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The Journal of Immunology, 1998, 161: 4709-4718.
Copyright © 1998 by The American Association of Immunologists

IL-4 in Combination with TGF-ß Favors an Alternative Pathway of Th1 Development Independent of IL-121

Karen Lingnau, Petra Hoehn, Saadia Kerdine, Stephan Koelsch, Christine Neudoerfl, Norbert Palm, Erwin Ruede and Edgar Schmitt2

Institute for Immunology, Johannes Gutenberg University, Mainz, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-4 was found to be the essential differentiation factor for Th2 cells and simultaneously to be a potent inhibitor of Th1 development that is induced by IFN-{gamma} and IL-12. Furthermore, it was demonstrated that TGF-ß can also inhibit Th1 development. In this work, we demonstrate that polyclonal activation of Mel-14highCD4+ T cells by immobilized anti-{alpha}ßTCR mAb together with a mixture of IL-4 and TGF-ß can lead to the development of both Th1 and Th2 cells, depending on the concentration of these cytokines. Additional experiments revealed that Th1 induction by a combination of IL-4 and TGF-ß depends on the presence of endogenous IFN-{gamma}, and that this alternative Th1 development is further enhanced by IL-12, but is not dependent on this cytokine. Moreover, naive OVA323–339-specific Th cells that were stimulated by APCs and OVA323–339 peptide differentiated toward Th1 cells after priming in the presence of IL-4 in combination with TGF-ß. Hence, this finding confirmed the results obtained by polyclonal activation of naive CD4+ Th cells and implicates that this alternative Th1 development may also occur in vivo under the influence of TGF-ß and IL-4 independently of the Th1-promoting effect of IL-12.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The crucial and often cross-regulatory role of Th1 and Th2 cells for the induction and regulation of Th cell-dependent immune responses is well established in the mouse as well as in the human system (1). Moreover, it was demonstrated convincingly that cytokines are the most efficient and important regulators for the development of naive Th cells toward Th1 and Th2 cells (2). The differentiation of Th1 cells is efficiently promoted by the coordinate action of IL-12 and IFN-{gamma} (3, 4, 5). The essential role of IL-4 for Th2 differentiation has been demonstrated in vitro and in vivo using a variety of different experimental approaches (6, 7, 8, 9). The cross-regulatory properties of IL-4 that strongly suppress the Th1-inducing capacity of IL-12 and IFN-{gamma} are also well established. For instance, treatment of mice in the model of experimental allergic encephalomyelitis with altered peptide ligands resulted in an amelioration of disease that was abrogated when endogenous IL-4 was neutralized (10). This demonstrates the profound Th1-inhibiting properties of IL-4 in vivo and its potential therapeutic properties in a Th1-mediated autoimmune model. In addition, in the same model, the induction of oral tolerance resulted in the emergence of Th cell clones that produce IL-4 or TGF-ß (11). Thus, IL-4 is a potent inducer of Th2 cell development and simultaneously a powerful inhibitor of Th1 development.

TGF-ß is a highly conserved homodimeric 25-kDa protein that has been reported to be an important immunomodulatory molecule that in general exerts immunosuppressive effects by inhibiting the growth of T cells, B cells, and hemopoietic cells (12, 13, 14, 15, 16, 17). Data with respect to the influence of TGF-ß on Th cell development are controversial. On the one hand, it has been published that TGF-ß strongly promotes the generation of Th1 cells in vitro, probably by enhancing the endogenous production of IFN-{gamma}, and, simultaneously, by suppressing the secretion of IL-4 by the respective T cells (18, 19, 20). On the other hand, it has been shown that TGF-ß strongly suppresses the development of Th1 cells from naive CD4+ T cells in vitro, even in the presence of the Th1 inducer IL-12 (4). In agreement with this finding, it has recently been published that the IL-12-induced IFN-{gamma} production of neonatal human T cells was strongly inhibited by TGF-ß (21). These discrepancies can at least partially be explained by the finding that TGF-ß inhibits Th1 development in the presence of low amounts of IL-2 and stimulates Th1 development in the presence of high amounts of IL-2 (22). As already mentioned above, studies dealing with the phenomenon of oral tolerance demonstrated that Th cells that suppressed a myelin basic protein-specific Th1-dominated autoimmune response secreted TGF-ß, implying that TGF-ß inhibits the development of Th1 cells in vivo. In a colitis model, neutralization of IL-12 or blocking of the CD40-CD40 ligand interaction could inhibit experimental colitis, and treatment with TGF-ß had the same effect, thus implying that TGF-ß is inhibitory by blocking the Th1-inducing capacity of IL-12 (23, 24). Hence, TGF-ß, at least in the presence of low concentrations of IL-2, and IL-4 seem to be strong inhibitors of the development of Th1 cells both in vitro and in vivo.

In this study, we demonstrate, however, that a combination of both cytokines present during the priming phase of naive CD4+ T cells led to the preferential development of Th1 cells. Detailed analyses of this unexpected finding revealed that this property of a combination of IL-4 and TGF-ß depends on the endogenous production of IFN-{gamma}, but is independent of IL-12.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice of strains C3H/He and DBA/1 were obtained from Charles River (Sulzfeld, Germany) and bred in our own animal facility. Males and females were used at the age of 6 to 12 wk. Mutant mice that were deficient for IL-12p40 were used at the age of 8 to 12 wk. The genetic background of these mice is BALB/c (25). Mice transgenic for the OVA323–339-specific TCR-{alpha}ß (26) on a BALB/c genetic background were identified by staining PBL with the anti-TCR clonotype-specific mAb KJ1-26 and used at the age of 8 to 12 wk.

