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The Journal of Immunology, 2002, 169: 1713-1720.
Copyright © 2002 by The American Association of Immunologists

Thymocyte Development in Early Growth Response Gene 1-Deficient Mice1

Matthew Bettini*, Hongkang Xi*, Jeffrey Milbrandt{dagger} and Gilbert J. Kersh2,*

* Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322; and {dagger} Departments of Pathology and Immunology and Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Early growth response gene 1 (Egr1) codes for a transcriptional regulator that contains a zinc-finger DNA binding domain. Egr1 expression is induced by a variety of extracellular stimuli including TCR-ligand interactions. Its pattern of expression in the thymus and dependence on ERK activation have led to speculation that it has a role in T cell development, but the exact nature of this role has been undefined. To more clearly define the role of Egr1 in thymocyte development, we have analyzed thymocytes from Egr1-deficient mice. We find that thymuses from Egr1-deficient mice contain twice as many cells as age-matched controls, and the increase in thymocyte number is apparent at the early CD4/CD8 double negative stage of development. Subsequent maturation to the CD4/CD8 double positive stage and survival of the double positive cells both appear normal in Egr1-deficient animals. We also find that Egr1 promotes positive selection of both CD4 and CD8 single positive cells without playing a major role in negative selection. Egr1 influences positive selection by enhancing expression of the helix-loop-helix inhibitor Id3 and the anti-apoptosis molecule bcl-2. Thus, Egr1 translates developmental signals into appropriate changes in gene expression at multiple stages of thymocyte development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T cell development is controlled at multiple stages by signals emanating from cell surface receptors. In CD4, CD8 double positive (DP)3 thymocytes, strong signals through the TCR (long TCR-ligand half-life) induce death, whereas weak signals (short TCR-ligand half-life) induce survival and differentiation to mature T cells (1). Integration of multiple signaling pathways in response to the quality and quantity of ligand binding determines whether the developing DP thymocyte undergoes positive or negative selection (2). It has been established that strong signals through the TCR result in activation of the mitogen-activated protein kinases (MAPKs), extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 and lead to negative selection, whereas weak signals stimulate only ERK activation and lead to positive selection (3, 4, 5). The phenomenon of positive selection is thought to include both survival and differentiation into either the CD4 or CD8 lineage. Therefore, changes in gene expression in response to weak TCR signals and ERK activation is an expected feature of positive selection. However, the transcription factors that translate ERK activation into changes in gene expression relevant to positive selection have not been described.

Early growth response gene 1 (Egr1) is a transcriptional regulator that contains a zinc-finger DNA binding domain. Egr1 is expressed in many different cell types and its expression is rapidly increased in response to a variety of extracellular stimuli (6, 7). Although Egr1 is expressed in many cell types and induced by different stimuli, it exerts cell type-specific effects based on its ability to transactivate a variety of genes in conjunction with cell type-specific factors. Egr1 is expressed in T cells and thymocytes and its expression is rapidly elevated in response to TCR signaling (8, 9). Furthermore, expression of Egr1 can be blocked by a MAPK/ERK kinase (MEK)-1 inhibitor, demonstrating that Egr1 is an ERK-dependent gene in T cells and thymocytes (8, 9, 10). Egr1 expression can also be induced by incubation of T cells with syngeneic APC presenting either an antagonist peptide or endogenous peptides, suggesting that weak TCR signals can induce Egr1 expression (M. Bettini and G. J. Kersh, manuscript in preparation). These results have led to the hypothesis that Egr1 may play a role in translating TCR-mediated ERK activation in DP thymocytes into positive and/or negative selection.

This hypothesis was supported by a study demonstrating that transgenic (tg) overexpression of Egr1 in the thymus led to positive selection of TCR tg T cells on backgrounds that were normally nonselecting (11). The conclusion was that tg Egr1 expression could alter the threshold of signaling required for positive selection. However, the precise role played by normal expression of Egr1 has been difficult to determine. In a more recent study, it was reported that in an in vitro model of CD8+ T cell development that uses an Ab against CD3, positive selection can proceed normally when Egr1 expression is blocked by a MEK-1 inhibitor (10). Thus, a definitive role for Egr1 in thymocyte development has not been determined.

We have sought to more precisely define the role of Egr1 in thymocyte development by a careful examination of thymocytes from Egr1-deficient mice. Mice that have a targeted disruption in the Egr1 gene have been produced and reported previously, and growth and differentiation in these mice is largely unperturbed (12). The most striking defect seen in these mice is infertility in the female mice due to an inability to produce luteinizing hormone (13). The only reported analysis of T cell development and function in Egr1-deficient mice showed that thymic architecture was normal and that peripheral T cells proliferate normally to anti-CD3 stimulation (12). In the current study, we have undertaken a more detailed examination of thymocyte development in Egr1-deficient mice using a normal TCR background and three different TCR tg backgrounds. Our studies demonstrate that Egr1 is a negative regulator of the size of the thymus. In addition, we have found that Egr1 promotes positive selection of both CD4+ and CD8+ thymocytes, whereas it seems to have a minimal role in regulating negative selection. Finally, we demonstrate a role for Egr1 in enhancing the expression of Id3 and bcl-2, two molecules that are expected to contribute to positive selection.


