MAP kinase phosphatase activity sets the threshold for thymocyte positive selection

  1. Matthew L. Bettini and
  2. Gilbert J. Kersh*
  1. Department of Pathology and Laboratory Medicine, 101 Woodruff Circle, Emory University School of Medicine, Atlanta, GA 30322

  1. Edited by Howard M. Grey, La Jolla Institute for Allergy and Immunology, La Jolla, CA, and approved August 20, 2007 (received for review June 6, 2007)

 
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Abstract

Phosphorylation of MAP kinases is important for proper translation of T cell antigen receptor (TCR) signals into thymocyte cell fates, but the role of MAP kinase phosphatase (MKP) activity in thymocyte development has not been characterized. To explore the role of MKP in thymocytes, we constructed a double mutant MKP-3 (DM-MKP3) that acts as a dominant-negative inhibitor of ERK- and JNK-specific MKP. Thymocytes developing in the presence of DM-MKP3 have enhanced frequencies of both CD4 and CD8 mature, single-positive cells and no increase in apoptosis. Expression of DM-MKP3 also results in an increased proportion of thymocytes with high levels of both CD69 and TCRβ, suggesting that the increased proportion of mature thymocytes is the result of an increased probability that CD4+CD8+ cells will be positively selected. Thus, MKP activity controls thymocyte cell fate by regulating the threshold of TCR signaling that is able to induce positive selection.

The development of α and β T cells in the thymus involves a series of cell-fate decisions that are dependent on the function of multiple cell-surface receptors, including the pre-T cell antigen receptor (TCR) and the TCR (1). Both pre-TCR and TCR signals in thymocytes result in the activation of MAP kinases, and this activation has been shown to be critical for the adoption of appropriate cell fates. ERK is phosphorylated in response to pre-TCR signals and is thought to be required for a proper transition from double negative (DN) to double positive (DP). ERK and JNK MAP kinases are all activated by phosphorylation downstream of TCR signaling in DP thymocytes. ERK is required for the positive selection of thymocytes (2), whereas JNK activation is thought to be important for the negative selection of thymocytes (3). The role of ERK activation in the negative selection of thymocytes has not been firmly established, although several studies that have considered this issue suggest that it is not required for negative selection (4–6). It also appears that the mechanism of ERK activation may be different downstream of positive- and negative-selecting ligands. Positive-selecting ligands activate ERK on the membrane of the Golgi for a sustained period, whereas negative-selecting ligands activate ERK more transiently at the plasma membrane (7, 8).

The importance of MAP kinase phosphorylation in thymocyte development suggests a role for MAP kinase phosphatases (MKPs) in the thymus. A gene family in mammals can be defined based on the presence of a domain homologous to the catalytic domain of the Vaccinia virus phosphatase VH1. There are at least 30 members of this so-called dual-specificity phosphatase (dusp) gene family in humans and mice, and many of these gene products have been shown to dephosphorylate MAP kinases (9). Eleven members of the dusp family have an N-terminal MAP kinase interaction motif and actively dephosphorylate with a conserved C-terminal phosphatase domain. However, other family members that lack the MAP kinase interaction motif also can dephosphorylate MAP kinases (9). Previously, we evaluated the expression of 26 dusp genes in the murine thymus and found seven thymic dusp genes (dusp 1, 2, 4, 5, 6, 7, and 10) that are expressed at all stages of thymocyte development and whose gene products are known to dephosphorylate MAP kinases (10). These MKPs are therefore candidates for regulation of cell fate in response to pre-TCR and TCR signals.