Cytokines, Abs, reagents, and Ags

Rat rIFN-{gamma} (HB B010A, Lot 51-14A03) was purchased from Laboserv GmbH (Gießen, Germany). Biologic activity of rat IFN-{gamma} on mouse cells was confirmed using the plaque reduction assay, as previously described (27); rat IFN-{gamma} proved to be as active as mouse IFN-{gamma} (mIFN-{gamma}3). Human rTGF-ß2 was given to us by Dr. G. Zenke (Novartis, Pharma-Ag, Basel, Switzerland). Human native TGF-ß1 was purchased from Pharma Biotechnology (Hannover, Germany). rmIL-4 was affinity purified using a column with anti-mIL-4 (11B11) mAb bound to Sepharose. Purified rmIL-12 (Lot MRB 91092) was a gift of Dr. M. K. Gately (Hoffmann-La Roche, Nutley, NJ). rmIL-9 and hamster anti-mIL-9 mAb C12 were given to us by Dr. J. Van Snick (Ludwig Institute, Brussels, Belgium). Rat anti-mIL-9 mAb 229.4 was generated by fusion of spleen cells of a Lewis rat immunized with reversed phase-HPLC-purified mIL-9. Anti-mIFN-{gamma} mAbs R4-6A2 (28) and AN18.17.24 (29) were gifts of Dr. M. Lohoff (Institut für Klinische Mikrobiologie, Erlangen, Germany). Anti-mIFN-{gamma} mAb XMG 1.2 (30) and anti-mIL-2 mAb S4B6.1 (31) were gifts of Dr. T. Mosmann (Department of Immunology, University of Alberta, Edmonton, Alberta, Canada). Hybridoma cells producing anti-CD4 mAb GK1.5 (32) were obtained from American Type Culture Collection (ATCC number, TIB 207; Manassas, VA); mAb were affinity purfied using protein G-Sepharose (Pharmacia, Freiburg, Germany) and coupled with FITC. Anti-mIL-4 mAb, 11B11, was a gift of Dr. W. Paul (National Institutes of Health, Bethesda, MD) (33). Anti-mIL-4 mAbs BVD4-1D11 and BVD6-24G2 and anti-mIL-2 mAbs JES6-1A12 and JES-5H4 (34) were gifts of Dr. A. O’Garra (DNAX Research Institute, Palo Alto, CA). In addition, the following mAbs were used: rat anti-mCD62L (L-selectin) mAb Mel-14 (biotinylated) (35); hamster anti-TCR-{alpha}ß mAb H57-597 (36). Mitomycin C was purchased from Sigma (M 0503; Deisenhofen, Germany). The antigenic OVA peptide (OVA323–339) was synthesized on an Applied Biosystems (Foster City, CA) peptide synthesizer.

Preparation of CD4+ T cells

CD4+Mel-14high T cells were isolated from spleen cells by positive selection using high-gradient magnetic cell separation in combination with MultiSort beads (MACS; Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany), according to the manufacturer’s instructions. The CD4 sort as well as the Mel-14 sort were performed twice. Mel-14highCD4+ T cells were enriched >99% and showed no proliferative response in the presence of Con A or soluble anti-{alpha}ßTCR mAb, which indicates negligible contamination with accessory cells.

Polyclonal primary and secondary stimulation of T cells

Culture medium was Iscove’s modified Dulbecco’s medium (Life Technologies, Grand Island, NY), supplemented with 2 mM L-glutamine, 5 x 10-5 M 2-ME, 10 IU penicillin, 100 µg/ml streptomycin, and 5% FCS, inactivated at 56°C. Primary stimulation was conducted by incubating 1 x 106 CD4+ T cells on anti-TCR-{alpha}ß mAb (5 µg/ml)-coated 24-well plates in a total volume of 1 ml of culture medium alone or with the addition of TGF-ß, IL-4, IL-12, rat IFN-{gamma}, or anti-mIFN-{gamma} mAb, as specified in the legends of the figures. After 96 h, an aliquot of the supernatant was used to determine primary IFN-{gamma}, IL-4, IL-2, and IL-9 production. The developing T cells were transferred to uncoated 24-well culture dishes, and 0.5 ml of culture medium was added. After an additional 48 h, the T cells were collected, washed, and restimulated by immobilized anti-TCR-{alpha}ß mAb (5 µg/ml) to determine their cytokine profile. After an additional 18 to 24 h, supernatants were collected and assayed for cytokines. Flow cytometry revealed that all T cell populations used for restimulation consisted of >99% CD4+ Th cells.

Stimulation of transgenic CD4+ T cells for cytokine production

Naive CD4+ Th cells (1 x 106/ml) were primarily stimulated using OVA323–339 peptide (10 ng/ml) and mitomycin C-treated (40 µg/ml/107 cells, 30 min, 37°C) A20 B tumor cells as APCs (1 x 105/ml) in a total volume of 1 ml in 24-well plates alone or in combination with TGF-ß and IL-4, as specified in Figure 9Go. After 4 days, 0.5 ml supernatant was replaced by an IL-2-containing culture medium (human IL-2, 2 ng/ml). After 6 days, the resulting Th cells were washed and restimulated (1 x 106/ml) using OVA323–339 peptide (5 µg/ml) and mitomycin C-treated A20 B tumor cells (1 x 105/ml). Supernatants were collected after 24 h for measurement of cytokines.



