets of effector Th cells can be functionally defined by their discrete cytokine expression profiles. Th1 cells secrete IFN-γ, IL-2, and TNF-α and mediate defense against infection with intracellular microbes and isotype switching to IgG2a and IgG2b, whereas Th2 cells secrete IL-4, IL-5, and IL-13 and promote humoral response to extracellular pathogens and isotype switching to IgG1 and IgE (1). Differentiation of Th subsets is regulated and coordinated by a complex network of transcriptional regulators, including T-box family transcription factor (T-bet)4 and GATA3 (2, 3). GATA3 is a master regulator of Th2 differentiation and activates Th2-specific cytokine genes primarily through epigenetic modification of the IL-4 locus (4). Ectopic expression of GATA3 can induce Th2 cell differentiation even under conditions in which STAT6, a transducer of IL-4 signals, is absent, a situation that otherwise promotes Th1 cell differentiation (5). Furthermore, GATA3 is involved in the autoactivation of GATA3 gene transcription (5). GATA3 is necessary for Th2-mediated immune responses in vivo, as demonstrated by the analysis of conditional GATA3 knockout mice (6, 7). T-bet is a master regulator of Th1 cell differentiation, and activates Th1-specific cytokine genes while repressing Th2-related cellular programs (8). When naive CD4+ T cells encounter Ag, GATA3 is transiently transcribed during the early response phase, even under Th1 conditions. However, at the protein level, T-bet negatively regulates GATA3 activity through a physical interaction with GATA3, and it functions as an antagonist of Th2 differentiation (9).

The Runt-related transcription factors (Runx) harbor an evolutionarily conserved 128-aa region termed the Runt domain, which is responsible for DNA binding (10, 11, 12). Runx proteins interact with coactivators or corepressors to activate or repress target genes in a context-dependent manner (13). In thymocyte development, Runx1 and Runx3 are essential factors in the generation of CD8+ cells. Runx1 and Runx3 have been shown to repress the expression of CD4 and to activate the expression of CD8 (14, 15, 16). Runx transcription factors are also involved in the regulation of Th cell differentiation. We previously reported that the forced expression of Runx1 in naive CD4+ T cells promotes Th1 cell differentiation even under conditions that promote Th2 differentiation. Conversely, introduction of a dominant negative form of Runx1 biases cytokine secretion toward the Th2 phenotype (17). Recently, Djuretic et al. (18) demonstrated that Runx3 expression is up-regulated during Th1 differentiation and functions in a positive feed-forward manner in Th1 differentiation. They also demonstrated that in Th1 cells, Runx3 interacts with T-bet and simultaneously attenuates IL-4 expression and augments IFN-γ expression. These effects are achieved by the binding of a Runx3/T-bet complex to the IL-4 silencer and IFN-γ promoter, respectively. Thus, the interaction of Runx3 and T-bet appears to augment the activity of each binding partner, which explains why Runx3-deficient T cells are prone to Th2 differentiation.

The relative levels of IFN-γ and IL-4 expression are also regulated by the Th2-associated transcription factor GATA3. In this study, we present evidence that Runx3 associates with GATA3 and attenuates GATA3 transcriptional activity, thereby affecting cell phenotypes. The overexpression of Runx3 converted CD4+ cells to a Th1-biased cell population and/or down-modulated Th2 responses, in part by neutralizing the activity of GATA3.

Mouse rIL-2, rIL-4, and rIL-12 were purchased from PeproTech. For T cell culture, anti-TCRβ (H57-597), anti-CD28 (37.51), anti-IL-4 (11B11), anti-IFN-γ (XMG1.2), and anti-IL-12 (C17.8) Abs were purchased from eBioscience. For intracellular cytokine staining, allophycocyanin-conjugated anti-IFN-γ (XMG1.2) and PE-conjugated anti-IL-4 (11B11) were purchased from BioLegend. The anti-Runx Ab was described previously (19). Anti-GATA3 (HG3-31), anti-α-tubulin (Ab-1), and anti-β-actin Abs were purchased from Santa Cruz Biotechnology and Calbiochem.

CD4+ T cells were purified from the spleens of C57BL/6J (B6) mice using an IMag Cell Separation System (BD Biosciences). The purity of the CD4+ fraction was >95% based on flow cytometry. Cells (2 × 105) were first stimulated with immobilized anti-TCRβ mAb (30 μg/ml) and soluble anti-CD28 mAb (10 μg/ml) in RPMI 1640 containing 10% (v/v) FBS for 2 days. To maintain cells under nonskewed (ThN) conditions, cells were cultured in the presence of IL-2 (30 U/ml) for an additional 7 days. For Th2 polarization, cells were cultured for 4 days in the presence of murine IL-4 (100 U/ml), anti-IL-12 Ab (10 μg/ml), and anti-IFN-γ Ab (10 μg/ml); for Th1 polarization, cells were cultured for 4 days in the presence of murine IL-12 (5 ng/ml) and anti-IL-4 Ab (10 μg/ml). Intracellular staining of IFN-γ and IL-4 was performed as described previously (17).

The murine Runx3 cDNA was inserted into the retroviral expression vector pMX-GFP (17) to generate pMX-Runx3-IRES-GFP. Mutated versions of Runx3 were also inserted into pMX-GFP, and they were Runx3/1–404, Runx3/R178Q, and Runt each. Runx3/1–404 was constructed by PCR, whereas Runx3/R178Q (20) was provided by Drs. K. Ito and Y. Ito (National University of Singapore, Singapore). Runt represents a Runt domain derived from murine Runx1 (10). Both Runx3/R178Q and Runt function dominant negatively against intact Runx1/3 proteins. The GATA3 cDNA was inserted into pMX-ratCD2 to generate pMX-GATA3-IRES-ratCD2. The packaging cell line PLAT-E was transfected with each plasmid using FugeneHD (Roche). After incubation for 24 h, the culture supernatant was harvested, concentrated, and used as a viral stock (21). CD4+ T cells that had been stimulated as described for 24 h were infected with retroviruses using the DOTAP Liposomal Transfection Reagent (Roche) and then incubated under ThN-, Th1-, or Th2-permissive conditions for an additional 6 days. Rat CD2+ cells were purified using a biotin-conjugated anti-rat CD2 Ab and an IMag Cell Separation System.

