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The Journal of Immunology, 1999, 163: 2432-2442.
Copyright © 1999 by The American Association of Immunologists

CD28 Costimulation Accelerates IL-4 Receptor Sensitivity and IL-4-Mediated Th2 Differentiation1

Masato Kubo2,*, Masakatsu Yamashita*,{dagger}, Ryo Abe*, Tomio Tada*, Ko Okumura{ddagger},§, John T. Ransom and Toshinori Nakayama*,{dagger}

* Division of Immunobiology, Research Institute for Biological Sciences, Science University of Tokyo, Noda City, Chiba, Japan; {dagger} Department of Molecular Immunology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba, Japan; {ddagger} Department of Immunology, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan; § CREST, Japan Science and Technology Corporation, Tokyo, Japan; and Cadus Pharmaceutical Corp., Tarrytown, NY 10591


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4R{alpha}-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T helper lymphocytes can be divided into two distinct subsets of effector cells based on their functional capacity and their cytokine production profile. The Th1 phenotype of CD4+ T cells secretes cytokines associated with cell-mediated immune responses, such as IL-2, IFN-{gamma}, and TNF-{alpha}. Th1 cells are responsible for defense against infectious intracellular microorganisms. The Th2 phenotype secretes cytokines that help the proliferation and differentiation of B cells, such as IL-4, IL-5, and IL-6, and are responsible for defense against extracellular pathogens and the development of allergic immune reactions (1, 2). The CD4+{alpha}+ß+ T cells leaving the thymus have a naive phenotype and produce IL-2 and a small amount of IL-3 and GM-CSF. The naive T cells differentiate into either Th1 or Th2 phenotypes following the appropriate activation signals.

There is much evidence that Th cell differentiation may reflect the nature of the antigenic stimulation and the cytokine environment to which the cells have been exposed (2, 3). Most work addressing the role of cytokines in T cell differentiation has used in vitro priming systems. IL-12 is thought to be a major factor for promoting Th1 differentiation because the presence of IL-12 during the priming stage directly augments Th1 differentiation (4, 5, 6). In contrast, the presence of IL-4 in the priming culture promotes Th2 differentiation (7, 8, 9, 10). A number of recent studies using mice with germline disruptions strongly supports the evidence accumulated with in vitro priming systems. For instance, disruption of either the IL-4 gene or the STAT6 gene resulted in mice that failed to generate Th2 cells (11, 12, 13). STAT6 is known to be a transcriptional factor involved in the IL-4-mediated Jak/STAT signaling pathway (14, 15). Recently, we have described that the activation status of STAT6 differs between Th1 and Th2 cells, and that Th2 cell-specific IL-4 expression is determined by the inhibition of a silencer gene caused by activated STAT6 (16). Therefore, the IL-4-mediated Jak/STAT signaling pathway plays an important role in determining the development of Th1 and Th2 cells. In contrast, little is known about the nature of TCR signaling events during the priming stage for Th cell differentiation. Targeted disruption of the CD4 gene selectively impairs Th2 differentiation (17). Similarly, we have reported a requirement of Lck activity for Th2 differentiation using mice that overexpress a dominant negative form of Lck (18). However, despite these findings, there remain questions such as the nature of the association between Ag stimulation through TCR and the IL-4R signaling pathway.

In this study, we focus on role of costimulatory signals on Th cell differentiation and on how both Ag stimulation and IL-4 mutually regulate Th2 generation. Th1/Th2 polarization has been analyzed previously in transgenic (Tg)3 TCR systems (7, 8, 9, 19). However, the combination of Tg TCR and Ag/MHC complexes in certain genetic backgrounds seems to automatically determine the polarization pattern. For example, naive CD4+ T cells from OVA-specific TCR Tg, DO11.10 mice with BALB background preferentially differentiate into Th2 cells by antigenic stimulation without the addition of IL-4 (19, 20, 21). To exclude those biases, we used nontransgenic congenic mice, and Th cell differentiation was initiated by polyclonal activators such as Con A or anti-TCR. Stimulation with anti-TCR was not sufficient for Th2 differentiation, while activation with Con A generated a significant number of Th2 cells. Costimulatory molecules such as CD28 influence this difference. In general, antigenic stimulation involves both TCR and costimulatory signals, and the coordination of these two signals strongly affects IL-4R function during the Th cell differentiation process. Most importantly, we have obtained evidence that an increase in the sensitivity of IL-4R to IL-4 is caused by coincidental TCR and CD28 signaling and is required for Th2 differentiation.


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

BALB/c and C57BL/6 mice were purchased from Clea (Tokyo, Japan). OVA-specific TCR{alpha}ß Tg mice (DO 11.10 Tg) were kindly distributed by Dr. Dennis Loh (Roche, Nutley, NJ). C57BL/6 STAT6 knockout mice were provided by Dr. Shizuo Akira (Hyogo College of Medicine, Hyogo, Japan) (13).

Cytokines and Abs

The reagents (for IL-2, JES6-1A12 and JES6-5H4 biotin; for IFN-{gamma}, R4-1A12 and XMG1.2 biotin; and for IL-4, BVD4-1D11 and BVD6-24G2 biotin) were purchased from PharMingen (La Jolla, CA) and used for ELISA. The reagents for cytostaining analysis, anti-IFN-{gamma} (XMG1.2) FITC and anti-IL-4 (11B11) PE, were obtained from PharMingen. Anti-IL-4R mAb (M-1) was purchased from Genzyme (Cambridge, MA). Anti-IL-4 mAb (11B11) was generously gifted by Dr. Waul (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). Anti-CD44 mAb (KM201) was kindly provided by Dr. K. Miyake (Saga Medical College, Saga, Japan). Anti-CD28 mAb (PV-1) was previously described (22). Mouse rIL-4 was purchased from PeproTech (London, U.K.). The antigenic OVA synthetic peptide (residues 323–339; ISQAVHAAHAEINEAGR) was synthesized by BEX (Tokyo, Japan).