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  		FIGURE 9. Naive OVA323–339-specific Th cells from TCR transgenic mice differentiated toward Th1 cells in the presence of a combination of IL-4 and TGF-ß. Naive CD4+ Th cells (1 x 106/ml) from TCR transgenic BALB/c mice were primarily stimulated using OVA323–339 peptide (10 ng/ml) and A20 B tumor cells as APCs (1 x 105/ml) alone or in combination with TGF-ß2 (10 ng/ml) and IL-4 (104 U/ml). After 6 days, the resulting Th cells were washed and restimulated using OVA peptide323–339 (5 µg/ml) and A20 B tumor cells. Supernatants were collected after 24 h for measurement of cytokines. Results are expressed as mean of triplicate determinations (SE of medium, ±5 U/ml; TGF-ß, ±14 U/ml; IL-4, ±10 U/ml; TGF-ß + IL-4, ±81 U/ml). Similar results were obtained in four independent experiments.

 
Cytokine assays

IFN-{gamma}, IL-2, IL-4, and IL-9 were assayed by specific two-site ELISA with reference standard curves using known amounts of the respective cytokines. One unit of IFN-{gamma} corresponds to 5 pg/ml (standard from PharMingen, San Diego, CA; 19301T), and 1 U/ml of IL-4 corresponds to 10 pg/ml (standard from R&D Systems, Minneapolis, MN; 404-ML-005). For the detection of cytokines, we used the following mAbs: IFN-{gamma}, mAbs R4-6A2 and AN18.17.24; IL-2, mAbs JES6-1A12 and JES-5H4; IL-4, mAbs BVD4-1D11 and BVD6-24G2; and IL-9, mAbs C12 and 229.4.

Intracellular fluorescence staining of cytokines

After restimulation, cells were harvested, washed twice with PBS, and fixed in 2% paraformaldehyde (Merck, Darmstadt, Germany) in PBS for 20 min at room temperature, washed with PBS, and resuspended in saponin buffer: PBS/0.5% BSA/0.01% NaN3/0.5% saponin (saponin from Quillaja Bark; Sigma). All following steps were conducted in saponin buffer at room temperature. Briefly, 5 x 105 cells were incubated for 15 min with anti-mIL-4 mAb BVD4-1D11 (10 µg/ml), washed twice, and subsequently incubated with 2.5 µg/ml FITC-conjugated mouse anti-rat IgG, AffiniPure F(ab')2 fragment (H+L; Jackson ImmunoResearch Laboratories, West Baltimore Pike, PA) for 20 min. Cells were washed twice and incubated for 30 min with 300 µg/ml of rat Ig to saturate free binding sites of the FITC-conjugated mouse anti-rat mAb. Subsequently, cells were incubated with biotinylated anti-mIFN-{gamma} mAb AN18.17.24 (10 µg/ml) for 15 min, washed twice, and stained for 10 min with 1.25 µg/ml R-phycoerythrin-conjugated streptavidin (Jackson ImmunoResearch Laboratories). Cells were washed twice and resuspended in PBS/0.5% BSA/0.01% NaN3 without saponin and stored at 4°C in the dark until flow-cytometric analysis.

For flow-cytometric analysis, data of 10,000 cells were analyzed using a FACScan and CellQuest software (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-4 stimulates the development of IFN-{gamma}-producing Th cells in the presence of TGF-ß

CD4+Mel-14high T cells were primed in the absence of accessory cells by plate-bound anti-{alpha}ßTCR mAb in the presence of IL-12, IL-4, and/or TGF-ß. After 6 days, the developing Th cells were restimulated solely by plate-bound anti-{alpha}ßTCR mAb to determine their secondary cytokine pattern and to assess the phenotype of the resulting Th cell populations.

It has been shown that priming with IL-12 strongly increases the secondary IFN-{gamma} production of developing Th cells as a marker for Th1 differentiation. By contrast, priming with IL-4 or TGF-ß profoundly inhibits the development of Th1 cells relatively to the medium control. Figure 1Go confirms these data and, in addition, illustrates that a combination of TGF-ß and IL-4 unexpectedly enhanced the production of IFN-{gamma} by the resulting Th cells. Thus, this intriguing finding implies that a combination of the Th1 inhibitors IL-4 and TGF-ß favors the development of Th1 cells. Consequently, we investigated the influence of IL-4 in combination with TGF-ß on Th cell differentiation in more detail. Titration solely of IL-4 shows that secondary IFN-{gamma} production as a marker for Th1 development is reduced in a concentration-dependent manner (Fig. 2GoA). However, in the presence of TGF-ß, priming with IL-4 has the opposite effect (Fig. 2GoB). Increasing amounts of IL-4 in combination with a fixed concentration of TGF-ß (10 ng/ml) during the priming phase led to an increased concentration of secondary IFN-{gamma}. This effect of IL-4 peaks at 300 U/ml, while higher amounts of IL-4 inhibit secondary IFN-{gamma} production. This finding was reproduced in at least 20 independent experiments. Moreover, it should be mentioned that there was no difference in the activities of TGF-ß1 and TGF-ß2 regarding their effect on IL-4-induced Th1 development (data not shown).



