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The Journal of Immunology, 1999, 163: 6053-6059.
Copyright © 1999 by The American Association of Immunologists

TCR {alpha}-Chain Repertoire in pT{alpha}-Deficient Mice Is Diverse and Developmentally Regulated: Implications for Pre-TCR Functions and TCRA Gene Rearrangement1

Stéphane Mancini*, Serge M. Candéias*, Hans Jorg Fehling{dagger}, Harald von Boehmer{ddagger}, Evelyne Jouvin-Marche* and Patrice N. Marche2,*

* Laboratoire d’Immunochimie, Commissariat à l’Energie Atomique-Grenoble, Département de Biologie Moléculaire et Structurale, Institut National de la Santé et de la Recherche Médicale Unit 238, Université Joseph Fourier, Grenoble, France; {dagger} Basel Institute for Immunology, Basel, Switzerland; and {ddagger} Institut Necker, Institut National de la Santé et de la Recherche Médicale Unit 373, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pre-TCR expression on developing thymocytes allows cells with productive TCRB gene rearrangements to further differentiate. In wild-type mice, most TCRA gene rearrangements are initiated after pre-TCR expression. However, in pT{alpha}-deficient mice, a substantial number of {alpha}ß+ thymocytes are still produced, in part because early TCR {alpha}-chain expression can rescue immature thymocytes from cell death. In this study, the nature of these TCR {alpha}-chains, produced and expressed in the absence of pre-TCR expression, have been analyzed. We show, by FACS analysis and sequencing of rearranged transcripts, that the TCRA repertoire is diverse in pT{alpha}-/- mice and that the developmental regulation of AJ segment use is maintained, yet slightly delayed around birth when compared with wild-type mice. We also found that T cell differentiation is more affected by pT{alpha} inactivation during late gestation than later in life. These data suggest that the pre-TCR is not functionally required for the initiation and regulation of TCRA gene rearrangement and that fetal thymocytes are more dependent than adult cells on pT{alpha}-derived signals for their differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thymic T lymphocyte precursor differentiation results in the regulated expression of cell surface molecules and the sequential rearrangement and expression of TCR genes (1). The most immature thymocytes are CD4-CD8- (DN).3 Expression of CD44 and CD25 further defines four DN subpopulations, respectively, CD44+25-, CD44+25+, CD44-25+, and CD44-25-, representing successive stages of maturation. CD44-25- cells are rapidly cycling and progress to the CD4+8+ [double positive (DP)] compartment, where they begin to express low levels of TCR-{alpha}ß. They are then selected according to their TCR specificity and differentiate into single positive (SP) CD4+ and SP CD8+ thymocytes that will ultimately exit the thymus and seed the periphery. Although TCR-{alpha}ß expression is first detected on DP thymocytes, TCRB locus rearrangement is initiated earlier, at the CD44+25+->CD44-25+ transition. Essentially all the DP thymocytes express a TCR ß-chain, despite the fact that V(D)J recombination generates out-of-frame junctions at a high frequency (66%) (2). The enrichment for in-frame rearrangements in DP thymocytes reflects the existence of a checkpoint called ß selection, whereby only cells able to express a rearranged TCR ß-chain as part of a pre-TCR complex are allowed to further differentiate (3).

The pre-TCR is composed of the TCR ß-chain covalently linked to the invariant pT{alpha} chain and noncovalently associated with the CD3 complex (4, 5, 6, 7). Pre-TCR expression on developing CD44-25+ thymocytes results in their differentiation through the CD44-25- stage to the DP compartment, a burst of proliferation (3, 8), and may facilitate {alpha}ß lineage commitment (3, 9). At the DNA level, pre-TCR expression induces the cessation of TCRB gene recombination (allelic exclusion) (10, 11, 12). Finally, pre-TCR expression and ß selection correlate with massive initiation of TCRA gene rearrangement (13). Mice unable to rearrange and/or express a TCR ß-chain (14) exhibit profoundly impaired DN->DP transition and severe thymic hypocellularity because of their inability to express a pre-TCR. Inactivation of the pT{alpha} chain also leads to a dramatic reduction of {alpha}ß+ thymocyte differentiation (15, 16). Only very low numbers of TCR{alpha}ß+ DP and SP thymocytes are produced in the thymus of pT{alpha}-/- animals. They are rescued from cell death either because early rearrangement of TCRA genes allows expression of an {alpha}ß heterodimer that can replace the pre-TCR, or under the influence of TCR{gamma}{delta}+ thymocytes (17). Thus, whereas in normal mice most TCRA rearrangements take place after pre-TCR expression, it seems that in pT{alpha}-/- mice, TCRA rearrangement in DN cells is required to generate TCR{alpha}ß-expressing thymocytes.