Because of potential redundancy among thymic MKPs, we chose a dominant-negative approach to evaluate the role of MKP activity in the thymus. Expression of a mutant form of murine dusp6 (MKP-3) that lacks phosphatase activity and the functional nuclear export sequence found in wild-type MKP-3 resulted in dominant-negative inhibition of ERK- and JNK-specific MKP. Expression of the mutant MKP-3 results in enhanced and sustained activation of ERK and JNK in response to TCR signals. Transduction of bone marrow progenitors with a retrovirus driving expression of dominant-negative MKP-3, followed by transfer into irradiated recipient mice, revealed that reduction of MKP activity allows more cells to be positively selected without any apparent effects on negative selection. Thus, MKP activity is important for setting the threshold for positive selection.

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Results

A Mutant Form of MKP-3 Acts as a Dominant Negative for ERK- and JNK-Specific MKP.

The murine dusp6 gene was cloned into the retroviral vector GFP-RV and then mutated such that the critical cysteine in the active site was changed to serine, a mutation that abolishes the phosphatase activity of the enzyme. A second mutation disrupted the consensus nuclear export sequence (NES) of MKP-3. Disruption of the NES has been shown to allow more of the MKP-3 protein to be present in the nucleus (11). We refer to the form of MKP-3 that contains both the active site and NES mutations as the double mutant MKP-3 (DM-MKP3). Based on previous studies that used similar mutations of MKP-3, DM-MKP3 was expected to bind to ERK and JNK and prevent the dephosphorylation of these MAP kinases by other active MKPs, thus performing as a dominant negative for all MKPs (11–13).

To test this idea, the 16610D9 DP thymocyte cell line (hereafter referred to as D9 cells) (14) was transduced with the empty GFP-RV vector, wild-type MKP-3, or DM-MKP3 so that >90% of the cells were GFP+. The cells were then stimulated with plate-bound anti-CD3ε for 5 min, followed by lysis in 0.2% Nonidet P-40. Analysis of lysates by immunoblot revealed that high expression of wild-type MKP-3 eliminated the induction of phospho-ERK (pERK) and phospho-JNK (pJNK) (Fig. 1 A). In contrast, cells expressing DM-MKP3 had more pERK and pJNK when compared with cells transduced with the empty GFP-RV vector. MKP-3 is primarily a cytoplasmic protein (15, 16), but overexpression resulted in high amounts in the nucleus, and similar effects of MKP-3 and DM-MKP3 were observed when nuclear protein extracts were analyzed by immunoblot probed with anti-pERK and anti-pJNK (data not shown).

Fig. 1.
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Fig. 1.

Wild-type MKP-3 prevents and DM-MKP3 enhances ERK and JNK activation. (A) D9 cells were transduced with empty vector (GFP-RV), MKP-3, or DM-MKP3 such that >90% of the cells were GFP+. The cells were stimulated with anti-CD3ε for 5 min, and the amounts of pERK, pJNK, MKP-3, and total ERK in 0.2% Nonidet P-40 lysates were determined by immunoblot. The blot shown is representative of at least three independent experiments. (B) Transduced D9 cells were stimulated with plate-bound anti-CD3ε for the indicated times and then fixed, permeabilized, and stained with anti-pERK. The dot plots show forward scatter versus pERK staining on GFP+ cells. These plots are representative of at least three independent experiments. (C) Transduced D9 cells (>90% GFP+) were stimulated with anti-CD3ε for 1 or 2 h, RNA was isolated, and egr1 expression (normalized to β-actin) was determined by quantitative real-time PCR. The data represent the mean ± SEM of five separate experiments. Differences between GFP-RV and MKP-3 (P = 0.03), as well as between GFP-RV and DM-MKP3 (P = 0.03), at 2 h were statistically significant as determined by Mann–Whitney U test.


