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Impairment in the Expression and Activity of Fyn During Differentiation of Naive CD4⁺ T Cells into the Th2 Subset¹

Toshiki Tamura^2,*, Osamu Igarashi^*, Ayako Hino^*, Hidehiro Yamane^*, Shinichi Aizawa, Takuma Kato^* and Hideo Nariuchi^3,*

^* Department of Allergology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and Department of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We previously showed that the amounts of Fyn protein in Th2 clones were approximately one-third to one-fifth of those in Th1 clones. In this study we examined the role of Fyn in the polarization of naive CD4⁺ T cells toward the Th2 subset using fyn^-/- mice. The fyn^-/- naive CD4⁺ T cells efficiently produced Th2 cytokines and polarized toward the Th2 subset even in the absence of IL-4 and IL-13. The expression of Fyn in wild-type CD4⁺ T cells decreased at a transcription level concomitant with polarization toward the Th2 subset. These results suggest that Fyn plays a role in the down-regulation of the differentiation of naive CD4⁺ T cells into the Th2 subset.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Murine activated CD4⁺ T cells can be classified into at least two subsets, Th1 and Th2, on the basis of cytokine production profiles (1). The distinction of cytokine production profiles appears to correlate with functional differences between Th1 and Th2 cells. Th1 cells secrete IL-2, IFN-, and TNF- to mediate cell-mediated immune responses such as delayed-type hypersensitivity, whereas Th2 cells mediate Ab production of B cells by secreting IL-4, IL-5, and IL-6 (2). Th cell subset differentiation has been well documented to be determined by cytokines in the environment, such as IL-4 and IL-12, during the primary Ag response of naive T cells (3). IL-12 secreted by APC such as macrophages and dendritic cells promotes the differentiation of naive T cells into Th1 cells (4, 5), while IL-4 produced by naive CD4⁺ T cells (6) and NK T cells (7) promotes the differentiation into Th2 cells. In addition to the cytokine environment, other mechanisms, such as type of APC, Ag dose, costimulation, and genetic background, can also be involved in the differentiation of naive CD4⁺ T cells into Th1 and Th2 cells (2, 8, 9).

It is generally accepted that nonreceptor-type protein tyrosine kinases (PTKs),⁴ such as Fyn, Lck, and ZAP-70, are involved in the TCR-mediated early activation signal transduction pathway in T cells (10). In our previous studies PTKs such as Fyn and ZAP-70 were not activated in Th2 clones by anti-CD3 stimulation in contrast to the activation of these molecules in Th1 clones, and herbimycin A treatment inhibited the elevation of the intracellular free Ca²⁺ concentration in Th1 clones, but not in Th2 clones. Moreover, the amounts of Fyn protein in Th2 clones were approximately one-third to one-fifth of those in Th1 clones (11). Taken together, these results indicate that the activation of these PTKs is not essential for the activation of Th2 clones and suggest a possibility that the impairment in the activation of PTKs results from a decrease in Fyn protein expression in Th2 clones. These results led us to examine the differentiation of naive CD4⁺ T cells into Th2 cells in fyn^-/- mice. We analyzed the cytokine production and differentiation of naive CD4⁺ T cells from fyn^-/- mice compared with those of cells from wild-type mice. Our results show that fyn^-/- naive CD4⁺ T cells efficiently produced Th2 cytokines and polarized toward the Th2 subset even in the absence of exogenous IL-4 and IL-13 in cultures. However, wild-type T cells produced only Th1 cytokines upon priming and polarized toward both Th1 and Th2 subsets. The synthesis of Fyn protein was decreased at the transcriptional level in wild-type CD4⁺ T cells during the polarization toward Th2 cells by the repetitive stimulation with anti-CD3 plus anti-CD28. These results suggest that Fyn mediates the inhibitory signal(s) against the production of Th2 cytokines and down-regulates the differentiation of naive CD4⁺ T cells into Th2 subset.

