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. 2005 Sep 20;102(38):13574-9.
doi: 10.1073/pnas.0505110102. Epub 2005 Sep 8.

A requirement for sustained ERK signaling during thymocyte positive selection in vivo

Lisa K McNeil  1 , Timothy K Starr, Kristin A Hogquist
Affiliations

A requirement for sustained ERK signaling during thymocyte positive selection in vivo

Lisa K McNeil et al. Proc Natl Acad Sci U S A. .

Abstract

It is unknown how the contrasting events of positive and negative selection can lead to the distinct biological outcomes of life or death. An increasing body of evidence suggests that the duration of extracellular signal-regulated kinase (ERK) signaling plays a role in thymocyte selection. However, it remains unclear what the kinetics of ERK activation are during positive selection in vivo. In this study, we examined the magnitude and duration of ERK signaling in intact murine thymic tissues cultured under conditions of negative or positive selection. We found that negative selection induced a rapid and robust ERK activation that is associated with death, whereas positive selection stimulated a lower intensity and brief ERK activation that quickly declined and then gradually increased and was sustained over several days. The expression pattern of Egr-1 (early growth response-1), a downstream ERK effector, correlates with the biphasic kinetics of ERK during positive selection. Id3 (inhibitor of differentiation/DNA binding 3) also exhibits biphasic kinetics but appeared to be independent of ERK signaling. Furthermore, inhibitors of T cell receptor ligation and ERK activation block maturation of CD8 single-positive thymocytes even when added after 24 h. These results demonstrate that the in vivo duration of ERK signaling must be sustained to support positive selection.

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Figures

Fig. 1.
Fig. 1.
The kinetics of positive and negative selection in the OT-I TAP° FTOC system. Fetal thymic lobes were incubated for the indicated times in media containing 1 μM OVAp (A), 2 μM P815p (B), or 2 μM βCATp (C). The lobes were then disrupted and stained with CD4 and CD8.
Fig. 2.
Fig. 2.
Addition of peptide to fetal thymic lobes results in rapid and transient ERK activation, which is sustained after 24 h under positive selection conditions. (A) Rapid phosphorylation of ERK in FTOC after the addition of peptides. Thymic lobes were prewarmed in media, and then peptide was added by basting the lobes. After various times of incubation, the lobes were fixed, permeabilized, and analyzed for intracellular pERK. Cells were gated on DP thymocytes at 2 min and 24 h but broadly gated to include DP and CD8 SP at the later time points. (B) Induction of phosphorylated ERK in OT-I TAP° fetal thymic lobes incubated for various times with the indicated peptides. DP thymocytes were gated and analyzed for the activation of ERK. The data are expressed as the mean fluorescence intensity (MFI) of pERK. Data are representative of at least three independent experiments. (C) Expression of phosphorylated ERK in OT-I TAP° fetal thymic lobes incubated for various times with βCATp or P815p. DP and CD8 SP thymocytes were gated. The MFIs of three lobes per time point were averaged, and the error bars represent the standard deviation. The difference between βCATp and P815p at 72 h was observed in more than five independent experiments.
Fig. 3.
Fig. 3.
Expression of Egr-1 and Id3 is biphasic in positively selected thymocytes and transient in negatively selected thymocytes. Egr-1 (A) or Id3 (B) expression in OT-I TAP° fetal thymic lobes incubated with OVAp, βCATp, or P815p for the indicated times. DP thymocytes were gated and analyzed for the expression of Egr-1 or Id3 by intracellular staining. The data are expressed as the MFI. Data are representative of at least three independent experiments.
Fig. 4.
Fig. 4.
Id3 up-regulation does not depend on ERK activation or expression of Egr-1. (A) Expression of Id3 and phosphorylated ERK in OT-I TAP° fetal thymic lobes. The lobes were incubated with βCATp with or without the addition of 20 μM U0126 (MEK inhibitor), gated on DP thymocytes, and analyzed for induction of phosphorylated ERK at 2 min and expression of Id3 at 1 h. The MFIs of three lobes per time point were averaged, and the error bars represent the standard deviation. (B) Western blot of Id3 protein expression in OT-I TAP° thymocytes. Thymocytes were stimulated with OVAp-pulsed B6.SJL splenocytes with or without the addition of 20 μM U0126 for 3 h. DP thymocytes were separated by magnetic-activated cell sorting bead separation, and whole-cell lysates were prepared. Expression of Id3 was analyzed in Western blots by using an Id3 antibody. Blots were probed with anti-ERK2 antibodies to control equal loading of protein. The numbers below the blot indicate the fold induction of Id3 expression.
Fig. 5.
Fig. 5.
Both phases of ERK activation are required for positive selection of OT-I TAP° thymocytes and depend on TCR/peptide-MHC interactions. (A) OT-I TAP° fetal thymic lobes were incubated with the indicated peptide with or without the addition of 20 μM U0126. Positive selection is reflected as the ratio of the percentage of cells in the CD8 SP gate to the percentage of DP thymocytes after 96 h of culture. The ERK inhibitor was added either at the beginning of the culture or 24 h after peptide was added to the lobes. The ratios of three lobes per time point were averaged. (B) Expression of phosphorylated ERK in OT-I TAP° fetal thymic lobes that were incubated for 2 min. The lobes were incubated with the indicated peptides with or without the addition of anti-Kb (MHC blockade) or anti-Db (control) antibodies, gated on DP thymocytes, and analyzed for expression of pERK. The MFIs of three lobes per time point were averaged. (C) The ratio of the percentage of CD8 SP thymocytes to the percentage of DP thymocytes in OT-I TAP° fetal thymic lobes after 96 h of culture. The lobes were incubated with the indicated peptide with or without the addition of anti-Kb or anti-Db antibodies and analyzed for the percentage of CD8 SP and DP thymocytes. The MHC antibodies were added either at the beginning of the culture or 24 h after peptide had been added to the lobes. The ratios of three lobes per time point were averaged. (D) Expression of phosphorylated ERK in OT-I TAP° fetal thymic lobes incubated for 72 h. After 72 h of incubation, the DP thymocytes were gated and analyzed for the expression of pERK.
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