293T cells were transfected with an expression vector, pcDNA3, encoding hemagglutinin (HA)-tagged Runx3 or GATA3 using the FugeneHD reagent (Roche). The Runt domain-deleted Runx3 and the Runt domain itself of Runx3 were also transfected into 293T cells. After incubation for 48 h, the cells were lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM NaVO4, 1 mM NaF, and 1 μg/ml aprotinin. The lysates were also prepared from splenic CD4+ T cells that had been cultured under Th2 conditions described previously. Each lysate was incubated with anti-HA (3F10; Roche) or anti-GATA3 (HG3-31) mAb, and the immunoprecipitates were adsorbed to protein G-Sepharose beads (GE Healthcare). The beads were washed five times with lysis buffer, and then proteins were eluted by boiling the beads in SDS sample buffer. As a control, whole-cell lysate was prepared by sonicating cells in SDS sample buffer. Proteins were resolved by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore). The membrane was probed with an appropriate primary Ab, followed by an alkaline phosphatase-conjugated secondary Ab (Promega). Immune complexes were detected using NBT-5-bromo-4-chloro-3-indolyl phosphate reagents (Promega).

RNA was isolated from cells using the ISOGEN reagent (Nippon Gene). First-strand cDNA was synthesized from RNA using Superscript II reverse transcriptase (Invitrogen Life Technologies). PCR amplification was performed under nonsaturating conditions using cDNA as the template and LA-Taq polymerase (Takara). The sequences of the sense and antisense primers were as follows: for T-bet, 5′-CTAAGCAAGGACGGCGAATGT-3′ and 5′-GGCTGGGAACAGGATACTGG-3′; for IFN-γ, 5′-GGTGACATGAAAATCCTGCAGAGC-3′and 5′-TCAGCAGCGACTCCTTTTCCGCTT-3′; for TNF-α, 5′-ATCAGTTCTATGGCCCAGACCCT-3′ and 5′-TCACAGAGCAATGACTCCAAAGTA-3′; for IL-12, 5′-GG GACATCATCAAACCAGACC-3′ and 5′-GCCAACCAAGCAGAAGACAGC-3′; for IL-18, 5′-ACTGTACAACCGCAGTAATAC-3′ and 5′-AGTGAACATTACAGATTTATCCC-3′; for GATA3, 5′-CTCCTTTTTGCTCTCCTTTTC-3′ and 5′-AAGAGATGAGGACTGGAGTG-3′; for IL-4, 5′-TCCACGGATGCGACAAAAAT-3′ and 5′-TTCTTCTTCAAGCATGGAGT-3′; for IL-5, 5′-GAGCACAGTGGTGAAAGAGACCTT-3′ and 5′-ATGACAGGTTTGGATAGCATTT-3′; for IL-13, 5′-AGTTCTACAGCTCCCTGGTTCTCT-3′ and 5′-CTTTGTGTAGCTGAGCAGTTTTGT-3′; for Runx1, 5′-ACTCTGCCGTCCATCTCCGACCCGC-3′ and 5′-CGTCGCTCTGGCTGGGGAGGCTGGG-3′; for Runx3, 5′-GCTGCAGAGCCTCACAGAGAGCCGC-3′ and 5′-GTCGGCTTCCACGCCATCAGGCTGG-3′; for β-actin, 5′-GATGACGATATCGCTGCGCTG-3′ and 5′-GTACGACCAGAGGCATACAGG-3′.

M12 cells were cotransfected with a luciferase reporter plasmid and an expression plasmid for Runx3 or GATA3 using FugeneHD (Roche). As an internal control, cells were also transfected with pRL-TK (Promega), and the activity of Renilla luciferase was used to normalize for transfection efficiency. The cells were incubated for 24 h after transfection and then lysed in a passive lysis buffer (Promega). Luciferase activity was measured using a luciferase substrate (Promega) and a LumatLB9507 (Berthold Technologies).

The procedure for the EMSA was described previously (15). The GATA binding sequence from the IL-5 promoter, 5′-GGTGTCCTCTATCTGATTGTT-3′, was used as a probe to detect GATA DNA-binding activity. GATA3, Runx3, and CBFβ polypeptides were synthesized in vitro using the TnT T7 Coupled Reticulocyte Lysate System (Promega) and each cDNA template in a pcDNA3 vector.

The transgenic mouse line expressing a murine Runx3 transgene was previously described (15). Litters of transgenic mice were backcrossed with B6 mice for 10 generations. B6 mice were purchased from Clea Japan. Stat6-deficient mice were obtained from Dr. S. Akira (Osaka University, Osaka, Japan) (22). Experiments were performed using 8- to 12-wk-old, age-matched mice. Mice were maintained and bred according to the guidelines defined by the Animal Facility of the Institute of Development, Aging and Cancer at Tohoku University (Sendai, Japan). All animal protocols used in this study were approved by our Institutional Animal Care and Use Committees.

Mice were injected s.c. with 10 μg of OVA (Sigma-Aldrich) in CFA or i.p. with 10 μg of OVA in aluminum hydroxide. Boosting injections were conducted at days 14 and 28. The titer of serum Ig was measured by ELISA. HRP-conjugated anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, and anti-mouse IgE were purchased from Bethyl. Spleens were isolated and fixed in 4% (w/v) paraformaldehyde in PBS for 18 h and then embedded in Tissue-Tek OCT compound (Sakura Finetechnical). Microsections of tissues on slides were stained by HRP-conjugated peanut agglutinin and counterstained with hematoxylin.

