Unexpectedly late expression of intracellular CD3
and TCR 
proteins during adult thymus development
Anne Wilson,
Myriam Capone and
H. Robson MacDonald
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne,Ch. Des Boveresses 155, 1066 Epalinges, Switzerland
Correspondence to:
A. Wilson
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Abstract
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During adult thymus development immature CD4CD8 [double-negative (DN)] precursor cells pass through four phenotypically distinct stages defined by expression of CD44 and CD25: CD44hiCD25 (DN1), CD44hiCD25+ (DN2), CD44loCD25+ (DN3) and CD44loCD25- (DN4). Although it is well established that the TCR ß,
and
genes are rearranged and expressed in association with the CD3 components in DN thymocytes, the precise timing of expression of the TCR and CD3 proteins has not been determined. In this report we have utilized a sensitive intracellular (ic) staining technique to analyze the expression of ic CD3
, TCR ß and TCR 
proteins in immature DN subsets. As expected from previous studies of TCR ß rearrangement and mRNA expression, icTCR ß+ cells were first detected in the DN3 subset and their proportion increased thereafter. Surprisingly, however, both icCD3
+ and icTCR 
+ cells were detected at later stages of development than was predicted by molecular studies. In particular icCD3
protein expression coincided with the transition from the DN2 to DN3 stage of development, whereas icTCR 
protein expression was only detected in a minor subset of DN4 cells. The implications of these findings for
ß lineage divergence will be discussed.
Keywords: cellular differentiation, FACS, gene rearrangement, thymus
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Introduction
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The two major lineages of T cells express mutually exclusive heterodimeric TCR (
ß or 
) associated with the CD3 signaling complex.
ß and 
T cells have been shown to arise from the same early thymic precursor population which has a surface phenotype CD4lo, CD90lo, CD24int, CD44hi, CD117+ and class Ihi (reviewed in 1). During subsequent thymus development, these precursors differentiate along a specific pathway, in which four CD4CD8 [double-negative (DN)] subsets expressing high levels of CD24 can be defined by the differential expression of CD44, CD25 and CD117 as follows: CD44hiCD25CD117+ (DN1), CD44hiCD25+CD117+ (DN2), CD44loCD25+CD117 (DN3) and CD44loCD25 (DN4) (reviewed in 24). During this time, the TCR
,
and ß genes are rearranged and expressed according to a precise developmental program (510). At the end of the DN stages,
ß precursors rapidly transit through a CD4CD8+CD3lo immature single-positive (ISP) stage to the CD4+CD8+ [double-positive (DP)] stage where TCR
is rearranged and expressed and mature single-positive (SP) cells are produced. In contrast, although mature 
T cells are also produced from the same CD4lo precursor population, little is known about the precise developmental pathway they follow (reviewed in 4). However, most of the DN populations have been shown either in vivo (1,5,11) or in vitro (1113) to produce mature cells of both lineages.
One of the major problems that has hampered analysis of the early stages of
ß and 
T cell development is the fact that readily detectable levels of TCR are only observed on relatively mature T cells. In the
ß lineage surface TCR expression can be reliably assessed by flow microfluorometry only on DP and SP subsets despite the fact that a pre-TCR complex (composed of a productively rearranged TCR ß chain, an invariant pT
chain and CD3) is already present on the surface of DN3 cells (reviewed in 14). The situation in the 
T cell lineage is even less clear, since only mature 
cells express detectable levels of TCR on the cell surface, thus precluding any identification of putative 
precursors.
To circumvent the lack of detectable surface TCR expression on immature thymic precursor cells, many groups have utilized molecular methods to follow rearrangement and/or expression of TCR ß,
and
genes (reviewed in 15,16) as well as CD3 genes (17) in defined subsets. Although these techniques have provided much useful information, they have two major drawbacks in that (i) they are generally not applicable to single cells and (ii) they do not measure the levels of the corresponding protein.
In this report we have attempted to overcome these technical problems by undertaking a detailed four-color flow cytofluorometric analysis of the expression of intracellular (ic) CD3
, TCR ß and TCR 
proteins at defined stages of adult thymus development. Although similar techniques have been utilized previously in the context of fetal thymus development (1820), the conclusions have been somewhat limited due to the general use of only two- (or occasionally three-) color analysis and to the fact that immature subsets are phenotypically less well defined in the fetal (as opposed to adult) thymus. Our data confirm and extend previous molecular analyses of the timing (and consequences) of icTCR ß expression during adult thymus development. Moreover, they provide novel information concerning the timing of both icCD3
and icTCR 
protein expression.
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Methods
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Mice and cell suspensions
Six-week-old C57BL/6 female mice were obtained from Harlan Olac (Bicester, UK). TCR ß/- and TCR
/- mice were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in our animal facility. DN and DN CD25 thymocytes were prepared as described previously (7,21). Briefly, thymocyte cell suspensions were incubated at 4°C in DMEM/1% FCS containing the appropriate concentrations of IgM isotype antibodies against CD4 (RL172.4), CD8 (31M) and CD25 (7D4) for 20 m. Rabbit complement (Saxon Europe, Suffolk, UK) and DNase I (Boehringer Mannheim, Mannheim, Germany), were added, and the incubation continued at 37°C for 45 min. After washing, viable cells were recovered after density-gradient centrifugation through Lympholyte M (Cedarlane, Hornby, Ontario, Canada). During subsequent FACS analysis and/or sorting, contaminating CD4+, CD8+, CD25+ (where required) and any CD3+ (
ß+ or 
+) cells were positively eliminated where appropriate by staining with a cocktail of FITC-conjugated mAb to these surface markers and `live' gating out all FITC+ cells.