Preparation of CD4+ naive T cells and induction of Th cells

To isolate CD4+ naive T cells, spleen cells were incubated with anti-CD8 mAb (53-6.72) at 4°C and the cells were incubated on the plate-coated anti-mouse Ig to eliminate B and CD8+ T cells. The CD4+-enriched T cells were incubated with anti-CD44 mAb (KM201), followed by the cytotoxic killing treatment with Low-Tox-M rabbit complement (Cederlane Laboratories, Hornby, Ontario, Canada). These CD4+ naive T cell preparations contain more than 80% CD4+, CD44- T cells. APCs were prepared from spleen cells by the cytotoxic killing treatment with anti Thy-1.2 mAb (30H12) and complement. The CD4+ naive T cells were stimulated with either antigenic peptide, Con A, anti-TCR (H57-597), or anti-TCR plus anti-CD28 mAb (PV-1). Ag and Con A stimulation was performed in the presence of the splenic APC, while the anti-TCR stimulation was performed with plate-bound Ab. After 5 days, the primed CD4+ T cells were repurified by panning with anti-CD8 mAb and anti-mouse Ig and with anti-TCR mAb to induce cytokine production.

Intracellular cytokine staining

To prevent release of cytokines, the naive CD4+ T cells were restimulated with anti-TCR mAb for 6 h in the presence of 4 µM monensin (Sigma, St. Louis, MO). Then, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton X. After blocking with PBS containing 3% BSA, cells were stained with anti-IFN-{gamma} (XMG1.2) FITC and anti-IL-4 (11B11) PE, as described previously (18). Flow-cytometric analysis was performed on FACSort and CellQuest software (Becton Dickinson, San Jose, CA).

Flow cytometry analysis

Cells were suspended in PBS supplemented with FCS and 0.1% sodium azide. In general, 106 cells were blocked with anti-FcR (2.4 G2) and stained using a standard method, as described previously (16). Flow cytometry analysis was performed with FACSort (Becton Dickinson) using CELL Quest software (Becton Dickinson).

Measurement of cytokine concentrations by ELISA

CD4+ naive T cells were stimulated with different stimuli, and after 24 h, the culture supernatants were harvested. Cytokine concentration in the supernatant was measured as described previously (18). Briefly, the supernatants were applied on the plastic plate coated with specific Ab against certain cytokines. After washing, the plate was probed with HRP-conjugated streptavidin (Zymed, San Francisco, CA) and developed with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Kirkegard & Perry Laboratories, Gaithersburg, MD). The 405 nm absorbance was measured by spectrophotometer (Bio-Rad Laboratories, Hercules, CA).

SDS-PAGE and Western blot analysis

Total cellular lysates of 2 x 106 cells were prepared in RIPA solution (50 mM Tris-HCl, 1% Nonidet P-40, 0.25% sodium-deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, aprotinin (1 µg/ml), leupeptin (1 µg/ml), pepstatin (1 µg/ml), 1 mM NaVO4, and 1 mM NaF) and separated by electrophoresis on 7.5% SDS-PAGE. Jak3, the IL-4R{alpha}-chain, and STAT6 were immunoprecipitated from the cell lysates of 8 x 106 cells using anti-Jak3 antisera (Upstate Biotechnology, Lake Placid, NY), anti-IL-4R{alpha}-chain mAb (M1) (Genzyme), and anti-STAT6 mAb (R&D Systems, Minneapolis, MN). The samples were loaded and run on 7.5% SDS-PAGE gel. After electrotransfer to polyvinylidene difluoride membrane, the blots were probed with HRP-conjugated anti-phosphotyrosine Ab (HRP-RC20) (Transaction Laboratory, Lexington, KY). To estimate protein concentration in lysates, the phospho-blots of total cellular lysates were stripped with phosphate buffer containing 2% SDS and 0.1 M 2-ME. The membranes were reprobed with either anti-STAT6 (Transaction Laboratory) or anti-Jak3 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) and developed with HRP-conjugated anti-mouse Igs and anti-rabbit Igs (Dako, Glostrup, Denmark).

Measurement of IL-4R affinity

T cells were resuspended in 4°C HBSS at 1 x 107 cells/ml. IL-4 was labeled by enzyme reaction (glucoseoxidase and lactoperoxidase) with 125I, and labeled IL-4 was purified with PD-10 column. Cells were incubated with 125I-labeled mouse rIL-4 (600 Ci/mmol) for 2 h at 4°C and added separating solution (8:1 mixture of di-n-butyl phthalate and olive oil). After centrifuge for 2 min at 4000 rpm, the reaction solution and half of separating solution were discarded, and the rest of the solution was counted by Auto well gamma system (Aloka, Tokyo, Japan).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The activation signal through TCR cross-linking using mAbs is insufficient for Th2 differentiation

First, it was important to characterize the Th development properties in the systems. Naive CD4+ T cells were obtained from OVA-specific TCR Tg (DO11.10) mice that were BALB/c background and stimulated with antigenic peptide, Con A, or immobilized TCR mAb (H57-597). Antigenic peptide and Con A stimulation was performed in the presence of splenic APC, while anti-TCR stimulation was performed with plate-bound Ab. After 5 days, the primed CD4+ T cells were restimulated with anti-TCR mAb for 6 h, and the Th1 and Th2 differentiation profile was assessed by intracellular cytokine staining. In this study, we defined Th1 cells as those producing IFN-{gamma} but not IL-4, and the Th2 cells as those producing IL-4 but not IFN-{gamma}.

