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International Immunology Advance Access originally published online on October 11, 2006
International Immunology 2006 18(12):1681-1690; doi:10.1093/intimm/dxl102
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Genetic background influences Th cell differentiation by controlling the capacity for IL-2-induced IL-4 production by naive CD4+ T cells

Junji Yagi1, Yutaka Arimura1,4, Hiroaki Takatori2, Hiroshi Nakajima2, Itsuo Iwamoto2 and Takehiko Uchiyama1,3

1 Department of Microbiology and Immunology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
2 Department of Allergy and Clinical Immunology, Chiba University School of Medicine, 1-8-1 Inohara, Chiba City, Chiba 260-8670, Japan
3 Institute of Laboratory Animals, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan
4 Present address: Inflammatory and Infectious Disease Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA

Correspondence to: J. Yagi; E-mail: jyagi1{at}research.twmu.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Comparative studies using Th2-prone BALB/c and Th1-prone C57BL/6 mice were performed to clarify the influence of genetic background on Th cell differentiation. The results showed IL-4, the production of which is induced by IL-2, to be much more abundantly produced by BALB/c naive CD4+ T cells than by C57BL/6 naive CD4+ T cells, thereby leading to a tendency for differentiation toward Th2 in BALB/c naive CD4+ T cells. This difference in IL-4 production between the two naive CD4+ T cells appeared to be attributable to specific intracellular signaling events. Signal transducer and activator of transcription 5 (STAT5) was preferentially activated by IL-2 in CD4+ T cells developing in BALB/c in contrast to the corresponding cells in C57BL/6. In addition, IL-4 also induced stronger STAT5 activation in CD4+ T cells developing in BALB/c than in those developing in C57BL/6, whereas STAT6 was equally activated in these two cells. Further results supported the involvement of STAT5 in the difference in Th cell differentiation between BALB/c and C57BL/6 naive CD4+ T cells. STAT5A–/– naive CD4+ T cells with the BALB/c genetic background showed markedly less IL-2-induced IL-4 production than BALB/c naive CD4+ T cells. Conversely, forced expression of the constitutively active forms of STAT5A and STAT5B in C57BL/6 naive CD4+ T cells promoted the differentiation of Th2 cells. Thus, our results indicate IL-2-induced IL-4 production by naive CD4+ T cells, in which STAT5 activation is involved and directly controlled by the genetic background, to influence Th cell differentiation in murine strains.

Keywords: BALB/c, C57BL/6, cytokines, STAT5, Th1/Th2 cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Naive CD4+ T cells are activated through recognition of the antigen peptide–MHC complex on antigen-presenting cells (APCs), and differentiate into functionally distinct effector cells, Th1 or Th2 (1, 2). Th1 cells secrete IL-2, IFN-{gamma} and TNF-ß, which induce cell-mediated immunity, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13, which mediate humoral immunity, thereby facilitating antibody production (3–5). In defense against infectious agents, Th1 cells eradicate intracellular micro-organisms, while Th2 cells act to protect the organism from extracellular pathogens such as helminths. Commitment of the immune response to either the Th1 or the Th2 lineage is primarily determined by cytokines present in the local environment. IL-4 or IL-12 drives antigen-stimulated naive CD4+ T cells toward the Th2 or the Th1 phenotype, respectively (6, 7). It is, additionally, known that a variety of factors such as antigen dose, affinity of the TCR for its antigen peptide–MHC complex, type of APC and co-stimulatory signals affect Th cell differentiation (8). When immune responses occur, genetic background also appears to regulate the above factors and influence Th cell differentiation, thereby leading to the preferential development of Th1 or Th2 cells, as reflected by the differences seen among murine strains (9, 10). The immune responses of BALB/c mice are Th2 prone, whereas those of C57BL/6, C3H and B10.D2 mice are Th1 prone (9, 10).

In regard to the mechanism underlying the influence of genetic background, Murphy and coworkers (9, 11) showed the difference in maintenance of IL-12 responsiveness of naive T cells to be a critical factor determining Th cell differentiation. BALB/c naive T cells lose responsiveness to IL-12 soon after antigen stimulation in vitro, whereas those of B10.D2 maintain their responsiveness to IL-12, allowing them to differentiate into Th1 cells. Differences in the levels of IL-4 produced by antigen-stimulated naive CD4+ T cells have also been shown among murine strains (12). In contrast to BALB/c naive CD4+ T cells producing substantial amounts of IL-4, those of B10.D2 secrete very little IL-4. The addition of exogenous IL-4 to the culture of B10.D2 naive CD4+ T cells promoted Th2 development, indicating that the genetic background influences Th cell differentiation via different abilities of naive CD4+ T cells to produce IL-4. Strain-specific differences among CD11b+ dendritic cells have been reported (13). In both parasite-specific and allospecific CD4+ T cell responses, B10.D2 and BALB/c dendritic cells differentially promote Th1 and Th2 differentiation in naive CD4+ T cells, respectively. Thus, in Th cell differentiation, various aspects of immune responses are influenced by the genetic background. However, the cellular and molecular bases of these influences are not fully understood.

