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TCRA Gene Rearrangement in Immature Thymocytes in Absence of CD3, Pre-TCR, and TCR Signaling¹

Stéphane J. C. Mancini^*, Serge M. Candéias^2,*, James P. Di Santo, Pierre Ferrier, Patrice N. Marche^* and Evelyne Jouvin-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 U548, Université Joseph Fourier, Grenoble, France; Unité des Cytokines et Développement Lymphoïde, Département d’Immunologie, Institut Pasteur, Paris, France; and Centre d’Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique, Marseille, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
During thymocyte differentiation, TCRA genes are massively rearranged only after productively rearranged TCRB genes are expressed in association with pT and CD3 complex molecules within a pre-TCR. Signaling from the pre-TCR via the CD3 complex is thought to be required to promote TCRA gene accessibility and recombination. However, ⁺ thymocytes do develop in pT-deficient mice, showing that TCR-chain genes are rearranged, either in CD4^-CD8^- or CD4⁺CD8⁺ thymocytes, in the absence of pre-TCR expression. In this study, we analyzed the TCRA gene recombination status of early immature thymocytes in mutant mice with arrested thymocyte development, deficient for either CD3 or pT and c expression. ADV genes belonging to different families were found rearranged to multiple AJ segments in both cases. Thus, TCRA gene rearrangement is independent of CD3 and c signaling. However, CD3 expression was found to play a role in transcription of rearranged TCR-chain genes in CD4^-CD8^- thymocytes. Taken together, these results provide new insights into the molecular control of early T cell differentiation.

	Introduction

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Tcell receptor gene rearrangements, sequential expression of particular cell surface molecules, and proliferation are important steps of thymic T cell maturation (reviewed in Ref. 1). The thymocytes first proceed through a double-negative (DN)³ CD4^-CD8^- stage of development. The regulated expression of CD44 and CD25 defines four DN subpopulations: the early T cell precursors, which are CD44⁺CD25^- (DN1), become successively CD44⁺CD25⁺ (DN2), CD44^-CD25⁺ (DN3), and CD44^-CD25^- (DN4). The CD44^-CD25^- thymocytes proliferate, then develop through CD4^low or CD8^low intermediates called immature single-positive (SP) cells, to finally enter the CD4⁺CD8⁺ double-positive (DP) stage. At this point, thymocytes begin to express low levels of TCR and are selected to become mature SP CD4⁺ or CD8⁺ according to the specificity of their TCR. A second subset of T cells bearing a TCR at the surface develop in the thymus from the same progenitor population (2). Unlike ⁺ T cells, ⁺ T cells differentiate along the DN stage of development and in general, do not express the CD4 or CD8 coreceptors (reviewed in Ref. 3).

TCRB, TCRD, and TCRG rearrangements are initiated in DN during a first wave of expression of recombination-activating gene (RAG)-1 and RAG-2 (4 and reviewed in Ref. 5). In adult mice, TCRB gene rearrangements are initiated at the transition between the DN2 and DN3 stage of development, whereas TCRD and TCRG rearrangements are initiated earlier in DN2 cells (6, 7). At the DN/DP transition, cells encounter a checkpoint, known as selection, which allows only those cells expressing in-frame TCR chains to survive and proceed further. This process is controlled by the pre-TCR, composed of the neosynthesized TCR chain associated to the invariant pT chain and the CD3 complex (8, 9, 10, 11). Pre-TCR expression at the surface of DN3 thymocytes induces their differentiation into DP cells and a burst of proliferation, which are impeded in the absence of a functional pre-TCR (12, 13, 14, 15, 16, 17, 18). The pre-TCR may also play an important role in vs commitment (19). It is generally considered that TCRA gene rearrangement is initiated after pre-TCR expression when the cells transit from DN to DP, once the second wave of expression of RAG-1 and RAG-2 is triggered (4, 20).

The TCRAD locus has the particularity to be composed of TCRA and TCRD gene segments, the TCRD genes being embedded between the TCRA genes. Between 75 and 100 ADV segments and 60 AJ segments are available for V(D)J recombination in mice (21, 22, 23). Accessibility of the TCR gene segments for V(D)J recombination is controlled in part by enhancer elements located in the TCR loci (reviewed in Refs. 24, 25, 26). A TCRA enhancer (E) situated just downstream of AC and a TCRD enhancer (E) located between DJ2 and DC have been identified (27, 28). Studies using E and E as part of transgenic artificial recombination substrates showed that these elements promote rearrangement of reporter genes after pre-TCR expression, in DN4 thymocytes, and in earlier DN cells, respectively (29, 30). These enhancers are thus implicated in the tissue- and stage-specific control of their respective substrates. However, it was later shown that TCRA and TCRD gene rearrangements are found in E^-/- and E^-/- mice, respectively, indicating that other elements are instrumental in the control of accessibility of the TCRAD locus (31, 32, 33). To explain the differential temporal use of the proximal TCRD and TCRA genes in DN and DP cells, respectively, it was proposed (in human) that blocking element 1, situated between the DC and AJ segments, prevents the upstream E to promote accessibility to the AJ genes in DN cells (34). In this context, the block over the AJ region would be released in late DN/early DP at the time of E activation, after pre-TCR signaling.

