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Allelic Exclusion at the TCR Locus and Commitment to Lineage: Different Modalities Apply to Distinct Human Subsets¹

Institut National de la Santé et de la Recherche Médicale Unité 463, Institut de Biologie, Nantes, France

	Abstract

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Expression of a -chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCR locus is unknown and the role of the TCR in lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCR, , and loci in human T cell clones, expressing either the TCR V1 or V2 variable regions, we show that the rate of partial rearrangements at the locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCR chains indicates that a TCR selection process is required for the commitment of T cell precursors to the lineage. Finally, while complete TCR rearrangements were observed in several V2 T cell clones, these were seldom found in V1 cells. This suggests a competitive / lineage commitment in the former subset and a precommitment to the lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the V1 and V2 subsets.

	Introduction

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Even before the mechanism of TCR and Ig gene rearrangement was discovered, the clonal distribution of Ag receptors was considered as a prerequisite for the control of acquired immune responses. It was later shown that rearrangements on one allele regulate rearrangements on the other, and that this control is linked to the expression of functional TCR or IgH chains. This translates into a block in endogenous TCR or IgH rearrangements in Ig H chain or TCR transgenic mice (1, 2). Analyses of TCR and Igµ rearrangements in normal lymphocytes showed that the production of a functional chain inhibits further rearrangements on the other allele and that rates of partial rearrangement are constant and consistent with the predicted theoretical values (3, 4). This mechanism of allelic exclusion has been thought to be the process responsible for the maintenance of clonal distribution of B and T cell Ag receptors. The observation that a functional rearrangement occurs on each allele in a large percentage of the T lymphocyte population and that the locus escapes from allelic exclusion, leading to the expression of two surface TCR, however, called into question the necessity of maintaining monospecificity (5, 6). Allelic exclusion then rather seemed to be the consequence of a particular mode of rearrangement arrest than an active monospecificity control mechanism. Allelic exclusion at the or µ locus, in either B or T lymphocytes, involves surface expression of the Ig H chain or TCR chain, associated with a surrogate 5-VpreB or preT chain, in the form of a pre-B cell receptor or pre-TCR, respectively (7, 8). This expression allows for the transduction of a signal that induces cessation of further rearrangements at the TCR or Igµ loci and leads these precursors to the next step of their development (9, 10). Rearrangements of TCR or L chain genes then come into play, allowing surface expression of the functional form of the Ag receptor.

As far as T lymphocytes are concerned, rearrangements occur independently on the two alleles, leading to the completion of rearrangements and to an allelic inclusion at this locus (11, 12). Further development of T cell precursors is then dependent on the specificity of the expressed TCR, which must interact with self-MHC molecules during the different steps of thymic selection. Moreover, this selection process, involving the specificity and the quality of the interaction of the TCR with its ligand, allows T lymphocyte differentiation to proceed with, notably, the acquisition of the CD4⁺ or the CD8⁺ phenotype (13, 14).

Concerning T lymphocytes, rearrangements at the and loci occur at the same developmental stage in T cell precursors, as opposed to rearrangements at the and loci (15). The existence of an invariant surrogate chain allowing independent expression of one of the TCR chains has never been shown and the mechanism of a thymic selection process allowing for development to proceed remains controversial (16, 17). With regard to mechanisms responsible for the cessation of rearrangement, it has been shown that the locus is subjected to allelic inclusion (18). The situation is, however, much less clear for the TCR locus. An analysis done on murine T hydridomas indicates that the percentage of cells showing two functional TCR rearrangements is consistent with an allelic inclusion at this locus. In these same hybridomas, the observed incompletion of TCR rearrangements, however, suggests that the occurrence of rearrangements is controlled, as opposed to the situation encountered at other loci subjected to allelic inclusion where rearrangement completion is observed (19). Consistent with this assumption, the phenotypic analysis of T lymphocytes in humans, using Abs specific for variable regions, allowed the detection of low percentages of cells expressing two TCR chains (20). The absence of complete TCR rearrangements in an allelic inclusion context in the mouse, as well as the low rate of T lymphocytes expressing two chains in humans, led us to wonder about the nature of the processes that control rearrangements at that locus. To address these questions, we characterized the status of the rearrangements at the and loci, as well as the reading frame of complete rearrangements, in a large sample of human peripheral blood T lymphocytes. A model for the mechanism that regulates cessation of rearrangements at the locus, consistent with results found in mice and humans, is proposed.