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  		FIGURE 1. IL-4 stimulates the development of IFN-{gamma}-producing Th cells in the presence of TGF-ß. Mel-14highCD4+ T cells (1 x 106/ml) from unimmunized C3H/He mice were stimulated with plate-bound anti-TCR-{alpha}ß mAb H57-597 (5 µg/ml), in the absence or presence of IL-12 (1000 U/ml), IL-4 (1000 U/ml), human TGF-ß2 (10 ng/ml), and a combination of IL-4 and TGF-ß2. T cells were cultured as described in Materials and Methods, and restimulated on day 6 after primary activation with immobilized anti-TCR-{alpha}ß mAb (5 µg/ml) alone. Supernatants from secondary stimulation (18 h) were tested for the presence of IFN-{gamma}, as described in Materials and Methods. Similar results were obtained in more than 20 independent experiments.

 


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  		FIGURE 2. Intermediate concentrations of IL-4 stimulate the development of IFN-{gamma}-producing Th cells in the presence of TGF-ß. Mel-14highCD4+ T cells (1 x 106/ml) from unimmunized C3H/He mice were stimulated with plate-bound anti-TCR-{alpha}ß mAb H57-597 (5 µg/ml), in the absence or presence of different concentrations of IL-4 (10–1000 U/ml) (A), human TGF-ß2 (10 ng/ml), and a combination of IL-4 and TGF-ß2 (B). T cells were cultured as described in Materials and Methods, and restimulated on day 6 after primary activation with immobilized anti-TCR-{alpha}ß mAb (5 µg/ml) alone. Supernatants from secondary stimulation (18 h) were tested for the presence of IFN-{gamma}, as described in Materials and Methods. Similar results were obtained in at least 10 independent experiments.

 
High concentrations of IL-4 favor the development of IFN-{gamma}- and IL-4-producing T cell populations

IL-4 is the essential differentiation factor for Th2 cells. Therefore, the secondary IL-4 production as a marker for the development of Th2 cells was also assessed in parallel to the production of IFN-{gamma}. Figure 3Go shows that intermediate (300 U/ml) and high concentrations (1000 U/ml) of IL-4 induce the simultaneous production of secondary IFN-{gamma} (Fig. 3GoA) and IL-4 (Fig. 3GoB). Still higher concentrations of IL-4 (3000 U/ml) during the priming phase resulted in a further reduction of secondary IFN-{gamma} production, whereas IL-4 production reached a plateau level (data not shown). Thus, intermediate concentrations of IL-4 (100–300 U/ml) together with TGF-ß (10 ng/ml) induced a comparatively high secondary IFN-{gamma} production and a relatively low IL-4 production, while high amounts of IL-4 led to a reduced secondary IFN-{gamma} production and to an increased IL-4 production, resulting in a Th cell population producing both IFN-{gamma} and IL-4. Therefore, the question arose as to whether this Th cell population consisted of a mixture of Th1 and Th2 cells or of so-called IFN-{gamma}/IL-4 double-producing Th0 cells.



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  		FIGURE 3. IL-4 in combination with TGF-ß favors the development of an IFN-{gamma}- and IL-4-producing Th cell population. Mel-14highCD4+ T cells (1 x 106/ml) from unimmunized C3H/He mice were stimulated with plate-bound anti-TCR-{alpha}ß mAb H57-597 (5 µg/ml), in the presence of human TGF-ß2 (10 ng/ml), or a combination of TGF-ß2 and different concentrations of IL-4 (10–1000 U/ml). T cells were cultured as described in Materials and Methods, and restimulated on day 6 after primary activation with immobilized anti-TCR-{alpha}ß mAb (5 µg/ml) alone. Supernatants from secondary stimulation (18 h) were tested for the presence of IFN-{gamma} (A) and IL-4 (B), as described in Materials and Methods. Similar results were obtained in at least 10 independent experiments.

 
IL-4 in combination with TGF-ß induces the codevelopment of Th1 and Th2 cells

The assessment of various cytokines in the supernatant of bulk cultures cannot reveal whether one of these cytokines is expressed alone or together with a second cytokine by an individual Th cell. Therefore, immunofluorescence double staining of intracellular IL-4 and IFN-{gamma} in combination with flow cytometry (FACS) was applied to determine the production of these cytokines at the single cell level.

Priming of naive CD4+ T cells in the absence of exogenous cytokines (Fig. 4GoA, medium) resulted in the development of a significant number of IFN-{gamma} single-producing Th cells (7.77%, Th1 cells). Priming exclusively with IL-4 induced a considerable development of IL-4 single producers (34.26 to 0.7%, Th2 cells) depending on the concentration of the added IL-4. Moreover, in the presence of high amounts of IL-4 (1000–3000 U/ml), the default pathway of Th1 development, which can be observed after priming naive cells in the absence of exogenous cytokines, was reduced. A low IL-4 concentration (30 U/ml) had no significant influence on Th2 or on Th1 development. These findings correspond to those obtained by measuring IL-4 and IFN-{gamma} production in the supernatants of the respective Th cell populations (Fig. 2GoA and data not shown).



