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Departments of
* Immunology and
Pathology, Duke University Medical Center, Durham, NC 27710
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and 
-chain TCRs and are
thus named T cells. The lineage T cells have been
operationally divided into the
CD4-CD8- (double negative
(DN)3),
CD4+CD8+ (double positive
(DP)), and CD4+ or CD8+
(single positive (SP)) stages according to their developmental
progression toward maturity. TCR and gene rearrangement and
expression occur sequentially in the DN stage and the DP stage,
respectively. DP cells expressing TCR undergo MHC-mediated positive and
negative selection before they further differentiate to become either
CD4 helper or CD8 cytotoxic SP cells. Frequent interactions between the
developing T cells and the thymic stromal cells provide developmental
cues to signal the immature DN cells to differentiate and to migrate
from the thymic cortex to the medulla, where they become mature SP
cells (1). Any genetic perturbation in either the
developing T cells or the thymic stromal cells may cause a
developmental block and/or unregulated cell proliferation, which often
leads to the formation of thymic lymphomas. Transcription factors encoded by the E2A gene have been implicated to play important regulatory roles in both B and T cell development (2). E2A gene products were first identified as non-tissue-specific transcription factors that bind to Ig gene enhancers and were subsequently classified as members of the basic helix-loop-helix (bHLH) protein family (3, 4). The E2A gene encodes two bHLH proteins, E12 and E47, through alternative splicing at two adjacent bHLH-encoding exons. Most bHLH proteins including E2A bind to a consensus DNA sequence, CANNTG, denoted by an E box, via the basic region located N-terminal to the HLH domain (3). The HLH domain mediates dimerization between two bHLH proteins, which is a prerequisite for DNA binding (4, 5). Functions of E2A in B and T cell development are best demonstrated in the studies of E2A knockout mice, which fail to produce any B-lineage cells and show impaired T cell development (6, 7). Biochemical studies have indicated that B cell development requires E2A homodimers (8), whereas T cell development requires both E2A homodimers and heterodimers of E2A and HEB, a homologue of E2A present at high levels in the thymus (9, 10).
The interaction between E2A and HEB has been shown to be important for
T cell-specific gene regulation and T cell development. A physical
interaction between the two bHLH proteins was first reported by Sawada
and Littman (9) in the study of CD4 enhancers. The CD4-3 E
box site, a functional enhancer of the CD4 gene, was found to be
predominantly occupied by the E2A-HEB heterodimers in thymocytes.
Subsequently, a much more complex role for E2A and HEB in T cell
development was revealed in the studies of mice carrying targeted
E2A or HEB mutations. E2A-deficient or
E47-deficient mice displayed a partial block at the
CD44+CD25- stage (the
earliest stage in thymic T cell development, also known as the DN1
stage) and thymic hypocellularity (11, 12). An accelerated
positive selection during the transition from the DP to the SP stage
was also observed in E47-deficient mice (13).
HEB-deficient (HEBko) mice
displayed a strong developmental block at the immature single positive
(ISP) stage, a transitional stage between DN and DP, and thymic
hypocellularity (14). The accumulation of ISP cells was
also observed in E2A and HEB compound
heterozygous mice, providing genetic evidence for a physical
interaction between E2A and HEB proteins at least during the ISP stage
of T cell development (15). The importance of E2A-HEB
heterodimers in T cell development was further demonstrated in the
study of mice carrying a dominant negative HEB allele named
HEBbm (10). The dominant
negative HEB proteins inactivate both E2A and HEB functions and block T
cell development at the DN3 stage, where TCR gene rearrangement and
selection occur. However, in the studies of both
HEBko and
HEBbm strains, no defects were seen in DN1
and DN2 stages of T cell development, suggesting that E2A-HEB
heterodimers only begin to function after the DN2 stage.
The importance of E2A in T cell development was further exemplified by the frequent development of malignant thymic lymphomas in E2A-deficient or E47-deficient mice (11, 16). Thymic T cell tumors are also observed in HEBko mice (Y. Zhuang, unpublished observations). Two possible mechanisms have been proposed to account for the observed tumor phenotypes. First, studies of these and other related T cell tumors have revealed a positive regulatory role for E2A in apoptosis (17, 18). The lack of E2A may promote the survival of cells that would have otherwise died during T cell selection. Second, a role for E2A in regulating transcription of the cell cycle inhibitor p21 gene has been indicated in the study of 3T3 fibroblasts (19). It is possible that the loss of E2A leads to enhanced cell proliferation and thus a higher probability of tumor formation.
