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* Laboratory of Cellular and Molecular Immunology, National Institutes of Health, Bethesda, MD 20892; and
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
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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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The first indication that Notch activity could promote CD8 single positive (SP)3 development came from analysis of the effect of activated Notch1 on thymic development (5). Transgenic mice expressing an activated form of Notch1 under the control of the Lck proximal promoter display an increase in CD8 SP thymocytes. In addition, activated Notch1 promotes CD8 SP development in MHC class I-deficient mice, but not in mice lacking both class I and II MHC. These results indicated that constitutive Notch activity could divert thymocytes bearing class II-specific TCRs into the CD8 lineage. A model for normal development was proposed in which MHC class I promotes the CD8 fate by up-regulating Notch activity.
Recently, a role for Notch in CD8 SP development has been called into question by analysis of a conditional Notch1 mutation (6). Deletion of the Notch1 locus by a CD4-driven CRE transgene does not lead to any alterations in the numbers or proportions of CD4 and CD8 SP thymocytes. However, thymocytes also express Notch2, Notch3, and Notch4 (7, 8), raising the possibility that multiple Notch family members might contribute to CD4 or CD8 SP development in a redundant fashion. Indeed, kinetic analysis of the development of thymocytes deficient for Notch1 reveals a delay in the appearance of CD8 SP, suggesting that Notch1 makes a nonessential contribution to CD8 SP development (6).
Further support for the idea that Notch promotes CD8 SP development comes from experiments in which Notch activity is blocked in fetal thymic organ culture (9, 10). Additions of inhibitors of presenilin, a family of proteins that are essential for Notch activity, inhibit the development of CD8 SP thymocytes. In addition, Notch1 antisense RNA expression and Notch1 Abs prevent the developmental progression of CD8 SP in a two-step culture system for thymic development (11).
Although all of these studies are consistent with a role for Notch signaling in promoting CD8 SP development, the effect of Notch activity on CD4 T cell development is less clear. The original description of Notch1 (Notch1IC) transgenic mice reported a decrease in the development of CD4 SP, but a recent study came to a different conclusion (12). In this study, which examined a slightly different version of the Notch1IC transgene, an increase in both CD8 SP and CD4 SP thymocytes was reported. Moreover, the Notch1IC transgene appeared to drive the development of CD4 and CD8 SP thymocytes in the absence of class I and II MHC. In addition to the apparent differences in CD4 and CD8 SP development, the modified Notch1IC transgene promoted early tumorigenesis. Thus, there was the possibility that these tumors might obscure effects of Notch on CD4 vs CD8 lineage commitment.
To clarify the effect of activated Notch1 on the development of CD4 and CD8 SP, we examined young mice before tumors appear. This analysis indicates that the two Notch1IC transgenic lines have very similar effects on thymic development, leading to extra CD8 SP thymocytes at the expense of CD4 SP. Activated Notch1 allows CD8 SP to develop in class I-deficient mice, but does not permit the development of CD4 SP thymocytes in class II MHC-deficient mice. Moreover, the enhanced development of CD8 SP thymocytes by activated Notch1 depends on the expression of either class I or II MHC. Thus, analysis of both versions of Notch1IC transgenic mice indicates that Notch activity favors CD8 SP and inhibits CD4 SP development. These results are consistent with a model in which Notch signaling regulates CD4 vs CD8 lineage determination.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) were obtained from the National Cancer Institute
(Frederick, MD) and MHC-deficient,
C57BL/6J-[KO]
2M-[KO]Ab
N17 (line 80; Ref. 13) from a National Institutes of
Allergy and Infectious Diseases (NIAID; Bethesda, MD) breeding contract
to Taconic Farms (Germantown, NY). Notch1IC mice (5) had
been maintained in a NIAID facility since 1996.
Notch1IC-transactivation domain (TAD) mice (12) were
kindly provided by M. Bevan (University of Washington, Seattle,
WA). Mice were bred, maintained, and used in NIAID Research
Animal Facilities according to American Association of Laboratory
Animal Care specifications and on protocols approved by NIAID Animal
Care and Use Committee. Transgenic mice were genotyped for
Notch1IC transgenes by PCR and for MHC by flow cytometry using standard
protocols.
