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Cell Growth & Differentiation Vol. 10, 829-838, December 1999
© 1999 American Association for Cancer Research

A Role for E2F1 in the Induction of ARF, p53, and Apoptosis during Thymic Negative Selection1

Jing W. Zhu, Deborah DeRyckere, Feng X. Li, Yisong Y. Wan and James DeGregori2

Departments of Biochemistry and Molecular Genetics and Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado 80262


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
E2F transcriptional activity controls the expression of many of the genes required for G1 to S phase progression. E2F1, one member of the E2F family, plays an important role in the induction of apoptosis. We have examined the role of the E2F1 transcription factor in apoptosis during T-cell maturation in the thymus. We show that E2F1 is required for the apoptosis of autoimmune immature T cells during thymic negative selection in vivo. This T-cell receptor-mediated apoptosis coincides with the E2F1-dependent increase of p19-ARF mRNA and p53 protein levels. In contrast, E2F1 is not required for the induction of apoptosis by glucocorticoids or DNA damage. These results demonstrate a specific role for E2F1, which triggers a pathway leading to ARF and p53 induction, in a physiological apoptosis pathway that is uncoupled from a normal proliferative event.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
To respond to virtually any foreign invader, the immune system must be capable of recognizing a vast range of antigens. To accomplish this, lymphocytes assemble antigen receptor genes by the somatic DNA rearrangement of families of gene segments, generating coding sequences with different variable (V) regions, which results in an enormous repertoire of potential reactivity toward antigens. However, because this process is relatively stochastic, it inevitably leads to the generation of lymphocytes with either nonfunctional or self-reactive antigen receptors. To prevent autoimmunity, self-reactive lymphocytes must be eliminated (1) . One mechanism by which this is accomplished is the induction of apoptosis, a genetically determined program of cell suicide, in immature self-reactive T cells during maturation in the thymus.

During the development of T cells, immature lymphocytes from the bone marrow migrate to the thymus (1) . These cells then undergo rearrangement of their genomic ß and then {alpha} TCR3 genes, followed by low levels of TCR expression together with the linked CD3 complex and the accessory CD4 and CD8 proteins. The CD3 complex mediates TCR signaling, in part through the recruitment of the ZAP70 and Src family kinases (2) . CD4 and CD8 facilitate the interaction of the TCR with the class II MHC and class I MHC, respectively, expressed on APCs. The fate of these immature CD4+CD8+ (DP) thymocytes is determined by interactions between their TCR and peptide/MHC complexes presented by thymic APCs, including epithelial and dendritic cells. Antigens are processed in these cells into peptides, and selected peptides are presented by the MHC on the cell surface. Thymocytes that are strongly reactive toward self-peptides in association with MHC proteins and are therefore potentially self-reactive are eliminated by apoptosis (negative selection; Refs. 1 and 3 ). DP thymocytes that either fail to express a functional TCR or express a TCR with insufficient affinity or avidity for self-MHC presented in the thymus die, due to the lack of signaling via the TCR. In contrast, T cells with moderate affinity for self-MHC (less than 5% of thymocytes) are induced to differentiate into mature CD4+CD8- or CD4-CD8+ T cells expressing high levels of TCR and CD3 (positive selection). These mature T cells migrate out of the thymus to form the peripheral T cell repertoire. The observation that type I diabetes appears to result in part from the failure to properly eliminate autoimmune T cells in the thymus underscores the importance of negative selection to normal physiology (4) .

E2F activity controls the transcription of a group of genes that are normally regulated at the G1-S-phase transition and encode proteins important for S-phase events including cyclin E, B-Myb, dihydrofolate reductase, DNA polymerase {alpha}, and Cdc6, a limiting component of the prereplication complex (5) . E2F transcriptional activity is composed of a variety of heterodimers formed by the association of one of at least six different E2F family members with one of at least three different DP proteins. E2F1, E2F2, and E2F3 associate specifically with Rb, and the expression of these E2Fs is growth-regulated in fibroblasts. E2F4 and E2F5 appear to associate with all three Rb family members, Rb, p107, and p130. Overexpression of E2F1, E2F2, E2F3, E2F4, or E2F5 indicates distinct abilities of these E2F family members to activate the transcription of different target genes, and E2F1 uniquely induces apoptosis in serum-starved fibroblasts (6) . Interestingly, disruption of E2F1 in the mouse results in an excess of mature T cells due to a maturation stage-specific defect in thymocyte apoptosis (7) as well as the genesis of a diverse range of tumors in older adults (8) . DP thymocytes from E2F1-/- mice exhibit reduced apoptosis when cultured in vitro (7) , suggestive of a defect in passive cell death. In addition, E2F1-/- thymocytes show reduced apoptosis in vivo (2.5% versus 3.5% for E2F1+/+ mice) in response to intrathymic injection of CD3{epsilon}-specific antibody. By binding to the TCR/CD3 complex, anti-CD3{epsilon} elicits TCR signaling, resulting in apoptosis primarily in DP thymocytes (9) .