To examine the expression of Runx proteins during Th cell differentiation, naive CD4+ cells were isolated from the spleens of mice and subjected to immunoblot analysis using an anti-pan-Runx Ab. The advantage of this Ab was that we could visualize the levels of each Runx family member simultaneously (the authenticity of Runx bands detected by the Ab had been confirmed previously in Refs. 15 and 19). As seen in Fig. 1, Runx1 was the sole major Runx component expressed in naive CD4+ cells (Fig. 1, lane 2). In contrast, in Th1-differentiated cells, Runx1 was undetectable, and Runx3 emerged as the principal Runx component (Fig. 1, lane 3). Th2-differentiated cells expressed equivalent amounts of Runx1 and Runx3 (Fig. 1, lane 6). In Stat6-deficient cells under Th2-promoting conditions, Runx expression was converted to a Th1-specific phenotype (Fig. 1, lane 5), which indicated that the expression of Runx proteins in wild-type Th2 cells is dependent on Stat6. When wild-type CD4+ cells were stimulated through the TCR using an anti-TCRβ Ab and then cultured under nonskewed conditions, the expression patterns of Runx proteins were similar to those of Th2 cells (Fig. 1, compare lanes 7 and 9). These results indicated that Runx3 is not a Th1-specific factor but is expressed in TCR-stimulated CD4+ cells as well as in Th2-differentiated cells.

FIGURE 1.

Expression of Runx proteins during Th cell differentiation. Immunoblot (IB) analysis of Runx proteins in naive CD4+ cells and in CD4+-derived Th cells cultured in vitro under ThN, Th1, or Th2 conditions for 7 days. CD4+ T cells were isolated from the spleens of wild-type (+/+), Stat6-deficient (Stat6−/−) and Runx3-transgenic mice, as indicated. Extracts were probed with an anti-pan-Runx Ab. Arrows, Runx1 and Runx3. In the third panel from the top, extracts were also probed with an anti-GATA3 Ab. β-Actin served as a loading control. Relative densities of Runx3 and GATA3 bands are indicated below the gel. The identity of the protein indicated by the asterisk is unknown; however, based on its apparent m.w., it might represent Runx2.

FIGURE 1.

Expression of Runx proteins during Th cell differentiation. Immunoblot (IB) analysis of Runx proteins in naive CD4+ cells and in CD4+-derived Th cells cultured in vitro under ThN, Th1, or Th2 conditions for 7 days. CD4+ T cells were isolated from the spleens of wild-type (+/+), Stat6-deficient (Stat6−/−) and Runx3-transgenic mice, as indicated. Extracts were probed with an anti-pan-Runx Ab. Arrows, Runx1 and Runx3. In the third panel from the top, extracts were also probed with an anti-GATA3 Ab. β-Actin served as a loading control. Relative densities of Runx3 and GATA3 bands are indicated below the gel. The identity of the protein indicated by the asterisk is unknown; however, based on its apparent m.w., it might represent Runx2.

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Previously, we reported that the expression of Runx1 in Th2 cells attenuates the exacerbation of the Th2 response (17). To determine whether Runx3 functioned in a similar manner, CD4+ cells were cultured under conditions that promoted Th2 differentiation, and then infected by a retrovirus that was engineered to express Runx3. The overexpression of exogenous Runx3 in Th2 cells was confirmed by RT-PCR and immunoblot analysis (Fig. 2, A and B). The expression of Runx3 protein in infected cells was ∼3.5-fold higher than in control Th2 cells (Fig. 2,B, compare lanes 1 and 2). Fig. 1,C shows the results of the analysis of intracellular IFN-γ and IL-4 levels in Th2 cells by flow cytometry. In control, pMX-infected cells, 45% of the cells were IL-4 positive, whereas only 1% were IFN-γ positive. In cells that overexpressed Runx3, this ratio was reversed, and 56% of the cells were IFN-γ positive whereas 8% were IL-4 positive. These results indicated that the overexpression of Runx3 can activate IFN-γ expression and silence IL-4 expression, even under conditions that promote Th2 differentiation. The level of GATA3 was not affected by the overexpression of Runx3 (Fig. 2 B, compare lanes 1 and 2). Thus, the observed effect of Runx3 on IFN-γ and IL-4 expression was not likely to be mediated through the modulation of GATA3 expression (however, the T-bet transcript was induced by the overexpressed Runx3; supplemental Fig. 1).5

FIGURE 2.

Effect of Runx3 expression on the expression of GATA3, and the relative levels of IFN-γ and IL-4. A, Semiquantitative RT-PCR analysis of Runx3 and GATA3 transcripts. Naive CD4+ cells were isolated from the spleens of wild-type (WT) mice, stimulated with TCR, and then infected by a retrovirus carrying pMX-IRES-rCD2, pMX-Runx3-IRES-rCD2, or pMX-GATA3-IRES-rCD2, as indicated. After 7 days in culture under Th2 conditions, rat CD2-positive cells were isolated by cell sorting, and RNA was isolated and converted to cDNA. Increasing amounts of cDNA were used as the template for the detection of Runx3 and GATA3 transcripts by PCR. β-Actin was analyzed as a control. B, Immunoblot analysis of Runx1, Runx3, and GATA3 in Runx3- or GATA3-expressing cells. Lanes are as described for A, with the exception that α-tubulin was analyzed as a loading control. C, Naive CD4+ cells were isolated from the spleens of wild-type mice, stimulated with TCR, and infected by a retrovirus carrying pMX-IRES-GFP, pMX-Runx3-IRES-GFP, or pMX-Runx3/1–404-IRES-GFP, as indicated. After 7 days in culture under Th2 conditions, the cells were restimulated and analyzed by intracellular staining for IFN-γ and IL-4, followed by flow cytometry. The fluorescence intensity of IFN-γ and IL-4 in the GFP-positive gated cell population is displayed. The numbers represent the percentage of cells in each quadrant. D, Effect of reducing endogenous Runx activity on IL-4 and IFN-γ expression. Experimental conditions were similar to those in C, except that cells were infected by a retrovirus carrying pMX-Runt-IRES-GFP and pMX-Runx3/R178Q-IRES-GFP and that the Th2 bias was far less prominent than in C (compare pMX-infected cells in C and D).

FIGURE 2.

