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Molecular and Cellular Biology, December 2001, p. 8547-8564, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8547-8564.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

E2F1 and E2F2 Determine Thresholds for Antigen-Induced T-Cell Proliferation and Suppress Tumorigenesis

Jing W. Zhu,1 Seth J. Field,2,dagger Lia Gore,3 Margaret Thompson,2 Haidi Yang,2,4 Yuko Fujiwara,2,4 Robert D. Cardiff,5 Michael Greenberg,2 Stuart H. Orkin,2,4 and James DeGregori1,3,6,*

Department of Biochemistry and Molecular Genetics,1 Integrated Department of Immunology,6 and Department of Pediatrics,3 University of Colorado Health Sciences Center, Denver, Colorado 80262; Howard Hughes Medical Institute4 and Division of Neuroscience, Children's Hospital,2 and Department of Neurobiology, Harvard Medical School and Dana Farber Cancer Institute, Boston, Massachusetts 02115; and Center for Comparative Medicine and Department of Pathology, School of Medicine, University of California at Davis, Davis, California 956165

Received 9 August 2001/Returned for modification 24 September 2001/Accepted 2 October 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

E2F activity is critical for the control of the G1 to S phase transition. We show that the combined loss of E2F1 and E2F2 results in profound effects on hematopoietic cell proliferation and differentiation, as well as increased tumorigenesis and decreased lymphocyte tolerance. The loss of E2F1 and E2F2 impedes B-cell differentiation, and hematopoietic progenitor cells in the bone marrow of mice lacking E2F1 and E2F2 exhibit increased cell cycling. Importantly, we show that E2F1 and E2F2 double-knockout T cells exhibit more rapid entry into S phase following antigenic stimulation. Furthermore, T cells lacking E2F1 and E2F2 proliferate much more extensively in response to subthreshold antigenic stimulation. Consistent with these observations, E2F1/E2F2 mutant mice are highly predisposed to the development of tumors, and some mice exhibit signs of autoimmunity.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

E2F activity controls the transcription of a group of genes that are normally regulated at the G1/S transition and that 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 (12). 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 two different DP proteins. E2F1, -2, and -3 associate specifically with the retinoblastoma protein (Rb). Based on the induction of these E2Fs in late G1 phase and the ability of overexpressed E2F1, E2F2, and E2F3 to transcriptionally activate positive cell cycle regulators and to induce S-phase entry in quiescent fibroblasts, E2F1, E2F2, and E2F3 are thought to function as positive regulators of cell cycle progression (12, 19). E2F4 and E2F5 appear to associate with all three Rb family members, Rb, p107, and p130, and these E2Fs are expressed throughout the cell cycle. The resulting Rb-E2F interactions not only block transcriptional activation by E2F but also form active transcriptional repressor complexes at promoters that can block transcription by recruiting histone deacetylase and remodeling chromatin (19).

In Drosophila melanogaster, mutation of either the dE2F or dDP gene results in attenuated S phases (11, 41). dE2F1 mutant embryos exhibit greatly reduced activation of E2F target genes (such as those for RNR2, cyclin E, and Mcm3), indicating an important role for dE2F in the transcriptional activation of some S-phase genes. The disruption of E2Fs in mice has revealed distinct roles for E2F family members in mouse development and physiology. The analyses of mouse embryo fibroblasts (MEFs) isolated from E2F3-/- mice reveal an important role for E2F3 in G1 to S phase progression and the efficient induction of multiple target genes, including the Cdc6 gene (22). In addition, the loss of E2F2 or E2F3 compromises S-phase entry in response to overexpressed c-Myc protein in MEFs (26).

Mutations in E2F4 and E2F5 have been less revealing in terms of the roles for these family members in the transcriptional regulation of cell proliferation. E2F5-/- mice develop nonobstructive hydrocephalus as newborns, but the proliferation of E2F5-/- MEFs appears normal (27). E2F4-/- mice are runted, have defective late-stage erythropoiesis, and show craniofacial defects (21, 40). However, proliferation and E2F target gene regulation in E2F4-/- MEFs and primary lymphocytes are unperturbed. Surprisingly, E2F4-/- E2F5-/- MEFs show normal serum starvation-induced growth arrest, proliferation kinetics following serum stimulation, and E2F target gene regulation, but are defective in their ability to arrest in response to p16-INK4A expression (16).

The pathway leading to E2F activation is deregulated in most human tumors either by potentiation of cyclin D-dependent kinase activity or by loss of the retinoblastoma tumor suppressor (36). Surprisingly, the disruption of E2F1 in the mouse results in the genesis of a diverse range of tumors in older adults (49); 19% of E2F1-/- mice develop tumors by 18 months of age, which is significantly increased over the expected tumor incidence of ca. 10% in wild-type mice. E2F1-/- mice also exhibit testicular atrophy with age (49). It is not clear how E2F1 loss contributes to testicular atrophy, which could result from increased apoptosis, decreased proliferation, decreased differentiation of progenitors, or various noncell autonomous effects.

Although E2F1-/- mice are tumor prone, E2F1 deficiency reduces pituitary and thyroid tumorigenesis in Rb+/- mice (48), possibly reflecting the critical role for E2F1 in promoting proliferation resulting from Rb inactivation. In addition, overexpression of E2F1 can contribute to tumorigenesis in mouse models of skin carcinogenesis (24, 39). Finally, E2F1 is required for the p53-dependent apoptosis and excess proliferation resulting from either the expression of transgenic polyomavirus large T antigen in the mouse choroid plexus epithelium (38) or the absence of the Rb gene product during mouse embryonic development (44). Whereas other E2F family members can largely compensate for the absence of E2F1 during mouse development, the absence of E2F1 appears to substantially compromise both the aberrant apoptosis and proliferation that result from Rb inactivation. Intriguingly, the loss of E2F3 abrogates both p53-dependent and, to a lesser extent, p53-independent apoptosis as well as excess proliferation resulting from Rb loss during embryogenesis (51), indicating that both E2F1 and E2F3 are required for the increased proliferation and apoptosis resulting from Rb loss.

When naive mature T cells are challenged by antigens, a series of signal transduction events are triggered, leading to T-cell proliferation and differentiation (1). T-cell responses are determined by interactions between their T-cell receptors (TCRs) and peptide/major histocompatibility complexes (MHCs) on the surface on antigen-presenting cells. T-cell activation requires additional signals beyond that provided by the interaction of MHC/peptide antigen-presenting cells with the TCR (1). These costimulatory signals include the interaction of CD28 on T cells with B7-1 and B7-2 on antigen-presenting cells, which amplifies TCR-generated signals and also activates distinct signaling pathways. Both the affinity and avidity of antigen for the T-cell receptor and the requirement for costimulation contribute to the proper discrimination of self from nonself and the maintenance of lymphocyte tolerance.

The expression levels of E2F1 and E2F2 are very low in naive T cells and are substantially induced in late G1 following T-cell activation (31). The loss of E2F1 does not affect either the basal or mitogen-stimulated proliferation of peripheral T cells or thymocytes from younger mice. However, older (6 to 12 months) E2F1-/- mice exhibit increased proliferation of thymocytes but not mature lymphocytes (13). The loss of E2F1 may indirectly affect thymocyte proliferation and eventually tumorigenesis, either due to the loss of E2F1 dependent apoptosis or in conjunction with other events that would normally reveal negative roles for E2F1 in proliferation control.