We observed genetic background to influence Th cell development during the course of a study on inducible co-stimulator (ICOS) expression (14, 15). The results indicated IL-4 in the primary culture of naive CD4+ T cells to be a key factor in the promotion of both ICOS expression on activated CD4+ T cells and Th2 differentiation. In keeping with the pioneer work by Paul and coworkers (16), IL-2 was shown to regulate IL-4 production in primary culture, thus suggesting that IL-2 triggers an immunoregulatory pathway linking IL-4 to ICOS in Th2 differentiation. Comparative studies using Th2-prone BALB/c and Th1-prone C57BL/6 mice raised the possibility that BALB/c may be more efficient than C57BL/6 mice in terms of IL-2-induced IL-4 production during primary stimulation of naive CD4+ T cells, leading to a deviation toward Th2 differentiation.

In the present study, we clarified the above possibility, attempted to uncover its molecular basis and found both IL-2 and IL-4 to induce signal transducer and activator of transcription 5 (STAT5) activation more strongly in CD4+ T cells developing in BALB/c than in their C57BL/6 counterparts. Additional results indicate STAT5 to be a regulator of IL-2-induced IL-4 production from naive CD4+ T cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals
BALB/c and C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan). STAT5A–/– mice with the BALB/c background were described previously (17). All mice were bred and used at 8–12 weeks of age in accordance with the guidelines of the ethics review committee for animal experiments, Tokyo Women's Medical University.

Preparation of cells
CD4+ T cells were obtained by treatment of spleen cells with mAbs to I-Ab,d [28-16-8S (18)], CD8 [83.12.5 (18)] and guinea pig complement. Naive CD4+ T cells were then enriched by treatment with a cocktail of mAbs to CD44 (IM7; BD PharMingen, San Diego, CA, USA), CD8 [53-6.7 (18)], TCR{gamma}{delta} (GL3; BD PharMingen), CD45R/B220 (RA3-6B2; BD PharMingen), CD11b (M1/70; BD PharMingen), CD16/CD32 (2.4G2; BD PharMingen) and mouse NK-T/NK cell antigen (U5A2-13; BD PharMingen) and depletion of mAb-coated cells using a mixture of sheep anti-rat and sheep anti-mouse IgG-bound magnetic beads (Dynabeads, Dynal Inc., Lake Success, NY, USA). The cells obtained were >92% CD4+ and contained <1% CD44high CD4+ T cells. Naive CD4+ T cells (1 x 106 ml–1) were cultured with plate-coated anti-CD3 mAbs (10 µg ml–1) [145-2C11 (18)] and soluble anti-CD28 mAbs (5 µg ml–1) [37.51 (19); a gift from Dr. J. Allison, University of California at Berkeley, Berkeley, CA, USA]. Developing CD4+ T cells were harvested at 48 h of culture, and starved for an additional 8 h in the presence of anti-murine IL-2 mAbs (30 µg ml–1) (S4B6; American Type Tissue Collection, Rockville, MD, USA). Activated CD4+ T cells were collected at 62 h of culture by applying the cell suspension to a Percoll (Pharmacia Biotech, Uppsala, Sweden) gradient (density, 1.075), and expanded with 100 U ml–1 of human rIL-2 (a gift from Takeda Chemical Industries, Ltd, Osaka, Japan) for 2 additional days.

Assay of responses in various T cell populations
For proliferative response and cytokine production, naive CD4+ T cells or developing CD4+ T cells (1 x 106 ml–1) were stimulated for the indicated times with plate-coated anti-CD3 mAbs (10 µg ml–1) and soluble anti-CD28 mAbs (5 µg ml–1), and activated CD4+ T cells (5 x 105 ml–1) were re-stimulated with plate-coated anti-CD3 mAbs (30 µg ml–1). For IL-2-induced proliferative response, developing CD4+ T cells (2 x 105 ml–1) were cultured for 40 h with titrated amounts of human rIL-2 in the presence of anti-murine IL-2 mAbs (30 µg ml–1). For intracellular cytokine staining, activated CD4+ T cells were re-stimulated as described above for 20 h, and with phorbol myristate acetate (Sigma–Aldrich, St Louis, MO, USA) (10 ng ml–1) and A23187 [GenBank] (Sigma–Aldrich) (0.4 µM) in the presence of monensin (GolgiStop, BD PharMingen) for the last 4 h. To assess proliferative responses, cells were pulsed with 0.5 µCi of [3H]thymidine ([3H]TdR) for the last 16 or 12 h, and the amount of [3H]TdR incorporated was measured. The IL-2 concentrations in culture supernatants were determined as the proliferation of IL-2-dependent CTLL-2 cells, as described previously (15). IL-4 and IFN-{gamma} were quantified by sandwich ELISA according to the manufacturer's instructions (BD PharMingen). Results are presented as the mean counts per minute or concentration ± SD of triplicate cultures. After cell-surface staining with PerCP–anti-Thy-1.1 (OX-7; BD PharMingen), intracellular cytokine staining was performed and analyzed as described previously (20).