	Materials and Methods

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Mice

Fyn^-/- mice, described previously (12, 13), were backcrossed 10 times onto the C57BL/6 background and housed in an environmentally controlled clean room at the animal breeding unit of our institute. IL-4^-/- mice on the C57BL/6 background and fyn^-/- (129S x C57BL/6)F₂ hybrid mice were purchased from The Jackson Laboratory (Bar Harbor, ME). fyn^-/--IL-4^-/- mice were generated by crossing fyn^-/- with IL-4^-/- mice in the clean room at the animal breeding unit of our institute. They grew to adults normally. C57BL/6 mice were purchased from Japan SLC (Hamamatsu, Japan).

Antibodies

Hybridoma 145-2C11 (anti-CD3 -chain, hamster IgG) (14) was provided by Dr. J. A. Bluestone (National Institutes of Health, Bethesda, MD). The mAb was purified from ascites on a protein A column. Anti-human p59^fyn (anti-Fyn; Fyn301, mouse IgG1) cross-reactive to mouse Fyn was purchased from Wako Pure Chemical Industries (Osaka, Japan). Purified anti-CD28 (PV-1, hamster IgG) (15) was provided by Dr. R. Abe (Research Institute for Biological Sciences, Science University of Tokyo, Chiba, Japan). Purified anti-human p56^lck (anti-Lck; MOL 171, mouse IgG1) (16) cross-reactive to mouse Lck was provided by Drs. Y. Koga and K. Nakamura (Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan). Anti-Thy1.2 (HO-13.4, mouse IgM) (17) was used in the form of ascites. Anti-IL-4 (11B11, rat IgG1) (18), anti-B7-2 (GL-1, rat IgG2a) (19), anti-CD8 (53.6.72, rat IgG2a) (20), and anti-MHC class II (M5/114, rat IgG2b) (21) were purified from ascites on a protein G column. Anti-heat-stable Ag (M1/69, rat IgG2a) (22) and anti-FcR (2.4G2, rat IgG2b) (23) were used in the form of culture supernatants. Anti-B7-1 (1G10, rat IgG2a), FITC-anti-CD44 (KM201, rat IgG2a), and rabbit antiserum reactive to human and mouse Lck were purchased from PharMingen (San Diego, CA). Anti-IL-13 (38213.11, rat IgG2b) was purchased from R&D Systems (Minneapolis, MN).

Preparation of naive CD4⁺ T cells

T cells were purified by passing spleen cells through nylon wool and Sephadex G-10 columns. The cells were incubated with a mixture of anti-CD8, anti-heat-stable Ag, anti-MHC class II, and anti-FcR and were treated with anti-rat IgG Microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), followed by passage through a MACS column according to the manufacturer’s instruction to enrich CD4⁺ T cells. The preparations contained >90% CD4⁺ cells, and CD8⁺ cells were undetectable in a flow cytometric analysis. CD44^low T cells were purified from the CD4⁺ T cells by sorting in a FACStar (BD Biosciences, Mountain View, CA) after staining with FITC-anti-CD44. The purities of the CD4⁺CD44^lowCD45RB^high population in CD44^low T cells were confirmed flow cytometrically to be >96% and were used as naive CD4⁺ T cells. We confirmed in preliminary experiments that there was no significant difference between fyn^-/- and wild-type naive CD4⁺ T cells in terms of TCR and CD3 expression in flow cytometry.

Preparation of T cell-depleted spleen accessory cells

Spleen cells from wild-type mice were depleted of T cells by the treatment with anti-Thy1.2 and 1-wk-old rabbit complement. TCR⁺ cells remaining in the preparations were confirmed to be <5% by flow cytometry. The preparation was used as accessory cells after irradiation with 40 Gy.

In vitro priming of naive CD4⁺ T cells

Naive CD4⁺ T cells (1 x 10⁶ cells/culture) were primed for 5 days with soluble anti-CD3 in the presence of accessory cells or with plate-coated anti-CD3 in the presence of soluble anti-CD28 in a flat-bottom 24-well microplate without the addition of any exogenous cytokine. Viable T cells were recovered by Ficoll-Hypaque centrifugation and used as once-primed T cells. The once-primed T cells were reprimed for 5 days under the same conditions as those used for the first priming, and viable T cells were recovered on Ficoll-Hypaque and used as twice-primed T cells. In some experiments, fyn^-/- naive CD4⁺ T cells were primed in the presence of 10 ng/ml of rIL-12 (provided by Genetics Institute, Cambridge, MA).

