The early growth response gene 1 (Egr-1) is induced during positive selection in the thymus and has been implicated in the differentiation of CD4+ thymocytes. Here, we show that signals that specifically direct CD8 lineage commitment also induce Egr-1 DNA-binding activity in the nucleus. However, we find that pharmacological inhibition of mitogen-activated protein kinase/extracellular signal-related kinase kinase activity potently inhibits Egr-1 DNA-binding function at concentrations that promote differentiation of CD8+ thymocytes, suggesting Egr-1 activity is not essential for CD8 commitment. To further determine the role of Egr-1 in thymocyte development, we compare steady-state Egr-1 DNA-binding activity in thymocytes from mice with defined defects in positive selection. The data indicate that the appearance of functional Egr-1 is downstream of signals induced by TCR/MHC engagement, whereas it is less sensitive to alterations in Lck-mediated signals, and does not correlate directly with proficient positive selection. Egr-1 is one of the earliest transcription factors induced upon TCR ligation on immature thymocytes, and plays a potential role in the transcription of genes involved in thymocyte selection.

The characterization of intracellular signaling cascades and transcription factors required for the differentiation of immature, double positive (DP)4 thymocyte precursors into mature, single positive (SP) T cells is essential for the understanding of thymocyte differentiation and lineage commitment at the molecular level. Gene knockout experiments have identified some key molecular players in thymocyte positive selection, the absence of which leads to developmental arrest at the DP stage (1). To date, this genetic approach has failed to clearly identify a single pathway or factor involved in the CD4/CD8 lineage decision, although some knockouts present with a phenotype affecting the normal CD4:CD8 ratio in the thymus (2, 3). Experimental evidence that signals downstream of the src kinase, lck, and the mitogen-activated protein kinase (MAPK) pathway may be differentially required for CD4 and CD8 differentiation comes from recent experiments by our own (4, 5, 6), as well as other laboratories (7, 8). Furthermore, it is clear that the signaling pathways engaged must diverge at some point to differentially activate transcription factors, thus inducing the expression of lineage-specific genes. The identification of transcription factors that are developmentally regulated during thymocyte differentiation and, in particular, those that are expressed at the DP to SP transition should allow the characterization of those gene products that regulate the CD4/CD8 lineage decision.

Recently, the expression of members of the early growth response (Egr) gene family was investigated in an immature DP thymocyte cell line and differentiation into mature, CD4 SP cells was shown to correlate with an increase in Egr gene expression (9). Egr-1 induction was shown in these experiments to require ras activity. Additionally, the same group showed that Egr-1 can be induced in immature thymocytes upon the in vitro induction of CD4 differentiation by sustained, low, protein kinase C-mediated MAPK activation, in the presence of ionomycin (10). A significant induction of Egr-1 binding activity was observed in normal C57BL/10 (B10) thymi, but not in thymocytes from MHCneg (9) and TCRαneg (10) mice, indicating a direct correlation between Egr-1 binding activity in the nucleus and positive selection. Collectively, these studies clearly showed that Egr gene expression is up-regulated during CD4 differentiation. However, the role of Egr genes during maturation of the CD8 lineage has not been addressed directly, although it was noted that transgenic overexpression of Egr-1 enhanced both CD4 and CD8 differentiation to a similar extent (11). We show in the present study that Egr-1 is also induced during CD8 differentiation, but that Egr-1 induction and binding activity is not required for commitment to the CD8 lineage. Using a variety of transgenic and knockout mouse lines, we also attempted a more detailed analysis of the requirements for Egr-1 induction in thymocytes. We provide evidence that Egr-1 is a very sensitive and early indicator of TCR ligation on DP thymocytes, yet Egr-1 binding activity neither correlates directly with lck activity nor with the efficiency of positive selection.

C57BL/10 mice were obtained from the specific pathogen-free breeding facility at the National Institute of Medical Research (London). The class I-restricted F5 TCR transgenic (12), RAG-1-deficient (13), β2-microglobulin (β2m)neg (14) mice have been described and were intercrossed to obtain F5/RAG-1neg or F5/RAG-1neg2mneg mice from which newborn thymus lobes were obtained. The class II-restricted A18 TCR transgenic mice (15) were crossed onto a RAG-1neg/CD4neg (16) background to obtain mice in which DP thymocytes expressing a class II-restricted TCR mature into the CD8 lineage (17). MHCnull mice were obtained by crossing β2mneg and I-Aβneg (18) mice. The pLGF mouse expressing medium levels of the dysregulated lckF505 transgene (line 3073) was a kind gift of Dr. Roger Perlmutter (Hoffman-LaRoche, Nutley, NJ) (19) and Zap70-deficient mice were provided by Dr. A. Weiss (University of Califorinia, San Francisco, CA) (20).