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

Egr1-deficient mice were generated as described and backcrossed to the C57BL/6 background >7 generations (12). The targeted Egr1 allele was then crossed two generations to B6.AKR and screened for the H-2k/k genotype to generate Egr1-deficient mice on the B6.AKR background. 3.L2tg mice were generated as described and maintained on a B6.AKR background (14). 3A9 TCR tg mice on the B6.AKR background were given to us by P. M. Allen (Washington University, St. Louis, MO), and OT-1 TCR tg mice were given to us by J. Kapp (Emory University, Atlanta, GA). CBA/J mice were purchased from the National Cancer Institute (Frederick, MD), and B6.AKR mice were purchased from The Jackson Laboratory (Bar Harbor, ME).

Flow cytometry

The following Abs used in this study were purchased from BD PharMingen (San Diego, CA): anti-CD44-biotin (IM7), anti-CD25-FITC (7D4), anti-CD4-PE (GK1.5), anti-CD5-FITC (53-7.3), anti-CD69-biotin (H1.2F3), anti-TCR-{beta}-biotin (H57), anti-V{beta}8.1,8.2-FITC (MR5-2), anti-V{alpha}2-biotin (B20.1), anti-CD24-FITC (J11d), anti-V{beta}5.1, 5.2 (MR9-4), and anti-bcl-2-FITC (3F11). The following reagents were purchased from Caltag Laboratories (Burlingame, CA): anti-CD8-TC (CD8a), streptavidin-TC, and streptavidin-FITC. The Ab against the 3.L2 TCR clonotype (Cab) was prepared as described (14). Thymocytes were surface stained using a buffer consisting of PBS, 0.02% sodium azide, and 0.5% BSA. Intracellular staining for bcl-2 was done using the cell permeabilization kit from Caltag Laboratories. Stained cells were analyzed using a BD Biosciences FACSort and CellQuest software (BD Biosciences, Mountain View, CA).

Bone marrow chimera

Bone marrow cells were isolated from the femurs of donor mice and washed in HBSS. Two to three million cells were then injected i.v. into recipients that had been gamma irradiated with 1060 rad 2 h before the time of injection. Six weeks later, thymocytes were analyzed by flow cytometry. Donor thymocytes were recognized by expression of the 3.L2 TCR.

Thymocyte stimulation

Single-cell suspensions of thymocytes were washed in RPMI and 10% FCS, and followed by incubation with anti-CD8-coated beads (Dynal Biotech, Great Neck, NY). CD8+ cells were collected with a magnet, followed by release of the beads with DNase according to the manufacturer’s protocol. This procedure resulted in thymocytes that were 90–95% CD4, CD8 DP, and 5–10% CD8 single positive (SP). These cells (10 x 106 per well) were then lightly centrifuged onto the wells of a six-well plate containing media only, or media plus a previous coating with anti-CD3 Ab (145-2C11; BD PharMingen). After 1 h of stimulation, the cells were harvested and RNA was isolated using TRIzol reagent (Invitrogen, San Diego, CA). A total of 4–6 µg of total RNA per lane were run on a formaldehyde/agarose gel. After transfer to a nylon membrane, blots were hybridized with the following 32P-labeled probes. The Id3 probe was a 200-bp fragment from the Id3 cDNA (9). This fragment was isolated from thymocyte RNA by RT-PCR using the following primers: 5'-CGCACTGTTTGCTGCTTTAGG-3' and 5'-GTAGCAGTGGTTCATGTCGTC-3'. The {beta}-actin probe was a 185-bp fragment isolated from thymocyte RNA by RT-PCR using the following primers: 5'-TGTTACCAACTGGGACGACA-3' and 5'-GGATGGCTACGTACATGGCT-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Egr1 regulates the size of the thymus

Our initial analysis of thymocyte development in Egr1-deficient mice showed that the percentages of CD4, CD8 double negative (DN) and DP cells in Egr1-/- mice were both similar to Egr1+/+ mice, but that the thymuses from Egr1-deficient mice were consistently larger than age-matched control thymuses. On average, the number of thymocytes in non-TCR tg Egr1-deficient thymuses was 1.75 ± 0.27 times the number in age- and sex-matched controls (Fig. 1Go). We have bred three different TCR tg lines onto the Egr1-deficient background, and also observed a similar increase in thymic cellularity. The 3A9 TCR tg, Egr1-deficient thymuses had a slightly larger increase than the other three backgrounds (2.6 ± 0.15). Overall, the Egr1-deficient thymuses were 1.99 ± 0.18 times as large as their age- and sex-matched Egr1+/+ controls (n = 16).



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  		FIGURE 1. Egr1-deficient mice have large thymuses, but a normal DN thymocyte phenotype. A, Single-cell suspensions of thymocytes were isolated from pairs of age- and sex-matched Egr1+/+ and Egr1-/- mice. Cells were counted and the ratio of the number of cells in the deficient thymus to the number of cells in the age-matched Egr1+/+ thymus was calculated. •, Ratio of a given pair of animals. The horizontal bars indicate the mean ratio for the different backgrounds. The mean values for the different backgrounds (± SEM) were as follows: B6.AKR, 1.75 ± 0.27; 3A9tg, 2.6 ± 0.27; 3.L2tg, 1.93 ± 0.3; OT-1, 1.57. Mice were analyzed between 5 and 15 wk of age. B, Thymocytes from Egr1+/+ and Egr1-/- mice on the B6.AKR background (left panels) or on the 3A9tg background (right panels) were stained for CD4, CD8, CD44, and CD25. The dot plots display expression of CD44 and CD25 on thymocytes that are negative for CD4 and CD8. To the lower right of each dot plot, the percentage of gated cells that fall into each quadrant is given.