In vitro stimulation of T cells

For the assessment of cytokine production, naive, once-primed, or twice-primed CD4⁺ T cells (1 x 10⁵ cells/culture) were stimulated with soluble anti-CD3 in the presence of accessory cells (5 x 10⁵ cells/culture) in a flat-bottom 96-well microplate, and the culture supernatants were assayed for IL-2, IL-4, IL-5, and IFN-. For the assessment of Fyn and Lck activities, the naive, once-primed, or twice-primed CD4⁺ T cells (1 x 10⁶ cells/100 µl/culture) from fyn^-/- and wild-type mice were added to the wells of 24-well flat-bottom plate and incubated at 37°C for 5 min, followed by the addition of T cell-depleted spleen accessory cells (5 x 10⁶ cells/100 µl/culture) incubated with 10 µg/ml anti-CD3 for 15 min at 4°C, and then warmed at 37°C for 5 min. The plate was immediately centrifuged at 200 x g for 30 s at room temperature, and then incubated at 37°C for 0, 5, 15, and 30 min.

Assay for cytokines

IL-2, IL-4, IL-5, and IFN- in culture supernatants were assayed by ELISA. All the mAbs specific for mouse IL-2, IL-4, IL-5, and IFN- used for capture and detection of cytokines were purchased from PharMingen. ELISA was performed following the instruction of PharMingen. The detection limits for IL-2, IL-4, IL-5, and IFN- were 25, 10, 5, and 150 pg/ml, respectively. The results are presented as the average of duplicate assays.

Immunoprecipitation

CD4⁺ T cells, naive, once-primed, or twice-primed, were solubilized in 1 ml cold TNE buffer consisting of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Nonidet P-40 containing 20 mM EDTA, 10 µg/ml aprotinin, 0.4 mM sodium vanadate, and 10 mM sodium pyrophosphate. The cell lysates were centrifuged at 10,000 x g for 5 min, and the supernatants were precleared with protein G-Sepharose. The lysates were then incubated with 1 µg anti-Fyn or anti-Lck at 4°C for 1 h, and the immune complexes were precipitated with protein G-Sepharose.

Immune complex kinase assay and immunoblotting

The immune complexes precipitated with protein G-Sepharose were washed four times with TNE buffer and then four times with kinase buffer (50 mM HEPES-NaOH (pH 7.4), 20 mM MnCl₂, and 10 mM MgCl₂). The immunoprecipitates were suspended in 10 µl of the kinase buffer containing 10 µCi [-³²P]ATP and incubated at 30°C for 30 min with continuous mixing in the presence of 1 µg denatured enolase for the assessment of kinase activity for an exogenous substrate. The reaction was stopped by the addition of 10 µl of 3x sample buffer (195 mM Tris-HCl (pH 6.8), 9% SDS, 15% 2-ME, and 30% glycerol). The mixture was then boiled for 3 min, subjected to SDS-PAGE under reducing conditions, and then transferred to polyvinylidene difluoride microporous membrane (Immobilon PVDF; Millipore, Bedford, MA), followed by autoradiography for the detection of kinase activity. To analyze Fyn and Lck protein expression, the membrane was blocked in 5% BSA/Tris-buffered saline (20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) and then incubated with anti-Fyn or rabbit antiserum reactive to human and mouse Lck. Immunoblots were incubated with appropriate biotinylated Ab, anti-mouse Ig (Amersham Pharmacia Biotech, Little Chalfont, U.K.), and anti-rabbit Ig (Amersham Pharmacia Biotech), and then incubated with streptavidin-alkaline phosphatase (Amersham Pharmacia Biotech). After the incubation the membrane was washed with TBS containing 0.1% Tween 20, and developed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate.