Previous work has shown that the induction of S phase by E2F1 overexpression in fibroblasts is accompanied by a p53-dependent induction of apoptosis (10, 11, 12, 13) , although overexpression of E2F1 can in some contexts induce apoptosis that is p53 independent (14 , 15) . The p53 gene is mutated in more than half of human tumors (16) . p53 is required for the induction of apoptosis in response to DNA damage from chemotherapeutic agents or radiation, both in T lymphocytes and in oncogenically transformed fibroblasts (17, 18, 19) . In addition, recent experiments have indicated a role for the ARF gene product (p19 in mice; p14 in humans) in oncogene-induced, p53-dependent apoptosis (20 , 21) . The ARF gene is translated from an alternative reading frame that overlaps the p16INK4A reading frame (22) in a locus frequently deleted in human tumors (23) . Overexpression of E2F1 activates the expression of ARF (6 , 24) , and in rodent fibroblasts, this activation is specific for the E2F1 and E2F2 family members (6) . Consensus E2F binding sites are present in the human ARF promoter, and E2F1 can activate minimal promoters containing these sites (24 , 25) . Recent work has shown that ARF antagonizes the activity of Mdm2, a protein known to target p53 for ubiquitin-mediated degradation. Thus, the activation of ARF leads to the stabilization of p53 and the potentiation of its activity (26, 27, 28, 29) . ARF appears to stabilize p53 in part by blocking the nuclear-cytoplasmic shuttling of Mdm2 and p53 (30, 31, 32) .

In this study, we demonstrate a specific role for E2F1 in the physiological apoptotic pathway mediating thymocyte negative selection. The levels of p19-ARF mRNA and p53 protein increase during antigen-induced TCR-mediated apoptosis. Importantly, this increase in ARF and p53 levels is dependent on E2F1. These results indicate a specific role for E2F1 and possibly for ARF and p53 in a physiological apoptosis pathway in vivo, suggesting a common pathway that controls the deletion of tumorigenic cells as well as autoimmune T cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
E2F1 Is Specifically Required for Antigen-induced Apoptosis of CD4+CD8+ Thymocytes in Vivo.
The study of thymocyte maturation has been greatly enhanced by the generation of TCR transgenic mice. Transgenic mice have been created that express {alpha} and ß chains for a TCR with known peptide/MHC specificity in nearly all of their T cells (33) . This has greatly facilitated the study of both positive and negative selection because an entire population of maturing T cells can be studied, rather than individual cells. In the background of the appropriate MHC but in the absence of antigen, TCR transgene-expressing thymocytes are positively selected. If the antigen recognized by the TCR is present in the thymus, either as an endogenous antigen or after its introduction into the thymus, then transgene-expressing thymocytes are eliminated by apoptosis.

We chose to examine the role of E2F1 in antigen-induced apoptosis using DO11.10 TCR transgenic mice. In these mice, most lymphocytes express {alpha} and ß chain transgenes encoding a TCR (DO TCR) specific for a chicken OVA (OVA 323–339) in the context of class II MHC I-Ad or I-Ab (3 , 34) . The introduction of OVA in vivo results in the elimination of the majority of thymocytes through the induction of apoptosis in CD4+CD8+ (DP) T cells. To assess whether E2F1 is required for thymic negative selection, we crossed the DO TCR transgene into mice disrupted for E2F1 (7) and further backcrossed both DO and E2F1 into the C57Bl/6 (MHC H-2b/b) genetic background for one to three additional generations. Where indicated, mice also contained disrupted Rag2 genes (35) . Rag2 is an essential component of the recombinase that mediates the assembly of either TCR or immunoglobulin chains from germ-line arrays of exons (36) . The expression of the DO TCR bypasses the maturation block imposed by the Rag2 deficiency; consequently, virtually all lymphocytes in DO+ Rag2-/- mice bear the DO TCR exclusively (34) . T and B cells are absent in E2F1+ and E2F1-/- mice that are Rag2-/- and nontransgenic for DO, indicating that E2F1 is not required for the developmental block imposed by the failure to rearrange antigen receptors (data not shown).

DO+ mice of the genotypes indicated in Fig. 1Citation were injected i.p. with OVA or control (CON) peptide. Thymuses were harvested 14 or 24 h pi, the number of cells in the thymus was determined, and thymocytes were analyzed for the expression of CD4, CD8, and the DO TCR by flow cytometry. The injection of CON peptide (ovalbumin residues 324–334), which is presented by I-A but does not stimulate the DO TCR (37) , did not result in a significant reduction of thymic cellularity in comparison with PBS-injected mice (Ref. 3 ; Fig. 1Citation ; data not shown). As expected, the injection of OVA caused a dramatic decrease in thymic cellularity in DO+ E2F1+ mice by 24 h pi. Results obtained from E2F1+/+ or E2F1+/- mice were similar for all of the experiments described in this study. In sharp contrast, no significant decrease in thymic cellularity was observed in DO+ E2F1-/- mice injected with OVA (Fig. 1)Citation . Flow cytometric analysis of thymocytes isolated from OVA-injected DO+ E2F1+ mice revealed a selective loss of DP thymocytes by 14 h pi, and the remaining DP cells exhibited reduced expression of CD4 and CD8 (Fig. 1B)Citation , which is characteristic of thymocytes undergoing TCR-mediated apoptosis (38) . In contrast, DO+ E2F1-/- thymocytes exhibit normal expression of CD4 and CD8 (Fig. 1B)Citation . Thymocytes from E2F1+ or E2F1-/- mice express similar levels of the DO TCR, as determined by staining with the clonotypic KJ26.1 monoclonal antibody (data not shown). Thus the absence of E2F1 appears to prevent or at least substantially delay the thymocyte cell death resulting from peptide-induced negative selection. Interestingly, OVA injection results in activation of peripheral DO+ CD4+ T cells, which is independent of E2F1 genotype (data not shown), indicating that E2F1 is not required for the TCR-activated proliferative response of mature T cells.



