By
From the Division of Molecular Immunology, Medical Research Council, National Institute for Medical Research, London NW7 1AA, United Kingdom
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Abstract |
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T cell development and selection in the thymus are shaped by the induction of apoptosis.
However, a direct role in T cell development and selection for any of the molecules known to
regulate apoptosis has remained controversial. We have studied the effect of bax and bcl-2 transgenes in recombination activation gene 1-deficient (RAG-1/
) mice transgenic for the major
histocompatibility complex class I-restricted F5 T cell receptor. Overexpression of a bax transgene in the thymus seriously impairs the production of mature T cells, whereas bcl-2 overexpression greatly promotes it. The effect of bax and bcl-2 overexpression on antigen-induced
negative selection was studied using fetal thymic organ cultures. This analysis showed that Bcl-2
strongly inhibits negative selection, whereas Bax does not affect it. Our data directly show
that Bcl-2 family members have specific roles in T cell selection and also lend support to the
hypothesis that Bax and Bcl-2 can antagonize each other's action in a certain apoptosis pathway
while in another they can be functionally nonreciprocal.
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Introduction |
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The development of T cells in the thymus is characterized by the production of large numbers of immature thymocytes that are then subjected to stringent selection criteria. Only those thymocytes expressing a TCR capable of interacting with self-peptides presented by MHC molecules on thymic epithelial cells are positively selected and exit into the periphery (1). However, thymocytes with receptors that bind to self-peptide-MHC complexes with high affinity are negatively selected and die (1, 5, 6). Although negative selection of thymocytes has been shown to be mediated by the induction of apoptosis (7), any direct role that molecules known to regulate apoptosis play in the outcome of this process has remained controversial.
Many different molecules that can regulate apoptosis have now been identified. Of particular interest with regards to T cell development are the members of the Bcl-2 family (for review see reference 8). This family consists of proteins that enhance cell survival, such as Bcl-2 or Bcl-XL, and those that promote cell death, such as Bax or Bad. Bax was isolated by virtue of its interaction with Bcl-2 with which it forms heterodimers (9). Thymocytes from mice expressing a bcl-2 transgene in their T cells are protected against apoptosis induced by dexamethasone (10, 11). Conversely, thymocytes from mice expressing a bax transgene in their T cells show accelerated cell death in response to treatment with dexamethasone (12). In the thymus the Bcl-2 protein is uniformly expressed in medullary thymocytes but only in a small fraction of cortical thymocytes (13, 14). This lack of Bcl-2 correlates with the fact that most cortical thymocytes undergo programmed cell death. Furthermore, nearly all CD4+ and CD8+ single positive (SP)1 thymocytes express Bcl-2, whereas the vast majority of CD4+CD8+ double positive (DP) thymocytes do not (15). DP thymocytes are the stage of T cell development during which the processes of positive or negative selection operate. These observations all serve to suggest that Bcl-2 upregulation in the final steps of thymocyte development may play a role in T cell selection.
Given that T cell maturation is contingent on positive
and negative selection, efforts have been made to study the
regulation of these processes by Bcl-2 with regard to negative selection in particular. Initial studies examined the effect of a bcl-2 transgene on thymocytes with receptors that
recognize self-superantigens. In some studies, Bcl-2 was
found to inhibit superantigen-mediated deletion of autoreactive thymocytes (11, 19), whereas another study failed to
demonstrate any effect of Bcl-2 on this process (10). Subsequently, two further studies sought to address the effect of
Bcl-2 on negative selection using TCR transgenic mice also expressing a bcl-2 transgene (20, 21). Both used mice transgenic for a TCR reactive with the male self-antigen
H-Y in the context of H-2Db. Recognition of the H-Y
antigen in male TCR transgenic mice of the H-2b haplotype results in the deletion of most DP thymocytes (5). Strasser et al. (20) found an approximately fourfold increase in the total number of thymocytes in H-Y TCR × bcl-2
double transgenic male mice over H-Y TCR male mice
alone. They concluded that Bcl-2 reduces the efficiency of
negative selection. However, Tao et al. (21) reached the
opposite conclusion since although they also found an increase in the total number of thymocytes in double transgenic male mice, a large fraction of the surviving DP thymocytes expressed endogenous TCR- chains and not the
chain of the H-Y TCR. This is at variance with Strasser
et al. (20), who showed that in their mice the majority of
surviving thymocytes did express the transgenic H-Y TCR
and that their survival could not be explained by the expression of endogenous TCR genes.