Detection of mRNA for fyn

To analyze the accumulation of mRNA for fyn, total RNA was isolated from CD4⁺ T cells using the acid guanidinium-phenol-chloroform method (24), and Northern blot analysis was conducted using a 0.7-kb BglI-HincII fragment of cDNA probe for fyn, pSN-2 (provided by Dr. T. Yamamoto, Department of Oncology of our institute) (25). The signal intensities were quantified by densitometry using an image analyzer BAS-2000II (Fuji Photo Film, Tokyo, Japan).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Naive CD4⁺ T cells from fyn^-/-mice produce both Th1 and Th2 cytokines

To evaluate the role of Fyn in cytokine production of naive CD4⁺ T cells, naive CD4⁺ T cells from fyn^-/- mice were stimulated with soluble anti-CD3 in the presence of T cell-depleted spleen cells from wild-type mice as accessory cells, and the culture supernatants were assayed for cytokines. The fyn^-/- naive CD4⁺ T cells produced comparable or even greater amounts of IL-2 compared with those produced by the cells from wild-type mice after stimulation with 0.1–10 µg/ml anti-CD3. In addition, fyn^-/- naive CD4⁺ T cells produced typical Th2 cytokines, IL-4 and IL-5, in response to 1 µg/ml (data not shown) and 10 µg/ml (Fig. 1) anti-CD3. The wild-type T cells produced neither IL-4 nor IL-5 for at least 120 h upon stimulation with 0.01–10 µg/ml anti-CD3 (data not shown). Naive CD4⁺ T cells from neither fyn^-/- nor wild-type mice produced any cytokine examined by the stimulation with soluble anti-CD3 in the absence of accessory cells (data not shown). It is possible that the above results could be obtained only in T cells from C57BL/6 background mice. Therefore, we examined the cytokine production of naive CD4⁺ T cells from both fyn^-/- and wild-type (129S x C57BL/6)F₂ hybrid mice, and essentially the same results were obtained (data not shown). Thus, fyn^-/- naive CD4⁺ T cells are shown to efficiently produce Th2 cytokines.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Cytokine production of fyn-/- and wild-type naive CD4+ T cells stimulated with anti-CD3 on accessory cells. Naive CD4+ T cells from fyn-/- and wild-type mice (1 x 105 cells/culture) were stimulated in a flat-bottom 96-well microplate for various time intervals with 10 µg/ml soluble anti-CD3 in the presence of accessory cells (5 x 105 cells/culture) from wild-type mice, and the culture supernatants were assayed for IL-2, IL-4, IL-5, and IFN- by ELISA. The results are presented as the average of duplicate assays. We obtained similar results in five repeated experiments.

In the above experiment we used T cell-depleted spleen cells as accessory cells. Therefore, it is possible that the costimulation with accessory cells plays an important role in the Th2 cytokine production of fyn^-/- naive CD4⁺ T cells. Therefore, we examined the effects of anti-B7-1 and anti-B7-2 on their cytokine production stimulated with soluble anti-CD3 in the presence of accessory cells. In the presence of anti-B7-2, IL-2 production of fyn^-/- T cells was remarkably reduced compared with that of wild-type T cells, and their IL-4 and IL-5 production was abrogated (Fig. 2). The inclusion of anti-B7-1 also suppressed the production of both IL-4 and IL-5 by fyn^-/- T cells, although IL-2 production was not affected. The production of IL-4 and IL-5 by fyn^-/- T cells was confirmed to be suppressed by anti-B7-1 or anti-B7-2 for 24–120 h after the stimulation with anti-CD3 (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Effect of anti-B7-1 or anti-B7-2 on the cytokine production of fyn-/- and wild-type naive CD4+ T cells stimulated with anti-CD3 on accessory cells. Naive CD4+ T cells from fyn-/- and wild-type mice (1 x 105 cells/culture) were stimulated with 10 µg/ml soluble anti-CD3 in the presence of accessory cells (5 x 105 cells/culture) in the presence of 30 ng/ml anti-B7-1, anti-B7-2, or control IgG. The culture supernatants obtained at 48 h for IL-2, 72 h for IL-4, and 120 h for IL-5 after the stimulation were assayed for IL-2, IL-4, and IL-5 by ELISA. The results are presented as the average of duplicate assays. The experiment was repeated three times with essentially the same results.