Bi- and mono-specific F(ab′)2 Abs dimerized through Fos or Jun leucine zippers were prepared as described previously (21). V-regions with specificity for CD3ε, CD4, or CD8α were derived from 145.2C11, GK1.5, and YTS169, respectively. mAbs were purified and conjugated to FITC or biotin in our own laboratory, unless stated otherwise. Egr-1 (sc-110 X)-specific rabbit polyclonal IgG reagent was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and control purified rabbit Ig from Sigma-Aldrich (Poole, U.K.).

Egr-1, -2, and -3-specific primers were as described by Shao et al. (9). GAPDH primers were 5′-GGGGTGAGGCCGGTGCTGAGTAT and 5′-CATTGGGGGTAGGAACACGGAAGG.

Freshly isolated thymocytes were stained with PE-conjugated anti-CD4 (PharMingen, San Diego, CA), FITC-conjugated anti-CD8 (YTS169, prepared in our laboratory), and biotinylated anti-CD69 (PharMingen, San Diego, CA) mAbs, followed by streptavidin-Red 670 (Life Technologies, Grand Island, NY). Cells were sorted on a FACSvantage (Becton Dickinson, San Diego, CA) to obtain between 0.5 and 1 × 106 cells, from which nuclear extracts were prepared as described below.

Neonatal thymus lobes from F5/RAG-1neg and F5/RAG-1neg2mneg mice were cultured in the presence of bi-specific CD3/CD4 Ab or a CD3-specific F(ab′)2 (CD3/CD3), as described (4, 22). In some experiments, lobes were cultured in the presence of 50 μM of the MEK1 inhibitor, PD98059 (Calbiochem, La Jolla, CA) or 9 μM of U0126 (kind gift of J. M. Trzaskos, DuPont Merck, Wilmington, DE) for 1–2 h before addition of the Abs to the culture medium. Inhibitors were replenished every 24 h to maintain sufficient levels during culture (6). Lobes were transfered to fresh filters and culture medium on day 4, and cultured for an additional 3 days before FACS analysis, as described (4, 22). Total RNA was extracted from single lobes at different time points during culture and analyzed by RT-PCR for gene expression. For EMSAs, three to four lobes were pooled, yielding 2–10 × 106 viable cells, from which nuclear extracts were prepared.

Total RNA was extracted from sorted cell populations and single lobes using 400 μl of RNAzol B solution per sample, according to the manufacturer’s instructions (Tel-Test, Friendswood, TX). RNA products were dissolved in 50–100 μl nuclease-free H2O and kept at −70°C until analysis.

RT-PCR was performed using the Promega Access RT-PCR system according to the manufacturer’s instructions (Promega, Southampton, U.K.). Control samples without RNA were always included and never produced a product. DNA contamination was controlled for by performing reactions under identical conditions in the absence of reverse transcriptase. Only minimal levels of DNA contamination could be observed in the occasional sample, and the results shown were from samples in which no DNA contamination could be detected. To provide a more accurate estimate of relative gene expression, 2 μl undiluted RNA was used per reaction and a cycle count performed over a range of 20–40 amplification cycles. The resulting PCR products were resolved on 1.5% agarose gels containing ethidium bromide, and band intensities quantitated using a digital camera. GAPDH expression was determined at the same time for each sample, run on the same gel, quantitated, and used as an internal control for RNA concentration. Results obtained in this way were reproducible between independent experiments.

Oligonucleotides containing overlapping Egr (underlined)/SP1 (bold) consensus binding sites, 5′-GGAGGAGCGGCGGGGGCGGGCGCCGG and 5′-CCGGCGCCCGCCCCGC (9), were annealed and 5 pmol labeled in a fill-in reaction catalyzed by the Klenow fragment of DNA Pol I (Promega), in the presence of 3.7 MBq [α32P]dCTP (Amersham Pharmacia Biotech, Buckinghamshire, U.K.), as described (23). A double-stranded Oct-1 consensus oligonucleotide (Promega), 5′-TGTCGAATGCAAATCACTAGAA was end labeled by incubating 5 pmol with T4 polynucleotide kinase (New England Biolabs, Hitchin, U.K.) in the presence of 12.3 MBq [γ32P]dATP (Amersham Pharmacia Biotech). Unincorporated nucleotides were removed by filtration through Sephadex G-25 microspin columns (Amersham Pharmacia Biotech) and 100 fmol labeled oligo, typically ∼15,000 cpm was used in each binding reaction. Thymic nuclear extracts were prepared from 5 × 106 thymocytes, as described by Shao et al. (9), and stored in aliquots at −70°C. The protein concentration of the extracts was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). For the binding reactions, 5 μl (30–40 μg protein) of nuclear extracts were mixed with the appropriate labeled oligonucleotide and incubated for 20 min at room temperature in a 20-μl reaction containing 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT (23), supplemented with 1 μg poly(dI:dC) (Amersham Pharmacia Biotech) and 5% glycerol. Binding reactions containing a 33-fold excess of unlabeled, double-stranded commercial oligonucleotodes with tandem consensus Egr-1 binding sites, 5′-GGATCCAGCGGGGGCGAGCGGGGGCGA, or a mutated version of it, 5′-GGATCCAGCTAGGGCGAGCTAGGGCGA (Santa Cruz Biotechnology), were included in experiments to control for specificity. The identity of the DNA binding proteins was determined by addition of 2 μg rabbit IgG specific for Egr-1 (sc-110) (Santa Cruz Biotechnology) or control, purified rabbit IgG (Sigma) to reactions after the initial 20 min binding, and incubated for an additional 10–20 min on ice. Protein-DNA complexes were separated from free oligonucleotides on a 7% nondenaturing polyacrylamide gel in 0.5× Tris borate-EDTA buffer containing 1% glycerol, whereafter the gel was fixed, dried, and exposed to x-ray film or phosphorimager screens and analyzed on a Storm 860 PhosphorImager (Molecular Dynamics, Chesham, U.K.).

