The IL-4 production capability of different strains of naive CD4+ T cells controls the direction of the Th cell response
Ryoji Yagi1,4,
Wataru Suzuki1,
Noriyasu Seki1,
Masako Kohyama2,
Tadahiro Inoue3,
Takao Arai4 and
Masato Kubo1
1 Divisions of Immunobiology and
2 Biotechnology, Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda City, Chiba 278-0022, Japan
3 Exploratory Research Group, Sumitomo Pharmaceuticals Research Center, Takarazuka City, Hyougo 665-0051, Japan
4 Department of Applied Biological Science, Science University of Tokyo, Noda City, Chiba 278-8510, Japan
Correspondence to:
M. Kubo
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Abstract
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The qualitative nature of an immune response raised against infectious pathogens depends upon the phenotypes of Th cell subsets, which secrete distinct types of cytokines. Genetic background is known to greatly influence the nature of the Th cell response. However, the precise nature of this influence still remains unclear. In the present study, we demonstrate that CD62L+, CD44low and CD4+ naive T cells from BALB/c mice are capable of producing significant amounts of IL-4, while naive T cells from B10.D2 mice exhibit no IL-4 production. The addition of exogenous IL-4 into the B10.D2 induction culture recovered Th2 development, thereby indicating that the potential of naive T cells to secrete IL-4 at primary activation is likely to substantially influence development of Th2. Regulation of the IL-4 gene in naive T cells differs from that in cells committed towards becoming Th2 cells, based on the observation that naive T cells from STAT6-deficient mice having a BALB/c background produce detectable amounts of IL-4. The IL-4 promoter region was found to be equally histone acetylated in both BALB/c and B10.D2 naive T cells by primary TCR activation. Interestingly, the expression levels of transcription factors NF-AT and GATA-3, which regulate promoter activity, differ between BALB/c and B10.D2 cells. These results suggest that the differences in expression level between the two transcriptional factors may affect the potential of naive T cells to secrete IL-4, which may subsequently influence the development of Th cell phenotypes.
Keywords: IL-4, naive T cell, NF-AT, strain difference, Th2
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Introduction
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Host defense against infectious pathogens requires the differentiation of naive CD4+ T cells to effector Th cells that secrete cytokines coordinating appropriate immune responses. The Th cells exhibit a characteristic cytokine secretion pattern that can divide them into at least two distinct subsets (14). Th1 cells produce IL-2, IFN-
and lymphotoxin, controlling cell-mediated effector responses resulting in the elimination of intracellular pathogens. Th2 cells produce IL-4, IL-5, IL-6, IL-10 and IL-13, which regulate humoral immune response against extracellular pathogens, such as helminths. A predominance of IL-4 and IL-5 production characterizes allergic responses via mast cells and eosinophils. The type of immune responses observed may be dictated by a number of contextual variable including; antigen dose, the route of invading pathogens, type of antigen-presenting cells (APC), the nature of co-stimulatory signals and the specific cytokines present in the developmental microenvironment (35).
The most well-defined factors mediating the Th cell development are IL-12 and IL-4. IL-12 secreted from the activated macrophage and dendritic cells plays an important role in Th1 development, at least after certain types of antigenic stimulation (69). IL-4 is a crucial for generating Th2 cells in various experimental systems (1012). The initial source of IL-4 is still subject to debate, although accumulating evidence shows that naive CD4+ T cells themselves directly contribute to supplement IL-4 (1317). However, the amount of IL-4 secreted from naive CD4+ T cells is insufficient by itself for the development of Th2 cells. The enhanced expression levels of IL-4 receptor and altered phosphorylation status by TCR and CD28 co-stimulation were also required for differentiation (18,19). Given the prominent role of primary IL-4, the regulation of their expression in naive CD4+ T cells is worthy of study.