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Fig. 1. E2F1 is required for antigen-induced apoptosis during negative selection in vivo. A, E2F1+ (E2F1+/+ or E2F1+/-) and E2F1-/- DO TCR transgenic littermates (H-2b/b; 20–24 days old) were injected i.p. with 0.5 mg of OVA (ISQAVHAAHAEINEAGR) or CON (SQAVTAAHAEI) peptides. Thymuses were isolated 24 h pi, and thymic cellularity was microscopically determined using a hematocytometer. For each set of littermates, the number of T cells in the thymus relative to E2F1+ mice injected with CON peptide is shown with the SE indicated. The data shown were derived from five experiments (in two of the experiments, the littermates were all Rag2-/-), except for the CON-injected E2F1 mutant sample, which was derived from two experiments. The average cellularity for the CON-injected DO+ E2F1+ mice was 4.34 x 107. The unpaired Student t test value for OVA-injected wild-type versus E2F1 mutant mice is P = 0.00054. B, DO TCR transgenic, Rag2-/- littermates of the indicated E2F1 genotypes were injected i.p. with CON or OVA, and thymus cells were isolated 14 h pi. Thymocytes were stained with fluorescent-labeled {alpha}-CD4 and {alpha}-CD8 antibodies and analyzed by flow cytometry. The percentage of DP thymocytes is indicated in the upper right corner of each panel. The total number of cells in each thymus is also shown.

 
E2F1 Mutant Mice Are Partially Deficient in the Deletion of T Cells Reactive with Endogenous Retroviral Superantigens.
E2F1 deficiency clearly prevents the deletion of DO TCR transgenic thymocytes after I-Ab presentation of OVA. We asked whether the deletion of nontransgenic T cells mediated by endogenous antigen might also be influenced by E2F1 genotype. In mice, a variety of superantigens are encoded in the 3' long terminal repeat of endogenous mouse mammary tumor viruses (reviewed in Ref. 39 ). These vSAGs bind to certain class II MHCs, particularly I-E MHC, facilitating their interaction with Vß segments on thymocytes and resulting in the negative selection of these thymocytes. By monitoring the expression of particular Vß chains using monoclonal antibodies, it was shown that thymocytes that are self-reactive with vSAGs are present at the developmental stage of low TCR expression but are eliminated before they express high levels of TCR (40 , 41) . Although CD4+ T cells are more efficiently deleted due to the association of vSAGs with class II MHC molecules, CD8+ T cells are also deleted (39) . Although the deletion of vSAG-reactive T cells is impaired in the absence of gp39-CD40 interaction (42) , other mutations that impair peptide-mediated T-cell deletion do not appear to affect vSAG-mediated deletion (see "Discussion").

Because the H-2b locus does not encode for I-E MHC, we bred the E2F1 mutation into the B10.D2 background (H-2d/d and I-E+, but otherwise congenic with C57Bl/10) for at least three generations. The I-Ed MHC efficiently mediates the deletion of Vß chains reactive with vSAGs, and T cells bearing Vß5, Vß7, Vß11, and Vß12 are normally deleted in B10.D2 mice, which express mouse mammary tumor proviruses Mtv 8, 9, and 17 (39) . By monitoring Vß expression on T cells from either E2F1+ or E2F1-/- mice, we further characterized the role of E2F1 in thymic negative selection without the expression of a transgenic TCR or the introduction of exogenous antigen. Lymphocytes were isolated from the peripheral LNs of 1–2-month-old E2F1+ or E2F1-/- mice and stained with fluorochrome-linked antibodies against CD4, CD8, and one of several different Vß chains. The cells were then analyzed by three-color flow cytometry. E2F1 mutant mice exhibited significantly greater numbers of T cells expressing Vß5, although the deletion of Vß12-bearing T cells was unimpaired (Fig. 2)Citation . In comparison, T cells displaying Vß6, which are not recognized by the vSAGs expressed in these mice, were not deleted. Similar results were obtained using thymocytes (data not shown).



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Fig. 2. E2F1 mutant mice are partially defective in the deletion of T cells bearing TCRs reactive with endogenous retroviral superantigens. Lymphocytes were isolated from the peripheral LNs of 1–2.5-month-old E2F1+ or E2F1-/- H-2d/d mice. Lymphocytes were stained with fluorescent-labeled antibodies against CD4, CD8, and either TCR Vß5, Vß6, or Vß12. The cells were then washed and analyzed by three-color flow cytometry. Cells were gated first for the expression of CD4 or CD8 and then analyzed for the expression of the indicated Vß chain. The number of lymphocytes isolated from LNs and the relative percentages of CD4+ and CD8+ T cells in LNs were similar in E2F1+ and E2F1-/- mice. A, flow cytometric profiles of Vß5 and Vß6 expression on CD8+ T cells from two 76-day-old E2F1+/- or E2F1-/- siblings. B, results obtained from at least four E2F1+ and five E2F1-/- mice (sets of littermates) expressed as the percentage of Vß+ cells among CD4+ or CD8+ cells. The SE is indicated. *, the unpaired Student t test value for the percentage of CD8+ T cells expressing Vß5 in wild-type versus E2F1 mutant mice is P = 0.011.