Effect of Runx3 expression on the expression of GATA3, and the relative levels of IFN-γ and IL-4. A, Semiquantitative RT-PCR analysis of Runx3 and GATA3 transcripts. Naive CD4+ cells were isolated from the spleens of wild-type (WT) mice, stimulated with TCR, and then infected by a retrovirus carrying pMX-IRES-rCD2, pMX-Runx3-IRES-rCD2, or pMX-GATA3-IRES-rCD2, as indicated. After 7 days in culture under Th2 conditions, rat CD2-positive cells were isolated by cell sorting, and RNA was isolated and converted to cDNA. Increasing amounts of cDNA were used as the template for the detection of Runx3 and GATA3 transcripts by PCR. β-Actin was analyzed as a control. B, Immunoblot analysis of Runx1, Runx3, and GATA3 in Runx3- or GATA3-expressing cells. Lanes are as described for A, with the exception that α-tubulin was analyzed as a loading control. C, Naive CD4+ cells were isolated from the spleens of wild-type mice, stimulated with TCR, and infected by a retrovirus carrying pMX-IRES-GFP, pMX-Runx3-IRES-GFP, or pMX-Runx3/1–404-IRES-GFP, as indicated. After 7 days in culture under Th2 conditions, the cells were restimulated and analyzed by intracellular staining for IFN-γ and IL-4, followed by flow cytometry. The fluorescence intensity of IFN-γ and IL-4 in the GFP-positive gated cell population is displayed. The numbers represent the percentage of cells in each quadrant. D, Effect of reducing endogenous Runx activity on IL-4 and IFN-γ expression. Experimental conditions were similar to those in C, except that cells were infected by a retrovirus carrying pMX-Runt-IRES-GFP and pMX-Runx3/R178Q-IRES-GFP and that the Th2 bias was far less prominent than in C (compare pMX-infected cells in C and D).

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The C-terminal five amino acid residues of Runx proteins comprise a characteristic VWRPY motif, with which a Groucho/TLE repressor interacts. To determine whether this motif contributed to Runx3-mediated IL-4 repression, we expressed a mutant of Runx3 in which this motif was deleted (Runx3/1–404). As seen in Fig. 2 C, 34% of cells that expressed Runx3/1–404 were IL-4 positive, whereas 7% were IFN-γ positive.

Endogenous Runx3 activity was then artificially reduced to see its effect on IL-4 and IFN-γ expression. In Fig. 2,D, CD4+ cells were cultured under Th2 conditions but with less efficacy (the ratio of IL-4+ to IFN-γ+ cells in pMX of Fig. 2,D was 2.1 as compared with 32 in pMX of Fig. 2 C) and were infected by a retrovirus carrying pMX-Runt-IRES-GFP and pMX-Runx3/R178Q-IRES-GFP. The Runt domain and Runx3/R178Q are known to function dominant negatively against endogenous Runx protein, thereby reducing Runx activity inside the cells (10, 20). Ratios of IL-4+ to IFN-γ+ cells were 8.0 for both Runt- and Runx3/R178Q-infected cells. Compared with pMX-infected control cells, reduction of Runx activity resulted in a significant increase of IL-4+ cells.

Runx1 has been shown to directly interact with GATA1 during megakaryocyte differentiation (23). As shown above, Runx3 was detected not only in Th1 but also in ThN and Th2 cells. To determine whether Runx3 interacted with GATA3, 293T cells were cotransfected with expression vectors for GATA3 and HA-tagged Runx3 and then subjected to immunoprecipitation using an anti-HA Ab, followed by immunoblot analysis using an anti-GATA3 Ab. As seen in Fig. 3, Runx3 and GATA3 coimmunoprecipitated with each other (Fig. 3,A, lane 4). The Runt domain of Runx1 is critical for its association with GATA1 (23). When we transfected cells with a mutant of Runx3 in which the Runt domain was deleted, Runx3 and GATA3 failed to coimmunoprecipitate (Fig. 3,B, lane 9). The Runx3 Runt domain alone, however, coimmunoprecipitated with GATA3 (Fig. 3 B, lane 10).

FIGURE 3.

Physical interaction of Runx3 and GATA3. A and B, 293T cells were transfected with the indicated expression vectors for GATA3 and HA-tagged Runx3 or Runx3 mutants (Runt domain-deleted Runx3 or the Runx3 Runt domain alone). Lysates were prepared and subjected to immunoprecipitation (IP) using anti-HA Ab-conjugated protein G-Sepharose, followed by immunoblot (IB) analysis using an anti-GATA3 Ab. Arrowhead, GATA3. C, Interaction of endogenous GATA3 and Runx3 proteins. CD4+ T cells were isolated from the spleen of wild-type mice, stimulated with TCR, and cultured under Th2 conditions for 7 days. The lysates were prepared and subjected to immunoprecipitation using anti-GATA3 mAb or control IgG. The precipitates were immunoblotted by anti-pan-Runx Ab. Arrowhead, Runx3.

FIGURE 3.

Physical interaction of Runx3 and GATA3. A and B, 293T cells were transfected with the indicated expression vectors for GATA3 and HA-tagged Runx3 or Runx3 mutants (Runt domain-deleted Runx3 or the Runx3 Runt domain alone). Lysates were prepared and subjected to immunoprecipitation (IP) using anti-HA Ab-conjugated protein G-Sepharose, followed by immunoblot (IB) analysis using an anti-GATA3 Ab. Arrowhead, GATA3. C, Interaction of endogenous GATA3 and Runx3 proteins. CD4+ T cells were isolated from the spleen of wild-type mice, stimulated with TCR, and cultured under Th2 conditions for 7 days. The lysates were prepared and subjected to immunoprecipitation using anti-GATA3 mAb or control IgG. The precipitates were immunoblotted by anti-pan-Runx Ab. Arrowhead, Runx3.

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Next, to determine whether endogenous GATA3 interacted with Runx3, cell lysates were prepared from CD4+ T cells that were cultured under Th2 conditions and subjected to immunoprecipitation using anti-GATA3 Ab (Fig. 3,C). Endogenous Runx3 coimmunoprecipitated together with endogenous GATA3 (see the band indicated by the arrowhead in Fig. 3 C, lane 13). These results indicated that Runx3 interacts with GATA3.