TCRADV genes can rearrange with any one of 61 AJ segments (18). Southern blot analysis of TCRA rearrangements in T cell hybridomas produced from fetal and adult thymocytes revealed the existence of two hot spots (HS) for recombination within the AJ region. HS1 contains segments from AJ61 to AJ48, and HS2 contains segments from AJ31 to AJ22 (19, 20). These HSs may represent two entry points used early and late during development, respectively. Sequence analysis of TCRA rearrangements expressed at different times of development further showed that not only AJ but also ADV gene segment utilization evolves with time (21). Because in normal mice the majority of TCRA rearrangements are initiated just after pre-TCR expression, we hypothesized that the pre-TCR may be implicated in the progressive and ordered utilization of the ADV and AJ segments during development.

In this study, we analyzed the TCRA repertoire expressed in pT{alpha}-deficient mice, both by FACS analysis and at the molecular level, to know whether thymocytes differentiating without pre-TCR expression use a special repertoire of TCR {alpha}-chains or whether TCRA gene rearrangements in DN thymocytes are similar to those found in wild-type animals. Our results show that pT{alpha}-/- mice have a diverse repertoire of TCRA rearrangements and that the pre-TCR is not required for the entire TCRAD locus to be accessible to VDJ recombinase. Furthermore, the regulated utilization of AJ segments found in wild-type thymocytes is maintained in pT{alpha}-/- animals, although slightly delayed. This delay may be related to a more severe impairment of DP thymocyte generation during gestation than later in life in the absence of pT{alpha} expression. As a whole, these results suggest that the pre-TCR functions in cellular expansion, allelic exclusion, and possibly TCR{alpha}ß lymphocyte lineage commitment, but is dispensable for the initiation and the regulation of TCRA gene rearrangement.


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

pT{alpha}-/ - mice on a C57BL/6 background (15) and C57BL/6 mice (purchased from IFFA CREDO, L’Arbresle, France) were maintained in the animal facilities of the Commissariat à l’Energie Atomique (Grenoble, France). pT{alpha}-deficient mice had been backcrossed on C57BL/6 background for eight generations at the beginning of the study. Detection of the vaginal plug was considered as day 1 of gestation.

Abs and FACS analysis

The following Abs were used for staining: FITC-conjugated anti-V{alpha}2 (B20.1), anti-V{alpha}3 (RR3-16), anti-V{alpha}8 (B21.14) and anti-V{alpha}11 (RR8-1), PE-conjugated anti-CD4 (GK1.5), Cy-Chrome-conjugated anti-CD8 (53-6.7), biotin-conjugated anti-TCR-ß (H57-597). All the Abs and Cy-Chrome-conjugated streptavidin were from PharMingen (San Diego, CA).

Single-cell suspensions of fetal day 16, fetal day 18, day 1, and day 28 thymocytes were prepared in PBS/3% FCS/0.16% NaN3. Cells were incubated for 15 min at room temperature with saturating concentration of the indicated Abs in the same medium, washed in PBS, and analyzed on a FACScalibur (Becton Dickinson, Pont de Claix, France) with CellQuest software. Before staining, cells were incubated with saturating concentration of anti-Fc receptor (Fc Block, PharMingen) to reduce nonspecific binding.

RNA isolation and RT-PCR

Thymocytes were isolated at day 18 of fetal life and 1 day and 28 days after birth. Two pools of fetal thymic lobes (for day 18 of gestation) or two thymi (for days 1 and 28) were analyzed independently. Total RNA and cDNA were prepared as described (22). Two consecutive nested PCR were conducted for each cDNA sample, with an equimolar mix of NW36, NW37, and NW38 (0.2 µM final) and C{alpha}3 in the first round, followed by a second round using NW primers and C{alpha}1 (Table IGo; Ref. 23). To facilitate cloning, SphI and EcoRI sites have been introduced into NW and C{alpha}1 primers, respectively. The conditions for the first PCR reaction were 5 min at 94°C followed by 5 cycles consisting of 30 s at 94°C, 30 s at 45°C, and 30 s at 72°C, 30 cycles consisting of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C, and finally 10 min at 72°C. The conditions for the second PCR reaction were 5 min at 94°C followed by 35 cycles consisting of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C, and 10 min at 72°C.