In addition to these results, most of the data available to date indicate that TCRA coding genes are not rearranged before the DN to DP transition. Because the complex structure of the TCRAD locus makes TCRA gene rearrangements difficult to study at the genomic level, most of these studies relied on Northern blot analysis (20, 35, 36), rather than direct analysis of rearrangement events at the DNA level. TCRA genes were found expressed only after the thymocytes did exit the DN compartment. Furthermore, mimicking pre-TCR signaling by injection of anti-CD3 Abs not only induces DP thymocyte differentiation, but also increases TCRA gene germline transcription, an event thought to reflect accessibility of these genes (37). In addition, TCRB, TCRD, and TCRG, but not TCRA genes were found rearranged and transcribed in CD3-deficient mice (15, 17, 38, 39). Collectively, these data were interpreted as evidence that pre-TCR-derived signals are required to induce accessibility and rearrangement of TCRA genes. However, TCR⁺ thymocytes still develop in pT-deficient mice, in absence of pre-TCR, and we showed that the TCR chains expressed in pT^-/- thymocytes display features identical to that found in wild-type (wt) animals (40). Pre-TCR-independent DP thymocyte differentiation can be achieved through the influence of TCR or TCR chain expression (41, 42). Nonetheless, analysis of pT x TCR double-deficient animals showed that DP thymocytes still develop in these mice, and that almost all of them have been selected (43). This phenomenon was postulated to happen through pre-TCR-independent early rearrangement and expression of TCR chains, able to substitute for pT and form a TCR that promotes the progression of TCR-expressing DN thymocytes to the DP and SP compartments. One other study found rearranged TCR chain genes in sorted immature DN3 wt thymocytes (44). However, it was later shown that a pre-TCR can potentially be expressed in DN2 thymocytes (45), and that the DN3 population includes thymocytes that have already undergone selection (46). Thus, TCRA rearrangements found in wt DN3 thymocytes may result from pre-TCR signaling. Therefore, it has not yet been formally demonstrated whether TCR chain genes can be rearranged before pre-TCR expression, and, if so, how they are induced.

In this study, we analyzed the occurrence of TCRA gene rearrangement in different models of genetically modified mice, in which thymocyte differentiation is blocked at or before the CD44^-CD25⁺ stage. Our results show that TCRA gene rearrangement does take place in immature thymocytes, independently of the main signals governing thymocyte differentiation, namely CD3, pre-TCR, TCR, and c expression and signaling. Furthermore, we also show that CD3 is required for TCRA rearrangements to be transcribed at these early stages of development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

CD3^5/5 (15) and RAG-2^-/- mice (13) were maintained in the animal facility of the Commissariat à l’Energie Atomique (Grenoble, France), and E^-/- x TCR^-/- (47, 48) and c^-/- x pT^-/- mice (45) in the animal facilities of the Center d’Immunologie de Marseille Luminy (Marseille, France) and of the Centre National de la Recherche Scientifique/Centre de Développement des Techniques Avancées (Orléans, France), respectively. All mice used were between 4 and 10 wk of age. C57BL/6 mice were used as wt control.

RNA preparation and RT-PCR

Total RNA and cDNA from E^-/- x TCR^-/- and CD3^5/5 thymi were prepared as previously described (40, 48). PCR were conducted using oligonucleotides specific for the ADV2, ADV3, or ADV7 family genes (CDNAU, ADV3UP, or ADV7UP, respectively) and for the AC region (MTA). Thy-1 cDNA was amplified with the Thy-1UP and Thy-1DO primers as a positive control. PCR products were migrated on 1.5% agarose gels and probed by Southern blot hybridization using ³²P-labeled primers specific for the AC region or Thy-1 (C2 and Thy-1UP2 primers, respectively). Primer sequences are shown in Fig. 1A.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. A, Sequence of the oligonucleotides used in this study. S, primer in transcription orientation; A, primer in reverse of transcription orientation. Artificial BamHI and EcoRI sites are underlined. B, Location of the AJ-specific oligonucleotides used to amplify ADV-AJ rearrangements. The AJ region is represented, between the TEA element (shaded diamond) and AC-coding exons (shaded box). The filled diamonds represent functional AJ segments; the open diamonds represent AJ pseudogenes.