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Effect of CD28 ligation on the cytokine production of fyn-/- and wild-type naive CD4+ T cells stimulated with plate-coated anti-CD3. Naive CD4+ T cells from fyn-/- and wild-type mice (1 x 105 cells/culture) were stimulated for various time intervals with 1 µg/well plate-coated anti-CD3 in the presence or absence of 1 µg/ml soluble anti-CD28, and the culture supernatants were assayed for IL-2, IL-4, IL-5, and IFN- by ELISA. The results are presented as the average of duplicate assays. The experiments was repeated three times with similar results.

Taken together, these results suggest that the costimulation signal through CD28 plays a critical role in inducing Th2 cytokine production of fyn^-/- naive CD4⁺ T cells stimulated with anti-CD3.

Role of Fyn in the polarization of naive CD4⁺ T cells toward the Th2 subset

To examine the role of Fyn in the differentiation of naive CD4⁺ T cells into the Th2 subset, we primed fyn^-/- naive CD4⁺ T cells with plate-coated anti-CD3 plus soluble anti-CD28. Five days after the priming, they were stimulated with anti-CD3 on accessory cells, and the culture supernatants were assayed for cytokines (Fig. 4). The fyn^-/- T cells produced only Th2 cytokines after stimulation, indicating that fyn^-/- naive CD4⁺ T cells were polarized toward the Th2 subset by the priming. In contrast, wild-type T cells primed in the same protocol produced both Th1 and Th2 cytokines after stimulation. Similar experiments were conducted by priming of wild-type CD4⁺ T cells with soluble anti-CD3 in the presence of accessory cells without addition of any exogenous cytokine or by using fyn^-/- naive CD4⁺ T cells from (129S x C57BL/6)F₂ hybrid mice. Essentially the same results as those described above were obtained (data not shown).

View larger version (24K): [in this window] [in a new window]   FIGURE 4. Cytokine production of primed fyn-/- and wild-type T cells after stimulation with anti-CD3 on accessory cells. Naive CD4+ T cells from fyn-/- and wild-type mice (1 x 106 cells/culture) were primed for 5 days with 1 µg/well plate-coated anti-CD3 plus 1 µg/ml soluble anti-CD28 in a flat-bottom 24-well microplate without addition of exogenous cytokine. The primed T cells (1 x 105 cells/culture) were stimulated for various time intervals with 10 µg/ml soluble anti-CD3 in the presence of accessory cells (5 x 105 cells/culture) in a flat-bottom 96-well microplate, and the culture supernatants were assayed for IL-2, IL-4, IL-5, and IFN- by ELISA. The results are presented as the average of duplicate assays. The experiment was repeated five times with similar results.

It is also possible that fyn^-/- Th1 cells are more susceptible to activation-induced cell death (AICD) than wild-type Th1 cells. Therefore, we compared the fyn^-/- and wild-type T cells primed with plate-coated anti-CD3 plus soluble anti-CD28 in terms of susceptibility to AICD. The AICD was assayed by flow cytometry using FITC-annexin V. We observed no difference in the susceptibility to AICD between the fyn^-/- and wild-type T cells. The percentages of cells positively stained with annexin V were 27.4 and 23.9% in fyn^-/- and wild-type T cells, respectively, within 24 h after restimulation with anti-CD3 on accessory cells. We used whole CD4⁺ T cells for the above assay, because IFN--producing T cells could not be detected in the fyn^-/- T cells in our system by intracellular staining (data not shown). Therefore, it is unlikely that the fyn^-/- Th1 cells are more susceptible to AICD than wild-type Th1 cells.