 
It is not clear why the absence of E2F1 differentially impairs the deletion of T cells bearing different vSAG-reactive Vß chains. The impaired deletion of Vß5-bearing lymphocytes in E2F1-/- mice is more apparent in the CD8 T cells than in the CD4 T cells. Because vSAG-mediated cross-linking of the TCR and I-E is facilitated by CD4 but not CD8 (39) , class I MHC restricted T cells (CD8+) may receive a weaker signal that is more easily impeded by E2F1 deficiency. In addition, although 2-fold more CD8+ T cells bearing Vß5 are present in the LNs of E2F1-/- mice, it does not appear that E2F1 deficiency completely eliminates the deletion of these self-reactive T cells. In C57Bl/6 mice lacking I-E expression, approximately 5% and 7% of CD4+ and CD8+ peripheral T cells express Vß5, respectively (data not shown). Thus, the absence of E2F1 reduces but does not eliminate negative selection mediated by endogenous superantigens. Our results do not imply that E2F1 is required for negative selection mediated by all MHC/antigen/TCR interactions. E2F1 deficiency may substantially increase the threshold for the affinity or avidity of MHC/antigen/TCR interactions that are required to induce apoptosis, such that T cells that would normally undergo negative selection survive. Nonetheless, T cells bearing TCRs that are highly reactive with self may still be eliminated in the absence of E2F1. Indeed, we have observed a reduced E2F1 requirement for negative selection induced by more robust TCR signals (data not shown). Perhaps stronger TCR signals induce an E2F1-independent apoptotic pathway(s) that can compensate for the absence of E2F1.

E2F1 Is Not Required for Thymocyte Apoptosis Induced by DNA Damage or GCs.
Either DNA damage or GCs can also stimulate thymocyte apoptosis, with only the former being dependent on p53 (17 , 18) . We asked whether E2F1 is required for thymocyte cell death mediated by either DNA damage or GC induced by treatment with Adr or Dex, respectively. Thymocytes from either E2F1+/+ or E2F1-/- mice (DO-; C57Bl/6 background) were cultured in the presence of Dex, Adr, or control DMSO and then assayed for apoptosis by staining with FITC-linked annexinV and PI. Apoptotic cells display phosphatidylserine in the outer cell membrane, which is bound by annexin V (43) . Either Dex or Adr induced apoptosis in both E2F1+/+ and E2F1-/- thymocytes (Fig. 3A)Citation . Whole body irradiation (50–500 cGy) of either E2F1+ or E2F1-/- mice also resulted in the selective deletion of DP thymocytes by 24 h, which was independent of the E2F1 genotype at all doses (Fig. 3B)Citation . Thus, the induction of cell death by either DNA damage or GC treatment is independent of E2F1 and is therefore distinct from the antigen-induced apoptosis that occurs during negative selection. These data suggest that E2F1 is not required for apoptosis in general but plays a specific role in the T-cell apoptotic signaling pathway activated by antigen.



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Fig. 3. E2F1 is not required for thymocyte apoptosis induced by DNA damage or GCs. A, thymus cells were isolated from wild-type and E2F1 mutant mice (H-2b/b) and cultured in the presence of 20 µM Adr (Sigma) for 6 h; 0.05 (L), 0.1 (M), or 0.2 µM (H) Dex (Sigma) for 3 h; or DMSO for 3 or 6 h (Con, left and right panels, respectively). Cells were harvested, stained with fluorescence-labeled annexin V (A) and PI, and examined by flow cytometry. The percentage of A+ (PI+ or PI-, as indicated) cells is shown. Adr fluorescence prevented the determination of PI incorporation. B, E2F1+ or E2F1-/- mice (1–2-month-old mice) were either mock-treated or irradiated in a Nordion Cobolt 60 gamma irradiator with 50, 100, 250, or 500 cGy. The mice were sacrificed 24 h later, and thymocytes were analyzed for the expression of CD4 and CD8 by flow cytometry (left panel). The percentage of DP thymocytes and thymic cellularity are indicated. The graph in the right panel shows the thymic cellularity (relative to the mock-treated E2F1+ littermate) and the percentage of DPs at the indicated radiation doses. Each data point is derived from the average of at least two experimental mice.

 
E2F1 Is Required for the Induction of p19-ARF and p53 upon TCR Stimulation.
To better understand the mechanism by which E2F1 is required for TCR-dependent negative selection, we analyzed the expression of various known modulators of apoptosis during negative selection. DO+ E2F1+ mice were injected i.p. with either CON or OVA, and thymuses were harvested at various time points for the analysis of cell number; CD4, CD8, and DO TCR expression; and mRNA/protein levels. At 10 h pi or earlier, OVA-stimulated thymocytes were indistinguishable from CON-treated thymocytes in terms of both cell number and CD4/CD8/DO TCR expression. OVA-stimulated thymocytes displayed the reduced expression of CD4 and CD8 characteristic of thymocytes undergoing TCR mediated apoptosis by 12 h pi, and reduced cellularity was evident by 16 h (data not shown).

