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The Journal of Immunology, 2000, 165: 6710-6715.
Copyright © 2000 by The American Association of Immunologists

MHC Recognition in Thymic Development: Distinct, Parallel Pathways for Survival and Lineage Commitment1

David Chang, Patricia Valdez, Thomas Ho and Ellen Robey2

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The molecular events triggered by MHC recognition and how they lead to the emergence of mature CD4 and CD8 lineage thymocytes are not yet understood. To address these questions, we have examined what signals are necessary to drive the development of CD8 lineage thymocytes in TCR{alpha}- mice in which TCR/MHC engagement cannot occur. We find that the combination of constitutive Notch activity and constitutive Bcl-2 expression are necessary and sufficient to allow the appearance of mature CD8 lineage thymocytes in TCR{alpha}- mice. In addition, Notch activity alone in TCR{alpha}- mice can induce the up-regulation of HES1, suggesting that thymocytes are competent to respond to Notch signaling in the absence of MHC recognition. These data indicate that survival and lineage commitment represent distinct, parallel pathways that occur as a consequence of MHC recognition, both of which are necessary for the development of mature CD8 lineage T cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During thymic development, thymocytes whose Ag receptors recognize MHC proteins on thymic epithelial cells with the appropriate affinity give rise to mature CD4 and CD8 lineage T cells: a process known as positive selection (reviewed in Refs. 1, 2). One consequence of positive selection is the rescue from programmed cell death. Preselection CD4+CD8+ precursors are short-lived cells, the majority of which are destined to die in the thymus, whereas postselection mature CD4+CD8- and CD4-CD8+ thymocytes are relatively long-lived cells that can eventually leave the thymus and take up residence in the periphery. This has led to the notion that positive selection provides a survival signal to thymocytes whose Ag receptors can engage MHC molecules with the appropriate affinity. A second consequence of positive selection is lineage commitment. The choice of CD4+CD8+ precursors to develop as CD4+CD8- (CD4 lineage) or CD4-CD8+ (CD8 lineage) T cells is guided by MHC recognition such that thymocytes whose Ag receptors recognize class I MHC proteins develop as CD8 lineage cells and thymocytes whose Ag receptors recognize class II MHC develop as CD4 lineage cells. Although both rescue from programmed cell death and lineage commitment result from TCR-MHC recognition, the relationship between these events is not yet clear.

Recent evidence indicates that the CD4 vs CD8 lineage decision is also influenced by Notch, an evolutionarily conserved receptor that controls binary cell fate decisions in many organisms (reviewed in Refs. 3, 4, 5, 6). Expression of an activated form of Notch overrides the normal requirement for class I MHC and allows CD8 lineage thymocytes to develop in class I MHC-deficient mice. However, activated Notch cannot permit CD8 cell development in the absence of both class I and class II MHC (7). These results imply that activated Notch does not override the requirement for positive selection, but rather alters the fate of developing T cells such that thymocytes whose Ag receptors recognize class II MHC develop as CD8 lineage cells rather than CD4 lineage cells.

These results imply that MHC recognition plays two distinct roles during thymic selection. One role normally regulates CD4 vs CD8 lineage commitment and can be overridden by an activated form of Notch in favor of the CD8 lineage. In addition, a general role for MHC recognition (either class I or class II) is required in a step that cannot be overridden by activated Notch. Does this general role for MHC recognition reflect a requirement to render a thymocyte responsive to the signals that direct lineage determination? Alternatively, does MHC recognition simply provide a permissive survival signal, without which mature CD4 or CD8 lineage cells cannot emerge? Are distinct signaling pathways driving survival and lineage commitment? To what extent can these events be separated?

To attempt to address these questions, we have asked what signals can drive the development of CD8 lineage thymocytes in TCR{alpha}- mice in which MHC recognition and positive selection cannot occur. We find that the combination of constitutive Notch activity and constitutive Bcl-2 expression are necessary and sufficient to allow the appearance of phenotypically and functionally mature CD8 lineage thymocytes in TCR{alpha}- mice. In addition, Notch activity alone in TCR{alpha}- mice can induce the up-regulation of HES1, suggesting that thymocytes are competent to respond to Notch signaling in the absence of MHC recognition. These data indicate that survival and lineage commitment represent distinct, parallel pathways that occur as a consequence of MHC recognition during positive selection, both of which are necessary for mature T cells to emerge.