 
The increase in the size of the Egr1-deficient thymuses could be due to an increase in differentiation from the DN stage to the DP stage, a developmental step that is accompanied by extensive proliferation. We have tested this possibility by staining DN thymocytes with Abs against CD44 and CD25. Cells enter the thymus as CD44+ and CD25-, and soon acquire expression of CD25 followed by loss of CD44 expression (15). The transition from CD44-CD25+ to CD44-CD25- is the step that requires expression of the pre-TCR and is coincident with extensive proliferation. Transition from CD25+ to CD25- is normal in Egr1-deficient mice in both the normal (B6.AKR) background and the 3A9 TCR tg background (Fig. 1GoB). The presence of the 3A9 transgene accelerates cells through this transition, but equally so in Egr1-/- and Egr1+/+ thymuses. This suggests that enhancement of the DN to DP transition is not responsible for the larger size of the Egr1-deficient thymuses. In support of this, analysis of thymocyte proliferation by 7-amino actinomycin D staining did not show a difference between Egr1-/- and Egr1+/+ thymuses (data not shown).

The increased cellularity of Egr1-deficient thymuses could be due to a failure of DP thymocytes to undergo apoptosis; however, we have found that DP thymocytes from Egr1-deficient mice undergo apoptosis at least as readily as DP thymocytes from Egr1+/+ mice. This is true in thymocytes either taken directly from the mouse, cultured overnight without stimulation, or cultured overnight in the presence of anti-CD3 Abs (data not shown). Therefore, the most likely explanation for the increased size of the Egr1-deficient thymuses is that there is an increased number of DN T cell precursors in the Egr1-deficient mice. A recent study has found that the number of DP cells generated in mice is proportional to the number of competent DN precursors present (16). The absolute number of DP thymocytes is increased almost 2-fold in Egr1-deficient mice, but the ratio of DP to DN cells remains the same because the number of DN cells is increased similarly. We have compared the number of DN thymocytes in 17 Egr1-/- mice to the number found in their age-matched Egr1+/+ counterparts, and found that there are 2.16 ± 0.16 times more DN cells in the Egr1-deficient mice. For example, three 9-wk-old 3A9tg, Egr1+/+ animals have an average of 7.48 ± 0.95 x 106 DN thymocytes compared with an average of 17.34 ± 1.51 x 106 DN thymocytes in three 9-wk-old 3A9tg, Egr1-/- animals. These increases in DN thymocyte number are similar to the increases in total thymocyte number observed in Egr1-/- mice. Therefore, we would argue that Egr1 plays a role in limiting the generation and/or survival of competent DN T cell precursors, or limits the entry of precursors into the thymus.

Egr1 regulates positive selection

Examination of CD4, CD8, and TCR-{beta} expression in Egr1-deficient thymuses revealed that although development of both CD4SP and CD8SP cells was not completely blocked, there was a reduction in the percentages of mature T cells in Egr1-deficient mice as compared with Egr1+/+ controls (Fig. 2Go). The percentage of CD4SP, TCRhigh cells is only 4.4% in Egr1-/- mice as compared with 7.3% in the Egr1+/+ littermate. Similarly, the percentage of CD8SP is reduced in the Egr1-/- thymus. Because of the increased number of thymocytes in the Egr1-deficient thymus, there is still a slight increase in the absolute number of CD4SP and CD8SP cells in these mice. However, the number of CD4 and CD8SP cells generated in the thymus has been found to be proportional to the number of DP precursors (16). Therefore, if positive selection were normal in the Egr1-deficient animal, the greater number of DP precursors should lead to a proportional increase in SP cells. The reduced percentage of mature cells in the Egr1-deficient thymus suggests that Egr1 plays a role in positive selection. Because positive selection is regulated by the avidity of the TCR-MHC/peptide interaction, defects in development of Egr1-deficient thymocytes could be somewhat compensated by selection of a unique TCR repertoire. To eliminate this variable, we have bred the Egr1-deficient mouse to three different TCR tg backgrounds, where selection of cells bearing a TCR of fixed specificity can be followed.



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  		FIGURE 2. Thymocyte development in Egr1-deficient mice on a B6.AKR background. Single-cell suspensions of thymocytes were isolated from a pair of age-matched Egr1+/+ and Egr1-/- mice and stained with Abs against CD4, CD8, and the {beta}-chain of the TCR. Left panels, CD4 and CD8 staining on total thymocytes, with the Egr1+/+ mouse shown in the upper panel and the Egr1-/- mouse in the lower panel. Middle panels, Staining for TCR-{beta} chain on total thymocytes. Right panels, A dot plot of CD4 and CD8 staining on cells that express high levels of the TCR. At the bottom right of the dot plots, the percentage of total thymocytes that fall into each quadrant is indicated.

