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

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.

Figure 1

Analysis of Txk expression of human Ag-specific T cell clones. (a) RT-PCR analysis of Txk expression of human Th1, Th2, and Th0 cell clones. Unstimulated T cell clones were used for analysis of Txk mRNA expression. T cell clones were stimulated for 12 h with the relevant Ag plus irradiated monocytes as APCs and analyzed for cytokine mRNA expression. Results of five Th1, five Th0, and five Th2 representative clones of various antigen specificities from several different donors are shown. (b) Representative Th1, Th2, and Th0 cell clones were stained by the immunocytochemical method for Txk protein expression (magnification 250). We studied a total of 20 Th1, 20 Th0, and 20 Th2 clones with various Ag specificities from several donors. All of the Th1 cell clones were strongly positive for Txk expression, whereas none of Th2 clones expressed it. All of the Th0 clones were positive for Txk expression with various staining intensities.

Figure 2

Effects of Txk transfection on IFN-γ production by Jurkat cells. (a) Jurkat cells were transfected with pME18S (mock) and pME18S-Txk (Txk). 48 h after transfection, the Jurkat cells were analyzed for Txk expression (molecular mass, 64 kD) by the immunoblotting method 28. (b) 48 h after transfection, Jurkat and Raji cells were stimulated with PHA plus PMA and PMA plus ionomycin, respectively. Cytokine production was measured by ELISAs. IL-4 production was not detected in any of the transfected Jurkat cell cultures. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. Without mitogenic stimulation, neither Jurkat nor Raji cells produced the cytokines tested, so the results were omitted. Results shown are representative of six independent experiments. ND, not detected. (c) Intracytoplasmic IFN-γ protein expression was assessed by immunofluorescence analysis with anti–IFN-γ mAb of the Jurkat cells. Txk- and mock-transfected Jurkat cells were cultured for 48 h to induce Txk expression. The transfected cells were stimulated with PHA plus PMA for 8 h or kept unstimulated and then analyzed for intracytoplasmic IFN-γ expression. (d) Limiting dilution RT-PCR analysis of IFN-γ mRNA expression. The transfected Jurkat T cells were stimulated with PHA plus PMA for 6 h. We used limiting dilution RT-PCR technique, where varying dilutions of cDNAs were subjected to PCR amplification to more precisely determine effects of Txk transfection on IFN-γ mRNA expression. Lanes 1, 2, and 3 represent 10, 5, and 2% of the total cDNA used for PCR amplification, respectively. Other PCR reaction conditions were exactly the same for lanes 1, 2, and 3. This was to show that PCR reaction ranges within the logarithmic phase of the PCR amplification. (e) Luciferase assay of the Jurkat cells. Jurkat cells were cotransfected with pIFN-γ(-538)-luciferase, pRSV-CAT, and pME18S-Txk or pME18S. 48 h after transfection, half of the cells were stimulated with PHA plus PMA for 8 h, and the remaining cells were kept unstimulated. pRSV-CAT (for pIFN-γ promoter-luciferase and pIL-2 promoter-luciferase) and pGL-3 control (for pIL-4 promoter-CAT) were used for estimating the transfection efficiency. According to the transfection efficiency, the luciferase and CAT activities of the promoter assays were corrected.