Cell preparation, Abs, and flow cytometry
Thymocytes were prepared in single-cell suspensions, stained
with labeled Abs, and analyzed by flow cytometry as described
(14, 15). Cells were pretreated with a culture supernatant
containing anti-FcR
III/IIR, 2.4G2, to block FcR binding by
labeled Abs. PBLs were used for some MHC typing. After staining with
labeled Abs, samples were depleted of RBCs with ACK lysing buffer (pH
7.4) and analyzed by flow cytometry. Labeled mAbs included:
anti-TCR-FITC (H57-597), -CD4-PE (RM4-5),
-CD8
-allophycocyanin (53-6.7), -CD25-FITC (7D4), -CD44-PE (IM7), and
-H-2Kb-FITC (AF6-88.5) obtained from BD
PharMingen (San Diego, CA); anti-Ab-FITC
(28-16-8S) and -CD45R/B220-PE (RA3-6B2) obtained from Caltag
Laboratories (Burlingame, CA). Flow cytometry was performed on a
FACSCalibur or FACStarPlus using CellQuest software (BD Biosciences,
Mountain View, CA). Dead cells were excluded by light scatter and
propidium iodide gating. A total of 150,000 events were collected for
three- and four-color analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
This region of Notch1 contains a TAD that contributes to the oncogenic
activity of Notch1IC (16). The complete TAD is included in
the construct of Deftos et al.(12), but is partially
deleted in the construct of Robey et al. (5). In this
study, we designate the Notch transgenic mice of Deftos et al.
(12) as Notch1IC-TAD to indicate this difference.
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high)
in young Notch1IC-TAD mice is increased (Fig. 2), and the vast majority of these CD8 SP
thymocytes are heat-stable Aglow (data not
shown). However, contrary to the Deftos et al. (12)
conclusion that Notch1IC promotes the development of CD4 as well as CD8
SP thymocytes, our analysis of young Notch1IC-TAD mice reveals that the
absolute number of CD4 SP thymocytes is diminished (Fig. 2).
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). Both
sets of mice give indistinguishable results in this analysis. In
contrast, our examination of older (>5 wk) NotchIC-TAD mice showed a
large mouse-to-mouse variation in the proportion of thymic subsets, and
in some cases a reduced
CD4+CD8+ population (data
not shown), consistent with the published data (12). In
addition, a significant proportion of these older Notch1IC-TAD
transgenic mice had tumors that appear to be of thymic origin, based on
their expression of CD4 and CD8 and their thymic location (data not
shown). The greater mouse-to-mouse variation seen in >5-wk-old
NotchIC-TAD transgenic mice compared with <3-wk-old mice, together
with indications that the majority of older NotchIC-TAD transgenic mice
are about to succumb to tumors (data not shown), suggests that
analysis of thymocyte populations at 3 wk provides a more reliable
indicator of the effect of NotchIC on the CD4/CD8 lineage decision.
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To further explore the role of MHC in T cell development in Notch1IC
transgenic mice, we crossed Notch1IC-TAD transgenic mice to
MHC-deficient mice and examined thymic subsets in mice from the second
back-cross. Again, the analysis was confined to young (10–21 days old)
mice to avoid the complicating effects of tumors. As shown in Fig. 4, CD8 SP thymocytes develop in class I-
and II-deficient mice, but not in mice deficient for both class I and
II MHC. Moreover, there is no increase in CD4 SP thymocytes in class
II-deficient mice. These results are in accord with previous analyses
of Notch1IC transgenic mice (5, 17), and suggest that
constitutive Notch activity can direct thymocytes bearing class I and
II-specific TCRs to the CD8 lineage, but does not override the
requirement for positive selection. We suspect that the CD4 and CD8 SP
cells seen in MHC-deficient mice reconstituted with NotchIC-TAD bone
marrow may represent tumors that arose in the NotchIC-TAD mice that
were used as bone marrow donors in these studies.