To examine the functional significance of the Runx3-GATA3 interaction, we conducted a luciferase reporter gene assay in which luciferase expression was driven by the IL-5 promoter. IL-5 is a well-characterized GATA3-responsive Th2-specific gene in reporter gene assays (24). A fragment of the IL-5 promoter spanning 1200 nt upstream of the transcription initiation site was ligated to the luciferase coding sequence (Fig. 4,A). The promoter fragment contained 2 Runx sites (at positions −1030 and −470 relative to the transcriptional start site) and 1 GATA site (−20 nt). M12 B cells were cotransfected with the IL-5-luciferase reporter construct and expression vectors for Runx3 and/or GATA3. As indicated in Fig. 4 B, the expression of GATA3 alone enhanced luciferase activity through the IL-5 promoter 4-fold as compared with mock, whereas Runx3 had only a marginal effect. However, coexpression of Runx3 and GATA3 completely attenuated the enhancement of luciferase activity by GATA3. Mutation of the GATA site of the 1200-nt IL-5 promoter fragment abolished its responsiveness to GATA3. A shorter fragment of the IL-5 promoter (460 nt), which did not contain either of the Runx sites, was similar to the longer 1200-nt promoter fragment in its responsiveness to GATA3 and/or Runx3.

FIGURE 4.

Effect of Runx3 and GATA3 on the activity of the IL-5 promoter. A, Schematic illustration of the luciferase (LUC) reporter plasmid, in which luciferase expression is driven by the murine IL-5 promoter. The 1200-nt region upstream of the transcriptional initiation site of IL-5 harbors 2 Runx sites (at nt −1030 and −470 relative to the transcriptional start site) and 1 GATA site (at nt −20). This 1200-nt fragment was ligated to a luciferase reporter gene. The IL-5 promoter in which the GATA site was mutated (mut), and a shorter 460-nt fragment of the IL-5 promoter were also ligated to the luciferase reporter gene. B, M12 cells were transfected with the indicated luciferase reporter plasmid together with pcDNA3, pcDNA3-GATA3, and/or pcDNA3-Runx3. Data are the means ± SD of three independent experiments. Statistically significant differences were determined by the t test and are indicated by brackets (p values are indicated for each panel). C, EMSA of GATA3 and Runx3 proteins to the IL-5 promoter sequence. GATA3, Runx3, and CBFβ proteins were synthesized from the respective cDNA in vitro, incubated with a radiolabeled oligonucleotide probe spanning the GATA-binding sequence of the IL-5 promoter, and processed to EMSA. Proteins were added as in the indicated combinations, whereas a cold probe means an excessive amount of nonlabeled oligonucleotide.

FIGURE 4.

Effect of Runx3 and GATA3 on the activity of the IL-5 promoter. A, Schematic illustration of the luciferase (LUC) reporter plasmid, in which luciferase expression is driven by the murine IL-5 promoter. The 1200-nt region upstream of the transcriptional initiation site of IL-5 harbors 2 Runx sites (at nt −1030 and −470 relative to the transcriptional start site) and 1 GATA site (at nt −20). This 1200-nt fragment was ligated to a luciferase reporter gene. The IL-5 promoter in which the GATA site was mutated (mut), and a shorter 460-nt fragment of the IL-5 promoter were also ligated to the luciferase reporter gene. B, M12 cells were transfected with the indicated luciferase reporter plasmid together with pcDNA3, pcDNA3-GATA3, and/or pcDNA3-Runx3. Data are the means ± SD of three independent experiments. Statistically significant differences were determined by the t test and are indicated by brackets (p values are indicated for each panel). C, EMSA of GATA3 and Runx3 proteins to the IL-5 promoter sequence. GATA3, Runx3, and CBFβ proteins were synthesized from the respective cDNA in vitro, incubated with a radiolabeled oligonucleotide probe spanning the GATA-binding sequence of the IL-5 promoter, and processed to EMSA. Proteins were added as in the indicated combinations, whereas a cold probe means an excessive amount of nonlabeled oligonucleotide.

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EMSA was then performed using a GATA binding sequence from the IL-5 promoter as a probe (Fig. 4,C). GATA3 and Runx3/CBFβ proteins were synthesized in vitro from the respective cDNAs and used. As expected, GATA3 but not Runx3 bound to the IL-5 promoter (Fig. 4,C, lanes 2 and 3). Coexistence of Runx3/CBFβ abolished DNA binding activity of GATA3 in a dose dependent manner (Fig. 4 C, lanes 4 and 5). These results indicated that Runx3 can interact with GATA3 and attenuate GATA3 transcriptional activity by inhibiting DNA-binding activity of GATA3. Thus, the mechanism of action of Runx3 and GATA3 is quite distinct from that of Runx3 and T-bet, in which the two factors interact with each other and augment the transcriptional activity of their binding partner (18).

To determine the effect of modulating the relative amounts of Runx3 and GATA3 on IFN-γ and IL-4 expression, isolated splenic CD4+ cells were stimulated with TCR and cultured under nonskewed conditions. The cells were then doubly infected by two retroviruses (Fig. 5), one encoding bicistronic Runx3 and GFP and the second encoding GATA3 and rat CD2. GFP and rat CD2 were used as markers for monitoring Runx3 and GATA3 expression, respectively. In noninfected cells, the IFN-γ- and IL-4-positive fractions were present in a 1:1 ratio (11 and 10%, respectively; Fig. 5, lower left). The overexpression of GATA3 decreased the ratio of IFN-γ and IL-4 to 0.4 (9 and 23%, respectively; upper left), whereas the overexpression of Runx3 increased the ratio of IFN-γ and IL-4 to 4.2 (23 and 5%, respectively; lower right). In doubly infected cells, the ratio of IFN-γ to IL-4 reverted to 1.1 (17 and 15%, respectively; Fig. 5, upper right). These results indicated that in TCR-stimulated cells, Runx3 and GATA3 can attenuate each other’s effect on the expression of IFN-γ and IL-4 (See supplemental Fig. 2 in which similar experiments as Fig. 5 was performed but under Th1 and Th2 conditions as well. Mutual countering effects of Runx3 and GATA3 were confirmed again.).