Further doubt has been cast on the significance of bcl-2
family members in negative selection from work using bcl-XL
transgenic mice (22, 23). Bcl-XL is a close homologue of
Bcl-2 that has similar protective effects against apoptotic
stimuli (24) and a role in the maturation of T cells as judged
from the block in T cell development found in bcl-XL/
chimeric mice (25). T cells from bcl-XL transgenic mice are protected against exposure to a variety of apoptosis-promoting stimuli. However, analysis of superantigen-mediated clonal deletion in bcl-XL transgenic mice, as well as
H-Y TCR × bcl-XL double transgenic male mice, suggests
that Bcl-XL has no effect on negative selection (22, 23, 26).
We sought to clarify the role of bcl-2 family members in thymic negative selection by studying the effects of bcl-2 and bax transgenes on peptide-induced negative selection in fetal thymic organ culture (FTOC). The FTOCs were derived from mice transgenic for the F5 TCR (27, 28), which recognizes an influenza virus nucleoprotein (NP)- derived nonamer peptide (NP68) in the context of MHC class I H-2Db (29). To exclude any effects due to endogenous TCR rearrangements, the mice used were also deficient for the recombination activation gene 1 (RAG-1). Negative selection of immature thymocytes in the FTOC was induced by addition of the cognate peptide and directly quantitated by measuring DNA fragmentation in the thymocytes. This approach has the advantage of allowing the direct quantitation of antigen-induced negative selection in isolation from other thymic selection events, effects on cell survival, and peripheral T cell activation.
Our study has shown that the apoptotic regulatory molecules Bax and Bcl-2 can have profound effects upon the selection of mature T cells bearing a single TCR. Bax impairs the production of mature SP T cells, whereas Bcl-2 enhances their production. We have also shown that Bcl-2 strongly inhibits negative selection, whereas Bax has no effect on this process. Therefore, for the first time we directly show that Bcl-2 can inhibit antigen-mediated negative selection and that Bax and Bcl-2 can be functionally nonreciprocal, as suggested by recent genetic studies (30).
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Materials and Methods |
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Mice.
The F5 RAG-1Reagents and Antibodies.
The nonamer peptide NP68, from the nucleoprotein of influenza virus A/NT/60/68 (NP366-374: amino acid sequence, ASNENMDAM), was synthesized on a peptide synthesizer (Applied Biosystems 430A; PE Applied Biosystems, Foster, CA). The dexamethasone used was purchased from Sigma Chemical Co. and anti-CD95 (anti-Fas) (Jo2) was purchased from PharMingen (San Diego, CA). The following mAbs and second layer reagents were used for flow cytometric analysis: FITC-conjugated YTS169.4 (anti-CD8Fetal Thymic Organ Culture.
Fetal thymic lobes were isolated from day 15 embryos, obtained from timed matings of homozygous F5/RAG-1Flow Cytometry.
For flow cytometric analysis, 1-5 × 105 cells were stained with various combinations of mAbs in PBS containing 1% BSA (Sigma Chemical Co.) and 0.1% sodium azide. For analysis of DNA content, cells were stained with 7-aminoactinomycin D (7AAD; Sigma Chemical Co.) in PBS containing 0.3% saponin (Sigma Chemical Co.), 2% FCS, and 0.1% sodium azide. Stained cells were analyzed on a FACScan® flow cytometer (Becton Dickinson, Mountain View, CA) using Cellquest software (Becton Dickinson). ![]() |
Results |
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To definitively study
the effects of apoptosis regulatory molecules on T cell development we generated mice having either a bax (12) or a
bcl-2 (32) transgene, an F5 TCR transgene (27) on a RAG-1/
background (31), as well as being homozygous for the
selecting H-2b or nonselecting H-2q MHC haplotypes.