E2A is broadly expressed and is suspected to function in many other cell types. In fact, E2A-deficient mice are growth retarded and die as neonates in high frequency (11, 16). The cause of these nonlymphoid-related phenotypes remains to be determined. The low survival rate of E2A-deficient mice and the unspecified functions of E2A in other cell types make it difficult to study the T cell phenotypes in detail. To circumvent the problems associated with the conventional gene targeting approach, we generated mice with the E2A gene specifically deleted in the T cell lineage. Mice lacking E2A in T cells are fully viable and fertile and thus provide an alternative model for investigating E2A function in T cell development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Galactosidase (-gal) enzyme histochemistry
Cultured cells or thymic lobes were fixed in 2%
paraformaldehyde and 0.125% glutaraldehyde for 5 or 25 min,
respectively. -Gal expression was detected by incubating postfixed
cells or thymi in 5-bromo-4-chloro-3-indolyl
-D-galactoside (X-gal; Sigma-Aldrich, St. Louis, MO)
staining solution (25 mM potassium ferrocyanide, 25 mM potassium
ferricyanide, 2 mM MgCl2, 0.02% Nonidet P-40,
0.01% sodium deoxycholate, and 1 mg/ml X-gal) for 14 h. For some
samples (e.g., Fig. 2
A), X-gal staining was conducted on
prefixed frozen sections.
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One lobe of the X-gal-stained thymus was postfixed in 10% Formalin and then subjected to routine tissue processing and paraffin embedding. The other lobe was embedded in OCT compound (Tissue-Tek; Miles, Elkhart, IN) and snap frozen in a dry ice and ethanol cryobath. Sections (8 µm) were prepared for immunohistochemical analysis. Sections were blocked with 10% goat serum in PBS before application of primary Abs. Primary Abs used included rat anti-mouse CD3 (BD PharMingen, San Diego, CA), rabbit anti-human von Willebrand factor (which cross-reacts with mouse Ags, DAKO, Carpenteria, CA), rat anti-mouse NLDC145 (Bachem, Torrance, CA), and rat anti-mouse MTS16 (BD PharMingen). Ab binding was detected using biotinylated goat anti-rabbit IgG or biotinylated goat anti-rat IgG (Southern Biotechnologiy Associates, Birmingham, AL), followed by an avidin-biotin-HRP complex (Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA) and 3,3'-diaminobenzidine tetrahydrochloride or 3-amino-9-ethyl-carbazole as chromogens. NLDC145 and MTS16 stainings were performed on frozen sections. All the other stainings were performed on paraffin-embedded sections.
Adoptive transfer experiment
C57BL/6 mice congenic for the pan-leukocyte marker CD45 (the Ly5A allotype) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred subsequently in-house. Host mice between 2 and 3 mo of age were irradiated with 1100 rad 1 day before bone marrow transfusion and were maintained on antibiotics in sterile bedding thereafter. Bone marrow cells (0.1–0.5 x 105) were delivered to the host in 0.2 ml PBS through tail vein injection. Two to five recipients were used for each experiment. Thymocytes were analyzed by FACS 1–3 mo postirradiation.
Gene targeting and transgenic mice
The E2Aloxp targeting construct was built
on a 9-kb KpnI-BamHI fragment of the
E2A genomic DNA isolated from the 129/sv strain. Linearized
targeting construct (25 µg) was electroporated into AK7 embryonic
stem cells (a gift from A. Imamoto and P. Soriano, Fred Hutchinson
Cancer Research Center, Seattle, WA). Cells were grown under
double selection with G418 and gancyclovir. Correct targeting events
were identified in 14 of 60 clones screened by PCR. Germline
transmission was obtained from two of three clones injected. Mice
carrying the E2Aloxp allele were intercrossed
and maintained in a specific pathogen-free environment at Duke
University animal facility throughout the experiment. The
Lck-Cretg construct was engineered using the
same strategy as described previously (20). The construct
was microinjected into fertilized (C56BL/6 x
SJL/J)F1 eggs. Founder lines were determined by
Southern blot analysis and bred to the C57BL/6 background twice before
crossing with E2Aloxp/+ mice. The various
E2A alleles used in this study are listed in Table I.