Given that the overexpression of E2F1 in quiescent fibroblasts results in stabilization of p53 and induction of apoptosis, which is usually p53 dependent (44 , 45) , we examined whether p53 up-regulation might be involved in TCR-mediated thymocyte apoptosis. Western blot analysis of cell lysates revealed increased p53 protein levels in OVA-treated DO+ E2F1+ thymocytes relative to CON-treated cells by 16 h pi (Fig. 4A)Citation . No change in p53 mRNA is evident (data not shown), indicating that changes in p53 levels are translational or posttranslational. Importantly, we do not observe any increase in p53 levels in OVA-treated DO+ E2F1-/- thymocytes (Fig. 4B)Citation . These results indicate that the accumulation of p53 protein in response to TCR signaling is dependent on the presence of E2F1. {gamma}-Irradiation of thymocytes has been previously shown to result in the accumulation of p53 protein (17) . Consistent with the fact that DNA damage-induced apoptosis is E2F1 independent, the accumulation of p53 in response to DNA damage was not dependent on E2F1 (Fig. 4C)Citation . As expected, p53 does not accumulate in response to treatment with Dex.



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Fig. 4. E2F1 is specifically required for the induction of p53 protein levels following TCR stimulation. DO TCR transgenic littermates (C57Bl/6, except in A) of the indicated E2F1 genotypes were injected with 0.5 mg of OVA or CON peptides, and thymus cells were isolated at the indicated times pi. A, p53 protein levels increase on TCR activation. Cell lysates were prepared from E2F1+, DO TCR transgenic mice (Balb/C background) and analyzed by Western blot using {alpha}-p53 and {alpha}-actin antibodies. B, p53 accumulation after TCR activation is E2F1 dependent. Thymus cells were isolated 24 h pi. Cell lysates were prepared and analyzed by Western blot as described in A. The levels of p53 normalized for actin expression and relative to that of the CON-injected mouse are shown. C, p53 accumulation after DNA damage is E2F1 independent. Thymocytes were isolated and cultured as described in Fig. 1CCitation . Cell lysates were prepared and subjected to Western blot analysis as described in A.

 
Recent work has suggested a pathway involving the ARF gene product, leading to p53 accumulation and apoptosis in response to oncogene activation (46) . ARF may be a transcriptional target of E2F1 that is critical for the accumulation of p53 and the induction of E2F1-dependent apoptosis. To investigate this possibility, we examined the expression of ARF in OVA-stimulated thymocytes by RPA. Indeed, increased ARF expression is observed by 6 h pi, with peak expression at 10 h pi (Fig. 5A)Citation , preceding any observable effect on thymocyte viability or CD4/CD8 expression. ARF expression returns to basal levels by 14 h pi. There is a notable delay between the induction of ARF message and the increase in p53 protein levels, which we do not presently understand. Importantly, whereas the expression of ARF was activated approximately 7-fold by 10 h in OVA-stimulated DO+ E2F1+ thymocytes, no increase was observed in DO+ E2F1-/- thymocytes (Fig. 5B)Citation . In OVA-treated E2F1-/- thymocytes, ARF induction is also not observed by 15 h, a time when apoptosis is already apparent in the OVA-treated E2F1+/+ thymocytes, and ARF expression has returned to basal levels. These data provide evidence that ARF expression is regulated during apoptosis in vivo and indicate that ARF is a physiological target of E2F1. Interestingly, OVA stimulation does not result in increased expression of other E2F target genes, such as cyclin E (Fig. 5C)Citation and cyclin A (Fig. 5D)Citation . Increased expression of cyclin A has been shown to coincide with increased proliferation in immature CD4-CD8- T cells (47) . Thus, TCR-stimulated thymocyte apoptosis is preceded by a specific, E2F1-dependent induction of ARF in the absence of a general increase in E2F-dependent transcription. These results demonstrate a role for E2F1 in apoptosis that is not associated with a proliferative event.



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Fig. 5. E2F1 is required for the up-regulation of ARF expression on TCR stimulation. DO TCR transgenic littermates (C57Bl/6, except in A) of the indicated E2F1 genotypes were injected with either 0.5 mg of CON peptide, 0.5 mg of OVA, 5 mg of ovalbumin protein (Sigma), or PBS, as indicated, and thymus cells were isolated at the indicated times pi. A, ARF mRNA levels increase on TCR activation. RNA was isolated from E2F1+ DO transgenic mice (B10.D2 background), and ARF and GAPDH RNA levels were determined by RPA. The identity of the ARF-specific protected fragments has been verified both by migration at the expected size (151 nucleotides) and the up-regulation of these fragments in p53-/- fibroblasts (data not shown), consistent with previous observations (21) . Double bands are not uncommon for RPA analysis and are presummed to be due to "breathing" at the ends of the RNA:RNA hybrid, which allows for limited RNase access. B, induction of ARF expression after TCR activation is E2F1 dependent. Thymus cells were harvested at either 10 or 15 h pi (two separate experiments). RNA was isolated and ARF and GAPDH RNA levels were determined by RPA. The levels of ARF RNA, normalized for GAPDH expression, relative to that of a CON-injected mouse are shown. At 10 h, we did not observe any reduction in thymic cellularity or CD4/CD8 staining in OVA-treated thymocytes. At 15 h, we observed a 2-fold reduction in the cellularity of the OVA-treated E2F1+/+ thymocytes and a reduced percentage of DP cells, but no reductions were observed in the OVA-treated E2F1-/- thymocytes. C, cyclin E expression is not induced on TCR activation. RNA was isolated from thymocytes from E2F1+ or E2F1-/- mice at the indicated times pi, and cyclin E and GAPDH RNA levels were determined by RPA. The levels of cyclin E RNA (normalized for GAPDH expression) relative to that of a CON-injected mouse are shown. D, cyclin A expression is not induced on TCR activation. RNA was isolated from thymocytes from E2F1+ mice at the indicated times pi, and cyclin A and GAPDH RNA levels were determined by RPA.