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

NotchIC-9 transgenic mice have been previously described (7). TCR{alpha} mutant mice (8) were obtained from The Jackson Laboratory (Bar Harbor, ME). Bcl-2 transgenic mice (9) were kindly provided by Stanley Korsmeyer (Harvard Medical School, Boston, MA). Transgenic offspring were identified by Southern blot and PCR typing. TCR{alpha} mutant offspring were identified by flow cytometry using anti-{alpha}ßTCR staining of thymocytes.

Flow cytometry

Thymus and lymph nodes (cervical, axillary, brachia, and mesenteric) were teased apart in cold M199 medium (Life Technologies, Grand Island, NY) supplemented with 2% FBS, and the cells were filtered through nylon mesh. A total of 106 cells were incubated with 10 µl Ab on ice for 20 min. Cells were then washed twice with staining buffer containing 1x HBSS (Fisher, Pittsburgh, PA), 0.2% sodium azide, and 0.2% bovine albumin (Sigma, St. Louis, MO). Data (50,000 events) were collected and analyzed using an Epics XL-MCL flow cytometer (Coulter, Hialeah, FL). Dead cells were excluded on the basis of forward and side scatter. Dot plot images were produced with the aid of WinMDI version 2.1.2 by Joseph Trotter (Scripps Research Institute, La Jolla, CA). Abs used were FITC-labeled anti-CD8{alpha} (53-6.7; PharMingen, San Diego, CA), FITC-labeled goat anti-mouse IgM (Caltag, South San Francisco, CA), RED613-labeled anti-CD4 (H129.19; Life Technologies), PE-labeled anti-TCR (H57-597; PharMingen), PE-labeled CD4 (Becton Dickinson, Mountain View, CA), FITC-labeled anti-{alpha}ßTCR (PharMingen), anti-heat stable Ag (HSA)3 (J11.d culture supernatant), R613-labeled anti-Rat Ig (Life Technologies), and rat {gamma}-globulin (Calbiochem, San Diego, CA), PE-labeled anti-{gamma}{delta} TCR (PharMingen), FITC-labeled anti-CD8ß (PharMingen), and FITC-labeled anti-Kb (PharMingen).

Functional assays

Thymocytes (~107) were stained with Abs against CD4 and CD8 and populations were isolated by FACS. Mature CD4+CD8- and CD4-CD8+ populations were preenriched before sorting by treating thymocytes with HSA and complement. Sorted double positive (DP) and CD4 single positive (SP) thymocytes were >98% pure. Sorted CD8 SP thymocytes were 80–97% pure. The contaminating population was primarily DP thymocytes, and there was <0.5% contamination with CD4 SP or double negative (DN) thymocytes. Sorted thymocytes (20,000/well) were cultured in round-bottom 96-well plates for 24 h in the presence of PMA (5 ng/ml) and ionomycin (A23187) (125 ng/ml). Culture supernatants were assayed for IL-2 using an ELISA kit (OptEIA; PharMingen) according to the manufacturer’s instructions. For measurement of thymocyte survival, cell suspensions of total thymocytes were cultured in RPMI containing 10% FCS at 37°C at initial cell concentrations of either 106/ml or 2 x 105/ml. Cell viability was measured after various times in culture using trypan blue exclusion.

Northern blot analyses

For preparation of thymocyte RNA, thymuses were teased in media and cell suspensions were filtered through nylon mesh. Cell suspensions prepared in this manner consist of >99% thymocytes and are free of stromal cells. RNA was isolated using Tripure Isolation Reagent (Boehringer Mannheim, Indianapolis, IN). For Northern blot analysis, 20 µg of total RNA was used (10). An equal amount of RNA was loaded in each lane based on ethidium bromide staining of the 18S and 28S ribosomal RNAs. The 1-kb EcoRI/DraI fragment from rat HES1 cDNA was used as a probe for HES1 (kindly provided by R. Kageyama, Kyoto University, Kyoto, Japan). The 350-bp EcoRI/HindIII fragment from the E47 cDNA was used as a probe for E2A (kindly provided by C. Murre, University of California, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Previous studies indicate that Notch activity can permit CD8 T cell development in the absence of class I MHC, but not in the absence of both class I and class II MHC (7). This requirement for MHC may reflect the need for a survival signal, independent of Notch signaling, to allow CD8 lineage T cells to emerge. Alternatively, MHC recognition might turn on components of the Notch signaling pathway, and thus render thymocytes competent to respond to activated Notch. If MHC recognition provides a survival signal independent of Notch signaling, it might be possible to supply this survival signal using a constitutive Bcl-2 transgene. Bcl-2 is an inhibitor of programmed cell death, and previous studies have shown that constitutive expression of Bcl-2 inhibits apoptosis in thymocytes (9, 11, 12). We therefore crossed both a constitutive Bcl-2 transgene (11) and an activated Notch transgene onto a TCR{alpha} mutant background (8) and examined thymic development in the offspring of this cross.