	Materials and Methods

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Generation of T cell clones

PBL were isolated by centrifugation on a Ficoll gradient of heparinized blood from one healthy individual. PBL were incubated with the TCR V region-specific mAb 389 (anti-V2) (21) or R9 (anti-V1C) (22) and sorted on magnetic beads coated with sheep anti-mouse Ig (Dynal, Oslo, Norway) as previously described (23). Bead-adherent 1-sorted cells were amplified by a polyclonal stimulation step before cloning. T cell clones were restimulated at 4-wk intervals.

Flow cytometry analysis

Clones were phenotyped by indirect immunofluorescence using TCR V-specific mAb 389 (anti-V2) and 360 (anti-V9) (21) for V2⁺ clones and 23D12 (anti-V2,3,4) (24), 56.3 (anti-V5,3) (25), R4.5 (anti-V8), R9 (anti-V1C), and p11-10b (anti-V3) for V1⁺ clones (22) along with FITC-conjugated rabbit anti-mouse IgG antiserum (BioAtlantic, Nantes, France). Cells were analyzed on a FACScan flow cytometer using the CellQuest software (BD Biosciences, Grenoble, France). 23D12 and 56.3 mAb were kindly provided by Dr. D. Kabelitz (Institute of Immunology, Christian-Albrechts University, Kiel, Germany).

Determination of the rearrangement status at the and loci

Rearrangement status at the locus. To determine the locus rearrangement status of the nonexpressed allele, two multiplex PCR were performed. The first one, termed the V-D/D-J PCR, combined the amplifications of a 341-bp noncoding genomic region between V2 and D1 (VD PCR product), a 266-bp noncoding genomic region between D3 and J1 (DJ PCR product), and a 226-bp C-positive control (Fig. 1). The second one, termed D-D PCR, combined the amplifications of a 303-bp noncoding genomic region between D1 and D2 (D1–2 PCR product), a 259-bp genomic noncoding region between D2 and D3 (D2–3 PCR product), and a 226-bp C-positive control. Each PCR experiment was performed on the Burkitt’s lymphoma Daudi cell line (Daudi) as a positive control. Negative controls were performed using water and the A22.19 T cell clone that lacks both the locus and the V11 downstream region. The VD/DJ PCR allowed the detection of partial VD or DJ rearrangements and the DD PCR detected the partials DD rearrangements. In case of rearrangement (by inversion), a VD amplification fragment remains at the V3 element due to its opposite transcriptional orientation. Thus, to detect potential rearrangements at the V3 element, a specific V3(D)J PCR was performed. Each PCR was performed directly on cell lysates: 10⁵ cells were washed once in PBS, resuspended in 15 µl of water, and incubated for 10 min at 100°C in a PCR tube. The final volume in each tube was then adjusted to 10 mM Tris-HCl (pH 9), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl₂, 1.25 U Taq DNA polymerase (Promega, Madison, WI), and 0.25 µM of each primer in a final volume of 50 µl. The amplification was performed in a 96-well thermocycler (PTC-100; MJ Research, Cambridge, MA) according to the following scheme: 94°C, 5 min; 60°C, 1 min 30 s; and 72°C, 1 min followed by 35 cycles: 94°C, 1 min; 60°C, 1 min 30 s; and 72°C 1 min. PCR products were analyzed on a 2% ethidium bromide-containing agarose gel.