Numerous studies indicate that genetic background is another important determinant influencing the type of immune responses. In genetic mapping of the human locus, phenotypic markers of atopic immune symptom, total serum IgE level and airway hyper-reactivity are linked genetically to a locus on the long arm of human chromosome 5 assembling many Th2 cytokine genes, e.g. IL-4, IL-13 and IL-5 (16,20,21). The importance of genetic background is also exemplified by the immune response against Leishmania major infection (2224) and against soluble antigen stimulation, such as ovalbumin (OVA), in mouse experimental system (25). Murphy and co-workers have accumulated the evidences demonstrating that a loss of functional IL-12 receptor (IL-12R) was implicated in the phenotype of Th cell development using TCR
ß transgenic (Tg) mouse (25). Interestingly, the status of IL-12R expression is mapped genetically to a locus on mouse chromosome 11 corresponding to the Th2 cytokine locus on human chromosome 5 (26). However, mouse genetic mapping of the appearance of IL-4-producing effector T cells has shown strong association with chromosome 7 and 16 (16). Therefore, the locus determining the phenotype of Th cell development remains controversial. Those previous observations suggest that the strain difference is not genetically controlled by a simple monogenic trait, which implicates several number of genes.
To identify the genetic factors controlling the strain-specific differences in Th cell development, we examined the functional relevance of naive T cell and APC using OVA-specific TCR
ß transgenic mice (DO11.10 Tg) having distinct genetic backgrounds, e.g. BALB/c and B10.D2. In this study, we demonstrate that naive CD4+ T cells, acting as a precursor of cytokine-producing Th cells, play an intrinsic role determining their own fate in secondary responses. The ability of naive T cells to produce IL-4 appeared to determine the phenotype of Th cell development among different strains of mice. Expression and nuclear localization pattern in NF-AT1 and 2, GATA-3 and c-Maf were investigated in both strains of mice. In BALB/c T cells, enhancement of NF-AT2 and GATA-3 expression was more prominent than those found in the T cells of B10.D2 mice. Our results revealed that primary TCR activation of naive T cells resulted in the induction of NF-AT2 expression as well as IL-4 production irrespective of the IL-4-mediated signaling. In contrast, the presence of IL-4 during primary activation was essential for induction of GATA-3 expression. These findings have important implications for the views regarding the molecular mechanisms how NF-AT regulates primary IL-4 production of naive T cells during the initial activation process.
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Methods
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Animals
BALB/c and B10.D2 mice were purchased from Clea (Tokyo, Japan). DO11.10 Tg with a BALB/c background was kindly distributed by Dr Kenneth Murphy (Washington University School of Medicine, St Louis, MO). The Tg mice have been backcrossed for six generations to B10.D2, generating DO 11.10 Tg with a B10.D2 background. STAT6-deficient mice having a C57BL/6 background were a gift from Dr Shizuo Akira (Osaka University, Osaka, Japan) (27). These mice have been backcrossed to BALB/c and B10.D2 for four generations, generating STAT6 knockout mice having a BALB/c and B10.D2 background respectively.
Cytokines and antibodies
The reagents for ELISA and intracellular cytokine staining, anti-IL-2 (JES6-1A12 and JES6-5H4)biotin, anti-IFN-
(R4-6A2 and XMG1.2)biotin), anti-IL-4 (BVD4-1D11 and BVD6-24G2)biotin, anti-IFN-
(XMG1.2)FITC and anti-IL-4 (11B11)phycoerythrin, were purchased from PharMingen (San Diego, CA). Anti-CD28 mAb (PV-1) was previously described (18). Mouse recombinant IL-4 was purchased from PeproTech (London, UK). The antibodies for Western blotting, anti-NFAT1 mAb (4G6-G5), anti-GATA-3 mAb (HG3-35), anti-c-Maf rabbit antiserum (M-153) and anti-OCT-1 mAb (C-1), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-NF-AT2 mAb (7A6) and anti-STAT6 mAb (clone 38) were purchased from Alexis (San Diego, CA) and Transduction Laboratories (Lexington, Kentucky)
Preparation of naive CD4+ T cells and induction of cytokine-producing effector T cells
Spleen cells were incubated with anti-CD8 mAb (3.155) at 4°C and then the cells were placed on plates coated with anti-mouse Ig (Cappel, Aurora, OH) to eliminate B and CD8+ T cells. This enrichment resulted in >80% CD4+T cells. The naive T cell population of CD4+CD62LhighCD44low cells was isolated by a FACS Vantage cell sorter (Becton Dickinson), yielding >95% purity. APC were prepared from spleen cells by the cytotoxic killing treatment with anti-Thy-1.2 mAb (30H12) and Low-Tox-M rabbit complement (Cederlane, Hornby, Ontario, Canada), and irradiated with 3000 rad before use.