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A). Biopsy of several
moribund NotchIC-TAD transgenic mice revealed the presence of thymic
tumor masses (data not shown). In contrast, Notch1IC mice develop
tumors later (2–6 mo) and with a much lower frequency. Also noted was
that the tumors of Notch1IC-TAD transgenic mice express CD4, CD8, and
high levels of CD25 (12). Interestingly, CD25 levels are
abnormally high, even on
CD4+CD8+ thymocytes of
young Notch1IC-TAD transgenic mice (Fig. 5B). Although CD25
expression is considerably lower on
CD4+CD8+ thymocytes of
Notch1IC compared with Notch1IC-TAD transgenic mice, CD25 expression is
abnormal in thymocytes of both lines when compared with nontransgenic
controls (Fig. 5B). Whether the abnormal expression of CD25
is related to transformation is not clear; however, there is a
correlation in the level of CD25 expression on
CD4+CD8+ thymocytes and the
degree of tumorigenesis in the two transgenic lines. High expression of
CD25 and early tumorigenesis have been reported also in transgenic mice
expressing Notch3IC in thymocytes (18).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We suspect that the sharp discrepancy between our results and those of Deftos et al. (12) is due to tumors arising in thymocytes of the Notch1IC-TAD transgenic mice. Many Notch1IC-TAD transgenic mice develop tumors by 4 wk, and Deftos et al. (12) analyzed mice at 5–12 wk of age. The presence of tumors could distort the results in a variety of ways. For example, transformation could lead to changes in the expression of surface molecules, and/or could cause the abnormal proliferation or expansion of subsets of thymocytes. Alternatively, the presence of tumors could indirectly distort thymocyte populations, because the stress associated with illness could lead to loss of CD4+CD8+ thymocytes, causing an increase in the proportion of SP thymocytes. Because the current study examines young mice before the onset of tumors, it is likely to provide a more accurate picture of the effect on activated Notch1 on T cell development. Confounding effects of tumors might also explain the lack of an obvious CD4/CD8 phenotype in transgenic mice expressing an activated form of NotchIC3 in thymocytes (18). Retroviral expression of Notch1IC has been reported to lead to diminished CD4 SP and CD8 SP development (19). This inhibition of both CD4 and CD8 SP development in this system may be due to higher levels of expression of activated Notch1 or the continued expression of activated Notch1 in SP thymocytes.
The differences between the two versions of Notch1IC focuses attention on the portion of the Notch1 intracellular domain from aa 2294–2444, residues that are included in the Notch1IC transgenic construct of Deftos et al. (12), but are missing from the construct of Robey et al. (5). This region of Notch1 forms part of a domain referred to as TAD, because it has been shown to contribute to the transactivation function of NotchIC (20). Although it appears that the TAD domain of Notch1 can contribute to Notch signaling, it does not appear to be essential. Expression of a minimal ankryn repeat region of Drosophila Notch or the related Caenorhabditis elegans proteins, glp-1 and lin-12, is sufficient to confer an activated Notch phenotype (21). In addition, the transgenic version of Notch1IC that lacks the full TAD domain still induces the up-regulation of the Notch target gene, hairy enhancer of split homolog 1 (HES1; Refs. 17 and 22). Finally, a similarly truncated form of Notch1 overrides the block in Notch signaling due to ectopic expression of lunatic fringe (3). Thus, it appears that the TAD domain provides an accessory function to Notch that may contribute to the level of Notch signaling, but is not essential for Notch activity.
It is noteworthy that the TAD region has also been identified as contributing to the oncogenic activity of Notch1IC (16). This finding provides a possible explanation for the more rapid development of tumors in Notch1IC-TAD compared with Notch1IC transgenic mice that do not contain the full TAD domain. However, we cannot exclude the possibility that transgene copy number or integration site could contribute to the difference in oncogenic activity of the two transgenes.
The issue of whether Notch activity inhibits or promotes CD4 SP development is related to the issue of whether Notch1IC exerts its effect on CD4/CD8 T cell development by altering lineage commitment or survival. Although the Notch signaling pathway is generally thought of as regulating cell fate decisions, in some experimental settings Notch can have pro- or anti-apoptotic effects (23, 24, 25). For example, thymocytes of Notch1IC transgenic mice show increased resistance to dexamethasone-induced apoptosis compared with thymocytes from nontransgenic mice. This observation led to the suggestion that Notch activity might promote the survival of CD4 and CD8 SP thymocytes during positive selection. However, mutations of the glucocorticoid receptor that abolish dexamethasone-mediated death of thymocytes have no apparent effect on positive or negative selection (26), raising questions about the relevance of the dexamethasone-induced death assay. Indeed, kinetic analysis of thymic development in Notch1IC transgenic mice revealed no alterations in thymocyte life span (5), indicating that Notch activity does not alter thymocyte survival in the presence of endogenous glucocorticoids.