We have previously described the in vitro differentiation of CD4 and CD8 SP thymocytes in response to bi- or monospecific F(ab′)2 reagents in TOC (4, 5, 22). A major advantage of this experimental system is that we can instruct the differentiation of DP thymocytes to either the CD4 or CD8 lineage, enabling us to address specific questions relating to the involvement of defined signaling cascades in the CD4/CD8 lineage decision. To evaluate the putative role of Egr genes during positive selection of the CD8 lineage, we initially investigated the induction of Egr genes in response to differentiation stimuli in vitro. Thymocytes from TCR transgenic mouse strains lacking selecting MHC molecules, e.g., F5/RAG-1neg2mneg, fail to differentiate beyond the DP stage. These DP cells were induced to differentiate in neonatal TOC (NTOC) and early changes in gene expression analyzed by RT-PCR at 18–24 h. FACS analyses were performed on day 7 after full differentiation had taken place to document the expected outcome of each experiment (Fig. 1 A).

FIGURE 1.

Egr-1, -2, and -3 gene expression is induced in NTOC upon treatment with CD3/CD4 in a MEK1-dependent fashion. A, Thymus lobes from neonatal F5/RAG-1neg2mneg mice were cultured for 3 days in the presence of Abs and/or inhibitors, as described (56 ) and for a further 4 days in medium before analysis for presence of mature SP cells. FACS profiles of gated mature (TCRhigh) cells are shown, confirming preferential differentiation of CD4+ cells after culture with CD3/CD4 BsAbs and of CD8+ cells after culture with CD3fos-F(ab′)2 (CD3/CD3). Incubation with PD98059 blocks CD4+ maturation and promotes CD8+ maturation as previously described (6 ). B, Total RNA was extracted after 18 h from equivalent thymus lobes in the same experiment, as shown in A, and Egr and GAPDH gene expression was determined by RT-PCR. PCR was performed for 15 and 20 cycles for GAPDH, and 25, 30, and 35 cycles for Egr genes, as indicated. RT-PCR products are shown for lobes cultured under the indicated conditions as well as for an ex vivo lobe. Densitometry was conducted on the bands obtained after 25 cycles for Egr genes, and the results are presented normalized relative to GAPDH expression after 15 cycles. Black bars indicate the presence of PD98059 in the cultures.

FIGURE 1.

Egr-1, -2, and -3 gene expression is induced in NTOC upon treatment with CD3/CD4 in a MEK1-dependent fashion. A, Thymus lobes from neonatal F5/RAG-1neg2mneg mice were cultured for 3 days in the presence of Abs and/or inhibitors, as described (56 ) and for a further 4 days in medium before analysis for presence of mature SP cells. FACS profiles of gated mature (TCRhigh) cells are shown, confirming preferential differentiation of CD4+ cells after culture with CD3/CD4 BsAbs and of CD8+ cells after culture with CD3fos-F(ab′)2 (CD3/CD3). Incubation with PD98059 blocks CD4+ maturation and promotes CD8+ maturation as previously described (6 ). B, Total RNA was extracted after 18 h from equivalent thymus lobes in the same experiment, as shown in A, and Egr and GAPDH gene expression was determined by RT-PCR. PCR was performed for 15 and 20 cycles for GAPDH, and 25, 30, and 35 cycles for Egr genes, as indicated. RT-PCR products are shown for lobes cultured under the indicated conditions as well as for an ex vivo lobe. Densitometry was conducted on the bands obtained after 25 cycles for Egr genes, and the results are presented normalized relative to GAPDH expression after 15 cycles. Black bars indicate the presence of PD98059 in the cultures.