The CD4+-enriched T cells from DO11.10 mice (1x106 cells/ml) were stimulated with OVA antigenic peptide (residues 323339; ISQAVHAAHAEINEAGR; BEX, Tokyo, Japan) in the presence of APC from syngeneic stains of mouse. Concanavalin A stimulation was performed at the dose of 5 µg/ml in the presence of APC from syngeneic stains of mice. The naive T cell population, CD4+CD62LhighCD44low cells (1x106 cells/ ml), was stimulated with plate-bound anti-TCR (H57-597) mAb (30µg/ml) plus the soluble form of anti-CD28 mAb (PV-1) (5 µg/ml). After 5 days, viable cells were collected and subjected to intracellular cytokine staining after stimulation with plate-bound anti-TCR mAb.
Intracellular cytokine staining
The activated CD4+ T cells were re-stimulated with anti-TCR mAb for 6 h in the presence of 2 µM monensin (Sigma, St Louis, MO) to prevent release of cytokines. Then, the cells were fixed with 4% paraformaldehyde and permeabilized with permeabilizing solution (50 mM NaCl, 5 mM EDTA and 0.02% NaN3, pH 7.5) containing 0.5% Triton X. After blocking with PBS containing 3% BSA, cells were stained with anti-IFN-
(XMG1.2)FITC and anti-IL-4 (11B11)phycoerythrin, as described previously (18). Flow cytometric analysis was performed on a FACSort and analyzed by CellQuest software (Becton Dickinson, San Jose, CA).
Measurement of cytokine concentrations by ELISA
The naive T cell population of CD4+CD62LhighCD44low cells (1x106 cells/ ml) was stimulated with plate-bound anti-TCR plus anti-CD28 mAb. After different time points, the culture supernatants were harvested, and the concentration of IL-2, IL-4 and IFN-
by analyzed ELISA. Briefly, the supernatants were applied on plastic plates coated with specific antibody for the cytokines. After washing, the plate was probed with the biotinylated specific antibody for the cytokines and horseradish peroxidase-conjugated streptavidin (Zymed, San Francisco, CA), and developed with 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (Kirkegard & Perry, Gaithersburg, MD). The absorbance at 405 nm was measured by a spectrophotometer (BioRad, Hercules, CA).
Separation of nuclear fraction and Western blot analysis
The CD4+-enriched T cells (3x106 cells) were stimulated with anti-TCR plus anti-CD28 mAb for 1248 h, and nuclear and cytoplasmic fractions were separated by NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL). The nuclear and cytoplasmic proteins (25 µg) were loaded on 6.512% SDSPAGE and then transferred to PVDF membranes by electroblotting. The blots were visualized with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit Ig (Dako, Glostrup, Denmark).
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Results
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The genetic background of naive T cells determines the development of Th cell phenotypes
Genetic background, apart from MHC, influences the direction of Th cell development (28). Indeed, DO11.10 TCR
ß transgenic mice (DO11.10 Tg) with BALB/c and B10.D2 genetic backgrounds were shown to have distinct differentiation profiles, despite the fact that Th cells were induced under identical TCR, antigen and MHC restriction circumstances. CD4+ T cells from BALB/c and B10.D2 mice developed into Th2 and Th1 cells respectively (Fig. 1A
). To exclude the possibility of influence from cytokines secreted from macrophages, naive CD4+ T cells from DO11.10 Tg and T cell-depleted splenic irradiated APC were exchanged between BALB/c and B10.D2 mice. As illustrated in Fig. 1(B)
, Th2 cells were only generated when T cells originated from BALB/c mice, suggesting that the genetic background of naive T cells, rather than the APC, contributed to the strain-specific differences in the appearance of IL-4-producing effector T cells.

