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Txk, a nonreceptor tyrosine kinase of the Tec family, is expressed in T helper type 1 cells and regulates interferon gamma production in human T lymphocytes.

Kashiwakura J, Suzuki N, Nagafuchi H, Takeno M, Takeba Y, Shimoyama Y, Sakane T - J. Exp. Med. (1999)

Bottom Line: We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential.Txk transfection did not affect IL-2 and IL-4 promoter activities.These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and the Department of Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan.

ABSTRACT
Differentiation of human T cells into T helper (Th)1 and Th2 cells is vital for the development of cell-mediated and humoral immunity, respectively. However, the precise mechanism responsible for the Th1 cell differentiation is not fully clarified. We have studied the expression and function of Txk, a member of the Tec family of nonreceptor tyrosine kinases. We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential. Txk transfection of Jurkat T cells resulted in a several-fold increase of IFN-gamma mRNA expression and protein production; interleukin (IL)-2 and IL-4 production were unaffected. Antisense oligodeoxynucleotide of Txk specifically inhibited IFN-gamma production of normal peripheral blood lymphocytes, antigen-specific Th1 clones, and Th0 clones; IL-2 and IL-4 production by the T cells was unaffected. Txk cotransfection led to the enhanced luciferase activity of plasmid (p)IFN-gamma promoter/enhancer (pIFN-gamma[-538])-luciferase-transfected Jurkat cells upon mitogen activation. Txk transfection did not affect IL-2 and IL-4 promoter activities. Thus, Txk specifically upregulates IFN-gamma gene transcription. In fact, Txk translocated from cytoplasm into nuclei upon activation and transfection with a mutant Txk expression plasmid that lacked a nuclear localization signal sequence did not enhance IFN-gamma production by the cells, indicating that nuclear localization of Txk is obligatory for the enhanced IFN-gamma production. In addition, IL-12 treatment of peripheral blood CD4(+) T cells enhanced the Txk expression, whereas IL-4 treatment completely inhibited it. These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

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Expression and localization of Txk in human T cells. (a) Nuclear translocation of Txk upon mitogenic stimulation. Peripheral blood T cells were cultured with IL-12 (1 ng/ml) or PHA (1 μg/ml) or kept unstimulated for various periods of time. Thereafter, the T cells were stained with anti-Txk Ab by the immunocytochemical method. Nuclear translocation of Txk was evident in T cells treated for 60 min with PHA but not in T cells treated with IL-12 for 60 min nor T cells incubated for 60 min in medium alone (unstimulated). Magnification of the results was 250. (b) Immunocytochemical staining of wild-type Txk- and nuclear localization sequence–deleted mutant Txk–transfected Jurkat cells. Jurkat cells were transfected with pME18S (mock), pME18S-Txk (wild type Txk), or pME18S-mutant (KRKP-deleted) Txk expression vector and cultured for 48 h. Thereafter, the cells were activated with PHA plus PMA for 1 h. Respective cytospin preparations were made and stained with anti-Txk Ab. Control Ab did not stain at all, so results of the control staining were omitted. The mutant Txk did not translocate into nuclei even upon activation. The results shown (magnification 250) are representative of four independent experiments with essentially the same results. (c) Effects of nuclear localization sequence–deleted mutant Txk transfection on IFN-γ production. Jurkat cells were transfected with pME18S (mock), pME18S-Txk (wild type), or mutant (KRKP-deleted) Txk expression vector and cultured for 48 h. Thereafter, the cells were activated with PHA plus PMA for 24 h. IFN-γ production by the transfected Jurkat cells was assessed by ELISA. The results shown are representative of four independent experiments with essentially the same results.
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Figure 3: Expression and localization of Txk in human T cells. (a) Nuclear translocation of Txk upon mitogenic stimulation. Peripheral blood T cells were cultured with IL-12 (1 ng/ml) or PHA (1 μg/ml) or kept unstimulated for various periods of time. Thereafter, the T cells were stained with anti-Txk Ab by the immunocytochemical method. Nuclear translocation of Txk was evident in T cells treated for 60 min with PHA but not in T cells treated with IL-12 for 60 min nor T cells incubated for 60 min in medium alone (unstimulated). Magnification of the results was 250. (b) Immunocytochemical staining of wild-type Txk- and nuclear localization sequence–deleted mutant Txk–transfected Jurkat cells. Jurkat cells were transfected with pME18S (mock), pME18S-Txk (wild type Txk), or pME18S-mutant (KRKP-deleted) Txk expression vector and cultured for 48 h. Thereafter, the cells were activated with PHA plus PMA for 1 h. Respective cytospin preparations were made and stained with anti-Txk Ab. Control Ab did not stain at all, so results of the control staining were omitted. The mutant Txk did not translocate into nuclei even upon activation. The results shown (magnification 250) are representative of four independent experiments with essentially the same results. (c) Effects of nuclear localization sequence–deleted mutant Txk transfection on IFN-γ production. Jurkat cells were transfected with pME18S (mock), pME18S-Txk (wild type), or mutant (KRKP-deleted) Txk expression vector and cultured for 48 h. Thereafter, the cells were activated with PHA plus PMA for 24 h. IFN-γ production by the transfected Jurkat cells was assessed by ELISA. The results shown are representative of four independent experiments with essentially the same results.

