31 research outputs found

    Different expressions of immune components in ascites of early- and advanced-stage ovarian cancer patients.

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    <p>(<b>A</b>) Representative figures of flow cytometric analyses of CD4<sup>+</sup> helper T cells, CD8<sup>+</sup> cytotoxic T lymphocytes, and CD4<sup>+</sup>CD25<sup>+</sup> regulatory T lymphocytes (Treg cells) in TAL. A1, CD4<sup>+</sup> helper T cells; A2, CD8<sup>+</sup> cytotoxic T lymphocytes; A3, CD4<sup>+</sup>CD25<sup>+</sup> Treg cells. (<b>B</b>) Percentages of CD4<sup>+</sup> helper T lymphocytes, CD8<sup>+</sup> cytotoxic T lymphocytes, and CD4<sup>+</sup>CD25<sup>+</sup> regulatory T lymphocytes (Treg cells) in TAL. B1, CD4<sup>+</sup> helper T cells; B2, CD8<sup>+</sup> cytotoxic T lymphocytes; B3, CD4<sup>+</sup>CD25<sup>+</sup> Treg cells. <i>Note</i>: The percentages of CD4<sup>+</sup> T cells were significantly higher in patients with advanced stage ovarian cancer. (<b>C</b>) Concentrations of various cytokines in ascites of ovarian cancer patients. C1, IL-4; C2, TNF-α; C3, TGF-β; C4, IL-6; C5, IL-10; C6, IFN-γ. <i>Note</i>: The IL-4 and TNF-α concentrations decreased while the TGF-β, IL-6, IL-10, and IFN-γ concentrations increased from early to advanced stage.</p

    Depletion of Regulatory T Lymphocytes Reverses the Imbalance between Pro- and Anti-Tumor Immunities via Enhancing Antigen-Specific T Cell Immune Responses

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    <div><h3>Background</h3><p>The regulatory T cells (Tregs) can actively suppress the immune responses. However, literature about detailed changes of host effective and suppressive immunities before and after depletion of Tregs in ovarian carcinomas, is rare.</p> <h3>Materials and Methods</h3><p>Ovarian cancer patients and the ascitogenic animal model were employed. Immunologic profiles with flow cytometric analyses, immunohistochemistric staining, RT-PCR, ELISA, and ELISPOT assays were performed. <em>In vivo</em> depletion of Treg cells with the mAb PC61was also performed in the animal model.</p> <h3>Results</h3><p>The cytokines, including IL-4 (<em>p</em> = 0.017) and TNF-α (<em>p</em> = 0.046), significantly decreased while others such as TGF-β (<em>p</em> = 0.013), IL-6 (<em>p</em> = 0.016), and IL-10 (<em>p</em> = 0.018) were elevated in ascites of ovarian cancer patients, when the disease progressed to advanced stages. The ratio of CD8<sup>+</sup> T cell/Treg cell in ascites was also lower in advanced diseases than in early diseases (advanced 7.37±0.64 vs. early 14.25±3.11, <em>p</em> = 0.037). The kinetic low-dose CD25 Ab depletion group had significantly lower intra-peritoneal tumor weight (0.20±0.03 g) than the sequential high-dose (0.69±0.06 g) and sequential low-dose (0.67±0.07 g) CD25 Ab deletion groups (<em>p</em> = 0.001) after 49 days of tumor challenge in the animal. The kinetic low-dose CD25 Ab depletion group generated the highest number of IFN-γ-secreting, mesothelin-specific T lymphocytes compared to the other groups (<em>p</em><0.001).</p> <h3>Conclusions</h3><p>The imbalance between effective and suppressive immunities becomes more severe as a tumor progresses. The depletion of Treg cells can correct the imbalance of immunologic profiles and generate potent anti-tumor effects. Targeting Treg cells can be a new strategy for the immunotherapy of ovarian carcinoma.</p> </div

    Alterations of systemic immune effector cells in splenocytes of mice challenged with PBS or WF-3 tumor cells.

