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TCR activation kinetics and feedback regulation in primary human T cells.

Poltorak M, Arndt B, Kowtharapu BS, Reddycherla AV, Witte V, Lindquist JA, Schraven B, Simeoni L - Cell Commun. Signal (2013)

Bottom Line: In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling.We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated.In summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Leipziger Str, 44, 39120, Magdeburg, Germany. luca.simeoni@med.ovgu.de.

ABSTRACT

Background: Signaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses.

Results: Here, we have analyzed signaling dynamics correlating with either unresponsiveness or proliferation induced upon TCR/CD28 ligation in primary human T cells. We used two widely employed methods to stimulate T cells in vitro, antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs). A comparative analysis of the signaling properties of iAbs and sAbs revealed that, under proliferation-inducing conditions, feedback regulation is markedly different from that leading to an unresponsive state. In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling. We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated.

Conclusions: In summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.

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Related in: MedlinePlus

An Erk-Lck feedback loop regulates TCR-mediated signaling. A) Purified human T cells were treated with iAbs alone for 30 min and then either DMSO or the MEK inhibitor U0126 was added and incubated for an additional 30 to 60 min. Samples were analyzed by Western blotting using the indicated Abs. B) Bands in A) were quantified and the values were normalized as described. Graphs show the mean of the phosphorylation levels of Erk1/2, ZAP70, and LAT or the level of p59 Lck as arbitrary units ± SEM of 4 independent experiments. C) Purified human T cells were preincubated either in the presence of DMSO or the MEK inhibitor U0126 and subsequently stimulated with sAbs for the indicated time points. Samples were analyzed by Western blotting using the indicated Abs. D) Bands in C) were quantified as described above and the data from at least two independent experiments are shown.
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Figure 4: An Erk-Lck feedback loop regulates TCR-mediated signaling. A) Purified human T cells were treated with iAbs alone for 30 min and then either DMSO or the MEK inhibitor U0126 was added and incubated for an additional 30 to 60 min. Samples were analyzed by Western blotting using the indicated Abs. B) Bands in A) were quantified and the values were normalized as described. Graphs show the mean of the phosphorylation levels of Erk1/2, ZAP70, and LAT or the level of p59 Lck as arbitrary units ± SEM of 4 independent experiments. C) Purified human T cells were preincubated either in the presence of DMSO or the MEK inhibitor U0126 and subsequently stimulated with sAbs for the indicated time points. Samples were analyzed by Western blotting using the indicated Abs. D) Bands in C) were quantified as described above and the data from at least two independent experiments are shown.

Mentions: To check whether the inhibition of Erk-mediated Lck phosphorylation also resulted in a reduction of its activity, we investigated phosphorylation levels of downstream signaling molecules that are substrates of Lck, such as the tyrosine kinase ZAP70 and the adaptor protein LAT whose phosphorylation depends on ZAP70. T cells were stimulated for 30 min with iAbs. Subsequently, Erk activity was blocked by the addition of the MEK inhibitor U0126. The data presented in (Figure 4A, B) show that the phosphorylation of both ZAP70 and LAT is reduced upon MEK inhibition, thus indicating that Erk-mediated Lck phosphorylation may enhance its response. Conversely, treatment of sAbs-stimulated T cells with the MEK inhibitor reduced Erk phosphorylation, as expected, but not ZAP70 or LAT phosphorylation (Figure 4C, D).


TCR activation kinetics and feedback regulation in primary human T cells.

Poltorak M, Arndt B, Kowtharapu BS, Reddycherla AV, Witte V, Lindquist JA, Schraven B, Simeoni L - Cell Commun. Signal (2013)

An Erk-Lck feedback loop regulates TCR-mediated signaling. A) Purified human T cells were treated with iAbs alone for 30 min and then either DMSO or the MEK inhibitor U0126 was added and incubated for an additional 30 to 60 min. Samples were analyzed by Western blotting using the indicated Abs. B) Bands in A) were quantified and the values were normalized as described. Graphs show the mean of the phosphorylation levels of Erk1/2, ZAP70, and LAT or the level of p59 Lck as arbitrary units ± SEM of 4 independent experiments. C) Purified human T cells were preincubated either in the presence of DMSO or the MEK inhibitor U0126 and subsequently stimulated with sAbs for the indicated time points. Samples were analyzed by Western blotting using the indicated Abs. D) Bands in C) were quantified as described above and the data from at least two independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3842781&req=5

Figure 4: An Erk-Lck feedback loop regulates TCR-mediated signaling. A) Purified human T cells were treated with iAbs alone for 30 min and then either DMSO or the MEK inhibitor U0126 was added and incubated for an additional 30 to 60 min. Samples were analyzed by Western blotting using the indicated Abs. B) Bands in A) were quantified and the values were normalized as described. Graphs show the mean of the phosphorylation levels of Erk1/2, ZAP70, and LAT or the level of p59 Lck as arbitrary units ± SEM of 4 independent experiments. C) Purified human T cells were preincubated either in the presence of DMSO or the MEK inhibitor U0126 and subsequently stimulated with sAbs for the indicated time points. Samples were analyzed by Western blotting using the indicated Abs. D) Bands in C) were quantified as described above and the data from at least two independent experiments are shown.
Mentions: To check whether the inhibition of Erk-mediated Lck phosphorylation also resulted in a reduction of its activity, we investigated phosphorylation levels of downstream signaling molecules that are substrates of Lck, such as the tyrosine kinase ZAP70 and the adaptor protein LAT whose phosphorylation depends on ZAP70. T cells were stimulated for 30 min with iAbs. Subsequently, Erk activity was blocked by the addition of the MEK inhibitor U0126. The data presented in (Figure 4A, B) show that the phosphorylation of both ZAP70 and LAT is reduced upon MEK inhibition, thus indicating that Erk-mediated Lck phosphorylation may enhance its response. Conversely, treatment of sAbs-stimulated T cells with the MEK inhibitor reduced Erk phosphorylation, as expected, but not ZAP70 or LAT phosphorylation (Figure 4C, D).

Bottom Line: In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling.We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated.In summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Clinical Immunology, Otto-von-Guericke University, Leipziger Str, 44, 39120, Magdeburg, Germany. luca.simeoni@med.ovgu.de.

ABSTRACT

Background: Signaling through the TCR is crucial for the generation of different cellular responses including proliferation, differentiation, and apoptosis. A growing body of evidence indicates that differences in the magnitude and the duration of the signal are critical determinants in eliciting cellular responses.

Results: Here, we have analyzed signaling dynamics correlating with either unresponsiveness or proliferation induced upon TCR/CD28 ligation in primary human T cells. We used two widely employed methods to stimulate T cells in vitro, antibodies either cross-linked in solution (sAbs) or immobilized on microbeads (iAbs). A comparative analysis of the signaling properties of iAbs and sAbs revealed that, under proliferation-inducing conditions, feedback regulation is markedly different from that leading to an unresponsive state. In fact, upon iAbs stimulation TCR-mediated signaling is prolonged by a positive feedback loop involving Erk, whereas sAbs strongly activate inhibitory molecules that likely terminate signaling. We additionally found that, by enhancing the phosphorylation of Src family kinases under proliferation-inducing conditions, signaling and T-cell activation are terminated.

Conclusions: In summary, our analysis documents TCR signaling kinetics and feedback regulation under conditions of stimulation inducing either unresponsiveness or proliferation.

Show MeSH
Related in: MedlinePlus