View larger version (33K): [in this window] [in a new window]   FIGURE 1. Determination of the , , and loci rearrangement status. A, Schematic representation of the , , and loci. HindIII (H) and EcoRI (R) restriction sites and position of probes at the and loci are shown. The nonfunctional and pseudo-V elements are indicated by filled rectangles. The position of the PCR products obtained with multiplex PCR using VJ, VD/DJ, and DD primers appear under the corresponding loci. EcoRI and HindIII digests were successively hybridized with C, D1, and D2 probes to unambiguously distinguish between partial DJ and complete VDJ rearrangements. pH 60 and C probes were hybridized with EcoRI and HindIII digests, respectively. A quantitative analysis of rearranged and germline bands on EcoRI/pH 60 and C/HindIII Southern blot have been performed to unambiguously determined whether rearrangement involved C1 or C2. B, Representative results obtained with multiplex PCR using VJ, VD/DJ, and DD primers are shown in the upper right panel. For each multiplex PCR, negative controls, i.e., without cells (no cell) or using an T cell clone with both alleles rearranged (A22.19), were included. The Burkitt’s lymphoma Daudi cell line (Daudi), which keeps both the and the locus in germline configuration, was used as positive control. In each multiplex PCR, an internal positive control, i.e., a C PCR product, was included (C). C, Reading frame analysis of rearranged genes in five CS1 clones is shown in the lower right panel. The length of each rearranged gene was assessed by primer extension and electrophoresis through a polyacrylamide-denaturing gel. The obtained fragments were compared with a ladder composed of in-frame rearrangements found in clones from our sample. The same approach was used for rearrangements.

Rearrangement status at the locus.

Analysis of TCR and rearrangement reading frame

Clones with both alleles rearranged were further characterized by PCR with primers specific for VI, V1P, V5P, V6P, V7P, V8, V9, V10, V11, and a mixture of JP, JP1, JP2, and J1/2 reverse primers for rearrangements. The rearrangements were characterized using primers specific for V1, V2, V3, and V elements, the often rearranged at locus (26), and a mixture of J1, J2, J3, and J4 reverse primers. The conditions were the same as those described for multiplex PCR. For each specific PCR, the V primer was used at 1 µM and each J-specific reverse primer at 0.25 µM.

Primer extension

Bands of interest from VI-J, V9-J, V10-J, V11-J, V1-J, and V2-J PCR were cut out from agarose gels, purified using the GFX PCR DNA and the Gel Band Purification kit (Amersham Pharmacia Biotech, Piscataway, NJ), and ethanol-precipitated. DNA pellets were resuspended in 10 µl of 1x Sequenase Rxn buffer (Amersham Pharmacia Biotech) with 1 µM of each of the appropriate purified nested primers specific for the VI-J, V9-J, V10-J, V11-J, V1-J, and V2-J PCR fragments. Extension was performed according to the previously described protocol for manual sequencing, but for the elongation mix, from which ddNTP were removed. The DNA ladders were established using clones that only displayed functional rearrangements. Extension products and ladders were separated on a 6% urea/6% acrylamide gel. Results were visualized on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) after a 6-h exposure.

Southern blot analysis

Fifteen micrograms of genomic DNA from the clones were digested with EcoRI and by HindIII restriction enzymes and size fractionated by electrophoresis on a 0.7% agarose gel. Gels were blotted onto Hybond N⁺ transfer membranes (Amersham Pharmacia Biotech) using a vacuum blotting system (LKB-Pharmacia, St. Quentin en Yvelines, France). Successive hybridizations and washes were performed as previously described (27). ³²P-Labeled multiprimed probes were prepared with the Rediprime II labeling system (Amersham Pharmacia Biotech). Hybridizations were detected using a PhosphorImager (Molecular Dynamics).

Hybridizations of EcoRI or HindIII digests with the C probe allowed the detection of rearrangements in the C1 and C2 region, respectively. Successive hybridizations of HindIII and EcoRI digests with D1 and D2 probes enabled us to distinguish VDJ from D1J1, D1J2, and D2J2 fragments.

Hybridization of the pH 60 probe (28) with EcoRI digests allowed the characterization of VJ rearrangements on J1 or J2 elements. Rearrangements involving other J elements (i.e., JP1, JP, and JP2) were not detected on EcoRI digests with pH 60 probe. The C probe on HindIII digests allowed the detection of the remaining constant region at the C locus (the C1 region is deleted when rearrangements occur in the C2 constant region) due to a HindIII RFLP in the C1 and C2 regions. To determine whether rearrangement involved C1 or C2, rearrangement patterns detected by EcoRI/pH 60 and HindIII/C Southern blots were quantified using the Image Quant software (Molecular Dynamics) and the results were compared with those from the V-specific PCR and cytofluorometric analyses.

Statistical analysis

All correlation analyses were evaluated using Fisher’s exact test and the SAS software (Cary, NC).