As shown in Fig. 1Go, A and B, wild-type mice contain a small population of CD4-CD8+ thymocytes, the majority of which express low levels of HSA, indicating that they are mature CD8 lineage thymocytes. Mice lacking the {alpha}ßTCR, due to a targeted mutation of the TCR{alpha} gene (8), display a block in the development of mature CD4 and CD8 lineage cells, and this is reflected in the reduced number of CD4-CD8+HSAlow thymocytes in these mice (Fig. 1Go, A and B). The presence of either a constitutive Bcl-2 transgene, or an activated Notch transgene alone, in TCR{alpha} mutant mice leads to only a small increase in the number of CD4-CD8+HSAlow thymocytes (Fig. 1Go, A and B). This is consistent with previous studies showing that neither the Bcl-2 transgene nor the activated Notch transgene are sufficient to permit the development of CD8 lineage thymocytes in the absence of both class I and class II MHC (9, 13). Strikingly, the combination of activated Notch and constitutive Bcl-2 leads to the appearance of a large population of thymocytes that are CD4-CD8+HSAlow. Similar to the CD4-CD8+ thymocytes from wild-type mice, the CD4-CD8+ population from TCR{alpha}- NotchIC, Bcl-2 transgenic mice lacks expression of {gamma}{delta}TCR, but expresses high levels of class I MHC (Fig. 1GoC). In addition, the CD4-CD8+ population from TCR{alpha}- NotchIC, Bcl-2 transgenic mice expresses CD8ß, a characteristic of conventional CD8 lineage thymocytes. The levels of both CD8{alpha} and CD8ß are somewhat lower on CD4-CD8+ cells from TCR{alpha}- NotchIC, Bcl-2 transgenic mice, compared with CD4-CD8+ population from wild-type mice, perhaps a result of inappropriately high Notch activity in these cells. Together, these analyses indicate that the combination of Notch activity and a survival signal are necessary and sufficient to permit the development of phenotypically mature CD8 lineage thymocytes in the absence of positive selection.



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  		FIGURE 1. Activated Notch and constitutive Bcl-2 lead to the appearance of phenotypically mature CD8 lineage thymocytes in TCR{alpha} mutant mice. Thymocytes from mice of the indicated genotype were analyzed by three-parameter flow cytometry using Abs to CD4, CD8, and HSA. A, Representative flow cytometric analysis showing CD4 and CD8 expression and HSA levels on total (ungated) thymocytes and HSA levels on gated CD4-CD8+ thymocytes. The numbers denote the % of total thymocytes within the indicated gates. B, The absolute number of mature CD4 and CD8 lineage thymocytes (CD4+CD8-HSAlow and CD4-CD8+HSAlow) was calculated using the gates indicated in A. Bars represent the mean values for mice of the indicated genotypes, and symbols represent values from individual mice. The absolute number of mature CD4 and CD8 lineage thymocytes for wild-type mice were 2 x 107 and 1 x 107, respectively. C, CD4-CD8+ thymocytes from TCR{alpha}- NotchIC, Bcl-2 transgenic mice were analyzed for expression of {gamma}{delta}TCR, CD8ß, and class I MHC by three-parameter flow cytometric analysis. Data from wild-type mice are shown for comparison. The gated population is indicated in the upper right hand corner of each histogram. The numbers indicate the % of gated thymocytes that are positive for the indicated marker. CD8 SP thymocytes (CD4-CD8+) from wild-type and double transgenic TCR{alpha} mutant mice are {gamma}{delta}TCR-, whereas DN thymocytes (CD4-CD8-) from wild-type mice contain a significant fraction of {gamma}{delta}TCR+ cells. CD8 SP thymocytes from wild-type and double-transgenic TCR{alpha} mutant mice express CD8 ß, whereas CD4 SP thymocytes (CD4+CD8-) from wild-type mice do not. CD8 SP thymocytes from wild-type and double-transgenic TCR{alpha} mutant mice express high levels of class I MHC, whereas DP thymocytes (CD4+CD8+) from wild-type mice are class I MHClow.