FIGURE 5.

Effect of coexpression of Runx3 and GATA3 on IFN-γ and IL-4 expression. Naive CD4+ cells were isolated from the spleens of wild-type mice, stimulated with TCR, and then doubly infected by retroviruses carrying pMX-Runx3-IRES-GFP and pMX-GATA3-IRES-ratCD2. After 7 days in culture under nonskewing conditions, the cells were restimulated and then analyzed by intracellular staining for IFN-γ and IL-4, followed by flow cytometry. The cells were gated into four subpopulations based on GFP- and rat CD2-specific fluorescence (left): GFP-negative/rat CD2-negative (bottom left); GFP-positive/rat CD2-negative (bottom right); GFP-negative/rat CD2-positive (top left); and GFP-positive/rat CD2-positive (top right). The fluorescence intensity of IFN-γ and IL-4 is shown for each subpopulation. Numbers represent the percentages of cells in each quadrant.

FIGURE 5.

Effect of coexpression of Runx3 and GATA3 on IFN-γ and IL-4 expression. Naive CD4+ cells were isolated from the spleens of wild-type mice, stimulated with TCR, and then doubly infected by retroviruses carrying pMX-Runx3-IRES-GFP and pMX-GATA3-IRES-ratCD2. After 7 days in culture under nonskewing conditions, the cells were restimulated and then analyzed by intracellular staining for IFN-γ and IL-4, followed by flow cytometry. The cells were gated into four subpopulations based on GFP- and rat CD2-specific fluorescence (left): GFP-negative/rat CD2-negative (bottom left); GFP-positive/rat CD2-negative (bottom right); GFP-negative/rat CD2-positive (top left); and GFP-positive/rat CD2-positive (top right). The fluorescence intensity of IFN-γ and IL-4 is shown for each subpopulation. Numbers represent the percentages of cells in each quadrant.

Close modal

To determine whether the effects we were observing in vitro in cultured CD4+ cells were also true in vivo, we analyzed cells from mice that expressed an Lck-driven Runx3 transgene (15). Fig. 1 shows the Runx expression profile of isolated Runx3-transgenic CD4+ cells that were stimulated through the TCR with anti-TCRβ Abs. The level of Runx3 in transgenic ThN cells was 1.5-fold higher than that in nontransgenic ThN cells and rather equivalent to that in nontransgenic Th1 cells (Fig. 1, compare lanes 7, 8, and 10). To determine whether Runx3-transgenic cells under nonskewed conditions also mimicked nontransgenic Th1 cells in terms of IFN-γ and IL-4 expression, we analyzed intracellular IFN-γ and IL-4 levels by flow cytometry. As seen in Fig. 6 A, under nonskewed conditions, Runx3-transgenic cells exhibited enhanced IFN-γ expression (73%) and marked repression of IL-4 expression (2%) as compared with nontransgenic cells (Transcription of Th2-type cytokines including IL-4, IL-5, and IL-13 were substantially reduced in Runx3-transgenic ThN cells. See supplemental Fig. 3.).

FIGURE 6.

Effect of transgenic (tg) Runx3 expression on IFN-γ and IL-4 expression. CD4+ cells were isolated from the spleens of nontransgenic (non-tg) and Runx3-transgenic mice, stimulated with TCR, and then infected by a retrovirus carrying pMX-GFP or pMX-GATA3-IRES-GFP. After 7 days in culture under nonskewing (A) or Th2 (B) conditions, the cells were re-stimulated and then analyzed by intracellular staining for IFN-γ and IL-4, followed by flow cytometry. The fluorescence intensity of IFN-γ and IL-4 in the GFP-positive population is displayed. Numbers represent the percentages of cells in each quadrant. The GATA3:Runx3 protein ratio in Th2-conditioned Runx3-transgenic cells was >1.0 (actually, it was 0.7:0.3 = 2.3 in Fig. 1, lane 12) as in the case of Th2-conditioned nontransgenic cells (1.7:0.5 = 3.4 in Fig. 1, lane 9). Therefore, it may not be unreasonable that Th2-conditioned Runx3-transgenic cells showed a Th2-like phenotype (B). In contrast, in Fig. 2, retrovirally introduced Runx3 overexpressing cells for which the Runx3:GATA3 ratio was 3.5:1.0 = 3.5 exhibited a Th1 phenotype even under a Th2 condition.

FIGURE 6.

Effect of transgenic (tg) Runx3 expression on IFN-γ and IL-4 expression. CD4+ cells were isolated from the spleens of nontransgenic (non-tg) and Runx3-transgenic mice, stimulated with TCR, and then infected by a retrovirus carrying pMX-GFP or pMX-GATA3-IRES-GFP. After 7 days in culture under nonskewing (A) or Th2 (B) conditions, the cells were re-stimulated and then analyzed by intracellular staining for IFN-γ and IL-4, followed by flow cytometry. The fluorescence intensity of IFN-γ and IL-4 in the GFP-positive population is displayed. Numbers represent the percentages of cells in each quadrant. The GATA3:Runx3 protein ratio in Th2-conditioned Runx3-transgenic cells was >1.0 (actually, it was 0.7:0.3 = 2.3 in Fig. 1, lane 12) as in the case of Th2-conditioned nontransgenic cells (1.7:0.5 = 3.4 in Fig. 1, lane 9). Therefore, it may not be unreasonable that Th2-conditioned Runx3-transgenic cells showed a Th2-like phenotype (B). In contrast, in Fig. 2, retrovirally introduced Runx3 overexpressing cells for which the Runx3:GATA3 ratio was 3.5:1.0 = 3.5 exhibited a Th1 phenotype even under a Th2 condition.