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  		Table I. Oligonucleotides used in PCR amplifications

 
Cloning and sequencing

The PCR products were cloned in the EcoRI and SphI sites of pBlueScribe KS- (Stratagene, La Jolla, CA), which was then used to transform TG1 competent bacteria. Positive clones obtained from pT{alpha}-/- and C57BL/6 thymus at days 1 and 28 were sequenced with the use of the Thermo Sequenase premixed cycle sequencing kit (Amersham, Les Ulis, France) and analyzed on a Vistra 725 DNA Sequencer (Vistra Systems, Molecular Dynamics, Bondoufle, France). For clones from day 18 fetal thymus, plasmids were prepared using the Wizard Plus Minipreps DNA Purification System (Promega, Charbonnières, France) and then sequenced by Genome Express (Grenoble, France). AJ gene segments were identified by comparison with the published sequences (18), and ADV gene segments were identified by comparison with genes contained in GenBank using BLAST 1.4.11 software. Pearson’s test was used to compare ADV gene utilization in pT{alpha}-/- and control mice. To compare AJ gene utilization in the different samples analyzed, the median value of AJ segment distribution was calculated, and AJ distributions in each population were compared by a Mann-Whitney nonparametric test. Statistical analyses were performed using SPSS6.0 software (SPSS, Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Diversity of the TCRA repertoire in absence of pT{alpha} expression

As a preliminary estimation of the TCRA repertoire diversity in pT{alpha}-/- mice, we analyzed by flow cytometry the expression of ADV2, ADV3, ADV8, and ADV11 gene families on the surface of TCR-{alpha}ß+ thymocytes and peripheral T lymphocytes in 4-wk-old animals. As can be seen in Fig. 1Go, these 4 gene families are expressed at similar levels in pT{alpha}-/- and age-matched C57BL/6 animals, both in the thymus and in lymph nodes. Although these results suggest that the repertoire of expressed TCR {alpha}-chains in pT{alpha}-deficient mice does not differ significantly from that of wild-type animals, FACS analysis cannot rule out the possibility that oligoclonal TCRA rearrangements account for the observed level of expression.Thus, to further investigate the complexity of the TCR {alpha}-chain repertoire in pT{alpha}-/- mice, rearranged TCRA transcripts were amplified by RT-PCR from total thymocytes isolated during late gestation (day 18 of fetal life), at birth (<24 h, day 1), and at 4 wk of age (day 28). Amplifications were performed with a mix of degenerate oligonucleotides designed to amplify multiple ADV genes (23) and an AC-specific primer, as described in Materials and Methods. PCR products were then cloned and sequenced to identify the ADV and AJ segments encoding the TCR {alpha}-chain variable region. Table IIGo summarizes all the ADV genes identified in pT{alpha}-/- and B6 mice during the course of this analysis. The degenerate primers clearly preferentially amplify members of the ADV3, ADV10, and ADV11 families in both types of mice. Altogether, 17 different genes (belonging to 5 families) were identified in 81 TCRA rearrangements sequenced from pT{alpha}-/- thymocytes, compared with 22 different genes (belonging to 7 families) for 92 rearrangements in corresponding C57BL/6 samples. No statistically significant differences were found when use of ADV3, ADV10, or ADV11 family members in pT{alpha}-/- and wt sequences were compared. Sequencing showed that ADV-AJ junctions are diversified in both type of mice, eliminating the hypothesis that TCRA rearrangements in pT{alpha}-deficient mice are only oligoclonal. Sequencing finally showed that the percentages of out-of-frame junctions and the distribution of CDR3 lengths were similar in pT{alpha}-/- and wild-type animals (Table IIIGo).



