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Cutting Edge: Role of IL-27/WSX-1 Signaling for Induction of T-Bet Through Activation of STAT1 During Initial Th1 Commitment

Atsunobu Takeda, Shinjiro Hamano, Atsushi Yamanaka, Toshikatsu Hanada, Tatsuro Ishibashi, Tak W. Mak, Akihiko Yoshimura and Hiroki Yoshida
J Immunol May 15, 2003, 170 (10) 4886-4890; DOI: https://doi.org/10.4049/jimmunol.170.10.4886
Atsunobu Takeda
*Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, and Departments of
† Ophthalmology and
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Shinjiro Hamano
‡ Parasitology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan;
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Atsushi Yamanaka
*Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, and Departments of
§ PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama, Japan; and
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Toshikatsu Hanada
*Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, and Departments of
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Tatsuro Ishibashi
† Ophthalmology and
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Tak W. Mak
¶ Ontario Cancer Institute and Departments of MedicalBiophysics and Immunology, University of Toronto, Toronto, Ontario, Canada
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Akihiko Yoshimura
*Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, and Departments of
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Hiroki Yoshida
*Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, and Departments of
§ PRESTO, Japan Science and Technology Corporation, Kawaguchi, Saitama, Japan; and
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  •            FIGURE 1.
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    FIGURE 1.

    Association of STAT1 with tyrosine-phosphorylated WSX-1. A, Lysates of Ba/F3-WSX-1 cell lines were incubated with anti-FLAG M2 Ab for precipitation of FLAG-tagged WSX-1 or with a control Ab. Precipitates or total cell lysates (TCL) were electrophoresed, blotted, and probed with indicated anti-JAK Abs. Reciprocal immunoprecipitation was conducted by treating cell lysates with anti-JAK1 Ab. Precipitates or total cell lysates were analyzed with anti-FLAG M2 Ab. B, Splenocyte lysates were incubated with GST-WSX-1 produced in either BL21 or TKB1 immobilized on glutathione-Sepharose beads. Bound proteins were electrophoresed, blotted, and visualized with anti-STAT1, -3, -4, or -5b Abs. The integrity and quality of the GST-fused WSX-1 protein were confirmed with anti-GST Ab (bottom panel). C, Ba/F3-WSX-1 cells were either untreated (−) or treated (+) with pervanadate. Immunoprecipitations were performed with anti-FLAG M2 Ab, and precipitates were examined for the coprecipitation of STAT1, -3, or -5b with indicated Ab. Ba/F3-WSX-1-STAT4 cells were treated likewise for the association of STAT4 with WSX-1 (bottom panel). Total cell lysates were examined for verification of the same expression level of each STAT after vanadate treatment. All experiments were repeated three times with similar results.

  •            FIGURE 2.
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    FIGURE 2.

    STAT1 activation by WSX-1 signaling. A, HEK293 cells were transfected with a plasmid mixture containing the GAS luciferase reporter gene, IL-12Rβ1-expressing constructs (β1), and either wild-type (β2/W) or Y824F mutant (β2/W-mt) chimeric receptor, along with β-galactosidase gene or untreated (mock). After 24 h, indicated transfected cells were incubated with (▪) or without (□) IL-12 (100 ng/ml for maximum induction of GAS-luciferase activity) for 12 h, and cell extracts were prepared for luciferase activity. ∗, p < 0.01 by paired t test. Data normalized with the β-galactosidase activity from triplicate experiments are shown. Experiments were repeated three times with similar results. B, CD4+ T cells from spleens of either WSX-1+/− or WSX-1−/− mice were activated with plate-bound anti-CD3ε Ab (10 μg/ml) for 24 h. The activated cells were washed twice, and then incubated in the presence of IL-27 with anti-IFN-γ neutralizing Ab (1 μg/ml) for the indicated periods. Phosphorylation of STAT1 in whole cell lysate was analyzed with anti-PY-STAT1 Ab. IFN-γ−/− CD4+ T cells were treated similarly, and STAT1 phosphorylation was observed. Filters were stripped and reprobed with anti-STAT1 Ab as internal controls.

  •            FIGURE 3.
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    FIGURE 3.

    IL-27 provides for IL-12-mediated IFN-γ production. Sorted CD4+CD45RBhigh naive T cells from WSX-1+/− (left) or WSX-1−/− (right) mice were stimulated with Con A, IL-27, and increasing doses of IL-12 in the presence of irradiated spleen cells for 72 h. Culture supernatants were collected and IFN-γ production determined in triplicate. One representative result of three experiments with similar results is shown.

  •            FIGURE 4.
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    FIGURE 4.

    Induction of T-bet expression by IL-27 stimulation. A, CD4+ T cells from WSX-1+/− or WSX-1−/− mice were stimulated with plate-bound anti-CD3ε Ab plus anti-CD28 Ab and anti-IFN-γ neutralizing Ab in the absence or presence of IL-27 for 48 h. Cell lysates were electrophoresed, blotted, and visualized with anti-T-bet Abs. The same filter was stripped and reprobed with anti-STAT5b Ab as internal controls. B, mRNAs were extracted from the cells similarly treated as in A. Same amounts of cDNA, adjusted to the amount of G3PDH measured by quantitative real-time PCR, were amplified for IL-12Rβ2 expression. Experiments were repeated three times with similar results.

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The Journal of Immunology: 170 (10)
The Journal of Immunology
Vol. 170, Issue 10
15 May 2003
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Cutting Edge: Role of IL-27/WSX-1 Signaling for Induction of T-Bet Through Activation of STAT1 During Initial Th1 Commitment
Atsunobu Takeda, Shinjiro Hamano, Atsushi Yamanaka, Toshikatsu Hanada, Tatsuro Ishibashi, Tak W. Mak, Akihiko Yoshimura, Hiroki Yoshida
The Journal of Immunology May 15, 2003, 170 (10) 4886-4890; DOI: 10.4049/jimmunol.170.10.4886

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Cutting Edge: Role of IL-27/WSX-1 Signaling for Induction of T-Bet Through Activation of STAT1 During Initial Th1 Commitment
Atsunobu Takeda, Shinjiro Hamano, Atsushi Yamanaka, Toshikatsu Hanada, Tatsuro Ishibashi, Tak W. Mak, Akihiko Yoshimura, Hiroki Yoshida
The Journal of Immunology May 15, 2003, 170 (10) 4886-4890; DOI: 10.4049/jimmunol.170.10.4886
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