The elimination of immature and mature autoreactive T cells via apoptosis is an essential mechanism to prevent autoimmunity. E2F1 has been shown to play a critical role in the elimination of self-reactive immature T cells during thymic negative selection (13, 15, 50). This TCR-mediated apoptosis coincides with the E2F1-dependent increase in p19-ARF mRNA and p53 protein levels (50). Furthermore, repeated antigenic stimulation of mature T cells results in activation-induced cell death (AICD) (45). Roles for E2F1 and p73 in AICD have recently been demonstrated (28) and may involve direct E2F1-dependent activation of p73 expression (23, 43).

In this paper, we show that E2F1 and E2F2 function redundantly to regulate hematopoietic cell proliferation and differentiation and to determine thresholds for antigen-induced T-cell proliferation. We propose that E2F1 and E2F2 function as components of a critical negative feedback loop that prevents S-phase entry following inappropriate antigenic stimulation. The important roles for E2F1 and E2F2 in limiting cell proliferation are underscored by the development of tumors and possibly autoimmunity in E2F1/E2F2 mutant mice.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Mice. Mice were housed in the University of Colorado Health Science Center (UCHSC) animal resource center in cages with microisolator lids. D011.10 (D0) TCRO transgenic mice were created by K. Murphy and D. Loh and Rag2-/- mice were created by F. Alt and obtained from P. Marrack. Mice were genotyped (for E2F1, E2F2, Rag2, and DO TCR genotype) by PCR analysis of DNA extracted from a small ear biopsy. Rag2 mutant mice were housed under sterile conditions. OVA peptide (ISQAVHAAHAEINEAGR) was produced by Research Genetics. All animal experiments were approved by the UCHSC Animal Care and Use Committee.

Histology. Autopsies were performed on morbid mice, and all major organs (and any tumors) were fixed in 10% formalin in phosphate-buffered saline (PBS) overnight. Tissues were then transferred into 70% ethanol until sectioning. Sections (5 µm) were cut and processed by routine hematoxylin and eosin (H&E) staining. CD4 immunostains were performed using anti-CD4 (1:200; Pharmingen RM4-5) and biotin-linked anti-rat immunoglobulin G (IgG) (1:200; Vector Labs), followed by streptavidin-linked horseradish peroxidase and development using the Elite ABC kit as per the manufacturer's instructions.

Cell culture. Single-cell suspensions obtained from spleens or lymph nodes were strained through nylon mesh, washed with PBS, and then cultured in RP10 (10% fetal bovine serum [FBS; Hyclone] in RPMI 1640 with 0.1 mM 2-mercaptoethanol and 1% penicillin-streptomycin [Gibco-BRL]) at 37°C in 5% CO2. Antigen (OVA peptide), Concanavalin A (ConA; Sigma), and/or interleukin-2 (IL-2; recombinant human) were added to the cultures at the concentrations indicated in the text. Bone marrow cells were cultured for 2 h in 10% FBS in Iscove's modified Dulbecco's medium (IMDM) (Gibco-BRL) with 10 µM bromodeoxyuridine (BrdU). Gamma interferon (IFN-gamma ) levels were determined by immunoassay using R46-A2 (BD-Pharmingen) as the capture antibody and biotinylated XMG1.2 (BD-Pharmingen) for detection. Streptavidin europium (Wallac) was added (500 ng/ml, final) for 30 min, plates were washed four times and blotted dry, and 200 µl of enhancement solution (Wallac) was added. Europium fluorescence was quantitated on a Wallac1232 Delfia fluorometer.

Flow cytometry. Hematopoietic single-cell suspensions were washed in PBS containing 5% FBS (FBS/PBS). Cells (106) were stained in 30 µl of antibody solution (1:100 of each antibody unless otherwise stated) for 45 min on ice. Cells were washed twice with 1 ml of FBS/PBS and resuspended in 400 µl of PBS. The following Pharmingen antibodies were used: phycoerythrin (PE)-linked alpha -CD4, Cy-Chrome-alpha -CD8, PE-alpha -CD25, biotin-alpha -CD25 (together with streptavidin-Cy-Chrome), allophycocyanin (APC)-linked alpha -B220, APC-alpha -Thy1.2, APC-alpha -GR-1, APC-alpha -Ter119, APC-alpha -CD34, PE-alpha -CD43, fluorescein isothiocyanate (FITC)-linked TCR variable-chain beta (Vbeta ) 5, FITC-TCR Vbeta 6, and FITC-CD44. FITC-conjugated KJ1.26 (anti-DO11.10 TCR) and B7.6 (anti-IgM) monoclonal antibodies were also used. BrdU incorporation was detected using FITC-linked anti-BrdU (Pharmingen) according to the manufacturer's protocols.

For propidium iodide (PI) staining, cells stained with antibodies as above (APC linked in each case) were fixed with 1% paraformaldehyde in PBS and then resuspended in 20 µg of PI per ml in PBS with 10 µg of RNase A. For carboxyfluorescein diacetate succinimidyl ester (CFSE) staining, 107 cells/ml were incubated with 3 µM CFSE (Molecular Probes) in PBS for 15 min at 37°C, washed with PBS, washed with RP10 for 30 min, and then cultured in RP10 for 3 days. Cells were harvested and stained with APC-linked alpha -B220. In each case, fluorescence was detected and analyzed using a Coulter Epics XL (Beckman Coulter) or FACSCalibur (Becton Dickinson) flow cytometer.

Western blotting and RNase protection assays. RNA and protein were prepared from cells using Trizol reagent (Gibco-BRL) according to the manufacturer's instructions. Antibodies used were alpha -E2F3 (SC-878 at 0.2 µg/ml) and alpha -tubulin (NeoMarkers MS-719-PI at 0.4 µg/ml). Western blots were performed as per the manufacturer's instructions except that 0.2% Tween 20 was included in the antibody solutions and washes. Levels of cell cycle regulator mRNAs were measured using the Pharmingen RiboQuant multiprobe RNase protection assay system and a custom template set. Dried radioactive polyacrylamide gels were exposed to Kodak X-Omat film.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

E2F1 and E2F2 regulate the development and proliferation of hematopoietic progenitors in bone marrow. In order to assess potential functional redundancy between E2F1 and E2F2, we bred a null E2F2 mutation into mice disrupted for E2F1, and these mutations were further backcrossed into the BALB/cJ background for two to four generations. The generation of the E2F1 and E2F2 knockout mice has been described previously (13, 26). The homozygous E2F1 and E2F2 disruptions result in the absence of detectable E2F1 and E2F2 protein expression, respectively, in embryo fibroblasts (26).

