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Anergy and cytokine-mediated suppression as distinct superantigen-induced tolerance mechanisms in vivo.

Miller C, Ragheb JA, Schwartz RH - J. Exp. Med. (1999)

Bottom Line: This state resulted from a combination of both clonal anergy and cytokine-mediated immunosuppression.Full suppression lasted for only 1 wk and involved both IL-10 and TGF-beta, but required additional unknown molecules for optimal effect.These experiments show that complex in vivo interactions of multiple peripheral tolerance mechanisms can now be dissected at both the cellular and molecular levels.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0420, USA.

ABSTRACT
Recombinant-activating gene 2 (RAG-2-/-) T cell receptor-transgenic mice repeatedly injected with the superantigen staphylococcal enterotoxin A entered a tolerant state in which splenic CD4+ T cells produced little interleukin (IL)-2, interferon gamma, or IL-4. This state resulted from a combination of both clonal anergy and cytokine-mediated immunosuppression. The anergy persisted for at least 3 wk and could be distinguished from the suppression by a decrease in IL-2 production per cell, a block in the activation of early response kinases, and a failure to be reversed with anti-transforming growth factor (TGF)-beta. Full suppression lasted for only 1 wk and involved both IL-10 and TGF-beta, but required additional unknown molecules for optimal effect. These experiments show that complex in vivo interactions of multiple peripheral tolerance mechanisms can now be dissected at both the cellular and molecular levels.

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CD4+ spleen cells from SEA-treated animals can suppress IL-2 production from stimulated naive spleen cells, and the effect diminishes with time. (A) 105 spleen cells alone from SEA-treated, PBS-treated, or mixtures of 105 or 5 × 104 PBS-treated plus SEA-treated spleen cells, were cultured with 1 μM PCC peptide in the presence of 5 × 105 T-depleted irradiated B10.A spleen cells for 48 h. Culture supernatants were harvested and IL-2 production was measured by a CTL-L assay. The experiment from day 4 was repeated 10 times, and those from days 5, 6, and 20 were repeated four times with similar results. (B) Spleen cells from SEA- or PBS-treated mice were stained with anti-CD4 or anti-CD8 antibodies and were FACS® sorted. Purified CD4+ or CD8+ T cells (105) from SEA-treated mice were cultured with 105 purified CD4+ T cells from PBS-treated mice in the presence of 1 μM PCC peptide and 5 × 105 T-depleted, irradiated spleen cells for 48 h (purity shown at top). Supernatants were then harvested and assayed for IL-2 production by CTL-L assay. Results from mixtures using unfractionated cells are shown for comparison. The CD4+ and CD8+ sorts are from separate experiments. Each experiment was performed three times.
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Figure 3: CD4+ spleen cells from SEA-treated animals can suppress IL-2 production from stimulated naive spleen cells, and the effect diminishes with time. (A) 105 spleen cells alone from SEA-treated, PBS-treated, or mixtures of 105 or 5 × 104 PBS-treated plus SEA-treated spleen cells, were cultured with 1 μM PCC peptide in the presence of 5 × 105 T-depleted irradiated B10.A spleen cells for 48 h. Culture supernatants were harvested and IL-2 production was measured by a CTL-L assay. The experiment from day 4 was repeated 10 times, and those from days 5, 6, and 20 were repeated four times with similar results. (B) Spleen cells from SEA- or PBS-treated mice were stained with anti-CD4 or anti-CD8 antibodies and were FACS® sorted. Purified CD4+ or CD8+ T cells (105) from SEA-treated mice were cultured with 105 purified CD4+ T cells from PBS-treated mice in the presence of 1 μM PCC peptide and 5 × 105 T-depleted, irradiated spleen cells for 48 h (purity shown at top). Supernatants were then harvested and assayed for IL-2 production by CTL-L assay. Results from mixtures using unfractionated cells are shown for comparison. The CD4+ and CD8+ sorts are from separate experiments. Each experiment was performed three times.

Mentions: 105 or 5 × 104 spleen cells from SEA-treated mice were mixed at a 1:1 ratio with PBS-treated control spleen cells in culture with peptide plus APCs to determine if a suppressive effect on IL-2 production was operative in the culture system. SEA-treated spleen cells almost completely abrogated IL-2 production by peptide-stimulated PBS control spleen cells (Fig. 3 A) or naive spleen cells (data not shown). In the experiment with 105 cells, the suppression was measured as a >500–1,000-fold decrease in IL-2 production from day 4 to 6, but then waned to only 3–10-fold by day 20 after immunization (Fig. 3 A). The effect was also seen at decreasing cell numbers: as few as 6,000 SEA-treated spleen cells per well still gave significant suppression (data not shown).