Close modal

As previously reported for Egr genes in MHCnull thymi (9), Egr-1, -2, and -3 were found to be expressed at low levels in F5/RAG-1neg2mneg thymi (Fig. 1,B, ex vivo). Culture in medium alone leads to some induction of Egr expression, most likely due to the presence of growth factors in the serum (Fig. 1,B, medium). Induction of CD4 differentiation with CD3/CD4 BsAb induced up-regulation of mRNA encoding all three Egr members within 24 h (Fig. 1, CD3/CD4), in line with previous reports (9). However, no appreciable Egr induction occurs in response to a CD8 differentiation stimulus, CD3fos-F(ab′)2 (CD3/CD3) (Fig. 1, CD3/CD3). The addition of pharmacological inhibitors of MEK1 has been shown to promote the differentiation of CD8+ thymocytes, even in the absence of the selecting MHC ligand or ligand mimic (6) (Fig. 1,A). NTOC cultures to which the inhibitor PD98059 was added (Fig. 1, +PD98059), showed reduced levels of Egr mRNA, indicating that induction of Egr transcripts occurs downstream of the ras/MAPK pathway in normal thymocytes. Because Egr expression was reduced in the presence of the MEK1 inhibitor, whereas CD8 differentiation proceeded unhindered, it seems that the induction of Egr gene expression may not be required for commitment to the CD8 lineage. The efficiency of mRNA up-regulation in response to our F(ab′)2 reagents seems less pronounced than reported for the stimulation of a DP cell line with CD3-specific Abs or cognate peptide (9). This discrepancy is perhaps not surprising, given that these latter stimuli are regarded as fairly potent stimuli for thymocytes.

Although RT-PCR analysis indicated that Egr mRNA need not be induced during selection of CD8+ thymocytes, we sought to establish whether functional Egr protein was required. To demonstrate directly the induction of Egr binding activity during positive selection, nuclear extracts were prepared from neonatal thymus lobes after 24–48 h culture with the F(ab′)2 reagents. Engagement of CD3ε (CD3/CD3), or coengagement of CD3ε with CD4 (CD3/CD4), induced significant up-regulation of Egr binding activity in these lobes (Fig. 2). Given that all three members of the Egr gene family are transcribed at increased levels upon CD3/CD4 stimulation (Fig. 1), we undertook to ascertain the identity of the Egr member(s) present in the nuclei of thymocytes after stimulation. The specificity of the Egr bandshifts in Ab-stimulated cells was confirmed by competing with a shorter oligonucleotide containing a tandem repeat of the consensus Egr-1 binding site (Fig. 2, Egr-1 consensus). The Egr bandshifts were unaffected by incubation with a similar excess of control Egr oligo in which an essential GG had been mutated to a TA (Fig. 2, Egr-1 mutant). Ab supershift experiments indicated that the DNA-binding activity was primarily due to the presence of Egr-1, because an Egr-1-specific Ab supershifted the DNA-protein complex (Fig. 2, αEgr-1). Densitometric analysis confirms that the Egr family member detected is Egr-1 as the presence of the anti-Egr-1 Ab reduces the observed binding activity to background levels (Fig. 2 C). We conclude that functional Egr-1 protein is induced in response to CD3/CD4 and CD3/CD3 stimulation.

FIGURE 2.

Both CD3/CD4 and CD3fos-F(ab′)2 (CD3/CD3) induce Egr-1 binding activity in TOC. Neonatal thymus lobes from nonselecting F5/RAG-1neg2mneg were cultured with CD3/CD4 or CD3/CD3 for 2 days, whereafter nuclear extracts were prepared. Egr-1, SP1 (A) and Oct-1 (internal loading control) (B) binding activities were determined by EMSA. The identity of the Egr-containing complex in nuclei from Ab-stimulated cells was demonstrated by competition with a 33-fold excess of unlabeled Egr-1 consensus oligo and not by a mutant form of the same sequence. The Egr bandshift was supershifted with an Egr-1-specific Ab and not by control rabbit Ig (RbIg), confirming that Egr-1 is responsible for most of the Egr binding activity detected. Oct-1 and SP1 bandshifts indicate comparable protein content in individual reactions. C, Quantitation of band intensities on a phosphorimager shows that the inducible Egr binding activity can be abrogated completely with an Egr-1 specific Ab (αEgr-1). Egr-1 binding activity is calculated relative to the corresponding SP1 binding in each lane to control for variations in protein content.

FIGURE 2.