E2F1 and E2F2 double-knockout (DKO) (E2F1-/- E2F2-/-) mice were born at the expected frequency and were similar in size and appearance to their littermates. We observed up to twofold-reduced cellularity in the bone marrow, thymus, lymph nodes, and spleens of DKO and, to a lesser extent, E2F1+/- E2F2-/- mice (data not shown). In all experiments in this study, we compared same-sex littermates unless otherwise noted. Also, the cells used in all of the experiments in this study were from young mice (4 to 7 weeks old) in order to avoid long-term complications from the mutations of E2F1 and E2F2. In many experiments in this study, E2F1+/- E2F2+/- cells and mice were used as the controls for DKO cells and mice. The chance of obtaining wild-type (WT) and DKO mice from breeding two E2F1+/- E2F2+/- mice is very small (1 in 256), and we only achieved this once (see Fig. 1B and Fig. 2A). Since it is very important that we compare littermates in our experiments, we obtained DKO mice by breeding E2F1+/- E2F2-/- and E2F1-/- E2F2+/- mice and used the resulting E2F1+/- E2F2+/- mice as controls. However, we observed at best a modest enhancement of proliferation in E2F1+/- E2F2+/- lymphoid cells compared to WT cells (Fig. 2A, Fig. 5C, and data not shown).

B cells and other hematopoietic progenitors develop in the bone marrow. Cells that are at different stages of development can be distinguished by the expression of characteristic cell surface markers (5). Cells committed to the B-cell lineage express B220 (CD45R), a membrane tyrosine phosphatase, at all stages of development, and the levels increase during the transition from the immature to the mature stage. The earliest B cells are characterized by expression of B220 and CD43 in the absence of detectable IgM and CD25 (pre-pro-B) (A and B in schemata in Fig. 1B). In response to cytokines and initial Ig chain rearrangement, these cells mature to the progenitor stage (C and C'). Thereafter, signals derived from the pre-B-cell receptor complex stimulate further maturation (D), eventually giving rise to immature B cells (E). These cells then migrate to peripheral lymphoid tissues, where they become mature B cells (F) (5).


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  		FIG. 1.   Loss of E2F1 and E2F2 results in reduced B-cell differentiation. (A) Bone marrow cells from E2F1+/- E2F2+/- and E2F1-/- E2F2-/- 5.5-week-old male littermates were harvested and stained with fluorescently labeled anti-B220, anti-CD25, anti-CD43, and anti-IgM. Cells were analyzed by flow cytometry. Bone marrow cells were gated for the expression of B220 and CD25 (left panels) or for B220 but not CD25 (right panels). The percentages of cells that are either CD43+ IgM- (more immature) or CD43- IgM+ (more mature) relative to total B220+ cells are indicated. (B) The fraction of cells in different stages of B-cell development as determined by flow cytometry (as in A) for 6-week-old male littermates of the indicated E2F1 and E2F2 genotypes is shown. A representative experiment is shown out of three experiments using littermates of all indicated genotypes and four additional experiments comparing bone marrow cells from E2F1+/- E2F2+/- and DKO littermates. The number of cells at each stage of B-cell development was determined for bone marrow cells isolated from the femurs and tibias of both hind legs. The indicated percentages represent the fraction of cells at each stage relative to the total B220+ population. The percentages do not add up to 100% because some B220+ cells did not clearly delineate into a defined subpopulation. The scheme of B-cell development is based on the model presented in Benschop et al. (5).

To examine the effects of the loss of E2F1 and E2F2 on hematopoietic cell development, bone marrow cells were harvested from mice of the indicated genotypes and analyzed for the expression of B220, CD25, CD43, and IgM (Fig. 1). While the cellularity of the more immature progenitors (pre-pro-B and pro-B) was not significantly reduced, at the later stages of B-cell development a substantial reduction in cellularity was evident in DKO bone marrow (ca. twofold reduction at the small pre-B-cell stage and a sixfold reduction at the IgM+ stage). Notably, E2F1+/- E2F2-/- mice consistently showed an intermediate reduction in B-cell maturation (Fig. 1B). Thus, the loss of E2F1 and E2F2 results in a reduction in the more mature B-cell progenitor cellularity during B-cell development. Decreased B-cell maturation could reflect roles for E2F1 and E2F2 in differentiation, perhaps in conjunction with Rb. Alternatively, changes in B-cell progenitor populations in DKO mice could result from increased apoptosis rates as B-cell progenitors mature.

Considering the critical roles of E2F transcription factors in regulating the cell cycle, we tested the effect of E2F1 and E2F2 loss on hematopoietic cell proliferation. Freshly isolated bone marrow cells from mice of the various E2F1/E2F2 genotypes were stained with fluorescently tagged antibodies that bind to cell surface proteins. The fixed and permeabilized cells were stained with PI. The DNA content of the cells was determined by flow cytometric analysis of PI intensity in cells gated for the expression of identifying hematopoietic cell surface markers (Fig. 2A). Surprisingly, we observed substantially increased percentages of cells in S phase of immature B cells (B220 low), mature B cells (B220 hi), erythroid (TER119+), myeloid (GR-1+) and multipotent progenitor cells (CD34+) in the bone marrow of DKO mice relative to WT and E2F1+/- E2F2+/- mice (Fig. 2A). This increased percentage of hematopoietic progenitors in S phase correlates with increased DNA synthesis, as measured by BrdU incorporation (Fig. 2B).


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  		FIG. 2.   Loss of E2F1 and E2F2 results in hyperproliferation of hematopoietic progenitors. (A) Bone marrow cells from 6-week-old male littermates of the indicated genotypes were harvested and stained with either fluorescently labeled anti-B220, anti-GR-1, anti-CD34, or anti-TER119, fixed, and then stained with PI. Cell cycle profiles (determined by PI intensity) of the different bone marrow subsets gated for the expression of the indicated marker are shown. The percentage of cells in either the G1 (first solid peak), S (hatched), or G2 (second solid peak) phase was determined using the ModFit program. The percentage of cells in S phase is indicated. The y axis represents cell number, and the x axis represents PI fluorescence intensity. This experiment is representative of more than four experiments. (B) Bone marrow cells from mice of the indicated genotypes were harvested and cultured with 1µM BrdU in 10% FBS-IMDM medium for 2 h. The cells were collected and stained with fluorescently labeled anti-BrdU. BrdU incorporation was detected by flow cytometry. In three similar experiments, DKO bone marrow cells exhibited a 2.5 ± 0.5 (standard error [SE])-fold increase in BrdU incorporation compared to bone marrow from E2F1+/- E2F2+/- littermates. (C) Bone marrow cells were harvested from Rag2-/- mice of the indicated E2F1/E2F2 genotypes (the right three panels are from littermates) and analyzed as in A for cell cycle profiles in the B220+ subset.

Interestingly, we consistently observed an intermediate increase in proliferation in E2F1+/- E2F2-/- but not E2F1-/- E2F2+/- mice (Fig. 2A and 2B). Thus, the loss of E2F1 and E2F2 increases the proliferation of hematopoietic progenitors. These results are surprising, as E2F1 and E2F2 are thought to positively regulate cell cycle progression. Indeed, overexpression of E2F1, E2F2, or E2F3 in quiescent fibroblasts is sufficient to activate the expression of a variety of positive cell cycle regulators and drive cells into S phase (10, 46).