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  		FIGURE 4. The IFN-{gamma}- and IL-4-producing CD4+ Th cell population consists mainly of a mixture of IFN-{gamma}-producing Th1 and IL-4-producing Th2 cells. Mel-14highCD4+ T cells (1 x 106/ml) from unimmunized DBA/1 mice were stimulated with plate-bound anti-TCR-{alpha}ß mAb H57-597 (5 µg/ml), in the presence or absence of different concentrations of IL-4 (30–3000 U/ml) (A), and by human TGF-ß2 (10 ng/ml), or by a combination of TGF-ß2 and different concentrations of IL-4 (30–3000 U/ml) (B). T cells were cultured as described in Materials and Methods, and restimulated on day 6 after primary activation with immobilized anti-TCR-{alpha}ß mAb (5 µg/ml) alone, harvested after 12 h, and stained for intracellular IL-4 and IFN-{gamma}. Quadrants were set on the basis of the corresponding cytokine-blocked negative controls. Similar results were obtained in three independent experiments.

 
Priming with TGF-ß alone did not induce Th2 cells, but strongly reduced the number of Th1 cells from 7.77% (Fig. 4GoA, medium) to 1.46% (Fig. 4GoB, TGF-ß). However, when naive CD4+ T cells were primed with various concentrations of IL-4 in combination with a fixed concentration of TGF-ß (10 ng/ml), a considerable number of Th1 cells was induced by IL-4 (Fig. 4GoB). A very high concentration of IL-4, which suppressed the default Th1 pathway if added alone, induced in combination with TGF-ß a relatively low but significant number of IFN-{gamma}-producing Th1 cells (8.83%: IL-4, 3000 U/ml). By reducing the concentration of IL-4, the percentage of Th1 cells increased. It reached an optimum at about 300 U/ml IL-4 (23.42%), and then declined until at 30 U/ml background values (6.21%) of Th1 cells were reached comparable with those observed in the absence of any exogenous cytokines (Fig. 4GoA, medium, 7.77%). Simultaneously, the IL-4-induced development of Th2 cells was suppressed significantly by copriming with TGF-ß as compared with IL-4 alone (see Fig. 4GoA). Furthermore, only a minimal number of IFN-{gamma}/IL-4 double producers was detectable, indicating that a combination of IL-4 and TGF-ß preferentially induced the codevelopment of Th1 and Th2 cells.

IFN-{gamma} is essential for the Th1-inducing capacity of a combination of IL-4 and TGF-ß

IFN-{gamma} was shown to mediate at least partially the Th1-inducing effect of IL-12 for naive CD4+ T cells (4, 37). Therefore, it has been tested whether endogenous IFN-{gamma} participates in the alternative Th1 development that is induced by a mixture of IL-4 and TGF-ß. Table IGo shows the Th1-inducing effect of a combination of IL-4 and TGF-ß in the presence or absence of neutralizing anti-mIFN-{gamma} mAb in three independent experiments. The addition of neutralizing anti-mIFN-{gamma} mAb during the priming phase completely abrogated the Th1-inducing capacity of a mixture of IL-4 and TGF-ß. This indicates that endogenous IFN-{gamma} is indispensable for the alternative Th1 development. To further substantiate this finding, we applied rat IFN-{gamma} in combination with TGF-ß, IL-4, and anti-mIFN-{gamma} mAb. Rat IFN-{gamma} is active on murine T cells and cannot be neutralized by the anti-mouse IFN-{gamma} mAb XMG1.2. Rat IFN-{gamma} completely compensates for the inhibitory effect of the anti-mIFN-{gamma} mAb, thus confirming the importance of endogenously produced IFN-{gamma} for alternative Th1 development.


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  		Table I. IFN-{gamma} is essential for alternative Th1 development

 
IL-12 promotes the alternative, IL-12-independent Th1 development in an additive fashion

IL-4 as well as TGF-ß were shown to strongly inhibit the IL-12-driven Th1 development of naive CD4+ Th cells (3, 4, 38). Therefore, we compared the Th1-promoting capacity of IL-12 with the alternative pathway of Th1 development, which is induced by a combination of IL-4 and TGF-ß. Figure 5GoA confirms the Th1-inducing activity of IL-4 in combination with TGF-ß. Figure 5GoB demonstrates the Th1-inducing activity of IL-12 (Fig. 5GoB, medium) and, in addition, the efficient inhibition of this effect of IL-12 by TGF-ß. However, IL-4 in combination with TGF-ß and IL-12 led to a strong development of Th1 cells in a concentration-dependent manner above the level reached with IL-12 alone. For instance, at 300 U/ml of IL-4, the negative influence of TGF-ß was not only abrogated, but the Th1 development was considerably enhanced above the IL-12-induced level. In addition, this result suggests that the alternative Th1 development is independent of IL-12. To confirm this assumption, we used naive CD4+ T cells isolated from IL-12p40-KO mice to induce the development of Th1 cells. Figure 6Go illustrates that IL-12p40-KO Th cells show an identical response pattern as compared with Th cells that are isolated from wild-type mice (see Fig. 2Go). Priming with IL-4 alone results in a slight inhibition of the Th1 development that can be observed after stimulating the Th cells in the presence of medium alone (Fig. 6GoA). Priming in the presence solely of TGF-ß completely abrogates this default Th1 development, whereas priming the Th cells in the presence of a combination of IL-4 and TGF-ß leads to the development of Th1 cells, which depends on the concentration of IL-4 (Fig. 6GoB). Since it can be excluded that IL-12 is produced by the IL-12p40-KO Th cells or by contaminating accessory cells, these data prove that the alternative Th1 development induced by a combination of IL-4 and TGF-ß is independent of IL-12.