DO11.10 T cells showed significant Th2 development in response to either antigenic peptide or Con A in the presence of BALB/c splenic APC (Fig. 1GoA). About 25% of the population were Th2 cells after stimulation by the antigenic peptide and Con A stimulation yielded a similar profile. Very little Th2 differentiation occurred following anti-TCR stimulation. To study the possibility that the distinct polarization was due to the absence of APC, the Th cells were induced by soluble anti-TCR in the presence of APC. However, these treatments were again insufficient for the generation of Th2 cells (Fig. 1Go, A and B).



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  		FIGURE 1. Th1/Th2 polarization pattern of CD4+ T cells induced by antigenic peptide, Con A, and anti-TCR mAb stimulation. A, Th1/Th2 polarization pattern of DO11.10 CD4+ T cells. Naive CD4+ T cells were purified from DO11.10 Tg mice and stimulated with antigenic peptide (1 µM), Con A (2.5 µg/ml), immobilized anti-TCR mAb (30 µg/ml), or soluble anti-TCR mAb (10% culture supernatant). After 5 days, the primed CD4+ T cells were restimulated with the anti-TCR mAb for 6 h in the presence of 4 µM monensin and stained for intracellular IL-4 and IFN-{gamma}. B, Th1/Th2 polarization pattern of BALB/c CD4+ T cells. Naive CD4+ T cells were purified from BALB/c mice and stimulated with Con A (2.5–10 µg/ml), immobilized anti-TCR mAb (100 µg/ml), immobilized anti-CD3 mAb (100 µg/ml), or soluble anti-TCR mAb (10% culture supernatant) with APC. The assessment of Th1 and Th2 generation was as described in the legend for A. Data in A and B are representative of three similar experiments.

 
To determine whether these profiles were consistent with nontransgenic mice, naive CD4+ T cells from normal BALB/c mice were activated by polyclonal T cell activators. Stimulation with Con A generated a significant number of Th2 cells (Fig. 1GoB), while initial activation by Abs against TCR or CD3 was insufficient for Th2 development (Fig. 1GoB). Similar frequencies of Th1 cells were detected after stimulation with a range of immobilized anti-TCR Ab from 1–300 µg/ml (data not shown), indicating that TCR cross-linking with mAb was sufficient for Th1 differentiation, but not for Th2 differentiation.

The addition of exogenous IL-4 (10 ng/ml) into the anti-TCR induction culture of BALB/c T cells resulted in the development of a significant number of Th2 cells (Fig. 2GoA). IL-4 itself was insufficient for Th2 generation, although all naive CD4+ T cells expressed significant amounts of IL-4R (Fig. 6GoA). We examined whether IL-4 was required before or after TCR cross-linking to promote Th2 differentiation. To test this, IL-4 was added at different time points into the anti-TCR induction culture of CD4+ T cells obtained from BALB/c and B10.D2 mouse. Significant numbers of Th2 cells were generated when IL-4 was added either simultaneously with or after the anti-TCR (Fig. 2GoB). The addition of IL-4 before TCR stimulation failed to promote Th2 differentiation. These results indicated that the sequence of a TCR activation signal followed by an IL-4 signal should be critical for Th2 differentiation and suggested that the TCR-mediated signal might modify the properties of the IL-4R.



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  		FIGURE 2. Requirement of exogenous IL-4 for Th2 differentiation induced by the TCR cross-linking. A, The addition of IL-4 initiated significant Th2 differentiation. Naive CD4+ T cells were purified from BALB/c mice and stimulated with immobilized anti-TCR mAb (100 µg/ml) in the presence or absence of exogenous IL-4 (10 ng/ml). Th1 and Th2 generation was assessed as described in the legend for Fig. 1GoA. Data are representative of three similar experiments. B, TCR activation signal followed by an IL-4 addition was critical for Th2 differentiation. Naive CD4+ T cells were purified from BALB/c and B10.D2 mice and stimulated with immobilized anti-TCR mAb (100 µg/ml). Exogenous IL-4 (10 ng/ml) was added at different time points into the induction culture. When T cells were cultured with IL-4 before TCR stimulation, exogenous IL-4 was washed out after cultivation with IL-4 at 37°C, then T cells were stimulated with immobilized anti-TCR mAb. Th1 and Th2 generation was assessed as described in the legend for Fig. 1GoA.

 


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  		FIGURE 6. CD28 costimulation increased tyrosine phosphorylation followed by IL-4 stimulation. A, TCR cross-linking and CD28 costimulation increased IL-4-dependent tyrosine phosphorylation of CD4+ T cells. BALB/c CD4+ naive T cells were stimulated with Con A (5 µg/ml), anti-TCR mAb (100 µg/ml), or anti-TCR mAb (100 µg/ml) plus 10% CD28 mAb in the presence of anti-IL-4R mAb (10 µg/ml). After 36 h, cells were washed with fresh medium and cultured at 37°C for 12 h for starvation. The activated 2 x 106 cells were stimulated in the presence or absence of IL-4 (10 ng/ml) for 10 min. Total cell lysates were loaded on 7.5% SDS-PAGE and were probed with HRP anti-phosphotyrosine mAb (RC20). The same blot was stripped and reprobed with anti-Jak3 and anti-STAT6. The arrow I indicated 160~170 kDa corresponding to the size of IRS-1 (165 kDa). The arrow II indicated 130~140 kDa corresponding to the size of Jak1, Jak3, and the IL-4R{alpha}-chain. The arrow III indicated unknown phosphorylation bands. The arrow II indicated about 100 kDa corresponding to the size of STAT6. The amount of Jak3 and STAT6 protein in the cells indicates under the Jak3 and STAT6 column as optimal density. B, TCR cross-linking and CD28 costimulation increased tyrosine phosphorylation of Jak3, the IL-4R{alpha}-chain, and STAT6. The Jak3, IL-4R{alpha}-chain, and STAT6 molecules were immunoprecipitated from the cellular lysates prepared in B. The immunoprecipitates were loaded on SDS-PAGE and were probed with HRP-RC20.