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    <p>(<b>A</b>) Diagrammatic representation of the collection of specimens in early and advanced diseases. (<b>B</b>) Representative figures of mice after WF-3 challenge at days 14 and 49. <i>Note</i>: Only small tumors (arrow) with little ascites were identified in mice of early disease. However, disseminated tumor implants (arrows) with bloody ascites within the whole peritoneal cavity were noted in mice with advanced disease. (<b>C</b>) Representative figures of flow cytometric analyses of various kinds of lymphocytes in splenocytes. (<b>D</b>) Percentages of various kinds of lymphocytes in splenocytes of naïve mice and in mice of early and advanced diseases. D1, CD4<sup>+</sup> helper T lymphocytes; D2, CD8<sup>+</sup> cytotoxic T lymphocytes; D3, NK1.1<sup>+</sup> natural killer cells; and D4, CD19<sup>+</sup> B lymphocytes. <i>Note</i>: All of the percentages of systemic immune effector cells significantly decreased as the tumor progressed from early to advanced stage.</p

    Kinetic changes of local immune effector cells in TACs of mice challenged with PBS or WF-3 tumor cells.

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    <p>(<b>A</b>) Percentages of various kinds of lymphocytes in TACs of naïve mice and mice of early and advanced diseases. A1, CD4<sup>+</sup> helper T lymphocytes; A2, CD8<sup>+</sup> cytotoxic T lymphocytes; A3, NK1.1<sup>+</sup> natural killer cells; and A4, CD19<sup>+</sup> B lymphocytes. <i>Note</i>: For helper and cytotoxic T lymphocytes, the percentages on TACs increased from early to advanced disease. However, the percentages of natural killer cells and lymphocytes in TACs significantly decreased with tumor progression. (<b>B</b>) CD223<sup>−</sup>CD8<sup>+</sup> lymphocytes in TACs of mice. B1: Representative figures of flow cytometric analyses of CD223<sup>−</sup>CD8<sup>+</sup> and CD223<sup>+</sup>CD8<sup>+</sup> lymphocytes in TACs. <b>B2:</b> Percentages of CD223<sup>−</sup>CD8<sup>+</sup> lymphocytes in TACs of mice. <i>Note</i>: Majority of CD8<sup>+</sup> cytotoxic T lymphocytes were non-activated as the disease progressed to advanced status. (<b>C</b>) The RT-PCR of various cytokines in splenocytes of mice challenged with PBS or WF-3 tumor cells. <i>Note</i>: The expression levels of IL-4, IL-12, TNF-α, and INF-γ decreased but the expression levels of IL-10 increased gradually as the disease progressed. (<b>D</b>) The concentrations of various cytokines by ELISA in ascites of mice challenged with PBS or WF-3 tumor cells. D1, IL-12; D2, TNF-α; D3, IFN-γ; D4, IL-6; D5, IL-10; D6, TGF-β. <i>Note</i>: The concentrations of IL-6, IL-10, and TGF-β were elevated but those of IL-12, TNF-α, and IFN-γ decreased as the tumor progressed.</p

    Kinetic changes of Treg cells in the splenocytes and TACs of mice challenged with PBS or WF-3 tumor cells.

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    <p>(<b>A</b>) Representative figures of CD4<sup>+</sup>FoxP3<sup>+</sup> Treg cells in the spleen of mice by immuno-histochemistry staining observed under confocal microscopy. A1, A single CD4<sup>+</sup>FoxP3<sup>+</sup> regulatory T cell (blue: nucleus, green: CD4, red: FoxP3); A2, PBS group; A3, WF-3 group with early disease; A4, WF-3 group with advanced disease (arrows: CD4<sup>+</sup>FoxP3<sup>+</sup> Treg cells). (<b>B</b>) CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> Treg cells in splenocytes. B1: Representative figures of flow cytometric analyses. B2: The bar figures of CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> Treg cells in various groups. B3: The ratios of CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> Treg cells. <i>Note:</i> Referenced with the naïve group, Treg cells of the advanced stage group were 1.5-fold higher in splenocytes. (<b>C</b>) CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> Treg cells in TACs. C1: Representative figures of flow cytometric analysese. C2: The bar figures of CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> Treg cells in various groups. C3: The bar figures of the ratios of CD4<sup>+</sup>CD25<sup>+</sup>FoxP3<sup>+</sup> Treg cells in various groups. <i>Note:</i> Referenced with the naïve group, Treg cells were 2.9-fold higher in TACs of the advanced stage group.</p

    The concentrations of cytokine profiles in ascites and kinetic changes of various lymphocytes in splenocytes of WF-3-challenged mice treated with PBS or CD25 monoclonal antibody.