Flow cytometric analysis
Purified rat IgG (RIgG) and anti-IL-2R{alpha} mAbs [7D4 (18)] were conjugated with biotin in our laboratory. To detect IL-2R or IL-4R expressions on the cell surface, developing CD4+ T cells were stained by incubation with biotinylated mAbs to IL-2R{alpha}, IL-2Rß (TM-ß1; BD PharMingen) or common {gamma} ({gamma}c) chain (TUGm2; BD PharMingen), or biotinylated mAbs to IL-4R{alpha} (mIL-4R-M1; BD PharMingen) or {gamma}c chain, followed by a cocktail of FITC–anti-CD4 mAb (RM4-5; BD PharMingen) and PE–streptavidin (BD PharMingen), respectively. Background stainings were performed with biotinylated RIgG. Samples of 10 000 viable cells gated for CD4+ T cells were analyzed with an Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA).

Immunoblotting and immunoprecipitation
The following antibodies were used: polyclonal antibodies to phospho-STAT5A/B (Tyr694/Tyr699), Janus kinase 1 (Jak1) and Jak3, mAb to phosphotyrosine (4G10) (Upstate Biotechnology, Lake Placid, NY, USA), polyclonal antibodies to STAT5A/B and actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal antibodies to phospho-extracellular signal-regulated kinase (phospho-ERK) (Thr202/Tyr204) and phospho-STAT6 (Tyr641) (Cell Signaling Technology, Beverly, MA, USA) and polyclonal antibodies to STAT6, STAT5A and STAT5B (R&D Systems, Minneapolis, MN, USA). Developing CD4+ T cells were stimulated with human rIL-2, murine rIL-4 (BD PharMingen) or plate-coated anti-CD3 and anti-CD28 mAbs for the indicated times at 37°C. Cells were lysed in TNE lysis buffer (10 mM Tris, pH 7.5, 0.15 M NaCl, 1 mM EDTA, 1 mM MgCl2, 1% NP-40, 1 mM Na3VO4 and 10% glycerol) containing a protease inhibitor cocktail (Sigma–Aldrich) for western blotting or in modified RIPA buffer (50 mM Tris, pH 7.4, 0.15 M NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1% NP-40, 1 mM Na3VO4 and 1 mM NaF) containing a protease inhibitor cocktail for immunoprecipitation. In some of the experiments, nuclear and cytoplasmic extracts were obtained using NE-PERTM Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA) supplemented with 1 mM Na3VO4 and the protease inhibitor cocktail. Preliminary assays indicated that amounts of protein in BALB/c and C57BL/6 cell lysates were equal when obtained from the same number of cells. Western blotting was conducted as described previously (14, 15), using lysates from the same numbers of BALB/c and C57BL/6 cells. For immunoprecipitation, the lysates were precleared with protein G-Sepharose beads (Pharmacia Fine Chemicals, Uppsala, Sweden) for 30 min at 4°C, and were incubated overnight with the indicated antiserum at 4°C. Immune complexes were collected from lysates after incubation with protein G-Sepharose beads for 2 h at 4°C, and analyzed by western blotting.

Construction of expression vectors and retrovirus-mediated gene transfer
The bicistronic retroviral vector pMXs-IG (21) and constitutively active forms of murine STAT5A- and STAT5B-inserted retroviral vectors (pMX-puro-STAT5A1*6 and pMX-puro-STAT5B1*6) (22) have been described, and were kindly provided by T. Kitamura (University of Tokyo, Tokyo, Japan). The full length of Thy1.1 cDNA was amplified with reverse transcription–PCR from total RNA of AKR spleen cells, and was replaced with the EGFP region in pMXs-IG (pMXs-IRES-Thy1.1). After digestion of pMX-puro-STAT5A1*6 or pMX-puro-STAT5B1*6 with EcoRI and NotI, the fragment was inserted into pMXs-IG or pMXs-IRES-Thy1.1. The culture supernatants containing recombinant retroviruses were collected from Plat-E packaging cells (a gift from T. Kitamura) (23) at 2 or 3 days after transfection with the above retrovirus vectors [12 µg of control empty vector, 12 µg of STAT5A1*6 vector, 12 µg of STAT5B1*6 vector or a mixture of 6 µg of STAT5A1*6 and 6 µg of STAT5B1*6 vectors/Plat-E packaging cell-seeded 10-cm dish (3003 Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA)] using the calcium phosphate method. Naive CD4+ T cells were cultured with anti-CD3 mAbs and anti-CD28 mAbs as described above, and infection with the recombinant virus was performed as described previously (15), replacing culture medium with supernatant from Plat-E cells supplemented with anti-CD28 mAbs (5 µg ml–1) and polybrene (10 µg ml–1) (Sigma–Aldrich). The infected cells were expanded with 100 U ml–1 of human rIL-2 for 2 additional days, stimulated and examined for cytokine productions. The efficiency of retrovirus infection was determined by flow cytometric analysis of EGFP+ or Thy1.1+ cells.