Figure 2

Effects of Txk transfection on IFN-γ production by Jurkat cells. (a) Jurkat cells were transfected with pME18S (mock) and pME18S-Txk (Txk). 48 h after transfection, the Jurkat cells were analyzed for Txk expression (molecular mass, 64 kD) by the immunoblotting method 28. (b) 48 h after transfection, Jurkat and Raji cells were stimulated with PHA plus PMA and PMA plus ionomycin, respectively. Cytokine production was measured by ELISAs. IL-4 production was not detected in any of the transfected Jurkat cell cultures. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. Without mitogenic stimulation, neither Jurkat nor Raji cells produced the cytokines tested, so the results were omitted. Results shown are representative of six independent experiments. ND, not detected. (c) Intracytoplasmic IFN-γ protein expression was assessed by immunofluorescence analysis with anti–IFN-γ mAb of the Jurkat cells. Txk- and mock-transfected Jurkat cells were cultured for 48 h to induce Txk expression. The transfected cells were stimulated with PHA plus PMA for 8 h or kept unstimulated and then analyzed for intracytoplasmic IFN-γ expression. (d) Limiting dilution RT-PCR analysis of IFN-γ mRNA expression. The transfected Jurkat T cells were stimulated with PHA plus PMA for 6 h. We used limiting dilution RT-PCR technique, where varying dilutions of cDNAs were subjected to PCR amplification to more precisely determine effects of Txk transfection on IFN-γ mRNA expression. Lanes 1, 2, and 3 represent 10, 5, and 2% of the total cDNA used for PCR amplification, respectively. Other PCR reaction conditions were exactly the same for lanes 1, 2, and 3. This was to show that PCR reaction ranges within the logarithmic phase of the PCR amplification. (e) Luciferase assay of the Jurkat cells. Jurkat cells were cotransfected with pIFN-γ(-538)-luciferase, pRSV-CAT, and pME18S-Txk or pME18S. 48 h after transfection, half of the cells were stimulated with PHA plus PMA for 8 h, and the remaining cells were kept unstimulated. pRSV-CAT (for pIFN-γ promoter-luciferase and pIL-2 promoter-luciferase) and pGL-3 control (for pIL-4 promoter-CAT) were used for estimating the transfection efficiency. According to the transfection efficiency, the luciferase and CAT activities of the promoter assays were corrected.

Figure 2

Effects of Txk transfection on IFN-γ production by Jurkat cells. (a) Jurkat cells were transfected with pME18S (mock) and pME18S-Txk (Txk). 48 h after transfection, the Jurkat cells were analyzed for Txk expression (molecular mass, 64 kD) by the immunoblotting method 28. (b) 48 h after transfection, Jurkat and Raji cells were stimulated with PHA plus PMA and PMA plus ionomycin, respectively. Cytokine production was measured by ELISAs. IL-4 production was not detected in any of the transfected Jurkat cell cultures. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. Without mitogenic stimulation, neither Jurkat nor Raji cells produced the cytokines tested, so the results were omitted. Results shown are representative of six independent experiments. ND, not detected. (c) Intracytoplasmic IFN-γ protein expression was assessed by immunofluorescence analysis with anti–IFN-γ mAb of the Jurkat cells. Txk- and mock-transfected Jurkat cells were cultured for 48 h to induce Txk expression. The transfected cells were stimulated with PHA plus PMA for 8 h or kept unstimulated and then analyzed for intracytoplasmic IFN-γ expression. (d) Limiting dilution RT-PCR analysis of IFN-γ mRNA expression. The transfected Jurkat T cells were stimulated with PHA plus PMA for 6 h. We used limiting dilution RT-PCR technique, where varying dilutions of cDNAs were subjected to PCR amplification to more precisely determine effects of Txk transfection on IFN-γ mRNA expression. Lanes 1, 2, and 3 represent 10, 5, and 2% of the total cDNA used for PCR amplification, respectively. Other PCR reaction conditions were exactly the same for lanes 1, 2, and 3. This was to show that PCR reaction ranges within the logarithmic phase of the PCR amplification. (e) Luciferase assay of the Jurkat cells. Jurkat cells were cotransfected with pIFN-γ(-538)-luciferase, pRSV-CAT, and pME18S-Txk or pME18S. 48 h after transfection, half of the cells were stimulated with PHA plus PMA for 8 h, and the remaining cells were kept unstimulated. pRSV-CAT (for pIFN-γ promoter-luciferase and pIL-2 promoter-luciferase) and pGL-3 control (for pIL-4 promoter-CAT) were used for estimating the transfection efficiency. According to the transfection efficiency, the luciferase and CAT activities of the promoter assays were corrected.