 
CD28 costimulation significantly enhances IL-4-mediated Th2 differentiation

CD28 costimulation is involved in Th2 differentiation and allows naive T cells to respond to lower levels of TCR occupancy (23, 24, 25, 26). CD28 costimulation was provided by the addition of anti-CD28 mAb (PV-1) into the anti-TCR induction culture. Soluble forms of this Ab activate the CD28-mediated signaling pathway (22). Purified PV-1 mAb (0.1–10 µg/ml) was added into the induction culture with plate-bound anti-TCR. As shown in Fig. 3GoA, CD28 costimulation with anti-TCR resulted in a significant number of Th2 cells (11–18%). In contrast, the Th1 frequency was decreased to almost half of that generated by anti-TCR mAb alone. Similarly, CD28 costimulation promoted significant enhancement of Th2 generation even in C57BL/6 (B6) strain, which favors Th1 response. The proportion of Th2 cells in B6 mice was constantly between 3 and 10%; thus, the enhancement was more pronounced in BALB/c mice (Fig. 3GoB, middle).



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  		FIGURE 3. The addition of a CD28 costimulation into TCR cross-linking initiated IL-4-mediated Th2 differentiation. A, Th2 development by the addition of a CD28 costimulation. CD28 costimulation was provided by adding purified anti-CD28 mAb (0.1–10 µg/ml) into the induction culture with immobilized anti-TCR mAb (30 µg/ml). Th1 and Th2 generation was assessed as described in the legend for Fig. 1GoA. Data are representative of three similar experiments. B, Th2 differentiation induced by CD28 costimulation is dependent on IL-4 and IL-4R signaling pathway. Naive CD4+ T cells were prepared from BALB/c, C57BL/6, and C57BL/6 STAT6 knockout mice and stimulated with anti-TCR mAb (30 µg/ml) and anti-CD28 mAb (10% culture supernatant). The effect of IL-4 was examined by the addition of either anti-IL-4 mAb (10 µg/ml) or exogenous IL-4 (10 ng/ml). Th1 and Th2 generation was assessed as described in the legend for Fig. 1GoA.

 
To eliminate the possibility that CD28 costimulation activated an unknown pathway other than the IL-4- and STAT6-mediated pathway, anti-IL-4 mAb (11B11) was added to the induction. The treatment with anti-IL-4 mAb completely blocked Th2 development in BALB/c cells (Fig. 3GoB, upper). Next, the effect of CD28 costimulation was examined in C57BL/6 (B6) STAT6 knockout mice. In STAT6 knockout mice, CD28 costimulation did not promote Th2 differentiation even after the addition of exogenous IL-4 (Fig. 3GoB, lower). Because Th1 generation occurred at the same level (14%) as that observed in B6 mice, abolishment of Th2 differentiation was not due to a defect in the initial activation signal. Taken together, these results indicate that the enhancement of Th2 generation initiated by CD28 costimulation was dependent on the IL-4R-mediated signaling pathway.

CD28 costimulation promotes Th2 differentiation by alteration of the sensitivity threshold for IL-4

Next, we asked whether the inability of TCR cross-linking with mAb to generate sufficient Th2 cells was due to the modest IL-4 production or the low sensitivity of the IL-4R. To examine quantitative differences in cytokine production promoted by different stimuli, naive CD4+ T cells were stimulated by various doses of Con A and anti-TCR mAb. To examine the effect of CD28 costimulation, anti-CD28 mAb was added into the anti-TCR stimulation. The amounts of IL-2, IL-4, and IFN-{gamma} in the culture supernatants were measured at 24 h after stimulation. As shown in Fig. 4GoA, T cells stimulated by Con A produced over 10 times more IL-2 than the T cells stimulated by 100 µg/ml of anti-TCR. The T cells were capable of producing detectable amounts of IL-4 following stimulation by Con A as well as anti-TCR. Stimulation with 10 µg/ml of Con A induced 120 pg/ml of IL-4, while the anti-TCR stimulation induced nearly 40 pg/ml. Stimulation with either Con A or anti-TCR induced very low amounts of IFN-{gamma}. There were no quantitative differences in IFN-{gamma} production between Con A and the anti-TCR. The addition of a CD28 costimulation dramatically augmented IL-2 production by 10- to 20-fold, while the same treatment modestly affected the IL-4 and IFN-{gamma} production. Because the enhancement of IL-4 production by the CD28 costimulation was only 2- to 3-fold, indicating that role of CD28 costimulation on Th2 development was not only an augmentation of IL-4 production.