 
The phenotype of CD8+CD4-HSAlow thymocytes in NotchIC, Bcl-2 transgenic, TCR{alpha} mutant mice suggests that they represent mature CD8 lineage T cells. To confirm this, we examined the ability of these cells to produce IL-2. We isolated CD4-CD8+HSAlow (CD8 SP), CD4+CD8-HSAlow (CD4 SP), or CD4+CD8+ DP thymocytes from wild-type mice and from NotchIC, Bcl-2 transgenic, TCR{alpha} mutant mice, cultured them in the presence of PMA and ionomycin, and measured IL-2 in the culture supernatants. As shown in Fig. 2Go, CD8+CD4- thymocytes from NotchIC, Bcl-2 transgenic TCR{alpha} mutant mice produce significant levels of IL-2, indicating that they are functionally, as well as phenotypically, mature. Interestingly, despite the evidence for functional maturity of CD8 cells in the thymus, CD8 T cells fail to accumulate in the lymph nodes of NotchIC, Bcl-2 transgenic, TCR{alpha} mutant mice (data not shown). The failure of these phenotypically mature CD8 thymocytes to migrate and survive in the periphery may be a result of the absence of {alpha}ßTCR expression on these cells and the known requirement for TCR-MHC interactions for the maintenance of peripheral T cells (14). An alternative explanation is that the NotchIC and Bcl-2 transgenes induce only a subset of the properties of normal mature CD8 T cells that allow them to accumulate in the periphery.



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  		FIGURE 2. CD8+CD4- thymocytes from TCR{alpha} mutant, NotchIC, Bcl-2 transgenic mice can produce IL-2. Thymocyte populations from either wild-type or TCR{alpha}- NotchIC and Bcl-2 transgenic (mutant) were isolated by FACS sorting based on expression of CD4 and CD8. Sorted populations were CD4+CD8+ (DP), CD4+CD8- (CD4 SP), or CD4-CD8+ (CD8 SP). Mature thymocytes were preenriched before sorting by treating with anti-HSA Abs and complement before sorting. Sorted thymocytes were cultured in the presence of PMA and ionomycin for 24 h, and IL-2 production in culture supernatants was measured. Data from two separate experiments are shown. The variation between the two experiments probably results from small variations in the stimulation conditions used for each experiment.

 
The appearance of CD8 lineage thymocytes in Bcl-2 and NotchIC transgenic, TCR{alpha} mutant mice implies that the down-stream events of Notch signaling can occur in the absence of MHC recognition. If this is the case, it might be possible to see indications of Notch signaling in thymocytes in the absence of the {alpha}ßTCR. We have previously shown that expression of activated Notch in thymocytes leads to the up-regulation of HES1 (15), a gene that encodes a basic helix-loop-helix transcription factor that is related to components of the Notch signaling pathway in Dro- sophila (16, 17). We find that up-regulation of HES1 mRNA also occurs in thymocytes from TCR{alpha} mutant, NotchIC transgenic mice (Fig. 3Go). This indicates that activated Notch can turn on theNotch signaling pathway in the absence of TCR-MHC engagement, although it is not sufficient to drive the development of CD8 lineage T cells.



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  		FIGURE 3. Activated Notch leads to HES1 up-regulation in the absence of positive selection. Northern analysis of HES1 mRNA in thymocytes from wild-type mice, NotchIC transgenic mice, TCR{alpha} mutant mice, and TCR{alpha} mutant, NotchIC transgenic mice. The same blot was reprobed with an E2A probe as a control for RNA loading.

 
In most systems, Bcl-2 acts to promote cell survival, whereas Notch affects cell fate decisions. There are reports that Notch activity can affect thymocyte survival in some experimental settings (18, 19). However, it seems unlikely that NotchIC is exerting its CD8-promoting effects via a survival signal, because Notch and Bcl-2 activity are jointly required to drive CD8 cell development in the absence of positive selection, and because Bcl-2 alone is known to deliver a potent survival signal. To explore this issue further, we directly compared the NotchIC transgene and the Bcl-2 transgene for their ability to promote thymocyte survival. Thymocytes from both normal and NotchIC transgenic mice die within a few days when placed in culture without thymic stromal cells (Fig. 4Go). In contrast, thymocytes from Bcl-2 transgenic mice show significantly enhanced survival under the same culture conditions (Fig. 4Go, and Ref. 20). We also note that the turnover rate of CD4+CD8+ thymocytes, as measured by the rate of 5-bromo-2'-deoxyuridine incorporation, is identical in NotchIC transgenic and wild-type mice (7). In contrast, the turnover of CD4+CD8+ cells from Bcl-2 transgenic mice is dramatically delayed compared with wild type (Ref. 21 , and data not shown). These data indicate that, unlike Bcl-2, the NotchIC transgene does not provide a general survival signal to thymocytes. Together, the data are most consistent with the interpretation that Notch and Bcl2 act in distinct ways to promote the CD8 fate: with Bcl-2 promoting survival, and NotchIC promoting the CD8 lineage choice.