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Single-cell suspensions of lymphocytes from the thymus, spleen,
bone marrow, and peripheral lymph nodes were prepared in ice-cold PBS
supplemented with 5% bovine calf serum. Splenocytes were depleted of
RBC by the ammonium chloride lysis treatment before use. Cells (1
x 106) were immediately stained with Abs and
analyzed on a FACSCalibur (BD Biosciences, Mountain View, CA). The Abs
used in this study included the following: APC- or FITC-conjugated
anti-B220 (RA3-6B2; Caltag Laboratories, Palo Alto, CA),
biotinylated or FITC-conjugated goat anti-mouse IgM
isotype-specific Abs (Southern Biotechnology Associates),
FITC-conjugated anti-CD4 (CT-CD4; Caltag Laboratories),
APC-conjugated anti-CD8 (CT-CD8b; Caltag Laboratories), and
PE-conjugated anti-CD5 (53-7.3; BD PharMingen). 7-amino actinomycin
D (7AAD) staining was used to eliminate dead and damaged cells
from the analysis. The enzymatic activity of -gal was determined as
previously described (7). Basically, single-cell
suspensions were first mixed with an equal volume of 2 mM fluorescein
di -D-galactopyranoside (FDG) at 37°C for 90 s.
The loading of FDG was stopped by adding 10 vol of ice-cold PBS. Cells
were then subjected to FACS analysis for detection of the fluorescent
intensity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate E2A function in the thymus, we first
examined E2A expression during thymopoiesis in mice carrying
an E2AGFP knockin allele (Table I
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E2AGFP allele contains the green fluorescent
protein (GFP) coding sequence inserted in-frame at the carboxyl end of
the E2A-coding sequence. E2AGFP
homozygous mice were phenotypically the same as their wild-type
littermates, suggesting that E2A-GFP fusion proteins must be
functionally equivalent to the wild-type E2A proteins (Y. Zhuang,
unpublished observations). Therefore, E2A expression in
individual thymocytes may be determined by the fluorescent intensity of
the E2A-GFP fusion proteins. Five-color flow cytometric analysis was
performed to determine E2A-GFP expression at various stages of T cell
development. We found that E2A was highly expressed at the
DN stage, was down-regulated at the DP stage, and was further
down-regulated at the SP stages (Fig. 1).
A low, but above background, level of E2A-GFP signal was detected
in SP cells in mice carrying two copies of
E2AGFP (data not shown). Within the DN
stage, E2A expression was first detected in a fraction of
DN1 cells (CD44+CD25-).
High level E2A expression was found in all DN2
(CD44+CD25+) cells and in
the majority of DN3
(CD44-CD25+) cells. DN4
(CD44-CD25-) cells showed
a broad pattern of E2A expression, indicating the
heterogeneity of cells in this population. This result is consistent
with the idea that E2A plays an important role at the DN
stage of T cell development (2).
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E2A expression in the thymus was further examined by
immunohistochemical analysis of thymic sections prepared from mice
carrying the E2Agal allele (7). The
E2A locus of this allele is disrupted due to an insertion of
a modified -gal gene which produces a nuclear form of -gal. The
-gal activity thus directly reflects that of the endogenous
E2A promoter. In comparison with the GFP method shown above,
the resolution of X-gal staining in most subpopulations of T-lineage
cells is poor after these cells are doubly stained with stage-specific
markers. However, X-gal staining is appropriate for examining
E2A expression in thymic stromal cells, since each thymic
stromal cell type has a unique morphology. Fig. 2 shows representative results from X-gal
staining of thymus sections and thymus-derived fibroblasts. X-gal
staining detected E2A-expressing cells in both thymic cortex
and medulla of E2Agal/gal or
E2Agal/+ mice (Fig. 2, A and
B, and data not shown). We found that X-gal activity was
undetectable in mature CD3+ thymocytes (Fig. 2C). In contrast, strong X-gal staining was detected in the
nuclei of endothelial cells in the blood vessels (Fig. 2D).