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  		FIGURE 4. Cytokine production and IL-4-dependent Th2 generation induced by various polyclonal activators. A, Cytokine production pattern induced by various polyclonal activators. BALB/c CD4+ naive T cells were stimulated with various concentrations of Con A, anti-TCR mAb, or anti-TCR mAb plus CD28 mAb (10% culture supernatant). After 24 h, the culture supernatants were harvested and the concentration of IL-2, IL-4, and IFN-{gamma} in the supernatant was measured by ELISA. B, IL-4 production at different time points after primary T cell stimulation. Naive T cells were stimulated with anti-TCR mAb (30 µg/ml) with or without anti-CD28 mAb (1 µg/ml). The culture supernatants were harvested at different time points after stimulation, and concentration of IL-4 was assessed by ELISA. C, The amount of IL-4 required for Th2 differentiation in the induction culture with TCR cross-linking. Naive T cells were stimulated with anti-TCR mAb (30 µg/ml) with or without anti-CD28 mAb (1 µg/ml). Exogenous IL-4 (0.1–100 ng/ml) was added into the induction culture with TCR cross-linking at day 0. Th1 and Th2 differentiation was assessed as described in the legend for Fig. 1GoA. Similar experiments were repeated more than three times and data are representative of one of those. D, IL-4-dependent Th2 differentiation. BALB/c or DO11.10 Tg CD4+ naive T cells were stimulated with anti-TCR mAb (30 µg/ml) at three different concentrations of anti-CD28 mAb (10–100 ng/ml of purified Abs). For DO11.10 Tg T cells, 0.1 µM was chosen for antigenic stimulation. Th2 differentiation was induced by exogenous IL-4 (0.1–100 ng/ml). Th2 differentiation was assessed as described in the legend for Fig. 1GoA.

 
To investigate this further, the same amount of IL-4 detected in the CD28 costimulation culture was supplied to the anti-TCR culture. TCR cross-linking resulted in 0.1–0.2 ng/ml of IL-4 at 36 and 72 h after stimulation, while the maximum amount of IL-4 produced by the addition of CD28 costimulation was 0.5 ng/ml at 36 h (Fig. 4GoB). However, the addition of 1 ng/ml of IL-4 into the induction culture with anti-TCR mAb fails to generate a significant number of Th2 cells (Fig. 4GoC). The addition of a 20- to 100-fold excess amount of IL-4 was required to bring the Th2 frequency to the level promoted by CD28 costimulation (Fig. 4GoC).

We directly compared the sensitivity of T cells to IL-4 after either anti-TCR or anti-TCR plus CD28 costimulation. Th2 cells were induced from BALB/c T cells by four different concentrations of exogenous IL-4 (0.01–10 ng/ml) in the presence of anti-TCR with or without CD28 costimulation. As shown in Fig. 4GoD, left, when the induction culture was performed with TCR cross-linking, 10 ng/ml of IL-4 was required for a significant number of Th2 cells (13.5%), while CD28 costimulation promoted Th2 development even without the addition of IL-4. To examine Th2 differentiation initiated by exogenous IL-4, the concentration of anti-CD28 mAb for the induction culture was reduced to 10 ng/ml. Stimulation with 10 ng/ml of anti-CD28 mAb generated 6.7% Th2 cells without exogenous IL-4. The addition of 1 ng/ml IL-4 increased the proportion of Th2 up to 13.2%, and 10 ng/ml IL-4 was sufficient for the maximum level of Th2 development (26.2%). Stimulation with anti-TCR required 100 times more IL-4 to generate the same number of Th2 cells as anti-TCR with 10 ng/ml of anti-CD28 mAb.

To mimic normal antigenic stimulation, Th2 cells were induced from DO11.10 Tg T cells by antigenic peptide in the presence of three different concentrations of IL-4. The effect of IL-4 in these cultures was very similar to that with the anti-TCR and CD28 costimulation (Fig. 4GoD, left and right). These results suggested that alteration of the sensitivity to IL-4 might be one function of CD28 costimulation in initial T cell activation, and this enhancement of the sensitivity to IL-4 might also occur in the antigenic activation process.

TCR cross-linking enhances the expression and binding property of the IL-4R

We next studied the role of CD28 costimulation on modification of IL-4R sensitivity. CD28 may simply enhance the cell surface expression of the IL-4R{alpha}-chain, which in turn increases the cell’s capacity. Therefore, BALB/c T cells were prestimulated with anti-TCR and anti-TCR plus anti-CD28 mAb for 36 h, and the expression of IL-4R was investigated. The naive CD4+ T cells expressed substantial levels of IL-4R, and the expression was increased after TCR cross-linking by almost 2-fold (Fig. 5GoA, left and middle). The addition of CD28 costimulation did not further enhance the expression level (Fig. 5GoA, right).



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  		FIGURE 5. TCR and TCR plus CD28 costimulation enhance the expression of IL-4R, CD25, and CD69 as well as the binding affinity of IL-4R. A, Enhancement of cell surface expression of IL-4R with TCR cross-linking. Naive T cells were stimulated with either anti-TCR (30 µg/ml) or anti-TCR plus CD28 mAb (1 µg/ml) in the presence of anti-IL-4 mAb for 36 h. Cell surface expression of IL-4R on CD4+ T cells was analyzed by flow cytometry using anti-IL-4R{alpha} and anti-CD4 mAb. The thick line indicates the anti-IL-4R{alpha} Ab staining. The thin line indicates the FITC-conjugated anti-rat Ig control staining. The numbers described in the box indicate mean fluorescence density. BG indicates the control staining. B, Binding affinity of IL-4R on the preactivated T cells. The binding affinity of IL-4R on preactivated CD4+ T cells was measured by Scatchard plot analysis using 125I-labeled IL-4. The open circle and dash line indicate the Scatchard plot for T cells activated with TCR cross-linking, while the closed circle and solid line indicate for T cells activated with anti-TCR plus CD28 mAb. Each symbol represents mean value of three independent experiments. C, Enhancement of activation marker expression, CD25 and CD69 with Con A, TCR cross-linking, and TCR plus CD28 costimulation. Naive T cells were stimulated with either Con A, TCR cross-linking, or TCR plus CD28 costimulation. After 36 h, CD4+ T cells were gated, and cell surface expression of CD25 and CD69 was analyzed by flow cytometry. BG indicates the control staining. Data are representative of three experiments.