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Fig. 1. Genetic background of the CD4+ T cells determined the strain difference in the Th cell differentiation profile. (A) Strain difference in the Th cell differentiation profile induced by antigen stimulation. CD4+CD62LhighCD44low T cells from DO11.10 Tg mice in either a BALB/c or B10.D2 background were stimulated with OVA peptide (1 µM) and irradiated T cell-depleted splenic APC. After 6 days, the primed CD4+ T cells were re-stimulated with the anti-TCR mAb for 6 h in the presence of 2 µM monensin, and stained for intracellular IL-4 and IFN- . (B) Genetic background of CD4+ T cells determined the nature of the Th cells. CD4+ CD62LhighCD44Iow T cells and APC were exchanged between BALB/c and B10.D2 mice, and stimulated with OVA peptide (1 µM). After 6 days, generation of Th1 and Th2 cells was assessed as described in (A). (C) The stimulation of CD4+CD62LhighCD44Iow T cells from BALB/c mice with anti-TCR/CD28 mAb resulted in the preferential Th2 development without APC. CD4+CD62LhighCD44low T cells from BALB/c and B10.D2 mice were stimulated with immobilized anti-TCR mAb (30 µg/ml) and soluble anti-CD28 mAb (1 µg/ml) in the presence or absence of exogenous IL-4 (10 ng/ml). After 6 days, generation of Th1 and Th2 cells was assessed as described in (A).
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To further rule out any effect from APC, purified CD4+CD62LhighCD44low T cells were stimulated with anti-TCR plus CD28 mAb in the absence of APC, in order to generate Th cells. The profile of Th cell differentiation was found to be very similar to that noted above. Again BALB/c and B10.D2 cells induced Th2 and Th1 development respectively (Fig. 1C
). This supported our hypothesis that the genetic background of the naive T cell itself was an important factor in determining differentiation.
The addition of IL-4 to B10.D2 T cells markedly changed the profile from Th1 to Th2, indicating that B10.D2 mice contain substantial numbers of precursor cells capable of generating Th2 cells (Fig. 1C
, bottom). In contrast, blockade of IL-4 function was found to completely abolish Th2 development in BALB/c mice (data not shown). These results would indicate that IL-4 secretion from naive T cells is critical for the generation of Th2 cells in the subsequent Th cell differentiation process.
Strain-specific differences in IL-4 production in naive T cells at initial activation.
A minor subset of the activated/memory phenotype is thought to be a major source of IL-4. Thus, the CD4+CD62LhighCD44low population was sorted as naive T cells from DO11.10 Tg mice with both BALB/c and B10.D2 genetic backgrounds. These T cells were stimulated with antigenic peptide in the presence of irradiated syngeneic APC, and the production of IL-2, IL-4 and IFN-
measured at various time points. The naive T cells from BALB/c mice showed readily detectable amounts of IL-4, and production increased gradually in a dose-dependent manner (Fig. 2A
). Moreover, stimulation with anti-TCR plus anti-CD28 mAb promoted significant increases in IL-4 at 48 h after primary activation (Fig. 2B
). In contrast, the naive T cells from B10.D2 mice showed very low levels of IL-4 production following TCR stimulation by antigen or by anti-TCR plus CD28 mAb (Fig. 2A and B
). However, this was not due simply to unresponsiveness against antigenic stimulation, because IL-2 production from B10.D2 T cells was found to be even higher than that from BALB/c T cells (Fig. 2A
, left).
Next we studied whether the strain-specific difference in primary IL-4 production could be due to the number of secreting cells or to the amount of IL-4 secreted from a single T cell. The numbers of IL-4-secreting cells were analyzed by intracellular IL-4 staining 48 h after TCR stimulation. Spleen cells from BALB/c mice were found to contain more CD4+ T cells secreting IL-4 when compared to those from B10.D2 mice (Fig. 2C
). Moreover, single BALB/c T cells produced 3 times more IL-4 than single B10.D2 T cells (Fig. 2C
). These results indicate that naive T cells from BALB/c mice were superior to those from B10.D2 mice, both in terms of the amount of IL-4 secreted per T cell and the number of cells secreting IL-4 at primary activation. It is worth noting that these observations of correlation between primary IL-4 production and Th2 development are not restricted to BALB/c and B10.D2 cells. We extended the same studies to C3H and B10.BR T cells, which favor the generation of Th2 and Th1 cells respectively. Therefore, the potential of naive T cells to secrete IL-4 could be an important genetic factor regulating the strain-specific differences observed in Th2 differentiation.
In order to analyze the genetic correlation between primary IL-4 production and the generation of Th2 cells, CD4+ T cells from (BALB/cxB10.D2)F1 mice were compared to those from parental strains. T cells from all four F1 mice showed a BALB/c phenotype (Fig. 3
, left). F1 mice were back-crossed to either F1 or parental strains. Of the eight F1xF1 mice analyzed, seven were found to have a BALB/c phenotype and the other, a B10.D2 phenotype. All five F1xBALB/c crosses showed a BALB/c phenotype, whilst four out of five F1xB10.D2 crosses had B10.D2 phenotypes (Fig. 3
, middle and right). These results demonstrate a tight correlation between primary IL-4 production capability and the generation of Th2 cells. However, the distribution of primary IL-4 production in F1 mice did not show the predicted 50% representation of each parental phenotype, suggesting that several genes may be involved.