Close modal

We next examined whether the magnitude of IFN-γ and IL-4 expression in Runx3-transgenic cells was also responsive to the balance of Runx3 and GATA3, as observed in wild-type CD4+ cells. The introduction of GATA3 into Runx3-transgenic cells resulted in a decrease in the percentage of IFN-γ-positive cells (37%), and an increase in the percentage of IL-4-positive cells (16%) as compared with the nonintroduction of GATA3 (Fig. 6,A). However, Runx3-transgenic cells remained to exhibit a Th2-like expression profile of IFN-γ and IL-4 even when cultured under Th2-permissive conditions (Fig. 6,B; there was no virus infection in this panel). The result in Fig. 6,B was in agreement with the relative expression levels of Runx3 and GATA3 proteins as detected in Fig. 1 (see the legend of Fig. 6 for detailed discussion on the Runx3:GATA3 or GATA3:Runx3 ratios).

These results collectively indicated that transgenic expression of Runx3 can activate IFN-γ expression and silence IL-4 expression, similar to its effects in vitro in cultured CD4+ cells.

When CD4+ cells were isolated from Runx3-transgenic spleens and analyzed immediately by immunoblot, an observed Runx expression profile was the same as that seen in Fig. 1, lane 10 (data not shown). This suggests that Runx3-transgenic naive CD4+ cells have already acquired a Th1 phenotype. To test this hypothesis, we compared the expression profiles of various Th1- and Th2-specific genes in naive CD4+ cells that were isolated from Runx3-transgenic and nontransgenic mice by semiquantitative RT-PCR analysis (Fig. 7). As a control, we also examined gene expression in CD8+ cells in parallel. As compared with nontransgenic cells, Runx3-transgenic cells expressed an excessive amount of Runx3 transcript, however, the levels of T-bet and GATA3 transcripts were unaffected. The levels of several Th1-type cytokine transcripts (IFN-γ, ΤΝFα, IL-12, and IL-18) were uniformly increased in Runx3-transgenic cells. In contrast, the levels of Th2-type cytokine transcripts (IL-4 and IL-13) were decreased in transgenic cells. These results indicated that Runx3-transgenic CD4+ cells are prone to a Th1 phenotype, even in the absence of TCR-stimulation.

FIGURE 7.

Cytokine expression profiles in naive Runx3-transgenic (tg) CD4+ cells. RNA was prepared from naive CD4+ and CD8+ cells from nontransgenic (non-tg) and Runx3-transgenic mouse spleens. Increasing amounts of cDNA were used for semiquantitative RT-PCR of cytokine gene transcripts under nonsaturating conditions. β-Actin was analyzed as a control.

FIGURE 7.

Cytokine expression profiles in naive Runx3-transgenic (tg) CD4+ cells. RNA was prepared from naive CD4+ and CD8+ cells from nontransgenic (non-tg) and Runx3-transgenic mouse spleens. Increasing amounts of cDNA were used for semiquantitative RT-PCR of cytokine gene transcripts under nonsaturating conditions. β-Actin was analyzed as a control.

Close modal

We next examined the Th1- and Th2-mediated immunological responses of Runx3-transgenic mice. Mice were immunized with OVA mixed with CFA, and serum Ab titers were measured (Fig. 8,A). After a second boosting injection (day 28), the titers of IgG2a and IgG2b were significantly higher in Runx3-transgenic mice than in nontransgenic mice. IgG2a and IgG2b are the Ig subclasses the production of which depends on a Th1 response. We also examined mice that were immunized with OVA mixed with aluminum hydroxide (Fig. 8 B). OVA mixed with aluminum hydroxide is a form of Ag that tends to trigger the Th2-dependent production of IgG1 and IgE. However, the production of these two subclasses was poor in Runx3-transgenic mice as compared with nontransgenic mice. These results demonstrated that Runx3-transgenic mice respond efficiently in a Th1-dependent manner, but that their Th2-mediated response is poor.

FIGURE 8.

Ig titers in the sera of immunized Runx3-transgenic (tg) mice. Nontransgenic (non-tg) and Runx3-transgenic mice were injected s.c. or i.p. with OVA mixed with CFA (A) or aluminum hydroxide (B), respectively. After the second boosting injection, sera were collected, and the titers of IgG1, IgE, IgG2a, and IgG2b were measured by ELISA. Horizontal lines represent the average OD450 for each group. A statistically significant difference was detected between nontransgenic and Runx3-transgenic mice (p values are indicated).

FIGURE 8.

Ig titers in the sera of immunized Runx3-transgenic (tg) mice. Nontransgenic (non-tg) and Runx3-transgenic mice were injected s.c. or i.p. with OVA mixed with CFA (A) or aluminum hydroxide (B), respectively. After the second boosting injection, sera were collected, and the titers of IgG1, IgE, IgG2a, and IgG2b were measured by ELISA. Horizontal lines represent the average OD450 for each group. A statistically significant difference was detected between nontransgenic and Runx3-transgenic mice (p values are indicated).

Close modal

During immunization, we noticed a striking feature of Runx3-transgenic mice. When spleens were isolated after a primary challenge of OVA-aluminum, the weight of the tissue was on average 135 mg in nontransgenic mice (Fig. 9, A and B), whereas the spleen weight in Runx3-transgenic mice was 80 mg. This corresponded to ∼60% of weight of the nontransgenic spleens. We prepared histological sections of spleens from transgenic and nontransgenic mice, and stained them using peanut agglutinin (Fig. 9 C). In nontransgenic spleens, a number of lectin-stained clusters were detected, but these clusters were scarcely detectable in Runx3-transgenic spleens. Because peanut agglutinin stains B cells in activated germinal centers, these results suggested that B cells were not sufficiently activated in Runx3-transgenic spleens following immunization.

FIGURE 9.

Impaired activation of B cells in Runx3-transgenic (tg) mice. A, Nontransgenic (non-tg) and Runx3-transgenic mice were immunized using OVA mixed with aluminum hydroxide, as described for Fig. 8. Animals were sacrificed after the second boosting injection, and the weight of the spleens was measured. Horizontal lines represent the averages of each group. B, Representative images of spleens. Bar, 1 cm. C, Nontransgenic and Runx3-transgenic mice were immunized once with OVA mixed with aluminum hydroxide, and the spleen were removed 1 wk after immunization. Spleen tissue sections were stained using HRP-conjugated peanut agglutinin (brown color).