Flow cytometry and cell sorting
Thymocyte subsets were analyzed for simultaneous detection of both surface and intracellular proteins as described previously (22), using FACScan (three color) or FACSCalibur (four color) flow cytometers and sorted with a FACStar Plus (Becton Dickinson, San Jose, CA). Briefly, surface staining was performed as usual for one, two or three colours followed by fixation in 2% paraformaldehyde. After permeabilization with 0.5% Saponin, intracellular proteins were detected with mAb directly conjugated to phycoerythrin (PE) and/or CyChrome. Specifically, surface staining was performed using a pool of FITC direct conjugates as described above, together with either direct TriColor or CyChrome conjugates, such as TCR 
TriColor, CD4TriColor and CD8
TriColor (Caltag, Burlingame, CA), CD44CyChrome (PharMingen, San Diego, CA), or biotinylated-CD25 revealed with TriColorstreptavidin, purchased from Caltag in FL3. For four-color staining, Cy5CD25, Cy5CD24 and CD117allophycocyanin (PharMingen) were used in FL4. All FITC, biotin and Cy5 conjugates were purified and conjugated from hybridomas grown in this laboratory, and have been described elsewhere (7). PE- and/or CyChrome-conjugates of mAb to TCR ß (H57-597), TCR 
(GL3), CD3
(17A2 and 145-2C11), and control rat and hamster Ig (PharMingen) were used for intracellular staining. PE conjugates of mAb to TCR V
1.1 [clone 11.2 (23)], TCR V
2 (UC3-10A6) and TCR V
4 (GL2) were prepared in this laboratory using the Phycolink PE-conjugation kit (Prozyme, San Leandro, CA). Surface TCR ß, TCR 
, TCR V
1.1, TCR V
2, TCR V
4 and CD3
were pre-blocked with unlabeled purified mAb from the same clones as used for intracellular staining. Analysis was performed using Lysys II or CellQuest software.
Cell culture
Purified and FACS-sorted CD44loCD25CD3 DN thymocytes were cultured in DMEM supplemented with HEPES buffer, Glutamine, ß2-mercaptoethanol and 5% FCS for 4 h at 37°C. After harvesting and counting the cells were stained and analyzed by four-color FACS either for surface expression of CD4, CD8, TCR ß and TCR 
or for intracellular expression of TCR ß and TCR 
after gating out any mature cells (CD4, CD8, CD3
, TCR ß or TCR 
) by surface staining with the FITC cocktail described above.
Cell cycle analysis
The DNA content of thymus subsets was determined by first sorting the selected populations defined by both surface and intracellular protein expression as described above. Propidium iodide (PI) staining of DNA was performed using standard procedures as described previously (22,24). Briefly, sorted cells were treated with 3N HCl to remove surface markers, washed in PBS and neutralized with 0.1 M Na2B4O7. RNA was removed by incubation in RNase A at 37°C before the addition of PI. Analysis was performed on the FACScan flow cytometer using the doublet discrimination module.
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Results
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Intracellular CD3
protein is first detected in CD25+CD44loCD117lo thymocytes
In order to establish the timing of CD3
protein expression during adult thymus development, DN thymocytes were purified by depletion and surface stained in three colors with a cocktail consisting of mAb to CD4, CD8 and CD3 (to remove residual contaminating mature T cells) together with CD25 and either CD44, CD24 or CD117. The fourth color was used for icCD3
. Surprisingly the CD25+ DN population contained a well-defined subset of icCD3
cells (Fig. 1A
). The proportion of icCD3
cells in the CD25+ DN subset was highly reproducible (6 ± 1 %, N = 20) and did not depend upon the mAb used to detect CD3
, since all four mAb used (145-2C11, 17A2, 13D5 and 500-A2) yielded similar results (data not shown). By four-color analysis the icCD3
subset of CD25+ DN cells homogeneously expressed much higher levels of CD44, CD25 and CD117 (c-kit R) as compared to the icCD3
CD25+ population (Fig. 1B
). However, expression levels of CD24 (HSA) were similar in both subsets. These data indicate that CD3
protein is expressed later in adult thymus development than was originally believed based on mRNA expression studies (17). Indeed the homogeneous phenotype of icCD3
CD25+ DN cells (CD44+CD117+) corresponds to the accepted definition of the DN2 subset (6), thus indicating that expression of CD3
marks the DN2 to DN3 transition. Analysis of icCD3
expression during subsequent stages of adult thymus development indicated that essentially 100% of cells were icCD3
+ as expected (Fig. 2
), with the exception of a small subset of DN4 cells that probably do not belong to the T cell lineage (see below).