Both CD3/CD4 and CD3fos-F(ab′)2 (CD3/CD3) induce Egr-1 binding activity in TOC. Neonatal thymus lobes from nonselecting F5/RAG-1neg2mneg were cultured with CD3/CD4 or CD3/CD3 for 2 days, whereafter nuclear extracts were prepared. Egr-1, SP1 (A) and Oct-1 (internal loading control) (B) binding activities were determined by EMSA. The identity of the Egr-containing complex in nuclei from Ab-stimulated cells was demonstrated by competition with a 33-fold excess of unlabeled Egr-1 consensus oligo and not by a mutant form of the same sequence. The Egr bandshift was supershifted with an Egr-1-specific Ab and not by control rabbit Ig (RbIg), confirming that Egr-1 is responsible for most of the Egr binding activity detected. Oct-1 and SP1 bandshifts indicate comparable protein content in individual reactions. C, Quantitation of band intensities on a phosphorimager shows that the inducible Egr binding activity can be abrogated completely with an Egr-1 specific Ab (αEgr-1). Egr-1 binding activity is calculated relative to the corresponding SP1 binding in each lane to control for variations in protein content.

Close modal

As we had shown that both CD4 and CD8 differentiation signals could induce Egr-1 binding activity in vitro, it was important to confirm the same observation in vivo. We addressed this question in mice expressing the same class II-restricted transgenic TCR, A18 on a RAG-1neg background. As has been reported for several class II-restricted TCRs (17), thymocytes in these mice normally differentiate into the CD4 lineage, whereas on a CD4-deficient background they differentiate to the CD8 lineage. Using thymocytes from these mice, we could rule out any changes in Egr induction resulting from affinity differences between individual TCRs. Egr-1 was found to be expressed at high levels in both A18/RAG-1neg and A18/RAG-1neg/CD4neg thymi in which thymocytes differentiate into the CD4 or CD8 lineages, respectively (Fig. 3). The identity of the binding complex was confirmed by competition with an excess of unlabeled consensus oligo and anti-Egr-1 supershifts (Fig. 3). We conclude that Egr-1 induction is associated with selection of thymocytes into either lineage and that this can occur independently of the CD4 coreceptor.

FIGURE 3.

High levels of Egr-1 are present in thymocytes undergoing selection into either the CD4 or the CD8 lineage, irrespective of TCR specificity. A, Steady-state Egr-1 levels were compared in nuclear extracts from CD4-selecting A18/RAG-1neg and CD8-selecting A18/RAG-1neg/CD4neg thymi. The Egr-1 bandshift is specific as indicated by competition with excess unlabeled Egr-1 consensus, but not mutant oligo. Anti-Egr-1 Ab supershifts the Egr-DNA complex, whereas control RbIg has no effect. B, Oct-1 bandshifts indicate a slight difference in loading that corresponds to the intensity of Egr-1 and SP1 bandshifts.

FIGURE 3.

High levels of Egr-1 are present in thymocytes undergoing selection into either the CD4 or the CD8 lineage, irrespective of TCR specificity. A, Steady-state Egr-1 levels were compared in nuclear extracts from CD4-selecting A18/RAG-1neg and CD8-selecting A18/RAG-1neg/CD4neg thymi. The Egr-1 bandshift is specific as indicated by competition with excess unlabeled Egr-1 consensus, but not mutant oligo. Anti-Egr-1 Ab supershifts the Egr-DNA complex, whereas control RbIg has no effect. B, Oct-1 bandshifts indicate a slight difference in loading that corresponds to the intensity of Egr-1 and SP1 bandshifts.

Close modal

By sorting thymocyte subpopulations from a class I-restricted TCR transgenic mouse, F5/RAG-1neg, we could show that Egr-1 binding activity is induced very early during CD8 selection, as it is evident in both CD69 and CD69+ DPs, and remains present in mature, CD8 SPs (Fig. 4). To exclude the possibility that these results may be biased toward early expression of Egr-1 due to the premature expression of the transgenic F5 TCR, we repeated the same analyses in nontransgenic Class II-deficient mice and confirmed that the same holds true when a polyclonal repertoire of TCRs is expressed under endogenous control (data not shown). We conclude that Egr-1 binding activity is present in the nuclei of DP thymocytes that differentiate into the CD8 lineage and that the appearance of nuclear Egr-1 binding activity preempts CD69 up-regulation.

FIGURE 4.

Egr-1 binding activity is induced early during thymocyte differentiation. F5/RAG-1neg thymocytes were electronically sorted into the CD69 and CD69+ DP, as well as CD8 SP subpopulations from which nuclear extracts were prepared and analyzed by EMSA for Egr-1, SP1 (A) and Oct-1 (B) activity.

FIGURE 4.