Cell sorting

The following Abs were used for staining: FITC-conjugated anti-CD25 (7D4), PE-conjugated anti-CD4 (GK1.5) and anti-CD44 (IM7), biotin-conjugated anti-B220 (RA3-6B2), anti-CD8 (53-6.7), anti-CD3 (2C11), anti-CD11b (M1/70), anti-I-A^b (25-9-17), and anti-TCR (H57-597). All the Abs and PE-conjugated streptavidin were purchased from BD PharMingen (Le Pont de Claix, France).

Single-cell suspensions of CD3^5/5 thymocytes were prepared and incubated with the indicated Abs, as previously described (40). CD25⁺ thymocytes were electronically sorted on a FACStar^Plus (BD Biosciences, San Diego, CA) using CellQuest software. The purity of sorted cells, assessed by reanalysis, was >99%.

Detection of TCRAD locus rearrangements

Genomic DNA was extracted and amplified using primers situated downstream of different AJ gene segments (AJ56, AJ48, AJ27, or AJ23) in combination with primers specific for the ADV2, ADV7, ADV8, or ADV10 family genes (CDNAU, ADV7UP, V8L, or ADV10UP, respectively). A control PCR was performed on the AC region using NM78 and MTCADO2 primers. The sequence of the primers and the location of AJ-specific oligonucleotides used to amplify ADV-AJ rearrangements are shown in Fig. 1. Multiplex PCR were performed with the Expend High Fidelity PCR system (Roche Diagnostics, Meylan, France), as follows: 5 min at 94°C, followed by 26 cycles consisting of 1 min at 94°C, 1 min at 58°C, 6 min at 72°C, and finally 10 min at 72°C. With 26 cycles, the PCRs were in the linear phase of amplification (data not shown). PCR products were migrated on 1.6% agarose gels and probed by Southern blot hybridization. The probes used to detect the TCRA rearrangements were specific of the AJ gene segments (AJ56p, AJ48p, AJ27p, AJ23p). Each of these oligonucleotides was first used individually to probe ADV2-AJ PCR products amplified from wt thymocyte DNA. In each case, only the expected were revealed. Thereafter, AJ56p, AJ48p, AJ27p, and AJ23p were used as a mix to probe ADV-AJ PCR products. The NM78/MTCADO2 PCR products were probed with the C2 primer.

To clone TCRA rearrangements using the AJ56, AJ48, AJ27, and AJ23 segments, DNA from CD3-deficient thymocytes was amplified by PCR using CDNAU or ADV7UP in combination with the AJ56, AJ48, AJ27, and AJ23 primers, respectively. For c^-/- x pT^-/- thymi, a second nested PCR was performed on the CDNAU/AJ48, CDNAU/AJ27, and CDNAU/AJ23 products obtained above to produce more material. For these reactions, PCR were performed with the AJ48, AJ27, or AJ23 primers in combination with the CDNAU2-Bam primer. The conditions used for the PCR were 5 min at 94°C, followed by 35 cycles consisting of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C, and finally 10 min at 72°C.

Cloning and sequencing

The PCR products from E^-/- x TCR^-/- mice were cloned in the EcoRI and BamHI sites of pBlueScribe KS^- (Stratagene, La Jolla, CA). AC-positive clones were sequenced using the Thermo Sequenase premixed cycle sequencing kit (Amersham, Les Ullis, France) and analyzed on a Vistra 725 DNA Sequencer (Vistra Systems; Molecular Dynamics, Bondoulfe, France). Rearrangements amplified from CD3-deficient and c x pT-double-deficient mice were ligated in the pSTBlue-1 vector using the Perfectly Blunt Cloning kit (Novagen, Madison, WI). ADV2- or ADV7-positive clones were sequenced by Genome Express S.A. (Grenoble, France). ADV and AJ gene segments were identified by comparison with the published sequences (22, 23).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Detection of numerous TCRA gene rearrangements by multiplex PCR assay