 
Additional RPA analysis of thymocyte RNA levels revealed an induction of the Bcl-2 family member Bfl-1/A1 by 4 h after OVA injection (Fig. 6A)Citation . The expression of Bfl-1 has recently been shown to be controlled by the Rel/NF-{kappa}B transcription factor (48 , 49) , and the Rel-dependent expression of Bfl-1 after antigen receptor ligation is necessary for B-cell survival (49) . The induction of Bfl-1 is similar in E2F1+ and E2F1-/- mice (Fig. 6B)Citation . Thus, antigen activation of immature T cells results in the activation of some transcriptional targets independent of E2F1. These results are important because they eliminate the possibility that the absence of negative selection in E2F1 mutant mice is due to a failure of OVA to accumulate and be presented in the thymus. As a further indication that apoptosis during negative selection is distinct from DNA damage- or GC-induced apoptosis, Bfl-1 message levels are not induced by either of the latter two stimuli. In fact, a reproducible 2-fold reduction in Bfl-1 mRNA levels was observed after Adr treatment (Fig. 6C)Citation . Although the Bax gene has been shown to be regulated by p53 (50) , the induction of p53 in the thymus by DNA damage or TCR stimulation did not result in substantial Bax activation (Fig. 6)Citation .



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Fig. 6. Induction of Bfl-1 expression on TCR stimulation is not E2F1 dependent. DO TCR transgenic littermates (C57Bl/6) of the indicated E2F1 genotypes were injected with 0.5 mg of OVA or CON peptides, and thymus cells were isolated at the indicated times after injection. A, Bfl-1 expression is induced on TCR activation. RNA was isolated from E2F1+ mice, and Bfl-1, Bax, and GAPDH RNA levels were determined by RPA. The levels of Bfl-1 and Bax mRNAs (normalized for GAPDH expression) relative to that of a CON-injected mouse are shown. The data shown here are from an experiment using Rag2-/- mice. B, Bfl-1 induction after TCR activation is E2F1 independent. Thymus cells were harvested 10 h pi (the same samples as in Fig. 5BCitation ), and Bfl-1, Bax, and GAPDH RNA levels were determined as described in A. C, Bfl-1 expression is not induced in thymus cells treated with Adr or Dex. Thymocytes were isolated and cultured as described in Fig. 3ACitation . Bfl-1, Bax, and GAPDH RNA levels were determined as described in A.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have shown that E2F1 functions upstream of ARF and p53 in a thymic apoptotic pathway that contributes to the elimination of autoimmune T cells. ARF mRNA and p53 protein levels are increased during negative selection, and this increase is dependent on the E2F1 transcription factor. Given the demonstrated roles for ARF and p53 in the induction of apoptosis, our data suggest that ARF and p53 may contribute to TCR-mediated thymocyte apoptosis. However, these data do not imply that ARF and p53 are required for negative selection. Other targets of E2F1 may redundantly contribute to this apoptosis because overexpression of E2F1 can induce apoptosis independent of p53 (14 , 15) , and ARF activation by the DMP1 transcription factor induces cell cycle arrest, not apoptosis (51) . Nonetheless, given the presence of potential E2F-responsive elements in the ARF promoter (24 , 25) and the demonstrated association of ARF with mdm2/p53, our data, together with the data of others, support the model shown in Fig. 7Citation . TCR stimulation and/or costimulatory signals result in the activation of E2F1 through an as yet undefined pathway. E2F1 transcriptionally activates the expression of ARF, which interferes with Mdm2 function, resulting in the stabilization and potentiation of p53 (46) . p53 then contributes to apoptosis by regulating gene transcription and possibly also through transcription-independent effects (16) . Antigen stimulation of immature thymocytes can also activate other pathways, such as the NF-{kappa}B pathway leading to the transcriptional activation of Bfl-1, that are independent of E2F1. DNA damage results in p53 accumulation and apoptosis (52) , independent of E2F1 (this work) and ARF (53) . Finally, more robust or prolonged TCR signals may also induce negative selection through pathways that do not require E2F1. Our data does not address how E2F1 activity is controlled during thymic negative selection. E2F1 activity may be regulated by Rb and G1 cdk activity analogous to its control during G1 to S phase in fibroblasts. Pharmacological inhibition of cdk2 activation blocks thymocyte apoptosis induced by TCR stimulation, irradiation, or GCs in vitro (54) . Inhibition of cdk2 activity also prevents caspase-mediated cleavage of Rb in response to Dex or irradiation. Further work will be required to delineate the pathway that controls E2F1 activation during thymic negative selection.