Intracellular TCR ß protein is first detected in the DN3 subset
Semi-quantitative PCR and Southern blotting techniques have shown that TCR ß rearrangement is initiated during the transition from the DN2 to DN3 stages of intrathymic development (6,10). However, the point at which productive TCR ß protein can be first detected has been difficult to determine by conventional FACS analysis due to the extremely low amount expressed at the surface of immature thymocytes. In order to define the earliest DN subset in which icTCR ß protein is expressed we have used four-color FACS analysis as described above. The results shown in Fig. 3
demonstrate clearly that icTCR ß protein is first detected in a subset of DN3 thymocytes and not at all in DN2 thymocytes. These results confirm and extend previous studies (6,8,10,17), which show that full-length VDJCß transcripts can be first detected by Northern blot analysis in the DN3 subset. As development proceeds, the percentage of icTCR ß+ cells increases rapidly from ~25% in DN3 to 80% at the DN4 stage, after which essentially 100% of the cells show high levels of icTCR ß (Fig. 2
).
Immature DN thymocytes expressing intracellular TCR ß are proliferating.
It is generally accepted that immature DN3 thymocytes which have undergone successful TCR ß rearrangement express the pre-TCR (TCR ß, pT
and the CD3 complex) and that subsequent signaling through this complex results in `ß-selection' (reviewed in 24,14,25). A major consequence of ß-selection is proliferation. In the absence of any one of the components of the pre-TCR, this proliferative effect is absent or severely reduced (reviewed in 1416). To determine directly whether expression of icTCR ß protein correlates with proliferation, icTCR ß+ and icTCR ß DN3 and DN4 thymocytes were purified by cell sorting and analyzed for DNA content by PI staining. As shown in Fig. 4
almost half (45%), of the icTCR ß+ cells in DN3 have a DNA content that corresponds to the S + G2/M phases of the cell cycle compared with only 10% in the icTCR ß DN3 population and 16% in the unfractionated DN3 population. Similar results were obtained for the DN4 subset where 50% of the icTCR ß+ cells are in cycle compared to only 12% in the icTCR ß subset and 38% in the unfractionated population (Fig. 4
). Thus there is a strict correlation between icTCR ß expression and proliferation in both the DN3 and DN4 subsets of adult thymocytes. A similar correlation was observed previously for thymocytes developing in fetal thymic organ culture (FTOC) (12,26).

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Fig. 4. Correlation between icTCR ß expression and proliferation in immature thymus subsets. Purified DN thymocytes were stained as for Figure 2 . DN3 and DN4 cells were sorted into icTCR ß+ or icTCR ß subsets and the percentage of cells in S + G2/M phases of the cell cycle was determined by PI staining. Control populations are total sorted DN3 and DN4 cells.
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Intracellular TCR 
protein is only detected in the DN4 subset
Until now, 
lineage precursor T cells have not been able to be distinguished from their
ß counterparts by surface phenotype due to the lack of a unique surface marker. To search for possible 
cell precursors we analyzed DN thymus subsets for icTCR 
expression in the same manner as for icTCR ß. As shown in Figs 2 and 3
, in contrast to icTCR ß expression, icTCR 
protein is detected in only a small percentage (~710%) of the DN4 subset and is undetectable in any subset either earlier or later in thymus development. Since the GL3 mAb used to detect icTCR 
protein in this study apparently only binds the TCR
chain when associated with a TCR
chain (27), we also stained DN3 and DN4 cells intracellularly with the available mAb against TCR V
and TCR V
chains (TCR V
1.1, V
2 and V
4). As shown in Fig. 5
, a significant percentage of the icTCR 
+ cells in the DN4 subset can be stained with each of the anti-V region mAb tested. However, none of the mAb showed detectable staining of cells in the DN3 subset.
TCR 
+ thymocytes can be produced from DN4 icTCR 
+ thymocytes in vitro
In order to determine whether icTCR 
+ thymocytes are indeed precursors of TCR 
+ cells, purified and FACS-sorted DN4 thymocytes were cultured at 37°C for 4 h. As it is not possible to culture cells after intracellular staining due to the fixation and permeabilization protocol, DN4 icTCR 
+ cells could not be specifically isolated to do a direct precursor to product study. Therefore the cultured DN4 cells were monitored in parallel for the appearance of surface TCR 
+ cells and the disappearance of icTCR 
+ cells. Cell loss after 4 h in culture was minimal (~15%). As shown in Fig. 6
, a significant proportion of surface TCR 
+ cells appeared after 4 h at 37°C and icTCR 
+ cells virtually disappeared. Importantly the absolute number of surface TCR 
+ cells produced in this time period corresponded to the number of icTCR 
+ cells that were present prior to culture and which had disappeared after 4 h at 37°C (Table 1
). These data provide compelling evidence that icTCR 
+ DN4 cells are immediate precursors of thymic 
T cells.