To analyze the TCRA gene rearrangement status at the DNA level, we designed a PCR assay allowing amplification of ADV genes rearranged to multiple successive AJ genes. This multiplex PCR assay is performed by using ADV-specific primers in combination with primers located downstream of different AJ gene segments (AJ56, AJ48, AJ27, and AJ23), as shown in Fig. 1B. The PCR products were then resolved on an agarose gel, transferred onto a nylon membrane, and hybridized to a mix of AJ-specific probes (AJ56p, AJ48p, AJ27p, AJ23p), as described in Materials and Methods. We chose AJ segments located in the hot spot (HS) 1 (AJ56 and AJ48), and in the HS2 (AJ27 and AJ23). These HS could represent entry points for recombination used early and late during development, respectively (40, 49, 50, 51). Wild-type thymus DNA was amplified using primers specific for the ADV2, ADV8, and ADV10 family members in combination with the four AJ primers. At least four rearrangements were easily detected in each reaction (Fig. 2). The same pattern is observed when an ADV-specific primer is used to probe amplified ADV-AJ rearrangements in place of the mix of AJ-specific probes (data not shown). The use of probes specific for AJ59, AJ51, AJ31, and AJ27, individually, and longer resolution of PCR products on agarose gels showed that the bands in the upper part of the gel are true TCRA gene rearrangements, with the exception of the top upper band in the AJ56 lane (Fig. 2 and data not shown). AJ60, AJ59, AJ51, AJ29, and AJ25 genes, described as pseudogenes, were never found, whereas rearrangements using the AJ61 pseudogene were. No rearrangements were found in RAG-2^-/- mice, as expected (Fig. 3). In our assay, the DNA input is 200 ng per PCR, or 800 ng for the set of four reactions. Cells contain 6 pg DNA. Thus, with a set of four PCR, we analyze ADV-AJ rearrangement in 1.3 x 10⁵ cells. To estimate the sensitivity of our assay, titration experiments were performed. DNA prepared from TCR⁺ SP wt thymocytes was serially diluted in RAG-2^-/- thymocytes/DNA and a constant amount of these dilutions (200 ng per PCR) were used as matrix. The PCR products were then resolved on an agarose gel, transferred onto a nylon membrane, and probed with the mix of four radiolabeled AJ probes, as described. ADV2-AJ rearrangements could be detected when as little as 0.3% TCR⁺ SP thymocyte DNA is present in the matrix (data not shown), which corresponds roughly to 400 cell equivalents (0.3% of 1.3 x 10⁵ cells).

View larger version (45K): [in this window] [in a new window]   FIGURE 2. Amplification of TCRA gene rearrangement from thymocyte genomic DNA. Thymic DNA from wt mice was amplified using ADV10 (A)-, ADV2 (B)-, and ADV8 (C)-specific primers in combination with primers located downstream of the AJ56, AJ48, AJ27, and AJ23 genes. The AJ primers used are indicated above each lane. Each of these amplifications was probed with a mix of radioactively labeled primers specific for the AJ56, AJ48, AJ27, and AJ23 genes, respectively. A, The identity of rearranged AJ genes is indicated. It was determined by comparing the migration distance of the bands with the expected size for a rearrangement, deduced from the AJ region sequence (MUSTCRA, GenBank accession no. M64239). C, The amplification yields three bands for each AJ segment rearranged (visible only for low m.w. products), because we use an ADV8-specific primer located in the first exon of ADV8 family members, which differ from each other by the size of the intron separating the two exons. *, Nonspecific signal.

View larger version (84K): [in this window] [in a new window]   FIGURE 3. TCRA genes are rearranged in CD35/5 mice. A, DNA from CD35/5 thymocytes on the BALB/c (TCRAD haplotype a/a) or C57BL/6 (TCRAD haplotype b/b) background and from RAG2-/- and wt thymocytes was amplified by multiplex PCR and probed as described in Fig. 2 legend, using the ADV10-specific primer (left panel). B, DNA from sorted CD35/5 CD25+ thymocytes, and from CD35/5, RAG-2-/-, and wt thymi was amplified as in A, using the ADV2-specific primer (left panel). A band migrating just above the one corresponding to AJ27 rearrangement is observed in the AJ27 lane of RAG-2-/-. This band was shown by sequencing to correspond to a nonspecific amplification of a region encompassing the unrearranged ADV2S6 gene (data not shown). A and B, the AJ primers used are indicated above each lane. To estimate the DNA loading, a control PCR was performed using AC-specific primers (right panels).

CD3

c

pT

TCRA genes are rearranged in CD3^5/5 mice

In CD3^5/5 mice, T cell differentiation is blocked at the DN3 stage. In a previous study, we were unable to detect rearranged TCRA gene transcripts in these mice (39), in accordance with previously published results (15). However, rearranged TCRB gene transcription is reduced in CD3^5/5 mice (15) and similarly, TCRA transcription could have been reduced to undetectable levels. We therefore performed multiplex PCR assay to determine whether the absence of rearranged TCRA transcripts in CD3^5/5 mice results from an absence of recombination at the DNA level.