These mice remove all problems of interpretation due to
endogenous TCR rearrangements since all the DP and SP
thymocytes in these mice express only the F5 TCR.
First, we studied F5/RAG-1/
/bax transgenics on the
H-2b/b background. As shown in Fig. 1 and Table 1, upregulated bax expression causes a dramatic decrease in total
thymus cellularity as well as mature SP T cell production.
Thymocytes were analyzed by three-color flow cytometry
with antibodies against CD4, CD8, and V
11 (recognizing
the F5 TCR-
chain) or CD69 (Fig. 1). The presence of
bax in the F5/RAG-1
/
mice results in a fall in total thymocyte numbers from 1.90 × 108 to 0.39 × 108 and decreases CD4
CD8hi cell numbers more than sixfold from
1.26 × 107 to 0.20 × 107 (Table 1). This is also reflected in
a fall in the percentage of CD8 SP (CD4
CD8hi) cells produced (Fig. 1). In the thymus of F5/RAG-1
/
mice, TCR
expression is upregulated (V
11hi) during the transition
from CD4hiCD8hi, through CD4loCD8hi, to CD4
CD8hi
thymocytes, as shown by the shaded region in Fig. 1. However, the presence of the bax transgene greatly diminishes this
upregulation, particularly in the transitional CD4loCD8hi
population, though even in the CD4hiCD8hi DP thymocytes
the small proportion of V
11hi cells is lost. A reduced level
of V
11 expression on CD4
CD8hi is also found on the
bax transgenic mice. The observed transient upregulation
of CD69 (35, 36), as thymocytes pass through the CD4lo
CD8hi compartment, is almost completely abolished in F5/
RAG-1
/
mice expressing the bax transgene (Fig. 1).
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To determine whether the reduction in total thymocyte
numbers in F5/RAG-1/
/bax mice was dependent on
the presence of selecting MHC molecules we crossed these
mice onto the H-2q/q background. The H-2q MHC haplotype is nonselecting for the F5 TCR and T cell development is blocked at the DP thymocyte stage in F5/RAG-1
/
/H-2q/q mice (37). The total number of thymocytes in
F5/RAG-1
/
/H-2q/q mice was reduced from 2.03 × 108
to 0.80 × 108 in the presence of the bax transgene (Table
1) showing that bax has an effect on the survival of thymocytes, which is independent of thymocyte selection.
Therefore, bax decreases the survival of thymocytes in the absence of positive selection and also has a direct effect on the selection of mature T cells expressing the MHC class I-restricted F5 TCR. The bax transgene leads to a great reduction in the expression levels of molecules (TCR and CD69) usually upregulated during positive selection and a substantial decrease in the number of mature T cells produced. We conclude that bax can directly affect the process of T cell selection.
Effects of Bcl-2 on T Cell Development.Our next question was to determine whether Bcl-2 could act in the opposite fashion to Bax and thus enhance T cell selection. Therefore, we examined the effect of a bcl-2 transgene on
F5/RAG-1/
mice with either the H-2b/b or H-2q/q
MHC background. As seen in Fig. 2 and Table 2, upregulated bcl-2 expression has a profound effect on the production of mature SP T cells. The bcl-2 transgene in F5/RAG-1
/
/H-2b/b mice results in a more than fourfold increase
in the total number of CD4
CD8hi cells from 2.64 × 107
to 11.37 × 107 (Table 2). Table 2 also shows that the presence of bcl-2 increases the total number of thymocytes from
2.48 × 108 to 4.98 × 108. The bcl-2 transgene also leads to
a greater than twofold increase in the percentage of CD8
SP (CD4
CD8hi) thymocytes produced (Fig. 2).
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In contrast to bax transgenic mice, the bcl-2 transgene
leads to enhanced upregulation of TCR and CD69 expression during T cell development (Fig. 2). A higher percentage of CD4hiCD8hi cells in the bcl-2 transgenic mice have
high levels of V11 and CD69 expression. Similarly, virtually all the CD4loCD8hi thymocytes are V
11hi and
CD69hi in the bcl-2 transgenic mice as opposed to the
control F5/RAG-1
/
mice. However, mature T cells in
both control and bcl-2 transgenic mice express similar levels
of the F5 TCR.