It is well documented that exposure to IL-4 drives naive CD4⁺ T cells to the Th2 subset in the initial immune response (3, 26). In the above experiment fyn^-/- naive CD4⁺ T cells produced a large amount of IL-4 during priming, suggesting that fyn^-/- T cells differentiated into Th2 cells in an environment with a large amount of autocrine IL-4. To examine this possibility, we generated fyn^-/--IL-4^-/- mice by crossing fyn^-/- with IL-4^-/- mice, and fyn^-/--IL-4^-/- naive CD4⁺ T cells were primed with plate-coated anti-CD3 plus soluble anti-CD28. Five days after the priming, they were stimulated with anti-CD3 on accessory cells, and the culture supernatants were assayed for IL-5 and IFN- (Fig. 5). The fyn^-/--IL-4^-/- T cells produced both IL-5 and IFN-, while IL-4^-/- T cells produced IFN-, but little IL-5 (Fig. 5), indicating that fyn^-/- naive CD4⁺ T cells could polarize toward Th2 cells even in the absence of IL-4. These results were confirmed by intracellular staining for these cytokines (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Effect of deficiency in IL-4 on cytokine production of primed fyn-/- T cells by the stimulation with anti-CD3 on accessory cells. Naive CD4+ T cells from fyn-/--IL-4-/- or IL-4-/- mice (1 x 106 cells/culture) were primed for 5 days with 1 µg/well plate-coated anti-CD3 plus 1 µg/ml soluble anti-CD28 in the presence of 10 µg/ml anti-IL-13 or control IgG in a flat-bottom 24-well microplate without addition of exogenous cytokine. The primed T cells (1 x 105 cells/culture) were stimulated for various time intervals with 10 µg/ml soluble anti-CD3 in the presence of accessory cells (5 x 105 cells/culture) in a flat-bottom 96-well microplate, and the culture supernatants were assayed for IL-5 and IFN- by ELISA. The results are presented as the average of duplicate assays. The experiment was repeated three times with similar results.

Role of Lck in the cytokine production and polarization of naive CD4⁺ T cells toward the Th2 subset

Activation of Lck kinase has been reported to be an important signal for the differentiation of Ag-stimulated naive CD4⁺ T cells into the Th2 subset (28). Therefore, we examined Lck kinase activity in fyn^-/- T cells stimulated with anti-CD3 on accessory cells compared with that in wild-type T cells by immune complex kinase assay. As shown in Fig. 6A, Lck kinase activity was not increased in naive CD4⁺ T cells from either fyn^-/- or wild-type mice by the stimulation in terms of either enolase phosphorylation or autophosphorylation, suggesting that the activation of Lck is not necessarily required for the polarization toward the Th2 subset in our system. In the T cells primed with plate-coated anti-CD3 plus soluble anti-CD28, Lck kinase activity in wild-type T cells was significantly increased 5 min after the stimulation with anti-CD3 on accessory cells and decreased thereafter; however, in fyn^-/- T cells the activity was not increased (Fig. 6B). No apparent difference was observed between the fyn^-/- and wild-type T cells in the amount of Lck immunoprecipitated from 5 x 10⁶ naive or primed T cells (Fig. 6). Taken together, these results suggest that Lck activity does not play a critical role in signaling for Th2 cytokine production, at least in our system.

View larger version (50K): [in this window] [in a new window]   FIGURE 6. Activation of Lck kinase after anti-CD3 stimulation in naive and primed CD4+ T cells from fyn-/- and wild-type mice. Naive (A) or primed T cells (B; 1 x 106 cells/culture) were stimulated for various time intervals with 10 µg/ml soluble anti-CD3 in the presence of accessory cells and lysed with TNE buffer. The lysates obtained from 5 x 106 T cells were immunoprecipitated with anti-Lck and subjected to immune complex kinase assay. Lck kinase activity was assayed by the phosphorylation of an exogenous substrate enolase and autophosphorylation. The positions of Lck and enolase are indicated by arrows. Lck protein expression was assayed by immunoblotting with rabbit antiserum reactive to human and mouse Lck. The positions of Lck protein are indicated by arrows. The value of each band for kinase activity is expressed as the ratio to the value at time zero normalized to Lck protein expression in densitometry. ND, Not detected. The experiment was repeated twice with essentially the same results.