In order to determine if the increased proliferation in DKO B-cell progenitors is due to increased percentages of the more immature pro-B cells, we bred the E2F1 and E2F2 disrupted genes into mice with mutations in the Rag2 gene (42). Rag2 is an essential component of the recombinase that mediates the assembly of either TCR or immunoglobulin chains from germ line arrays of exons (37). Thus, T- and B-cell development is blocked prior to the VDJ DNA rearrangement stage, and therefore, there are no mature T and B cells in Rag2-/- mice. B-cell development is similarly arrested at the pro-B cell stage in Rag2-/- mice with or without mutation of E2F1 and E2F2 (data not shown). Importantly, we still observed increased pro-B-cell proliferation in DKO Rag2-/- and E2F1+/- E2F2-/- Rag2-/- mice (Fig. 2C), indicating that the increased B-cell progenitor proliferation caused by loss of E2F1 and E2F2 is not stage specific.

Loss of E2F1 and E2F2 results in deregulated T-cell proliferation. Due to heterogeneity of the cells in the bone marrow as well as possible influences of the bone marrow microenvironment on cell proliferation, we used peripheral T cells from DKO mice to further study the roles of E2F1 and E2F2 in the regulation of cell proliferation. We first examined the cellular composition of the thymus and peripheral lymphoid organs. Lymphocytes were harvested from either the thymus, spleen, or lymph nodes (combined submandibular, axillary, peri-aortic, mesenteric, and inguinal). The cells were stained with fluorescent compound-linked antibodies to cell surface proteins in order to distinguish distinct subsets of T cells.

The percentage of immature CD4+ CD8+ thymocytes in E2F1+/- E2F2-/- mice was modestly lower than in control littermates (Fig. 3A, right panels). The composition of the peripheral lymphoid organs (spleen and lymph nodes) was very similar regardless of E2F1 and E2F2 genotypes (Fig. 3A, left panels; shown for lymph nodes, but similar for spleen). The percentages of singly CD4- and CD8-positive cells in the periphery were indistinguishable between mice of different E2F1/E2F2 genotypes. The percentage of spleen cells expressing B220 (B cells) and the distribution of these cells expressing IgM and IgD was also not affected by E2F1/E2F2 genotype (data not shown). Furthermore, the expression of the TCR (shown for the transgenic TCR DO11.10), lymphocyte activation markers (CD25 and CD69), and a marker of memory T cells (CD44) was similar on T cells from mice of the different E2F1 and E2F2 genotypes (Fig. 3B; see also Fig. 4 and 5). Thus, the loss of E2F1 and E2F2 does not significantly affect the expression of lymphocyte lineage and activation markers on peripheral T cells.


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  		FIG. 3.   Loss of E2F1 and E2F2 does not significantly affect the expression of T-cell markers. (A) Lymphocytes were harvested from the lymph nodes (left four panels) or the thymus (right four panels) from 6-week-old male littermates of the indicated genotypes, stained with fluorescent compound-linked antibodies to CD4 or CD8, and analyzed by flow cytometry. The percentage of cells positive for the expression of CD4 and/or CD8 is shown, representing the average ± SE of three experiments for lymph nodes and six experiments for thymus using sex-matched littermates. The differences in percentages of CD4+ CD8+ thymocytes was significant when comparing E2F1+/- E2F2+/- littermates to E2F1+/- E2F2-/- littermates (P = 0.019), but not when comparing E2F1+/- E2F2+/- littermates to DKO littermates (P = 0.051). Observed differences in the percentages of CD4+ or CD8+ singly positive T cells in either the thymus or lymph nodes were not statistically significant. (B) Lymph node T cells processed as in A and gated for the expression of CD4 were analyzed for the expression of either the DO11.10 TCR or CD44 by flow cytometry. Mice in the left panel are DO11.10 transgenic. The average fluorescence intensity of the cells expressing CD44 is indicated, and the average intensity of CD44 expression in E2F1+/- E2F2+/-, E2F1+/- E2F2+/-, and E2F1+/- E2F2+/- CD4+T cells was 22.1, 21.0, and 20, respectively (flow profile not shown for these littermates).

We examined T-cell proliferation ex vivo, removed from other complicating effects of the mutations in vivo. We first analyzed the proliferation of T cells of the various E2F1/E2F2 genotypes in response to stimulation with the lectin ConA, which crosslinks T-cell surface proteins and thereby stimulates proliferation. Lymphocytes were isolated from mice of the indicated genotypes and cultured in the presence of ConA and IL-2. BrdU was added for the indicated time interval, the cells were harvested, and BrdU incorporation was determined by flow cytometry using a FITC-linked anti-BrdU antibody.

Following ConA stimulation, a substantially greater percentage of DKO lymphocytes entered S phase during each time interval than E2F1+/- E2F2+/- lymphocytes, as measured by BrdU incorporation (Fig. 4A). The deregulated proliferation of DKO T cells was also clearly evident by a direct measure of the extent of division of individual T cells using CFSE labeling (Fig. 4B). Due to the approximately twofold decrease in CFSE fluorescence after each cell division, we were able to assess the extent of division of individual T cells stained with CFSE (29). After harvesting, lymphocytes of the indicated genotypes were labeled with CFSE and cultured with ConA for 64 h. The cells were then stained with fluorescent-linked anti-B220 to distinguish B cells from T cells, and CFSE fluorescence was determined in T cells by flow cytometry.


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  		FIG. 4.   DKO T cells hyperproliferate in response to mitogenic stimulation. (A) Lymphocytes from 7-week-old male littermates of the indicated genotypes were isolated from the lymph nodes and cultured with ConA (4µg/ml) and IL-2 (50 U/ml) in RP10 medium. During the indicated time windows, BrdU was added, and the cells were harvested at the end of the window. BrdU incorporation was determined by immunofluorescence and flow cytometry. (B) T lymphocytes were isolated from the spleens of 5-week-old female littermates of the indicated genotypes. Cells were stained with CFSE and then cultured without (control) or with ConA (4 µg/ml) in RP10 for 64 h. Cells were harvested and stained with APC-alpha -B220 antibody. CFSE fluorescence was determined in T cells (B220-) by flow cytometry. The Proliferation Wizard software was used to identify cells in different generations as indicated (P, parental). IFNgamma levels (in picograms per milliliter) in the culture medium of the same cells after 64 h are indicated. Stimulated DKO cultures had three to four times as many cells after 64 h as control cultures, perhaps accounting for some of the increased IFN-gamma production. (C) Lymphocytes from the experiment in B were cultured with ConA (or unstimulated [control]) for 40 h and stained with fluorochrome-linked anti-B220 and anti-CD69 antibodies. The expression of CD69 was determined in T cells (B220- lymphocytes) by flow cytometry.

In the absence of ConA stimulation (control), T cells of all E2F1/E2F2 genotypes maintained quiescence. Almost half of the E2F1+/- E2F2+/- T cells did not proliferate at all upon ConA stimulation, and those that did progressed variably through one to four divisions. In contrast, almost all DKO T cells stimulated with ConA progressed through three to four cell divisions. These data indicate that the loss of E2F1 and E2F2 increases both the percentage of T cells that proliferate in response to mitogen and the extent of proliferation. Notably, E2F1+/- E2F2-/- lymphocytes showed an intermediate increase in S-phase entry (Fig. 4A).

