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Stat5 synergizes with T cell receptor/antigen stimulation in the development of lymphoblastic lymphoma.

Kelly JA, Spolski R, Kovanen PE, Suzuki T, Bollenbacher J, Pise-Masison CA, Radonovich MF, Lee S, Jenkins NA, Copeland NG, Morse HC, Leonard WJ - J. Exp. Med. (2003)

Bottom Line: Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that mediate a wide range of actions induced by cytokines, interferons, and growth factors.The rate of lymphoma induction was markedly enhanced by immunization or by the introduction of TCR transgenes.These data demonstrate the oncogenic potential of dysregulated expression of a STAT protein that is not constitutively activated, and that TCR stimulation can contribute to this process.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology, National Heart and Blood Institute, National Cancer Institute, Bethesda, MD 20892-1674, USA.

ABSTRACT
Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that mediate a wide range of actions induced by cytokines, interferons, and growth factors. We now report the development of thymic T cell lymphoblastic lymphomas in transgenic mice in which Stat5a or Stat5b is overexpressed within the lymphoid compartment. The rate of lymphoma induction was markedly enhanced by immunization or by the introduction of TCR transgenes. Remarkably, the Stat5 transgene potently induced development of CD8+ T cells, even in mice expressing a class II-restricted TCR transgene, with resulting CD8+ T cell lymphomas. These data demonstrate the oncogenic potential of dysregulated expression of a STAT protein that is not constitutively activated, and that TCR stimulation can contribute to this process.

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Analysis of lymphoblastic lymphoma cells from Stat5 transgenic mice. (A) Augmented CD4+/CD8+ and/or CD8+ T cells in Stat5b transgenic mice. Flow cytometric analysis of cells from thymus (panels a, e, and i), spleen (panels b, f, and j), and cervical lymph node (panels c and g). Also shown is the CD4/CD8 profile of subcutaneous cervical lesions in a RAG/γc double KO mouse 3 wk after subcutaneous injection of fresh tumor cells (panels d and h). (B) Vβ2-FITC and Vβ4-PE staining in two representative tumors demonstrating a single population of positively staining cells. Although Onco 12 had a substantial Vβ2-negative population, staining for Vβ3–6, 8, 10, 11, and Vα11 was negative. Tumor masses resulting from the injection of lymphoma cells into RAG/γc double KO mice maintained the same expression profile as the donor cells (panels g vs. a and h vs. d). (C) No constitutive phosphorylation of Stat5a or Stat5b, as evaluated by anti-phospho-Stat5 Western blotting, is seen in the tumor lysates (lanes 4–7), whereas the positive control (Stat5b transgenic splenocytes stimulated with IL-2 for 30 min) confirmed the function of the anti-phosphoStat5 antibody (lane 11). (D) No constitutive phosphorylation of Stat3 was detected by anti-phospho-Stat3 Western blotting in tumor lysates (lanes 2–5), whereas the positive control (tumor lysates stimulated with IL-6 for 30 min) confirmed the function of the anti-phospho-Stat3 antibody (lane 7). (E) EMSAs using a β-casein probe and the indicated samples. Cells from WT (lanes 1–3, 8, 9, 15, 16, 21, 22), transgenic (TG; lanes 10, 11), or mice with tumors (Onco; lanes 4–7, 12–14, 17–20) were not stimulated or stimulated with IL-2 or IL-7, and DNA binding activity measured. Supershifting with anti-Stat5 or Stat5b is shown in lanes 19 and 20, respectively.
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fig3: Analysis of lymphoblastic lymphoma cells from Stat5 transgenic mice. (A) Augmented CD4+/CD8+ and/or CD8+ T cells in Stat5b transgenic mice. Flow cytometric analysis of cells from thymus (panels a, e, and i), spleen (panels b, f, and j), and cervical lymph node (panels c and g). Also shown is the CD4/CD8 profile of subcutaneous cervical lesions in a RAG/γc double KO mouse 3 wk after subcutaneous injection of fresh tumor cells (panels d and h). (B) Vβ2-FITC and Vβ4-PE staining in two representative tumors demonstrating a single population of positively staining cells. Although Onco 12 had a substantial Vβ2-negative population, staining for Vβ3–6, 8, 10, 11, and Vα11 was negative. Tumor masses resulting from the injection of lymphoma cells into RAG/γc double KO mice maintained the same expression profile as the donor cells (panels g vs. a and h vs. d). (C) No constitutive phosphorylation of Stat5a or Stat5b, as evaluated by anti-phospho-Stat5 Western blotting, is seen in the tumor lysates (lanes 4–7), whereas the positive control (Stat5b transgenic splenocytes stimulated with IL-2 for 30 min) confirmed the function of the anti-phosphoStat5 antibody (lane 11). (D) No constitutive phosphorylation of Stat3 was detected by anti-phospho-Stat3 Western blotting in tumor lysates (lanes 2–5), whereas the positive control (tumor lysates stimulated with IL-6 for 30 min) confirmed the function of the anti-phospho-Stat3 antibody (lane 7). (E) EMSAs using a β-casein probe and the indicated samples. Cells from WT (lanes 1–3, 8, 9, 15, 16, 21, 22), transgenic (TG; lanes 10, 11), or mice with tumors (Onco; lanes 4–7, 12–14, 17–20) were not stimulated or stimulated with IL-2 or IL-7, and DNA binding activity measured. Supershifting with anti-Stat5 or Stat5b is shown in lanes 19 and 20, respectively.

