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Divisions of
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Molecular and
Cellular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 TCR
neg (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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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), ß2-microglobulin (ß2m)neg (14) mice have been described and were intercrossed to obtain F5/RAG-1neg or F5/RAG-1neg/ß2mneg 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 ß2mneg and I-Aßneg (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).
Antibodies
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 CD3
,
CD4, or CD8 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.).
PCR primers for RT-PCR
Egr-1, -2, and -3-specific primers were as described by Shao et al. (9). GAPDH primers were 5'-GGGGTGAGGCCGGTGCTGAGTAT and 5'-CATTGGGGGTAGGAACACGGAAGG.
Cell sorting
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 x 106 cells, from which nuclear extracts were prepared as described below.
Thymus organ cultures (TOC)
Neonatal thymus lobes from F5/RAG-1neg and F5/RAG-1neg/ß2mneg 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 x 106 viable cells, from which nuclear extracts were prepared.
RNA extraction
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.
Semiquantitative RT-PCR
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.
EMSA
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
[32P]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 [
32P]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 x 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 0.5x
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.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-1neg/ß2mneg,
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).
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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. 1B, 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. 1A). 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. Egr-1 binding activity is induced in response to positive selection stimuli
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 CD3 (CD3/CD3), or coengagement of CD3
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, Egr-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. 2C). We conclude that functional
Egr-1 protein is induced in response to CD3/CD4 and CD3/CD3
stimulation.
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). 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.
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). 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.
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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.
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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-1neg/ß2mneg
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.
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),
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-1neg/ß2mneg
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. 6C). 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
(TCRneg, pLGF), its ligands
(MHCnull,
F5/RAGneg/ß2mneg),
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 TCRß 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.
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Acknowledgments |
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Footnotes |
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2 Current address: Institute for Neurodegenerative Diseases, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143-0518.
3 Address correspondence and reprint requests to Dr. Rose Zamoyska, Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
4 Abbreviations used in this paper: DP, double positive; SP, single positive; Egr, early growth response; TOC, thymus organ culture; NTOC, neonatal TOC; ß2m, ß2-microglobulin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; MEK, MAPK/ERK kinase.
Received for publication March 13, 2000. Accepted for publication June 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and ZAP-70. J. Immunol. 158:5773.[Abstract]
B pathways. Proc. Natl. Acad. Sci. USA 96:3035.This article has been cited by other articles:
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  K. Cante-Barrett, E. M. Gallo, M. M. Winslow, and G. R. Crabtree Thymocyte Negative Selection Is Mediated by Protein Kinase C- and Ca2+-Dependent Transcriptional Induction of Bim J. Immunol., February 15, 2006; 176(4): 2299 - 2306. [Abstract] [Full Text] [PDF]
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 L. K. McNeil, T. K. Starr, and K. A. Hogquist A requirement for sustained ERK signaling during thymocyte positive selection in vivo PNAS, September 20, 2005; 102(38): 13574 - 13579. [Abstract] [Full Text] [PDF]
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A. Singh, J. Svaren, J. Grayson, and M. Suresh CD8 T Cell Responses to Lymphocytic Choriomeningitis Virus in Early Growth Response Gene 1-Deficient Mice J. Immunol., September 15, 2004; 173(6): 3855 - 3862. [Abstract] [Full Text] [PDF]
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D. DeRyckere, D. L. Mann, and J. DeGregori Characterization of Transcriptional Regulation During Negative Selection In Vivo J. Immunol., July 15, 2003; 171(2): 802 - 811. [Abstract] [Full Text] [PDF]
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H. Xi and G. J. Kersh Induction of the Early Growth Response Gene 1 Promoter by TCR Agonists and Partial Agonists: Ligand Potency Is Related to Sustained Phosphorylation of Extracellular Signal-Related Kinase Substrates J. Immunol., January 1, 2003; 170(1): 315 - 324. [Abstract] [Full Text] [PDF]
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M. Bettini, H. Xi, J. Milbrandt, and G. J. Kersh Thymocyte Development in Early Growth Response Gene 1-Deficient Mice J. Immunol., August 15, 2002; 169(4): 1713 - 1720. [Abstract] [Full Text] [PDF]
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