Increased proliferation of E2F1+/- E2F2-/- lymphocytes was observed in multiple experiments, but not in others, and we do not currently understand this variable penetrance. In contrast, E2F1-/- E2F2+/- T cells consistently showed reduced S-phase entry and proliferation in response to ConA (Fig. 4A and 4B). Thus, E2F1 and E2F2 are not equivalent. We propose that the loss of E2F1 results in loss of a positive cell cycle E2F function, while the negative cell cycle function of E2F1 is compensated for by E2F2. In contrast, the loss of both E2F1 and E2F2 (or to a lesser extent the loss of E2F2 only) results in a complete loss of negative function which is dominant to the loss of positive functions.

Substantially increased effector cytokine (IFN-gamma ) production was also observed following TCR activation of DKO T cells (Fig. 4B), presumably as a consequence of increased passage through multiple S phases, which appears necessary for the maximal transcriptional activation of cytokines such as IL-4 and IFN-gamma (6, 17). Thus, DKO T cells not only proliferate more extensively, but also show increased effector T-cell function. In contrast, DKO cultures do not show enhanced production of IL-2 (data not shown), the expression of which is not dependent on cell cycle passage. Importantly, despite dramatic differences in proliferation, very similar increases in the expression of the activation marker CD69 occurred following TCR stimulation of T cells of the different genotypes (Figure 4C), suggesting that proximal TCR signaling pathways are similar in T cells of the different genotypes.

Loss of E2F1 and E2F2 decreases the threshold for antigen-induced T-cell proliferation. To reduce variability associated with the large repertoire of T cells bearing different TCRs and to assess proliferation in response to bona fide antigen presentation, we analyzed proliferation of lymphocytes transgenic for the DO11.10 (DO) TCR in response to presentation of the OVA peptide (chicken ovalbumin residues 323 to 339) by I-Ad-bearing antigen-presenting cells (35). We first asked whether the loss of E2F1 and E2F2 influenced the kinetics of S-phase entry following antigenic stimulation. Lymphocytes from DO transgenic E2F1+/- E2F2+/- or DKO mice were cultured with 2 µM OVA peptide and labeled with BrdU for the indicated time intervals, and BrdU incorporation was determined by immunofluorescence.

In the absence of antigen, very little S phase entry was observed in T cells from either E2F1+/- E2F2+/- or DKO mice (Fig. 5A and 5B). However, in response to OVA stimulation, DKO lymphocytes showed a more rapid entry into S phase than E2F1+/- E2F2+/- lymphocytes (Fig. 5A). Thus, the loss of E2F1 and E2F2 appears to result in a shorter G1 period prior to S-phase entry. Significantly, we did not observe obvious differences in the apoptosis of peripheral lymphocytes of the different E2F1/E2F2 genotypes following antigenic stimulation (data not shown).



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  		FIG. 5.   Loss of E2F1 and E2F2 decreases thresholds for antigen-induced T-cell proliferation. (A) Lymphocytes from 6-week-old male E2F1+/- E2F2+/- and DKO littermates were isolated from the lymph nodes and cultured with 2 µM OVA in RP10 medium. During the indicated time windows, BrdU was added, and the cells were harvested at the end of the window. BrdU incorporation was detected by flow cytometry. We found that 5.5% of E2F1+/- E2F2+/- and 4.9% of DKO lymphocytes cultured without antigen and labeled with BrdU from 8 to 42 h were positive for BrdU incorporation. (B) Lymphocytes from lymph nodes of DO11.10 TCR transgenic 6-week-old female littermates of the indicated genotypes were harvested and cultured with the indicated concentrations of antigenic peptide (OVA) and 1 µM BrdU for 36 h. BrdU incorporation was detected by flow cytometry. The y axis represents cell number. The percentages of cells that were positive for BrdU incorporation are indicated. (C) Lymphocytes from the spleens of 6.5-week-old female mice of the indicated genotypes were harvested, stained with CFSE, and cultured with OVA at the indicated concentrations for 72 h. The DKO and E2F1+/- E2F2+/- mice were littermates, and the WT mouse was female and age matched. The number of cell divisions (as indicated for divisions 1 to 5 above plots; P, parental) in T cells was determined by the intensity of CFSE. The position of the parental (P) peak is based on the analysis of the same cells cultured without antigen, as shown in Fig. 4B. The * peak reflects nonlymphocytes that contaminated the T-cell gate. (D) Lymphocytes from DO transgenic littermates of the indicated genotypes cultured with 0.2 µM OVA for 24 h (or unstimulated; 0 h) were stained with fluorochrome-linked anti-Thy1.2, anti-CD25, and anti-CD69 antibodies. The expression of CD69 and CD25 was determined in T cells (Thy1.2+ lymphocytes) by flow cytometry, and the percentages of cells that upregulated both CD69 and CD25 expression are indicated.

In order to determine how the E2F1/E2F2 genotype influences proliferation in response to varying antigenic dose, DO transgenic lymphocytes of the indicated E2F1/E2F2 genotypes were harvested from lymph nodes and cultured with various concentrations of the OVA peptide. In response to the various concentrations of OVA, DKO T cells showed greatly enhanced S-phase entry, as determined by BrdU incorporation, relative to T cells from littermates of other genotypes (Fig. 5B). For example, DKO T cells showed as much BrdU incorporation at 0.2 µM OVA as control T cells exhibited at 5 µM OVA. DO+ E2F1-/- E2F2+/- T cells consistently failed to show reduced proliferation when stimulated with OVA, in contrast to our results with ConA stimulation. Furthermore, in the experiment shown here, DO+ E2F1+/- E2F2-/- T cells did not show enhanced proliferation. However, in other experiments using cells from different mice, substantially enhanced proliferation was observed (data not shown). The reasons for the variable effects on proliferation in the single-KO mice is currently not clear.

The deregulated proliferation in DKO mice is also evident by CFSE labeling. After harvesting, DO transgenic T cells of the indicated genotypes were labeled with CFSE and cultured with either subthreshold (0.05 or 0.2 µM) or above-threshold (2 µM) concentrations of the OVA peptide for 72 h (Fig. 5C). At the high antigen concentration, T cells of all genotypes proliferated extensively, with DKO T cells proliferating somewhat better. However, at low antigen (0.2 µM), most WT T cells did not proliferate, whereas DKO T cells proliferated extensively, virtually as much as with high antigen (Fig. 5C). At an even lower antigen concentration (0.05 µM OVA peptide), WT T cells did not proliferate detectably, while a significant fraction of DKO T cells proliferated. E2F1+/- E2F2+/- T cells may exhibit a modest enhancement of proliferation compared to WT T cells.

Again, despite dramatic differences in proliferation in response to low antigen concentrations, the upregulation of CD69 and CD25 expression in response to 0.2 µM OVA was indistinguishable in T cells from E2F1+/- E2F2+/- and DKO mice (Fig. 5D). Finally, by combining unlabeled DKO lymphocytes with CFSE-labeled WT lymphocytes prior to antigen activation (or vice versa), we demonstrated that the increased proliferation of DKO T cells is cell autonomous (data not shown). The presence of DKO lymphocytes did not enhance the proliferation of WT T cells, nor did the presence of WT lymphocytes hinder the proliferation of DKO T cells. These results indicate that the combined loss of E2F1 and E2F2 results in decreased TCR signaling thresholds for antigen-induced T-cell proliferation.