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  		FIGURE 4. The Bcl-2 transgene, but not the NotchIC transgene, promotes thymocyte survival in culture. Total thymocytes from the indicated mice were cultured in RPMI containing 10% FCS at 37°C and cell viability was measured after various times in culture using trypan blue exclusion. Data for two independent cell samples from each genotype are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The generation of mature CD4 or CD8 lineage thymocytes from CD4+CD8+ precursors is driven by recognition of MHC proteins by TCR expressed by thymocytes, but the nature of the signaling pathways that contribute to this process is poorly understood. Here, we show that the combination of a constitutive Notch signal and Bcl-2 up-regulation is necessary and sufficient to drive the development of phenotypically and functionally mature CD8+ thymocytes in the absence of TCR-MHC engagement. We also provide evidence that the Notch signaling pathway can be activated in the absence of TCR-MHC engagement, but that this is not sufficient to allow the emergence of mature CD8+ thymocytes in the absence of a survival signal. Given the well-established roles for Bcl-2 in survival and for Notch in lineage commitment, these data support a model (Fig. 5Go) in which TCR-MHC engagement during positive selection of thymocytes activates two distinct signaling pathways, one which regulates survival and one which regulates lineage commitment. The survival signal is required for the emergence of both CD4 and CD8 lineage T cells, but does not control lineage choice. The lineage commitment signal differentially modulates Notch signaling in response to class I or class II MHC recognition. Thus, class I MHC recognition (Fig. 5GoA) would both up-regulate Notch signaling and provide a survival signal, leading to the development of CD8 lineage T cells. Class II MHC recognition would both decrease Notch signaling and provide a survival signal, thus leading to the development of CD4 lineage T cells (Fig. 5GoB). In the absence of MHC recognition (Fig. 5GoC), CD8 cells can be generated by providing both a lineage commitment signal in the form of an activated Notch transgene, and a survival signal in the form of a Bcl-2 transgene.



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  		FIGURE 5. Distinct pathways for survival and lineage commitment. According to this model, one consequence of class I MHC recognition (A) would be a survival signal, perhaps supplied in part by up-regulation of Bcl-2 (9 22 23 ). In addition, class I MHC recognition would lead to increased Notch signaling that would promote the CD8 cell fate, in part by causing up-regulation of HES1 (15 ). The combination of the survival signal and increased Notch signaling would lead to the appearance of mature CD8 lineage T cells. Class II MHC recognition (B) would also produce a survival signal, but would lead to decreased Notch signaling. The combination of a survival signal and decreased Notch signaling would lead to the appearance of mature CD4 lineage cells. In the absence of MHC recognition (C) the combination of constitutive Bcl-2 expression and constitutive Notch activity could mimic class I MHC recognition and lead to the appearance of mature CD8 lineage cells. Italics are used to denote steps, which may contribute to, but are not absolutely required for, the indicated pathway. See text for discussion.

 
Although our data show that Notch and Bcl-2 transgenes can provide survival and lineage commitment signals in the absence of positive selection, they leave open the question of which molecules provide these signals during normal positive selection. There is evidence that CD4+CD8+ thymocytes up-regulate Bcl-2 in response to positive selection signals (9, 22, 23), suggesting that Bcl-2 may contribute to a survival signal generated during positive selection. However, positive selection occurs normally in mice lacking Bcl-2 (24, 25), indicating that other anti-apoptotic molecules may also contribute to this process. In the case of the lineage commitment signal, the effects of an activated form of Notch on the CD4 vs CD8 lineage choice implicate the Notch signaling pathway; however, it is not yet clear which endogenous proteins are normally involved. A conditional disruption of the Notch1 gene produces a very early block in thymic development (26), precluding an examination of the effect of the mutation on the CD4 vs CD8 lineage choice. The effect of blocking Notch1 function in a thymic organ culture system provides evidence that Notch1 is involved in the developmental progression of CD8 lineage, but not CD4 lineage, thymocytes (27). However, Notch1, Notch2, and Notch3 are expressed by thymocytes (28), suggesting the possibility that multiple Notch homologues may contribute to the CD4 vs CD8 lineage choice. With regard to proteins acting downstream of Notch, we have identified one possible candidate, the basic helix-loop-helix transcription factor, HES-1. The observation that HES-1 is up-regulated in response to Notch activity in thymocytes (15), together with the fact that HES-1 related genes function downstream of Notch in other systems (16, 17), suggests that HES-1 up-regulation by Notch may contribute to the CD4 vs CD8 lineage choice.