Mentions: Because Txk has a hypothetical nuclear localization signal sequence 161729, we examined nuclear translocation of the Txk protein in response to activation signals. Jurkat cells were stimulated with either PHA or IL-12, and subsequent localization of Txk was assessed by immunocytochemical staining (Fig. 3 a). Unstimulated Jurkat cells showed cytoplasmic localization of Txk. Txk protein accumulated in the nuclei of Jurkat cells after treatment for 1 h with PHA. The nuclear accumulation of Txk was specific for PHA, because Txk protein remained in the cytoplasm of Jurkat cells treated with IL-12. The results suggest that Txk itself translocates into nuclei and enhances IFN-γ gene transcription in T cells.


Txk, a nonreceptor tyrosine kinase of the Tec family, is expressed in T helper type 1 cells and regulates interferon gamma production in human T lymphocytes.

Kashiwakura J, Suzuki N, Nagafuchi H, Takeno M, Takeba Y, Shimoyama Y, Sakane T - J. Exp. Med. (1999)

Expression and localization of Txk in human T cells. (a) Nuclear translocation of Txk upon mitogenic stimulation. Peripheral blood T cells were cultured with IL-12 (1 ng/ml) or PHA (1 μg/ml) or kept unstimulated for various periods of time. Thereafter, the T cells were stained with anti-Txk Ab by the immunocytochemical method. Nuclear translocation of Txk was evident in T cells treated for 60 min with PHA but not in T cells treated with IL-12 for 60 min nor T cells incubated for 60 min in medium alone (unstimulated). Magnification of the results was 250. (b) Immunocytochemical staining of wild-type Txk- and nuclear localization sequence–deleted mutant Txk–transfected Jurkat cells. Jurkat cells were transfected with pME18S (mock), pME18S-Txk (wild type Txk), or pME18S-mutant (KRKP-deleted) Txk expression vector and cultured for 48 h. Thereafter, the cells were activated with PHA plus PMA for 1 h. Respective cytospin preparations were made and stained with anti-Txk Ab. Control Ab did not stain at all, so results of the control staining were omitted. The mutant Txk did not translocate into nuclei even upon activation. The results shown (magnification 250) are representative of four independent experiments with essentially the same results. (c) Effects of nuclear localization sequence–deleted mutant Txk transfection on IFN-γ production. Jurkat cells were transfected with pME18S (mock), pME18S-Txk (wild type), or mutant (KRKP-deleted) Txk expression vector and cultured for 48 h. Thereafter, the cells were activated with PHA plus PMA for 24 h. IFN-γ production by the transfected Jurkat cells was assessed by ELISA. The results shown are representative of four independent experiments with essentially the same results.
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Related In: Results  -  Collection

