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Requirements for peptide-induced T cell receptor downregulation on naive CD8+ T cells.

Cai Z, Kishimoto H, Brunmark A, Jackson MR, Peterson PA, Sprent J - J. Exp. Med. (1997)

Bottom Line: These stringent requirements did not apply to TCR downregulation.Thus, TCR downregulation seemed to depend solely on TCR/peptide/interaction and did not require either CD8 or B7-1 expression; ICAM-1 potentiated TCR downregulation, but only with limiting doses of peptides.TCR downregulation was most prominent with high affinity peptides and appeared to be neither obligatory nor sufficient for T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, IMM4, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
The requirements for inducing downregulation of alpha/beta T cell receptor (TCR) molecules on naive major histocompatibility complex class I-restricted T cells was investigated with 2C TCR transgenic mice and defined peptides as antigen. Confirming previous results, activation of 2C T cells in response to specific peptides required CD8 expression on the responder cells and was heavily dependent upon costimulation provided by either B7-1 or ICAM-1 on antigen-presenting cells (APC). These stringent requirements did not apply to TCR downregulation. Thus, TCR downregulation seemed to depend solely on TCR/peptide/interaction and did not require either CD8 or B7-1 expression; ICAM-1 potentiated TCR downregulation, but only with limiting doses of peptides. TCR downregulation was most prominent with high affinity peptides and appeared to be neither obligatory nor sufficient for T cell activation. In marked contrast to T cell activation, TCR downregulation was resistant to various metabolic inhibitors. The biological significance of TCR downregulation is unclear, but could be a device for protecting T cells against excessive signaling.

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Metabolic requirements for TCR downregulation. CD8+  2C cells (1 × 106) were cultured with 5 × 106 CD8-depleted B10.D2  spleen cells in the presence or absence of 10 μM QL9 peptides for 12 h.  Inhibitors were added at the beginning of the cultures as indicated. Cells  were harvested and stained for the expression of TCR (1B2) and CD69 as  described in Materials and Methods. Staining for TCR and CD69 expression is shown in the form of histograms (A) and MFI (B) and was obtained by gating on CD8+ cells. In A, the histograms at the bottom refer  to staining of control 2C cells cultured at 4°C in the absence of APCs. In  B, the inhibitors were added at 200 μM for genistein, 4 μg/ml for CCD,  and 0.2% for sodium azide.
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Figure 7: Metabolic requirements for TCR downregulation. CD8+ 2C cells (1 × 106) were cultured with 5 × 106 CD8-depleted B10.D2 spleen cells in the presence or absence of 10 μM QL9 peptides for 12 h. Inhibitors were added at the beginning of the cultures as indicated. Cells were harvested and stained for the expression of TCR (1B2) and CD69 as described in Materials and Methods. Staining for TCR and CD69 expression is shown in the form of histograms (A) and MFI (B) and was obtained by gating on CD8+ cells. In A, the histograms at the bottom refer to staining of control 2C cells cultured at 4°C in the absence of APCs. In B, the inhibitors were added at 200 μM for genistein, 4 μg/ml for CCD, and 0.2% for sodium azide.

Mentions: The capacity of various metabolic inhibitors to impair TCR downregulation was measured with B10.D2 spleen cells as APCs ± QL9 peptide (10−5 M); drugs were dissolved in DMSO and TCR expression on CD8+ 2C cells was examined at 12 h. As shown in Fig. 7, relatively high concentrations of cycloheximide (100 μg/ml, inhibitor of protein synthesis), colchicine (20 μg/ml, inhibitor of microtubule polymerization; 42), CCD (see above), genistein (200 μM, inhibitor of tyrosine-specific protein kinase; 43) or sodium azide (0.2%, cytochrome oxidase inhibitor) had no detectable effect in inhibiting TCR downregulation at the time point studied. However, with the exception of colchicine, each drug prevented CD69 upregulation (though in accordance with the data in Fig. 6, the inhibition induced by CCD was prominent only when the B10.D2 APCs were not supplemented with QL9 peptide). Higher concentrations of drugs, e.g., 1% sodium azide, did reduce TCR downregulation, but substantially impaired cell viability, implying that the effects were nonspecific. The only approach tested that prevented TCR downregulation without causing cell damage was to culture cells at 4°C (Fig. 7 B).


Requirements for peptide-induced T cell receptor downregulation on naive CD8+ T cells.