Primers

Multiplex PCR primers. In all multiplex PCR, a C-positive control amplified with SC (5'-AGTCAGCCTCATACCAAACCATCC-3') and ASC (5'-CGTGTTGAACTGAACATGTCACTG-3') was included.

V-D/D-J PCR. VD PCR product was amplified with VDS (5'-ATCATAGCTCACTGCAGCCTCACA-3') and VDAS (5'-ACTGACCATGTCTATCACCACAA-3') and DJ PCR product was amplified with SDJ (5'-TCAGGAGACAAACACAGCAAGCAG-3') and ASDJ (5'-GAAATTCCGTATGAGGTGGCTCAT-3').

D-D PCR. D1–2 PCR product was as follows: SD1–2 (5'-TTTTACCAACATCTTGCCCACATT-3') and ASD1–2 (5'-CCATCCCAATACCCCTGAAACTC-3'); D2–3 PCR product was as follows: SD2–3 (5'-GCACACCGTCACTTCCACTCATA-3') and ASD2–3 (5'-TGAGCTCCTTCTAGTGCTGGTGAC-3').

V-J PCR. VJ PCR product SVJ (5'-GGCATCCATCCAAGGCTTTAGCAG-3') and ASVJ (5'-TGACAACTCGAACACTGTGGTGCC-3') were used.

Characterization of and rearrangements

V-specific PCR. VI (5'-GYTKTTCCCAYTGCAGCCAGTCAG-3'), specific for all V elements of the VI group, V1P (5'-CAGGAGGGGAAGGCCCCACAGT-3'), and V5P6P7P (5'-AGGMGGGAAGRCCCCACAGCA-3'), specific for the pseudo-V elements of VI group, V9 (5'-TGTCCATTTCATATGACGGCACTG-3'), V10, and V11 (5'-CATWCACTGGTACYGGCAGAAACC-3') were used. Each V PCR was performed with a specific V sense and a mix of JP (5'-CTTTGTTCCGGGACCAAATACC-3'), JP1 (5'-TACTATGAGCTTAGTCCCTTCAGC-3'), JP2 (5'-TACTATGAGCCTAGTCCCTTTTGC-3'), and J1/2 (5'-MAGTGTTGTTCCACTGCCAAAGAG-3') reverse primers.

V-specific PCR. The previously described V1, V2, V3, and V PCR primers (26) were used (50 pM) in combination with a mix of J1 (5'-CTGCCTCCTTAGATGGAGGATGC-3'), J2 (5'-TTCAGGACTACGCTACAACAACCC-3'), J3 (5'-TTATAATGCAAACTGGATTGCAGC-3'), and J4 (5'-CCTACTGGAGCACTGCTTCCTAAC-3') reverse primers.

Primer extension. The V or V-specific PCR amplification products VI-J, V9-J, V10-J, V11-J, V1-J, and V2-J were amplified using the following corresponding nested primers: TCRGVISEQ (5'-CKKMTACAYCCACTGTACCT-3'), TCRGV9SEQ (5'-GAATCCGGCATTCCGTCAGGC-3'), TCRGV10SEQ (5'-ATGGGTAAGACAAGCAACAAAGTG3-'), TCRGV10–11S (5'-CATWCACTGGTACYGGCAGAAACC-3'), TCRDV1SEQ (5'-GGTCGCTATT-CTGTCAACTTC-3'), and TCRDV2SEQ (5'-CACAATGACTTTCATATACCG-3').

Probes

D1 probe. A 335-bp PCR fragment amplified with 5' DB1S (5'-AAGTCATAGCTTAAAACCCTCCGA-3') and 5' DB1AS (5'-AGAGCCTGGGAGAGACCACCAGAG-3') primers that corresponds to a noncoding region upstream of the D1 element was used.

D2 probe. A 433-bp PCR fragment amplified with 5' DB2s (5'-TGTGATGTGTTGTTATCAACTTCC-3') and 5' DB2AS (5'-GGTTGCATGGAGGTAAGTTTATTC-3') primers that corresponds to a noncoding region upstream of the D2 element was used.