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  		FIGURE 1. Similar expression of ADV genes on pT{alpha}-/- and wild-type T lymphocytes. Single-cell suspensions of thymus and lymph nodes from six 4-wk-old C57BL/6 and pT{alpha}-/- mice were stained with FITC-conjugated anti-V{alpha}2, anti-V{alpha}3, anti-V{alpha}8, or anti-V{alpha}11 and biotinylated anti-TCR-ß labeled with streptavidin-Cy-Chrome. Values represent the mean ± SD percentage of TCR-ßhigh cells which are V{alpha}2+, V{alpha}3+, V{alpha}8+, and V{alpha}11+ in thymus (A) and lymph nodes (B).

 

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  		Table II. ADV gene segments identified in C57BL/6 and pT{alpha}-/- micea

 

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  		Table III. Percentage of in-frame TCRA rearrangements and CDR3 length during development in pT{alpha}-/- and control mice

 
Thus, ADV genes used to form TCR {alpha}-chain in pT{alpha}-deficient mice are as diverse as in C57BL/6 mice. These ADV genes are rearranged with a large panel of different AJ segments, distributed on the entirety of the 65-kb-long AJ region, with no restriction regarding their position within this region (Fig. 2Go). Therefore, even in the absence of pT{alpha}, the AJ region is completely accessible for rearrangement. Altogether, our results show that in spite of the absence of pre-TCR expression, the level of complexity of TCRA rearrangement is similar in pT{alpha}-/- and wild-type mice.



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  		FIGURE 2. AJ gene segment use is not restricted in pT{alpha}-/- mice. AJ gene segments were identified by sequencing as described in the legend of Table IIGo. The position of all the AJ segments (in productive and nonproductive transcripts) identified in C57BL/6 and pT{alpha}-/- mice is shown on the AJ region, represented to scale. The number of occurrences of each AJ segments is indicated in the circles (identical sequences are scored only once). Unshaded symbols correspond to pseudogenes. AC, TCRA constant region gene.

 
Progressive utilization of the AJ gene segments in pT{alpha}-/- mice

Having established that the TCR {alpha}-chain repertoire is diverse in pT{alpha}-/- mice, we next wanted to know whether AJ segment use was still developmentally regulated in the absence of pT{alpha}. We therefore compared the pattern of AJ use in productive TCRA gene rearrangements at the different time points analyzed. A graphic representation of this analysis is shown in Fig. 3Go.



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  		FIGURE 3. Progressive AJ gene segment use during development in pT{alpha}-/- mice. The AJ segments identified in productive TCRA rearrangements from C57BL/6 and pT{alpha}-/- thymus at day 18 of fetal life (A), day 1 (B), and day 28 (C) are represented. The sequences were obtained as described in the legend of Table IIGo. The number of sequences is indicated on each panel (identical sequences are scored only once). In this representation, the TCRAJ region is represented on the x-axis and graduations from 61 to 1 represent the 61 AJ segments, in the same order as on the TCRAD locus. Each bar represents one TCRA transcript. The bars begin at the beginning of the AJ region and stop at the position of the AJ segment used in this particular TCRA rearrangement. Their length indicates the position of the segment within the AJ region : the shortest are the most ADV proximal, the longest the most AC proximal. The overall pattern is indicative of the regularity of AJ segment distribution in the sample. A sharp decline indicates that numerous AJ segments in close proximity are used in distinct rearrangements. A regular decline indicates that the AJ segments used are evenly spaced in the AJ region. Shaded regions from AJ61 to AJ48 and from AJ31 to AJ22 correspond to HS1 and HS2, respectively. AC, TCRA constant region gene.