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    <p>(<b>A</b>) Concentrations of various cytokines in ascites. A1, IL-6; A2, IL-12; A3, TNF-α; A4, IFN-γ; A5, TGF-β. <i>Note</i>: Compared to mice treated with PBS only, the concentrations of cytokines like IL-6, IL-12, TNF-α, and IFN-γ were significantly elevated in mice treated with kinetic low-dose CD25 antibody than those in the other groups. The concentrations of TGF-β significantly decreased in the kinetic low-dose CD25 Ab group compared to the other groups. (<b>B</b>) Percentages of various lymphocytes. B1, CD3<sup>+</sup>CD4<sup>+</sup> help T lymphocytes. <i>Note</i>: The percentages of CD4<sup>+</sup> T lymphocytes in the splenocytes were not different between the PBS and CD25 Ab depletion groups on day 14 after WF-3 tumor challenge. However, the kinetic low-dose CD25 Ab depletion group had higher percentages of CD4<sup>+</sup> helper T cells than the PBS and the other two sequential CD25 Ab depletion groups after 49 days of tumor challenge. B2, CD3<sup>+</sup>CD8<sup>+</sup> cytotoxic T lymphocytes. <i>Note</i>: The percentages of CD8<sup>+</sup> T cells were not different between the PBS and CD25 Ab depletion groups on 14 days after tumor challenge. The kinetic low-dose CD25 Ab depletion group had higher percentages of CD8<sup>+</sup> cytotoxic T cells than the other groups on day 49 after tumor challenge. B3, CD4<sup>+</sup>CD25<sup>+</sup>Foxp3<sup>+</sup> Treg cells. <i>Note</i>: The percentages of Treg cells in the splenocytes were not different among these groups on day 14 after tumor challenge. However, the kinetic low-dose CD25 Ab depletion group had lower percentages of Treg cells than the other groups on day 49. B4, ratios of cytotoxic T lymphocytes/Treg cells. <i>Note</i>: The ratios of CD8<sup>+</sup> cytotoxic T cells/Treg cells in the kinetic low-dose CD25 Ab depletion group were highest among the four groups. (<b>C</b>) Mesothelin-specific IFN-γ ELISPOT assays of splenocytes in the PBS and CD25 Ab-treated groups. C1, the representative figures of mesothelin-specific IFN-γ ELISPOT assays of splenocytes; C2, bar figures of the numbers of IFN-γ-secreting, mesothelin-specific T lymphocytes in various groups. <i>Note:</i> The kinetic low-dose CD25 Ab depletion group generated the highest numbers of IFN-γ-secreting, mesothelin-specific T lymphocytes compared to the other groups. (<b>D</b>) Survivals of mice treated with PBS, neutralizing TGF-β, neutralizing IL-10 or CD25 monoclonal antibodies. D1, Diagrammatic representation of the different treatment protocols of neutralizing TGF-β, neutralizing IL-10 or CD25 monoclonal antibody; D2, Survival curves of mice treated with PBS, neutralizing TGF-β, neutralizing IL-10, neutralizing TGF-β and neutralizing IL-10 or CD25 monoclonal antibody. <i>Note:</i> The mice treated with kinetic low-dose CD25 Ab had the longest survival time after WF-3 tumor challenge when compared to the other mice treated with PBS, neutralizing TGF-β, neutralizing IL-10 or neutralizing TGF-β and neutralizing IL-10 monoclonal antibodies.</p

    Comparative experiments between mice immunized with various numbers of PE(ΔIII)/E6 and/or PE(ΔIII)/E7 protein vaccines.

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    <p>Numbers of (<b>A</b>) E6-specific CD8<sup>+</sup> T precursors and (<b>B</b>) E7-specific CD8<sup>+</sup> T precursors in the mice vaccinated with varying frequencies of fusion protein vaccines. <i>Note</i>: The mice vaccinated more times with PE(ΔIII)/E6+PE(ΔIII)/E7 protein vaccines generated higher numbers of E6- and E7-specific CD8<sup>+</sup> T precursors. (<b>C</b>) Percentages of tumor-free mice in <i>in </i><i>vivo</i> prevention experiments of the mice vaccinated once with various fusion protein vaccines. <i>Note</i>: All of the naïve mice and mice vaccinated once with PE(ΔIII)/E6 and/or PE(ΔIII)/E7 protein vaccines had tumorigenesis within 14 days after TC-1 tumor challenge. (<b>D</b>) Percentages of tumor-free mice in <i>in </i><i>vivo</i> prevention experiments of mice vaccinated twice with various fusion protein vaccines. <i>Note</i>: 20%, 60%, and 100% of the mice vaccinated twice with PE(ΔIII)/E6, PE(ΔIII)/E7, and PE(ΔIII)/E6+PE(ΔIII)/E7 protein vaccines were tumor-free after 60 days of TC-1 tumor challenge. (<b>E</b>) Subcutaneous tumor volumes of the mice in <i>in </i><i>vivo</i> therapeutic experiments of mice vaccinated thrice with various fusion protein vaccines. <i>Note</i>: The mice vaccinated with the PE(ΔIII)/E6+PE(ΔIII)/E7 fusion protein vaccines had the smallest tumor volumes 55 days after TC-1 tumor challenge (<i>p</i><0.001, one-way ANOVA).</p