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  		FIGURE 5. IL-12 promotes alternative Th1 development in an additive manner. Mel-14highCD4+ T cells (1 x 106/ml) from unimmunized C3H/He mice were stimulated with plate-bound anti-TCR-{alpha}ß mAb H57-597 (5 µg/ml), in the absence or presence of human TGF-ß2 (10 ng/ml), or a combination of TGF-ß2 and different concentrations of IL-4 (30, 300, 3000 U/ml, A). In addition, priming was performed in the presence of IL-12 (1000 U/ml, B). T cells were cultured as described in Materials and Methods, and restimulated on day 6 after primary activation with immobilized anti-TCR-{alpha}ß mAb (5 µg/ml) alone. Supernatants from secondary stimulation (18 h) were tested for the presence of IFN-{gamma}, as described in Materials and Methods. Similar results were obtained in five independent experiments.

 


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  		FIGURE 6. Alternative Th1 development induced by a combination of IL-4 and TGF-ß is independent of IL-12. Mel-14highCD4+ T cells (1 x 106/ml) from IL-12p40-KO mice were stimulated with plate-bound anti-TCR-{alpha}ß mAb H57-597 (5 µg/ml), in the absence or presence of different concentrations of IL-4 (30–1000 U/ml) (A), human TGF-ß2 (10 ng/ml), and a combination of IL-4 and TGF-ß2 (B). T cells were cultured as described in Materials and Methods, and restimulated on day 6 after primary activation with immobilized anti-TCR-{alpha}ß mAb (5 µg/ml) alone. Supernatants from secondary stimulation (18 h) were tested for the presence of IFN-{gamma}, as described in Materials and Methods. Similar results were obtained in three independent experiments.

 
The inhibition of primary IL-2 production by TGF-ß is compensated for by IL-4

TGF-ß strongly inhibits the proliferation and IL-2 production of T cells (39, 40, 41), whereas IL-4 enhances the production of IL-2 by freshly isolated CD4+ T cells after activation via plate-bound anti-CD3 Abs (42). Therefore, we tested whether IL-4 can compensate for the inhibitory effect of TGF-ß. Figure 7GoA illustrates that TGF-ß strongly reduced the primary IL-2 production of the control (medium), while IL-4 slightly increased it. Priming naive Th cells with a mixture of IL-4 and TGF-ß led to a complete abrogation of the inhibitory effect of TGF-ß by IL-4 in a concentration-dependent manner and induced a primary IL-2 production that still exceeded the level of the control (medium). One may argue that IL-4 simply blocks the effect of TGF-ß on Th cells, for instance by down-regulating its receptor. That this is obviously not the case is illustrated in Figure 7GoB, which shows primary IL-9 production of naive Th cells. IL-9 production was weakly stimulated in the presence of TGF-ß, while IL-4 had no effect. In agreement with published data (43), a combination of IL-4 and TGF-ß strongly enhanced the production of IL-9. Since IL-4 alone had no effect on the production of IL-9, this indicates that TGF-ß was still acting on the T cells in the presence of even high concentrations (1000 U/ml) of IL-4. Thus, IL-4 does not inhibit, but modulates the effect of TGF-ß on naive Th cells with regard to the production of IL-9 and simultaneously with respect to their differentiation toward Th1 cells.



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  		FIGURE 7. IL-4 compensates for the inhibition of primary IL-2 production by TGF-ß. Mel-14highCD4+ T cells (1 x 106/ml) from unimmunized C3H/He mice were stimulated with plate-bound anti-TCR-{alpha}ß mAb H57-597 (5 µg/ml), in the absence or presence of human TGF-ß2 (10 ng/ml), or a combination of TGF-ß2 and different concentrations of IL-4 (10–1000 U/ml). T cells were cultured as described in Materials and Methods, and primary IL-2 (A) and IL-9 (B) production was tested after 96 h, as described in Materials and Methods. Similar results were obtained in more than 20 independent experiments.

 
IL-4 increases Th1 development in the presence of a combination of TGF-ß and saturating amounts of IL-2

We have shown previously that TGF-ß inhibits Th1 differentiation in the presence of low amounts of IL-2, and that it promotes Th1 differentiation in the presence of high amounts of IL-2 (22). Therefore, restoration of the endogenous IL-2 production by IL-4 in the presence of TGF-ß might be one mechanism that leads to the promotion of Th1 differentiation by a combination of IL-4 and TGF-ß. Thus, naive CD4+ T cells were primed in the presence of a saturating concentration of human IL-2 in combination with anti-mouse neutralizing IL-2 Abs to make sure that the different amounts of primary IL-2 secreted in the presence of TGF-ß, IL-4, or a mixture of TGF-ß and IL-4 cannot differentially influence Th cell development in this experiment. Figure 8Go demonstrates that IL-4 in combination with TGF-ß and IL-2 can raise Th1 development strongly above that level, which can be reached by a mixture of TGF-ß and IL-2, indicating that the stimulation of Th1 development after priming by a combination of IL-4 and TGF-ß is mainly an effect of IL-4 and only partially mediated by endogenous IL-2.