Statistical analysis
Results were analyzed by variance using a one- or two-way analysis of variance, as appropriate, followed by Tukey–Kramer's test for multiple comparisons, and considered significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The efficiency of IL-2-induced IL-4 production determines Th cell differentiation in BALB/c versus C57BL/6 mice
Consistent with the well-known difference in Th cell differentiation, BALB/c activated CD4+ T cells produced ~25-fold more IL-4, but a slightly lower amount of IFN-{gamma} than C57BL/6 activated CD4+ T cells upon re-stimulation (Fig. 1A). As reported previously in a different experimental system (15), BALB/c naive CD4+ T cells were stimulated to produce a substantial amount of IL-4, whereas C57BL/6 naive CD4+ T cells produced >10-fold less IL-4 (Fig. 1A). The amounts of IL-2 and IFN-{gamma} produced by BALB/c naive CD4+ T cells were also higher than those produced by C57BL/6 naive CD4+ T cells (Fig. 1A). It is, however, likely that a similar level of cell activation was induced in the two naive CD4+ T cells as shown by the equal degree of proliferation (Fig. 1A). BALB/c activated CD4+ T cells obtained from a primary culture with neutralizing anti-IL-4 mAbs secreted much less IL-4, but a slightly greater amount of IFN-{gamma} upon re-stimulation (Fig. 1B), indicating a critical role for IL-4 produced from naive CD4+ T cells in Th2 development. Thus, these results indicate that the amount of IL-4 produced from naive CD4+ T cells is a determinant of the difference in Th cell differentiation between BALB/c and C57BL/6 mice. As described by Ben-Sasson et al. (16), this IL-4 production is regulated by IL-2, since the presence of neutralizing anti-IL-2 mAbs inhibited IL-4 production almost completely in primary culture with a slight down-regulatory effect on proliferation (Fig. 1C). In response to exogenous IL-2, BALB/c naive CD4+ T cells produced a much higher (~10-fold at 100–900 U ml–1 of IL-2) amount of IL-4 than C57BL/6 CD4+ T cells with similar levels of proliferation in these two cells (Fig. 2A). IFN-{gamma} production was also regulated by IL-2, and BALB/c naive CD4+ T cells produced higher amounts of IFN-{gamma} than C57BL/6 cells (Fig. 2A). Furthermore, despite the similar levels of IL-2R expression on their cell surfaces, as shown by equal IL-2R{alpha}, ß and {gamma}c chain expressions (Fig. 2B), BALB/c developing CD4+ T cells obtained at 48 h of primary culture showed increased IL-4 production in response to exogenous IL-2 (~3-fold at 33 U ml–1), while the corresponding C57BL/6 cells showed only a slight increase (Fig. 2C). Thus, BALB/c naive and developing CD4+ T cells produce IL-4 in response to IL-2 very efficiently, as compared with the corresponding C57BL/6 cells.


Figure 1
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  		Fig. 1 The amount of IL-4 produced by naive CD4+ T cells is a key factor determining Th cell differentiation in BALB/c and C57BL/6 mice. (A) BALB/c and C57BL/6 naive CD4+ T cells were stimulated for 72 h with anti-CD3 mAbs and anti-CD28 mAbs as described in Methods, and cytokine productions and uptakes of [3H]TdR were assayed. BALB/c and C57BL/6 activated CD4+ T cells were re-stimulated for 24 h with anti-CD3 mAbs as described in Methods, and cytokine productions were determined. (B) BALB/c naive CD4+ T cells were stimulated for 62 h in the absence or presence of RIgG or anti-IL-4 mAbs (3 µg ml–1) (11B11; American Type Tissue Collection). Activated CD4+ T cells were examined for cytokine productions. (C) BALB/c naive CD4+ T cells were stimulated for 62 h in the absence or presence of titrated amounts of RIgG or anti-murine IL-2 mAbs, and IL-4 productions and uptakes of [3H]TdR were assayed. *P < 0.05; **P < 0.01 as compared with BALB/c (A), control cultures in the absence and presence of RIgG (B) or control cultures with RIgG (C).

 

Figure 2
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  		Fig. 2 IL-2-induced IL-4 production is much higher in BALB/c naive or developing CD4+ T cells than in the corresponding C57BL/6 cells. (A) BALB/c and C57BL/6 naive CD4+ T cells were stimulated with anti-CD3 mAbs and anti-CD28 mAbs for 62 h with RIgG (300 µg ml–1) alone (#), or anti-murine IL-2 mAbs (300 µg ml–1) in the absence or presence of titrated amounts of human rIL-2, and cytokine productions and uptakes of [3H]TdR were assayed. (B) Cell-surface expression of IL-2R components on developing CD4+ T cells obtained at 48 h of primary culture. Background staining results are shown as filled curves with a solid line (BALB/c) and open curves with a dashed line (C57BL/6). (C) CD4+ T cells developing in BALB/c and C57BL/6 were stimulated as in (A) for 48 h with anti-murine IL-2 mAbs (30 µg ml–1) in the absence or presence of titrated amounts of human rIL-2, and cytokine productions were determined. *P < 0.05; **P < 0.01 as compared with BALB/c.