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Fig. 7. Model for the E2F1-dependent pathway controlling negative selection. TCR stimulation and/or costimulatory signals result in the activation of E2F1, which transcriptionally activates the expression of ARF. ARF interferes with Mdm2 function, resulting in the stabilization and potentiation of p53. DNA damage results in p53 accumulation and apoptosis, independent of E2F1 and ARF. TCR signaling can also induce apoptosis in thymocytes through pathways that do not require E2F1.

 
Multiple factors that influence apoptosis during thymic negative selection have been identified. TNF-R-mediated signaling appears to serve as a costimulatory signal for negative selection (55) . The interaction of gp39 with CD40, a TNF-R family member expressed on APCs, is required for negative selection mediated by either vSAGs or TCR transgene recognition of endogenously expressed antigen (42) . However, inhibition of gp39/CD40 interaction did not affect negative selection mediated by TCR recognition of exogenous antigen. Fas, another TNF-R family member, may also play a role in thymic negative selection (38) , particularly at a higher antigen dose (56) , although the role of Fas in negative selection is still controversial. The CD30 TNF-R family member also contributes to the apoptosis of autoreactive immature thymocytes. Whereas CD30-deficient mice are resistant to thymocyte negative selection induced by the endogenous male antigen recognized by the H-Y transgenic TCR, negative selection of thymocytes that recognize endogenous vSAGs is unperturbed (57) . In contrast, the deletion of thymocytes bearing Vß5 chains reactive with vSAGs is reduced in E2F1 mutant mice. E2F1 deficiency does not completely abrogate this deletion, perhaps due to the chronic nature of the antigen or because of the reported differences in the TCR signaling pathways activated by vSAGs versus peptide/MHC (58 , 59) .

The involvement of other transcription factors in negative selection has also been demonstrated. A dominant negative Nur77 transgene, which inhibits all three known members of the Nur77 orphan steroid receptor family, reduced negative selection induced by transgenic TCR recognition of a peptide antigen but did not affect vSAG-mediated thymocyte deletion (60) . In contrast to its traditional role in mediating cell survival, NF-{kappa}B appears to be required for thymic negative selection because the expression of an I{kappa}B transgene abrogates anti-CD3-induced apoptosis in the thymus (61) . Whereas anti-CD3 can activate the TCR, antigen presentation by thymic stromal cells normally entails multiple interactions between additional cell surface molecules, such as B7/CD28, LFA-1/intercellular adhesion molecule 1, and tumor necrosis factor/TNF-R family members, that can contribute to apoptotic signaling (42 , 62, 63, 64) . Indeed, whereas TNF-R I/II-deficient mice showed impaired thymocyte apoptosis in response to anti-CD3, antigen-mediated clonal deletion was unimpaired (65) . Thus, the requirement for different players in negative selection appears to vary, depending on the nature of the recognized antigen, the MHC class, and the acute versus chronic presence of the antigen (66) . Whether deletion occurs in immature DP thymocytes in the thymic cortex or in late-stage DP and immature CD4 single positive thymocytes in the medulla may in part underlie the differential requirement for particular signaling components. Thus, negative selection is not as simple as each TCR model implies.

The important role of the E2F1-ARF-p53 pathway in the control of lymphocyte cell death is underscored by the high susceptibility of mice disrupted in E2F1 (8) , ARF (53) , or p53 (67 , 68) to the development of lymphomas and the frequent mutation of ARF and p53 in human tumors (16 , 46) . Although E2F1-/- mice are tumor prone, E2F1 deficiency reduces pituitary and thyroid tumorigenesis in Rb+/- mice (69) , possibly reflecting the critical role for E2F1 in promoting proliferation resulting from Rb inactivation. Recent experiments have also demonstrated a requirement for E2F1 in p53-dependent apoptosis and excess proliferation resulting from either the expression of transgenic polyoma virus large T antigen in the mouse choroid plexus epithelium (70) or the absence of the Rb gene product during mouse embryonic development (71) . Whereas the other E2F family members can largely compensate for the absence of E2F1 during mouse development, the absence of E2F1 alone appears to substantially compromise the aberrant proliferation that results from Rb inactivation. In addition, p19ARF/p16INK4A deficiency attenuates the apoptosis that occurs in the Rb-/- mouse lens without decreasing proliferation (28) , suggesting that E2F1 targets other than ARF contribute to the proliferative role of E2F1. The data presented here suggest that the E2F1-ARF-p53 pathway that functions to eliminate transformed cells may also contribute to the elimination of autoimmune T cells. Indeed, E2F1-/- mice that exhibit lymphoproliferation syndrome also display glomerulonephritis of the kidneys (8) , which is indicative of autoimmunity. Despite a defect in thymic negative selection, E2F1-/- mice may avoid a more severe autoimmune response as a result of peripheral mechanisms that induce tolerance.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Mice.
Mice were housed in the University of Colorado Health Sciences Center animal resource center in cages with micro-isolator lids. RAG2-/- and DO transgenic mice were created by the F. Alt (Harvard Medical School, Boston, MA) and D. Loh (Nippon Roche Research Center, Kanagawa, Japan) laboratories, respectively, and obtained from P. Marrack (National Jewish Hospital, Denver, CO). E2F1-/- mice were provided by S. Fields and M. Greenberg (Harvard Medical School). Immunocompromised RAG2-/- mice were maintained under sterile conditions. Mice were genotyped (for E2F1, RAG2, DO TCR, and H-2 genotype) by PCR analysis of DNA extracted from a small ear biopsy. Genomic DNA was isolated as described previously (72) . Peptides were produced by the Molecular Resources Center at the National Jewish Hospital.