C probe. A 424-bp PCR fragment amplified with TCRBCSONDES (5'-CGAGGTCGCTGTGTTTGAGCC-3') and TCRBCSONDEAS (5'-CATTTTCCCTGGTAGCTGGTC-3') primers was used. This probe hybridized with C1 and C2 elements.

J probe. The pH 60 probe is a 700-bp HindIII–EcoRI genomic restriction fragment from the J1 region (28) which hybridizes with the J1 and J2 regions.

C probe. A 422-bp PCR fragment amplified with TRGEX1S (5'-GTACTGTGCAGCCTATCCTGG-3') and TRGEX1AS (5'-CGATACATCTGTGTTCT TTGTCC-3') primers which hybridizes with the C1 and C2 elements.

	Results

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Selection of T cell subsets and analysis of rearrangements at the TCR, , and loci

This analysis was done on clones from the two most important peripheral T lymphocyte subsets which typically express V1⁺ or V2⁺ TCR. The V2⁺ subset represents 12% of all thymocytes at birth and 60–95% of the peripheral blood T lymphocyte pool in the adult. This increase is attributed to a postnatal amplification process following recognition of recurrent Ags. This translates into a highly restricted junctional and combinatorial diversity (23, 29). Indeed, most V2⁺ lymphocytes express V9 JP C1 TCR chain and share conserved patterns at the VDJ and VJ junctions. The V1⁺ subset represents nearly 65% of all thymocytes at birth. As opposed to V2⁺ lymphocytes, V1⁺ lymphocytes maintain a naive T lymphocyte phenotype in the peripheral blood after birth and express TCR exhibiting a high combinatorial and junctional diversity (30).

To avoid a representation bias due to the amplification of V2⁺ lymphocytes, peripheral lymphocytes from an adult individual were sorted using Abs against V2 and V1 variable regions. Isolated cell lines were cloned by limiting dilution, and rearrangement status at the , , and loci were characterized for each clone obtained. For and rearrangements, the reading frame was determined by PCR-RFLP (Fig. 1). Seventy-nine V1⁺ and 32 V2⁺ T cell clones with a distinct TCR rearrangement pattern were characterized. They are listed in Table I.

View this table: [in this window] [in a new window]   Table I. TCR , , and locus rearrangement status from V1+ and V2+ peripheral T cell clonesa

Incomplete TCR rearrangements in human T cell clones

The percentage of distinct clones exhibiting incomplete rearrangement in the V1⁺ and V2⁺ lymphocyte subsets were 65.8 and 81.6%, respectively (Table II). These rates suggest that there is a more drastic control of recombination at this locus in humans than in mice. In the latter, only 30% of cells exhibit an incomplete rearrangement (19). The rate of incomplete rearrangements detected in our series of clones was of the same order, and in the case of V2⁺ lymphocytes, significantly superior (p < 0,02) to those observed at the locus of T lymphocytes in an allelic exclusion context (Table II). Conversely, the frequency of clones exhibiting two functional rearrangements seems to be comparable to that expected in an allelic inclusion context (4). Among the 20 distinct V1⁺ clones for which the reading frame was determined on each allele, 4 (20%) exhibited two in-frame rearrangements (Table I). The high frequency of clones that exhibited two functional rearrangements and the limited TCR rearrangement completion are not consistent with the established models of allelic inclusion, in which completion of rearrangement is always observed. These observations suggest that recombination-independent processes are involved in the control of rearrangements arrest. We wondered about the possibility that a competitive engagement of T cell precursors toward / lineage could affect the rate of partial rearrangements at the locus. The occurrence of functional rearrangements and expression of a pre-TCR when and rearrangements take place might limit the time window during which T cell precursors have the opportunity to make rearrangements. According to and rearrangement kinetics (31), some precursors that produced a nonfunctional rearrangement after a first trial may be confronted with the concomitant activation of the V to DJ rearrangement at the second allele and V To DJ rearrangement at the locus. At the population level, this would favor cells that successfully produced a functional -chain after the first V to DJ rearrangement trial (i.e., with a partial rearrangement at one allele).