 
Differential activation of downstream molecules in IL-2-induced signaling in CD4+ T cells from BALB/c and C57BL/6 mice
The signaling pathway may be differentially activated by IL-2 in BALB/c and C57BL/6 CD4+ T cells. To investigate this possibility, developing CD4+ T cells were analyzed for the activation of signaling molecules in response to exogenous human IL-2. It was recently reported that STAT5, a downstream effector molecule of IL-2R-associated kinases, Jak1 and Jak3, is a key molecule promoting Th2 differentiation (17, 24, 25). Therefore, we examined the phosphorylation of STAT5 to compare the activation levels of this molecule. As shown in Fig. 3(A), STAT5 was activated by IL-2 more strongly in CD4+ T cells developing in BALB/c than in C57BL/6 cells with the corresponding dose responses, and at each time point examined. A representative molecule in another signaling cascade triggered by IL-2, p44 ERK, was also more strongly activated in BALB/c than in C57BL/6 cells, whereas differences in p42 ERK activation were barely detectable in these two cells (Fig. 3A). Ligation of IL-2R induced similar levels of Jak1 and Jak3 phosphorylation in BALB/c and C57BL/6 cells (Fig. 3B), ruling out the possibility of these kinases being involved in the different activation levels of STAT5. These signaling events have little effect on the overall activation, since their proliferative responses to IL-2 were equivalent (Fig. 3C). TCR stimulation did not elicit STAT5 activation in developing CD4+ T cells (Fig. 3D). Thus, differential activation of STAT5 is induced by IL-2 in CD4+ T cells developing in BALB/c versus C57BL/6.


Figure 3
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  		Fig. 3 Higher activation of STAT5 is induced by IL-2 in CD4+ T cells developing in BALB/c than in C57BL/6 cells. (A) CD4+ T cells developing in BALB/c and C57BL/6 were stimulated with titrated amounts of human rIL-2 for 10 min, or with 100 U ml–1 of human rIL-2 for the times indicated. The lysates were subjected to western blotting with the specific antibodies indicated. Numbers below each band or those above the p44 phospho-ERK band show arbitrary densitometric units. (B) CD4+ T cells developing in BALB/c and C57BL/6 were stimulated with 100 U ml–1 of human rIL-2 for the times indicated. Immunoprecipitates from the lysates with anti-Jak1 or anti-Jak3 antibodies were subjected to western blotting with anti-phosphotyrosine mAbs and anti-Jak1 antibodies or anti-phosphotyrosine mAbs and anti-Jak3 antibodies, respectively. (C) IL-2-induced proliferation of developing CD4+ T cells was assessed as described in Methods. (D) CD4+ T cells developing in BALB/c were stimulated with human rIL-2 (100 U ml–1) or plate-coated anti-CD3 (10 µg ml–1) and anti-CD28 (10 µg ml–1) mAbs for 10 min in the presence of anti-murine IL-2 mAbs (30 µg ml–1). The lysates were blotted with the specific antibodies indicated.

 
Differential activation of downstream molecules in IL-4-induced signaling in CD4+ T cells from BALB/c and C57BL/6 mice
Binding of IL-4R on naive CD4+ T cells by IL-4 can lead to Th2 differentiation via STAT6 activation (26–28). In addition, several reports have shown STAT5 to be activated through IL-4-induced signaling (29, 30), and Yamashita et al. (30) reported that STAT5 plays an important role in IL-4-induced expansion of developing Th2 cells. Therefore, we next investigated the activation levels of STAT5 and STAT6 in developing CD4+ T cells after stimulation with exogenous IL-4. IL-4R appeared to be expressed at similar levels on the surfaces of BALB/c and C57BL/6 cells (Fig. 4A). Binding of IL-4R with titrated amounts of IL-4 resulted in much stronger activation of STAT5 in CD4+ T cells developing in BALB/c than in the corresponding C57BL/6 cells (Fig. 4B), whereas differences in STAT6 activation between these two cells were essentially undetectable (Fig. 4B). Kinetics also showed much stronger activation of STAT5 in BALB/c cells than C57BL/6 cells with equivalent levels of STAT6 activation (Fig. 4B). IL-4R-associated kinases, Jak1 and Jak3, were phosphorylated at equivalent levels in BALB/c and C57BL/6 cells after stimulation with IL-4 (Fig. 4C). Thus, the results indicate that IL-4 drives the signaling pathway toward different activations of STAT5, but not STAT6, in CD4+ T cells developing in BALB/c versus C57BL/6.


