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The Journal of Immunology, 2004, 173: 340-348.
Copyright © 2004 by The American Association of Immunologists

Sustained Early Growth Response Gene 3 Expression Inhibits the Survival of CD4/CD8 Double-Positive Thymocytes1

Hongkang Xi and Gilbert J. Kersh2

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the absence of selection, CD4+, CD8+ double-positive (DP) thymocytes will die after 3–4 days. The mechanism for regulating the life span of DP cells is unknown. Previously, we demonstrated that the zinc finger transcription factor, early growth response gene 3 (Egr3), promotes proliferation during the transition from double negative (DN) to DP. In this study we demonstrate a novel role for Egr3 in controlling DP thymocyte survival in mice. Constitutive transgenic expression of Egr3 in thymocytes increases apoptosis among DP cells and shortens their survival in vitro. In addition, DP cells in Egr3 transgenic mice have poor expression of TCR{alpha}, and based on the predominant usage of 3' V{alpha} and 5' J{alpha} gene segments, the low level of TCR{alpha} expression is a result of DP death soon after the initiation of TCR{alpha} rearrangements. Constitutive transgenic expression of Egr3 results in poor expression of Bcl-xL and the thymic isoform of retinoic acid receptor-related orphan receptor {gamma} (ROR{gamma}t) in DP thymocytes, two molecules that are required in DP cells for normal life span. Egr3 expression induced by pre-TCR signals in nontransgenic mice is transient and returns to background levels before ROR{gamma}t or Bcl-xL is induced. The data support a model in which Egr3 must be transiently induced in response to pre-TCR signals, so that the expression of the prosurvival molecules, ROR{gamma}t and Bcl-xL, can be elevated only after the proliferative signal provided by Egr3 has subsided.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Intrathymic {alpha}{beta} T cell maturation involves multiple well-defined stages, each characterized by distinct expression of cell surface proteins. Immature CD4–CD8– double-negative (DN)3 cells, which are also TCR negative, can be further divided into four sequential developmental stages based on CD44 and CD25 expression: DN1 (CD44+CD25–), DNII (CD44+CD25+), DNIII (CD44–CD25+), and DNIV (CD44–CD25–) (1). It is at the DNIII stage that rearrangements of the TCR{beta} genes occur, and a successfully rearranged and expressed TCR{beta} associates with the pre-TCR{alpha} and CD3 chains to form the pre-TCR complex (2). The transition from DNIII to DNIV has been called the {beta} selection checkpoint, as the process only allows cells with a functional TCR{beta} to proceed. Consequences of pre-TCR signaling include enhanced survival, allelic exclusion at the TCR{beta} locus, a reduction in CD25 expression, and extensive proliferation (3). After the DNIV stage, immature thymocytes first up-regulate CD8 expression and become immature single-positive (ISP) cells, followed by up-regulation of CD4 expression, thus becoming CD4+CD8+ double-positive (DP) cells.

Once the resulting DP thymocyte population has stopped proliferating, it has an intrinsic life span of 3–4 days, during which DP thymocytes undergo multiple rounds of TCR{alpha} rearrangements to maximize the chances of forming a functional TCR {alpha}{beta} heterodimer (4, 5). However, the majority of DP thymocytes fail to form a TCR that interacts productively with self peptide/MHC complexes and die after their 4-day life span has expired (6). The normal life span of DP thymocytes requires the proper signals derived from the pre-TCR. These signals ultimately regulate the expression of Bcl-2 family members in DP cells that control the susceptibility of DP cells to apoptosis. Bcl-2 is expressed in DN and SP thymocytes, but only at very low levels in DP cells (7). In contrast, Bcl-xL is most highly expressed in DP thymocytes, where it is thought to promote the survival of DP cells (8). Although the expression of Bcl-2 and Bcl-xL in DP thymocytes is thought to be regulated by pre-TCR signals, the molecular basis for this regulation is not known. In addition, the mechanism by which DP thymocytes that are unable to produce a functional {alpha}{beta} TCR initiate apoptosis is not known.

The transcription factors that are induced by pre-TCR signals and regulate the life span of DP thymocytes are largely unknown. Among the transcription factors thought to be regulated by pre-TCR signals are the early growth response (Egr) genes, which code for a family of zinc finger-containing proteins, including Egr1, -2, -3, and -4. Previous studies have implicated the Egr family members in both {beta} selection and positive selection of {alpha}{beta} T cells (9, 10, 11, 12, 13). Egr1, -2, and -3 are all rapidly induced by both pre-TCR and TCR activation in thymocytes. Retroviral transduction of Egr1, -2, or -3 in CD3{gamma}-deficient fetal thymocytes, which are blocked at the DNIII stage, is able to promote some differentiation to the DNIV stage (13). Overexpression of Egr1 or Egr3 in RAG-deficient thymocytes allows DN cells to differentiate into immature CD8 SP cells (9, 14). In a previous study we found that Egr3 played a significant role in promoting thymocyte proliferation during the transition from the DN stage to the DP stage (14). Egr3-deficient mice exhibited reduced thymic cellularity due to poor proliferation in response to pre-TCR signals. Transgenic overexpression of Egr3 in RAG-deficient thymocytes not only promoted some differentiation, but also led to extensive cellular expansion.