View this table: [in this window] [in a new window]   Table II. Frequency of V1+ and V2+ with a partial rearrangement and comparison to the theoretical frequency expected for allelic exclusion

Influence of TCR rearrangements on the completion of TCR rearrangements

The analysis of the TCR rearrangement status in T lymphocyte clones allows for an estimation of the rearrangement progression at this locus at the time when precursor cells commit to the lineage. In our subset of clones, ordered rearrangements are observed at the and loci: the number of and (i.e., DD to VDJ on the unexpressed allele) rearrangements increase concomitantly (Fig. 2). This suggests that partial DD-type rearrangements would be the first rearrangement events to occur at the locus, then followed by D to J rearrangements or, on rare occasions, V to D rearrangements. Differences in terms of distribution of rearrangements between V1⁺ and V2⁺ subsets (p < 0,01) are observed (Table III). The V2⁺ subset is characterized by a strong proportion of clones exhibiting two alleles in germline configuration (55.2%) as compared with the V1⁺ subset (26.8%). Moreover, although a majority of V1⁺ clones exhibit at least one partially rearranged allele (68.3%), this rearrangement status is only found in one-quarter of the V2⁺ clones (27.5%). Paradoxically, the proportion of alleles exhibiting a complete VDJ rearrangement is higher among the V2⁺ (17.2%) than the V1⁺ (4.9%) subset (Table III).

View larger version (19K): [in this window] [in a new window]   FIGURE 2. TCR rearrangement pattern distribution in the V1 and V2 subsets according to the rearrangement status at the locus of the unexpressed allele. The results are expressed in percentage of alleles with a given rearrangement status in the V1+ and V2+ subsets as a function of rearrangement status at the nonexpressed allele. The n value on each histogram corresponds to the number of rearranged alleles for each rearrangement status of the nonexpressed allele in the V1+ and V2+ subsets. Because of their limited number (n = 4 for both V1 and V2 subset), VD rearrangements were pooled with DJ rearrangements.

View this table: [in this window] [in a new window]   Table III. locus rearrangement status in V1+ and V2+ peripheral T cell clonesa

The high percentage (81.3%) of V2⁺ clones exhibiting a partial rearrangement on one of their alleles and one allele in germline configuration (12%) (Table I) is consistent with the hypothesis that incompletion of rearrangements may be related to a competition between commitment to one or the other T lineage. Conversely, in the V1⁺ subset, competition for commitment to the or lineage seems to have but very little impact on the completion of and rearrangements.

Clones with two in-frame rearrangements are favored in the V1⁺ subset

The partial rearrangement rate observed in the V1⁺ subset (65.8%) thus seems essentially dependent on the rearrangement process and remains consistent with a cessation of rearrangements following expression of a functional -chain comparable to a strict allelic exclusion process. The high percentage of clones exhibiting two functional rearrangements is, however, hard to explain in this context. If the production of a functional chain does not induce systematic cessation of rearrangements, one should expect rearrangements at that locus to proceed, but this would be inconsistent with the observed limited completion of rearrangements.

Alternatively, the high frequency of clones exhibiting two functional -chains may be the result of a T cell precursor selection process that would occur within the thymus, independently of the process that regulates cessation of rearrangement. We have indeed observed a great variability in the expression rates of different types of TCR in distinct clones expressing the same combinations of VJC and VDJ elements (Fig. 3) (18). This indicates that the junctional diversity plays an important role in the stability of TCR. The possibility for T precursors to form several TCR with different - and -chain combinations may give them the opportunity to express TCR whose expression rate or specificity for an actual thymic ligand may confer a selective advantage (32). As these rearrangements occur at the same stage of development (31, 33), a T lymphocyte selection process based on the expression of TCR, and favoring clones exhibiting two functional -chains, may also have an impact on the rate of cells expressing two functional -chains. Since the locus is subjected to an allelic inclusion, 20% of clones are expected to express two rearrangements in an open reading frame due to the recombination process only (4, 19). The percentage of V2 cells with two in-frame alleles (18.2%) is consistent with the expected theoretical percentage. Conversely, the percentage of V1 cells exhibiting two functional rearrangements reaches 47.3%, which significantly differs from 20% (p < 0,001; Table IV). The fact that the percentage of cells with two in-frame rearrangements at the end of the recombination process goes from 20 to 47.3%, implies that the T precursors that were able to produce a functional TCR are subjected to a drastic selection.