DNA from CD3^5/5, RAG-2^-/-, and wt thymi was amplified to identify ADV10 family member rearrangement (Fig. 3A). In CD3^5/5, but not RAG-2^-/- mice, several bands were detected, indicating the presence of TCRA rearrangements. The patterns of utilization were not identical between two CD3^5/5 mice, but were quite diverse in each case (detection of 8 and 10 bands in CD3^5/5 of 20 possible in wt mice). AJ segments located in the HS2 are used, indicating that the entire AJ region is accessible for recombination in DN thymocytes. Finally, TCRA rearrangements are detected in CD3e^5/5 indifferently under either a BALB/c or a C57BL/6 background.

The presence of TCR gene rearrangement in immature B cells was previously described (52). To ensure that the TCRA gene rearrangements observed in CD3-deficient thymi were produced in T and not B cells, DNA from sorted CD3^5/5 CD25⁺ thymocytes (purity >99%) was analyzed. Again we could detect various TCRA gene rearrangements using ADV2 family members (Fig. 3B). We detect 10 ADV2-AJ rearrangements in 1.3 x 10⁵ CD25⁺ DN CD3^5/5 thymocytes. Our assay allows us to determine the rearrangement status of 20 AJ segments of 60. One can then extrapolate that for the complete AJ region, 3 times as many rearrangements are potentially present. Similarly, because 10 rearrangements are detected for the ADV2 family, it can be estimated that 22 times as many rearrangements could be detected if one were to test all the ADV families. From these extrapolations, it results that 10 x 22 x 3 = 660 rearrangements are potentially present in 1.3 x 10⁵ CD25⁺ DN CD3^5/5 thymocytes, which indicates that 0.5% of these cells do have rearranged TCRA genes.

TCRA rearrangements found in CD3-deficient mice are diverse

To characterize the molecular nature of TCRA rearrangements found in CD3-deficient mice, TCRA rearrangements using either the ADV2 or ADV7 family members, rearranged to AJ56, AJ48, AJ27, or AJ23, were cloned and sequenced. Sequencing showed that the ADV2 and ADV7 members used in the CD3^5/5 mouse analyzed are diverse (Fig. 4). Multiple junctions were identified, which present nucleotide addition and/or deletion. Insertions of >13 nucleotides at the ADV-AJ junction are observed for three sequences. One of these is clearly DD2 gene segment. In three other junctions, a stretch of four or five nucleotides matching DD1 or DD2 sequence can also be identified. Rearrangement of a DD segment to ADV and AJ segments was found in other studies conducted in wt animals (unpublished results). Finally, analysis of the sequences indicates that the thymocytes were not selected for in-frame TCRA rearrangements, as expected because of absence of surface expression.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Sequence of TCRA gene rearrangements from CD35/5 mice. TCRA gene rearrangements using AJ56, AJ48, AJ27, or AJ23 segments in combination with ADV2 or ADV7 family members were amplified, cloned, and sequenced from CD35/5 thymocytes. The extremities of ADV and AJ genes, the DD1 or DD2 genes, and the N and P nucleotides are shown. ADV and AJ segments identified are indicated. P nucleotides are written in small letters. Sequences were considered as DD1 or DD2 genes in the case of homologies of at least four nucleotides. In-frame (+) and out-of-frame (-) sequences are indicated.

5/5

TCRA genes are rearranged in absence of the c chain and pT

Overlapping signals derived from growth factors, cytokines, and Ag receptors are essential for early T cell differentiation. Mice deficient for the common cytokine receptor -chain (c) and pT exhibit thymic hypoplasia (4 x 10⁴ thymocytes) and a complete block of development at the CD44⁺25⁺ stage. Despite this low cellularity and early arrest, limited TCRB gene rearrangements could nonetheless be found in total thymic DNA from these mice (45). Thus, we next analyzed whether ADV2-AJ recombination is also present.

Thymic DNA from c x pT double-deficient mice was amplified using the AJ primers in combination with the ADV2 family-specific primer (Fig. 5). A clear signal was obtained with the two of three c^-/- x pT^-/- mice shown. Although certain mice did not present TCRA rearrangements involving ADV2 family members, a signal was obtained using either ADV7- or ADV10-specific oligonucleotides (Fig. 5 and data not shown). Only one sample of five tested negative for both ADV2 and ADV10 (Fig. 5 and data not shown), a result that does not preclude the presence of rearrangements involving other ADV families.