In this study we define a new role for Egr3 in thymocyte development by examining transgenic mice that constitutively express Egr3 under the control of the CD2 promoter and enhancer (Egr3 transgenic (Egr3TG) mice). Surprisingly, we found that on a normal background Egr3TG mice have reduced thymic cellularity despite the fact that Egr3 positively regulates thymocyte proliferation. The reduced thymic cellularity of Egr3TG mice is due to poor survival of DP cells, with reduced expression of Bcl-xL. Constitutive Egr3 expression also leads to impaired TCR{alpha} rearrangements, which results in a skewed TCR{alpha} repertoire and lowered TCR{alpha} expression. Consequently, the production of mature CD4 and CD8 SP thymocytes is severely impaired in Egr3TG mice. Finally, we demonstrate that Egr3TG mice have reduced expression of retinoic acid receptor-related orphan receptor {gamma} (ROR{gamma}), an orphan nuclear receptor involved in promoting DP cell survival (15). The results are consistent with a model in which Egr3 promotes proliferation in response to pre-TCR signals, but must be down-regulated in the hours after pre-TCR signaling for ROR{gamma} and Bcl-xL expression to increase and promote the normal life span of DP cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Generation of Egr3TG mice that constitutively express a FLAG-tagged rat Egr3 driven by CD2 promoter in T cell lineage was previously described (14). The Egr3TG mice were maintained on a B6.AKR background. RAG1-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

Flow cytometry

Abs and reagents used in flow cytometry were either obtained from BD Pharmingen (San Diego, CA; anti-CD4-PE, anti-CD4-FITC (GK1.5), anti-CD5-FITC (53-7.3), anti-TCR{beta}-biotin (H57), anti-heat-stable Ag-FITC (M1/69)) or from Caltag (Burlingame, CA; anti-CD8{alpha}-TC (Ly-2), anti-CD3-FITC (CD3{epsilon}), and streptavidin-FITC). For analysis of thymocyte apoptosis, thymocytes were stained with annexin V-FITC and propidium iodide (PI) using the annexin V-FITC apoptosis detection kit I (BD Pharmingen) according to the manufacturer’s instructions. For intracellular staining of Bcl-xL, thymocytes were first surface-stained with anti-CD4-FITC and anti-CD8{alpha}-FITC. The cells were fixed and permeabilized using the Fix and Perm Cell Permeabilization kit (Caltag Laboratories, Burlingame, CA) according to the manufacturer’s instruction. The cells were then incubated with 1/1000 diluted rabbit anti-Bcl-xL Ab (BD Pharmingen) or isotype control Ab in the permeabilization medium at room temperature for 30 min. The cells were washed with PBS and incubated with 1/1000 diluted goat F(ab')2 anti-rabbit IgG-PE (Southern Biotechnology Associates, Birmingham, AL) in the permeabilization medium at room temperature for 30 min. After washing, the cells were resuspended in 1% formaldehyde in PBS and analyzed by flow cytometry. All cell analyses were performed with a FACSort flow cytometer and CellQuest software (BD Immunocytometry Systems, San Jose, CA).

Cell sorting and preparation of whole cell lysate and total RNA

DP thymocytes were isolated using Ab-coated Dynabeads (Dynal Biotech, Oslo, Norway). Thymocyte suspension was first incubated with CD4 Dynabeads at 4°C for 30 min with rotation. After the rosetted cells (containing DP and CD4SP thymocytes) were washed three times with the washing buffer (RPMI 1640 with 1% FCS), the cells were detached from the beads by incubation with the CD4 detach Ab at room temperature for 1 h with rotation. The beads were then washed three times with the washing buffer, and the detached cells were collected. The cells were incubated with CD8 Dynabeads at 4°C for 30 min with rotation. The DP thymocytes attached to the beads were separated from the CD4SP thymocytes by applying the mixture to a magnet. The DP cells were released from the beads by incubating the beads with the CD8 releasing buffer at room temperature for 30 min with rotation. The isolated DP cells were used for preparation of whole cell lysate and total RNA as described previously (14).

Western blot analysis

Whole cell lysates (50 µg) from thymocytes were separated by 10% SDS-PAGE, and the separated proteins were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The membrane was probed sequentially with the following Abs: rabbit anti-Bcl-xL (BD Pharmingen), anti-BIM, anti-BAX, anti-BAD (R&D Systems, Minneapolis, MN), mouse anti-Fas ligand (BD Pharmingen), and mouse anti-{beta}-actin (AC-15; Sigma-Aldrich, St. Louis, MO). The expressions of TCR{alpha}, TCR{beta}, and CD3{epsilon} were determined by probing the membrane sequentially with anti-TCR{alpha} (H28-710), anti-TCR{beta} (H57), and anti-CD3{epsilon} (500A2) mAb (BD Pharmingen). Immune complexes were detected with appropriate HRP-conjugated Abs (Jackson ImmunoResearch Laboratories, West Grove, PA) and ECL reagents (Amersham Pharmacia Biotech).