Egr-1 binding activity is induced early during thymocyte differentiation. F5/RAG-1neg thymocytes were electronically sorted into the CD69 and CD69+ DP, as well as CD8 SP subpopulations from which nuclear extracts were prepared and analyzed by EMSA for Egr-1, SP1 (A) and Oct-1 (B) activity.

Close modal

RT-PCR experiments indicated that pharmacological inhibition of MEK1 inhibits the induction of Egr gene transcription. Similarly, MEK1 inhibitors, PD98059 (data not shown) and U0126, completely abrogated any Egr-1 binding activity induced by the coligation of CD3 with CD4 on the surface of thymocytes in NTOC (Fig. 5, lanes 1 and 2). An identical result was obtained with CD3/CD3 stimulation (Fig. 5, lanes 3 and 4), demonstrating that the induction of Egr-1 binding activity in the nuclei of DP thymocytes requires MEK1 activity. The culture of lobes in the presence of these inhibitors not only prevented Ab-induced Egr-1 up-regulation, but also completely abolished constitutive Egr-1 present at low levels in nonselecting lobes (Fig. 5, lanes 5 and 6) and at high levels in selecting, F5/RAG-1neg lobes (Fig. 5, lanes 7 and 8). Because CD8 lineage commitment is enhanced under these conditions, we can conclude from these experiments that neither Egr-1 mRNA up-regulation, nor DNA binding activity is required for commitment of thymocytes to the CD8 lineage. In contrast, Egr-1 induction is associated with CD4 differentiation signals, and both these events are sensitive to MEK1 inhibition.

FIGURE 5.

Egr-1 induction in response to CD3/CD4 and CD3/CD3 stimulation of thymocytes in NTOC requires MEK1 activity. Neonatal thymus lobes from nonselecting F5/RAG-1neg2mneg thymocytes were stimulated for 40 h with Abs in the presence of MEK1 inhibitor U0126 (9 μM) where indicated. Thymocytes from cultures incubated in medium only (medium) and thymus lobes from selecting F5/RAG-1neg mice were cultured and analyzed under identical conditions. Egr-1, SP1 (A), and Oct-1 (internal loading control) (B) binding activity in thymic nuclear extracts were determined by EMSA. The identity of the Egr-containing complex in nuclei was confirmed by supershifting by an Egr-1-specific Ab (αEgr-1). Oct-1 and SP1 bandshifts indicate comparable protein content in individual reactions.

FIGURE 5.

Egr-1 induction in response to CD3/CD4 and CD3/CD3 stimulation of thymocytes in NTOC requires MEK1 activity. Neonatal thymus lobes from nonselecting F5/RAG-1neg2mneg thymocytes were stimulated for 40 h with Abs in the presence of MEK1 inhibitor U0126 (9 μM) where indicated. Thymocytes from cultures incubated in medium only (medium) and thymus lobes from selecting F5/RAG-1neg mice were cultured and analyzed under identical conditions. Egr-1, SP1 (A), and Oct-1 (internal loading control) (B) binding activity in thymic nuclear extracts were determined by EMSA. The identity of the Egr-containing complex in nuclei was confirmed by supershifting by an Egr-1-specific Ab (αEgr-1). Oct-1 and SP1 bandshifts indicate comparable protein content in individual reactions.

Close modal

Results presented so far indicate that, although receptor ligations that generate CD8 differentiation signals induce Egr-1, the complete inhibition of this inducible Egr-1 binding activity does not adversely affect CD8 differentiation. We asked whether Egr-1 induction is always associated with positive selection and what the requirements are for induction in immature thymocytes. To address these questions we compared the steady-state levels of Egr-1 binding activity in thymocytes from different mouse lines that either can or cannot positively select due to defects in key signaling molecules.

As shown earlier, Egr-1 expression in the thymus requires TCR ligation by MHC, because thymi from nonselecting F5/RAG-1neg2mneg mice express much reduced levels of Egr-1 (Fig. 6, A and C), compared with the positively selecting F5/RAG-1neg thymi (Fig. 6, A and C), supporting the assumption that Egr-1 is associated with positive selection.

FIGURE 6.

Egr-1 induction in the thymus requires TCR expression on DP thymocytes and ligation by the appropriate MHC ligands, but does not require CD45 or full maturation to SP thymocytes. Steady-state Egr-1, SP1 (A), and Oct-1 (B) binding activity in thymocytes from different mouse strains were compared by EMSA. A summary of accumulated data from three experiments is presented in C. Band intensities were determined on a PhosphorImager and Egr-1 activity quantitated relative to SP1 activity as an internal loading control.

FIGURE 6.

Egr-1 induction in the thymus requires TCR expression on DP thymocytes and ligation by the appropriate MHC ligands, but does not require CD45 or full maturation to SP thymocytes. Steady-state Egr-1, SP1 (A), and Oct-1 (B) binding activity in thymocytes from different mouse strains were compared by EMSA. A summary of accumulated data from three experiments is presented in C. Band intensities were determined on a PhosphorImager and Egr-1 activity quantitated relative to SP1 activity as an internal loading control.