Most icTCR 
+ DN4 thymocytes are not `ß-selected'
To determine if icTCR 
+ cells within the DN4 subset also expressed icTCR ß and could therefore be `ß-selected', we performed two-color intracellular staining for these two TCR proteins after gating out all non-DN4 and mature T cells with the third color. The contour plot shown in Fig. 7
demonstrates that the majority of icTCR 
+ DN4 cells are icTCR ß, although a small but reproducible fraction (~15%) are icTCR ß+. The four DN4 subsets defined by icTCR ß and icTCR 
staining were then sorted and analyzed for their cell cycle status by PI staining (Fig. 7
). Interestingly, only 10% of icTCR 
+ß cells were found to be in the S + G2/M phases of the cell cycle, whereas 23% of icTCR 
+ß+ cells were in cycle. As expected from analyses of the total DN4 subset (Fig. 4
), 46% of icTCR 
ß+ cells were in cycle as compared to 3% of icTCR 
ß cells. Taken together these data indicate that icTCR 
+ DN4 cells are in general icTCR ß and not cycling, so therefore not `ß-selected'.
Increased frequency of icTCR 
+ DN4 thymocytes in TCR ß-deficient mice
Mice deficient for either the TCR ß chain or pT
have essentially normal development of 
cells despite a dramatic reduction (50- to 100-fold) in
ß T cell development (28,29). It was therefore of interest to investigate whether the percentage and absolute number of icTCR 
+ DN4 cells present in TCR ß/ mutant mice differs from wild-type animals. As shown in Fig. 8
, the proportion of icTCR 
+ cells in the DN4 subset of TCR ß-deficient mice is greatly increased (~58%). However, the number of DN4 thymocytes is correspondingly decreased in the absence of TCR ß, such that the absolute number of icTCR 
+ DN4 cells is similar to that found in wild-type mice (data not shown).
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Discussion
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Timing of CD3
expression during normal adult T cell development
The data presented here demonstrate clearly that icCD3
protein is not expressed at detectable levels until both CD44 and CD117 are down-regulated in CD25+ DN adult thymocytes. Thus icCD3
expression marks the transition between the DN2 and DN3 thymocyte subsets. Previous studies of icCD3
expression in the fetal thymus have been controversial. Whereas essentially all CD25+ fetal thymocytes were found to be icCD3
+ in one report (18), ~40% of CD25+CD117+ fetal thymocytes were shown to be icCD3
in another study (20). The apparent discrepancy between these data and our own may be related to differences in the timing of CD3
expression in the fetal and adult thymus. Alternatively, since CD25 expression was not directly correlated with both CD44 and CD117 in the earlier studies, it is possible that a discrete (but homogeneous) subset of icCD3
CD25+CD44+ CD117+ fetal thymocytes was overlooked.
The relatively late expression of icCD3
protein observed in this study is surprising in view of our previous analysis of CD3
mRNA expression in adult thymus subsets (17). Whereas CD3
mRNA could be detected in a large fraction of DN1 and DN2 thymocytes by single-cell in situ hybridization, no detectable icCD3
protein can be observed prior to the DN3 stage. This discrepancy could reflect an intrinsic instability of the CD3
protein in more immature thymocytes. Alternatively it is formally possible that icCD3
protein can only be detected by the mAb used here (17A2 and 145-2C11) when complexed to other proteins first expressed at the DN3 stage such as TCR ß and/or pT
. However, the latter explanation seems unlikely in view of the fact that icCD3
can be readily detected in immature thymocytes of TCR ß/- and pT
/- mice (data not shown).
Although icCD3
protein expression strictly correlates with the DN2 to DN3 transition in the adult thymus it does not represent a critical check-point for further T cell development. Indeed in CD3
-deficient mice development does not arrest until the DN3 stage, and TCR ß,
and
genes undergo apparently normal rearrangement (30). Nevertheless it is possible that signaling via CD3
may influence the level of expression of rearranged TCR ß genes, as suggested by a recent report (31).
Timing of icTCR ß expression during normal adult T cell development
Although it has been known for some time that icTCR ß protein is expressed early during fetal thymus development (18,19,26), the data presented here provide the first quantitative analysis of icTCR ß protein expression at defined stages of normal adult thymus development. Previous mRNA studies at the population level have shown that 1.3 kb TCR ß transcripts (corresponding to VDJß rearrangements) are first detected at the DN3 stage (5,6,8,17), although shorter (DJß) and longer (presumably germline) transcripts can already be observed in DN2 cells (6,17). Intracellular staining directly confirms and extends these earlier studies to the single-cell level by demonstrating that TCR ß protein is undetectable in DN2 cells but present in a fraction (~25%) of DN3 cells. Moreover, icTCR ß+ cells increase in frequency during subsequent thymus development, reaching levels of ~80% in DN4 cells, and 100% in the subsequent ISP and DP stages.
The heterogeneous expression of icTCR ß protein observed in the DN4 subset reflects the presence of several distinct cell populations. In particular, the 20% icTCR ß DN4 cells are composed of ~10% icCD3
cells (presumably non-T cell contaminants resulting from the negative gating criteria used to define this subset) and ~10% icTCR 
+ cells, presumably 
lineage precursors (see below). Thus it appears that essentially all
ß lineage-committed cells in the DN4 subset express icTCR ß.