Figure 2

Effects of Txk transfection on IFN-γ production by Jurkat cells. (a) Jurkat cells were transfected with pME18S (mock) and pME18S-Txk (Txk). 48 h after transfection, the Jurkat cells were analyzed for Txk expression (molecular mass, 64 kD) by the immunoblotting method 28. (b) 48 h after transfection, Jurkat and Raji cells were stimulated with PHA plus PMA and PMA plus ionomycin, respectively. Cytokine production was measured by ELISAs. IL-4 production was not detected in any of the transfected Jurkat cell cultures. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. Without mitogenic stimulation, neither Jurkat nor Raji cells produced the cytokines tested, so the results were omitted. Results shown are representative of six independent experiments. ND, not detected. (c) Intracytoplasmic IFN-γ protein expression was assessed by immunofluorescence analysis with anti–IFN-γ mAb of the Jurkat cells. Txk- and mock-transfected Jurkat cells were cultured for 48 h to induce Txk expression. The transfected cells were stimulated with PHA plus PMA for 8 h or kept unstimulated and then analyzed for intracytoplasmic IFN-γ expression. (d) Limiting dilution RT-PCR analysis of IFN-γ mRNA expression. The transfected Jurkat T cells were stimulated with PHA plus PMA for 6 h. We used limiting dilution RT-PCR technique, where varying dilutions of cDNAs were subjected to PCR amplification to more precisely determine effects of Txk transfection on IFN-γ mRNA expression. Lanes 1, 2, and 3 represent 10, 5, and 2% of the total cDNA used for PCR amplification, respectively. Other PCR reaction conditions were exactly the same for lanes 1, 2, and 3. This was to show that PCR reaction ranges within the logarithmic phase of the PCR amplification. (e) Luciferase assay of the Jurkat cells. Jurkat cells were cotransfected with pIFN-γ(-538)-luciferase, pRSV-CAT, and pME18S-Txk or pME18S. 48 h after transfection, half of the cells were stimulated with PHA plus PMA for 8 h, and the remaining cells were kept unstimulated. pRSV-CAT (for pIFN-γ promoter-luciferase and pIL-2 promoter-luciferase) and pGL-3 control (for pIL-4 promoter-CAT) were used for estimating the transfection efficiency. According to the transfection efficiency, the luciferase and CAT activities of the promoter assays were corrected.

Figure 2

Effects of Txk transfection on IFN-γ production by Jurkat cells. (a) Jurkat cells were transfected with pME18S (mock) and pME18S-Txk (Txk). 48 h after transfection, the Jurkat cells were analyzed for Txk expression (molecular mass, 64 kD) by the immunoblotting method 28. (b) 48 h after transfection, Jurkat and Raji cells were stimulated with PHA plus PMA and PMA plus ionomycin, respectively. Cytokine production was measured by ELISAs. IL-4 production was not detected in any of the transfected Jurkat cell cultures. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. Without mitogenic stimulation, neither Jurkat nor Raji cells produced the cytokines tested, so the results were omitted. Results shown are representative of six independent experiments. ND, not detected. (c) Intracytoplasmic IFN-γ protein expression was assessed by immunofluorescence analysis with anti–IFN-γ mAb of the Jurkat cells. Txk- and mock-transfected Jurkat cells were cultured for 48 h to induce Txk expression. The transfected cells were stimulated with PHA plus PMA for 8 h or kept unstimulated and then analyzed for intracytoplasmic IFN-γ expression. (d) Limiting dilution RT-PCR analysis of IFN-γ mRNA expression. The transfected Jurkat T cells were stimulated with PHA plus PMA for 6 h. We used limiting dilution RT-PCR technique, where varying dilutions of cDNAs were subjected to PCR amplification to more precisely determine effects of Txk transfection on IFN-γ mRNA expression. Lanes 1, 2, and 3 represent 10, 5, and 2% of the total cDNA used for PCR amplification, respectively. Other PCR reaction conditions were exactly the same for lanes 1, 2, and 3. This was to show that PCR reaction ranges within the logarithmic phase of the PCR amplification. (e) Luciferase assay of the Jurkat cells. Jurkat cells were cotransfected with pIFN-γ(-538)-luciferase, pRSV-CAT, and pME18S-Txk or pME18S. 48 h after transfection, half of the cells were stimulated with PHA plus PMA for 8 h, and the remaining cells were kept unstimulated. pRSV-CAT (for pIFN-γ promoter-luciferase and pIL-2 promoter-luciferase) and pGL-3 control (for pIL-4 promoter-CAT) were used for estimating the transfection efficiency. According to the transfection efficiency, the luciferase and CAT activities of the promoter assays were corrected.