 
To examine selection into the CD4 lineage, we used the 3A9 TCR tg mouse (3A9tg) and the 3.L2 TCR tg mouse (3.L2tg). The 3A9 TCR is specific for a peptide derived from hen egg lysozyme (48–61) presented by I-Ak (17). The 3.L2 TCR is specific for a peptide derived from the d allele of murine hemoglobin (Hb; 64–76) presented by I-Ek (14). Both tg lines are maintained on the B6.AKR background (H-2k), and in both cases, cells bearing the tg receptor are selected into the CD4 lineage. As displayed in Fig. 3Go, A and B, a comparison of selection of the cells bearing these tg receptors on the Egr1+/+ vs Egr1-/- backgrounds reveals that positive selection of cells bearing these specific class II MHC-restricted TCRs is significantly impaired in the absence of Egr1. In the 3A9tg mice on a normal background, CD4SP, TCRhigh cells make up 34.8% of the thymus, whereas these cells only constitute 9.7% of the thymus on the Egr1-/- background. Similarly, 3.L2 TCRhigh, CD4SP cells normally make up 6.1% of the thymus, but in the absence of Egr1, these cells only constitute 2.1% of thymocytes. These results demonstrate that Egr1 plays a significant role in positive selection of CD4SP cells.



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  		FIGURE 3. Thymocyte development in Egr1-deficient mice bred to three different TCR tg backgrounds. Egr1-deficient mice were bred to 3A9tg mice (A), 3.L2tg mice (B), and the OT-1 TCR tg (C). Single-cell suspensions of thymocytes were isolated from pairs of TCR transgene positive, age-matched Egr1+/+ and Egr1-/- mice, and stained with Abs against CD4, CD8, and the tg TCRs. The panels in A–C are all arranged in a similar manner. Left panels, CD4 and CD8 staining on total thymocytes, with the Egr1+/+ mouse shown in the upper panel and the Egr1-/- mouse in the lower panel. Middle panels, Staining for the tg TCR on total thymocytes. Right panels, A dot plot of CD4 and CD8 staining on cells that express high levels of the TCR. At the bottom right of the dot plots, the percentage of total thymocytes that fall into each quadrant is indicated. For each TCR tg background, three to seven age-matched pairs of mice were compared.

 
It has been shown that TCR-dependent induction of Egr1 is dependent on ERK activation (8, 10), and it has been suggested that higher levels of ERK activity favor the development of CD4SP cells at the expense of CD8SP cells (18). Furthermore, it has also been suggested that induction of Egr1 is not required for the development of CD8SP cells (10). To directly test the role of Egr1 in development of CD8SP cells, we bred the Egr1-deficient mice to the class I-restricted TCR tg mouse OT-1 (19). The OT-1 TCR is specific for an OVA peptide presented by Kb; and therefore, we compared selection of OT-1 CD8SP cells on the C57BL/6 background in the presence and absence of Egr1. The results show that Egr1 does play a significant role in the selection of OT-1 CD8SP cells (Fig. 3GoC). V{alpha}2high, CD8SP cells make up 10.9% of thymocytes on the Egr1+/+ background vs 5.4% on the Egr1-/- background. The effect on OT-1 CD8SP development is less dramatic than the effect we observed on CD4SP development in the two class II-restricted tgs, leaving open the possibility that CD4 development is more sensitive than CD8 development to alterations in ERK activity and Egr1 expression.

We have also used expression of CD24 (heat-stable Ag) to determine the percentage of cells undergoing positive selection in Egr1-/- and Egr1+/+ animals. CD24 is expressed at high levels on immature DP thymocytes, and after DP cells become CD4 or CD8SP, expression of CD24 decreases. Therefore, cells that are CD24low and CD4 or CD8SP have been positively selected. We have gated on thymocytes from 3A9tg, 3.L2tg, and OT-1 mice that are CD24low and displayed the CD4 and CD8 expression on these cells (Fig. 4GoA). In agreement with the analysis in Fig. 3Go that used TCR levels as an indicator of positive selection, CD24low, CD4SP cells are reduced from 11.8% in 3A9tg, Egr1+/+ mice to 3.0% in 3A9tg, Egr1-/- mice. Similarly, CD24low CD4SP cells are reduced from 9.0% in 3.L2tg, Egr1+/+ mice to 3.8% in 3.L2tg, Egr1-/- mice. In agreement with the analysis in Fig. 3Go, OT-1, Egr1-/- mice only had a ~2-fold reduction in CD24low, CD8SP cells (Fig. 4GoA).



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  		FIGURE 4. Inefficient production of mature SP cells in Egr1-deficient thymocytes, but normal expression of activation markers. A, Thymocytes from 3A9tg, 3.L2tg, and OT-1 mice on an Egr1+/+ or Egr1-/- background were isolated and stained for CD24, CD4, and CD8. The dot plots display staining for CD4 and CD8 after gating on CD24 low cells. The numbers on the dot plots indicate the percentage of total thymocytes that fall into each quadrant. B, left panels, CD5 and CD69 staining on thymocytes from Egr1+/+ (thin line) and Egr1-/- (thick line) mice on the B6.AKR background. Middle panels, Staining for CD5 on thymocytes from 3A9tg, Egr1+/+ thymocytes (upper panel) and 3A9tg, Egr1-/- thymocytes (lower panel). Right panels, Staining for CD4 and CD8 after gating on thymocytes that high levels of CD5 in the 3A9tg, Egr1+/+ (upper panel) and Egr1-/- (lower panel) mice. The numbers on the dot plots indicate the percentage of gated thymocytes that fall into each quadrant.