Recently, Deftos et al. (19, 29) have put forth a very different view of the role of Notch in which Notch provides survival signals that promote the development of both CD4 and CD8 cell development. Their view is based in part on the observation that Notch activity can induce Bcl-2 expression in a thymocyte cell line and inhibit glucocorticoid-mediated cell death in thymocytes, suggesting the possibility that the effect of activated Notch on the CD4 vs CD8 lineage choice is an indirect consequence of disregulating Bcl-2 expression. The data presented here, showing that activated Notch and Bcl-2 perform distinct and separable functions during positive selection, argue strongly against this view and are most compatible with the notion that Notch exerts its effect directly on lineage commitment. Interestingly, whereas the Bcl-2 transgene is not sufficient to allow the positive selection of CD8 T cells in MHC or TCR{alpha}-deficient mice, it can allow CD8 cell development in class I MHC-deficient mice (9). Although the explanation for this is not yet clear, one possibility is that constitutive Bcl-2 expression leads to disregulation of Notch, causing thymocytes that recognize class II MHC to receive an inappropriately high Notch signal and directing them to the CD8 lineage.

With regard to the question of whether Notch activity promotes the CD4 fate as well as the CD8 fate, we see no evidence for the induction of CD4 lineage development by activated Notch in the absence of positive selection, either with or without the Bcl-2 transgene. This is in contrast to a recent report that activated Notch can permit CD4 and CD8 lineage T cell development upon transfer of Notch transgenic bone marrow cells to MHC-deficient hosts (29). Although the reason for this discrepancy is not yet clear, it is may reflect differences in the oncogenic potential of the different forms of activated Notch used in the two studies. The C-terminal transactivation domain of Notch, which is contained in the transgenic Notch construct of Deftos et al., but is partially deleted in ours, has been shown to contribute to the oncogenic potential of Notch (30). This may explain the more rapid onset of leukemia in the Notch transgenic mice described by Deftos et al. (4 wk, compared with >12 wk in our Notch transgenic line). Given the early onset of leukemia, it is possible that the mature T cells observed in the thymus upon transfer of Notch transgenic bone marrow into MHC-deficient hosts, do not arise in the thymus, but are due instead to the transfer of preleukemic T cells from the Notch transgenic bone marrow. This issue could be addressed by using fetal liver rather than bone marrow as a source of donor cells, or by crossing the Notch transgene onto a TCR{alpha} or MHC-deficient background and examining thymic development in young mice.

It is interesting to note that Bcl-2 expression in TCR{alpha} mutant mice is insufficient to allow the development of either CD4 or CD8 lineage T cells. According to our model, one might have expected that simply providing a survival signal in the absence of a Notch signal would be sufficient to allow CD4 lineage T cells to develop. The fact that this does not occur suggests that thymocytes might experience a low level of Notch signaling before positive selection and that class II MHC recognition plays an active role in turning down Notch signaling, thus allowing CD4 cells to develop. The question of how MHC recognition promotes survival and regulates Notch signaling is an important area for future investigation.


   Acknowledgments
 
We thank Stanley Korsmeyer for providing Bcl-2 transgenic mice, Hector Nogales for expert assistance with flow cytometry, and B. J. Fowlkes for comments on the manuscript.


   Footnotes
 
1 This work was supported by the National Institutes of Health (AI42033 and AI32985). P.V. is a recipient of a fellowship from the Ford Foundation. Back

2 Address correspondence and reprint requests to Dr. Ellen Robey, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720. Back

3 Abbreviations used in this paper: HSA, heat stable Ag; DP, double positive; SP, single positive; DN, double negative. Back

Received for publication May 7, 2000. Accepted for publication September 11, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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