The larger amounts of pERK and pJNK that we observed on immunoblots could be due to increases in the amount of pERK and pJNK per cell or an increase in the number of cells generating pERK and pJNK. Previous reports have suggested that TCR signaling activates ERK in a digital and all-or-none manner, but have not investigated the role of MKP in the ability of TCR signaling to activate ERK (17). A time course of ERK activation was examined at a single-cell level in transduced D9 cells by measuring pERK by flow cytometry. D9 cells were transduced with GFP-RV, MKP-3, or DM-MKP3 and then stimulated with plate-bound anti-CD3 for different periods of time. The analysis of pERK staining in GFP+ cells is shown in Fig. 1 B. Expression of wild-type MKP-3 reduced the percentage of pERK-positive cells over an extended period. In contrast, expression of DM-MKP3 increased the percentage of pERK-positive cells at all time points. The main source for the increase in pERK was the greater number of pERK-positive cells, although there was a slight increase in mean fluorescence intensity among pERK-positive cells in the presence of DM-MKP3. More cells were able to activate ERK for longer periods when MKP activity was inhibited by DM-MKP3. Thus, inhibition of MKP activity in this thymocyte cell line increases the probability that a TCR stimulus will activate ERK.

To determine whether expression of MKP-3 or DM-MKP3 could have an impact on MAP kinase-dependent gene expression, induction of the gene encoding the transcription factor Egr1 was examined in D9 cells stimulated through the TCR. We have previously shown that the duration of ERK activation determines the induction of Egr1 expression in response to TCR signals (18), and that induction of Egr1 is required for normal positive selection (6). Thus, egr1 induction indicates the ability of ERK to induce downstream effectors relevant for thymocyte development. D9 cells were transduced with the empty GFP-RV vector, wild-type MKP-3, or DM-MKP3 so that >90% of the cells were GFP+. The cells were then stimulated with anti-CD3 for 1 or 2 h, RNA was isolated, and the fold induction of Egr1 RNA compared with unstimulated cells was determined by quantitative real-time PCR. As shown in Fig. 1 C, in the presence of the empty GFP-RV vector, egr1 is rapidly induced (54-fold at 1 h and 60-fold at 2 h). In contrast, the presence of wild-type MKP-3 results in only a 6.7-fold induction at 1 h and 9.6-fold at 2 h. Thus, overexpression of MKP-3 inhibits the induction of this ERK target gene. Expression of the putative dominant-negative DM-MKP3 results in enhanced egr1 induction, with 76-fold at 1 h and 195-fold at 2 h. Thus, expression of DM-MKP3 results in enhanced induction of a MAP kinase-dependent gene, which is consistent with its ability to act as a dominant-negative inhibitor of multiple MKPs.

Bone Marrow Progenitors That Overexpress MKP-3 Do Not Develop in Vivo.

To determine the influence of MKP activity on thymocyte development in vivo, B6.AKR (Thy1.2+) bone marrow was isolated and depleted of lineage-positive cells and then transduced with the empty GFP-RV vector, wild-type MKP-3, or DM-MKP3. Transduced bone marrow cells were then injected i.v. into lethally irradiated B6.AKR (Thy1.1+) mice. After 6 weeks, the bone marrow, spleen, and thymus were harvested from the recipient mice and analyzed. When we analyzed the thymocytes of mice reconstituted with MKP-3- transduced bone marrow, we found that, although these mice had thymuses comprised of >90% donor cells (Thy1.2+), most of the mice had few GFP+ thymocytes (Fig. 2 A), and the GFP+ cells were generally GFPlo. This result is despite the fact that the bone marrow cells injected into irradiated recipients were typically 10–20% GFP+ and contained many GFPhi cells. Analysis of bone marrow cells in mice that had received MKP-3-transduced bone marrow 6 weeks previously revealed that these cells also had few GFP+ cells, and that these cells were generally GFPlo. This finding suggests that the failure to find GFPhi cells in the thymuses of mice reconstituted with MKP-3-transduced bone marrow is due to a requirement for MAP kinase phosphorylation for the survival of progenitors in the bone marrow. When bone marrow was transduced with empty GFP-RV vector or DM-MKP3-containing GFP-RV, 10–30% GFP+ cells were typically obtained. Six weeks after transfer, thymocytes (Fig. 2 A) and bone marrow (data not shown) of the recipients were also 10–30% GFP+.