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  		FIGURE 8. IL-4 promotes Th1 development in the presence of a combination of TGF-ß and saturating amounts of human IL-2. Mel-14highCD4+ T cells (1 x 106/ml) from unimmunized C3H/He mice were stimulated with plate-bound anti-TCR-{alpha}ß mAb H57-597 (5 µg/ml), in the presence of a combination of anti-mIL-2 mAb (S4B6.1, 20 µg/ml) and human IL-2 (100 ng/ml) alone, or together with TGF-ß2 (10 ng/ml), IL-4 (300 U/ml), or a mixture of TGF-ß2 and IL-4. T cells were cultured as described in Materials and Methods, and secondary IFN-{gamma} production was tested as described in Materials and Methods. Results are expressed as mean of fivefold determinations (SE of medium, ±12 U/ml; TGF-ß, ±65 U/ml; IL-4, ±12 U/ml; TGF-ß + IL-4, ±168 U/ml). Similar results were obtained in four independent experiments.

 
Naive OVA323–339-specific Th cells from TCR transgenic mice differentiate toward Th1 cells in the presence of a combination of IL-4 and TGF-ß

Although rather artificial, polyclonal activation of naive Th cells in the absence of APCs clearly reveals the differentiation potency of such cells independently of the complex influence of APC-dependent costimuli mediated, for instance, via CD40, CD80, CD86, and several APC-derived cytokines, among them IL-1, IL-6, IL-10, IL-12, and different members of the chemokine family. Furthermore, the type of APC (dendritic cell, macrophage, B cell) and the concentration of the Ag as well as the affinity of the TCR to a given peptide/MHC combination were shown to affect the development of Th cells (44, 45, 46). Therefore, we tried to choose conditions for the Ag-specific stimulation of naive Th cells that do not favor the development of Th1 cells per se. For this purpose, the B cell tumor line A20 was used as a well-defined source of APC that does not produce IL-12 (47). As Ag, the OVA323–339 peptide was used at a concentration of 10 ng/ml that has been shown not to promote the development of Th1 cells (45).

The primary Ag-specific activation of naive CD4+ Th cells led to a Th cell population that produced rather low amounts of IFN-{gamma} after restimulation (Fig. 9Go, medium). Priming of such Th cells in the presence of TGF-ß or IL-4 alone did not significantly alter secondary IFN-{gamma} production. However, priming with a combination of IL-4 and TGF-ß strongly enhanced the secondary IFN-{gamma} production of the resulting Th cell population, thus confirming the results obtained by polyclonal TCR activation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cytokines play a pivotal role for the differentiation of naive CD4+ T cells toward the Th1 and Th2 subtypes. For the development of Th1 cells, it was convincingly shown that IL-4 and TGF-ß, at least in the presence of low amounts of IL-2, are potent negative regulators both in vitro and in vivo. Therefore, it has been unexpected to find that the combination of two Th1 inhibitory agents resulted in the promotion of Th1 development.

Since highly purified naive CD4+ Th cells or, alternatively, naive IL-12p40-KO Th cells were used for these experiments, it can be excluded that endogenously produced IL-12 plays any role, suggesting that the combination of IL-4 and TGF-ß may induce an alternative, IL-12-independent pathway of Th1 development. The balance between the action of IL-4 and TGF-ß is obviously of critical importance. Low concentrations of IL-4 (10–30 U/ml) had no effect on Th cell differentiation in the presence of a fixed concentration of TGF-ß (10 ng/ml), whereas high concentrations of IL-4 (1000–3000 U/ml) led in most cases to a comparatively poor Th1 development and simultaneously favored the development of Th2 cells. Only intermediate concentrations of IL-4 (100–300 U/ml) induced a maximum Th1 development in combination with TGF-ß (10 ng/ml). With lower concentrations of TGF-ß, optimal Th1 development, although less pronounced than with 10 ng/ml of TGF-ß, also required less IL-4, and concentrations of TGF-ß above 20 ng/ml inhibited Th1 development in general (data not shown).

Using IL-12 in combination with a mixture of IL-4 and TGF-ß increased Th1 development in an additive manner if an optimal combination of IL-4 and TGF-ß was applied. Since an alternative Th1 development could also be observed with naive IL-12p40-KO Th cells, these data suggest that IL-12 and the mixture of IL-4 and TGF-ß mediate Th1 development via independent pathways (Fig. 10Go).



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  		FIGURE 10. IL-4 in combination with TGF-ß favors an alternative pathway of Th1 development independent of IL-12.

 
Concerning the mechanism underlying the Th1-promoting effect of the mixture of TGF-ß and IL-4, one may speculate that additional endogenously produced cytokines that are regulated by IL-4 and TGF-ß might be involved. A good candidate is IL-2, which has been shown to be down-regulated by TGF-ß and up-regulated by IL-4 (39, 42). The assessment of the production of IL-2 by naive Th cells 4 days after primary activation revealed that TGF-ß abrogated this IL-2 production, and that addition of IL-4 compensated for this inhibitory effect of TGF-ß. Furthermore, we have shown previously that on the one hand, TGF-ß inhibits Th1 differentiation in the presence of low amounts of IL-2, and on the other hand, it promotes Th1 differentiation in the presence of high amounts of IL-2 (22). Thus, restoration of the endogenous IL-2 production by IL-4 in the presence of TGF-ß might be one mechanism that leads to the promotion of Th1 differentiation by a combination of IL-4 and TGF-ß. However, priming naive Th cells by a combination of saturating amounts of IL-2 and TGF-ß in the presence or absence of IL-4 revealed that a combination of IL-2 and TGF-ß induced a comparatively low Th1 development that is strongly enhanced by the addition of IL-4 (see Fig. 8Go). This indicates that the Th1-promoting effect of IL-4 in combination with TGF-ß is mediated only to marginal extent by endogenous IL-2.