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Fig. 3. Genetic correlation between primary IL-4 production and Th2 differentiation. CD4+ T cells were prepared from (BALB/cxB10.D2)F1, F1xF1, F1xBALB/c and F1xB10.D2, and stimulated with a combination of anti-TCR and anti-CD28 mAb. Generation of Th2 cells was assessed by intracellular staining after 6 days, and the primary IL-4 production level and the percentage of the Th2 population was plotted for individual mice.
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Regulation of IL-4 gene expression in naive T cells.
The above results clearly indicate that primary IL-4 production in naive T cells is quite polymorphic between the different strains of mice studied. Although much attention has been given towards understanding the regulation of IL-4 gene expression during general Th cell differentiation, the specific regulation of IL-4 in naive T cells has been largely not studied. We therefore focused in this study on the regulation of IL-4 transcription in naive T cells and, in particular, on the requirement of IL-4-mediated signaling events for primary IL-4 production in naive T cells.
Naive T cells were prepared from STAT6-deficient mice in either a BALB/c or B10.D2 background. After stimulation, IL-4 levels were measured at various time points. As illustrated in Fig. 4(A)
, disruption of the STAT6 gene did not affect IL-4 production in naive T cells, thereby indicating that primary IL-4 production is unrelated to the appearance of Th2 cells. Under these circumstances, naive T cells from a BALB/c background revealed markedly higher IL-4 production levels than those from a B10.D2 background (Fig. 4A
). It would therefore appear that the observed strain difference in primary IL-4 production is determined by an intrinsic capability of naive T cells. Significant evidence has been accumulated to suggest that transcriptional regulation of the IL-2 gene is controlled by its promoter region (29,30). We therefore hypothesized that the promoter region may also play a significant role in regulating primary IL-4 production. The promoter region spanning 800 bp upstream of the IL-4 gene is the most characterized region in this locus (3133). However, we compared the IL-4 promoter region sequences of BALB/c and B10.D2 mice, and found no differences (data not shown).

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Fig. 4. Regulation of primary IL-4 production in naive T cells. (A) Primary IL-4 production in naive T cells was independent of the STAT6-dependent signaling pathway. Naive T cells were prepared from STAT6-deficient mice in either a BALB/c or B10.D2 background. After stimulation with a combination of anti-TCR and anti-CD28 mAb, the amounts of IL-4 in culture supernatant were measured at various time points. (B) Acetylation status of IL-4 promoter region in primary activation. CD4+ T cells were stimulated with a combination of anti-TCR and anti-CD28 mAb for 48 h. The nuclear fractions were prepared from naive (1, 3, 5 and 7) and activated T cells for 48 h (2, 4, 6 and 8). After immunoprecipitation with anti-acetyl Histone H4 mAb (IP), acetylation in the promoter and silencer region of the IL-4 gene was assessed by PCR amplification. Lanes 1, 2, 5 and 6 represent BALB/c. Lanes 3, 4, 7 and 8 represent B10.D2.
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Histone acetylation is a pre-requisite for binding of transcription factors to the cis-acting elements in the promoter, eventually leading to gene activation. We examined whether the IL-4 promoter region is accessible to transcriptional factors at primary TCR stimulation. To do this, the degree of Histone H4 acetylation was studied using the chromatin immunoprecipitation (ChIP) assay. Since Th2-specific DNase I hypersensitive sites are located at the 3' untranslated region of the IL-4 gene, corresponding to the IL-4 silencer region (34), the differentiated Th2 cells showed clear acetylation in both the promoter and silencer regions (data not shown). In the primary activated CD4+ T cells, the promoter regions from both mouse strains were found to be acetylated to the same degree, while the silencer regions showed no acetylation (Fig. 4B
). These results would indicate that the IL-4 promoter becomes functionally active as a result of primary TCR stimulation, irrespective of any strain difference in the potential for naive T cells to secrete IL-4.