Fig. 2.
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Fig. 2.

Expression of MKP-3 and DM-MKP-3 in vivo. (A) Bone marrow progenitors from B6.AKR (Thy1.2) mice were transduced with GFP-RV, MKP-3, or DM-MKP3 so that 10–20% of the cells were GFP+. The transduced progenitors were then injected i.v. into lethally irradiated B6.AKR (Thy1.1) mice. Six weeks after bone marrow transfer, thymocytes and bone marrow cells were analyzed by flow cytometry. The graph shows mean percentages ± SEM for GFP+ thymocytes. GFP-RV, n = 11; MKP-3, n = 9; DM-MKP3, n = 7. The difference between GFP-RV and MKP-3 was significant (P = 0.01) as determined by Mann–Whitney U test. (B) Bone marrow progenitors transduced with DM-MKP3 were injected into an irradiated recipient mouse. Six weeks after bone marrow transfer, thymocytes were stained with a mixture of lineage markers, plus anti-CD117 and anti-CD25. Lineage-negative cells were divided into GFP− and GFP+ populations, and CD117 versus CD25 dot plots are shown.


Therefore, this system allowed for the analysis of thymocyte development under conditions where endogenous MKP activity was inhibited. DM-MKP-3 chimeric thymocytes were analyzed by flow cytometry and divided into GFP+ and GFP− populations. Staining of lineage-negative cells with anti-CD117 (c-kit) and anti-CD25 did not reveal any consistent differences between GFP− and GFP+ populations, even when cells with the highest levels of GFP were considered (Fig. 2 B). This finding suggests that MKP regulation of MAP kinase activity is not critical for the early stages of thymocyte development. Although there is evidence that ERK activation plays a role in the transition from DN3 to DN4 (2, 19), we did not observe a change in that transition when DM-MKP3 was expressed. It is possible that MAP kinase activity is in vast excess during this transition, as suggested by ERK-deficient mice (2).

Enhanced Positive Selection in Thymocytes Expressing DM-MKP3.

Analysis of later stages of thymocyte development revealed significant changes brought about by the inhibition of MKP activity. There was a higher frequency of CD4 and CD8 single-positive (SP) thymocytes within the GFPhi cell populations in comparison with GFP− donor thymocytes (Fig. 3). It has been demonstrated that, with a bicistronic expression vector that uses GFP as a marker gene, an increase in the expression of inserted cDNA correlates with increased GFP expression (20). This result allows us to titrate the amount of DM-MKP3 by gating on different levels of GFP in the chimeric thymuses. This approach showed that CD4SP and CD8SP percentages correlated with the intensity of GFP fluorescence (Fig. 3). In this chimera, GFP− cells were 4.16% CD4SP, but the two lowest levels of GFP intensity had a CD4SP percentage of 9.27 and 8.31. This effect was enhanced with greater GFP expression; the two highest GFP intensities had 21.7% and 45.4% CD4SP cells. CD8SP percentages also were increased, but only at the two highest levels of GFP. In these cases, the percentage of CD8SP cells went to 4.5% and 10.6%, compared with 2.1% in GFP cells. The more pronounced phenotype that is observed with increasing levels of DM-MKP3 expression is expected of a molecule that is acting as a dominant-negative inhibitor. Chimeric animals made with empty GFP-RV vector had normal percentages of CD4SP and CD8SP cells that did not vary with the GFP intensity (data not shown). These data indicate that a reduction in MKP activity results in increased percentages of CD4SP and CD8SP thymocytes.

Fig. 3.
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Fig. 3.