The identity of these X-gal-positive endothelial cells was confirmed by
double staining with Abs specific for von Willebrand factor (data not
shown). Strong X-gal staining was also observed in a few
NLDC145-expressing cells (Fig. 2E), but not in
cytokeratin-expressing epithelial cells (data not shown). Because
NLDC145 recognizes both thymic cortical epithelial cells and dendritic
cells, the NLDC145 and X-gal DP cells were most likely dendritic cells.
Additionally, a fraction of the extracellular matrix (ECM)-expressing
cells (Fig. 2F, MTS16 staining) were positive for X-gal.
Finally, we cultured thymic stromal cells from both
E2Agal/+ and E2Agal/gal
mice. Cells with fibroblast morphology were obtained and analyzed for
-gal activity. As shown in Fig. 2, G and H,
fibroblasts derived from both E2Agal/+ and
E2Agal/gal thymi were positive for -gal
activity. Because E2Agal/gal mice do not produce
functional E2A proteins, the development of these fibroblasts must be
independent of E2A function.
An adoptive transfer test of E2A function in T cell development
Previous studies have shown that disruption of the E2A
gene in mice resulted in abnormal T cell development and thymic T cell
lymphoma (11, 13, 16). However, it is not clear whether
these defects are cell autonomous because E2A is disrupted
in both T-lineage cells and thymic stromal cells. We attempted to
address this question by an adoptive transfer test. Bone marrow
hemopoietic stem cells from E2A mutant mice were transferred
into lethally irradiated E2A wild-type mice congenic for the
common leukocyte Ag CD45. These mice were analyzed 1 or 3 mo after
transfer to evaluate the presence of donor cells and the possible
development of T cell lymphomas. The success of the transfer experiment
was determined by the survival of the hosts and the presence of
donor-derived hemopoietic cells. Consistent with previous findings
(15), E2A-deficient donor cells completely
failed to give rise to B-lineage cells in wild-type recipients (data
not shown). In contrast, high degrees of contribution of donor T cells
were detected in all wild-type recipients. Abnormal T cell development
was partially recapitulated after adoptive transfers (Fig. 3A). The total thymic
cellularity recovered from E2A-deficient donors was 5- to
10-fold lower than that of E2A heterozygous donors. A
proportional increase in mature SP cells was also observed. Both the
hypocellularity and the skewed SP population resemble the
E2A-deficient T cell phenotypes before their transfer. T
cell lymphoma was not observed up to 3 mo after transfer (six mice
analyzed at 1 mo, three mice at 2 mo, and four mice at 3 mo). Although
the chimeric mice only partially recapitulate the T cell phenotypes
reported in the E2A-deficient mice, these studies clearly
demonstrate a cell autonomous role of E2A in T cell
development.
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B). To avoid possible complications of tumor
development in the E47bm hosts, we chose
2-mo-old healthy E47bm homozygous mice as
recipients in the adoptive transfer experiments. Mice reconstituted
with CD45-congenic E2A wild-type cells were analyzed 2 mo
after transfer (Fig. 3C). Analysis of three separate
recipients shows various degrees of disproportional increase in CD4 SP
cells, a phenotype similar to, but less severe than, that observed in
age-matched E47bm mice (Fig. 3B).
This result raises the possibility that a role for E2A
outside the T cell lineage may affect proper T cell development. The
effect of E2A gene disruption on tumor development was not
studied in this adoptive transfer test due to the limitation of sample
size and other inadvertent effects of irradiation to the
E47bm hosts. T cell-specific inactivation of the E2A gene
One caveat of the adoptive transfer experiments is that the
analysis is based on reconstitution of the entire hemopoietic system
rather than the T cell lineage alone. Consequently, other undefined
hemopoietic cells derived from the donors may influence the outcome of
the adoptive transfer experiment. To explicitly determine the functions
of E2A in individual cell lineages and to clarify the
results of the adoptive transfer experiments, we established an
E2A conditional disruption mouse model. A new E2A
allele, named E2Aloxp, was generated by
introducing two identically oriented loxP sites into the E2A
locus (Fig. 4A). These loxP
sites serve as targets for Cre recombinase-mediated, lineage-specific
ablation of the E2A gene. The 5' loxP site was inserted into
an intron upstream of the bHLH-encoding exons for both E12 and E47. The
3' loxP site and selection markers were placed downstream of the
E2A gene. Positive selection for gene-targeting events was
provided by the neomycin resistance gene driven by the phosphoglyceryl
kinase gene promoter (PGKneo). PGKneo was placed in front of the 3'
loxP site but outside of the E2A gene, such that it would
not affect the transcription and translation of the E2A
gene. -Gal was used as an enzyme marker to monitor Cre-mediated
recombination in individual cells. The -gal sequence driven by an
internal ribosomal entry site was placed downstream of the PGKneo gene
and the 3' loxP site. -Gal will be transcribed as a bicistronic
transcript only after Cre-mediated recombination, which removes all
transcription stop sites preceding the -gal sequence. To increase
the stability of the chimeric transcript, a splice acceptor site and a
3' transcription stop signal were also built into the internal
ribosomal entry site--gal expression cassette. Successful targeting
of the E2A gene was confirmed by Southern blotting analysis
(Fig. 4B). Mice homozygous for the
E2Aloxp allele were completely viable and
fertile without any visible developmental abnormalities.