A strain-specific difference in nuclear expression of NF-AT1, NF-AT 2, GATA-3 and c-Maf at primary activation
The IL-4 promoter region in both strains of mice was found to be accessible to transcription factors. We therefore examined whether the strain-specific difference in IL-4 production might be due to differences in the amount of transcription factors present in the nucleus that could bind to the IL-4 promoter following TCR activation. CD4+ T cells from BALB/c and B10.D2 spleen cells were stimulated by a combination of antibodies against TCR and CD28, and nuclear and cytoplasmic extracts were obtained at different time points. Expression of NF-AT1, NF-AT2, GATA-3 and c-Maf was assessed by Western blotting analysis. The expression levels were found to be below detection or very low in pre-activated CD4+ T cells. However, TCR stimulation of naive T cells resulted in increased expression of all four factors examined in this study, although expression levels at 24 h after stimulation showed no difference between BALB/c and B10.D2 cells. At 48 h, NF-AT2 and GATA-3 expression was markedly increased in BALB/c cells, while expression of these factors remained unchanged in B10.D2 cells (Fig. 5A
), indicating that NFAT2 and GATA-3 may both play a role in controlling IL-4 promoter activity in primary T cell activation.

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Fig. 5. Expression and nuclear translocation of transcriptional factors in primary activation of CD4+ T cells. Expression and nuclear translocation of NF-AT1, NF-AT2, GATA-3 and c-Maf. CD4+ T cells from either BALB/c or B10.D2 mice were stimulated with a combination of anti-TCR and anti-CD28 mAb. Cells were harvested at different time points (048 h), and nuclear and cytoplasmic fractions were separated as described in Methods. Protein expression in nuclear and cytoplasmic fractions was assessed by anti-NF-AT1 mAb (4G6-G5), anti-NF-AT2 mAb (7A6), anti-GATA-3 mAb (HG3-35) and anti-c-Maf rabbit antisera. The blots were developed with either horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit Ig.
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All four transcription factors gradually appeared in the nuclei following TCR stimulation. NF-AT1, NF-AT2 and GATA-3 from nuclear extracts showed gel mobility shifts following migration into the nuclei, although the mobility of c-Maf was unchanged even after nuclear migration (Fig. 5A
). It is well documented that the nuclear form of NF-AT1and NF-AT2 decreases its mobility as a result of dephosphorylation by the protein phosphatase, calcineurin (35). In this study, the nuclear form of GATA-3 showed a decrease in mobility. Competition analysis clearly showed that the shifted smaller band observed in the nuclei was specific for GATA-3 (data not shown), thereby indicating that the mobility shift in GATA-3 observed here may be correlated with nuclear translocation. The nuclear fraction from BALB/c T cells showed a 2-fold higher expression of NF-AT2 and GATA-3 than that observed in B10.D2 T cells, while the expression of c-Maf and NF-AT1 remained at almost the same level in the two types of cells, even after 48 h.
To further examine the correlation between NF-AT2 level and the primary IL-4 production, T cells from (BALB/cxB10.D2)F1 mice were stimulated by anti-TCR and CD28 mAb for 48 h, and the secreted IL-4 and the nuclear expression levels of NF-AT2 and GATA-3 were analyzed. Two out of four F1 mice (F1-1 and -2) revealed a BALB/c phenotype in the IL-4 production, while the two other mice (F1-3 and -4) revealed an intermediate phenotype (Fig. 6A
). Among these four mice, the primary IL-4 production level was correlated with the nuclear expression levels of NF-AT2 and GATA-3. The nuclear expression levels of NF-AT2 and GATA-3 in the mice representing an intermediate phenotype in the IL-4 production were lower than those in the BALB/c phenotype mice (Fig. 6A
), although the difference in the expression levels of NF-AT2 and GATA-3 was very small. We therefore studied the correlation between NF-AT2 level and the primary IL-4 production in different strains of mice. C3H and B10.BR T cells showed a similar relation to BALB/c and B10.D2 T cells. Naive T cells from C3H mice produced higher levels of IL-4 at primary responses compared to the level in B10.BR T cells. In C3H and B10.BR, the primary IL-4 production levels were consistently correlated with the nuclear expression levels of NF-AT2. These results suggest that the expression levels of NF-AT2 and GATA-3 at primary T cell activation may influence the potential to secrete IL-4 in naive T cells, subsequently resulting in the differences in Th2 development among different strains of mice.