Cai Z, Kishimoto H, Brunmark A, Jackson MR, Peterson PA, Sprent J - J. Exp. Med. (1997)

Metabolic requirements for TCR downregulation. CD8+  2C cells (1 × 106) were cultured with 5 × 106 CD8-depleted B10.D2  spleen cells in the presence or absence of 10 μM QL9 peptides for 12 h.  Inhibitors were added at the beginning of the cultures as indicated. Cells  were harvested and stained for the expression of TCR (1B2) and CD69 as  described in Materials and Methods. Staining for TCR and CD69 expression is shown in the form of histograms (A) and MFI (B) and was obtained by gating on CD8+ cells. In A, the histograms at the bottom refer  to staining of control 2C cells cultured at 4°C in the absence of APCs. In  B, the inhibitors were added at 200 μM for genistein, 4 μg/ml for CCD,  and 0.2% for sodium azide.
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Related In: Results  -  Collection

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Figure 7: Metabolic requirements for TCR downregulation. CD8+ 2C cells (1 × 106) were cultured with 5 × 106 CD8-depleted B10.D2 spleen cells in the presence or absence of 10 μM QL9 peptides for 12 h. Inhibitors were added at the beginning of the cultures as indicated. Cells were harvested and stained for the expression of TCR (1B2) and CD69 as described in Materials and Methods. Staining for TCR and CD69 expression is shown in the form of histograms (A) and MFI (B) and was obtained by gating on CD8+ cells. In A, the histograms at the bottom refer to staining of control 2C cells cultured at 4°C in the absence of APCs. In B, the inhibitors were added at 200 μM for genistein, 4 μg/ml for CCD, and 0.2% for sodium azide.
Mentions: The capacity of various metabolic inhibitors to impair TCR downregulation was measured with B10.D2 spleen cells as APCs ± QL9 peptide (10−5 M); drugs were dissolved in DMSO and TCR expression on CD8+ 2C cells was examined at 12 h. As shown in Fig. 7, relatively high concentrations of cycloheximide (100 μg/ml, inhibitor of protein synthesis), colchicine (20 μg/ml, inhibitor of microtubule polymerization; 42), CCD (see above), genistein (200 μM, inhibitor of tyrosine-specific protein kinase; 43) or sodium azide (0.2%, cytochrome oxidase inhibitor) had no detectable effect in inhibiting TCR downregulation at the time point studied. However, with the exception of colchicine, each drug prevented CD69 upregulation (though in accordance with the data in Fig. 6, the inhibition induced by CCD was prominent only when the B10.D2 APCs were not supplemented with QL9 peptide). Higher concentrations of drugs, e.g., 1% sodium azide, did reduce TCR downregulation, but substantially impaired cell viability, implying that the effects were nonspecific. The only approach tested that prevented TCR downregulation without causing cell damage was to culture cells at 4°C (Fig. 7 B).

Bottom Line: These stringent requirements did not apply to TCR downregulation.Thus, TCR downregulation seemed to depend solely on TCR/peptide/interaction and did not require either CD8 or B7-1 expression; ICAM-1 potentiated TCR downregulation, but only with limiting doses of peptides.TCR downregulation was most prominent with high affinity peptides and appeared to be neither obligatory nor sufficient for T cell activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, IMM4, The Scripps Research Institute, La Jolla, California 92037, USA.

ABSTRACT
The requirements for inducing downregulation of alpha/beta T cell receptor (TCR) molecules on naive major histocompatibility complex class I-restricted T cells was investigated with 2C TCR transgenic mice and defined peptides as antigen. Confirming previous results, activation of 2C T cells in response to specific peptides required CD8 expression on the responder cells and was heavily dependent upon costimulation provided by either B7-1 or ICAM-1 on antigen-presenting cells (APC). These stringent requirements did not apply to TCR downregulation. Thus, TCR downregulation seemed to depend solely on TCR/peptide/interaction and did not require either CD8 or B7-1 expression; ICAM-1 potentiated TCR downregulation, but only with limiting doses of peptides. TCR downregulation was most prominent with high affinity peptides and appeared to be neither obligatory nor sufficient for T cell activation. In marked contrast to T cell activation, TCR downregulation was resistant to various metabolic inhibitors. The biological significance of TCR downregulation is unclear, but could be a device for protecting T cells against excessive signaling.

Show MeSH
Related in: MedlinePlus