View larger version (32K): [in this window] [in a new window]   FIGURE 3. Single-color flow cytometry analysis of two V1+ T cell clones expressing two surface TCR with identical combinatorial diversity using V- and V-specific mAb. The rearrangement status at the TCR locus in clones CS12.4 and CS1.49 was determined using a combination of PCR, Southern blot, and flow cytofluorometric analysis. Shaded histograms, Staining with anti-V1 (R9 mAb), anti-V9 (389 mAb), and anti-V2–4 (23D12 mAb). Open histograms, Staining with control mAb.

View this table: [in this window] [in a new window]   Table IV. Frequency of V1+ and V2+ with two functional in-frame V rearrangements and comparison to the theoretical frequency expected for a locus without allelic exclusion

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Differences observed between two human lymphocyte subsets belonging to the same T lineage and inconsistencies with previous observations done in a murine model puzzled us. Mechanisms of T lymphocyte differentiation in humans and mice are similar. In both species, perception by the cell of a signal that induces cessation of rearrangement would depend on the expression of a surface TCR (16). It is unlikely that a surrogate chain allowing the separate selection of a - or -chain, comparable to the selection of or Ig H chains, is involved.

As shown in Fig. 3, TCR stability is greatly influenced by junctional diversity and the nature of the rearranged V(D)J elements. Moreover, it seems that accessibility to V(D)J elements for the recombination machinery is regulated throughout the T cell precursor differentiation process, as shown by the differences observed between the V1 and V2 subsets in terms of rearrangements. It is thus important to keep the characteristics of each population studied in perspective with the various differentiation modalities of V2⁺ and V1⁺ lymphocytes.

Distinct features for distinct human subsets

For the most part, the lymphocyte subsets that we analyzed seemed to originate from adult precursors. No preferential use of a single D3 element with limited N addition and nucleotide nibbling, typical from subsets of cells originating from early embryonic development, could be found in the -chain sequences of the V2 clones studied (data not shown and Ref. 34). The high frequency of loci in the germline configuration in V2 clones, as compared with V1 clones, might be linked to a preferential accessibility of the V2 element at an earlier stage of the precursor differentiation, irrespective of the thymus age. This would be consistent with a kinetics of rearrangements, at different TCR chain loci, overlapping later stages of precursor differentiation (16, 31, 35, 36). Moreover, studies of the rearrangements during adult thymic development in mice revealed a differential accessibility to variable elements depending on the T cell precursor differentiation stage (37). Such a regulation of the accessibility to TCR gene loci dependent on the precursor differentiation stage would be consistent with the ordered and rearrangements observed and with the preferential use of the C2 constant region by V1 lymphocytes and not V2 lymphocytes (Table I). The drop in the number of complete rearrangements in the V1 subset, as compared with that in the V2 subset, indicates that V1 cells might have originated from late-stage precursors that have lost the capacity to activate complete V to DJ rearrangements. This would also concur with the idea of a progressive loss of the capacity of T precursors to commit to one or the other T lineage (17).

Differential accessibility of and loci regions has notable consequences on the structure of V1 and V2 lymphocyte TCR. Indeed, as opposed to the C1 constant chain, the C2 chain is deprived of the cysteine residue that forms the disulfide bridge between - and -chains and exhibits a duplication or triplication on the third exon (38). The absence of this covalent bond decreases TCR stability, making TCR more sensitive to constraints due to pairing of - and -chains related to the junctional and combinatorial diversity. Conversely, in all clones of the V2 subset, the presence of a C1 chain stabilizes the TCR structure and thus limits the effect of these constraints; this may account for the fact that unlike for the V1 subset, there is no selection bias favoring the expression of different combinations of TCR in the V2 subset. This selection process does not seem directly related to the mechanism responsible for the cessation of rearrangements. If that were the case, the incapacity of certain combinations of -chains to form a TCR able to produce a signal that induces cessation of rearrangement would allow for rearrangements at the locus to proceed normally, which is inconsistent with the incomplete rearrangements observed. This suggests that the selective advantage of cells expressing different in-frame - and, to a lesser extent, -chains seems to be the result of a process that occurs after cessation of rearrangements and that affects the pool of thymocytes that were able to produce a functional TCR.