FIGURE 9.

Impaired activation of B cells in Runx3-transgenic (tg) mice. A, Nontransgenic (non-tg) and Runx3-transgenic mice were immunized using OVA mixed with aluminum hydroxide, as described for Fig. 8. Animals were sacrificed after the second boosting injection, and the weight of the spleens was measured. Horizontal lines represent the averages of each group. B, Representative images of spleens. Bar, 1 cm. C, Nontransgenic and Runx3-transgenic mice were immunized once with OVA mixed with aluminum hydroxide, and the spleen were removed 1 wk after immunization. Spleen tissue sections were stained using HRP-conjugated peanut agglutinin (brown color).

Close modal

Runx3 was originally identified as a critical transcription factor in CD8 single-positive thymocyte differentiation that induced CD8 and repressed CD4 gene expression (14, 15, 16, 25). Recently, a novel function of Runx3 as a feed-forward regulator of T-bet during Th1 cell differentiation of peripheral CD4+ Th cells was described (18). Runx3 is barely detectable in naive CD4+ T cells, but its expression is markedly up-regulated under Th1-skewing conditions. Runx3 induction is dependent on T-bet, and T-bet and Runx3 form a regulatory complex that activates the IFN-γ promoter and suppresses IL-4 expression, at least in part, by binding to an IL-4 silencer. In agreement with this novel function of Runx3, the derepression of IL-4 expression is observed in Th1 cells that are deficient in CBFβ (26), which has been shown to be necessary for Runx function (27).

Under nonskewed culture conditions, TCR-stimulated CD4+ cells express both T-bet and GATA3. The cells are more or less differentiated along a Th1- or Th2-specific lineage, and the two subpopulations are maintained in a kind of equilibrium. As for GATA3, it is detected even under Th1 conditions, although to a substantially lower extent than Th2. Here, we demonstrated that Runx3 physically interacts with GATA3, and suppresses the transcriptional activity of GATA3. Thus, under nonskewed as well as Th1 conditions, Runx3 appears to negatively regulate Th2 cytokine expression by antagonizing GATA3 activity, contributing to the augmentation or stabilization of Th1 responses. In agreement with this hypothesis, double infection of nonskewed CD4+ cells with retroviruses that expressed Runx3 and GATA3 polarized the cells toward IFN-γ-only or IL-4-only expression, depending on the balance of the two factors. We also demonstrated that Runx3-transgenic CD4+ cells were prone to the Th1 phenotype, even in the absence of TCR stimulation. However, Runx3-transgenic cells were still able to differentiate along either lineage, given that the introduction of GATA3 or culturing of cells under Th2 conditions could redirect the cells to be IL-4 producers. In Runx3-transgenic cells, the protein levels of Runx3 and GATA3 were 1.5-fold higher and 0.7-fold lower, respectively, than in nontransgenic cells. In contrast, in cells in which Runx3 was ectopically overexpressed up to 3.5-fold over wild-type cells, culturing under Th2 conditions failed to restore IL-4 production, which suggests that this level of Runx3 might be above a certain threshold level that can be neutralized by IL-4-induced GATA3. We propose that one mechanism of regulating the Th1/Th2 preference of TCR-stimulated cells involves the balance of Runx3 and GATA3.

In one sense, the role of Runx3 resembles that of T-bet, which represses IL-4 expression through a direct association with GATA3 and inhibition of the DNA binding property of GATA3 (9). We demonstrated that Runx3/1–404, a Runx3 mutant that is devoid of the Groucho-binding domain, failed to repress IL-4 expression. Groucho/TLE is an evolutionarily conserved corepressor of Runx family proteins, and functions by recruiting histone deacetylases (28, 29, 30). Our results suggest that the repression of IL-4 expression by the GATA3/Runx3 complex may be mediated through the recruitment of histone deacetylase activity by Groucho.

In nontransgenic Th1 cells, Runx3 was the principal Runx component, and Runx1 was barely detectable. Similarly, naive CD4+ cells isolated from Runx3-transgenic mice contained only a trace amount of Runx1. The low level of Runx1 seen in these cells probably reflects the transcriptional repression of the Runx1 promoter by an excess of the Runx3 transcription factor (31). In contrast, in nontransgenic Th2 cells, equivalent amounts of Runx3 and Runx1 were present. Runx3 in this cell population might prevent the exacerbation of the Th2 response, as was previously suggested for Runx1. We actually obtained results that are in agreement with this notion. When endogenous Runx activity was reduced by introducing dominant negative forms of Runx3, relative expression ratios of IL-4 to IFN-γ under Th2 conditions were substantially enhanced. Runx1 also interacted with GATA3, similar to Runx3 (data not shown), which suggests that the Runx1-GATA3 complex likely exerts a similar biological effect as the Runx3-GATA3 complex.

T cell-specific Runx3-deficient mice spontaneously develop asthma-related symptoms, including elevated serum IgE, which is a hallmark of a Th2 bias (26, 32). We showed that Runx3-transgenic mice exhibit a Th1-biased phenotype, including elevated titers of serum IgG2a and IgG2b following immunization. These results support the hypothesis that Runx3 is a critical regulator of Th1 and Th2 responses in vivo. A rather unexpected observation in the present study was that B cells failed to respond quickly to immunization in Runx3-transgenic mice, as revealed by the poor staining of B cells by peanut agglutinin in Runx3-transgenic spleens. Impairment of B cell activation may be due either to the reduction of IL-4 production or an insufficiency of follicular T cells, both of which are crucial factors for B cell activation (33).

We thank Dr. S. Akira for Stat6-targeted mice, Drs. K. Ito and Y. Ito for Runx3/R178Q plasmid, and M. Kuji for secretarial assistance.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported in part by research grants from the Ministry of Education, Science, Sports, Culture and Technology, Japan. M.S. is a participant in the Global COE Program Network Medicine at Tohoku University.

4

Abbreviations used in this paper: T-bet, T-box family transcription factor; HA, hemagglutinin; Runx, Runt-related transcription factor; ThN, Th cells under nonskewed conditions.

5

The online version of this article contains supplemental material.

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