Interferon- Activates Multiple STAT Proteins and Upregulates Proliferation-Associated IL-2R, c-myc, and pim-1 Genes in Human T Cells by Sampsa Matikainen, Timo Sareneva, Tapani Ronni, Anne Lehtonen, Päivi J. Koskinen, and Ilkka Julkunen Blood Volume 93(6):1980-1991 March 15, 1999 ©1999 by American Society of Hematology

Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

Dose-dependent activation of IL-2R, c-myc, andpim-1 gene expression by type I IFNs. T cells were stimulated with different doses of IFN- or IFN-β for 3 hours, the cells were collected, and total cellular RNA was isolated. Dose-dependent activation of IL-2R, c-myc, andpim-1 gene expression by type I IFNs. T cells were stimulated with different doses of IFN- or IFN-β for 3 hours, the cells were collected, and total cellular RNA was isolated. RNA samples (20 μg) were size-fractionated on agarose gels, transferred to nylon membranes, and hybridized with IL-2R, c-myc, and pim-1 cDNA probes. EtBr-stained gel is shown to verify equal RNA loading. Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

Effect of IFN- pretreatment on IL-2–induced T-cell proliferation. Effect of IFN- pretreatment on IL-2–induced T-cell proliferation. T lymphocytes were activated with anti-CD3-antibodies and expanded in the presence of IL-2, after which IL-2–containing medium was removed. The cells were then left untreated or treated with 100 IU/mL of IFN- for 24 hours. The cells were collected, and an equal number of (□) untreated or (▪) IFN-–primed T cells was applied in microtiter plates. Different doses of IL-2 were added for 18 hours, followed by further incubation of 6 hours in the presence of 1 μCi/well of 3H-labeled thymidine. After harvesting the cells, the proliferation index was determined as described in Materials and Methods. The mean proliferation index (±SD) of six individual donors is shown. Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

(A) STAT DNA binding to the IL-2R GAS-c/GAS-n in response to cytokine stimulation. (A) STAT DNA binding to the IL-2R GAS-c/GAS-n in response to cytokine stimulation. T cells were stimulated with IFN-, IL-2, IL-12, or IL-15 as indicated; nuclear extracts were prepared from the cells; and the STAT DNA binding was analyzed by EMSA. (B) STAT DNA binding to the IL-2R GAS-c/GAS-n in response to IFN- and IL-2. T cells were stimulated with IFN- or IL-2 for 30 minutes, after which nuclear extracts were prepared. Nuclear extracts were incubated for 1 hour on ice with STAT antibodies indicated, followed by binding to 32P-labeled IL-2RGAS-c/GAS-n probe. (C) STAT DNA binding to the IL-2RGAS-c/GAS-n in response to IL-12 and IL-15. T cells were stimulated with IL-2 or IL-15 for 30 minutes, and nuclear extracts were prepared and incubated for 1 hour on ice with different anti-STAT antibodies followed by binding to 32P-labeled IL-2RGAS-c/GAS-n probe. The results are representative of three separate experiments. Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

(A) Multiple STAT complexes bind to the pim-1 GAS element in response to cytokine stimulation. (A) Multiple STAT complexes bind to the pim-1 GAS element in response to cytokine stimulation. T cells were stimulated with IFN-, IL-2, IL-12, or IL-15 for the indicated times, and nuclear extracts were prepared from the cells. The extracts were incubated with 32P-labeled pim-1 GAS probe, and the STAT DNA binding was analyzed by EMSA. (B) IFN- induced STAT1, STAT3, and STAT4 DNA binding to pim-1 GAS. T cells were stimulated with IFN- for 30 minutes, and nuclear extracts were prepared. The extracts were incubated for 1 hour on ice with anti-STAT antibodies, followed by binding to 32P-labeledpim-1 GAS probe. Comparable data were obtained in three independent experiments, each consisting of pooled T cells from two different donors. Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

(A) STAT binding to the IRF-1 GAS by IFN-, IL-2, IL-12, or IL-15. (A) STAT binding to the IRF-1 GAS by IFN-, IL-2, IL-12, or IL-15. T cells were stimulated with IFN-, IL-2, IL-12, or IL-15 for the times indicated, and nuclear extracts were prepared. The extracts were incubated with 32P-labeledIRF-1 GAS probe, and the DNA binding activity was analyzed by EMSA. (B) IFN-–induced STAT1, STAT3, and STAT4 DNA binding toIRF-1 GAS. T cells were stimulated with IFN- for 30 minutes, and nuclear extracts were prepared and incubated for 1 hour on ice with anti-STAT antibodies, followed by binding to 32P-labeledIRF-1 GAS. The experiment was repeated three times with similar results. Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

(A) IFN- induces tyrosine phosphorylation of STAT1 and STAT4 in human T lymphocytes. (A) IFN- induces tyrosine phosphorylation of STAT1 and STAT4 in human T lymphocytes. T cells were treated with IFN- or IL-12 for 15 minutes, and T-cell lysates were prepared and immunoprecipitated with anti-STAT1, anti-STAT3, or anti-STAT4 antibodies. Proteins were separated on 10% SDS-PAGE, transferred to membranes, and immunoblotted with antiphosphotyrosine antibody. The membranes were stripped and reblotted with anti-STAT antibodies. (B) IFN- induces tyrosine phosphorylation of STAT5a and STAT5b. T cells were treated with IFN- or IL-2 for 15 minutes and T-cell lysates were prepared. The lysates were immunoprecipitated with anti-STAT5a or anti-STAT5b antibodies, followed by immunoblotting with antiphosphotyrosine antibody. Membranes were then stripped and reblotted with anti-STAT antibodies, as indicated. Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

IFN-–induced STAT5 DNA binding to the IFP53GAS element. IFN-–induced STAT5 DNA binding to the IFP53GAS element. T cells were stimulated with IFN- or IL-2 for 30 minutes, and nuclear extracts were prepared and incubated with anti-STAT1 or anti-STAT5 antibodies for 1 hour on ice, followed by EMSA with 32P-labeled IFP53 GAS probe. Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology

IFN- enhances IL-2–induced STAT1 and STAT5 binding to the IRF-1 GAS IFN- enhances IL-2–induced STAT1 and STAT5 binding to the IRF-1 GAS. T cells were left untreated or treated with IFN- (100 IU/mL) for 24 hours. IFN- enhances IL-2–induced STAT1 and STAT5 binding to the IRF-1 GAS. T cells were left untreated or treated with IFN- (100 IU/mL) for 24 hours. The cells were washed and resuspended in fresh medium, and different concentrations of IL-2 (0, 3, 10, or 30 IU/mL) were added. After 30 minutes of incubation, nuclear extracts were prepared and analyzed in EMSA with IRF-1 GAS probe. The experiment was repeated three times with similar results. Sampsa Matikainen et al. Blood 1999;93:1980-1991 ©1999 by American Society of Hematology