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-gal
activity in total thymocytes was determined with the FDG assay (Fig. 5A). The majority of
thymocytes from E2Aloxp/loxpCretg
mice were FDG positive, indicating that Cre-mediated deletion had
occurred in these thymocytes. Second, a PCR-based analysis was used to
detect E2A disruption in FACS-sorted thymocyte
subpopulations (Fig. 5, B and C). The
Cre-mediated deletion was detected as early as the
CD44+CD25- DN1 stage (Fig. 5B). However, the undeleted allele was also detected up to
the CD44-CD25-DN4 stage
(Fig. 5B), but not in the DP and SP stages (Fig. 5C). No Cre-mediated deletion was detected in other
cell types, including B220+ cells,
Mac1+ cells, and Gr1+ cells
isolated from bone marrow or spleen (data not shown). These results
indicate that the Cre-mediated E2A deletion was initiated
after cells have committed to the T cell lineage and was completed
before they reach the DP stage of thymopoiesis.
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The effect of T cell-specific E2A disruption on the
development of T and other hemopoietic cells was evaluated by FACS
analysis of thymocytes, splenocytes, and bone marrow cells in 4- to
10-wk-old E2Aloxp/loxpCretg mice and
their E2Aloxp/loxp littermates. The development
of myeloid cells (determined by Mac1 and Gr-1 markers), NK cells
(determined by NK1.1 and or DX5 markers), B cells, and T cells was
normal in E2Aloxp/loxpCretg mice
(Fig. 6, and data not shown). The absence
of any developmental defect in the B cell lineage confirms that the
E2A function in B cells has not been affected by the
introduction of loxP sites into the E2A gene locus and the
presence of the Cre transgene. However, normal T cell
development in these mice was unexpected. In contrast to
E2A-deficient mice (Fig. 6A, top),
neither a reduction of total thymocytes nor a decrease in DP thymocytes
and an increase in SP thymocytes was observed (Fig. 6A,
bottom).
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, top). We further examined
TCR-mediated positive selection by introducing the AND TCR transgene
(22) into
E2Aloxp/loxpCretg mice. Our analysis
showed that AND-mediated positive selection to CD4 SP cells was not
affected by E2A deletion (Fig. 7, bottom).
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50% of
the E2A-deficient mice starting from the age of 3 mo
(11, 16). An effect of E2A disruption on T cell development revealed by a genetic interaction between E2A and HEB
A genetic interaction between E2A and HEB
has been shown to occur during the transition between DN and DP stages
(14, 15). This interaction was demonstrated by an increase
in ISP cells in fetal thymus double heterozygous for both
HEB knockout and E2A knockout mutations
(15). To further evaluate the functional impact of
Cre-mediated E2A disruption on T cell development, we tested
the conditional E2A disruption on the HEB
knockout background. Due to a low survival rate for
HEBko mice, we analyzed neonates from
day 0 to 2 wk after birth from breeding among
HEBko/+E2Aloxp/+Cretg
mice. To avoid age-related phenotypic changes, FACS analysis was
performed exclusively on littermates containing informative genotypes.