The effect of the bcl-2 transgene was also determined on
the nonselecting H-2q MHC haplotype. The total number
of thymocytes was increased in the F5/RAG-1/
/bcl-2
transgenic mice from 1.78 × 108 to 3.60 × 108 (Table 2).
Bcl-2 can thus increase the survival of DP thymocytes even
in the absence of positive selection. However, Bcl-2 also increases the number of mature SP T cells produced when
positive selection does take place, in mice of the H-2b MHC
haplotype. This latter effect is associated with elevated levels
of TCR and CD69 expression during T cell development.
Thus, bcl-2 can directly affect the process of T selection.
Since thymocyte selection involves both positive and negative selection, the question arises whether Bax and Bcl-2 are
affecting either or both of these processes. To address this,
we analyzed the effect of these molecules on negative selection in isolation from other thymic selection events.
As described
above, previous studies have provided contradictory evidence as to the influence of Bcl-2 on negative selection. We wanted to unambiguously determine the role of apoptotic regulatory molecules such as Bax and Bcl-2 on negative selection so we chose to study negative selection in
FTOC. The use of FTOCs to study antigen-induced negative selection has been previously described (38). We have
modified this system in order to directly quantitate the extent of apoptosis induced in DP thymocytes after acute exposure to antigenic peptide or other apoptotic stimuli. Fetal
thymic lobes from F5/RAG-1/
mice, with bax or bcl-2
transgenes, were taken at day 15 of gestation. The genotype of the thymic lobes was determined by PCR analysis of DNA from the individual embryos from which they
originated. Thymic lobes were cultured for 4 d in vitro, after which the apoptotic stimuli were added to the cultures
and 11 h later the percentage of DP thymocytes undergoing apoptosis was determined (Fig. 3).
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Since at this stage of the culture most of the thymocytes have differentiated into immature DP cells, the FTOC enabled us to analyze responses of DP thymocytes at early stages of thymic differentiation. Administration of antigen to adult TCR transgenic mice leads to thymocyte apoptosis as an indirect result of mature T cell activation as well as recognition of the antigenic peptide by the thymocytes themselves (39, 40). At day 4 of culture the thymic lobes were virtually devoid of mature T cells, therefore, apoptosis of thymocytes was solely due to recognition of the antigenic peptide. Although FTOCs are performed in vitro, they create the possibility to analyze the responses of thymocytes in an intact thymic microenvironment, which is lost in suspension culture systems (41, 42). Another important benefit of the FTOC approach is that we can discard the contribution of survival effects on thymocyte viability in culture to the overall level of apoptosis. The differentiated FTOCs are grown under optimal conditions in vitro and show very low levels (<1%) of apoptosis, regardless of the expression of the bax or bcl-2 transgenes. Therefore, the levels of apoptosis found are directly due to the stimuli and the influence of bax or bcl-2 on the pathways they invoke and not to factors associated with culture conditions. Thus, we compared thymocyte apoptosis due to antigen, dexamethasone, and treatment with anti-CD95 (anti-Fas) antibody, and the influence of Bax and Bcl-2 in each of these apoptotic pathways.
Bax and Negative Selection.First, we examined negative
selection in FTOCs generated from F5/RAG-1/
H-2b/b
mice with or without the bax transgene. Thymocyte apoptosis was examined in response to NP68 (antigenic peptide)
as well as dexamethasone and anti-CD95 antibody (Jo2).