In contrast to DN4, the DN3 subset contains 75% icTCR ß cells that are homogeneously icCD3
+ and icTCR 
. Many of these icTCR ß DN3 cells most likely represent
ß lineage-committed thymocytes that have either not yet attempted VDJß rearrangement or undergone non-productive rearrangement on one or both alleles. Since under normal circumstances 44% (four of nine) of precursor cells that attempt VDJß rearrangement on both alleles are unsuccessful, the simplest interpretation of our data is that ~45% of DN3 cells have already undergone VDJß rearrangement (25% productively and 20% non-productively) while the remaining 55% have not. Obviously this estimate does not take into account the dynamics of cell death and proliferation within the DN3 subset (9,3234). In this context DN3 cells with non-productive VDJß rearrangements might be preferentially eliminated via programmed cell death, whereas selective proliferation of productively rearranged DN3 cells (see below) might lead to an overestimate of their frequency. Irrespective of these caveats it appears clear that VDJß rearrangement in immature thymocytes does not initiate synchronously during the DN2 to DN3 transition, but rather occurs progressively throughout the DN3 stage. Progression in VDJß rearrangement during thymus development has also been shown by quantitative Southern blot analysis using a Vß probe (10); however, the estimated frequency of VDJß rearrangements in DN3 cells in that study was too low to account for our observed frequency of icTCR ß+ cells.
Intracellular TCR ß expression and `ß-selection'
The concept that productive TCR ß rearrangement and the ensuing expression of a pre-TCR (composed of TCR ß, pT
and CD3) allows progression of DN3 thymocytes to the DN4 stage as well as their subsequent expansion is based on two independent lines of evidence. First analysis of mutant mice lacking the ability to form a pre-TCR (TCR ß/-, RAG/-, pT
/- and CD3
/-) has revealed that development is largely (or completely) blocked at the DN3 stage (2830,35,36). Moreover re-introduction of a TCR ß transgene in mice deficient for TCR ß (TCR ß/- or RAG/-) releases this block and allows development to proceed (reviewed in 3,15). In a second approach PCR-restriction length fragment polymorphism analysis of the frequency of productive (in-frame) VDJß rearrangements in DN3 and DN4 cells has revealed a clear progression from essentially random to preferentially productive rearrangements (9,10,37). Moreover separation of DN3 thymocytes on the basis of forward scatter has revealed that large (cycling) DN3 cells exhibit preferentially in-frame VDJß rearrangements, whereas small (non-cycling) DN3 cells exhibit random VDJß joints (38). Taken together these results have led to the conclusion that DN3 thymocytes expressing productive TCR ß rearrangements (and hence a pre-TCR) are selected to proliferate and proceed to the DN4 stage, a process which has been termed `ß-selection' (2).
The data presented here on the relationship between proliferation and icTCR ß protein expression in adult thymocyte subsets together with related studies carried out in FTOC (12,19,26) provide unequivocal evidence that `ß-selection' plays a critical role in thymus development. In particular icTCR ß+ cells in both DN3 and DN4 subsets were enriched by 4- to 5-fold in cycling cells as compared to their icTCR ß counterparts. Interestingly, the icTCR ß+ DN3 subset had a similar proportion of cells in cycle as the icTCR ß+ DN4 subset despite the fact that only a minority of DN3 cells express icTCR ß. This result demonstrates that the consequences of `ß-selection' (at least insofar as cell cycle progression is concerned) are manifested rapidly as soon as icTCR ß is expressed. This in turn implies that icTCR ß expression in DN3 cells is most likely the limiting component in the assembly of a pre-TCR complex and consequently that pT
must already be present. In this regard we have previously observed that pT
mRNA (assessed by Northern blot) is first detectable at the DN3 stage (17).
In contrast to
ß T cell development the potential significance of ß-selection in 
lineage cells remains controversial (reviewed in 4,39). In this context it is interesting to note that a small but distinct subset of icTCR 
+ DN4 thymocytes expresses TCR ß. Moreover, the frequency of cycling cells in this icTCR ß+
+ subset was 2-fold higher than in the corresponding icTCR ß
+ DN4 population. Assuming that icTCR 
+ DN4 thymocytes belong to the 
lineage (see below), these data imply that expression of icTCR ß protein confers a proliferative advantage on immature 
cells. Interestingly a similar correlation between icTCR ß expression and cell cycle has been observed for mature thymic 
cells (22,40). Whether the increased cycling of icTCR ß+ 
lineage cells reflects pre-TCR signaling or some other unknown function of the TCR ß chain remains to be established. Clearly analysis of pT
expression in these cells will be required to resolve this issue.
ß/
lineage divergence
An important new finding in this study is the identification of a distinct subset of thymocytes that express 
protein intracellularly but not on the cell surface. Interestingly icTCR 
+ thymocytes are found exclusively in the DN4 subset. Although it is formally possible that icTCR 
+ DN4 thymocytes represent mature 
T cells which have internalized the 
TCR or `
-selected'
ß lineage-committed cells (41,42), that do not express a pre-TCR but have been rescued by expression of in-frame TCR
and TCR
rearrangements, we consider it much more likely that they are the immediate precursors of mature thymic 
T cells for the following reasons. (i) Mature thymic 
cells and icTCR 
+ DN4 cells are indistinguishable in surface phenotype (except for 
TCR expression) (43). (ii) Although present in the DN4 subset, icTCR 
+ cells are completely absent in the subsequent ISP and DP subsets that are universally accepted to be
ß lineage committed. (iii) In contrast to what would be expected for `
-selected'
ß cells, icTCR 
+ DN4 cells contain a significant fraction of cells that actually co-express icTCR ß. Importantly a similar proportion of icTCR ß+ cells (~1015%) is found among mature 
cells in both mice (22,40) and rats (44). (iv) Short-term culture of purified DN4 thymocytes results in the appearance of surface TCR 
+ cells and a concomitant reduction in the number of icTCR 
+ cells. The latter argument in particular provides compelling support for the hypothesis that a precursorproduct relationship exists between icTCR 
+ DN4 cells and mature thymic 
cells. If icTCR 
+ thymocytes are indeed the immediate precursors of 
cells, it is of considerable interest that they are apparently restricted to the DN4 subset. In this regard, although the precise timing of TCR ß,
and
rearrangements during adult thymus development remains controversial (reviewed in 1,4,10, 15,37,45), it is generally accepted that all three TCR loci are extensively rearranged in DN3 cells. Nevertheless no icTCR 
protein was observed in the DN3 subset.