We next examined whether the priming of wild-type naive CD4⁺ T cells with the costimulation of anti-CD3 and anti-CD28 decreases the activity and expression of Fyn kinase. When naive T cells were primed with two cycles of costimulation with anti-CD3 and anti-CD28, they produced IL-4, but little IFN-, upon stimulation with anti-CD3 on accessory cells (Fig. 7A); that is, naive CD4⁺ T cells polarized toward the Th2 subset by the two cycles of the priming as previously shown (29). Then, aliquots of T cells primed once or twice under the same conditions as described above were assayed for Fyn kinase activity by immune complex kinase assay after the stimulation with anti-CD3 on accessory cells. The kinase activity peaked 5–15 min after the stimulation in terms of autophosphorylation and also of enolase phosphorylation in naive and once-primed T cells. However, no Fyn activity was detected for 30 min in the twice-primed T cells; that is, no autophosphorylation band was detected for 30 min, and the phosphorylation of enolase was not increased by the stimulation (Fig. 7B). In our stimulation procedure it is difficult to define time zero. Therefore, we also examined the Fyn activity in twice-primed T cells 60 min after the stimulation, and no activity of Fyn was confirmed (data not shown).

View larger version (38K): [in this window] [in a new window]   FIGURE 7. Activation of Fyn kinase after anti-CD3 stimulation in naive, once-primed, and twice-primed CD4+ T cells from wild-type mice. A, Polarization of wild-type naive CD4+ T cells toward the Th2 subset by priming with plate-coated anti-CD3 plus soluble anti-CD28. Naive CD4+ T cells from wild-type mice (1 x 106 cells/culture) were primed twice at a 5-day interval with 1 µg/well plate-coated anti-CD3 plus 1 µg/ml soluble anti-CD28 in a flat-bottom 24-well microplate without addition of exogenous cytokine. The cells (1 x 105 cells/culture) were stimulated with 10 µg/ml soluble anti-CD3 in the presence of accessory cells (5 x 105 cells/culture) in a flat-bottom 96-well microplate, and the culture supernatants were harvested at various time intervals and assayed for IL-4 and IFN- by ELISA. The results are presented as the average of duplicate assays. The experiment was repeated three times with similar results. B, Activation of Fyn kinase in naive, once-primed, and twice-primed T cells from wild-type mice stimulated with anti-CD3 on accessory cells. Naive, once-primed, and twice-primed T cells (1 x 106 cells/culture) were stimulated for various time intervals with 10 µg/ml soluble anti-CD3 in the presence of accessory cells and lysed with TNE buffer. The lysates obtained from 5 x 106 T cells were immunoprecipitated with anti-Fyn and subjected to immune complex kinase assay. Fyn kinase activity was assayed by the phosphorylation of an exogenous substrate enolase and autophosphorylation. The positions of Fyn and enolase are indicated by arrows. Fyn protein expression was analyzed by immunoblotting with anti-Fyn. The positions of Fyn protein are indicated by arrows. The values of kinase activity for each band are expressed as the ratios to the value at time zero normalized to Fyn protein expression by densitometry. The experiment was repeated three times with essentially the same results.

View larger version (31K): [in this window] [in a new window]   FIGURE 8. Expression of Fyn and fyn mRNA in naive, once-primed, and twice-primed T cells from wild-type mice. A, Fyn protein expression in naive, once-primed, and twice-primed T cells. Fyn protein was immunoprecipitated from 5 x 106 naive, once-primed, and twice-primed T cells and analyzed by immunoblotting with anti-Fyn. The positions of Fyn protein are indicated by arrows. The value of each band is expressed as the ratio to that of naive cells by densitometry. The experiment was repeated three times with similar results. B, fyn mRNA expression in naive, once-primed, and twice-primed T cells. Total cellular RNAs from naive, once-primed, and twice-primed T cells were analyzed by Northern blotting for fyn mRNA. The values are expressed as the ratio of Fyn to -actin by densitometry. The positions of fyn and -actin are indicated by arrows.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The fyn^-/- naive CD4⁺ T cells produced a large amount of IL-4 after stimulation with anti-CD3 plus anti-CD28. It is well documented that IL-4 promotes Th2 subset differentiation (3). Therefore, it is possible that fyn^-/- naive CD4⁺ T cells were polarized toward the Th2 subset by exposure to a high concentration of autocrine IL-4. However, fyn^-/--IL-4^-/- naive T cells could also polarize toward the Th2 subset even in the absence of exogenous IL-4 in cultures. These results suggest that Th2 subset differentiation of fyn^-/- naive T cells could be induced by an IL-4-independent mechanism. Consistent with our results, human naive CD4⁺ T cells were reported to differentiate into Th2 cells in the presence of neutralizing anti-IL-4 after priming with anti-CD3 plus anti-CD28 (32). T cells from IL-4^-/- mice infected with Leishmania major were also shown to produce Th2 cytokines by Ag restimulation in vitro (33).