 
The failure of DP cells to efficiently undergo positive selection in the absence of Egr1 could be due to a defect in the responsiveness of the DP cells to TCR signaling in general, or due to a specific defect in differentiation in response to TCR signaling. We investigated these possibilities by staining for CD5 and CD69 on thymocytes from Egr1+/+ and Egr1-/- mice. We found that CD5 and CD69 expression are normal on Egr1-/- thymocytes (Fig. 4GoB). Because up-regulation of these two markers on thymocytes is dependent on TCR signaling (20, 21), this result shows that TCR signaling is functional in the Egr1-deficient thymocytes, but that events downstream of TCR signaling are inefficient. This is perhaps most strikingly shown in the panels of Fig. 4GoB that show CD4 and CD8 expression on CD5high cells in 3A9tg Egr1+/+ and Egr1-/- mice. Because of the expression of the tg TCR, 80–85% of the 3A9tg thymocytes have high levels of CD5, and this is not altered in the absence of Egr1. However, if we gate on the CD5high cells and look at their CD4 and CD8 expression, 55% of the CD5high cells are DP in the Egr1+/+ thymus, with ~45% differentiating to CD4SP. In contrast, 88% of CD5high cells in the Egr1-/- thymus are DP, and only ~11% are differentiating to CD4SP. Similar results were seen when we examined CD4 and CD8 expression on CD69high cells, and when thymocytes from the 3.L2 and OT-1 tg backgrounds were analyzed in the same manner (data not shown). These data demonstrate that the DP thymocytes in Egr1-deficient mice are receiving a TCR-mediated signal that is normal in some respects, but that differentiation and/or survival of differentiating cells is impaired when Egr1 is absent.

Negative selection is efficient in Egr1-deficient mice

ERK activation has been shown to be a specific regulator of positive selection in thymocytes, but the role of ERK activation in negative selection has been somewhat controversial (22, 23, 24, 25). Because Egr1 induction is dependent on ERK activation, it is of interest to define the role of Egr1 expression in negative selection. Therefore, we have analyzed negative selection in the Egr1-deficient mice by taking advantage of the fact that the agonist ligand for the 3.L2 TCR is an allelic variant of murine Hb (Hbbd allele). This TCR is positively selected on the B6.AKR background (Hbbs allele, H-2k), and negatively selected in the presence of Hbbd and I-Ek (26). We have used CBA/J mice as a negatively selecting background because they are H-2k and express the Hbbd allele of Hb.

The experimental approach was to isolate bone marrow from 3.L2tg, Egr1+/+ and Egr1-/- mice on the B6.AKR background and transfer it into lethally irradiated B6.AKR or CBA/J hosts. The resulting bone marrow chimeras were analyzed 6 wk after transfer, and Fig. 5GoA displays staining for the 3.L2 TCR on thymocytes for the four combinations that were tested. As expected, transfer of Egr1+/+ bone marrow into a B6.AKR recipient resulted in a phenotype that is very similar to typical 3.L2tg mice. A total of 15.5% of the thymocytes expressed high levels of the 3.L2 TCR, and of the total thymocytes, 6.1% were CD4SP and 3.L2 TCRhigh. In contrast, when Egr1-/- bone marrow was used as a donor for a B6.AKR recipient, only 5.6% of the thymocytes expressed high levels of the 3.L2 TCR, and 2.4% were CD4SP and 3.L2 TCRhigh. This reduction in positive selection of cells expressing the 3.L2 TCR was similar to that observed when the 3.L2tg mice were bred to the Egr1-deficient background, and indicates that the defect in positive selection in Egr1-/- mice is due to a lack of Egr1 expression in a bone marrow-derived cell, and not in the thymic stroma.



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  		FIGURE 5. Negative selection is not impaired in Egr1-deficient mice. A, Bone marrow from 3.L2tg, Egr1+/+ mice (left panels) or 3. L2tg, Egr1-/- (right panels) mice was transferred into lethally irradiated B6.AKR (upper panels) or CBA/J (lower panels) hosts. The histograms display staining for the 3.L2 TCR on thymocytes analyzed 6 wk after transfer. The CBA/J mice express the d allele of the Hb {beta}-chain as well as I-Ek; and therefore, have a ligand that prevents the development of cells expressing high levels of the 3.L2 TCR. B, Thymocytes were analyzed from Egr1+/+ and Egr1-/- mice on the C57BL/6 background and on the B6.AKR background. Both backgrounds express a superantigen that deletes V{beta}5-expressing cells in the presence of I-E, but only the B6.AKR background expresses an I-E molecule. Thymocytes were isolated and stained for CD4, CD8, and either V{beta}5 or V{beta}8.3. The data are representative of three separate comparisons.

 
When 3.L2tg, Egr1+/+ bone marrow was transferred into CBA/J recipients, negative selection was observed as expected (Fig. 5GoA). 3.L2 TCRhigh cells made up only 6.4% of thymocytes in this chimera, and of total thymocytes, only 0.9% were CD4SP and 3.L2 TCRhigh. When 3.L2tg, Egr1-/- cells were used as the donor for CBA/J recipients, negative selection was entirely functional, 3.L2 TCRhigh cells made up only 2.3% of the thymus, and CD4SP, 3.L2 TCRhigh cells made up only 0.04% of thymocytes. The data demonstrate that Egr1 is not required for negative selection of thymocytes in this model.