RT-PCR

cDNA was synthesized with Superscript II reverse transcriptase (Invitrogen) with random hexamers according to the manufacturer’s instructions. One tenth of the cDNA was used in the PCR. TCR V{alpha}3 or V{alpha}6 to C{alpha} PCR was performed as follows: 94°C for 2 min and then 30 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by 72°C for 10 min. Oligonucleotide sequences for the V{alpha}3 and V{alpha}6 forward primers and the C{alpha} reverse primer were previously described (16, 17). The expression of RAG1, RAG2, TCR V{alpha}19, TCR C{alpha}, and hypoxanthine phosphoribosyltransferase (HPRT) was analyzed by semiquantitative PCR with serially diluted cDNA (1/5) as follows: 94°C for 2 min, then 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min, followed by 72°C for 10 min. The oligonucleotide sequences of PCR primers are as follows: RAG1, 5'-CCAAGCTGCAGACATTCTAGCACTC and 5'-CTGGATCCGGAAAATCCTGGCAATG; RAG2, 5'-CACATCCACAAGCAGGAAGTACAC and 5'-GGTTCAGGGACATCTCCTACTAAG; and HPRT, 5'-GCTGGTGAAAAGGACCTCT and 5'-CACAGGACTAGAACACCTGC. The PCR primers for TCR V{alpha}19 and TCR C{alpha} were previously described (16, 17). PCR products were separated on a 1.5% agarose gel, transferred to a Hybond N nylon membrane (Amersham Pharmacia Biotech), and analyzed by Southern blot using the oligonucleotide probes as follows: TCR-C{alpha}, 5'-ACATCCAGAACCCAGAACCTGC; RAG1, 5'-GCTGCCTCCTTGCCGTCTACC; RAG2, 5'-AAAATGTCCCTGCAGATGGTAAC; and HPRT, 5'-TACGAGGAGTCCTGTTGATGTTGCCA. The oligonucleotide sequences of J{alpha}-specific probes were previously described (16, 18). RT-PCR of Egr3 and the thymic isoform of ROR{gamma} (ROR{gamma}t) mRNA was described previously (14, 19). PCR of Bcl-xL was performed as follows: 94°C for 2 min, then 40 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min, followed by 72°C for 10 min. The Bcl-xL PCR primers are as follows: 5'-TCGCTCGCCCACATCCCAGCTTCACATAACCCC and 5'-CCACCAACAAGACAGGC.

Northern blot analysis

Northern blot was performed with 10 µg of total RNA isolated from thymocytes as previously described (12). The membrane carrying the RNA was sequentially probed with a full-length mouse ROR{gamma}t cDNA probe and a GAPDH cDNA probe. The full-length mouse ROR{gamma}t cDNA was cloned by RT-PCR using B6.AKR mouse thymus RNA and was verified by sequencing.

Transient transfection and reporter assays

The ROR{gamma}t promoter luciferase construct pROR{gamma}t-Luc was generated by PCR amplification of a 1.4-kb DNA fragment upstream of the first ROR{gamma}t exon using mouse thymus genomic DNA. The DNA fragment was cloned into the luciferase reporter vector pGL3 Basic (Promega, Madison, WI). The 16610D9 CD4+, CD8+ cell line (20) was a gift from S. Hedrick (University of California, San Diego, La Jolla, CA). 16610D9 cells (10 x 106) were transfected by electroporation in 0.5 ml of serum-free RPMI 1640 medium with 10 µg of pROR{gamma}t-Luc, 0.5 µg Renilla luciferase control vector (pRL-TK; Promega), and various amounts of an Egr3 expression vector. The total amount of transfected DNA was kept constant by adding the appropriate amount of empty vector. The cells were electroporated at 240 V and 950 µF. After electroporation, the cells were incubated at room temperature for 10 min and transferred into 10 ml of growth media and incubated at 37°C with 5% CO2 for 40–48 h. Dual luciferase assays were performed using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Reduced cellularity and perturbed thymic development in Egr3 transgenic mice

As described in our previous report, Egr3TG mice express a FLAG-tagged Egr3 transgene at a level 2–3 times that of endogenous Egr3 in the thymus (14). The expression of the transgene under the control of a CD2 promoter was detected in thymocytes at the DNIII stage through the mature CD4 and CD8 SP stages by RT-PCR or Northern blot analysis using sorted cells from each population (data not shown). Our previous study demonstrated that Egr3 positively regulates thymocyte proliferation (14). Whereas Egr3-deficient mice had reduced thymus cellularity and proliferation in response to pre-TCR signaling, overexpression of Egr3 in RAG1-deficient mice not only promoted thymocyte differentiation past the {beta} selection checkpoint, but also led to increased cellularity.

To further understand the role of Egr3 in thymocyte development, we have analyzed Egr3TG thymocytes in more detail on a normal (B6.AKR) background. Surprisingly, we have found that Egr3TG mice exhibit reduced thymus cellularity. As shown in Fig. 1A, thymuses from Egr3TG mice have, on the average, only 18% of the number of thymocytes in age-matched non-TG littermate controls (NLC). Analysis of the cellularity of each thymocyte compartment revealed that although the numbers of DN and CD8SP cells of Egr3TG mice appear normal compared with those of NLC mice, the numbers of DP and CD4SP cells of Egr3TG mice are significantly reduced. The Egr3TG mice have only 11 and 9% the numbers of DP and CD4SP cells, respectively, found in NLC mice. As DP cells constitute ~85% of the total thymocyte population in NLC mice, the reduction of thymus cellularity in Egr3TG mice is mostly due to the absence of DP thymocytes. Examination of CD4, CD8, and TCR{beta} expression in Egr3TG thymuses revealed abnormal distribution of cells among DN, DP, and SP compartments (Fig. 1B). The percentage of DP cells is only 51.7% in Egr3TG mice compared with 84.7% in control mice. The percentage of CD4SP cells in Egr3TG mice is also reduced (compare 5.6% in Egr3TG mice with 8.6% in NLC). However, Egr3TG mice have increased percentages of DN (29.4%) and CD8SP (13.3%) cells compared with control mice (3.7 and 3.0%, respectively). Because of the reduced total number of thymocytes in Egr3TG mice, the absolute numbers of DN and CD8SP in these mice are comparable to those in control mice. Further examination of CD4SP and CD8SP compartments of Egr3TG thymuses by analysis of TCR{beta} expression revealed that the percentages of mature CD4SP and CD8SP are even more reduced in Egr3TG thymuses. The percentage of CD4SP, TCRhigh cells is only 2.7% in Egr3TG mice compared with 7.1% in NLC. Whereas NLC thymuses have 2.1% CD8SP, TCRhigh cells, Egr3TG thymuses have only 0.4%. Thus, although Egr3TG thymuses have an increased percentage of CD8SP cells, the majority (97%) of this population expresses low levels of the TCR.