Mentions: Of the 12 lymphomas from Table I analyzed by flow cytometry, 10 had populations of both CD4+CD8+ double positive and CD8+ single positive cells (Table I, Onco 2, 4, 9–13, 15, 19, and 20; Fig. 3 A, Onco 12, panels a, b, and c) and two had predominantly CD8+ cells (Table I, Onco 3 and 6; Fig. 3 A, Onco 3, panels e, f, and g) in thymus, spleen, and lymph nodes. Of eight mice examined for TCR-β usage, one exhibited two prominent populations and seven a single prominent population based on Vβ staining (e.g., Onco 12 cells stained only for Vβ2 (Table I and Fig. 3 B, panels a versus b), while Onco 3 had only Vβ4+ staining (Table I, and Fig. 3 B, panels c versus d). We confirmed the monoclonal nature of the tumor in two lymphomas by PCR analysis of TCRβ rearrangements (reference 28; unpublished data). Subcutaneous injection of malignant cells into γc/RAG2 double KO mice resulted in tumor formation at the site of injection; cells from these tumor masses had flow cytometric profiles indistinguishable from the original transplanted donor cells (Fig. 3 A, panels d versus a, b, c and h versus e, f, g; and Fig. 3 B, panels g versus a, and h versus d). Thus, both Stat5a and Stat5b transgenes predisposed to development of T cell lymphoblastic lymphoma.


Stat5 synergizes with T cell receptor/antigen stimulation in the development of lymphoblastic lymphoma.

Kelly JA, Spolski R, Kovanen PE, Suzuki T, Bollenbacher J, Pise-Masison CA, Radonovich MF, Lee S, Jenkins NA, Copeland NG, Morse HC, Leonard WJ - J. Exp. Med. (2003)