View larger version (48K): [in this window] [in a new window]   FIGURE 5. TCRA gene rearrangements in c-/- x pT-/- thymi. DNA from c-/- x pT-/-, RAG-2-/-, and wt thymi was amplified by multiplex PCR performed as in Fig. 4 using the ADV2-specific primer (left panel). The AJ primers used are indicated above each lane. The lanes corresponding to the c-/- x pT-/- and RAG-2-/- mice were exposed longer for better visualization. The control PCR was performed as in Fig. 2 (right panel).

View larger version (43K): [in this window] [in a new window]   FIGURE 6. Sequence of TCRA gene rearrangements from c-/- x pT-/- mice. TCRA gene rearrangements using AJ48, AJ27, or AJ23 segments in combination with ADV2 family members were amplified, cloned, and sequenced from c-/- x pT-/- thymi. The extremities of the ADV and AJ genes, and the N and P nucleotides are shown. ADV and AJ segments identified are indicated. P nucleotides are written in small letters. In-frame (+) and out-of-frame (-) sequences are indicated.

TCRA-rearranged transcripts are expressed in E x TCR double-deficient mice

To date, no TCRA transcripts have been found in CD3^5/5 mice (39). The results presented in this study show nonetheless that TCRA genes are rearranged in these mice, revealing an unsuspected level of transcriptional regulation that could be controlled either by the pre-TCR, or by CD3 alone, as CIC.

To investigate these possibilities, we analyzed TCRA gene transcription in E x TCR double-deficient mice. In these animals, thymocytes are blocked at the DN3 stage of development. Absence of TCRB gene rearrangement and absence of TCR chain expression preclude pre-TCR, TCR, and TCR expression (47, 48), but CIC should not be affected. Rearranged TCRA transcripts using ADV2, ADV3, and ADV7 family members were analyzed by RT-PCR (Fig. 7). Transcripts were detected for the three ADV families studied in E^-/- x TCR^-/- and wt mice. As expected, no rearranged transcripts were detected in CD3^5/5 and RAG2^-/- mice. Thus, rearranged TCRA genes can be transcribed in DN thymocytes in absence of pre-TCR and TCR expression. Cloning and sequencing of ADV2-AC amplification products revealed a high proportion of ADV-DD-DJ rearrangements spliced onto the AC segment (data not shown), as described in mice bearing the same mutation in the DC segment and wt mice (53). Nonetheless, bona fide ADV-AJ-AC transcripts (15–35%) were also identified, and sequencing showed utilization of different AJ gene segments and ADV2 family members in the two E^-/- x TCR^-/- mice analyzed (data not shown). Thus, rearranged TCRA genes can be transcribed in immature DN thymocytes, in absence of pre-TCR and TCR expression. Together with the absence of TCR chain transcripts in CD3^5/5, this result strongly argues in favor of a role for CIC complexes in the control of this transcription.

View larger version (70K): [in this window] [in a new window]   FIGURE 7. Rearranged TCR genes are transcribed in E-/- x TCR-/- mice. Rearranged TCRA transcripts from 4-wk-old CD35/5, E-/- x TCR-/-, RAG-2-/-, and C57BL/6 mice were amplified by RT-PCR using primers specific for three different ADV families in combination with an AC-specific primer. A control PCR was performed using Thy-1-specific oligonucleotides. The lane "no cDNA" corresponds to the same PCR amplifications performed without cDNA as a negative control. The PCR products were probed with radioactively labeled primers specific for the AC region or Thy-1.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Until now, most of the available data indicated that TCRA gene rearrangement only begins after pre-TCR signaling, at the DN/DP transition. Our results now show that ADV-AJ recombination is not so bluntly regulated, but is rather modulated during thymocyte differentiation. This event already takes place in a fraction of thymocytes during early DN stages, before being induced in most of the cells while they mature to the DP stage. In CD3-deficient animals, we estimated that 0.5% of immature thymocytes do have rearranged TCRA genes. This low abundance and the randomness of ADV-AJ recombination probably explain why different patterns of ADV-AJ rearrangements are observed in different CD3^5/5 mice. However, this figure may be higher in wt mice, as we cannot exclude that CD3, although not necessary, participates in the regulation of gene accessibility when expressed. In addition, it must be stressed out that this number is only an estimate, since 1) all ADV gene families and all AJ segments may not rearrange to the same extent (it is known, for example, that ADV2 genes are used in 12% of peripheral T cells), and 2) in our calculation, each PCR product corresponds to only one rearrangement, whereas ADV-AJ rearrangements are diverse (Fig. 4).