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  		FIGURE 1. Phenotypic analysis of Egr3TG thymocytes. A, Egr3TG mice have reduced thymic cellularity. The numbers of total thymocytes and DN, DP, and SP compartments of 6- to 8-wk-old Egr3TG and NLC mice are shown. The data represent the mean ± SEM of eight pairs of mice. B, Representative analysis of thymic CD4, CD8, and TCR{beta} expression in a pair of age-matched Egr3TG and NLC mice. Total numbers of thymocytes are shown in parentheses. The numbers shown to the right of the dot blots represent the percentage of total cells in each quadrant. The percentages of mature CD4 and CD8 SP thymocytes were determined by analyzing the CD4 and CD8 expression on TCR{beta} high cells.

 
The increased TCRlow CD8SP population in Egr3TG thymuses could result from abnormal differentiation of TCRlow DP cells into CD8SP cells due to overexpression of Egr3. Alternatively, the CD8SP cells in Egr3TG mice could be CD8+CD4–CD3low ISP cells. To distinguish between these two possibilities, we examined CD8SP cells by analysis of CD3, CD24, and CD5 expression (Fig. 2). Mature CD8SP cells, such as those found in NLC mice, are CD3highCD24low, and the majority have high levels of CD5. The CD8SP cells in Egr3TG thymuses are mostly CD3lowCD24highCD5low, a phenotype characteristic of ISP cells. Thus, very few DP or mature SP cells are found in Egr3TG mice.



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  		FIGURE 2. The majority of CD8 SP thymocytes in Egr3TG mice are ISP cells. Representative analysis of CD3{epsilon}, CD24 (heat-stable Ag) and CD5 expression on Egr3TG CD8 SP thymocytes. Thymocytes were stained with anti-CD4 and anti-CD8 in combination with anti-CD3{epsilon}, anti-CD24, or anti-CD5. CD8 SP thymocytes were gated and analyzed for the expression of CD3{epsilon}, CD24, and CD5. The numbers in the histograms represent the percentages of CD8 SP thymocytes in each gated region. For comparison, CD24 expression in DP cells, which express intermediate levels of CD24 (med), is also shown. Note that CD24med DP cells partially overlap with CD24low and CD24high CD8 SP cells.

 
Impaired cell survival and reduced Bcl-xL expression in Egr3TG thymuses

The small number of DP thymocytes in Egr3TG mice could be the result of poor cell proliferation or survival. However, DP cells in Egr3TG mice exhibited increased cell proliferation compared with those in control mice, as examined by BrdU incorporation (14). To determine whether Egr3TG thymocytes have poor survival, we first performed ex vivo apoptosis analysis on freshly isolated thymocytes using annexin V and PI staining. Although only 8% of thymocytes from control mice are undergoing either early or late apoptosis, the number increases to 29% in Egr3TG thymocytes (Fig. 3A). In addition, the majority (65%) of the apoptotic cells are DP cells (Fig. 3B). We also examined thymocyte survival by culturing thymocytes from Egr3TG and control mice and measuring the percentage of live cells remaining at various times over 24 h. Egr3TG thymocytes displayed a faster rate of death compared with control thymocytes (Fig. 3C). We previously reported that Egr3TG thymocytes had reduced p27kip1 levels and increased cyclin-dependent kinase 2 (CDK2) activity (14). High CDK2 activity is associated with entry into the cell cycle, but it has also been shown to contribute to apoptosis (21). To investigate whether high CDK2 activity in Egr3TG thymocytes was associated with increased cell death, we cultured Egr3TG thymocytes in the presence of a CDK2 inhibitor, roscovitine. The results show that roscovitine partially rescues Egr3TG thymocytes from spontaneous apoptosis (Fig. 3C). Thus, the increased cell death of Egr3TG thymocytes is due at least in part to high CDK2 activity.



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  		FIGURE 3. Impaired survival of Egr3TG thymocytes. A, Annexin V and PI staining of freshly isolated thymocytes from age-matched Egr3TG and NLC mice. The numbers represent the percentage of cells in each quadrant. B, Freshly isolated thymocytes were stained with anti-CD4 and anti-CD8, followed by staining with annexin V. CD4 and CD8 expression of apoptotic cells (annexin Vhigh) is shown. The numbers represent the percentage of DP cells among annexin Vhigh cells. C, Thymocytes from age-matched Egr3TG and NLC mice were cultured alone, with 10 µM roscovitine, or with 0.1 µM dexamethasone for the indicated times. Thymocyte viability was analyzed by staining the thymocytes with annexin V and PI. The percent viability represents the percentage of live cells (negative for annexin V and PI). The data represent three independent experiments. D, Reduced Bcl-xL expression of Egr3TG thymocytes. The expression of Bcl-xL as well as other apoptosis-related proteins in total and DP thymocytes was analyzed by immunoblot. The data are representative of four experiments. E, Flow cytometric analysis of Bcl-xL expression in Egr3TG thymocytes. Thymocytes were stained with anti-CD4 and anti-CD8, followed by intracellular staining with anti-Bcl-xL or control Ab. The histograms display Bcl-xL expression in total and DP thymocytes.