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Figure 3: Expression and localization of Txk in human T cells. (a) Nuclear translocation of Txk upon mitogenic stimulation. Peripheral blood T cells were cultured with IL-12 (1 ng/ml) or PHA (1 μg/ml) or kept unstimulated for various periods of time. Thereafter, the T cells were stained with anti-Txk Ab by the immunocytochemical method. Nuclear translocation of Txk was evident in T cells treated for 60 min with PHA but not in T cells treated with IL-12 for 60 min nor T cells incubated for 60 min in medium alone (unstimulated). Magnification of the results was 250. (b) Immunocytochemical staining of wild-type Txk- and nuclear localization sequence–deleted mutant Txk–transfected Jurkat cells. Jurkat cells were transfected with pME18S (mock), pME18S-Txk (wild type Txk), or pME18S-mutant (KRKP-deleted) Txk expression vector and cultured for 48 h. Thereafter, the cells were activated with PHA plus PMA for 1 h. Respective cytospin preparations were made and stained with anti-Txk Ab. Control Ab did not stain at all, so results of the control staining were omitted. The mutant Txk did not translocate into nuclei even upon activation. The results shown (magnification 250) are representative of four independent experiments with essentially the same results. (c) Effects of nuclear localization sequence–deleted mutant Txk transfection on IFN-γ production. Jurkat cells were transfected with pME18S (mock), pME18S-Txk (wild type), or mutant (KRKP-deleted) Txk expression vector and cultured for 48 h. Thereafter, the cells were activated with PHA plus PMA for 24 h. IFN-γ production by the transfected Jurkat cells was assessed by ELISA. The results shown are representative of four independent experiments with essentially the same results.
Mentions: Because Txk has a hypothetical nuclear localization signal sequence 161729, we examined nuclear translocation of the Txk protein in response to activation signals. Jurkat cells were stimulated with either PHA or IL-12, and subsequent localization of Txk was assessed by immunocytochemical staining (Fig. 3 a). Unstimulated Jurkat cells showed cytoplasmic localization of Txk. Txk protein accumulated in the nuclei of Jurkat cells after treatment for 1 h with PHA. The nuclear accumulation of Txk was specific for PHA, because Txk protein remained in the cytoplasm of Jurkat cells treated with IL-12. The results suggest that Txk itself translocates into nuclei and enhances IFN-γ gene transcription in T cells.

Bottom Line: We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential.Txk transfection did not affect IL-2 and IL-4 promoter activities.These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and the Department of Medicine, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8511, Japan.

ABSTRACT
Differentiation of human T cells into T helper (Th)1 and Th2 cells is vital for the development of cell-mediated and humoral immunity, respectively. However, the precise mechanism responsible for the Th1 cell differentiation is not fully clarified. We have studied the expression and function of Txk, a member of the Tec family of nonreceptor tyrosine kinases. We found that Txk expression is restricted to Th1/Th0 cells with IFN-gamma producing potential. Txk transfection of Jurkat T cells resulted in a several-fold increase of IFN-gamma mRNA expression and protein production; interleukin (IL)-2 and IL-4 production were unaffected. Antisense oligodeoxynucleotide of Txk specifically inhibited IFN-gamma production of normal peripheral blood lymphocytes, antigen-specific Th1 clones, and Th0 clones; IL-2 and IL-4 production by the T cells was unaffected. Txk cotransfection led to the enhanced luciferase activity of plasmid (p)IFN-gamma promoter/enhancer (pIFN-gamma[-538])-luciferase-transfected Jurkat cells upon mitogen activation. Txk transfection did not affect IL-2 and IL-4 promoter activities. Thus, Txk specifically upregulates IFN-gamma gene transcription. In fact, Txk translocated from cytoplasm into nuclei upon activation and transfection with a mutant Txk expression plasmid that lacked a nuclear localization signal sequence did not enhance IFN-gamma production by the cells, indicating that nuclear localization of Txk is obligatory for the enhanced IFN-gamma production. In addition, IL-12 treatment of peripheral blood CD4(+) T cells enhanced the Txk expression, whereas IL-4 treatment completely inhibited it. These results indicate that Txk expression is intimately associated with development of Th1/Th0 cells and is significantly involved in the IFN-gamma production by the cells through Th1 cell-specific positive transcriptional regulation of the IFN-gamma gene.

Show MeSH