Increased positive selection of CD4SP and CD8SP thymocytes in the presence of high levels of DM-MKP3. B6.AKR (Thy1.2+) bone marrow was transduced with DM-MKP3 and injected into irradiated B6.AKR (Thy1.1+) mice. Six weeks later, thymocytes were analyzed by flow cytometry. Four gates were drawn on GFP+ cells. The CD4 versus CD8 as well as the CD69 versus TCRβ dot plots were determined for GFP− cells and the four groups of GFP+ cells. The data shown are representative of seven different chimeric mice.


The increase in SP thymocyte percentage could be due to preferential loss of DP thymocytes or more efficient positive selection. Cells in the process of positive selection express high levels of CD69 and TCRβ. Therefore, we stained cells from DM-MKP3 chimeras for CD69 and TCRβ and evaluated the expression in populations that expressed different levels of GFP. As shown in Fig. 3, GFP− cells had 3.15% of thymocytes that were CD69hi, TCRβhi. In contrast, the percentages of CD69hi, TCRβhi cells were 7.71, 6.69, 15.6, and 33.9 in populations with increasing expression of DM-MKP3. No significant changes in the percentage of CD69hi, TCRβhi cells were observed in GFP+ cells from mice transduced with GFP-RV empty vector (data not shown).

Thymocytes expressing high levels of DM-MKP3 have a reduced percentage of DP cells. Based on the data in Fig. 3, this result is likely because of enhanced differentiation of the cells to the SP stages. However, enhanced death of the DP population could be playing a role. The DM-MKP3 construct leads to enhanced JNK activation, and JNK has been shown to play a role in promoting the negative selection of thymocytes (3). However, we have examined apoptosis of thymocytes in DM-MKP3 chimeras by using annexin V staining and found that there are similar amounts of apoptosis in GFP− and GFP+ cells, both directly ex vivo and after 5 and 24 h of culture (Fig. 4 A). This result suggests that enhancement of ERK and JNK phosphorylation in DP thymocytes does not lead to more apoptosis.

Fig. 4.
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Fig. 4.

DM-MKP3 does not alter apoptosis of thymocytes, but increases the percentage of mature thymocytes. (A) B6.AKR (Thy1.2+) bone marrow was transduced with DM-MKP3 and injected into irradiated B6.AKR (Thy1.1+) mice. Six to 10 weeks later, thymocytes were analyzed by flow cytometry. Thymocytes were stained with annexin V directly ex vivo or after 5 or 24 h in culture. The numbers show the percentages of annexin V-positive cells after gating on GFP− and GFP+. These data are representative of the analysis of four chimeric mice. (B) DM-MKP3 chimeric mice were analyzed 6 to 10 weeks after bone marrow transfer. Cells were electronically divided into GFP− and GFP+ populations, and the TCRβ staining on DP, CD4SP, and CD8SP subsets is shown. The numbers on the histograms show the percentages of each subset that is TCRβhi. The numbers in parentheses show the percentages of cells within each GFP gate that are TCRβhi and fall within the subset. These data are representative of the analysis of five chimeric mice. (C) DM-MKP3 chimeric mice were analyzed as in B, with staining for CD24 instead of TCRβ. These data are representative of the analysis of five chimeric mice.


The increased percentages of SP thymocytes in the presence of DM-MKP3 are suggestive of a greater number of cells going through positive selection. However, many of the cells in the SP populations are not truly mature, particularly in the CD8SP population, which contains immature SP cells that are in the transition from DN to DP. To determine the maturation status of SP cells, we used two different markers: TCRβ, which increases as cells mature from DP to SP, and CD24, which decreases as cells mature from DP to SP. Bone marrow cells were transduced with DM-MKP3 and transferred to irradiated hosts; after 6 weeks, thymocytes were separated into GFP− and GFP+. Use of TCRβ expression as a maturation marker showed that CD4SP cells are uniformly TCRβhi regardless of GFP expression, but a higher percentage of DM-MKP3-expressing cells are both CD4SP and TCRβhi (Fig. 4 B). For the CD8SP cells, the GFP+ cells have a greater percentage of TCRβhi cells compared with GFP−. Thus, a greater percentage of the CD8SP cells are mature when DM-MKP3 is expressed. Data shown in Fig. 4 C by using CD24 as a marker for maturity fully support the conclusions reached by using TCRβ. Thus, inhibition of MKP activity results in more efficient production of mature thymocytes from the immature DP thymocyte pool.