We have also examined a second model of negative selection in Egr1-deficient mice. In mice that express I-E and the endogenous provirus Mtv9, T cells expressing V{beta}5 undergo clonal deletion. Therefore, we have examined V{beta}5 expression on CD4SP thymocytes from Egr1+/+ and Egr1-/- mice on the C57BL/6 and B6.AKR backgrounds. Both backgrounds contain the Mtv9 provirus, but B6.AKR (H-2k/k) mice express I-Ek, whereas C57BL/6 (H-2b/b) mice do not express I-E. Accordingly, on the H-2b/b background, 4.3 and 5.5% of CD4SP thymocytes express V{beta}5 in Egr1+/+ and Egr1-/- mice, respectively (Fig. 5GoB). In contrast, V{beta}5 bearing cells are efficiently deleted from both Egr1+/+ and Egr1-/- CD4SP cells on the H-2k/k background. CD4SP cells from H-2k/k, Egr1+/+ mice are 0.4% V{beta}5 positive, and 0.9% V{beta}5 positive in H-2k/k, Egr1-/- mice. This deletion is specific for V{beta}5, as V{beta}8.3 expressing cells are not deleted (Fig. 5GoB). Thus, in this second model of negative selection, Egr1 does not play a major role.

Egr1 is a positive regulator of Id3 expression in thymocytes in vivo

To understand the mechanism by which Egr1 regulates positive selection of DP thymocytes, the target genes of Egr1 in these cells need to be identified. A strong candidate for an Egr1 target gene that could play a role in regulation of positive selection is the transcriptional regulator Id3. The Id3 gene product can act as a dominant negative inhibitor of the basic helix-loop-helix E proteins (the E2A gene products), and it has been shown that the two E2A gene products E12 and E47 both play a role in regulating T cell development (27, 28). Furthermore, induction of Id3 expression by TCR stimulation is dependent on ERK activation, and transfection of Egr1 into certain cell lines results in an increase in Id3 expression (9). In addition, Id3-deficient mice have a reduction in positive selection similar to Egr1-deficient mice (29). Therefore, we have evaluated the role of Egr1 in regulation of Id3 expression in DP thymocytes by stimulating DP cells from Egr1+/+ and Egr1-/- animals and assaying for Id3 gene expression by Northern blot. As shown in Fig. 6Go, stimulation of normal DP thymocytes with anti-CD3 for 1 h results in an 8-fold increase in Id3 mRNA. In contrast, stimulation of Egr1-deficient DP thymocytes results in a much more modest increase in Id3 mRNA levels (2-fold). This result establishes Egr1 as a positive regulator of TCR-dependent Id3 expression in vivo, and suggests that the positive selection defect seen in Egr1-/- mice can partially be explained by a failure to up-regulate Id3 expression in response to TCR signaling. However, the normal size of Id3-deficient thymuses (29) suggests that the increased cellularity in Egr1-deficient thymuses cannot be explained by reduced Id3 expression.



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  		FIGURE 6. Efficient up-regulation of Id3 expression in response to TCR signaling requires Egr1. DP thymocytes were isolated from Egr1+/+ and Egr1-/- mice on a B6.AKR background using anti-CD8-coated magnetic beads. RNA was isolated from these cells either directly after purification, or after a 1-h culture in tissue culture media (RPMI with 10% FCS), or after a 1-h culture in media on a dish coated with anti-CD3. RNA was analyzed by Northern blot using probes against Id3 and {beta}-actin.

 
Egr1 regulates TCR-mediated bcl-2 up-regulation in DP thymocytes

Although Id3 and Egr1-deficient thymuses are similar, it is likely that additional Egr-1-dependent genes are induced by TCR signaling during positive selection. Expression of the anti-apoptotic molecule bcl-2 is increased in response to TCR signals in DP thymocytes (30). Most DP thymocytes express low levels of bcl-2, but after they receive a TCR signal, DP thymocytes increase expression of bcl-2, and SP thymocytes express relatively high levels. Therefore, it is thought that bcl-2 plays an important role in the survival of positively selected thymocytes. It is important to note that bcl-2 expression is also somewhat increased by signals that lead to negative selection (31, 32), but that strongly signaled DP thymocytes nevertheless undergo apoptosis. bcl-2 expression may be able to inhibit apoptosis of DP thymocytes to some degree (33), but in most cases, it is unable to stop the process of negative selection (34, 35, 36).

Because up-regulation of bcl-2 in response to TCR stimulation has been shown to be dependent on ERK activation (32), we have evaluated the role of Egr1 in bcl-2 up-regulation by stimulating thymocytes from 3A9tg, Egr1+/+ and Egr1-/- mice with anti-CD3 in culture overnight, and measuring levels of bcl-2 by flow cytometry. The results are displayed in Fig. 7Go, and show that overnight culture in the absence of TCR signaling results in essentially no increase in intracellular bcl-2 expression in both Egr1+/+ and Egr1-/- thymocytes. In contrast, overnight culture with plate-bound anti-CD3 results in high expression of bcl-2 in 38% of CD4lowCD8low thymocytes from Egr1+/+ mice, but only 21% of CD4lowCD8low thymocytes in the Egr1-deficient mice. CD4lowCD8low thymocytes were examined specifically because it has been shown that DP thymocytes stimulated with anti-CD3 in culture will reduce expression of both CD4 and CD8 (37). Therefore, we gated on these cells to enrich for TCR-signaled DP thymocytes. The results indicate that Egr1 is required for efficient up-regulation of bcl-2, and this suggests that the defect in positive selection observed in the Egr1-deficient mice may be in part due to poor survival of thymocytes after they have received a TCR signal.