Close modal

We showed that Egr-1 activity can be induced in a CD4-independent manner in A18 class II-restricted TCR transgenic mice (Fig. 3), suggesting that coreceptor-mediated recruitment of lck to the TCR is not essential. To ask more directly whether lck-mediated signals were important for Egr activity, we thought it informative to determine the Egr-1 content of thymocytes from mice expressing a dysregulated, constitutively active lckF505 transgene (pLGF) (19). Thymocytes in pLGF mice exhibit a pronounced defect in positive selection, in part due to the almost total absence of TCR expression on DP thymocytes. We found that these thymi contain significantly reduced levels of Egr-1, comparable to those in F5/RAG-1neg2mneg thymi (Fig. 6, A and C). To ask whether the reduction in Egr-1 expression in these thymocytes resulted from their failure to express significant levels of TCR, we introduced a F5 TCR transgene into these mice, and found that Egr-1 expression was restored (Fig. 6). Positive selection can occur in F5pLGF thymi and they contain similar numbers of CD8 SP thymocytes to F5/RAG-1neg thymi (not shown), confirming the link between TCR-transduced signals and Egr-1 induction during thymocyte differentiation.

CD45-deficient thymocytes exhibit a severe positive selection defect (24, 25) that has been attributed to the abnormal regulation of src kinase activity (26, 27). However, thymi from F5/RAG-1neg mice still express high levels of Egr-1 in the absence of CD45 (Fig. 6). This observation is not an artifact of transgenic TCR expression, because polyclonal CD45neg thymi contain comparable levels of Egr-1 protein (Fig. 6). Thus, even though CD45-deficient thymocytes fail to mature fully, it is clear that ligation of their TCRs generates signals of sufficient potency to result in the induction of Egr-1 binding activity. Therefore, Egr-1 induction is not a necessary indicator of positive selection, because in CD45-deficient thymocytes the strength or nature of signals that allow induction of Egr-1 activity are not sufficient to ensure thymocyte differentiation.

The protooncogene Vav and tyrosine kinase ZAP70 are key mediators of TCR-induced signaling in thymocytes (1), and mice lacking these molecules (20, 28) exhibit positive selection defects of comparable severity to CD45-deficient mice (24, 25). In contrast to CD45neg thymi, thymocytes from Vavneg and ZAP70neg mice contain considerably reduced levels of Egr-1 (Fig. 6 C). As we fail to find a direct correlation between the capacity to positively select and Egr-1 binding activity, we conclude that Egr-1 induction is not always associated with efficient positive selection, but rather represents a very sensitive indication of TCR ligation on double positive thymocytes. The much reduced Egr-1 binding activity in Vav- and ZAP70-deficient thymocytes may indicate that Egr-1 induction is downstream of Vav and ZAP70 signaling pathways, but is rather insensitive to alterations in CD45/Lck activity.

Egr-1 is an immediate early gene that encodes a Zn finger transcription factor that is induced in many cell types by a variety of growth and differentiation stimuli. Recent studies clearly implicated Egr-1 in thymocyte selection (9, 10, 11); however, many questions remain as to the exact role of Egr-1 during positive selection. Previous studies on Egr-1 induction in the thymus relied on experimental systems in which thymocytes differentiate to the CD4 lineage. However, the ability of transgenic Egr-1 expression to reduce the threshold for selection seems to impact on CD4 and CD8 lineages to a similar extent (11), suggesting that the effect of Egr-1 may not be lineage-specific.

In the present report, we show that CD4 differentiation signals induce Egr-1, -2, and -3 gene expression in primary thymocytes, giving rise to the appearance of functional Egr-1 protein in the nuclei of these cells. Despite the fact that Egr-2 and Egr-3 transcripts were induced in stimulated thymocytes, all of the Egr-DNA complexes observed in EMSA experiments supershifted with an Egr-1-specific Ab. The major Egr component present in the nuclei of thymocytes from normal mice in which selection is ongoing, also corresponds to Egr-1 protein. A similar observation was reported by Shao et al. (9), who suggested that the failure to detect Egr-2 and Egr-3 binding may be due to lower expression of these proteins or suboptimal binding conditions employed in EMSA experiments that favor Egr-1 complexes. The oligonucleotide we and Shao et al. used contains an additional 3′ G in the consensus sequence that has been reported to be required for optimal Egr-2 binding (29). Furthermore, we specifically employed binding conditions that have been reported to optimize Egr-2 binding to the consensus sequence (29), yet, despite this, we failed to detect Egr-2 complexes. Therefore, we feel that our failure to detect Egr-2 complexes is more likely to reflect lower expression of these proteins than inappropriate assay conditions. Given the neonatal lethality of the Egr-2 knockout mouse, it is unclear at present whether Egr-2 plays a role during this late stage of thymocyte differentiation. In contrast, thymocyte differentiation in an Egr-1-deficient mouse occurs normally (30) and analyses of dominant negative Egr transgenics may be required to resolve whether this lack of phenotype is due to other family members being able to compensate for Egr-1 deficiency despite lower levels of expression.