Analysis of lymphoblastic lymphoma cells from Stat5 transgenic mice. (A) Augmented CD4+/CD8+ and/or CD8+ T cells in Stat5b transgenic mice. Flow cytometric analysis of cells from thymus (panels a, e, and i), spleen (panels b, f, and j), and cervical lymph node (panels c and g). Also shown is the CD4/CD8 profile of subcutaneous cervical lesions in a RAG/γc double KO mouse 3 wk after subcutaneous injection of fresh tumor cells (panels d and h). (B) Vβ2-FITC and Vβ4-PE staining in two representative tumors demonstrating a single population of positively staining cells. Although Onco 12 had a substantial Vβ2-negative population, staining for Vβ3–6, 8, 10, 11, and Vα11 was negative. Tumor masses resulting from the injection of lymphoma cells into RAG/γc double KO mice maintained the same expression profile as the donor cells (panels g vs. a and h vs. d). (C) No constitutive phosphorylation of Stat5a or Stat5b, as evaluated by anti-phospho-Stat5 Western blotting, is seen in the tumor lysates (lanes 4–7), whereas the positive control (Stat5b transgenic splenocytes stimulated with IL-2 for 30 min) confirmed the function of the anti-phosphoStat5 antibody (lane 11). (D) No constitutive phosphorylation of Stat3 was detected by anti-phospho-Stat3 Western blotting in tumor lysates (lanes 2–5), whereas the positive control (tumor lysates stimulated with IL-6 for 30 min) confirmed the function of the anti-phospho-Stat3 antibody (lane 7). (E) EMSAs using a β-casein probe and the indicated samples. Cells from WT (lanes 1–3, 8, 9, 15, 16, 21, 22), transgenic (TG; lanes 10, 11), or mice with tumors (Onco; lanes 4–7, 12–14, 17–20) were not stimulated or stimulated with IL-2 or IL-7, and DNA binding activity measured. Supershifting with anti-Stat5 or Stat5b is shown in lanes 19 and 20, respectively.
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fig3: Analysis of lymphoblastic lymphoma cells from Stat5 transgenic mice. (A) Augmented CD4+/CD8+ and/or CD8+ T cells in Stat5b transgenic mice. Flow cytometric analysis of cells from thymus (panels a, e, and i), spleen (panels b, f, and j), and cervical lymph node (panels c and g). Also shown is the CD4/CD8 profile of subcutaneous cervical lesions in a RAG/γc double KO mouse 3 wk after subcutaneous injection of fresh tumor cells (panels d and h). (B) Vβ2-FITC and Vβ4-PE staining in two representative tumors demonstrating a single population of positively staining cells. Although Onco 12 had a substantial Vβ2-negative population, staining for Vβ3–6, 8, 10, 11, and Vα11 was negative. Tumor masses resulting from the injection of lymphoma cells into RAG/γc double KO mice maintained the same expression profile as the donor cells (panels g vs. a and h vs. d). (C) No constitutive phosphorylation of Stat5a or Stat5b, as evaluated by anti-phospho-Stat5 Western blotting, is seen in the tumor lysates (lanes 4–7), whereas the positive control (Stat5b transgenic splenocytes stimulated with IL-2 for 30 min) confirmed the function of the anti-phosphoStat5 antibody (lane 11). (D) No constitutive phosphorylation of Stat3 was detected by anti-phospho-Stat3 Western blotting in tumor lysates (lanes 2–5), whereas the positive control (tumor lysates stimulated with IL-6 for 30 min) confirmed the function of the anti-phospho-Stat3 antibody (lane 7). (E) EMSAs using a β-casein probe and the indicated samples. Cells from WT (lanes 1–3, 8, 9, 15, 16, 21, 22), transgenic (TG; lanes 10, 11), or mice with tumors (Onco; lanes 4–7, 12–14, 17–20) were not stimulated or stimulated with IL-2 or IL-7, and DNA binding activity measured. Supershifting with anti-Stat5 or Stat5b is shown in lanes 19 and 20, respectively.
Mentions: Of the 12 lymphomas from Table I analyzed by flow cytometry, 10 had populations of both CD4+CD8+ double positive and CD8+ single positive cells (Table I, Onco 2, 4, 9–13, 15, 19, and 20; Fig. 3 A, Onco 12, panels a, b, and c) and two had predominantly CD8+ cells (Table I, Onco 3 and 6; Fig. 3 A, Onco 3, panels e, f, and g) in thymus, spleen, and lymph nodes. Of eight mice examined for TCR-β usage, one exhibited two prominent populations and seven a single prominent population based on Vβ staining (e.g., Onco 12 cells stained only for Vβ2 (Table I and Fig. 3 B, panels a versus b), while Onco 3 had only Vβ4+ staining (Table I, and Fig. 3 B, panels c versus d). We confirmed the monoclonal nature of the tumor in two lymphomas by PCR analysis of TCRβ rearrangements (reference 28; unpublished data). Subcutaneous injection of malignant cells into γc/RAG2 double KO mice resulted in tumor formation at the site of injection; cells from these tumor masses had flow cytometric profiles indistinguishable from the original transplanted donor cells (Fig. 3 A, panels d versus a, b, c and h versus e, f, g; and Fig. 3 B, panels g versus a, and h versus d). Thus, both Stat5a and Stat5b transgenes predisposed to development of T cell lymphoblastic lymphoma.

Bottom Line: Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that mediate a wide range of actions induced by cytokines, interferons, and growth factors.The rate of lymphoma induction was markedly enhanced by immunization or by the introduction of TCR transgenes.These data demonstrate the oncogenic potential of dysregulated expression of a STAT protein that is not constitutively activated, and that TCR stimulation can contribute to this process.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Immunology, National Heart and Blood Institute, National Cancer Institute, Bethesda, MD 20892-1674, USA.

ABSTRACT
Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that mediate a wide range of actions induced by cytokines, interferons, and growth factors. We now report the development of thymic T cell lymphoblastic lymphomas in transgenic mice in which Stat5a or Stat5b is overexpressed within the lymphoid compartment. The rate of lymphoma induction was markedly enhanced by immunization or by the introduction of TCR transgenes. Remarkably, the Stat5 transgene potently induced development of CD8+ T cells, even in mice expressing a class II-restricted TCR transgene, with resulting CD8+ T cell lymphomas. These data demonstrate the oncogenic potential of dysregulated expression of a STAT protein that is not constitutively activated, and that TCR stimulation can contribute to this process.

Show MeSH
Related in: MedlinePlus