As illustrated in Fig. 3, after staining with 7AAD as well as
anti-CD4 and CD8, FACS® was used to determine the
percentage of DP thymocytes containing hypodiploid DNA
and thus undergoing apoptosis. To obtain statistically significant results, four to six individual lobes were assayed for
each value shown in Fig. 4. These experiments were repeated on four independent occasions and similar data were
obtained each time. After negative selection induced by
NP68, the level of apoptosis in F5/RAG-1
/
/bax FTOCs
was not higher than those from the FTOCs without bax transgene, in fact it was slightly reduced. The level of
NP68 induced apoptosis in bax FTOCs was also examined
at 4 and 6 h to confirm that no effect was detectable at earlier time points in the culture (data not shown). However,
the thymocytes of bax FTOCs show significantly higher
apoptosis than non-bax FTOCs after incubation with dexamethasone (Fig. 4) as previously seen in primary thymocytes from bax transgenics (12). The bax transgene has no significant effect on CD95-induced apoptosis, which
also accords with previous data from primary bax thymocytes (43). We conclude that the bax transgene can act
in FTOCs to increase apoptosis in response to dexamethasone but that in the case of cognate antigen induced negative selection it has no effect. This suggests that Bax has no
role in the apoptotic pathway activated by antigen induced
negative selection.
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We then studied the induction of apoptosis in thymocytes of FTOC from F5/
RAG-1/
mice, with or without the bcl-2 transgene. After negative selection induced by NP68 the level of apoptosis in bcl-2 FTOCs was greatly reduced, four- to fivefold,
over those without bcl-2. This demonstrates a strong protective effect of Bcl-2 on antigen-induced negative selection. The bcl-2 transgene also caused a threefold reduction in dexamethasone induced apoptosis. Interestingly, we also
found a moderate protective effect of Bcl-2 on CD95 induced apoptosis, as previously described for primary bcl-2
thymocytes (44). However, in our system this could not be
explained by enhanced survival of bcl-2 transgenic thymocytes (see above). Thus, Bcl-2 can indeed partially inhibit CD95-induced apoptosis.
Our data show that Bcl-2 can strongly inhibit antigen-specific thymic negative selection, whereas Bax, an antagonist of Bcl-2, has no effect on negative selection.
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Discussion |
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Apoptosis has been clearly demonstrated to be a key
mechanism in negative selection (7). However, the role of
individual molecules known to regulate apoptosis, in particular Bcl-2, has remained an area of controversy. Bcl-2
has been shown either to have no effect (10) or to inhibit
(11, 19) superantigen-mediated deletion of autoreactive
thymocytes. Studies on mice doubly transgenic for the
H-Y TCR and bcl-2 have reached different conclusions, namely that deregulated bcl-2 expression reduces the efficiency of negative selection (20) or has no effect (21). Similar types of studies in bcl-XL transgenic mice have concluded that Bcl-XL has no effect on negative selection (22,
23). We sought to address the role of Bcl-2 in T cell development and negative selection using MHC class I-restricted
F5 TCR transgenic mice on a RAG-1/
background to
produce mice whose T cells express a single TCR. We also
studied the effect of a bax transgene within the same context, as Bax can act antagonistically of Bcl-2 function and therefore, might be expected to have opposite functional
consequences.
Expression of the bax transgene in F5/RAG-1/
mice
reduced the size of the thymus in the context of both selecting and nonselecting MHC. This suggests that Bax can
exert a proapoptotic effect on thymocytes independently of
thymic selection. However, with selecting MHC the number of CD8 SP thymocytes produced is greatly reduced and
the upregulation of TCR and CD69 expression normally
associated with maturation is also severely reduced. Precisely the converse is found with the bcl-2 transgene, where
thymus size is increased regardless of MHC. On the selecting MHC background, such mice exhibit increased numbers of thymocytes with upregulated TCR and CD69, coinciding with elevated numbers of CD8 SP thymocytes.
These observations suggest that while Bax and Bcl-2 confer
pro- and antiapoptotic signals, respectively, they also exert
effects on the processes involved in T cell selection. Therefore, these findings unambiguously confirm previous suggestions about bcl-2 and T cell selection that were complicated by a lack of complete allelic exclusion and the
presence of endogenous TCR rearrangements.