Finally, a recent report of the combined effects of constitutive Notch1
and Bcl2 argue against a model in which Notch promotes the CD8 T cell
fate by providing a survival signal (26). Although neither
a Notch1IC transgene or a constitutive Bcl2 transgene alone can allow
the development of SP thymocytes in TCR-deficient mice, the
combination of both transgenes leads to the appearance of
phenotypically and functionally mature CD8 SP thymocytes. The effect of
Notch1IC in this setting is unlikely to be mediated by survival because
Bcl2 alone provides a potent survival signal. In addition, CD8 SP, but
not CD4 SP, was observed in TCR mutant mice expressing both Notch1IC
and Bcl2 transgenes. Thus, the effects of activated Notch1 on CD4/CD8 T
cell development cannot be readily explained by effects on survival,
but are most consistent with models in which Notch activity regulates
CD4/CD8 lineage commitment.
A separate line of evidence used to support the notion that Notch activity promotes both CD4 and CD8 SP development comes from analysis of Notch-induced genes (12). A number of genes that are expressed at higher levels in Notch1IC transgenic mice compared with nontransgenic mice are also expressed at higher levels in CD4 and CD8 SP thymocytes compared with DP thymocytes. If these genes represent direct targets of the Notch signaling pathway, their expression in CD4 and CD8 SP thymocytes could be taken as evidence for Notch signaling in both lineages. However, it is unclear whether these genes are direct targets of Notch signaling; therefore, their induction may not provide a reliable measure of Notch activity. Mature CD4 and CD8 T cells display very similar patterns of gene expression, and there are likely to be a large number of "T cell maturation genes" that are turned on in both lineages. If Notch activity induces CD8 maturation, it would turn on this set of T cell maturation genes, but signals that lead to CD4 maturation would turn on this same set of genes. Changes in gene expression due to Notch signaling may involve a limited set of genes and may lead to very transient induction or repression of gene expression.
Of the Notch-induced genes examined, the one most likely to be a direct target of the Notch signaling pathway is HES1. HES1 is related to the Drosophila Notch target genes encoded by the enhancer of split complex. The HES1 promoter is regulated by CBF1, a transcription factor that is activated by Notch signaling (27). Of relevance, the expression pattern of the Notch-target gene enhancer of split complex in the developing nervous system of Drosophila is highly dynamic (28). Moreover, there is evidence that HES1 represses its own transcription (29). Thus, induction of HES1 following Notch signaling could be a transient event. Careful kinetic analysis of direct targets of Notch signaling pathway during CD4 and CD8 cell differentiation is needed to address this issue.
Although this report clarifies the effect of constitutively activated Notch1 on CD4 and CD8 T cell development, the question of how endogenous Notch signaling participates in this development remains unanswered. Further analysis of the role of endogenous Notch signaling during CD4 and CD8 T cell development may help to resolve this question.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. B. J. Fowlkes, Laboratory of Cellular and Molecular Immunology, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 111, Bethesda, MD 20892-0420. E-mail address: bfowlkes{at}nih.gov
3 Abbreviations used in this paper: SP, single positive; HES1, hairy enhancer of split homolog 1; TAD, transactivation domain.
Received for publication April 15, 2002. Accepted for publication June 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity in hemopoietic progenitor cells. J. Immunol. 167:4458.-secretase activity modulates thymocyte development. Proc. Natl. Acad. Sci. USA 98:9312.-Secretase inhibitors repress thymocyte development. Proc. Natl. Acad. Sci. USA 98:7487.B and T-cell leukemia/lymphoma in Notch3 transgenic mice. EMBO J. 19:3337.[Medline]
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) and nontransgenic
(Tg-; 
) littermates. Number of mice analyzed in each
group: wild type, Tg-, n = 9,
Tg+, n = 10; class Io,
Tg-, n = 11, Tg+,
n = 11; class IIo, Tg-,
n = 6, Tg+, n = 10;
MHCo, Tg-, n = 10,
Tg+, n = 9.
,
The age range of mice analyzed in the study of Deftos et al.
(
indicates the age range of mice analyzed in
the current study. B, Analysis for CD25 expression
by CD4+CD8+ thymocytes. Thymocytes from young
(17–18 days old) Notch1IC transgenic mice were analyzed for expression
of CD4, CD8, and CD25 by three-color flow cytometry. Histograms display
relative fluorescence from analysis of thymocytes for CD25,
gating for CD4+CD8+ of Notch1IC-TAD
(solid black line), Notch1IC-TAD nontransgenic (nonTg) littermate
(dotted black line), Notch1IC (solid gray line), and Notch1IC
nontransgenic (dashed gray line) mice. Data are representative of four
litters, containing 3–5 mice of each genotype.