 
There was no difference in IL-4R expression between anti-TCR and anti-TCR plus CD28 mAb, but this did not address whether CD28 altered the binding properties of the IL-4R. Naive T cells were stimulated with either anti-TCR or anti-TCR plus CD28 mAb in the presence of anti-IL-4 mAb for 36 h. Then, we measured the binding affinity of IL-4R on the preactivated T cells by Scatchard plot analysis using 125I-labeled IL-4. Naive T cells revealed undetectable levels of binding affinity against IL-4, while the T cells activated by TCR cross-linking showed relatively high binding affinity. However, no difference was observed between TCR cross-linking and the addition of CD28 costimulation in the binding affinity for IL-4, which showed mean Kd of 416.7 and 625 pM, respectively (Fig. 5GoB). Taken together, activation by TCR cross-linking was sufficient to enhance the expression, and the binding property of the IL-4R and CD28 costimulation did not affect these properties further.

The expression of CD25 and CD69 as activation markers was markedly up-regulated by Con A, anti-TCR, and anti-TCR plus anti-CD28 mAb. The expression of CD69 reached its maximum level with TCR cross-linking, and the effect of CD28 costimulation was very slight (Fig. 5GoC). The maximum level of CD25 expression was induced by Con A and TCR plus CD28 stimulation, and up-regulation by TCR cross-linking was lower than that by the other two stimuli (Fig. 5GoC). The difference between TCR cross-linking and TCR plus CD28 stimulation in CD25 expression may be explained by the amount of IL-2 that was induced by these two stimuli. IL-2 regulates CD25 expression via STAT5 activation. The additional up-regulation by CD28 may be due to the striking enhancement of IL-2 production by CD28 stimulation. The protein expression of Jak3 and STAT6 had the same profile as CD69 and IL-4R. The expression of Jak3 and STAT6 was increased by TCR cross-linking between 1.2- to 2-fold and 8.3- to 24-fold in their densities, respectively (Fig. 6GoA, bottom panels). Again, their expression was not enhanced any further by CD28 costimulation (Fig. 6GoA, bottom panels; Fig. 7GoA, bottom panels). Thus, activation by TCR cross-linking was sufficient to result in the maximum protein expression of IL-4R, CD69, Jak3, and STAT6.



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  		FIGURE 7. The IL-4-dependent tyrosine phosphorylation was increased by the CD28 mAb as well as antigenic stimulation. A, The IL-4-dependent tyrosine phosphorylation was increased by the CD28 mAb. BALB/c CD4+ naive T cells were stimulated with anti-TCR mAb (100 µg/ml) plus different amount of anti-CD28 mAb. Tyrosine phosphorylation was examined as described in the legend for Fig. 6GoA. B, Correlation between the IL-4-mediated tyrosine phosphorylation and the Th2 differentiation. The cells were also cultured for another 3 days, and Th1 and Th2 differentiation was assessed as described in the legend for Fig. 1GoA. C, The IL-4-dependent tyrosine phosphorylation was increased by antigenic stimulation. CD4+ naive T cells were prepared from DO11.10 Tg mice and stimulated with antigenic peptide (0.3–3 µM) in the presence of anti-IL-4R mAb (10 µg/ml). The tyrosine phosphorylation of total cell lysates (2 x 106 cells) was examined as described in the legend for A. The same blot was stripped and reprobed with anti-STAT6.

 
CD28 costimulation increased the tyrosine phosphorylation of the IL-4R{alpha}-chain, Jak3, and STAT6 initiated by IL-4

We further tested the tyrosine phosphorylation profiles initiated by IL-4 after the initial activation. Naive CD4+ T cells from BALB/c mice were prestimulated with Con A, anti-TCR, and anti-TCR plus anti-CD28 mAb. All cultures were performed in the presence of anti-IL-4R mAb to avoid the effect of IL-4 secreted from naive CD4+ T cells after the primary stimulation. After 36 h, the cells were harvested and then cultured another 12 h in the absence of exogenous cytokines. The naive and the primed CD4+ T cells were activated with 10 ng/ml of IL-4 for 5–10 min, and the tyrosine phosphorylation patterns were studied.

The naive CD4+ T cells did not reveal detectable tyrosine phosphorylation, while the stimulated T cells showed significant phosphorylated bands when followed by IL-4 stimulation (Fig. 6GoA). Strong phosphorylation bands were observed at about 130–140 kDa and 100 kDa (Fig. 6GoA, arrows II and IV) after prestimulation with either Con A or anti-TCR plus anti-CD28. However, cells prestimulated with anti-TCR showed weaker phosphorylation, indicating that TCR cross-linking increased the phosphorylation status of IL-4R on naive T cells to a detectable level. Addition of CD28 costimulation further augmented the phosphorylation (Fig. 6GoA).

Tyrosine phosphorylations of the IL-4R{alpha}-chain, Jak3, and STAT6 among the same preparations in Fig. 6GoA were further examined by immunoprecipitation (Fig. 6GoB). Either Con A or anti-TCR with CD28 costimulation phosphorylated Jak3, while anti-TCR stimulation initiated very little phosphorylation. The tyrosine phosphorylation of Jak3 was increased 12.6-fold by the addition of a CD28 costimulation as compared with that in TCR cross-linking alone. IL-4R{alpha} and STAT6 were phosphorylated by all three stimuli, but phosphorylation by anti-TCR with CD28 costimulation was 1.5- and 6.3-fold more than that with TCR cross-linking alone (Fig. 6GoB). The induction culture with Con A that provided a significant Th2 generation resulted in 7.6-, 1.2-, and 2.7-fold higher phosphorylation of Jak3, the IL-4R{alpha}-chain, and STAT6 compared with TCR cross-linking alone.