In our results the fyn^-/- naive CD4⁺ T cells were polarized toward the Th2 subset, but wild-type T cells were polarized toward both Th1 and Th2 subsets after the priming. It has been demonstrated that Th1 cells were more susceptible than Th2 cells to AICD (34). However, there was no difference in the susceptibility to AICD between the fyn^-/- and wild-type T cells in our system. Therefore, it is unlikely that fyn^-/- CD4⁺ T cells were easily polarized toward the Th2 subset because Th1-polarizing cells in fyn^-/- mice are more susceptible to AICD than those in wild-type mice.

Our results showed that wild-type T cells polarized toward the Th2 subset concomitant with the reduction in Fyn protein expression, and Fyn kinase activity was not detected in the cells that produced Th2 cytokines after anti-CD3 stimulation. These results are consistent with our previous findings that Th2 clones expressed a low level of Fyn protein (approximately one-fourth to one-sixth of Th1 clones) and the Fyn kinase activities in those clones were not detectable after TCR stimulation (11). Fc receptor-nonbinding anti-CD3 induced IL-4 production of Th2 clones without phosphorylation of TCR-, ZAP-70, or mitogen-activated protein kinase, although it stimulated IL-2 production of Th1 clones with low efficiency (35). Taken together, these results indicate that the activation of PTKs such as Fyn and ZAP-70 is not required for the production of Th2 cytokines. The most important finding in our present experiments may be the reduction of Fyn protein expression in T cells during polarization toward the Th2 subset. Recent studies showed that stimulation with only TCR cross-linking induced the differentiation of naive CD4⁺ T cells into Th1 cells, but not Th2 cells, while costimulation with anti-CD28 was shown to induce differentiation into Th2 cells by accelerating the sensitivity of naive CD4⁺ T cells to IL-4 (36). Therefore, it is possible that CD28 mediates the signal(s) for reduction of Fyn protein expression. Costimulation with anti-CD28 induced IL-4 production in naive CD4⁺ T cells (32), and it was shown to be essential for the differentiation of naive CD4⁺ T cells to the Th2 subset (37). The requirement for IL-4R-mediated signal for the differentiation of naive CD4⁺ T cells into Th2 cells has been demonstrated by targeted disruption of the IL-4 and STAT6 genes (38, 39, 40). Therefore, it is also possible that IL-4R mediates the signal(s) to reduce Fyn protein expression. Indeed, treatment with anti-IL-4 inhibited the reduction of Fyn protein expression in wild-type T cells induced by priming with anti-CD3 plus anti-CD28 (data not shown).

In summary, our results suggest that the following mechanisms may be operative in the differentiation of naive CD4⁺ T cells to the Th2 subset. CD3 plus CD28 signals directly or indirectly down-regulate the expression of Fyn protein and enhance IL-4 production, resulting in the differentiation of naive CD4⁺ T cells into the Th2 subset.

	Footnotes

 
¹ This work was supported in part by a grant-in-aid for Scientific Research on Priority Areas and a grant-in-aid for Scientific Research (B) from the Ministry of Education, Science, Sports, and Culture, Japan.

² Address correspondence and reprint requests to Dr. Toshiki Tamura, Department of Allergology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address: toshikit{at}ims.u-tokyo.ac.jp

³ Present address: Ogata Institute for Medical and Chemical Research, Inc., Chiyoda-ku, Tokyo 101-0031, Japan.

⁴ Abbreviations used in this paper: PTK, protein tyrosine kinase; AICD, activation-induced cell death; anti-Fyn, anti-human p59^fyn; anti-Lck, anti-human p56^lck.

Received for publication July 17, 2000. Accepted for publication May 31, 2001.

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