 
In C57BL/6 mice (Fig. 3Go, left), AJ segments used at day 18 of gestation are predominantly located between AJ61 and AJ32, i.e., in HS1 and between this point and HS2, confirming that HS1 is the only active entry point at that time. At day 1, we observe a clear increase in the number of transcripts containing AJ segments located in HS2 or downstream, which now represent about one-half of the rearrangements. At day 28, the AJ segments used are distributed almost evenly over the entire AJ region. This progressive widening of AJ usage in TCRA transcripts results in a shift of the value of the median point of AJ distribution, from AJ42 in d18 fetal thymus to AJ32 and AJ33 at day 1 and 4 wk, respectively. AJ distributions were statistically different between fetal day 18 and day 1 (p = 0.01). These results show that in C57BL/6 mice, AJ use evolves during development in much the same way as in BALB/c mice (21), with a progressively larger use of AJ segments from day 18 of gestation until 4 wk of age. In pT{alpha}-/- mice, the situation is somewhat different. At day 18 of gestation, AJ segments are spread from AJ58 to AJ13. About one-third (5 of 16) are located in or downstream of HS2. At birth, the rearrangements now involve AJ segments from AJ56 to AJ23, located mainly upstream of HS2, giving an overall pattern similar to that of C57BL/6 thymocytes at day 18 of gestation. At 4 wk, we observe a widening of the set of AJ segments used, as in age-matched B6 mice. Despite the wider use of AJ segments at day 18 than at day 1 (AJ58 to AJ13 vs AJ56 to AJ23, respectively), these distributions are not significantly different (p > 0.05), and both have their median point located at AJ39. However, differences in AJ distributions were statistically significant between day 1 and 4 wk of age (p = 0.048), with a shift of the median point from AJ39 to AJ31, respectively. Thus, as in wild-type mice, AJ use is developmentally regulated, increasing with age, in pT{alpha}-/- mice, but this program seems to be delayed in the absence of the pT{alpha} chain, starting only after birth rather than during late gestation.

T cell development in pT{alpha}-/- and wild-type mice

The delay in AJ utilization in pT{alpha}-deficient mice prompted us to analyze and compare the kinetics of T cell differentiation in fetal and adult pT{alpha}-/- and wt mice (Fig. 4Go). At day 16 of gestation, similar numbers of thymocytes were recovered from pT{alpha}-/- and wt mice (~2.2 x 105), all of them CD4-8- (Fig. 4Go, A and E). Thus, pT{alpha} inactivation seems to have no effect on T cell differentiation until at least day 16 of fetal life, as judged by FACS analysis and cellularity, even if it was shown that TCR-ß and pT{alpha} transcripts are present as early as day 15 of gestation (25, 26) and that a pre-TCR can potentially be expressed on a fraction of DN CD44+25+ thymocytes (27). However, we were unable to amplify rearranged TCRA transcripts from 6 independent cDNA preparations of fetal day 16 pT{alpha}-/- thymocytes in two successive nested PCR reactions, whereas they were easily detected, in one round of amplification, in two of four cDNA preparations of age-matched control thymocytes. These results suggest that TCRA gene rearrangement is already affected at that early stage of development.



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  		FIGURE 4. T cell differentiation in pT{alpha}-/- and wt mice during development. C57BL/6 (A, B, C, D) and pT{alpha}-/- (E, F, G, H) thymocytes isolated at fetal day 16 (A, E), fetal day 18 (B, F), day 1 (C, G), and day 28 (D, H) were analyzed for CD4 and CD8 expression. From 4 to 14 thymus and/or pooled thymic lobes were analyzed for each point. The number of cells (±SD) per thymus and the percentage (±SD) of DP thymocytes are indicated for each panel. For fetal day 18 pT{alpha}-/- thymocytes, the percentage of DP cells was calculated from 6 individual thymi and 1 pool of 4 thymic lobes, whereas cellularity was calculated only from the pool of 4 lobes.