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Fig. 6. Correlation between the primary IL-4 production and the nuclear expression of NF-AT2 and GATA-3. (A) The nuclear expression of NF-AT2 and GATA-3 in (BALB/cxB10.D2)F1 T cells. CD4+ T cells from either BALB/c, B10.D2 or (BALB/cxB10.D2)F1 mice were stimulated with a combination of anti-TCR and anti-CD28 mAb for 48 h, and nuclear and cytoplasmic fractions were separated as described in Methods. The nuclear fraction was probed with antibodies against either NF-AT2 or GATA-3. The cytoplasmic fraction was probed with anti-STAT6 mAb. (B) The primary IL-4 production, and the nuclear expression of NF-AT2 in C3H and B10.BR mice. CD4+ T cells from C3H and B10.BR mice were stimulated with a combination of anti-TCR and anti-CD28 mAb for 48 h, and nuclear fractions were probed with anti-NF-AT2 mAb. (C) The presence of IL-4 was required for GATA-3 expression. Expression of NF-AT1, NF-AT2 and GATA-3 in BALB/c CD4+ T cells was assessed as described in Fig. 4(A) . The effect of IL-4 was examined by the addition of either anti-IL-4 mAb (10% culture supernatant) or exogenous IL-4 (10 ng/ml).
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However, a previous report has provided evidence that GATA-3 expression during Th cell differentiation is initiated by IL-4 signaling (36). Thus, it is likely that GATA-3 expression at the early activation stage is induced by the IL-4 secreted from naive T cells. To examine whether IL-4 may be involved in the enhancement of GATA-3 expression at primary T cell activation, naive T cells from BALB/c mice were stimulated with anti-TCR and CD28 mAb in the presence or absence of antibody against IL-4, and the nuclear expression of NF-AT1, NF-AT2 and GATA-3 was assessed 48 h after stimulation. The blockade of IL-4 function markedly inhibited GATA-3 expression but not NF-AT1 and 2 expression (Fig. 6C
). These results indicated that GATA-3 expression at primary stimulation was regulated by a small amount of IL-4 from naive T cells that was secreted independent of the IL-4 signaling.
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Discussion
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In present study, we provide evidence that IL-4 production capability in naive CD4+ T cells dictates the generation of IL-4-producing effector T cells in different strains of mice. At primary stimulation, naive CD4+ T cells from BALB/c contained large number of cells secreting significant amounts of IL-4. In contrast, IL-4 production in B10.D2 T cells was below detectable levels, so that these T cells showed very little Th2 differentiation. The promoter region of the IL-4 gene became active in both BALB/c and B10.D2 T cells at primary TCR stimulation. Strain differences were rather observed in the expression and nuclear translocation level of transcriptional factors regulating IL-4 promoter activity. We propose that the expression level of NF-AT2 may influence the STAT6-independent IL-4 production in naive T cells.
The presence of IL-4 during the priming stage is required for the subsequent appearance of IL-4-producing effector T cells. We propose here the existence of a mechanism in which naive T cells are an important source of the requisite early IL-4, causing naive T cells to commit to a Th2 lineage. Other sources of IL-4 have been proposed in previous reports. V
14 NKT cells and CD4+CD62LlowCD44high memory-type T cells produce relatively high amounts of IL-4 after TCR stimulation (3739). However, a recent report clearly indicates that the presence of activated V
14 NKT cells during the priming stage predominantly drives Th1 lineage responses (40), indicating that the importance of these cells as a source of IL-4 for Th2 development is far from established (41). In various experimental systems, accumulating evidence supports our notion that naive T cells themselves contribute as a source of IL-4 (1317). In fact, the purified CD4+CD62LhighCD44low population from BALB/c T cells produced readily detectable amounts of IL-4 (Fig. 2
), although the levels secreted from naive T cells were 1000 times less than that from the committed Th2 cells. We observed a similar relationship between the primary IL-4 production and Th2 development in other strains of mice, such as C3H/HeJ and B10.BR which lead to Th2 and Th1 development respectively (data not shown). Since all naive T cells express detectable levels of receptor for IL-4 (18,19), the IL-4 secreted from naive T cells may act on Th2 lineage commitment in an autocrine manner. Thus, it is reasonable to speculate that naive T cells could be a important source of IL-4 during the early developmental process of Th cells.