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.

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.

Figure 4

Effects of Txk antisense ODN on cytokine production by human T cells. (a) Txk expression of normal peripheral blood T cells treated with Txk antisense ODN was analyzed. Txk expression was specifically reduced when treated with Txk antisense ODN, but not sense ODN (both 10 μM), for 12 h. The result shown (magnification 250) is representative of three independent experiments with essentially the same results. (b) Normal peripheral blood T cells were cultured in the presence of Txk antisense ODN for 12 h to reduce Txk expression. Thereafter, the T cells were stimulated with PHA. The result shown is representative of three independent experiments with essentially the same results. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. (c) PPD- and Crj-1-specific T cell clones pretreated with the ODNs (5 μM, which inhibits Txk expression of the clones) were stimulated with the relevant Ag plus irradiated autologous PBMCs. The similar results were reproduced in the experiments using cells from different donors and different Ag-specific T cell clones. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted.

Figure 4

Effects of Txk antisense ODN on cytokine production by human T cells. (a) Txk expression of normal peripheral blood T cells treated with Txk antisense ODN was analyzed. Txk expression was specifically reduced when treated with Txk antisense ODN, but not sense ODN (both 10 μM), for 12 h. The result shown (magnification 250) is representative of three independent experiments with essentially the same results. (b) Normal peripheral blood T cells were cultured in the presence of Txk antisense ODN for 12 h to reduce Txk expression. Thereafter, the T cells were stimulated with PHA. The result shown is representative of three independent experiments with essentially the same results. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. (c) PPD- and Crj-1-specific T cell clones pretreated with the ODNs (5 μM, which inhibits Txk expression of the clones) were stimulated with the relevant Ag plus irradiated autologous PBMCs. The similar results were reproduced in the experiments using cells from different donors and different Ag-specific T cell clones. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted.

Figure 4

Effects of Txk antisense ODN on cytokine production by human T cells. (a) Txk expression of normal peripheral blood T cells treated with Txk antisense ODN was analyzed. Txk expression was specifically reduced when treated with Txk antisense ODN, but not sense ODN (both 10 μM), for 12 h. The result shown (magnification 250) is representative of three independent experiments with essentially the same results. (b) Normal peripheral blood T cells were cultured in the presence of Txk antisense ODN for 12 h to reduce Txk expression. Thereafter, the T cells were stimulated with PHA. The result shown is representative of three independent experiments with essentially the same results. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted. (c) PPD- and Crj-1-specific T cell clones pretreated with the ODNs (5 μM, which inhibits Txk expression of the clones) were stimulated with the relevant Ag plus irradiated autologous PBMCs. The similar results were reproduced in the experiments using cells from different donors and different Ag-specific T cell clones. Mean of triplicate cultures is shown. SEM never exceeded 10% of the mean and was thus omitted.

Figure 5

Effects of cytokine treatment on Txk expression. Purified peripheral blood CD4⁺ T cells were cultured with various concentrations of recombinant cytokines for 4 h. Thereafter, intracytoplasmic Txk expression of the T cells was analyzed by immunofluorescence staining using anti-Txk Ab. IL-12 (1 ng/ml) enhanced whereas IL-4 (10 ng/ml) reduced Txk expression of the T cells. IL-2 (2 ng/ml) treatment did not significantly affect Txk expression. The results shown are representative of five independent experiments with essentially the same results.