Anergy and cytokine-mediated suppression as distinct superantigen-induced tolerance mechanisms in vivo.

Miller C, Ragheb JA, Schwartz RH - J. Exp. Med. (1999)

CD4+ spleen cells from SEA-treated animals can suppress IL-2 production from stimulated naive spleen cells, and the effect diminishes with time. (A) 105 spleen cells alone from SEA-treated, PBS-treated, or mixtures of 105 or 5 × 104 PBS-treated plus SEA-treated spleen cells, were cultured with 1 μM PCC peptide in the presence of 5 × 105 T-depleted irradiated B10.A spleen cells for 48 h. Culture supernatants were harvested and IL-2 production was measured by a CTL-L assay. The experiment from day 4 was repeated 10 times, and those from days 5, 6, and 20 were repeated four times with similar results. (B) Spleen cells from SEA- or PBS-treated mice were stained with anti-CD4 or anti-CD8 antibodies and were FACS® sorted. Purified CD4+ or CD8+ T cells (105) from SEA-treated mice were cultured with 105 purified CD4+ T cells from PBS-treated mice in the presence of 1 μM PCC peptide and 5 × 105 T-depleted, irradiated spleen cells for 48 h (purity shown at top). Supernatants were then harvested and assayed for IL-2 production by CTL-L assay. Results from mixtures using unfractionated cells are shown for comparison. The CD4+ and CD8+ sorts are from separate experiments. Each experiment was performed three times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195559&req=5

Figure 3: CD4+ spleen cells from SEA-treated animals can suppress IL-2 production from stimulated naive spleen cells, and the effect diminishes with time. (A) 105 spleen cells alone from SEA-treated, PBS-treated, or mixtures of 105 or 5 × 104 PBS-treated plus SEA-treated spleen cells, were cultured with 1 μM PCC peptide in the presence of 5 × 105 T-depleted irradiated B10.A spleen cells for 48 h. Culture supernatants were harvested and IL-2 production was measured by a CTL-L assay. The experiment from day 4 was repeated 10 times, and those from days 5, 6, and 20 were repeated four times with similar results. (B) Spleen cells from SEA- or PBS-treated mice were stained with anti-CD4 or anti-CD8 antibodies and were FACS® sorted. Purified CD4+ or CD8+ T cells (105) from SEA-treated mice were cultured with 105 purified CD4+ T cells from PBS-treated mice in the presence of 1 μM PCC peptide and 5 × 105 T-depleted, irradiated spleen cells for 48 h (purity shown at top). Supernatants were then harvested and assayed for IL-2 production by CTL-L assay. Results from mixtures using unfractionated cells are shown for comparison. The CD4+ and CD8+ sorts are from separate experiments. Each experiment was performed three times.
Mentions: 105 or 5 × 104 spleen cells from SEA-treated mice were mixed at a 1:1 ratio with PBS-treated control spleen cells in culture with peptide plus APCs to determine if a suppressive effect on IL-2 production was operative in the culture system. SEA-treated spleen cells almost completely abrogated IL-2 production by peptide-stimulated PBS control spleen cells (Fig. 3 A) or naive spleen cells (data not shown). In the experiment with 105 cells, the suppression was measured as a >500–1,000-fold decrease in IL-2 production from day 4 to 6, but then waned to only 3–10-fold by day 20 after immunization (Fig. 3 A). The effect was also seen at decreasing cell numbers: as few as 6,000 SEA-treated spleen cells per well still gave significant suppression (data not shown).

Bottom Line: This state resulted from a combination of both clonal anergy and cytokine-mediated immunosuppression.Full suppression lasted for only 1 wk and involved both IL-10 and TGF-beta, but required additional unknown molecules for optimal effect.These experiments show that complex in vivo interactions of multiple peripheral tolerance mechanisms can now be dissected at both the cellular and molecular levels.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-0420, USA.

ABSTRACT
Recombinant-activating gene 2 (RAG-2-/-) T cell receptor-transgenic mice repeatedly injected with the superantigen staphylococcal enterotoxin A entered a tolerant state in which splenic CD4+ T cells produced little interleukin (IL)-2, interferon gamma, or IL-4. This state resulted from a combination of both clonal anergy and cytokine-mediated immunosuppression. The anergy persisted for at least 3 wk and could be distinguished from the suppression by a decrease in IL-2 production per cell, a block in the activation of early response kinases, and a failure to be reversed with anti-transforming growth factor (TGF)-beta. Full suppression lasted for only 1 wk and involved both IL-10 and TGF-beta, but required additional unknown molecules for optimal effect. These experiments show that complex in vivo interactions of multiple peripheral tolerance mechanisms can now be dissected at both the cellular and molecular levels.

Show MeSH
Related in: MedlinePlus