 
The survival of DP thymocytes is regulated by the overall ratio of anti- and proapoptotic Bcl-2 family members (22, 23). We examined the expression of several Bcl-2 family members in Egr3TG thymocytes by Western blot analysis (Fig. 3D). We found that Bcl-xL expression, which is expressed mostly in DP cells in NLC mice, is profoundly reduced in Egr3TG thymocytes and DP cells purified from Egr3TG mice. Similar results were obtained by flow cytometric analysis of Bcl-xL expression (Fig. 3E). The expression of Bcl-2, another antiapoptotic protein in thymocytes, is normal in Egr3TG thymocytes, as determined by flow cytometry (data not shown). There is no apparent difference in the expression of several proapoptotic Bcl-2 family members such as BIM, BAX, and BAD between Egr3TG and NLC thymocytes. Because Egr3 has been shown to be a positive regulator of FasL expression (24) and the Fas-FasL interaction can induce cell death in thymocytes, we examined the expression of FasL in Egr3TG thymocytes. We found that FasL expression in Egr3TG thymocytes is comparable to that in control thymocytes (Fig. 3D). The above data suggest that transgenic expression of Egr3 leads to impaired survival of DP thymocytes resulting from high CDK2 activity and reduced Bcl-xL expression.

Defective TCR{alpha} expression in Egr3TG thymocytes

Egr3TG mice have reduced numbers of mature CD4 and CD8 SP thymocytes (Fig. 1) and reduced expression of CD5 and CD69 on DP thymocytes (data not shown). An explanation that is compatible with these observations is that Egr3TG mice have poor expression of TCR{alpha}{beta}. We tested this possibility by looking at the expression of the TCR{alpha} and -{beta} proteins by immunoblot (Fig. 4A). The expressions of TCR{beta} and CD3{epsilon} are normal in Egr3TG thymocytes, whereas the amount of TCR{alpha} protein in Egr3TG thymocytes is only ~20% that in NLC thymocytes. Thus, the Egr3 transgene results in a specific defect in the expression of TCR{alpha} protein. To rule out the possibility that the reduced TCR{alpha} expression in unfractionated thymocytes could simply reflect the low numbers of DP cells in Egr3TG thymuses, we sorted DP thymocytes from Egr3TG and NLC mice and examined TCR{alpha} expression by immunoblot (Fig. 4B). Similar to unfractionated thymocytes, the amount of TCR{alpha} protein is low in Egr3TG DP cells. We also examined the steady state level of TCR{alpha} transcripts in DP thymocytes from Egr3TG mice by Northern blot analysis and found that Egr3TG DP thymocytes have reduced TCR{alpha} mRNA compared with control thymocytes (data not shown).



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  		FIGURE 4. Reduced TCR{alpha} expression in Egr3TG thymocytes. The expression of TCR{alpha}, TCR{beta}, and CD3{epsilon} in total (A) and purified DP (B) thymocytes of Egr3TG and NLC mice was analyzed by immunoblot. The data are representative of four experiments.

 
The low numbers of mature T cells in Egr3TG mice and the low expression of CD5 and CD69 are therefore probably the result of low TCR expression. In support of this, Egr3TG mice bred to an I-Ek-restricted TCR transgenic mouse (3.L2tg) (25) have restoration of TCR expression to normal levels and markedly increased expression of CD5 (Fig. 5). In Egr3TG/3.L2tg mice, there are a normal percentage of mature T cells, but the cellularity of the thymus remains small (similar to Egr3TG mice). Thus, the introduction of a rearranged transgene alleviates the defect in TCR{alpha} expression caused by transgenic Egr3, but not the poor survival of DP thymocytes. This suggests that the defect in TCR{alpha} expression in Egr3TG mice may be due to inefficient TCR{alpha} rearrangement, and that poor TCR{alpha} rearrangement could be a consequence of the impaired DP survival in Egr3TG mice.



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  		FIGURE 5. A TCR transgene restores TCR and CD5 expression in Egr3TG mice. Egr3TG, 3.L2tg, or Egr3TG/3.L2tg double-transgenic mice were stained for the TCR{beta}-chain and CD5. The histograms display staining on total thymocytes. The data displayed are representative of four separate comparisons.

 
One way that transgenic Egr3 could regulate TCR{alpha} rearrangements is by controlling expression of the rag1 and rag2 genes. To test this possibility, we examined rag1 and rag2 expression by semiquantitative RT-PCR using sorted DP thymocytes from Egr3TG and NLC. The expression of rag1 and rag2 in DP thymocytes was reduced substantially in Egr3TG mice compared with NLC DP thymocytes (Fig. 6A). Thus, transgenic Egr3 expression results in low rag gene expression in DP thymocytes. The rag gene expression in normal mice is reduced after pre-TCR signaling, so that the cells can go through several rounds of cell division without the detrimental effects of recombination during DNA synthesis (26). For initiation of TCR{alpha} rearrangements, the rag genes are expressed again at the DP stage after the cells stop proliferating. Thus, low rag gene expression in Egr3TG mice could be due to rapid death of DP cells after they have stopped proliferating, rather than to direct control of rag gene transcription by Egr3.