 
The impact of pT{alpha} inactivation on T cell differentiation becomes evident 2 days later. At day 18 of gestation, thymic cellularity is reduced 2.5-fold on average in pT{alpha}-deficient mice, and, more importantly, the percentage of CD4+8+ cells is decreased 11 times (4.0% vs 43.6%) when compared with age-matched control mice (Fig. 4Go, B and F). At birth and at 4 wk of age, the difference in cellularity increases (3.6 and 19 times, respectively), whereas the difference in the percentage of DP thymocytes diminishes at day 1 when compared with day 18 of gestation (only 6 times) and remains stable over the considered period (Fig. 4Go). Thus, pT{alpha} inactivation has a more profound effect on DP thymocyte differentiation during late gestation than later in life. This differential effect of pT{alpha} inactivation on fetal vs adult T cell development may then explain the slower regulation of AJ segment use observed in pT{alpha}-deficient mice around birth.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we analyzed the TCRA repertoire expressed in pT{alpha}-/- mice at different developmental stages. Despite the inactivation of the pT{alpha} chain, DP and SP thymocytes are nonetheless generated in these animals, either because early rearrangement and expression of TCRA genes allow the cells to progress beyond the CD44-25+ stage of differentiation, or under the influence of TCR-{gamma}{delta}+ thymocytes (17). Alternatively, DP thymocytes can also be generated in pT{alpha}-deficient mice through {delta} selection (28) or by the expression of a {gamma}{delta} TCR (29). In pT{alpha}-/- x TCR-{delta}-/- double-deficient mice, nearly all DP and SP thymocytes have been ß selected; whereas in pT{alpha}-/- x TCR{alpha}-/- double-deficient animals, no evidence of ß selection could be found in DP thymocytes (17). Thus, TCR {alpha}-chain expression is required for ß selection in the absence of pT{alpha}. These findings exclude the possibility that thymocyte development in pT{alpha}-/- mice results from the expression of an " incomplete " pre-TCR that would comprise only a TCR ß-chain and the CD3 complex, because expression of such a structure would result in at least some degree of ß selection in pT{alpha}-/- x TCR{alpha}-/- double-deficient animals. TCRA rearrangements in pT{alpha}-/- mice can have any of three origins. They can originate either from {alpha}ß- or {gamma}{delta}-expressing thymocytes. Furthermore, in {alpha}ß lineage, even though it is clear that some of them have been generated during the DN stages of differentiation, we cannot exclude that a low level of TCRA gene rearrangement is induced during the DN to DP transition driven by {gamma}{delta}+ thymocytes even in the absence of ß selection (17) or in DP thymocytes generated through {gamma} and/or {delta} selection (28, 29). Regardless of their origin, TCRA rearrangements analyzed in our study are generated in the absence of any pre-TCR signaling, whereas in wild-type mice, most of the rearrangements are initiated after pre-TCR expression. Therefore, it was of interest to determine whether TCRA rearrangements present in pT{alpha}-deficient mice exhibit a special pattern, and how they are regulated.

ADV genes belonging to different families as well as multiple members of a same family were identified in TCRA transcripts amplified from pT{alpha}-deficient thymocytes. Although the exact physical map of the C57BL/6 TCRAD locus is currently unknown, it probably evolved through successive duplications of more or less conserved blocks encompassing several ADV genes belonging to different families, as the BALB/c TCRAD locus (30, 31). Therefore, it is likely that the different ADV3, ADV10, and ADV11 family members identified in pT{alpha}-/- mice are not clustered together but are interspersed with other ADV genes belonging to different families and widely spread within the TCRADV region. Our results therefore suggest that most of this region is accessible for VDJ recombination and used in pT{alpha}-/- mice. These ADV genes were rearranged with a wide array of AJ segments, encompassing most of the AJ region. Formally, we cannot rule out that TCRA rearrangements in pT{alpha}-deficient mice are initially limited to a small number of thymocytes and that this "initial" repertoire is then diversified by cellular expansion and secondary ADV-AJ recombination. However, in that case, one would expect an underrepresentation of ADV-proximal AJ genes caused by secondary rearrangements, and this is clearly not the case in our pT{alpha}-/- samples (Fig. 3Go). Altogether, these findings indicate that in pT{alpha}-/- mice, the TCRAD locus is widely if not completely open and accessible for rearrangements and that the expressed TCRA repertoire is diversified and shows no signs of restriction. Therefore, pre-TCR signaling is required neither to confer accessibility to the TCRAD locus nor to generate a diverse repertoire of TCRA gene rearrangements. However, one must not forget that pre-TCR expression results in sustained thymocyte proliferation and selects cells that have successfully rearranged their TCRB genes. Hence, the pre-TCR participates in increasing {alpha}ß+ lymphocyte diversity and makes T cell differentiation more efficient, by generating a large number of thymocytes that have already succeeded in rearranging their TCRB genes and are capable of rearranging their TCRA genes.