At least three hypotheses could explain our failure to detect icTCR 
+ DN3 cells. First, it is possible that the vast majority of DN3 cells represent
ß lineage-committed precursors that do not express TCR
protein due to the activation of a lineage-specific transcriptional silencer (4650). The few remaining bona fide 
lineage-committed DN3 cells that productively rearrange and transcribe both TCR
and TCR
genes may be too infrequent to be detected by icTCR 
staining. Alternatively the formation of a 
TCR in 
lineage-committed DN3 cells may signal a rapid down-regulation of CD25, thus resulting in the observed DN4 phenotype of icTCR 
+ thymocytes. This latter scenario would be consistent with the reported under-representation of productive TCR
rearrangements in DN3 cells (51). A third formal possibility would be that 
cells may not follow the same DN1 to DN4 developmental sequence as
ß cells. For example, immature 
precursor cells could branch off from this pathway at the DN1 and/or DN2 stages. Following productive TCR 
rearrangement and down-regulation of CD44 and/or CD25 these precursor cells would subsequently give rise to an icTCR 
+ DN4 developmental intermediate and ultimately to mature 
cells.
Finally it should be noted that the unexpectedly late expression of icCD3
protein during adult thymus development places certain constraints upon models of
ß/
lineage divergence that depend upon TCR signaling events. In this regard recent data indicate that TCR
and TCR
rearrangements initiate at the DN2 stage of adult thymus development (10,52,53). However, the absence of icCD3
in the DN2 subset implies that such TCR rearrangements presumably cannot impinge upon the lineage commitment process until a later developmental stage.
 |
Acknowledgments
|
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The authors wish to thank Pablo Pereira for providing the anti-V
1.1 mAb, Isabel Ferrero for helpful discussions, Pierre Zaech for FACS sorting and Céline Maréchal for excellent technical assistance. This work was in part supported by a HFSP grant (A. W.).
 |
Abbreviations
|
---|
ic | intracellular |
DN | double negative |
DP | double positive |
FTOC | fetal thymic organ culture |
ISP | immature single positive |
PE | phycoerythrin |
PI | propidium iodide |
SP | single positive |
 |
Notes
|
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Transmitting editor: T Hüning
Received 6 April 1999,
accepted 16 June 1999.
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References
|
---|
-
Shortman, K. and Wu, L. 1996. Early T lymphocyte progenitors. Annu. Rev. Immunol. 14:29.[ISI][Medline]
-
Godfrey, D. I. and Zlotnik, A. 1993. Control points in early T cell development. Immunol. Today 14:547.[ISI][Medline]
-
Fehling, H. J. and von Boehmer, H. 1997. Early
ß T cell development in the thymus of normal and genetically altered mice. Curr. Biol. 9:263.
-
Kang, J. and Raulet, D. H. 1997. Events that regulate the differentiation of
ß TCR+ and 
TCR+ T cells from a common precursor. Semin. Immunol. 9:171.[Medline]
-
Pearse, M., Wu, L., Egerton, M., Wilson, A., Shortman, K. and Scollay, R. 1988. A murine early thymocyte developmental sequence is marked by transient expression of the interleukin 2 receptor. Proc. Natl Acad. Sci. USA 1989:1614.