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  		FIGURE 6. Impaired rag gene expression and restricted 3' TCR J{alpha} and 5' TCR V{alpha} usage in Egr3TG mice. A, The expressions of rag1 and rag2 were determined by semiquantitative RT-PCR using RNA isolated from DP thymocytes of two pairs of Egr3TG and NLC mice. PCR was performed with serially (1/5) diluted cDNA samples. PCR products were analyzed by Southern blotting with rag1 and rag2 oligonucleotide probes. RT-PCR of HPRT was used to normalize the input RNA. B, Profiles of J{alpha} transcripts in thymuses and spleens of three pairs of age-matched Egr3TG and NLC mice. RT-PCR was performed with primers specific for V{alpha}3 and C{alpha} or V{alpha}6 and C{alpha} using RNA from purified DP thymocytes and splenocytes. PCR products were probed sequentially with specific J{alpha} oligonucleotide probes as indicated. Probing with an internal C{alpha} probe was used to normalize the input RNA. C, TCR V{alpha} usage in DP thymocytes from two pairs of age-matched Egr3TG and NLC mice was analyzed by semiquantitative RT-PCR using primers specific for V{alpha}19 and C{alpha} or V{alpha}3 and C{alpha}. PCR was performed with serially (1/5) diluted cDNA samples. PCR products were probed with an internal C{alpha} oligonucleotide probe. RT-PCR of HPRT was used to normalize the input RNA.

 
If this is true, then the successful TCR{alpha}-chain rearrangements that are detected in Egr3TG mice should have a distinctive pattern. It has been shown that TCR{alpha} rearrangements proceed in a bidirectional way in which J{alpha} rearrangements proceed 5' to 3', whereas V{alpha} rearrangements proceed 3' to 5' (17, 27). The rearrangements of shorter distances are attempted first (the most 3' V{alpha}s rearranging to the most 5' J{alpha}s), progressing to more and more distant V{alpha} to J{alpha} combinations. Therefore, if transgenic Egr3 expression is causing death of DP thymocytes shortly after they begin TCR{alpha} rearrangements, then a bias toward usage of the 3' V{alpha} gene segments and 5' J{alpha} gene segments should be observed. To test this possibility, we compared J{alpha} usage between Egr3TG and NLC mice using RNA from DP thymocytes and total splenocytes with a PCR-based method previously described (16). RT-PCR was performed with a primer specific for either the V{alpha}3 or V{alpha}6 family in conjunction with a C{alpha} primer. J{alpha} usage was analyzed by sequentially probing the RT-PCR products with a series of oligonucleotide probes, each representing a specific J{alpha} segment ranging from the most 5' to the most 3' J{alpha}. Comparison of J{alpha} profiles between Egr3TG and NLC mice revealed that the J{alpha} usage in Egr3TG mice is skewed toward the 5' J{alpha} segments (Fig. 6B). The J{alpha} segments from 58 to 45 were over-represented, whereas the J{alpha} segments from 34 to 2 were under-represented in Egr3TG mice compared with NLC mice. The J{alpha} segments from 44 to 35 were similarly represented between Egr3TG and control mice. Thus, the bias toward usage of only the most 5' J{alpha} gene segments in Egr3TG mice is consistent with the death of DP cells shortly after the initiation of TCR{alpha} rearrangement.

A shortened life span of Egr3TG DP thymocytes should also bias the usage of the V{alpha} gene segments toward the 3' end of the locus (17). To verify this prediction, we performed semiquantitative RT-PCR using a primer specific for either V{alpha}19 (the very 5' end of V{alpha} locus) or V{alpha}3 (the very 3' end of V{alpha} locus) along with a C{alpha} primer. The PCR products were probed with an internal C{alpha} oligonucleotide probe. Whereas the V{alpha}3 transcripts were equally present in both Egr3TG and NLC, the V{alpha}19 transcript was not detected in Egr3TG thymuses (Fig. 6C). These results indicate that constitutive overexpression of Egr3 results in rapid death of DP thymocytes after the initiation of TCR{alpha} rearrangements, resulting in reduced expression of the TCR{alpha}-chain and a limited repertoire of TCR{alpha} rearrangements.

Egr3 reduces expression of ROR{gamma}t

In an effort to understand how transgenic expression of Egr3 was reducing the survival of DP thymocytes, we considered that the thymic phenotype of Egr3TG mice is very similar to that of ROR{gamma}-deficient mice. Both strains of mice have reduced thymic cellularity, similar distribution of thymic subsets, low Bcl-xL expression, high CDK2 activity, low p27kip expression, poor survival of DP thymocytes, and impaired TCR{alpha} rearrangements (14, 15, 17, 28). It has been shown previously that in a T cell hybridoma, ectopic expression of ROR{gamma}t (ROR{gamma}t is the thymic isoform of ROR{gamma}) has no effect on endogenous Egr3 expression (19). These data suggest that either Egr3 and ROR{gamma}t act in parallel, or Egr3 acts upstream of ROR{gamma}t and inhibits its expression. In the thymus, ROR{gamma}t is expressed at the highest level in DP cells, but it is also expressed at low levels in DN cells and is induced by pre-TCR signals (29). We examined the expression of ROR{gamma}t in Egr3TG thymocytes by Northern blot analysis using RNA from either unfractionated thymocytes or sorted DP cells. The data show that ROR{gamma}t expression is substantially reduced in Egr3TG thymuses (Fig. 7A). These results suggest that Egr3 is an upstream regulator of ROR{gamma}t and that overexpression of Egr3 inhibits ROR{gamma}t expression.