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  		FIGURE 7. Up-regulation of bcl-2 in response to anti-CD3 stimulation is reduced in Egr1-deficient mice. Thymocytes were isolated from 3A9tg, Egr1+/+ and 3A9tg, Egr -/- mice, and then cultured for 22 h in tissue culture media on plates coated with anti-CD3 or uncoated plates. The cells were then harvested and surface stained for CD4 and CD8, followed by intracellular staining for bcl-2. The histograms show staining for bcl-2 after gating on CD4lowCD8low thymocytes. The CD4lowCD8low cells have low levels of bcl-2 in the absence of TCR stimulation, and up-regulate bcl-2 after overnight TCR stimulation. The thick lines depict bcl-2 levels in Egr1+/+ mice and the thin lines depict bcl-2 levels in Egr1-/- mice. In the anti-CD3-treated thymocytes, 38.8% of cells are bcl-2 positive in Egr1+/+ mice, and 21.5% are positive in Egr1-/- mice. A similar percentage of cells became CD4lowCD8low in Egr1+/+ and Egr1-/- mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have investigated the role of Egr1 in thymocyte development. By analysis of Egr1-deficient mice on a normal, polyclonal TCR background and three different TCR tg backgrounds, we have found the following. First, Egr1 regulates the size of the thymus. The overall cellularity of the thymus is increased in Egr1-deficient mice, and this effect is observed starting at the DN stage of thymocyte development. This suggests that Egr1 functions to limit the generation and/or survival of DN thymocytes. Second, we have found that Egr1 promotes positive selection of both CD4 and CD8SP cells. This is based on the fact that the percentage of TCRhigh, CD4SP thymocytes is reduced by ~3-fold in Egr1-deficient mice on two different class II MHC-restricted TCR tg backgrounds, and the percentage of TCRhigh, CD8SP thymocytes is reduced by ~2-fold on a class I MHC-restricted TCR tg background. Third, we have found that negative selection is unimpaired in Egr1-deficient mice. Fourth, Egr1 is a positive regulator of Id3 and bcl-2 expression in response to TCR signaling in DP thymocytes. The functions of the Id3 and bcl-2 gene products suggest that Egr1 regulates positive selection by promoting both the survival and differentiation of TCR-signaled DP thymocytes.

The first phase of thymic development begins as precursors enter the thymus with a CD44+CD25- cell surface phenotype. These cells soon acquire expression of CD25 followed by loss of CD44 expression (15). This early stage of thymic development is dependent on growth factors such as IL-7 and c-kit (38). Cells that then successfully rearrange TCR-{beta} will form a pre-TCR and transition from CD44-CD25+ to CD44-CD25-. Previous studies that evaluated the involvement of Egr1 in DN thymocyte development have shown that Egr1 is expressed at low levels in CD44-CD25+ DN thymocytes, and that expression is increased in the CD44-CD25- population, suggesting that Egr1 can be induced by pre-TCR signaling (39, 40). Furthermore, in mice that have constitutive overexpression of Egr1 in thymocytes (39), and in thymocytes retrovirally transduced with Egr1 (40), some cells can develop to the CD44-CD25- stage in the absence of pre-TCR signaling. These results have led to the hypothesis that Egr1 may be important for translating the pre-TCR signal and promoting the DN to DP transition. However, in Egr1-deficient mice, the DN to DP transition appears normal, suggesting that Egr1 is not essential for this developmental transition. It is possible that the phenotype observed after overexpression of Egr1 does not occur with normal levels of Egr1, or that in the Egr1-deficient mice there is compensation by other Egr family members, or other unknown factors.

However, it is clear that Egr1 does have some role to play in early thymocyte development: the thymuses in Egr1-deficient mice contain more cells than their littermate controls, and this increase in thymic cellularity extends to the earliest stages of thymocyte development. This result suggests that normal expression of Egr1 limits the size of the DN thymocyte compartment. This could either be through controlling survival of DN thymocytes during the growth factor dependent stage of development, or by controlling the number of precursors that enter the thymus. Several growth factors have been reported to induce Egr1, such as nerve growth factor, IL-3, and GM-CSF (6, 41), but whether Egr1 can be regulated by IL-7 or c-kit has not been reported. One study found that pre-B cells derived from fetal liver and cultured in IL-7 did express Egr1, but it was not determined whether the Egr1 expression was directly related to IL-7R signal transduction (42). We conclude that there is some role for Egr1 in the early, TCR-independent phase of T cell development, and this will be more clearly defined by further study.

DP thymocytes have a limited lifespan, and for these cells to be rescued from death they must receive a signal through the TCR. However, strong signals through the TCR will result in rapid apoptosis of the DP cell. Therefore, there is a distinction between signals that lead to apoptosis and signals that lead to positive selection. We favor a model where positive selection signals are essentially a subset of those signals that result in apoptosis. When a complete signal is induced by a high-affinity TCR ligand, the induction of death is dominant. When the TCR interacts with a lower affinity ligand, the signals for death are not induced, but signals that allow survival and differentiation are induced. Data in support of this model come from analysis of TCR-dependent induction of MAP kinases. It has been demonstrated that high-affinity TCR ligands that induce death of DP thymocytes will activate ERK, JNK, and p38 MAPKs, whereas ligands for positive selection will activate only ERK (3, 4, 5). Importantly, it has been shown that when activation of ERK is blocked by a dominant negative form of MEK-1 in vivo, an MEK-1 inhibitor in fetal thymic organ culture, or by a targeted mutation in the ERK-1 gene, positive selection is inhibited (18, 22, 43, 44). In addition, use of a p38 inhibitor in fetal thymic organ culture and a dominant negative JNK1 transgene both block negative selection (43, 45).