Figure 4
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  		Fig. 4 Higher STAT5 activation is induced by IL-4 in CD4+ T cells developing in BALB/c than in those developing in C57BL/6 with equal STAT6 activations in these two cells. (A) CD4+ T cells developing in BALB/c and C57BL/6 were examined for cell-surface expression of IL-4R components. Background staining results are shown as filled curves with a solid line (BALB/c) and open curves with a dashed line (C57BL/6). (B) CD4+ T cells developing in BALB/c and C57BL/6 were stimulated with titrated amounts of IL-4 for 10 min, or with 20 ng ml–1 of IL-4 for the times indicated. The lysates were subjected to western blotting using the specific antibodies indicated. (C) The levels of Jak1 and Jak3 activation induced by 20 ng ml–1 of IL-4 in CD4+ T cells developing in BALB/c and C57BL/6 were assessed as in Fig. 3(B).

 
Activation of STAT5 is involved in IL-2-induced IL-4 production from naive CD4+ T cells
The difference in STAT5 activation is suggested to be involved in different efficiencies of IL-2-induced IL-4 production in BALB/c versus C57BL/6 naive CD4+ T cells. To examine this possibility, STAT5A–/– mice were used, since inductions of STAT5 activation are markedly reduced in the mammary tissues (31) and splenocytes (32) of these mice. As shown in Fig. 5(A), IL-4 production from naive CD4+ T cells from STAT5A–/– mice with the BALB/c background was greatly reduced in primary culture as compared with that from BALB/c naive CD4+ T cells. STAT5A–/– naive CD4+ T cells also produced a smaller amount of IFN-{gamma} with a little stronger proliferation than BALB/c cells (Fig. 5A). Upon re-stimulation, STAT5A–/– activated CD4+ T cells produced very little IL-4 as compared with the corresponding BALB/c cells, whereas substantial IFN-{gamma} productions were detected in these two cells (Fig. 5A), indicating Th1 deviation of Th cell differentiation of STAT5A–/– naive CD4+ T cells. IL-4 production from BALB/c CD4+ T cells in response to IL-2 increased in a dose-dependent manner to a level exceeding that from control cells cultured with RIgGs, whereas the increase in IL-4 production from STAT5A–/– CD4+ T cells was minimal (Fig. 5B). Net proliferative responses at each IL-2 dose and the corresponding degree of the IL-2 dose-dependent increase were equivalent in these two CD4+ T cells (Fig. 5B). Thus, the efficiency of IL-2-induced IL-4 production in STAT5A–/– naive CD4+ T cells is extremely low as compared with that in BALB/c CD4+ T cells, indicating STAT5 to be critically involved in this Th2-promoting pathway.


Figure 5
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  		Fig. 5 STAT5A is involved in IL-2-induced IL-4 production and Th 2 differentiation of naive CD4+ T cells. (A) BALB/c and STAT5A–/– naive CD4+ T cells were stimulated for 62 h, and cytokine productions and uptakes of [3H]TdR were assayed. Activated CD4+ T cells were stimulated to determine cytokine productions. (B) BALB/c and STAT5–/– naive CD4+ T cells were cultured as in Fig. 2(A), and IL-4 production and proliferation were assayed. *P < 0.05; **P < 0.01 as compared with BALB/c.

 
Constitutively active STAT5 promotes Th2 differentiation in C57BL/6 CD4+ T cells
The effect of forced expression of active STAT5 on Th cell development was next examined. The STAT5A and STAT5B mutants which are designated STAT5A1*6 and STAT5B1*6, respectively, were shown to be constitutively phosphorylated on tyrosine residues, localized in the nucleus, and transcriptionally active (22). We introduced STAT5A1*6-GFP and/or STAT5B1*6-GFP expression constructs into C57BL/6 CD4+ T cells during primary stimulation of naive CD4+ T cells using retroviral infection. STAT5A1*6-GFP- and/or STAT5B1*6-GFP-infected cells expressed phosphorylated STAT5 in both the cytoplasm and the nucleus, whereas control cells exhibited an undetectable level of STAT5 phosphorylation, before (data not shown) and after re-stimulation (Fig. 6A). Upon re-stimulation, STAT5A1*6-GFP-, STAT5B1*6-GFP- or STAT5A1*6-GFP- plus STAT5B1*6-GFP-infected cells produced ~3-fold higher amounts of IL-4 with essentially the same IFN-{gamma} productions as compared with control cells (Fig. 6B). The efficiency of retrovius infection in STAT5A- and/or STAT5B-infected cells was lower than that in control cells in repeated experiments. Therefore, STAT5A1*6-Thy1.1 and/or STAT5B1*6-Thy1.1 expression constructs were introduced into C57BL/6 CD4+ T cells, and after re-stimulation, cytokine-producing cells were examined in infected cells identified by Thy1.1 cell-surface expression. The results showed that the percentage of IL-4- but not IFN-{gamma}-producing cells was ~10-fold higher in infected cells with the constitutively active form of STAT5A and/or STAT5B than that in control cells (Fig. 6C). In contrast, the percentage of IFN-{gamma}- but not IL-4-producing cells was conversely decreased in these active STAT5-infected cells (Fig. 6C). Thus, the activation of STAT5A and STAT5B by phosphorylating tyrosine residues results in equal promotion of Th2 differentiation in C57BL/6 CD4+ T cells.