Fig. 8A shows that
Cre-mediated disruption of two copies of
E2Aloxp, but not one copy of
E2Aloxp, on
HEBko/+ background causes a dramatic
accumulation of DN and ISP cells in 2-wk-old neonates. The identity of
the ISP cells detected in these mice was confirmed by the reduced level
of CD5 and TCR expression relative to that in the mature
CD8+ single-positive cells (Fig. 8A,
R3 histogram, and data not shown). The CD5 level in DP cells has also
been shown to be correlated with the combined gene dosage of
E2A and HEB (14, 15). Indeed, a
progressive reduction of CD5 expression on DP thymocytes is observed as
the copy number of E2A decreased from two to one and from
one to zero (Fig. 8A, R4 histogram). Fig. 8B
further shows in a separate litter that Cre-mediated disruption of
single copy of E2Aloxp on
HEBko/ko background is able to enhance the
developmental block between DN and ISP stages (comparing the
lower left with the lower right panel). These
results are consistent with the PCR-based assay that deletion of the
E2Aloxp allele has completed in most
developing T cells during the transition between DN to DP stage of
development.
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C shows
that E2A expression, revealed by E2AGFP levels, remains
relatively stable in DP and ISP thymocytes regardless of HEB
genotype. Thus, it appears that E2A expression is not
dependent on HEB during early thymocyte development. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using the E2AGFP knockin allele, we were able to examine E2A protein expression at the single-cell level in live thymocyte populations. In this knockin allele, GFP was inserted into the last exon of the E2A gene, which encodes a common sequence for both E12 and E47 proteins. The GFP sequence directly fused to the carboxyl end of E2A proteins without any disruption of E2A-coding sequences. Mice homozygous for E2AGFP are completely viable and fertile and lack any developmental phenotypes normally associated with conventional E2A gene disruption (data not shown). The appropriate expression level and the function of E2A-GFP fusion proteins are further demonstrated by the fact that B cell lineage development is unaffected by this fusion allele. Therefore, we believe that E2A-GFP expression in E2AGFP mice is a good approximation of the levels of endogenous E2A proteins in wild-type mice. Our data show that E2A is highly expressed in most DN cells. A small fraction of DN1 and DN4 cells express a lower level of E2A. The nature of these cells and their significance to T cell development require further investigation. E2A expression remains high in ISP cells and down-regulated in DP cells. A further down-regulation in E2A expression occurs in the conversion from DP to SP cells. Our observation is consistent with a recent report, which showed intracellular E2A protein staining in a flow assay (23). The mechanism underlying these stepwise E2A down-regulations is not known. To date, only Id3 has been implicated to negatively regulate E2A protein activity (24, 25). However, there is no evidence on how this negative regulation may lead to down-regulation of E2A gene expression. The use of E2AGFP mice provides a simple way to study E2A gene regulation during thymocyte development. In this regard we have determined that E2A expression is not dependent on HEB. This type of study may be expended to evaluate other relevant genes for their roles in E2A expression.
E2A function in thymocyte development
The involvement of E2A in thymocyte development was first revealed in studies of mice carrying germline targeted E2A mutations. An increase in CD44+CD25- DN1 cells, a decrease in DP thymocytes, and an increase in the percentage of CD4 and CD8 SP cells were reported in E2A-deficient mice (11, 12). The phenotypic discrepancy between E2A-deficient mice and T cell-specific E2A knockout mice is most likely due to the timing of T cell-specific E2A disruption in E2Aloxp/loxpCretg mice. Although Cre-mediated E2A deletion is detected as early as the CD44+CD25- DN1 stage, the nondeleted E2Aloxp allele is still detectable up to the DN4 stage. Thus, it is likely that a significant amount of E2A protein is still present in the DN stage. This might explain why no block in the transition from the CD44+CD25- DN1 to the CD44+CD25+ DN2 stage was observed in these mice (data not shown). However, it is not clear why the DP and SP phenotypes were not seen in the conditional knockout mice. PCR assay of sorted thymocytes indicates that E2A disruption is completed in most cells reaching the DP stage. A genetic test of the HEB knockout background further shows a strong effect of E2A disruption on T cell development during the transition from DN to DP. It is formally possible that a residue amount of E2A proteins persists in DP cells. The positive and negative selections in the development of DP to SP may need only a very small amount of E2A proteins. Alternatively, it is equally possible that a T cell extrinsic role of E2A may be partially responsible for the defects in DP and SP cell development reported in E2A germline knockout mice. Further investigations are needed to distinguish these possibilities.
