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Kinetics and extent of T cell activation as measured with the calcium signal.

Wülfing C, Rabinowitz JD, Beeson C, Sjaastad MD, McConnell HM, Davis MM - J. Exp. Med. (1997)

Bottom Line: Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal.The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response.The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide-MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5402, USA.

ABSTRACT
We have characterized the calcium response of a peptide-major histocompatibility complex (MHC)-specific CD4(+) T lymphocyte line at the single cell level using a variety of ligands, alone and in combination. We are able to distinguish four general patterns of intracellular calcium elevation, with only the most robust correlating with T cell proliferation. Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal. We have also measured the time between the first T cell-antigen-presenting cell contact and the onset of the calcium signal. The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response. The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide-MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.

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Effect of anti-CD4 antibody GK1.5 on the type and onset of  the calcium signals. (A) Percentages of each signal type (as explained in  Fig. 3). MCC or 102S were diluted 1:1,000 into the neutral peptide  MCC93E99T102A, resulting in 25 nM concentration. The type of peptide and the absence or presence of the antibody GK1.5 are indicated under the columns. The antibody concentration is 100 ng/ml. (B) Cumulative representation of % cells activated (irrespective of the type of signal)  during a specific onset time interval. Time axis as in Fig. 4 B. Conditions  are as in A and are indicated in the header.
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Figure 5: Effect of anti-CD4 antibody GK1.5 on the type and onset of the calcium signals. (A) Percentages of each signal type (as explained in Fig. 3). MCC or 102S were diluted 1:1,000 into the neutral peptide MCC93E99T102A, resulting in 25 nM concentration. The type of peptide and the absence or presence of the antibody GK1.5 are indicated under the columns. The antibody concentration is 100 ng/ml. (B) Cumulative representation of % cells activated (irrespective of the type of signal) during a specific onset time interval. Time axis as in Fig. 4 B. Conditions are as in A and are indicated in the header.

Mentions: To determine the effect of low concentrations of stimulating peptide in a defined manner, the MCC and 102S peptides were each serially diluted into the neutral peptide 93E99T102A, such that the total peptide concentration during loading of the APCs is kept constant at 25 μM. As shown in Fig. 3, diluting the MCC peptide 1:1,000, resulting in a 25 nM MCC concentration, has no effect on the frequency or the type of the calcium signal. At a 1:10,000 dilution of the MCC (2.5 nM), more than 85% of the cells still respond with a full signal (see Fig. 5 A). When 102S is diluted into MCC93E99T102A in a dilution series ranging from 25 μM to 25 nM, the signaling frequency throughout the series is still almost 100% (Fig. 3). However, at a 25 nM (1:1,000 dilution), 70% of the cells respond with a reduced signal, as opposed to 30% at 25 μM.


Kinetics and extent of T cell activation as measured with the calcium signal.

Wülfing C, Rabinowitz JD, Beeson C, Sjaastad MD, McConnell HM, Davis MM - J. Exp. Med. (1997)

Effect of anti-CD4 antibody GK1.5 on the type and onset of  the calcium signals. (A) Percentages of each signal type (as explained in  Fig. 3). MCC or 102S were diluted 1:1,000 into the neutral peptide  MCC93E99T102A, resulting in 25 nM concentration. The type of peptide and the absence or presence of the antibody GK1.5 are indicated under the columns. The antibody concentration is 100 ng/ml. (B) Cumulative representation of % cells activated (irrespective of the type of signal)  during a specific onset time interval. Time axis as in Fig. 4 B. Conditions  are as in A and are indicated in the header.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196319&req=5

Figure 5: Effect of anti-CD4 antibody GK1.5 on the type and onset of the calcium signals. (A) Percentages of each signal type (as explained in Fig. 3). MCC or 102S were diluted 1:1,000 into the neutral peptide MCC93E99T102A, resulting in 25 nM concentration. The type of peptide and the absence or presence of the antibody GK1.5 are indicated under the columns. The antibody concentration is 100 ng/ml. (B) Cumulative representation of % cells activated (irrespective of the type of signal) during a specific onset time interval. Time axis as in Fig. 4 B. Conditions are as in A and are indicated in the header.
Mentions: To determine the effect of low concentrations of stimulating peptide in a defined manner, the MCC and 102S peptides were each serially diluted into the neutral peptide 93E99T102A, such that the total peptide concentration during loading of the APCs is kept constant at 25 μM. As shown in Fig. 3, diluting the MCC peptide 1:1,000, resulting in a 25 nM MCC concentration, has no effect on the frequency or the type of the calcium signal. At a 1:10,000 dilution of the MCC (2.5 nM), more than 85% of the cells still respond with a full signal (see Fig. 5 A). When 102S is diluted into MCC93E99T102A in a dilution series ranging from 25 μM to 25 nM, the signaling frequency throughout the series is still almost 100% (Fig. 3). However, at a 25 nM (1:1,000 dilution), 70% of the cells respond with a reduced signal, as opposed to 30% at 25 μM.

Bottom Line: Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal.The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response.The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide-MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5402, USA.

ABSTRACT
We have characterized the calcium response of a peptide-major histocompatibility complex (MHC)-specific CD4(+) T lymphocyte line at the single cell level using a variety of ligands, alone and in combination. We are able to distinguish four general patterns of intracellular calcium elevation, with only the most robust correlating with T cell proliferation. Whereas all three antagonist peptides tested reduce the calcium response to an agonist ligand, two give very different calcium release patterns and the third gives none at all, arguing that (a) antagonism does not require calcium release and (b) it involves interactions that are more T cell receptor proximal. We have also measured the time between the first T cell-antigen-presenting cell contact and the onset of the calcium signal. The duration of this delay correlates with the strength of the stimulus, with stronger stimuli giving a more rapid response. The dose dependence of this delay suggests that the rate-limiting step in triggering the calcium response is not the clustering of peptide-MHC complexes on the cell surface but more likely involves the accumulation of some intracellular molecule or complex with a half-life of a few minutes.

Show MeSH
Related in: MedlinePlus