Our analysis of Egr1 also supports such a model. Egr1 can be induced in thymocytes and T cells by both strong and weak signals through the TCR, and the induction of Egr1 has been shown to be ERK-dependent using an inhibitor of MEK-1 (8, 10). Thus, Egr1 expression is induced downstream of a pathway that is stimulated by weak TCR signals. Although Egr1 is also responsive to strong TCR signals, its primary function in thymocytes seems to be the up-regulation of genes important for differentiation and survival. Products of these survival genes (e.g., bcl-2) could slightly inhibit negative selection, but they do not overcome the death effector pathways that are induced by strong TCR signals. Therefore, the data in this study suggest that Egr1 is a link between activation of ERK and changes in gene expression that are important for positive selection. Although some previous studies have proposed that activation of ERK is also important for negative selection (24, 25), our model of in vivo negative selection suggests that ERK induction of Egr1 does not play a role in negative selection.

Failure to up-regulate bcl-2 could be the explanation for why on a negatively selecting background 3.L2tg, Egr1-/- thymocytes are reduced to a greater extent than 3.L2tg, Egr1+/+ thymocytes. Negative selection of Egr1-deficient 3.L2tg thymocytes results in only 0.04% of thymocytes that are CD4SP and 3.L2 TCRhigh, as compared with 0.9% of Egr1+/+ thymocytes. This result is consistent with the idea that bcl-2 expression in response to a negative selection signal has a modest inhibitory effect on negative selection, but still allows negative selection to result in apoptosis. Studies on the role of bcl-2 in negative selection have generated conflicting conclusions, but most are consistent with this scenario; bcl-2 expression reduces the efficiency of negative selection (33, 34, 35, 36). Our results are consistent with a model where TCR signaling induces ERK activation which leads to a marked increase in Egr1 expression. bcl-2 expression is then increased in a manner dependent on Egr1, and survival of signaled thymocytes is favored. In the case of weak signals, positive selection ensues, whereas strong signals overcome the bcl-2 up-regulation and lead to death.

Our data indicate that Id3 and bcl-2 up-regulation by TCR signaling is dependent on Egr1, but it is unknown whether the enhancement of Id3 and bcl-2 expression by Egr1 is due to a direct action of Egr1 on the promoters of these genes. Regulation of Id3 by Egr1 has been suggested previously by in vitro experiments (9). Overexpression of Egr1 in a DP thymocyte tumor cell line resulted in increased levels of Id3 mRNA; however, it was not determined whether this was due to a direct activation of the Id3 promoter. The kinetics of the expression of the two genes suggests that the relationship may be direct. Egr1 expression is increased 15 min after stimulation of ERK, and Id3 expression is induced 45 min after ERK activation (9), making it unlikely that an intermediate factor induced by Egr1 stimulates Id3 expression. However, an analysis of the Id3 promoter, and demonstration that Egr1 can transactivate this promoter directly, has not been reported.

The role of Egr1 in regulating bcl-2 expression has not been extensively investigated. One study used B cell lymphomas that did not contain a translocation of the bcl-2 gene, and showed that Egr1 expression can decrease expression of bcl-2, suggesting that Egr1 plays a negative regulatory role for bcl-2 expression in B cells (46). Sites in the bcl-2 promoter (human) that bound purified Egr1 were identified, but it is not clear whether these sites are important for regulated expression of bcl-2. The data suggest that Egr1 has different effects on bcl-2 expression depending on the cell type and stage of differentiation, but it is not clear whether these effects are mediated by a direct interaction of Egr1 with the bcl-2 promoter. We favor a model where Egr1 regulates bcl-2 expression in DP thymocytes in an indirect manner, as Egr1 is induced minutes after TCR stimulation in DP thymocytes, but bcl-2 expression increases only after several hours.

In conclusion, our results demonstrate that induction of Egr1 expression is a mechanism by which activation of ERK can lead to positive selection. In turn, induction of Egr1 leads to increased levels of Id3 and bcl-2, resulting in efficient differentiation and survival of thymocytes. In addition, we have unexpectedly found that Egr1 plays a role in limiting the number of T cell precursors. The identification of Egr1 target genes that mediate control of thymocyte number awaits further investigation.


   Acknowledgments
 
We thank Jeremy Boss and Peter Jensen for critical review of the manuscript.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant AI-48784 and by an award from the University Research Committee of Emory University. Back

2 Address correspondence and reprint requests to Dr. Gilbert J. Kersh, Department of Pathology, Emory University, 7301 Woodruff Memorial Building, 1639 Pierce Drive, Atlanta, GA 30322. E-mail address: gkersh{at}emory.edu Back

3 Abbreviations used in this paper: DP, double positive; DN, double negative; Egr1, early growth response gene 1; SP, single positive; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; Hb, hemoglobin; tg, transgenic. Back

Received for publication March 13, 2002. Accepted for publication June 6, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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