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  		FIGURE 7. Egr3 reduces ROR{gamma}t expression in thymocytes and in a DP cell line. A, ROR{gamma}t expression was analyzed by Northern blotting with total RNA isolated from total or DP thymocytes from age-matched Egr3TG and NLC mice. The blots were probed with a full-length cDNA of the thymic isoform of ROR{gamma} (ROR{gamma}t). The blots were reprobed with a GAPDH probe to normalize the RNA loading. The three different sizes of ROR{gamma}t transcripts are made by alternative polyadenylation (35 ). B, The DP cell line 16610D9 was cotransfected with pROR{gamma}t-Luc and increasing amounts of an Egr3 expression vector. As a control, the pGL3-CMV luciferase vector (that uses the CMV promoter) was cotransfected with increasing amounts of the Egr3 expression vector. The total amount of transfected DNA was kept constant. The pGL3-basic vector shows the luciferase activity in the absence of a promoter. Luciferase activities were measured 48 h post-transfection. The relative luciferase activities shown are the mean ± SD of triplicate determinations and are representative of four independent experiments. C, ROR{gamma}t expression was analyzed by Northern blotting with total RNA isolated from age-matched Egr3+/+ and Egr3–/– thymocytes.

 
We also cloned the 1.4-kb sequence upstream of the first ROR{gamma}t exon and placed it in front of the luciferase gene. We found that this sequence could function as a good promoter and drive the expression of luciferase in the DP thymocyte cell line 16610D9. Cotransfection of increasing amounts of an Egr3 expression plasmid resulted in a decrease in ROR{gamma}t promoter activity (Fig. 7B). In contrast, cotransfection of Egr3 had little effect on the activity of the CMV promoter used to drive luciferase expression in a control plasmid (Fig. 7B). We conclude that Egr3 can reduce ROR{gamma}t expression by inhibiting the activity of the ROR{gamma}t promoter.

This conclusion is supported by the observation that the expression of ROR{gamma}t is elevated in thymocytes from mice that lack Egr3. RNA was isolated from Egr3+/+ and Egr3–/– thymocytes, and a Northern blot probed for ROR{gamma}t. Fig. 7C shows that ROR{gamma}t mRNA levels are elevated in thymocytes from Egr3–/– mice. Levels of Bcl-xL mRNA were also elevated in Egr3–/– thymocytes (data not shown).

The transient nature of Egr3 expression in response to pre-TCR signaling is essential for normal development of thymocytes

During the transition from the DN to the DP stage in normal mice, both Egr3 and ROR{gamma}t are induced by pre-TCR signaling, and both proteins seem to be required for normal progression from DN to DP. Thus, it is not clear why overexpression of Egr3 results in low levels of ROR{gamma}t expression and impedes the survival of DP cells. A possible explanation is that in normal mice, Egr3 and ROR{gamma}t could each have distinct time courses for expression. To test this hypothesis, we performed time-course analysis of the expression of Egr3, ROR{gamma}t, and Bcl-xL induced by pre-TCR stimulation. This was done by injecting rag1-deficient mice with anti-CD3{epsilon} Ab, which mimics the pre-TCR stimulation (30). As shown in Fig. 8A, Egr3 expression was rapidly increased after stimulation and remained high at 6 h, followed by a rapid decrease. At 3 days, Egr3 expression decreased to the basal level. The expression of both ROR{gamma}t and Bcl-xL started to increase at 36 h and reached a maximum 4 days after stimulation. During this period, Egr3 expression remained at a low level. The above results indicate that in normal mice, Egr3 expression decreases to the basal level well before ROR{gamma}t and Bcl-xL are induced by pre-TCR signals. Therefore, we conclude that Egr3 expression (which promotes proliferation during the DN to DP transition) must dissipate for the expression of ROR{gamma}t and Bcl-xL to increase. Sustained expression of Egr3, as in Egr3TG thymuses, results in inhibition of ROR{gamma}t and Bcl-xL expression and thus is detrimental to thymocyte survival.



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  		FIGURE 8. Time-course analysis of Egr3, ROR{gamma}t, and Bcl-xL expression in response to pre-TCR stimulation. A, RAG1-deficient mice were injected i.p. with 100 µg of anti-CD3{epsilon} Ab. Total RNA was isolated from the thymuses at various times as indicated and analyzed by RT-PCR for the expression of Egr3, ROR{gamma}t, and Bcl-xL. RT-PCR of HPRT was used to normalize the input RNA. B, Schematic representation of temporal regulation of Egr3, ROR{gamma}t, and Bcl-xL in response to pre-TCR stimulation. Egr3 is transiently induced by pre-TCR signaling in thymocytes and is down-regulated preceding the expression of ROR{gamma}t and Bcl-xL.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previously we had shown that Egr3 promotes proliferation in response to pre-TCR signals, and we have now shown that sustained, high level expression of Egr3 leads to low levels of ROR{gamma}t and Bcl-xL and poor survival of DP thymocytes. Taken together, these results demonstrate that a single transcription factor that is induced by pre-TCR signals can promote one consequence of pre-TCR signaling (proliferation) yet be inhibitory to another (DP survival). The fact that proliferation and survival are regulated in an opposing fashion by Egr3 results in a temporal distinction between proliferation and survival in response to pre-TCR signals. Egr3 induces proliferation, but long term survival of proliferating cells will not occur unless at least one component of the proliferative signal subsides. In support of this, in rag1-deficient mice injected with anti-CD3 antibody, the prosurvival factors ROR{gamma}t and Bcl-xL are not highly expressed until near the end of the proliferative burst that follows pre-TCR signaling, well after Egr3 levels have dissipated.

This scenario provides a possible mechanism by which an unregulated proliferative signal induced by Egr3 would be inhibited from promoting neoplasia because its sustained expression does not favor survival of the cellular progeny. Indeed, we have not observed the overt thymic lymphomas in young Egr3TG mice that may be expected of mice that have a high level of proliferation after pre-TCR signals. For example, in a line of mice that expresses a transgene encoding a constitutively active form of Lck, all the progeny get thymic lymphomas by 6 wk of age (31). We have not yet observed any lymphomas in Egr3TG mice and have followed a few mice out to ~14 wk of age. This does not mean that the promotion of thymic lymphomas by Egr3 is not possible, but that it may take somewhat longer and be a more rare event than in the lck transgenic mice. The ability of Egr3 to inhibit the survival of DP thymocytes is likely to significantly inhibit lymphomagenesis, but may not prevent it entirely. In support of this view, ROR{gamma}-deficient mice, which have a nearly identical thymic phenotype compared with Egr3TG mice, have a high incidence of thymic lymphoma in older mice (32).