Figure 6
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  		Fig. 6 Forced expression of the activated forms of STAT5A and STAT5B promotes Th2 differentiation. (A) A control empty construct, a STAT5A1*6-GFP construct, a STAT5B1*6-GFP construct or a mixture of STAT5A1*6-GFP and STAT5B1*6-GFP constructs was introduced into C57BL/6 CD4+ T cells, as described in Methods. Activated CD4+ T cells were examined for the indicated protein expressions in nuclear and cytoplasmic extracts obtained at 6 h after re-stimulation with anti-CD3 mAbs. (B) Activated CD4+ T cells obtained as in (A) were re-stimulated, and cytokine productions were determined. The efficiencies of retrovirus infection assessed by the percentage of EGFP+ cells were 52.5, 28.8, 29.7 and 38.2% in control RV-, STAT5A1*6-, STAT5B1*6- and STAT5A1*6 plus STAT5B1*6-infected cells, respectively. (C) A control empty construct, a STAT5A1*6-Thy1.1 construct, a STAT5B1*6-Thy1.1 construct or a mixture of STAT5A1*6-Thy1.1 and STAT5B1*6-Thy1.1 constructs was introduced into C57BL/6 CD4+ T cells. Activated CD4+ T cells were re-stimulated, followed by surface Thy1.1 and intracellular cytokine staining. The efficiencies of retrovirus infection were 32.8, 34.5, 27.2 and 29.7% in control RV-, STAT5A1*6-, STAT5B1*6- and STAT5A1*6 plus STAT5B1*6-infected cells, respectively. The profiles for cytokine-producing cells among Thy1.1+ cells are presented. Numbers indicate the percentage of positive cells in each quadrant. *P < 0.05; **P < 0.01 as compared with control RV.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results showed IL-2-induced IL-4 production from naive CD4+ T cells to be a Th-differentiating pathway influenced by genetic background, since it was induced much more efficiently in BALB/c naive CD4+ T cells than in C57BL/6 cells, thereby promoting Th2 differentiation in BALB/c CD4+ T cells (Figs 1 and 2). A recent report by Paul and coworkers (24) clearly showed IL-2 to play a crucial role in Th2 development. IL-2 maintains accessibility at DNase I hypersensitivity regions II and III in the Il4 gene, and STAT5A associates with these regions, during development of IL-4-producing cells. In keeping with their results, binding of IL-2R by IL-2 elicited stronger activation of STAT5 in CD4+ T cells developing in BALB/c than in C57BL/6 cells (Fig. 3A). Thus, a different level of STAT5 activation appears to be involved in the differential capacity of IL-2-induced IL-4 production from the two naive CD4+ T cells. This notion is further supported by the observations that (i) a STAT5A deficiency decreased IL-4 production from naive CD4+ T cells to a minimal level and induced a polarized Th1 response (Fig. 5A), (ii) IL-4 production from STAT5A–/– naive CD4+ T cells in response to IL-2 was minimal (Fig. 5B) and (iii) forced expression of constitutively active STAT5A or STAT5B in C57BL/6 naive CD4+ T cells during primary stimulation promoted Th2 differentiation (Fig. 6).

Despite a 50–60% reduction in activation of STAT5 induced by IL-2 in CD4+ T cells developing in C57BL/6 as compared with BALB/c (Fig. 3A), IL-2-induced IL-4 productions from C57BL/6 naive and developing CD4+ T cells were markedly reduced as compared with those of their BALB/c counterparts (Fig. 2). It is, therefore, plausible that other signaling pathways may be involved in the difference in IL-2-induced IL-4 production, acting synergistically with or being induced by the Jak1 and Jak3–STAT5 pathway. In this regard, it would be of interest to examine whether the SLAM-SAP-Fyn cascade, which is triggered by TCR engagement and up-regulates the production of Th2 cytokines such as IL-4 and IL-13 by naive CD4+ T cells (33, 34), is activated differentially in BALB/c and C57BL/6 naive CD4+ T cells. The observation that IL-2-induced IL-4 production from STAT5A–/– naive CD4+ T cells was very slight (Fig. 5B) suggests that such pathways may be dependent on STAT5. Obviously, future studies are needed to clarify the dependency of Th2-promoting pathways on STAT5.