We examined the expression of transcriptional targets of E2F and Myc in antigen-activated control and DKO lymphocytes at 24 h poststimulation, prior to significant proliferation. E2F3 expression was robustly activated with both subthreshold and above-threshold levels of antigen stimulation and was independent of the E2F1 and E2F2 genotype of the T cells, despite dramatic differences in subsequent proliferation (Fig. 6A). Furthermore, the mRNA expression of c-myc and its transcriptional target cyclin D2 were also increased similarly at both antigen doses independent of E2F1 and E2F2 (Fig. 6B). Similar regulation was observed for the E2F target genes for cyclin E, Cdk2, and Cdk1/Cdc2, although a modest increase in the expression of these genes was evident at 2 µM versus 0.2 µM OVA. Importantly, the activation of these E2F target genes is not affected by the loss of E2F1 and E2F2, despite the fact that the subsequent proliferation is markedly affected by E2F1/E2F2 genotype. Either E2F1 and E2F2 are not involved in the regulation of these target genes or, more likely, other E2F activities compensate for the loss of E2F1 and E2F2 in the regulation of these targets.


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  		FIG. 6.   Loss of E2F1 and E2F2 does not affect the regulation of at least some Myc-and E2F-dependent target genes. (A) Lymphocytes from mice of the indicated genotypes were harvested and cultured with the indicated concentrations of OVA for 24 h. Total cell protein was prepared and analyzed by Western blotting sequentially with alpha -E2F3 and alpha -tubulin antibodies (20 µg of protein/lane). The E2F1- E2F2- samples are from lymphocytes from two different DKO mice processed independently, and the E2F1+ E2F2+ lymphocytes are from an E2F1+/- E2F2+/- littermate. Similar results were obtained by comparing lymphocytes from DKO and E2F1+/+ E2F2+/+ mice (data not shown). The band labeled with an asterisk represents residual signal remaining after detection of E2F3. Ag, antigen. (B) Lymphocytes were treated as described in A. RNA was isolated and 2 µg of total RNA was used to determine the mRNA expression of the indicated genes by RNase protection assay. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is substantially increased following T-cell activation (2).

The identity of the specific targets of E2F1 and E2F2 that underlie the negative roles for these E2Fs in cell cycle progression is currently unknown. By analyzing gene arrays for genes that are differentially regulated in lymphocytes with and without E2F1 and E2F2, we have identified several E2F1/E2F2-dependent candidate genes, although the roles of these genes in lymphocyte proliferation have not been examined.

E2F1 and E2F2 mutant mice are highly predisposed to development of tumors and autoimmunity. Of our group of E2F1/E2F2 mutant mice born at least 15 months ago, 17% (1 of 6) of E2F1+/- E2F2+/-, 86% (12 of 14) of E2F1-/- E2F2+/-, 63% (10 of 16) of E2F1+/- E2F2-/-, and 100% (19 of 19) of DKO mice died before reaching 15 months of age. The postmortem analysis of E2F1/E2F2 mutant mice is shown in Table 1, which includes mice that were born and died within the last 15 months. Of the deaths, 5 of 9 E2F1+/- E2F2+/-, 6 of 17 E2F1-/- E2F2+/-, 2 of 3 E2F1+/+ E2F2-/-, and 14 of 17 E2F1+/- E2F2-/- mice exhibited tumors. E2F2-/- mice exhibited a particularly high frequency of tumors, starting at 4 months of age, and over half of these mice die from tumors within 15 months of age (Fig. 7 and Table 1).

                              
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  		TABLE 1.   Premature deaths among E2F1/E2F2 mutant micea



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  		FIG. 7.   E2F1/E2F2 mutant mice develop tumors. (A) Survival plot of mice of the indicated genotypes. A value of 1.0 represents 100% surviving mice of the indicated age in days. Survival curves for E2F1-/- E2F2+/-, E2F1+/- E2F2-/-, and E2F1-/- E2F2-/- mice are statistically different from the E2F1+/- E2F2+/- survival curve (P < 0.001 in each case). (B) Bronchial adenoma (indicated with an arrow) in the lung of a 6.5-month-old E2F1+/- E2F2-/- mouse (original magnification of H&E stain is ×10). The pattern is papillary, with a tall columnar, ciliated epithelium with abundant cytoplasm. (C) Histiocytic sarcoma of the uterus in 21-month-old E2F1+/- E2F2-/- mouse (×10; H&E). The sections of uterus show a spindle cell mass which infiltrates the uterine muscle. The neoplasm was composed of spindle cells with variable amounts of eosinophilic cytoplasm and large pleomorphic hyperchromatic nuclei. (D) A myeloid (Mac1+) leukemia infiltrating the liver of a 15-month-old E2F1+/- E2F2-/- mouse (×25; H&E). Leukemic cells are indicated with an arrow. (E and F) Flow cytometric analysis of the expression of CD4 and CD8 on lymphoma cells from a 4-month-old E2F1+/- E2F2+/- male mouse (E) and a 4-month-old E2F1+/- E2F2-/- female mouse (F). The lymphomas are weakly CD4+ and CD8+, respectively. (G) Flow cytometric analysis of the expression of CD8 and TCR Vbeta chains on lymphoma cells from a 5-month-old E2F1-/- E2F2+/- female mouse. The tumor is mostly Vbeta 5 positive and Vbeta 6 negative. Vbeta 5 and Vbeta 6 are normally expressed on about 1 and 10%, respectively, of peripheral T cells from mice of the H-2d MHC haplotype (50). The expression of Vbeta s 3, 7, 8, 11, and 12 on this lymphoma was similar to that of Vbeta 6 (data not shown).

Most of these mice were E2F1+/- E2F2-/-. Of the eight E2F1+/+ E2F2-/- mice that lived at least 1 year, four died within the first year, and two of these had confirmed lymphomas (Table 1; we did not recover the carcass for one dead E2F1+/+ E2F2-/- mouse, and we could not identify the cause of death for the other one). In contrast to E2F2-/- mice, we observed a lower incidence of tumors in E2F1-/- E2F2+/- mice. Only 22% of E2F1-/- E2F2+/- mice allowed to age for at least 15 months died from tumors, which is similar to the cancer predisposition shown for E2F1-/- mice in the Yamasaki et al. study (49).

In the tumors examined from E2F1+/- E2F2+/-, E2F1-/- E2F2+/-, and E2F1+/- E2F2-/- mice, the remaining WT E2F1 and E2F2 alleles were not detectably lost (even from lymphoma cells purified by flow cytometry), and E2F1 and E2F2 protein expression was maintained in tumors heterozygous for these alleles, suggesting that a reduction of E2F1/E2F2 activity may be sufficient to contribute to tumor development (data not shown). We have not observed any tumors in DKO mice. However, all DKO mice die within about 1 year of age (average, 4.2 months) of diabetes mellitus (data not shown), hindering the analysis of spontaneous tumorigenesis.

E2F1-/- mice in the Yamasaki et al. study showed a predominance of reproductive tract sarcomas and also developed lung adenocarcinomas (49). It is important to point out that the E2F1/E2F2 mutant mice examined in our study have been crossed for several generations into the BALB/cJ background, while the mice used for the Yamasaki study were in the 129/C57BL6 background. Lymphocytic malignancies are relatively rare in BALB/cJ mice, representing only 2.2% of spontaneous tumors in a large colony of BALB/cJ mice (7). In all, only 0.34% of mice in this colony (age range, 1 to 15 months) developed neoplasms, with an average age of onset of 7 months.