    Immunologic profiles of the vaccinated mice using intracellular cytokine staining and ELISA.

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    <p>(<b>A</b>) The number of IFN-γ-secreting CD4<sup>+</sup> T cells with (open columns) or without (filled columns) the corresponding E6 protein (aa1-151). <i>Note</i>: The numbers of E6-specific, IFN-γ-secreting CD4<sup>+</sup> T cells in the PE(ΔIII)/E6 (219.5±18.9) and PE(ΔIII)/E6+PE(ΔIII)/E7 (260.0±22.4) groups were significantly higher than those in the other groups (<i>p</i><0.01, one-way ANOVA). (<b>B</b>) The number of IFN-γ-secreting CD4<sup>+</sup> T cells in with (open columns) or without (filled columns) the corresponding E7 protein (aa1-98). <i>Note</i>: The numbers of E7-specific, IFN-γ-secreting CD4<sup>+</sup> T cells in the PE(ΔIII)/E7 (258.0±18.2) and PE(ΔIII)/E6+PE(ΔIII)/E7 (239.5±17.5) groups were significantly higher than those in the other groups (<i>p</i><0.01, one-way ANOVA). (<b>C</b>) The number of IFN-γ-secreting CD8<sup>+</sup> T cell precursors with (open columns) or without (filled columns) the corresponding E6 peptide (aa 50-57). <i>Note</i>: The PE(ΔIII)/E6 (247.5±18.2) and PE(ΔIII)/E6+PE(ΔIII)/E7 (392.5±19.6) groups generated higher numbers of E6-specific, IFN-γ-secreting CD8<sup>+</sup> T precursors than the other groups (<i>p</i><0.01, one-way ANOVA). (<b>D</b>) The number of IFN-γ-secreting CD8<sup>+</sup> T cell precursors with (open columns) or without (filled columns) the corresponding E7 peptide (aa 49-57). <i>Note</i>: The PE(ΔIII)/E7 (588.5±16.8) and PE(ΔIII)/E6+PE(ΔIII)/E7 (778.0±18.9) groups also generated higher numbers of E7-specific, IFN-γ-secreting CD8<sup>+</sup> T precursors than the other groups (<i>p</i><0.01, one-way ANOVA). ELISA demonstrated (<b>E</b>) E6-specific and (<b>F</b>) E7-specific Abs in the mice vaccinated with various protein vaccines. <i>Note</i>: The titers of E6-specific Abs were higher in the PE(ΔIII)/E6 and PE(ΔIII)/E6+PE(ΔIII)/E7 groups than in the other groups (<i>p</i><0.01, one-way ANOVA). The PE(ΔIII)/E7 and PE(ΔIII)/E6+PE(ΔIII)/E7 also generated significantly higher titers of E7-specific Abs than the other groups (<i>p</i><0.01, one-way ANOVA).</p

    <i>In vivo</i> tumor prevention and Ab depletion experiments in mice vaccinated with PE(ΔIII)/E6 and/or PE(ΔIII)/E7 protein vaccines.

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    <p><i>In </i><i>vivo</i> tumor protection experiments of the mice vaccinated with (<b>A</b>) respective E6 protein vaccines and (<b>B</b>) E6 and/or E7 fusion protein vaccines. <i>In </i><i>vivo</i> Ab depletion experiments of mice vaccinated with (<b>C</b>) PE(ΔIII)/E6 protein vaccine, (<b>D</b>) PE(ΔIII)/E7 vaccine, and (<b>E</b>) PE(ΔIII)/E6+PE(ΔIII)/E7 protein vaccines.</p
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