Since polyclonal activation of naive CD4+ Th cells by immobilized anti-TCR mAb is a rather artificial approach, we also used an Ag-specific system. Naive OVA323–339-specific Th cells were activated under conditions that do not favor Th1 development per se by using IL-12-nonproducing A20 B tumor cells and a low OVA323–339 peptide concentration (10 ng/ml). Ag-specific priming of naive Th cells in the presence of a combination of IL-4 and TGF-ß resulted in considerably enhanced Th1 development as compared with the medium control or the development of Th1 cells after priming solely with IL-4 or TGF-ß. Thus, these data corroborated the results obtained by polyclonal activation of naive CD4+ Th cells and suggest that the alternative, IL-12-independent Th1 development may also play a role in vivo.

Nevertheless, TGF-ß and IL-4 were both found to inhibit Th1 differentiation in vivo in different experimental systems (48, 49, 50). Therefore, it might still be debatable whether the data presented herein were of any in vivo relevance. However, it recently has been published that treatment of rats with IL-4 in an experimental autoimmune uveoretinitis model resulted in an aggravation of the disease (51). Although it has been shown that experimental autoimmune uveoretinitis is a typical Th1-dependent disease (52), analysis of the cytokines produced in response to the immunizing Ag by splenocytes ex vivo revealed that the concentrations of IFN-{gamma} and TNF-{alpha} were up-regulated following IL-4 treatment. Also in vitro, IL-4 stimulated the production of IFN-{gamma} by Con A-activated splenocytes isolated from naive mice and, in analogy to our results, rather low concentrations of IL-4 enhanced the production of IFN-{gamma}, whereas it was inhibited at high concentrations of IL-4. These authors speculated that suboptimal concentrations of IL-4 enhance Th1 development and the production of inflammatory cytokines, while high concentrations of IL-4 promote a Th2-dominated immune response. Concerning the effects of TGF-ß, it was shown that this cytokine plays a crucial role in immune-privileged sites, especially in the intraocular microenvironment, where significant quantities of TGF-ß could be detected (53, 54). Hence, in the light of our findings, it is conceivable that low amounts of IL-4 aggravate experimental autoimmune uveoretinitis at least partially by enhancing a pathologic Th1 response in combination with the locally produced TGF-ß.

In addition, IL-4 was found to have exacerbating effects in a murine Ag-induced arthritis model (55). Treatment with neutralizing anti-IL-4 mAb inhibited the disease efficiently, indicating that in this model IL-4 has proinflammatory properties. It was discussed that Th2 cells themselves could promote a proinflammatory response, as demonstrated by Müller et al. (56), or that IL-4 could act as a costimulator of Th1 cells. In view of our data, we would like to favor the latter possibility. Moreover, it was shown that in IL-4 transgenic mice, the basic expression of IFN-{gamma} in the spleen is up-regulated in vivo (57), suggesting that endogenous IL-4 directly stimulates the production of IFN-{gamma} by T cells and/or indirectly favors IFN-{gamma} production by inducing the development of Th1 cells. Finally, it has recently been published, IL-4 is required for the development of a protective Th1 response to Candida albicans (58). These authors described that the IFN-{gamma} production of CD4+ Th splenocytes was reduced in IL-4 KO mice in response to virulent C. albicans. On the one hand, they presumed that the defective production of IL-12 by neutrophils in IL-4 KO mice was at least partially responsible for the impairment of Th1 development. On the other hand, they demonstrated that IL-4 also directly promotes the development of Th1 cells presumably independent of IL-12.

In summary, our data provide evidence for an ambivalent influence of IL-4 on the development of Th2 and Th1 cells. The combination of certain concentrations of the Th2 promoter IL-4 and TGF-ß led to the development of Th1 cells alternative and additive to that induced by IL-12 (Fig. 10Go). Thus, a mixture of differentiation factors can have biologic effects opposite to those of the single components. Finally, these results exemplify the plasticity of Th cell development that depends on an integration of signals mainly induced by different cytokines.


   Acknowledgments
 
We thank our colleagues Drs. M. Gately, M. Lohoff, F. Mattner, H. Mossmann, W. Müller, A. O’Garra, W. Paul, J. v. Snick, and G. Zenke for their generous gifts of antibodies, cytokines, and cell lines.


   Footnotes
 
1 This work was supported by Deutsche Forschungsgemeinschaft, SFB 311. Back

2 Address correspondence and reprint requests to Dr. Edgar Schmitt, Institute for Immunology, Johannes Gutenberg University, 55101 Mainz, Germany. E-mail address: Back

3 Abbreviations used in this paper: m, mouse; KO, knockout. Back

Received for publication December 8, 1997. Accepted for publication July 1, 1998.


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