Uncoupling cessation of rearrangement and the T cell selection process

Because of the stochastic aspect of junctional diversification, the rearrangement mechanism in itself imposes fixed rates of in-frame and out-of-frame rearrangements. In lymphocyte subsets and in an allelic inclusion context, where rearrangements occur independently on each allele of a given locus, constraints related to rearrangements lead to a fixed rate of clones exhibiting two functional rearrangements of 20% (4). This was confirmed experimentally for the locus in wild-type mice and mice deficient for the TCR chain (39). The 47% rate of cells expressing two functional rearrangements in the V1 population, however, cannot be explained by the cessation of rearrangements, thus implying that an additional selection mechanism exists. Thymic selection mechanisms have been documented in mice based on a transgenic model (40, 41). Recent observations also indicate that intracytoplasmic expression of - and -chains strongly increases at the DN4 stage, which is late considering the intense recombination activity that takes place at the and loci at the DN3 stage (42). It is, however, impossible to distinguish whether this increase in expression reflects a general mechanism or whether it is the result of a selection of lymphocytes that are able to express rates of TCR that are high enough to allow for their development to proceed toward the T lineage. The uncoupling of signals required for allelic exclusion and those that allow the development of T precursors to proceed was demonstrated by dissecting the transduction pathways of the pre-TCR in mice expressing activated forms of lck, fyn, and Ras proteins and mice deficient in these proteins (43, 44, 45). Other authors also showed that even though they allow for normal expression rates, certain mutations in the constant region affect the transduction pathways necessary for the development of T precursors to proceed normally without precluding allelic exclusion at the locus (46). We propose that a baseline expression of TCR may be sufficient to allow for cessation of the rearrangements at the locus which would be consistent with the observed incomplete rearrangements. Following rearrangement, clones expressing different functional - and, to a lesser extent, -chains would have a better chance to express a TCR selectable by a thymic ligand or to reach expression levels superior to a certain threshold required for the detection of a signal that induces development to proceed toward the lineage.

Model for the cessation of rearrangements at the locus in mice and humans

The detection of a signal that commands cessation of rearrangements, generated by the expression of a functional TCR, is consistent with the kinetics of rearrangements at the and loci. The first rearrangement events that occur at the and loci are detected at the same stage of differentiation of the precursor (31). However, because one to three rearranged D elements are used, the production of a -chain is delayed in comparison to that of a -chain, in which each recombination event leads to the formation of complete rearrangements. The fact that -chains are already rearranged by the time -chains are expressed would in itself be sufficient to induce cessation of further rearrangements. In humans, this control seems as drastic as that exerted by the pre-TCR on the completion of rearrangements. In these conditions, the high rate of rearrangement completion observed in mice would be the consequence of a delay in the completion of rearrangements. Thus, in the precursors that cannot produce a functional rearrangement on either allele (i.e., 55% of them) the first time, the particular structure of the murine locus and the absence of an isotype exclusion at this locus allows for rearrangements to proceed on another VJC cluster (47). This delay in the expression of a surface TCR leaves enough time for a second complete rearrangement at the locus, whether or not the first rearrangement was functional. In humans, however, despite the presence of two constant regions, the possibilities of secondary rearrangements seem fairly limited. Indeed, there is no difference in the frequency of use of the closest V elements to J elements, such as pseudogenes V10 and 11, which should definitely be deleted should secondary rearrangements occur within constant region C2 (Table I).

Differences in the kinetics of and rearrangements in T cell precursors could thus have distinct consequences in terms of lineage commitment, as observed between V2 and V1 lymphocyte subsets in humans. They may also account for differences in the control of rearrangements between humans and mice, although the signal that induces cessation of rearrangements, which is contingent upon the expression of a functional TCR, is the same for both species.

	Footnotes

 
¹ This work was supported by a grant from the Association pour la Recherche sur le Cancer. C.C. was supported by a grant from the Ministère de la Recherche.

² Address correspondence and reprint requests to Dr. François Davodeau, Institut National de la Santé et de la Recherche Médicale Unité 463, Institut de Biologie, 44093 Nantes, Cedex 1, France. E-mail address: davodeau{at}nantes.inserm.fr

Received for publication October 2, 2003. Accepted for publication February 23, 2004.

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