We recently showed during a comprehensive study of TCRADV2 gene rearrangements in BALB/c thymocytes that use of AJ segments changes during development (21). The present study shows that AJ use is similarly regulated from day 18 of gestation to 4 wk of age in C57BL/6 mice, even if the AJ segments considered here are rearranged with a wide array of different ADV genes. In pT{alpha}-/- mice, we found that evolution of the distribution of AJ segments used over the same time frame is slower and that the widening of AJ gene use starts only after birth, and not from day 18 of gestation as in wild-type mice. This observation may be related to our finding that late fetal thymocytes have a more stringent requirement for pT{alpha}, and thus pre-TCR, derived signals for development than their adult counterparts. One possible explanation for this finding is that the alternate, pre-TCR independent mechanisms driving DN to DP maturation, including early TCRA gene rearrangement and expression, are not yet fully functional at that time, and it is only around birth, when "adult type" precursors begin to develop in the thymus, that these mechanisms are activated. Differences between fetal and adult thymocyte development have been documented in many instances. They have, for example, differential requirement for p56lck (32) and the IL-7 receptor {alpha}-chain (33). These changes may also be related to changes in the thymic microenvironment.

The delay in AJ use in pT{alpha}-/- mice could also stem from the fact that DP thymocytes generated in the absence of a pre-TCR do not pass through a phase of intense proliferation. Cell cycle progression involves many different chromatin structure changes that may be required to establish a proper control of accessibility of TCRA genes in wild-type mice, i.e., open and close different regions of the locus at different times. In addition, one must not forget that VDJ recombination activity is also regulated throughout cell cycle progression (34, 35). Therefore, in the absence of chromatin remodeling and VDJ recombinase regulation during the cell cycle, it is possible that the factors providing and/or controlling accessibility of the TCRAD locus function more progressively, thereby leading to a different (slower) regulation of accessibility of the different domains of the TCR AJ region. Also, it has been shown that CD3-mediated signals activate transcription of TCRA genes in immature thymocytes (36). This transcription may in some as yet undefined way influence the pattern of VDJ recombination at the TCRAD locus.

Two non-mutually exclusive hypotheses can be proposed to explain that pre-TCR signaling is not necessary for developmental regulation of AJ segment use. The progressive opening of the TCRAD locus may be dependent on a cell autonomous mechanism related to the maturity of T cell precursors entering the thymus at different times. Alternatively, it could be the result of extracellular signals, delivered by thymic stroma by cell-cell interaction or by soluble mediators, independently of pre-TCR expression.

Collectively, our results show that the TCRA repertoire in pT{alpha}-/- mice is limited only in terms of cellularity and not in terms of diversity. These findings suggest that the main functions of the pre-TCR are to promote TCRB allelic exclusion, to increase thymocyte number, and probably to engage thymocytes in the {alpha}ß+ lymphocyte lineage but not to induce and control TCRA gene rearrangement. The requirements for TCRA gene rearrangement are currently poorly defined. The T early {alpha} (TEA) element has been shown to play a role in the control of accessibility of the first AJ segments (37). These segments are rearranged in pT{alpha}-/- mice, and we also found significant levels of TEA transcription (data not shown). Thus, pre-TCR expression is also dispensable for this event. However, different pre-TCR mutations are not equivalent in terms of their influence on TCRA rearrangement; no ADV-AJ gene rearrangements were found in CD3{epsilon}-deficient mice (22, 38), whereas the rearrangements are found in the absence of expression of the TCR ß-chain (39). Therefore, even if the nature of the molecular events leading to TCR {alpha}-chain expression remains to be determined, it is likely that CD3{epsilon} plays a role in their regulation, maybe independently of its functions in a pre-TCR complex.


   Acknowledgments
 
We thank Dr. V. Leroy for help with statistical analyses, M. Gallagher for comments on the manuscript, and K. Vallon and V. Giraud for animal care. The Basel Institute for Immunology is supported by Hoffman-La Roche (Basel).


   Footnotes
 
1 This work was supported by institutional grants from Commissariat à l’Energie Atomique, Institut National de la Santé et de la Recherche Médicale and Université Joseph Fourier. S.M. was supported by a predoctoral fellowship from Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche. Back

2 Address correspondence and reprint requests to Dr. Patrice N. Marche, Laboratoire d’Immunochimie, Commissariat à l’Energie Atomique-Grenoble, Département de Biologie Moléculaire et Structurale, Institut National de la Santé et de la Recherche Médicale Unit 238, F-38054 Grenoble cedex 9, France. E-mail: Back

3 Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; HS, hot spot; TEA, T early {alpha}. Back

Received for publication February 25, 1999. Accepted for publication September 14, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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