In pT^-/- mice, 40% of the few DP cells that can develop express an intracellular TCR chain. The proportion of -selected cells is nearly null in pT^-/- x TCR^-/- mice, but raises to 95% in pT^-/- x TCR^-/- animals, indicating that pT^-/- thymocytes that are not selected differentiate under the influence of T cells, and that early expression of a TCR compensates for the absence of pre-TCR and promotes DN/DP progression and selection (43). We previously found that rearranged TCRA genes expressed in pT^-/- mice are diverse, as in wt mice (40). However, in this study, we could not formally rule out that the TCR chain transcripts sequenced were produced in those DP thymocytes that did differentiate under the influence of ⁺ thymocytes. The results presented in this work demonstrate for the first time, at the DNA level, the occurrence of diverse TCRA gene rearrangement in a small subpopulation of immature pre-TCR-deficient thymocytes. In addition, we show that TCRA gene rearrangement is independent not only of pre-TCR expression, but also of expression of either the CD3 complex, as CIC or associated with a TCR, or c-containing cytokine receptors. Altogether, these findings provide strong support to the hypothesis that TCRA rearrangements produced in DN thymocytes are responsible for DP thymocyte development in absence of pT, at least partially in pT^-/- animals, and probably totally in pT x TCR double-deficient mice. Furthermore, the low abundance of thymocytes having rearranged TCRA genes probably explains why DP thymocyte number in pT^-/- mice is only 2% of the wt level (14). Because all of the T lymphocytes produced in pT^-/- x TCR^-/- animals use a TCR chain generated in early thymocytes, these mice constitute an ideal model to determine whether T lymphocytes using these chains fully participate in all immune functions, or whether they constitute a T lymphocyte subset with special homing or functional properties. In a normal situation, the contribution of this differentiation pathway to T lymphocyte development is probably low compared with the pre-TCR pathway. Indeed, we must keep in mind that, in wt mice, the majority of TCRA rearrangements take place in DP cells and, even if our results show that the pre-TCR is not required for TCRA gene rearrangements in DN thymocytes, pre-TCR-induced proliferation vastly increases the number of cells that will rearrange their TCRA genes.

TCRA gene rearrangement in early thymocytes does, however, impact T lymphocyte development. In adult mice, ⁺ and ⁺ thymocytes are generated from the same precursor population (2). Numerous studies have tried to decipher the mechanisms responsible for the dual fate of these precursors, and two major models have emerged (see Ref. 3 for review). In the separate lineage (or stochastic) model, lineage decision is imposed first, and TCR genes rearrange later. If the TCR isotype then expressed matches the lineage (e.g., TCR in thymocytes and pre-TCR in thymocytes), the cell survives and differentiates. Otherwise, it dies. In the competitive rearrangement (or instructive) model, the choice is dictated by the nature of the TCR isotype expressed, either or pre-TCR, that transduces different signals to the developing thymocytes. In both models, only the TCR and the pre-TCR receptors are thought to be instrumental for cell fate, differentiation, and/or survival. The finding that TCRA genes can be rearranged and expressed in the same DN2/DN3 population(s) as TCRD, G, and B genes now offers the distinct possibility that an TCR can also play a role in lineage commitment, at least in two ways. First, it gives developing thymocytes a new pathway to become an T lymphocyte, either by rescuing committed precursors from death (stochastic), or by delivering differentiation signals (instructive). Second, it reduces the probability for a precursor to become a T lymphocyte. Indeed, the unique genomic organization of the TCRAD locus is such that any ADV-AJ rearrangement event will excise TCRD genes from the chromosome. The progenitors will therefore be allowed only one attempt to rearrange TCRD genes on the remaining allele, and have only one chance of three to succeed, compared with five of nine if both alleles are available. Unlike thymocyte development, which includes a phase of intense proliferation, T lymphocyte development is linear. Thus, in both cases, and irrespective of the lineage commitment model considered, the net result of TCRA gene rearrangement in early thymocytes will be a depletion of T lymphocytes. However, it should be noted that occurrence of ADV-AJ recombination in a T cell progenitor does not per se preclude its development toward the lineage (44). Finally, in addition to their biological significance, our findings do have another important consequence regarding the study of lineage commitment: since TCRA rearrangements can be detected as early as the DN2 stage of differentiation, they can no longer be considered a marker for divergence operating after the pre-TCR.