Having established these effects on T cell selection the question arises as to how Bax and Bcl-2 exert their actions. Do they act on positive or negative selection separately or on both processes in opposite directions? We addressed this question from the perspective of negative selection using fetal thymic organ culture as our model system. FTOCs have numerous advantages for this type of study as outlined above. Our experiments show that upregulated bax expression while accelerating glucocorticoid-induced apoptosis, has no effect on negative selection (Fig. 4). However, increased bcl-2 expression leads to substantial inhibition of negative selection, three- to fourfold, while also inhibiting glucocorticoid-induced apoptosis and having a marginal effect on anti-CD95-induced apoptosis (Fig. 5). The marginal inhibitory effect of bcl-2 on CD95-induced apoptosis has been noted previously on primary thymocytes in culture (44) and attributed to a survival effect, i.e., that the bcl-2 transgene simply confers greater viability in culture. However, as the FTOC system negates the survival effect of Bcl-2 in culture it would seem that Bcl-2 can partially inhibit anti-CD95 induced apoptosis. This may be by partially blocking one or more parts of the caspase cascade activated by anti-CD95 treatment (reviewed in reference 45).
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Overall, our study suggests that Bax has no role in the apoptotic pathway activated by antigen-induced negative selection, whereas Bcl-2 can act to block this pathway. Several important points can be made as a result of this finding. First, it establishes that a specific apoptosis regulatory molecule, Bcl-2, can also regulate negative selection. It remains to be seen whether apoptosis regulatory molecules can similarly influence positive selection and this may be directly measurable using an FTOC approach. For instance, the bax transgene leads to a decline in mature SP T cell production but does not increase negative selection. Therefore, it may act to decrease positive selection, as suggested by the decreased expression of CD69 on intermediate CD4loCD8hi thymocytes of bax transgenic mice. Thus, Bax may directly inhibit positive selection or reduce its efficiency indirectly by reducing the viability of thymocytes (12), effectively reducing the number of thymocytes available for positive selection. In contrast the bcl-2 transgene leads to an increase in the number of intermediate CD69hi CD4loCD8hi and mature CD8 SP thymocytes as well as an increase in the total number of thymocytes in the presence of selecting and nonselecting MHC molecules. These results suggest that Bcl-2 increases the efficiency of positive selection. Thus, in thymocytes Bax and Bcl-2 appear to be functionally antagonistic in the decision between death by neglect and positive selection, whereas Bcl-2 acts independently of Bax in inhibiting negative selection. A model summarizing these properties of Bax and Bcl-2 is shown in Fig. 6. Although Bax does not have a direct role in negative selection, it is very likely that other proapoptotic members of the Bcl-2 family do, in order to counteract Bcl-2 (Fig. 6). Therefore, a further task will be to determine which ones act in negative selection. Another aspect of the data is that whereas Bcl-2 has a strong inhibitory effect on negative selection it has very little effect on CD95-induced apoptosis. This would support the idea that the CD95 pathway has no great role in thymic negative selection (46, 47) rather than the view that CD95 modulation is an integral part of the negative selection of thymocytes (48).
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Our study establishes peptide antigen-induced deletion in FTOCs as a model system to directly study apoptosis as a result of negative selection. Due to the intrinsically negligible levels of background cell death in the system it is also a very useful method to study other apoptotic stimuli in T cells. We hope to further develop this method to study apoptosis regulatory molecules in positive selection.
Recently, it has been proposed that, although Bax and Bcl-2 can form heterodimers and act to antagonize each other's function, they also regulate apoptosis independently of one another (30, 49). We further validate this hypothesis by showing Bax and Bcl-2 do not have reciprocal effects within the same system. Bax and Bcl-2 exert their opposing effects on glucocorticoid-induced apoptosis, whereas Bcl-2 inhibits negative selection and Bax has no effect. Hence, Bax and Bcl-2 can function independently of their physical interaction with each other in the cell (30).
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Footnotes |
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Address correspondence to Hugh J.M. Brady, Division of Molecular Immunology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK. Phone: 44-181-959-3666, ext. 2193; Fax: 44-181-913-8531; E-mail: h-brady{at}nimr.mrc.ac.uk
Received for publication 19 May 1998.
This work was supported by the Medical Research Council. O. Williams is the recipient of a fellowship from the Leukaemia Research Fund.
Abbreviations used in this paper 7AAD, 7-aminoactinomycin; DP, double positive; FTOC, fetal thymic organ culture; NP, nucleoprotein; RAG-1, recombination activation gene 1; SP, single positive.
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