To examine the correlation between IL-4-mediated tyrosine phosphorylation and the Th2 differentiation caused by CD28 costimulation, naive T cells were prestimulated with anti-TCR and three different amounts of anti-CD28 mAb (1–10% of the culture supernatant containing 5 µg/ml of PV-1 mAb). After 36 h, the cells were harvested and then cultured for another 12 h in the absence of exogenous cytokines. The cells were divided into two groups, and half of the cells were stimulated with IL-4 to study the tyrosine phosphorylation patterns. The rest of cells were cultured for another 3 days to study Th2 development. As shown in Fig. 7Go, A and B, tyrosine phosphorylation followed by IL-4 stimulation correlated with the amount of anti-CD28 mAb, and the presence of CD28 costimulation resulted in significant Th2 generation in a dose-dependent manner.

Augmentation of IL-4-dependent tyrosine phosphrylation was also observed in DO11.10 Tg CD4+ T cells initially activated with 1 µM antigenic peptide (Fig. 7GoC). These results indicated that initial activation by Ag was also able to augment the receptor sensitivity against IL-4, and that the coordination of TCR and CD28 signals might be involved in those processes.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that qualitative differences in T cell activation signals can influence the pattern of T cell differentiation. Th cell differentiation was induced using a TCR cross-linking, or Con A, or Ag in the presence of APC. Stimulation with either Con A or Ag was able to promote Th2 differentiation, but anti-TCR mAb was not sufficient for Th2 differentiation. The in vitro TCR cross-linking system provided us a mean to examine Th2 differentiation and allowed us to control the cytokine environment. An additional activation signal from CD28 enhanced the sensitivity of naive CD4+ T cells to IL-4 in terms of their ability to differentiate into Th2 cells. This was accompanied by increases in the phosphotyrosine content of proteins associated with IL-4R-mediated signaling.

Our observations demonstrated that anti-TCR stimulation generated a detectable number of Th2 cells only if high concentrations of IL-4 were added. This prompted us to speculate that there could be quantitative differences in the magnitude of T cell activation by anti-TCR as compared with the initial activation in which costimulatory molecules might be involved. The Th1/Th2 polarization profile initiated by TCR cross-linking with mAb was consistent with previous reports indicating that the initial activation with anti-CD3 mAb was able to initiate the mRNA expression of Th1 cytokines, but not that of Th2 cytokines (27, 28). The initiation of Th1 dif-ferentiation appears to be less dependent on a CD28 costimulatory signal because Th1 responses could be developed in CD28-deficient mice or in mice in which CD28 costimulation was blocked by CTLA4Ig (29, 30).

In contrast, Th2 differentiation appears to be dependent on CD28 costimulation in an in vitro TCR cross-linking differentiation system. The addition of CD28 costimulation promoted the generation of a significant number of Th2 cells (Fig. 3Go). Our result was consistent with previous reports demonstrating that CD28 costimulation was required for the Th2 responses (29, 31, 32, 33, 34). Abbas (35) and Thompson (36) have proposed a possible explanation for how CD28 costimulation could affect the initial commitment of naive CD4+ T cells. Th1/Th2 polarization was determined by the strength of the initial activation signal. Low Ag concentrations and low-dose infections tend to induce Th1 differentiation preferentially, while high doses induce Th2 differentiation (35, 36). CD28 costimulation could increase the strength of the initial activation signal. This is also supported by recent observations that CD28 costimulation is required for the full activation of naive T cells in a TCR Tg system (37). Our data were consistent with the concept that CD28 costimulation is required for Th2 differentiation and that it enhances the strength of the initial activation signal.

Previous reports have shown that polarization of the Th1 and Th2 cells may reflect both the cytokine environment to which the cells are exposed and the nature of the antigenic stimulation (35). The requirement for IL-4- and IL-4R-dependent signals to drive Th2 differentiation has been demonstrated by targeted disruption of the IL-4 and STAT6 genes and by using recombinant cytokines (7, 8, 9, 10). We have provided evidence that STAT6 activation is essential to up-regulate IL-4 transcription in Th2 cells (16). In con-trast, the signaling mechanisms initiated by TCR stimulation that could influence IL-4R function are not well understood. All naive T cells express significant numbers of IL-4R without prior activation signals (Fig. 6GoA) (38, 39). The IL-4R expressed on naive T cells is functionally active because IL-4 significantly up-regulated IL-4R expression (38) and naive T cells can proliferate with IL-4 in the presence of PMA (40). However, our results showed that neither IL-4 stimulation alone nor IL-4 before antigenic stimulation could initiate Th2 differentiation (Fig. 2GoB). Stimulation with the TCR cross-linking increased expression levels of IL-4R (Fig. 6GoA). Only the addition of IL-4 simultaneously with or within several days after TCR stimulation was able to support Th2 development (Fig. 2GoB). Thus, our data indicated that the number and the nature of the IL-4R expressed on naive CD4+ T cells are different from those on the primed T cells. We propose that initial T cell activation events may be responsible for the alteration of the sensitivity of the IL-4R to IL-4 and that this may be at the level of the Jak/STAT signaling pathway, which is activated by IL-4. This hypothesis was supported by observations that the total phosphotyrosine protein content and the phosphotyrosine content of the IL-4R{alpha}-chain, Jak3, and STAT6 generated by IL-4 were substantially increased by initial T cell activation (Fig. 6Go, B and C).