Given its critical role in supporting the development of Th2 cells, the manner in which primary IL-4 expression is regulated becomes an important question. We demonstrated that naive T cells were capable of producing IL-4 in the absence of the STAT6-mediated signaling pathway (Fig. 4A
), suggesting that control of the primary IL-4 production may be distinct from the regulation of IL-4 expression during commitment into the Th2 lineage. Our study using the ChIP assay provides evidence that the promoter plays an intrinsic role in regulating IL-4 production in naive T cells. Several transcriptional factors have been implicated in the commitment to the Th2 lineage through regulation of the IL-4 promoter activity. Both GATA-3 and c-Maf are predominantly expressed in Th2 cells, but not Th1 cells (42,43). A recent report using gene disruption has shown that the c-maf gene is particularly relevant in the activation of the IL-4 promoter (44). However, both BALB/c and B10.D2 strains of mice showed no difference in nuclear translocation of c-Maf protein during the priming process, even though IL-4 production capability of BALB/c T cells was much higher than that of B10.D2 T cells. Thus, the presence of c-Maf may be important for the activation of IL-4 promoter, but c-Maf is unlikely to contribute to the strain difference in primary IL-4 production.
The strain differences in primary IL-4 production between BALB/c and B10.D2 mice were correlated with the appearance of NF-AT2 and GATA-3 in nuclei. The promoter region of the IL-4 gene contains several possible recognition sites for NF-AT and the deletion of those sequences abrogates the promoter activity (47,48). Alternatively, the gene disruption of NF-AT2 results in a remarkable reduction of IL-4 production in committed Th2 cells (49,50). In contrast, IL-4 mRNA levels in NF-AT1-deficient mice show less decline at later time points of primary activation, leading to subsequent enhancement of IL-4 production and Th2-dominant responses (51,52). The present study showed that the enhancement of NF-AT2 expression at early activation appeared to be more pronounced than that of NF-AT1, indicating that significant amounts of NF-AT2 may be required by naive T cell to secrete IL-4 at primary activation. The difference in primary IL-4 production between BALB/c and B10.D2 was >10-fold, whereas the difference in NF-AT2 expression was only 2- to 3-fold. Thus, it is unlikely that the significant differences in primary IL-4 production were determined by NF-AT2 expression level alone. Some additional factors might be involved in the regulation of primary IL-4 production.
GATA-3 plays a role in the termination of Th1 development by abrogation of IL-12Rß2 chain expression (45), and overexpression of GATA-3 by a retrovirus system is shown to directly activate IL-4 and IL-5 gene expression through DNA remodeling (36,46). This evidence confirms that GATA-3 is a prominent transcription factor promoting Th2 development. Consistently, the migration of GATA-3 to the nucleus in BALB/c T cells is markedly enhanced in the early activation process. However, this enhanced GATA-3 migration was completely dependent upon the presence of IL-4 and STAT6 (Fig. 4A
) (36), supporting the possibility that the enhanced GATA-3 expression during early activation occurred as a result of the presence of IL-4 secreted from naive T cells (Fig. 6C
). The IL-4 production in naive T cells is independent of the IL-4-mediated signaling, suggesting that GATA-3 may be responsible for late phase of the IL-4 secretion. Thus, a certain level of NF-AT2 expression may be required for generating the primary IL-4 in naive T cells and the primary IL-4 may result in significant GATA-3 expression. Therefore, NF-AT2 expression may, at least partly, be a genetic factor to regulate the strain-specific differences in primary IL-4 secreted from naive T cells.
 |
Acknowledgments
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The authors are very grateful to Dr Kenneth Murphy and Dr Shizuo Akira for the generous gift of DO11.10 Tg andC57BL6 STAT6-deficient mice. This work was supported by a grant from the Ministry of Education, Science and Culture (Japan), by the Takeda Science Foundation, by the Novartis Foundation (Japan), and by the Motida Memorial Foundation.
 |
Abbreviations
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APC antigen-presenting cell |
ChIP chromatin immunoprecipitation assay |
DO11.10 Tg OVA-specific TCR transgenic mouse |
IL-12R IL-12 receptor |
OVA ovalbumin |
Tg transgenic |
 |
Notes
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Transmitting editor: T. Watanabe
Received 23 March 2001,
accepted 21 September 2001.
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