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Inhibition of Cell Cycle Progression by Rapamycin Induces T Cell Clonal Anergy Even in the Presence of Costimulation

Jonathan D. Powell, Cara G. Lerner and Ronald H. Schwartz
J Immunol March 1, 1999, 162 (5) 2775-2784;
Jonathan D. Powell
Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Cara G. Lerner
Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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Ronald H. Schwartz
Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
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  • FIGURE 1.
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    FIGURE 1.

    A, Rapamycin but not CSA inhibits proliferation of A.E7 cells to exogenous IL-2. A.E7 cells were incubated with 50 U/ml of IL-2 in the presence of increasing doses of either CSA or rapamycin. After 48 h, [3H]thymidine was added to each well and the cells were harvested and assayed for proliferation after an additional 16 h of culture. Comparable data were obtained in three other experiments. B, CSA but not rapamycin inhibits IL-2 production. A.E7 cells were incubated overnight with plate-bound anti-TCR and soluble anti-CD28 in the presence of increasing doses of either CSA or rapamycin. Supernatant fluids were harvested and assayed for IL-2 by ELISA.

  • FIGURE 2.
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    FIGURE 2.

    A, CSA inhibits anergy induction. A.E7 cells were incubated overnight with plate-bound anti-TCR alone (signal 1) or plate-bound anti-TCR plus soluble anti-CD28 (signal 1 plus 2) in the presence or absence of CSA. The cells were harvested, washed, and rested for 7 days in fresh medium. The cells were then tested for their ability to proliferate to Ag and APCs. Note, the CSA is only present during the induction phase of anergy and not during the rest period or rechallenge phase. In this and subsequent figures, the legend refers to the conditions of the induction phase. B, Rapamycin augments anergy induction even in the presence of costimulation. In the same experiment as shown in A, A.E7 cells were incubated overnight with plate-bound anti-TCR alone or plate-bound anti-TCR plus soluble anti-CD28 in the presence or absence of rapamycin. The signal 1 and signal 1 plus 2 groups are the same as in A to facilitate easy comparison. Once again, the rapamycin was only present during the induction of anergy and not during the rest or rechallenge phases. C, Rapamycin-induced anergic cells proliferate to exogenous IL-2. The cells that were anergized in A and B were tested for their ability to proliferate to 50 U/ml of exogenous IL-2. A.E7 cells were cultured for 48 h and pulsed with [3H]thymidine for an additional 16 h and then harvested and assayed for incorporation of radioactivity into DNA. Proliferation of cells in the absence of IL-2 was <1000 cpm for all conditions except signal 1 plus 2, which was 2400 cpm (not shown). Comparable data were obtained in three other experiments.

  • FIGURE 3.
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    FIGURE 3.

    CSA inhibits rapamycin-induced anergy. A.E7 cells were anergized as in Fig. 2. In an additional culture, A.E7 cells were incubated overnight with plate-bound anti-TCR, soluble anti-CD28, and 100 nM of both CSA and rapamycin. The cells were rested and rechallenged as described in Fig. 2. These data are representative of two separate experiments.

  • FIGURE 4.
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    FIGURE 4.

    A, Stimulation with signal 1 plus 2 can result in hypoproliferation upon rechallenge. A.E7 cells were anergized as described in Materials and Methods. However, this time a mock culture (A.E7 cells that were cultured without any Abs) was included during the induction phase. These results are representative of three experiments. B, Induction of anergy in the presence of rapamycin as well as signal 1 plus 2 alone results in decreased IL-2 production upon rechallenge. The cells were anergized, rested, and then tested for their ability to produce IL-2 upon rechallenge by incubating them in the presence of plate-bound anti-TCR and soluble anti-CD28 for 16 h. Supernatant fluids were harvested and assayed for IL-2 using a CTLL bioassay as described in Material and Methods. This pattern of results was obtained in three other experiments (see Fig. 5B for example).

  • FIGURE 5.
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    FIGURE 5.

    Culturing cells in IL-2 rescues them from anergy. A, After being anergized, the cells were washed and split into two cultures containing fresh medium with or without 50 U/ml of IL-2 and cultured for 12 days. The cells were washed and tested for their ability to proliferate to Ag. B, Cells were anergized and cultured in the presence or absence of IL-2 and then tested for their ability to produce IL-2. Comparable results were obtained in three separate experiments. Note, in both A and B, “+ IL-2” refers to the presence of IL-2 during the rest culture before rechallenge.

  • FIGURE 6.
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    FIGURE 6.

    Rapamycin-induced anergy results in a block in the MAP kinase pathway. Extracts were derived from cells anergized under various conditions and then either left unstimulated or stimulated with plate-bound anti-TCR for 10 min. The upper panel was blotted with anti-phospho-ERK, while the bottom panel is the same blot stripped and reblotted with anti-ERK. The upper and lower arrows indicate ERK 1 and 2, respectively. Extracts from nonanergic A.E7 cells are denoted by “Mock,” conventionally anergized cells are denoted as “1 alone,” partially anergized cells are denoted as “1 + 2,” and rapamycin-anergized cells are denoted as “1 + 2 + R.”

  • FIGURE 7.
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    FIGURE 7.

    Rapamycin-induced anergy results in decreased IFN-γ and IL-3 production. Cells were anergized, rested in fresh medium, and stimulated with plate-bound anti-TCR and anti-CD28 ascites. Supernatant fluids were assayed by ELISA for IFN-γ (A) and IL-3 production (B). Cytokine production was <2.5 pg/ml of IL-3 and undetectable for IFN-γ for all unstimulated cultures (only unstimulated “Mock” culture is shown). Comparable results were observed in three experiments.

  • FIGURE 8.
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    FIGURE 8.

    A, Hydroxyurea inhibits IL-2-mediated T cell proliferation. A.E7 T cells were incubated with 50 U/ml IL-2 and increasing concentrations of hydroxyurea in a 96-well plate. The cultures were pulsed with [3H]thymidine after 48 h and harvested at 64 h. In a separate culture, A.E7 cells were stimulated with plate-bound anti-TCR and anti-CD28 in the presence of increasing concentrations of hydroxyurea. After 16 h, supernatant fluid was harvested and IL-2 production was determined using ELISA. B, The effect of hydroxyurea on anergy induction. A.E7 T cells were stimulated overnight with either anti-TCR alone or anti-TCR plus anti-CD28 in the presence or absence of hydroxyurea, rapamycin, or both. The cells were washed and rested 7 days and then rechallenged with anti-TCR and anti-CD28 for 16 h. Supernatant fluid was collected and assayed for IL-2 production using the CTLL bioassay.

  • FIGURE 9.
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    FIGURE 9.

    Model of anergy induction. This model adapts the kinetic proofreading model of T cell activation (35, 36) to incorporate a number of observations concerning anergy induction in CD4+ T cell clones. See Discussion for details.

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The Journal of Immunology: 162 (5)
The Journal of Immunology
Vol. 162, Issue 5
1 Mar 1999
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Inhibition of Cell Cycle Progression by Rapamycin Induces T Cell Clonal Anergy Even in the Presence of Costimulation
Jonathan D. Powell, Cara G. Lerner, Ronald H. Schwartz
The Journal of Immunology March 1, 1999, 162 (5) 2775-2784;

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Inhibition of Cell Cycle Progression by Rapamycin Induces T Cell Clonal Anergy Even in the Presence of Costimulation
Jonathan D. Powell, Cara G. Lerner, Ronald H. Schwartz
The Journal of Immunology March 1, 1999, 162 (5) 2775-2784;
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