DP Thymocytes That Express DM-MKP3 Are More Likely to Induce Egr1 in Response to TCR Cross-Linking.

Our interpretation of these data is that inhibition of MKP activity increases the probability that a given DP thymocyte can activate ERK in response to TCR signals, and that this results in positive selection of DP thymocytes that would die by neglect under normal circumstances. To test whether DP thymocytes have an increased probability of ERK activation when DM-MKP3 is expressed, total thymocytes were isolated from DM-MKP3 chimeric mice and stimulated with plate-bound anti-CD3 for 1 or 2 h, followed by cell-surface staining for CD4 and CD8 and intracellular staining for Egr1. After gating on DP cells, Egr1 staining between GFP+ and GFP− cells was compared. The results show that a higher percentage of DP cells induce Egr1 in response to TCR stimulation when MKP activity is inhibited by DM-MKP3 (Fig. 5 A). We also measured ERK phosphorylation in DP thymocytes from DM-MKP3-transduced chimeras after anti-CD3 stimulation for 10 or 20 min. Similar to the induction of Egr1, the percentage of cells with elevated ERK phosphorylation was increased in GFP+ cells (Fig. 5 B).

Fig. 5.
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Fig. 5.

DM-MKP3 enhances Egr1 induction in DP thymocytes. (A) Six to 10 weeks after bone marrow transfer, DM-MKP3 chimeric thymocytes were stained with anti-CD4 and anti-CD8 and then fixed, permeabilized, and stained intracellularly with anti-Egr1 directly ex vivo (0 h). Thymocytes also were stimulated for 1 and 2 h with plate-bound anti-CD3ε and then stained similarly. After gating on DP thymocytes, the percentage of Egr1-positive cells was determined in GFP− and GFP+ populations. The data shown are the mean ± SEM for seven different mice. The percentages of GFP+ cells that are Egr1+ are statistically significantly different from the corresponding values among GFP− cells at both 1 h (P = 0.0008) and 2 h (P = 0.0013) as determined by Student's t test. (B) Five weeks after bone marrow transfer, DM-MKP3 chimeric thymocytes were stained for anti-CD4, anti-CD8, and phospho-ERK. The data show the percentage of DP thymocytes that were phospho-ERK-positive in the GFP+ and GFP− populations. Staining was done directly ex vivo or after 10 or 20 min of anti-CD3ε stimulation. The data shown are the mean ± SEM for three different mice.


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Discussion

These studies demonstrated that MKP-3 can limit ERK and JNK activation in response to TCR signaling, and that the DM-MKP3 protein can act as a dominant-negative inhibitor of MKP activity. DM-MKP3 was therefore used as a tool to probe the role of MKP activity in thymocyte development. Expression of the dominant-negative MKP in the thymus did not have a significant effect on early thymocyte development or β selection. The main effect of inhibiting MKP activity was to increase the percentage of mature SP thymocytes.

The prevailing model for thymocyte development is that the fate of DP thymocytes depends on the interaction with self-peptide–MHC complexes. Many of the TCRs on DP thymocytes have a low or nonexistent affinity for self-peptide–MHC complexes, and these DP cells die without a sufficient signal through their TCR. The data presented here support a role for MKP activity in setting the threshold of TCR–ligand affinity that is required to induce positive selection. When MKP activity is inhibited, we find that more cells are positively selected. Our interpretation is that the extra mature thymocytes are DP thymocytes that, under ordinary circumstances, have an affinity that is too low to mediate positive selection. However, with reduced MKP activity, these extremely low-affinity TCR–ligand interactions are able to activate ERK and promote positive selection. This model is supported by a recent study demonstrating that the microRNA miR-181a controls the threshold of T cell responses by reducing the expression of multiple phosphatases, including dusp5 and dusp6 (21).