We have previously described a method for the exclusive generation of CD8 SP thymocytes in vitro (4), which allowed us to address directly the role of Egr-1 induction during CD8 differentiation in the thymus. Analyses of nuclear extract from thymus lobes that had been cultured in the presence of CD3fos-F(ab′)2 indicate that CD3 ligation by this reagent induces high levels of Egr-1 binding activity. Importantly, this reagent was found to be poor at inducing Egr-1 gene transcription under similar conditions (Fig. 1), suggesting perhaps that transcriptional induction may not be the only way in which Egr-1 binding activity can be induced. Egr-1 binding activity has been shown to be regulated by ser/thr phosphorylation (31), and it is possible that Egr-1 binding activity may be enhanced by ERK-mediated phosphorylation. We could detect no difference in Egr-1 protein in selecting (B10) and nonselecting (MHCnull) thymocytes by intracellular staining and immunoblotting (data not shown), further lending support to the notion that an increase in gene expression may not be the only mechanism by which Egr-1 activity is up-regulated in thymocytes. As we were measuring DNA binding and not transcriptional activity, it is also possible that the Egr-1 complexes induced during CD4 vs CD8 differentiation have distinct effects on gene transcription, for example, by associating with different co-activator or repressor complexes.

Commitment to the CD8 lineage is less dependent on the ERK/MAPK pathway than CD4 commitment (6), and because Egr induction has been shown to require ras activity (9), we sought to determine whether Egr induction is required for selection into the CD8 lineage. We show that pharmacological inhibition of MEK1 can completely abrogate Egr-1 induction in intact thymi in response to CD3 ligation by Ab or by TCR ligation with endogenous ligands. This inhibition is associated with more efficient commitment of DP thymocytes to the CD8 lineage as evidenced by full CD4 and heat-stable Ag down-regulation (6), confirming that Egr-1 induction is not strictly required for this step in thymocyte differentiation. We showed in a previous report that the final maturation of thymocytes that involved up-regulation of the TCR requires release from MEK1 inhibition (6) and it is possible that Egr-1 is required during the process of TCR up-regulation.

A systematic analysis of Egr-1 binding activity in thymocytes from several transgenic and knockout mouse strains indicated that Egr-1 induction occurs in response to TCR-mediated signals. Deficiencies impacting on the expression of the TCR (TCRαneg, pLGF), its ligands (MHCnull, F5/RAGneg2mneg), or signaling capacity (ZAP70neg, Vavneg), translates to defects in Egr-1 induction. The reduced Egr-1 activity in Vav-deficient cells is consistent with the observed defect in Erk activation (32). In contrast, modulation of lck signaling status by genetic removal of the protein tyrosine phosphatase CD45, or expression of a constitutively active form of lck, F505, had little effect on Egr-1 activity, as long as a TCR is expressed.

The first steps in positive selection probably involve ligation of the successfully rearranged TCRαβ heterodimer by endogenous MHC-peptide complexes in the thymus. It is likely that Egr-1 is one of the earliest factors induced upon TCR ligation, given the observation that high levels of Egr-1 are already present in the CD69 DP population. The exact role of Egr-1 in this differentiation process is unknown but it has been proposed to lower the threshold required for positive selection. This may be the result of inducing genes required for the differentiation process or even those encoding survival factors, but only the direct identification of such genes will provide the answer.

We thank Dimitris Kioussis and Gitta Stockinger for TCR transgenic mice and all the laboratories that made knockout mice available to us (B. Stockinger, E. Spanopoulou, D. Mathis, C. Benoist, N. Killeen, D. Littman, A. Weiss); James Trzaskos for the gift of U0126; Trisha Norton, Keith Williams, and Pauline Travel for their efforts in maintaining the animal colony, Chris Atkins for cell sorting, Helen Coope and Monica Belich for advice on EMSAs, and Matthew Lovatt and Gitta Stockinger for critical comments on the manuscript.

1

This work was supported by the Medical Research Council (U.K.) and grants from the Leukemia Research Fund.

4

Abbreviations used in this paper: DP, double positive; SP, single positive; Egr, early growth response; TOC, thymus organ culture; NTOC, neonatal TOC; β2m, β2-microglobulin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; MEK, MAPK/ERK kinase.

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