IFN-{gamma} production from naive CD4+ T cells is also regulated by IL-2 as shown by the IL-2 dose-dependent increase in IFN-{gamma} production (Fig. 2). Since STAT5A–/– naive CD4+ T cells produced significantly less IFN-{gamma} than BALB/c cells in a primary culture (Fig. 5A), STAT5 activation seems to also be involved in this pathway. Supporting this possibility, IFN-{gamma} production was more efficiently induced by IL-2 in BALB/c naive CD4+ T cells than in C57BL/6 cells (Fig. 2). However, it has not yet been determined in this experimental system whether a higher amount of IFN-{gamma} is involved in the promotion of Th2 differentiation as was recently reported (35), or inhibits Th2 development as previously described (36).

Binding of IL-4R by IL-4 also induced much stronger activation of STAT5 in BALB/c CD4+ T cells than in those from C57BL/6 (Fig. 4B). The extent to which IL-4-induced STAT5 activation is involved in IL-4 production from naive CD4+ T cells has not been determined. It was reported that IL-4 stimulates developing CD4+ T cells to phosphorylate STAT5 at a lower level than does IL-2 (25). However, if IL-4 and STAT5 form a positive feedback loop, IL-4-induced STAT5 activation may also affect the difference in the efficiency of IL-2-induced IL-4 production from naive CD4+ T cells. In contrast, binding of IL-4R by IL-4 induced equal activation of STAT6, which has been well characterized as a key molecule leading to Th2 differentiation (26–28), in CD4+ T cells from BALB/c and C57BL/6 (Fig. 4B). Thus, the activation capacity of STAT6 is comparable between CD4+ T cells from these two murine lines. Since STAT6 activation is dependent on the amount of IL-4 (Fig. 4B), a much higher level of STAT6 activation is likely induced due to a higher amount of IL-4 in CD4+ T cells developing in BALB/c than in C57BL/6 cells. Thus, STAT6 appears to be involved indirectly in the difference in Th differentiation between BALB/c and C57BL/6 CD4+ T cells.

Questions arise as to the mechanism underlying the difference in activation of STAT5 in BALB/c versus C57BL/6 CD4+ T cells. Since IL-2R and IL-4R appear to be expressed at similar levels on cell surfaces (Figs 2B and 4AGo) and their ligation by IL-2 or IL-4 induced similar degrees of activation in receptor-associated kinases, Jak1 and Jak3, in CD4+ T cells developing in BALB/c and C57BL/6 (Figs 3B and 4CGo), the upstream molecules for STAT5 are likely triggered at similar levels by IL-2 and IL-4 in these two cells. Accumulating evidence indicates that STAT activations are regulated by several molecules and signaling cross-talk among the Jak–STAT pathways or between Jak–STAT and other pathways (37). Thus, regulators may be involved in the difference in activation of STAT5 between these two cells. One group of such regulatory molecules is the protein tyrosine phosphatases (PTPs), and SH2 domain-containing PTP2 and PTP1B are reportedly PTPs, the substrate of which is STAT5 (38, 39). It is reasonable to assume that activated STAT5 with phosphorylated tyrosine residues would be dephosphorylated and deactivated by these PTPs more efficiently in C57BL/6 CD4+ T cells than in BALB/c cells. To date, protein levels of SH2 domain-containing PTP2 and PTP1B have been found to be equal in these two cells (data not shown). Experiments are currently underway to clarify the activation levels of these PTPs in developing CD4+ T cells. Involvement of other regulatory mechanisms such as ubiquitin-mediated protein degradation (40) is also possible, but as yet uncertain. A lower activation level of p44 ERK by IL-2 in CD4+ T cells developing in C57BL/6 than in BALB/c (Fig. 3A) suggests that differences in activation of signaling molecules may be mediated by IL-2 and IL-4 in these two CD4+ T cells involving pathways separate from the Jak1 and Jak3–STAT5 pathway and with different STAT5 cross-talk, affecting the activation state of STAT5.

In conclusion, the results of the present study provide additional evidence for the influence of genetic background on Th cell differentiation. Takatori et al. (41) recently showed STAT5 to be involved in allergic airway inflammation in mice. Taken together, we assume that manipulation of STAT5 activation would be a therapeutic approach to alleviating human Th2-mediated diseases such as asthma and atopy. In order to establish this approach, further basic research is required to elucidate the biology of STAT5.


   Acknowledgements
 
We would like to thank J. Ihle for helpful comments on the immunoprecipitation. We would also like to thank Hisako Yagi and Naoko Kodama for technical help, and Masamichi Yoshikawa for animal care. We are grateful for the donation from Emeritus Shigeko Nakanishi, Department of Microbiology and Immunology, Tokyo Women's University School of Medicine. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan, the Ministry of Public Welfare of Japan.


   Abbreviations
 
APC, antigen-presenting cell
ERK, extracellular signal-regulated kinase
{gamma}c, common {gamma}
ICOS, inducible co-stimulator
Jak, Janus kinase
[3H]TdR, [3H]thymidine
PTP, protein tyrosine phosphatase
RIgG, rat IgG
STAT, signal transducer and activator of transcription

   Notes
 
Transmitting editor: K. Yamamoto Back

Received 22 June 2006, accepted 13 September 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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