The dominant-negative MKP-3 construct that we have used enhances activation of both ERK and JNK. Published data (15, 16) suggest that MKP-3 is more effective at ERK dephosphorylation, and our experience agrees with this assessment. However, at the concentrations that we achieve with retroviral expression, the DM-MKP3 protein seems to be equally effective at enhancing ERK and JNK phosphorylation in response to TCR signaling. It is therefore an open question whether the effect that we see is because of increased ERK or JNK activation, or both. However, dominant-negative inhibition of JNK activity impairs negative, but not positive, selection (3). In contrast, several experiments have made it clear that ERK activation is required for positive selection (2, 22). Hence, it is more likely that the effects that DM-MKP3 has on ERK activation are the critical changes that lead to more mature thymocytes, but a role for enhanced JNK activation cannot be eliminated.

One could argue that DM-MKP3 also should change the threshold of signaling required for negative selection. In this scenario, ligands that normally induce positive selection could be converted into negative-selecting ligands when the probability of JNK activation is increased by DM-MKP3. Our results with the DM-MKP3 construct suggest that this scenario is not the case. The greater percentage of mature thymocytes shows that we are seeing a net increase in the cells that are getting a positive-selection signal, and we do not see more cells undergoing apoptosis. This finding suggests two possibilities. The first is that JNK may be necessary, but not sufficient, for negative selection, and therefore an increase in JNK activation in the absence of other signals cannot convert positive-selecting signals into negative. The second option is that the sustained activation of ERK at the Golgi in response to positive-selection signals inhibits JNK-mediated negative selection.

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Methods

Mice, Cell Lines, and Plasmids.

B6.AKR mice were purchased from The Jackson Laboratory (Bar Harbor, ME). B6.AKR (Thy1.1) mice were generated by breeding B6.AKR mice to B6.PL mice (The Jackson Laboratory) for two generations and selecting for H-2K- and Thy1.1-positive offspring. All animal procedures were approved by the Emory University Institutional Animal Care and Use Committee. 16610D9, a DP thymocyte cell line derived from a p53−/− mouse, was provided by S. Hedrick (University of California at San Diego, La Jolla, CA). The Phoenix ecotropic packaging cell line was purchased from American Type Culture Collection (Manassas, VA) with the permission of G. Nolan (Stanford University, Stanford CA). The bicistronic retroviral vector used in these studies, GFP-RV, was a generous gift from K. Murphy (Washington University, St. Louis, MO).

Antibodies and Flow Cytometry.

Antibodies were obtained from the following sources: BD PharMingen (San Diego, CA), anti-mouse CD3 (2C11-145), anti-mouse CD4 PE (GK1.5), anti-mouse CD8a PerCP (53-6.7), anti-mouse CD69 biotin (H1.2F3), and anti-mouse CD90.2/Thy1.2 PE (53-2.1); eBioscience (San Diego, CA), anti-mouse CD24 biotin (M1/69); Caltag/Invitrogen (Carlsbad, CA), anti-mouse TCRα/β APC (H57-597) and anti-mouse CD8a Tri-Color (CT-CD8a); Cell Signaling Technology (Beverly, MA), anti-pERK (phospho-p44/42 MAPK), anti-pJNK (phospho-SAPK/JNK), and anti-EGR1 (44D5); Promega (Madison, WI), anti-ERK (anti-ERK 1/2 pAb); Southern Biotechnology (Birmingham, AL), goat anti-rabbit IgG-PE; Santa Cruz Biotechnology (Santa Cruz, CA), anti-MKP-3 (C-20); and Jackson ImmunoResearch (West Grove, PA), peroxidase-conjugated AffiniPure goat anti-rabbit IgG, and peroxidase-conjugated AffiniPure mouse anti-goat IgG. All cell analyses were performed with a BD FACS Calibur flow cytometer (BD Immunocytometry Systems, San Jose, CA) and FlowJo software (Tree Star, Inc., Ashland, OR).