Flow Cytometry.
Single cell suspensions obtained from thymuses or LNs were strained through nylon mesh and washed in PBS containing 5% fetal bovine serum (FBS/PBS). Cells (5 x 106) were stained in 30 µl of CD4/CD8/DO TCR or CD4/CD8/Vß antibody solution 1:100 PE-{alpha}-CD4 (PharMingen #09005B), 1:100 Cy-Chrome-{alpha}-CD8 (PharMingen #01048A), 1:200 {alpha}-Fc{gamma} III/II receptor (PharMingen #01241A), and 8 µg/ml FITC-conjugated KJ1.26 monoclonal antibody or 1:100 FITC-conjugated {alpha}-Vß antibody (PharMingen) in FBS/PBS for 45 min on ice. Cells were washed twice with 1 ml of FBS/PBS and resuspended in 400 µl of PBS. For annexin V/PI staining, thymocytes were cultured in RP10 [10% FBS (Hyclone) in RPMI 1640 with 0.1 mM 2-mercaptoethanol and 1% penicillin-streptomycin (Life Technologies, Inc.)] in the presence of Dex, Adr, or DMSO; harvested; and washed once in PBS. Cells (1 x 106) were stained in 100 µl of binding buffer [10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2] containing 0.5 µg of PI (Boehringer Mannheim) and 1.0 µg of FITC-annexin V (Caltag Laboratories) for 15 min on ice. Samples were then diluted with an additional 200 µl of binding buffer. Fluorescence was detected and analyzed using a Coulter Epics XL flow cytometer (Beckman Coulter).

Western Blotting and RPAs.
RNA and cell lysates were prepared using Trizol reagent (Life Technologies, Inc.) according to the manufacturer’s instructions. {alpha}-p53 (#sc6243) and {alpha}-actin (#sc1616) antibodies were purchased from Santa Cruz Biotechnology and used at 0.4 and 0.2 µg/ml, respectively. Western blots were performed as per the manufacturer’s instructions, except that 0.2% Tween 20 was included in the antibody solutions and washes. Quantitation of the resulting autoradiograms was performed using a Molecular Dynamic densitometer. Levels of cyclin E RNA, cyclin A RNA, or Bfl-1/Bax RNA were measured using the PharMingen RiboQuant Multi-Probe RPA System and the mCYC-2, mCYC-1, or mAPO-2 template sets, respectively. ARF RNA levels were determined by the same method using the pCDNA3 plasmid (Invitrogen) containing ARF exon 1 sequences (nt 4–155 of the ARF ORF) as a template for the ARF probe and the mGAPDH probe (PharMingen) as an internal control. Dried radioactive polyacrylamide gels were exposed to Kodak X-OMAT film (shown in figures) or exposed and analyzed on a PhosphorImager to quantitate mRNA levels.


    Acknowledgments
 
We thank S. Fields, M. Greenberg, T. Mitchel, D. Hildeman, P. Marrack, J. Kappler, M. McHeyzer-Williams, R. Leon, K. Murphy, C. Sherr, R. Sclafani, M. Kimbrough, D. Wegmann, and J. Nevins for important reagents, assistance, and/or advice; K. Helm, P. Schor, and M. Ashton of the Cancer Center Flow Cytometry Core (supported by Grant 2 P30 CA 46934-09); and P. Skavlen and Center for Laboratory Animal Care for excellent veterinary care.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 D. D. is supported by a NIH postdoctoral fellowship. J. D. is supported by grants from the V Foundation, the Howard Hughes Medical Institute, and the NIH (Grant RO1 CA77314-01). Back

2 To whom requests for reprints should be addressed, at Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, BRB802, Mail Box C229, 4200 East Ninth Avenue, Denver, CO 80262. Phone: (303) 315-5792; Fax: (303) 315-3244; E-mail: james.degregori{at}uchsc.edu Back

3 The abbreviations used are: TCR, T-cell receptor; vSAG, viral superantigen; pi, postinjection; DP, double positive; Rb, retinoblastoma protein; OVA, ovalbumin peptide; Vß, TCR ß chain variable region; RPA, Ribonuclease Protection Assay; GC, glucocorticoid; Dex, dexamethasone; PI, propidium iodide; Adr, Adriamycin; LN, lymph node; APC, antigen-presenting cell; NF-{kappa}B, nuclear factor {kappa}B; cdk, cyclin-dependent kinase; TNF-R, tumor necrosis factor receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back

Received for publication 9/17/99. Accepted for publication 11/ 1/99.


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