-
Godfrey, D. I., Kennedy, J., Mombaerts, P., Tonegawa, S. and Zlotnik, A. 1994. Onset of TCR-ß rearrangement and selection during thymocyte development in adult mice. J. Immunol. 152:4783.[Abstract/Free Full Text]
-
Wilson, A., Held, W. and MacDonald, H. R. 1994. Two waves of recombinase gene expression in developing thymocytes. J. Exp. Med. 179:1355.[Abstract]
-
Wilson, A., de Villartay, J.-P. and MacDonald, H. R. 1996. T cell receptor
gene rearrangement and T Early
(TEA) expression in immature
ß lineage thymocytes: implications for
ß/
lineage commitment. Immunity 4:37.[ISI][Medline]
-
Petrie, H. T., Livak, F., Burtrum, D. and Mazel, S. 1995. T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production. J. Exp. Med. 182:121.[Abstract]
-
Tourigny, M., Mazel, S., Burtrum, D. B. and Petrie, H. T. 1997. T cell receptor (TCR)-ß gene recombination: dissociation from cell cycle regulation and developmental progression during T cell ontogeny. J. Exp. Med. 185:1549.[Abstract/Free Full Text]
-
Petrie, H. T., Scollay, R. and Shortman, K. 1992. Commitment to the T cell receptor-
ß or -
lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes. Eur. J. Immunol. 22:2185.[ISI][Medline]
-
Falk, I., Levelt, C. N. and Eichmann, K. 1993. Lineage relationships of the fetal thymocyte subset that expresses the ß chain of the interleukin-2 receptor. Eur. J. Immunol. 23:3373.[ISI][Medline]
-
Godfrey, D. I., Kennedy, J., Suda, T. and Zlotnik, A. 1993. A developmental pathway involving four phenotypically and functionally distinct subsets of CD3CD4CD8 triple-negative adult mouse thymocytes defined by CD44 and CD25 expression. J. Immunol. 150:4244.[Abstract/Free Full Text]
-
von Boehmer, H. and Fehling, H. J. 1997. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol. 15:433.[ISI][Medline]
-
Fehling, H. J., Gilfillan, S. and Ceredig, R. 1999.
ß/
lineage commitment in the thymus of normal and genetically manipulated mice. Adv. Immunol. 71:1.[Medline]
-
Rodewald, H.-R. and Fehling, H. J. 1998. Molecular and cellular events in early thymocyte development. Adv. Immunol. 69:1.[ISI][Medline]
-
Wilson, A. and MacDonald, H. R. 1995. Expression of genes encoding the pre-TCR and CD3 complex during thymus development. Int. Immunol. 7:1659.[Abstract]
-
Levelt, C. N., Carsetti, R. and Eichmann, K. 1993. Regulation of thymocyte development through CD3. II. Expression of T cell receptor ß CD3
and maturation to the CD4+8+ stage are highly correlated in individual thymocytes. J. Exp. Med. 178:1867.[Abstract]
-
Falk, I., Biro, J. Kohler, H. and Eichmann, K. 1996. Proliferation kinetics associated with T cell receptor-ß chain selection of fetal murine thymocytes. J. Exp. Med. 184:2327.[Abstract/Free Full Text]
-
Hozumi, K., Kobori, A., Sato, T., Nishimura, T. and Habu, S. 1996 Transcription and demethylation of TCR ß gene initiate prior to the gene rearrangement in c-kit+ thymocytes with CD3 expression: evidence of T cell commitment in the thymus. Int. Immunol. 8:1473.[Abstract]
-
Wilson, A., Petrie, H. T., Scollay, R. and Shortman, K. 1989. The acquisition of CD4 and CD8 during the differentiation of early thymocytes in short-term culture. Dev. Immunol. 2:85.
-
Wilson, A. and MacDonald, H. R. 1998. A limited role for `ß-selection' during

T cell development. J. Immunol. 161:5851.[Abstract/Free Full Text]
-
Pereira, P., Gerber, D., Huang, S. Y. and Tonegawa, S. 1995. Ontogenic development and tissue distribution of V
1-expressing 
T lymphocytes in normal mice. J. Exp. Med. 182:1921.[Abstract]
-
Renno, T., Hahne, M. and MacDonald, H. R. 1995. Proliferation is a pre-requisite for bacterial superantigen-induced T cell apoptosis in vivo. J. Exp. Med. 181:2283.[Abstract]
-
Malissen, B. and Malissen, M. 1996. Functions of TCR and pre-TCR subunits: lessons from gene ablation. Curr. Biol. 8:383.
-
Parkin, I. G., Owen, J. J. T. and Jenkinson, E. J. 1988. Proliferation of thymocytes in relation to T-cell receptor ß chain expression. Immunology. 64:97.[ISI][Medline]
-
Goodman, T. and LeFrancois, L. 1989. Intraepithelial lymphocytes: anatomical site, not T cell receptor form, dictates phenotype and function. J. Exp. Med. 170:1569.[Abstract]
-
Mombaerts, P., Clarke, A. R., Rudnicki, M. A., Iacomini, J., Itohara, S., Lafaille, J. J., Wang, L., Ichikawa, Y., Jaenisch, R., Hooper, M. L. and Tonegawa, S. 1992. Mutations in T-cell antigen receptor genes
and ß block thymocyte development at different stages. Nature 360:225.[ISI][Medline]
-
Fehling, H. J., Krotova, A., Saint-Ruf, C. and von Boehmer, H. 1995. Crucial role of the pre-T cell receptor
gene in the development of
ß but not 
T cells. Nature 375:795.[ISI][Medline]
-