Retroviral Transduction.

Phoenix packaging cells were transfected with GFP retroviral vectors by using Lipofectamine 2000 (Invitrogen). After 24 h, supernatant was removed and replaced with fresh DMEM containing 15% FCS without antibiotics. After another 24 h, the fresh viral supernatant was filtered, and polybrene (Sigma–Aldrich, St. Louis, MO) was added to a final concentration of 5 μg/ml, followed by a 10-min incubation on ice. Bone marrow cells were enriched for progenitors by using SpinSep mouse progenitor isolation kit (Stem Cell Technologies, Vancouver, BC, Canada). Enriched bone marrow cells were washed and resuspended in viral supernatant with 5 μg/ml polybrene plus 50 ng/ml mSCF, 50 ng/ml hIL-6, and 20 ng/ml mIL-3 (Peprotech, Rocky Hill, NJ) at 0.5 × 106 cells per ml in 12-well plates. Cells were spun at 2,500 × g for 90 min at 26°C; 48 h later, transduced bone marrow cells were injected i.v. into lethally irradiated (1,100 rads) congenic B6.AKR (Thy1.1) mice. After adoptive transfer, mice were supplied with water containing 500 mg/ml Neomycin Sulfate and 12.5 mg/ml Polymyxin B Sulfate Salt (Sigma–Aldrich). For transduction of D9 cells, viral supernatants mixed with polybrene were added to 0.5 × 106 cells per well in 12-well plates. D9 cells were spun at 2,500 × g for 30 min at 26°C.

RNA and Protein Isolation.

Stimulated cells were lysed in TRIZOL (Invitrogen) for RNA isolation per the manufacturer's instructions. For immunoblot, D9 cells were lysed in 0.2% Nonidet P-40 lysis buffer containing 40 mM Hepes (pH 7.9), 2 mM EDTA, 2 mM EGTA, 0.5 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and phosphatase inhibitor mixtures 1 and 2 (Sigma–Aldrich). Lysates were incubated on ice for 5 min and centrifuged at 13,200 × g for 30 sec. Supernatant was taken for cytoplasmic protein.

Quantitative Real-Time PCR.

RNA from isolated cells was used to make cDNA by using random primers and SuperScript II (Invitrogen). For quantitative real-time PCR, SYBR Green PCR Master Mix (Invitrogen) was used according to the manufacturer's instructions. Oligos were Egr-1 forward, 5′ CCACAACAACAGGGAGACCT-3′, Egr-1 reverse, 5′-ACTGAGTGGCGAAGGCTTTA-3′; β-Actin forward, 5′-AAGTGTGACGTTGACAT CCGTAA-3′, and β-Actin reverse, 5′-TGCCTGGGTACATGGTGGTA-3′.

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Acknowledgments

We thank Mindy Tanzola for cloning of the dusp6 gene. This work was supported by National Institutes of Health grants and the American Cancer Society.

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Footnotes

  • *To whom correspondence should be addressed. E-mail: gkersh{at}emory.edu

  • Author contributions: M.L.B. and G.J.K. designed research; M.L.B. performed research; M.L.B. and G.J.K. analyzed data; and M.L.B. and G.J.K. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • Abbreviations:
    MKP,
    MAP kinase phosphatase;
    DM-MKP3,
    double mutant MKP-3;
    DN,
    double negative;
    DP,
    double positive;
    NES,
    nuclear export sequence;
    SP,
    single positive;
    TCR,
    T cell antigen receptor.
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  1. Published online before print September 27, 2007, doi: 10.1073/pnas.0705321104 PNAS October 9, 2007 vol. 104 no. 41 16257-16262
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