It is well documented that thymocyte development requires cell-cell interaction between bone marrow-derived T cell precursors and thymic stroma (1). Stromal cells regulate not only the initial differentiation of DN cells, but also the maturation process of DP cells by providing MHC-peptide/TCR interaction and instructing the proper adhesion and migration of thymocytes from cortex to medulla. ECM has been implicated in guiding the migration of CD3highCD69high postselected DP thymocytes from cortex to medulla (26). In our study we observed high level expression of E2A in thymic stromal cells, including a subpopulation of ECM-secreting cells. These E2A-expressing cells are scattered in both the cortex and medulla (data not shown). It is conceivable that E2A may play a role in the proper development and function of these stromal cells. Conditional disruption of E2A in these stromal cells will be required to test this hypothesis in the future.
What is the role of E2A in T cell tumor formation?
The study of E2A-deficient mice in the past has revealed a high incidence of T cell lymphoma in the thymus. This phenotype is believed to be partially due to the important roles of E2A in regulating cell proliferation and apoptosis (17, 18). During the early G1 phase of the cell cycle, the E box binding activities of E2A proteins are transiently depressed at a time coincident with the peak induction of Id proteins (27, 28). Forced expression of E2A in many cell types, including myoblasts, 3T3 cells, and 293T cells, leads to a growth arrest, possibly by enhancing the transcription of several cyclin-dependent kinase inhibitor genes, including p21CIP/WAF1, p15INK4B, and p16INK4B (19, 29). The involvement of E2A proteins in apoptosis was also suggested. Reintroduction of E2A proteins into E2A-deficient lymphomas promoted the death of tumor cells (17). Growth arrest and apoptosis were observed upon restoration of E2A activity in T cell acute lymphoblastic leukemia cells (18). These observations led to the current dogma that the loss of E2A in E2A-deficient mice may promote cell survival and/or proliferation, which predisposes these mice to tumor formation.
Although about 50% of E2A-deficent mice developed thymic lymphoma between 3 and 10 mo of age (11, 16), none of the 26 E2Aloxp/loxpCretg mice analyzed from 4 to 16 mo of age developed malignant tumors. Although the origin of T cell tumors in E2A-deficent mice is not precisely determined, most of them phenotypically resemble DP or SP cells. The most likely scenario is that the tumors found in E2A-deficient mice originated from DN cells and acquired CD4 and/or CD8 expression during or after their transformation. Since E2A is not completely inactivated in E2Aloxp/loxpCretg DN cells, its tumor suppression activity may prevent the transformation of these DN cells. We cannot rule out the possibility that a T-independent function of E2A may be required for thymic homeostasis. This is supported by a recent study by Lyden et al. (30), who found that Id1 and Id3 proteins are expressed in endothelial cells and are required for angiogenesis and vascularization of tumor xenografts. The fact that E2A is highly expressed in thymic endothelial cells makes E2A a potential candidate to regulate the function of Id proteins in tumorigenesis. Finally, the loss of E2A function in both T cells and thymic stromal cells may have a synergistic effect on tumor formation in E2A-deficient mice. Future studies are required to distinguish these possibilities.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yuan Zhuang, Department of Immunology, Box 3010, Duke University Medical Center, Durham, NC 27710. E-mail address: yzhuang{at}acpub.duke.edu
3 Abbreviations used in this paper: DN, double negative; bHLH, basic helix-loop-helix; DP, double positive; ECM, extracellular matrix; FDG, fluorescein di -D-galactopyranoside; -gal, -galactosidase; GFP, green fluorescent protein; ISP, immature single positive; PGKneo, phosphoglyceryl kinase gene promoter driven neomycin resistance gene; SP, single positive; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactoside; 7AAD, 7-amino actinomycin D.
Received for publication September 21, 2001. Accepted for publication February 5, 2002.
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T-cell development and to rapid development of T-cell lymphomas. Mol. Cell. Biol. 17:4782.[Abstract]
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. The probe used for Southern
blot analysis is shown below the E2Awt
construct. Restriction enzymes and selection markers are abbreviated as
follows: X, XbaI; B, BamHI; PGK,
phosphoglycerate kinase gene promoter; TK, thymidine kinase gene; neo,
neomycin resistance gene; sa, splicing acceptor; ires, internal
ribosomal entry site; 