TCR cross-linking was unable to promote significant Th2 differentiation. Anti-TCR stimulation resulted in the production of a detectable amount of IL-4 but, in comparison with the CD28 costimulation cultures, the addition of a 20- to 100-fold excess amount of IL-4 was required to generate Th2 cells. TCR cross-linking enhanced the protein expression of Jak3 and STAT6, but only a minor alteration occurred in IL-4R-mediated tyrosine phosphorylation. TCR cross-linking with CD28 costimulation apparently enhanced the magnitude of the initial activation event, as evidenced by increased IL-2 and IL-4 production (Fig. 4GoA). CD28 costimulation enhanced the IL-4 production induced by anti-TCR 2- to 5-fold. Moreover, even when the shortage in the IL-4 amount was supplemented, the number of Th2 cells in the anti-TCR cultures did not reach the level that generated in TCR plus CD28 stimulation (Fig. 4GoC). Therefore, it is unlikely that the difference in Th2 development with or without CD28 costimulation was due only to the difference in IL-4 production. TCR cross-linking markedly up-regulates the protein synthesis of the IL-4R{alpha}, CD69, Jak3, and STAT6, but CD28 does not further enhance the expression of those molecules. Moreover, there is no difference between anti-TCR and anti-TCR plus CD28 in the cell surface expression of the IL-4R and the binding affinity for IL-4. Therefore, major role of CD28 costimulation may be an alteration of the IL-4R-mediated tyrosine phosphorylation property. Strong phosphorylations were consistently observed in the IL-4R{alpha}-chain, Jak3, and STAT6 by the addition of CD28 costimulation (Fig. 6Go, B and C, and Fig. 7GoA), indicating that these stimuli together influenced the IL-4R-mediated Jak/STAT cascade. Our data were consistent with a previous report that CD28 costimulation was necessary for the IL-4 responsiveness of Th2 (41) and provided direct evidence at the biochemical level to explain the effect of CD28 costimulation on the IL-4R-mediated Jak/STAT cascade. However, we could not directly address whether the modification of the IL-4-dependent phosphorylation properties was simply consequence of the high magnitude of activation signal or this was a specific outcome for CD28 costimulation. These questions remain to be answered for further investigation.

Recent reports demonstrated that overexpression of Lck in a T cell lymphoma led to constitutive activation of Jak1, Jak2, STAT3, and STAT5 (42), and that TCR activation resulted in the phosphorylation of Jak3 in these cells (T. Saito, personal communication). These findings also address the possibility that the initial T cell activation process might affect the activation status of the Jak/STAT cascade. Moreover, we and others have recently described that the activation status of STAT6 in Th2 cells is distinct from that in Th1 cells (16, 43). The Jak/STAT signaling cascade is impaired in murine Th1 cells, while it is functional in Th2 cells. This was consistently observed in cloned lines and in induced Th1 and Th2 cells. These results are also consistent with the hypothesis that a qualitative difference in the IL-4R signaling pathway can influence the differentiation pathway of Th cells. Indeed, the generation of Th2 cells by CD28 costimulation was totally dependent on the IL-4-mediated pathway because Th2 cells were not generated in the situation that IL-4 and IL-4-mediated signaling were blocked (Fig. 3GoB). Thus, CD28 costimulation may be required for promoting Th2 generation at the IL-4 concentration that naive T cells can produce.

In our hypothesis, the presence of IL-4 at the initial activation process is essential for Th2 differentiation. This raises the question of which cells are the initial source of IL-4. One such source of primary IL-4 is a small population of NK1.1+ cells, which may recognize Ags that are associated with CD1 (44). However, the importance of these cells in in vivo Th2 responses to specific Ags is still unclear (3, 45). Because many types of cells express IL-4R in the resting state, it is possible that a small amount of secreted IL-4 was immediately utilized by the cells expressing IL-4R. Thus, we assayed IL-4 production from naive T cells in the presence of neutralizing Ab against IL-4R. Under these conditions, detectable amounts of IL-4 (50–100 pg/ml) were secreted by naive T cells (Fig. 4GoA), although we could not exclude the possibility that our preparation contained CD4+NK1.1+ cells. Moreover, some previous reports have indicated that naive T cells express IL-4 mRNA and can produce a small amount of IL-4 upon primary activation (46, 47, 48, 49). Further evidence that a Th2 phenotype is not essential for IL-4 production by naive T cells is that T cells from STAT6 knockout mice that are deficient in Th2 responses can secrete IL-4 upon primary stimulation (unpublished data). Therefore, it appears likely that naive CD4+ T cells are one possible source of primary IL-4 and that an IL-4-dependent autocrine pathway is necessary for Th2 differentiation.

In summary, our observations suggest that the following novel mechanisms may be operative during Th2 differentiation. Costimulatory molecules, in addition to the TCR signal, may play an important role in generating Th2 cells. The CD28 plus TCR cross-linking signals provide an optimal initiation signal to the naive T cells and influence the IL-4R-mediated Jak/STAT signaling cascade. We hypothesize that as a result of the alterations in IL-4R signaling molecules, the sensitivity of the IL-4R is enhanced so that it can respond to the small amount of IL-4 produced by naive CD4+ T cells. Only under all of those circumstances the IL-4R may generate sufficient signals in the primed naive T cells to drive Th2 differentiation.


   Acknowledgments
 
We thank Dr. Shizuo Akira for generous gift of C57BL/6 STAT6 knockout mice and Dr. Fidel Zavala for critical comments on the manuscript. We also thank Ryoji Yagi and Yohichi Seki for excellent technical support.


   Footnotes
 
1 This work was supported by grants from the Kanae Foundation for Life and Socio-medical Science and research grants from the Science University of Tokyo. This work was also supported by grants provided by the Ichiro Kanehara Foundation, the Kato Memorial Bioscience Foundation, and the Uehara Memorial Foundation. Back

2 Address correspondence and reprint requests to Dr. M. Kubo, Division of Immunobiology, Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda City, Chiba 278-0022, Japan. E-mail address: Back

3 Abbreviations used in this paper: Tg, transgenic; Jak, Janus kinase. Back

Received for publication October 14, 1998. Accepted for publication June 10, 1999.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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