We have observed only one histiocytic sarcoma of the uterus and one lung adenoma in E2F2-/- mice (Fig. 7B and 7C). In fact, E2F2-/- mice (E2F1+/+ or E2F1+/-) predominantly develop hematopoietic malignancies, including myeloid leukemias and T-cell lymphomas (Table 1). Splenic, lymph node, and thymic cellularity in these mice ranged from 20- to 100-fold higher than normal. We have observed both B-cell and T-cell lymphomas, and the latter have expressed CD4, CD8, or both. Flow cytometric analysis of the expression of CD4 and CD8 in lymphoma cells revealed that the CD4+ and CD8+ lymphomas had replaced other lymphocyte populations, as B cells and other T cells were not detected (Fig. 7E and 7F). These leukemia cells have a large, blastic morphology characteristic of such malignancies (Fig. 7D and data not shown). In addition, the analysis of the expression of the TCR Vbeta chains revealed that the CD8+ lymphoma shown in Fig. 7G is monoclonal, as the malignant cells primarily expressed Vbeta 5 but not other Vbeta chains (shown for Vbeta 6). Vbeta 5 is normally expressed on only about 1% of peripheral CD8+ T cells in mice of the H-2d haplotype (50). Thus, the normally diverse expression of different Vbeta chains is virtually eliminated.

In the E2F1+/- E2F2-/- mouse with a lung adenoma, expansion of immature lymphoid cells in the spleen was also observed (the spleen was enlarged severalfold), suggesting that this mouse also developed an independent lymphoma or lymphoproliferative disorder. Myeloid hyperplasia was also evident in the E2F1+/- E2F2-/- mouse with a uterine sarcoma (Table 1). Most of these leukemias appeared to be very aggressive. For example, in the myeloid leukemia observed in an E2F1+/- E2F2-/- mouse, shown in Fig. 7D, leukemic infiltration into the liver, lung, pancreas, and intestine was observed. In summary, while the loss of E2F1 results in a modest increase in cancer incidence, E2F2 mutation results in a dramatic increase in tumorigenesis, with a particularly high incidence of lymphomas.

Although cancer accounted for the majority of the premature deaths of E2F1+/- E2F2-/- mice and about a fourth of the premature deaths of E2F1-/- E2F2+/- mice, the other morbid mice showed no evidence of tumors. Many morbid E2F1-/- E2F2+/- mice exhibited severe pancreatic atrophy but, unlike DKO mice, only rarely developed diabetes (Table 1 and data not shown). Notably, the development of diabetes in the DKO mice was not autoimmune mediated (data not shown). We frequently observed hugely distended intestines (particularly in the cecum) with large quantities of undigested material in both morbid DKO and E2F1-/- E2F2+/- mice (and one E2F1-/- E2F2+/+ mouse). We speculate that this phenotype results from pancreatic atrophy and the consequent loss of appropriate digestive enzymes and neutralizing bases normally secreted into the intestine. The roles of E2F1 and E2F2 in pancreatic physiology will be described elsewhere (J. W. Zhu, F. X. Li, and J. DeGregori, unpublished data). In addition, histological examination of several of these tumor-free E2F1/E2F2 mutant mice revealed lymphocytic infiltration into the pancreas, lungs, liver, intestines, and salivary glands (Fig. 8A, 8B, and 8C and data not shown), suggesting that lymphocyte-mediated autoimmune destruction of some tissues contributed to the morbidity of these mice. These mice do not demonstrate any signs of lymphocytic malignancy, such as increased hematopoietic cellularity or blastic morphology. Immunohistochemistry for the detection of CD4+ cells verified that infiltrating mononuclear cells include T cells (Fig. 8E). However, the experiments presented in Fig. 8 are not sufficient to conclude that self-reactive T cells contributed to the observed pathology in these E2F1/E2F2 mutant mice.


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  		FIG. 8.   E2F1/E2F2 mutant mice show signs of autoimmunity. (A and B) Lymphocytic infiltration (indicated with arrows) in the lungs and salivary gland of a 10-month-old E2F1-/- E2F2+/- mouse, respectively (original magnifications of H&E stains are ×10 and ×25). (C) Cluster of polymorphic mononuclear cells (PMNs) (indicated with arrow) in the liver of an 11.5-month-old E2F1-/- E2F2+/- mouse (×40; H&E). (D and E) Immunohistochemistry for the expression of CD4 in the liver of a 10-month-old E2F1-/- E2F2+/- mouse (E) and a control liver (D). Brown indicates CD4+ cells.

In conclusion, our data indicate that E2F1 and E2F2 function to negatively regulate hematopoietic cell proliferation and the loss of E2F1 and E2F2 contributes to increased cancer and, to a lesser extent, autoimmunity. We believe that our observation that E2F1 and E2F2 play a role in determining antigenic thresholds for primary T-cell proliferation provides an explanation for the subsequent development of cancer and possibly autoimmunity in E2F1/E2F2 mutant mice.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Regulation of antigenic thresholds for T-cell proliferation. Our studies reveal a surprising negative role for E2F1 and E2F2 in cell cycle progression. Our hypothesis is that subthreshold antigenic signals activate an E2F1/E2F2-dependent negative feedback loop that limits further cell cycle progression. E2F1 and E2F2 may activate the expression of negative cell cycle regulators, so that the loss of E2F1 and E2F2 results in decreased expression of these inhibitors of proliferation. Alternatively, E2F1 and E2F2 could function in quiescent T cells, perhaps together with Rb, to repress the expression of positive cell cycle regulators. Thus, the loss of E2F1/E2F2 could result in increased expression of positive cell cycle regulators, perhaps as the consequence of reduced target gene repression by Rb/E2F1 and Rb/E2F2. We also propose that strong TCR signaling activates pathways that counteract the E2F1/E2F2-dependent suppression of cell cycle progression, facilitating G1 to S progression. Thus, with an above-threshold signal, the derepression of genes inhibited by Rb-E2F together with the activation of E2F1-, E2F2-, and E2F3-dependent transcription results in the expression of positive regulators of cell cycle progression, like Cdc6 and cyclin E, the latter of which promotes a positive feedback loop by enhancing Rb phosphorylation. In DKO T cells, E2F3 may compensate for the loss of positive E2F1 and E2F2 functions in cell cycle progression. However, the increased proliferation observed in DKO lymphocytes does not appear to result from upregulated expression of E2F3.

Interestingly, we observed a striking lack of correlation between the activation of myc and at least some E2F-dependent transcription in T cells and subsequent proliferation, indicating that the transcriptional activation of these genes does not necessitate progression into S phase. Furthermore, E2F1 and E2F2 do not appear to influence antigen-induced signaling pathways that regulate the expression of CD69 and CD25 (including Ras, NF-kappa B, and NFAT activation). Thus, T cells with similar levels of activation of CD69, CD25, Myc, E2F3, and some E2F targets exhibited markedly different propensities to enter S phase and proliferate depending on the presence of E2F1 and E2F2.