Our data also demonstrate that the life span of DP thymocytes is critical for normal T cell development. The shortened life span of DP thymocytes that we have observed is detrimental to T cell development primarily because of the negative effects that a short life span has on TCR{alpha} rearrangements. Very few mature T cells were found in Egr3TG mice, but the introduction of an already rearranged transgenic TCR restored the production of mature T cells. The survival window of DP thymocytes seems to be linked to the time it takes for multiple rearrangements to occur at the TCR{alpha} locus. Thus, the short life span of DP cells in Egr3TG mice results in usage of only the most 5' J{alpha} gene segments. To be rescued from cell death, the DP cells in these mice probably only have one chance to produce a functional TCR{alpha}-chain that pairs with the TCR{beta} in that cell and recognizes self peptide/MHC with the appropriate affinity. In normal mice, it is likely that DP cells will attempt multiple, sequential rearrangements at the TCR{alpha} locus during the DP life span (4, 5). The low TCR{alpha} protein expression in Egr3TG mice is most likely due to the fact that the majority of the V{alpha}-J{alpha} rearrangements that occur on the first try are nonfunctional, and the ones that do produce a functional TCR have a low probability of inducing positive selection. Thus, a short DP life span results in low TCR{alpha} protein expression and a low number of thymocytes that make it through positive selection.

Our results also demonstrate a previously unrecognized relationship between Egr3 and ROR{gamma}t. The strikingly similar phenotypes of Egr3TG mice and ROR{gamma}-deficient mice suggest that these two molecules influence the same downstream targets. This could be because Egr3 acts in parallel with ROR{gamma}t and turns off genes that are turned on by ROR{gamma}t and/or vice versa. However, the data are probably better explained by a model in which Egr3 inhibits the expression of ROR{gamma}t. The following data support this interpretation: 1) in response to pre-TCR signals, Egr3 expression is elevated and returns to basal levels before ROR{gamma}t expression is induced; 2) Egr3TG mice have reduced amounts of ROR{gamma}t mRNA; 3) a previous study reported that ectopic expression of ROR{gamma}t in a T cell hybridoma did not have an effect on Egr3 expression (19); and 4) Egr3 inhibits ROR{gamma}t promoter activity in a DP cell line.

How does Egr3 inhibit ROR{gamma}t expression? We are currently evaluating the ROR{gamma}t promoter in an effort to understand how Egr3 can reduce its function. One model is that Egr3 promotes the expression of Id genes that act as negative regulators of E proteins. It has been previously demonstrated that Egr1 activates Id3 transcription (12, 33), and it is possible that Egr3 can also induce Id3. The Id3 gene product is able to form a heterodimer with E proteins and block their ability to bind DNA and activate transcription. Although the role of E proteins in activation of ROR{gamma}t transcription is not currently known, we have identified two potential high affinity E protein binding sites in the ROR{gamma}t promoter. Thus, it is possible that Egr3 inhibits ROR{gamma}t by inducing Id gene expression. A similar scenario could be proposed for the reduced rag gene expression that we have observed in Egr3TG mice. The expression of rag genes is thought to be regulated by E proteins (34), and if Egr3 is able to induce Id3, this could also explain the low expression of rag1 and rag2 in Egr3TG mice. In support of these ideas, we have observed elevated levels of Id3 mRNA in Egr3TG thymocytes (data not shown).

Although this model is attractive, our observations cannot rule out the possibility that Egr3 acts in opposition to ROR{gamma}t on common target genes. A potentially critical target gene for Egr3 and ROR{gamma}t is Bcl-xL. Bcl-xL expression is low in both ROR{gamma}-deficient mice and Egr3TG mice (15). The poor survival of DP thymocytes in ROR{gamma}-deficient mice can be rectified by breeding the ROR{gamma}-deficient mice to Bcl-xL transgenic mice (15). Thus, Egr3 and ROR{gamma}t probably both regulate DP thymocyte survival at least in part by controlling the expression of Bcl-xL, either directly or indirectly.


   Acknowledgments
 
We thank Aron Lukacher for critical review of the manuscript, and Steve Hedrick for the 16610D9 cell line.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI48784 and a grant from the university research committee of Emory University. Back

2 Address correspondence and reprint requests to Dr. Gilbert J. Kersh, Department of Pathology and Laboratory Medicine, Emory University, 101 Woodruff Circle, Room 7311 Woodruff Memorial Building, Atlanta, GA 30322. E-mail address: gkersh{at}emory.edu Back

3 Abbreviations used in this paper: DN, double negative; CDK2, cyclin-dependent kinase 2; DP, double positive; Egr, early growth response gene; Egr3TG, Egr3 transgenic; HPRT, hypoxanthine phosphoribosyltransferase; ISP, immature single positive; Luc, luciferase; NLC, non-TG littermate control; PI, propidium iodide; ROR{gamma}, retinoic acid receptor-related orphan receptor {gamma}; ROR{gamma}t, thymic isoform of ROR{gamma}; SP, single positive; TG, transgenic. Back

Received for publication January 16, 2004. Accepted for publication April 30, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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