Malissen, M., Gillet, M., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E. and Malissen, B. 1995. Altered T cell development in mice with a targeted mutation of the CD3-
gene. EMBO J. 14:4641.[Abstract]
-
Wurch, A., Biro, J., Potocnik, A. J., Falk, I., Mossmann, H. and Eichmann, K. 1998. Requirement of CD3 complex-associated signaling functions for expression of rearranged T cell receptor ß VDJ genes in early thymic development. J. Exp. Med. 188:1669.[Abstract/Free Full Text]
-
Egerton, M., Shortman, K. and Scollay, R. 1990. The kinetics of immature thymocyte development in vivo. Int. Immunol. 2:501.[ISI][Medline]
-
Shortman, K., Egerton, M. and Scollay, R. 1990. The generation and fate of thymocytes. Semin. Immunol. 2:3.[Medline]
-
Penit, C., Lucas, B. and Vasseur, F. 1995. Cell expansion and growth arrest phases during the transition from precursor (CD48) to immature (CD4+8+) thymocytes in normal and genetically modified mice. J. Immunol. 154:5103.[Abstract/Free Full Text]
-
Mombaerts, P., Iacomini, J., Johnson, R. S., Herrup, K., Tonegawa, S. and Papaioannou, V. E. 1992. Rag-1 deficient mice have no mature B and T lymphocytes. Cell 68:869.[ISI][Medline]
-
Shinkai, Y., Rathburn, G., Lam, K.-P., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A. M. and Alt, F. W. 1992. Rag-2 deficient mice lack mature lymphocytes owing to their inability to initiate V(D)J rearrangemant. Cell 68:855.[ISI][Medline]
-
Dudley, E. C., Petrie, H. T., Shah, L. M., Owen, M. J. and Hayday, A. C. 1994. T cell receptor ß chain gene rearrangement and selection during thymocyte development in adult mice. Immunity 1:83.[ISI][Medline]
-
Hoffman, E. S., Passoni, L., Crompton, T., Leu, T. M. J., Schatz, D. G., Koff, A., Owen, M. J. and Hayday, A. C. 1996. Productive T-cell receptor ß-chain gene rearrangement: coincident regulation of cell cycle and clonality during development in vivo. Genes Dev. 10:948.[Abstract]
-
MacDonald, H. R. and Wilson, A. 1998. The role of the T-cell receptor (TCR) in
ß/
lineage commitment: clues from intracellular staining. Immunol. Rev. 165:87.[ISI][Medline]
-
Aifantis, I., Azogui, O., Feinberg, J., Saint-Ruf, C., Buer, J. and von Boehmer, H. 1998. On the role of the pre-T cell receptor in
ß versus 
T lineage commitment. Immunity 9:649.[ISI][Medline]
-
Livak, F., Wilson, A., MacDonald, H. R. and Schatz, D. G. 1997.
ß lineage-committed thymocytes can be rescued by the 
T cell receptor (TCR) in the absence of TCR ß chain. Eur. J. Immunol. 27:2948.[ISI][Medline]
-
Passoni, L., Hoffman, E. S., Kim, S., Crompton, T., Pao, W., Dong, M.-Q., Owen, M. J. and Hayday, A. C. 1997. Intrathymic
selection events in 
cell development. Immunity 7:83.[ISI][Medline]
-
Zorbas, M. I. and Scollay, R. 1995. The

T lymphocytes of the perinatal murine thymus. Dev. Immunol. 4:93.[ISI][Medline]
-
Bischof, A., Park, J.-H. and Hünig, T. 1999. Expression of T-cell receptor ß chain mRNA and protein in
/
T cells from euthymic and athymic rats: implications for T cell lineage divergence. Dev. Immunol., in press.
-
Zuniga-Pflücker, J. C. and Lenardo, M. J. 1996. Regulation of thymocyte development from immature progenitors. Curr. Opin. Immunol. 8:215.[ISI][Medline]
-
Bonneville, M., Ishida, I., Mombaerts, P., Katsuki, M., Verbeek, S., Berns, A. and Tonegawa, S. 1989. Blockage of
ß T-cell development by TCR 
transgenes. Nature 342:931.[ISI][Medline]
-
Ishida, I., Verbeek, S., Bonneville, M., Itaharo, S., Berns, A. and Tonegawa, S. 1990. T cell receptor

and
transgenic mice suggest a role of a
gene silencer in the generation of
ß T cells and programmed rearrangements of 
TCR genes. Cell 72:337.
-
Haas, W. and Tonegawa, S. 1992. Development and selection of

T cells. Curr. Opin. Immunol. 4:147.[ISI][Medline]
-
Sim, G.-K., Olsson, C. and Augustin, A. 1995. Commitment and maintenance of the
ß and 
T cell lineages. J. Immunol. 154:5821.[Abstract/Free Full Text]
-
Kang, J., Fehling, H. J., Laplace, C., Malissen, M., Cado, D. and Raulet, D. H. 1998. T cell receptor
gene regulatory sequences prevent the function of a novel TCR
/pT
pre-T cell receptor. Immunity 8:713.[ISI][Medline]
-
Dudley, E. C., Girardi, M., Owen, M. J. and Hayday, A. C. 1995.
ß and 
T cells can share a late common precursor. Curr. Biol. 5:659.[ISI][Medline]
-
Capone, M., Hockett, R. D., Jr and Zlotnik, A. 1998. Kinetics of T cell receptor ß,
and
rearrangements during adult thymic development: T cell receptor rearrangements are present in CD44+ CD25+ pro thymocytes. Proc. Natl Acad. Sci. USA 95:12522.[Abstract/Free Full Text]
-
Livak, F., Tourigny, M., Schatz, D. G. and Petrie, H. T. 1999. Characterization of TCR gene rearrangements during adult murine T cell development. J. Immunol. 162:2575.[Abstract/Free Full Text]