How a T cell distinguishes a subthreshold signal from an above-threshold signal is a critical question in immunology. T cells have a phenomenal ability to proliferate in response to foreign antigen, and particular CD8+ T cells in a mouse can undergo an almost 105-fold amplification in response to a pathogen in less than a week (34). For this reason, the prevention of proliferation in response to subthreshold antigen stimulation is critical for the maintenance of T-cell tolerance and tumor suppression. A sustained T-cell proliferative response requires antigen of sufficient affinity and concentration as well as costimulatory signals.

Antigen activation of T lymphocytes is largely regulated at the level of the TCR and costimulatory receptor signaling complexes which, upon association with peptide/MHC of sufficient affinity, form an "immunological synapse" between the T cell and the antigen-presenting cell (32, 47). Signaling pathways proximal to the TCR, both positive and negative, also control the proper discrimination of low- and high-affinity antigens by the cell (20). For example, the Clb-b adaptor protein negatively regulates lymphocyte activation downstream of the antigen receptors, and deletion of Clb-b in mice results in lymphocyte hyperresponsiveness to antigen and predisposition to autoimmunity (3, 9). Proximal TCR signaling resulting in increased expression of CD69 and CD25 is not affected by E2F1/E2F2 disruption, suggesting that E2F1 and E2F2 determine how a T cell responds to these signals but do not influence TCR signaling. We suggest that there are at least two hurdles to T-cell proliferation. The first is at the level of TCR/antigen/MHC interaction and efficient synapse formation. The second E2F1/E2F2-dependent hurdle is responsive to the strength of the first signal, preventing progression into S phase in response to insufficient antigenic stimulation. Both hurdles set thresholds for TCR signaling-induced proliferation.

Pathways downstream of TCR signaling can also modulate antigen-induced T-cell proliferation. Reduction in Ikaros activity in T cells results in reduced thresholds for TCR activation-induced proliferation, perhaps related to the role of Ikaros proteins in maintaining higher-order chromatin structures (2). Mutations in cell cycle regulators can also affect activation-induced T-cell proliferation. p18-/- lymphocytes show increased DNA synthesis in response to mitogenic stimulation with lectins (14, 25). While primary p21-/- lymphocytes proliferate normally in response to stimulation, these lymphocytes show a proliferative advantage after prolonged IL-2 stimulation. Indeed, p21-/- mice lose tolerance to nuclear antigens and develop a lupus-like autoimmune disease (4). Furthermore, Rb-/- T cells, generated by Rag2-/- blastocyst complementation with Rb-/- embryonic stem (ES) cells, develop normally but show enhanced DNA synthesis in response to ConA stimulation (8). Finally, while p107 mutant T cells proliferate normally in response to mitogens, p107-/- p130-/- T cells are hypersensitive to ConA stimulation (33).

Roles for E2F1 and E2F2 in limiting tumorigenesis and autoimmunity. E2F1/E2F2 mutant mice are highly predisposed to tumorigenesis, perhaps as a consequence of reduced signaling thresholds for proliferation. These results further highlight the involvement of the E2F pathway in tumorigenesis and suggest the possibility that E2Fs might be directly targeted during the genesis of human malignancies. Indeed, the human E2F2 gene maps to chromosome 1p36.11, near the familial prostate/brain cancer susceptibility locus and a region frequently lost in human tumors such as neuroblastomas (18, 30).

Consistent with the differential effects of E2F1 and E2F2 loss on hematopoietic cell proliferation, the loss of either E2F1 or E2F2 results in very different predispositions to cancer. E2F2-/- mice show a very high incidence of tumors, substantially higher than either we or others have observed in E2F1-/- mice (49). The prevalence of hematopoietic malignancies in E2F2-/- mice also correlates with the increased proliferation of hematopoietic progenitors and mature T cells observed in young E2F2-/- mice. In addition, potential effects of the loss of E2F1 and E2F2 on differentiation, as reflected by decreased B-cell maturation, could also contribute to increased tumorigenesis. Our failure to observe increased tumorigenesis in DKO mice is also interesting, perhaps suggesting that while a reduction in E2F1/E2F2 activity promotes tumorigenesis, some E2F1/E2F2 activity may be required for cancer formation. However, since most DKO mice die from diabetes before most tumors are observed in E2F2-/- mice, it is difficult to determine how the complete loss of E2F1 and E2F2 affects tumorigenesis.

Given our demonstration that the loss of E2F1 and E2F2 reduces the threshold for antigen activation of T cells, it is not surprising that some mice with mutations in E2F1 and E2F2 develop indications of autoimmunity. These data suggest that the deregulation of pathways controlling T-cell proliferation can contribute to the development of autoimmunity. Experiments in the Zubiaga lab have also demonstrated that E2F2 mutant mice develop autoimmunity, as evidenced by both widespread inflammatory infiltrates and antinuclear antibodies (34a). Other labs have shown that T helper cell effector functions such as cytokine (IL-4 or IFN-gamma ) secretion are dependent on T-cell proliferation (6, 17). The increased production of IFN-gamma that we observed for stimulated DKO T cells is consistent with these reports and suggests that increased proliferation of T cells in E2F1/E2F2 mutant mice may contribute to autoimmune disease by increasing T-cell effector functions. Finally, our results indicate that the pathways that limit the development of both autoimmunity and cancer are overlapping. E2F1 and E2F2 are required for both tumor suppression and the maintenance of tolerance, probably mediated through the regulation of target genes that function to limit proliferation in response to inappropriate signals.


    ACKNOWLEDGMENTS

S.J.F. is supported by a Howard Hughes Medical Institute (HHMI) Postdoctoral Fellowship. S.H.O. is an investigator of HHMI. M.G. is supported by NIH grants R01 CA43855 and P30-HD18655 from the Mental Retardation Center. R.D.C. is supported, in part, by grant 5JB-0014 from the State of California Breast Cancer Research Program. J.D. is supported by grants from the NIH (RO1 CA77314-01) and the American Cancer Society (RSG LIB-101051) and by a Scholar Award from the Leukemia and Lymphoma Society.

We thank the following individuals for critical review of the manuscript: P. Marrack, D. Bentley, T. Van Dyke, J. Hagman, A. Gutierrez-Hartmann, J. Nevins, D. DcRyckere, and N. Jones. We also thank K. Helm, P. Schor, and M. Ashton of the Cancer Center Flow Cytometry Core (supported by grant 2 P30 CA 46934-09), P. Skavlen and CLAC for excellent veterinary care, J. Torvik and D. Wegman for cytokine measurements, and J. Cambier and B. Benschop for reagents and advice concerning B-cell differentiation. We thank Ana Zubiaga for sharing unpublished data. We also thank Leslie Bloomquist for histological processing, supported by Diabetes Endocrinology Research Center grant P30 DR 57516.


    FOOTNOTES

* Corresponding author. Mailing address: University of Colorado Health Sciences Ctr. BRB802, 4200 E. Ninth Ave., Denver, CO 80262. Phone: (303)-315-5792. Fax: (303)-315-3244. E-mail: james.degregori{at}uchsc.edu.

dagger Present address: Harvard Institutes of Medicine, Beth Israel Deaconess Hospital, and Division of Endocrinology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Molecular and Cellular Biology, December 2001, p. 8547-8564, Vol. 21, No. 24
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.24.8547-8564.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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