TCRA rearrangements found in pT^-/- mice are normal (40). Recombination of TCRA genes also takes place in TCR^-/- animals. In this study, in contrast to previously published studies conducted in CD3^5/5 mice (15, 54), we show that TCRA genes are also rearranged in absence of CD3 complex expression. Malissen and coworkers (15, 39, 54) might have failed to detect TCRA gene rearrangement in these animals because they did use an ADV8-specific oligonucleotide and, in our hands, this family seems to be rearranged at a very low level, if at all, in CD3-deficient mice (N. Pasqual, S. J. C. Mancini, S. M. Candéias, and E. Jouvin-Marche, unpublished results). Despite the presence of TCRA rearrangements, TCR chain transcripts were not detected in CD3^5/5 mice (Fig. 7), indicating that either the pre-TCR, the TCR, or CIC complexes are required for the transcription of rearranged TCRA genes. In contrast, TCRA transcripts were found in E^-/- x TCR^-/- mice, which lack conventional pre-TCR and TCR expression. An alternative pre-TCR, composed of a TCR chain associated with pT and the CD3 complex, was recently described (55). However, its expression would be expected to induce at least a low level of DP thymocyte differentiation. As no DP population was observed among E^-/- x TCR^-/- thymocytes, TCR/pT complex expression was ruled out (48). It seems therefore that no clonotypic TCR or pre-TCR can be assembled in E^-/- x TCR^-/- mice. One can argue that ADV-AJ rearrangements in these mice result from perturbations in the local control of accessibility, because of the Neo cassette insertion near E and blocking element -1. Even though, any ADV-AJ rearrangement event will excise the Neo cassette from the TCRAD locus, and identical pattern of E occupancy was found in E^-/- x TCR^-/- and RAG-1^-/- thymocytes (56). Therefore, transcriptional control of rearranged TCRA genes in E^-/- x TCR^-/- thymocytes is as in nonmanipulated animals. The presence of rearranged TCR chain transcripts in E^-/- x TCR^-/- mice then suggests that the absence of TCRA transcripts in CD3^5/5 thymocytes results from CD3 deficiency. Thus, the CD3 complex is not necessary for induction of TCRA gene rearrangement, but rather seems to be involved in the transcriptional control of rearranged TCRA genes, as previously suggested in the case of TCRB genes (57).

It is clear in our study that TCRA rearrangements are both pre-TCR and TCR independent. The CD3 complex is also dispensable, as CIC. Furthermore, although it was shown that the pre-TCR provides c-independent signal allowing the T cell differentiation in c^- mice (45), the induction of TCRA gene rearrangement is independent of overlapping c and pre-TCR signals. Thus, none of the main molecules known to regulate thymocyte development are required. What then are the signals responsible for ADV-AJ recombination in early thymocytes? One possibility is that they appear as a byproduct of TCRD gene rearrangement, because of a faulty control of accessibility. The other possibility is of course that TCRA genes are made accessible to the recombination machinery only after a specific signal is delivered to developing immature thymocytes. In the context of the current model of enhancers as accessibility regulators, ADV/AJ gene rearrangement in early thymocytes would suggest that E is already active in these cells and its activation is independent of CD3- and c-derived signals. However, E is dispensable for ADV/AJ recombination. Its inactivation only impairs, but does not abolish TCR gene rearrangement and expression (31, 32), showing that at least one other yet unidentified element is able to promote ADV-AJ recombination. Whether this element is responsible for ADV-AJ rearrangement in immature thymocytes must await its identification and elucidation of its regulation. In any case, TCRA gene rearrangements are found as early as the CD44⁺25⁺ stage of development, at the time of TCRD gene rearrangement, in mice in which T lymphocyte differentiation is blocked at an immature stage because of genetic inactivation of different signaling pathways. It remains to be formally demonstrated whether the same phenomenon also takes place during normal thymocyte development in wt mice.

	Acknowledgments

 
We thank N. Pasqual for communication of unpublished results; V. Collin for excellent cell sorting; E. Borel, V. Giraud, and S. Bama for animal care; and Dr. R. Ceredig for critical reading of this manuscript.

	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, Unité 548, and Université Joseph Fourier. S.J.C.M. was supported by a predoctoral fellowship from the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche, and by a grant from La Ligue Nationale contre le Cancer.

² Address correspondence and reprint requests to Dr. Serge M. Candéias, Commissariat à l’Energie Atomique-G, Département de Biologic Moléculaire et Structurale/Laboratoire d’Immunochimie, Institut National de la Santé et de la Recherche Médicale Unité 548, 17 rue des martyrs, 38054 Grenoble, Cedex, France. E-mail address: immuno{at}dsvsud.cea.fr

³ Abbreviations used in this paper: DN, double negative; CIC, clonotype-independent CD3 complex; DP, double positive; HS, hot spot; RAG, recombination-activating gene; SP, single positive; wt, wild type.

Received for publication June 29, 2001. Accepted for publication August 15, 2001.

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