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The Journal of Immunology, 2000, 165: 1364-1373.
Copyright © 2000 by The American Association of Immunologists

T Cell Development in TCRß Enhancer-Deleted Mice: Implications for {alpha}ß T Cell Lineage Commitment and Differentiation1

Isabelle Leduc, William M. Hempel2, Noëlle Mathieu, Christophe Verthuy, Gaëlle Bouvier, Françoise Watrin3 and Pierre Ferrier4

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
 
T cell differentiation in the mouse thymus is an intricate, highly coordinated process that requires the assembly of TCR complexes from individual components, including those produced by the precisely timed V(D)J recombination of TCR genes. Mice carrying a homozygous deletion of the TCRß transcriptional enhancer (Eß) demonstrate an inhibition of V(D)J recombination at the targeted TCRß locus and a block in {alpha}ß T cell differentiation. In this study, we have characterized the T cell developmental defects resulting from the Eß-/- mutation, in light of previously reported results of the analyses of TCRß-deficient (TCRß-/-) mice. Similar to the latter mice, production of TCRß-chains is abolished in the Eß-/- animals, and under these conditions differentiation into cell-surface TCR-, CD4+CD8+ double positive (DP) thymocytes depends essentially on the cell-autonomous expression of TCR{delta}-chains and, most likely, TCR{gamma}-chains. However, contrary to previous reports using TCRß-/- mice, a minor population of TCR {gamma}{delta}+ DP thymocytes was found within the Eß-/- thymi, which differ in terms of T cell-specific gene expression and V(D)J recombinase activity, from the majority of TCR-, {alpha}ß lineage-committed DP thymocytes. We discuss these data with respect to the functional role of Eß in driving {alpha}ß T cell differentiation and the mechanism of {alpha}ß T lineage commitment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The T lymphocytes belong to distinct lineages that express either {alpha}ß or {gamma}{delta} TCRs on their cell surface and, within {alpha}ß T cells, either the CD4 or CD8 coreceptors. T cell development in the thymus proceeds from a common precursor (1) and requires the ordered rearrangements of TCR genes (i.e., through V(D)J recombination) as well as a series of selection events through which newly assembled TCR complexes signal for cell survival, proliferation, and differentiation (2). Due to the inherent imprecision of the DNA joining step during V(D)J recombination (3), only rearrangements that generate an open reading frame across the recombined TCR gene(s) (i.e., productive rearrangements) allow for expression of the corresponding polypeptide(s) and ensuing selection. It is not yet clear whether TCR complexes, once assembled, instruct the developing thymocytes to commit to and differentiate along one or the other lineages ("instructive" model of lineage commitment) or, alternatively, whether lineage commitment occurs stochastically before receptor assembly ("stochastic selection" model) (4, 5, 6).

During the past few years, a comprehensive view of {alpha}ß T cell development in the mouse thymus has started to emerge. TCRß gene recombination, starting with Dß-to-Jß rearrangement, is first detected within a subpopulation of CD4-CD8- double negative (DN)5 cells expressing the CD25 and CD44 markers (DN CD44+CD25+); Vß-to-DJß assembly is completed within the subsequent DN CD44-CD25+ cell stage (7, 8, 9). Productive rearrangements allow for the expression of a TCRß-chain which, when associated with the pT{alpha}-chain and signal-transducing CD3 proteins, forms the pre-TCR (10). Pre-TCR-expressing cells possess a selective advantage to differentiate along the {alpha}ß developmental pathway, resulting in cell populations in which in-frame TCRß rearrangements are thus overepresented (11, 12). Passage through this check-point, referred to as ß selection, is coupled to the down-modulation of CD25, massive cell proliferation, the arrest of TCRß gene rearrangement to mediate allelic exclusion, and the onset of TCR{alpha} gene rearrangement. Cells emerging from these processes are CD4+CD8+ double positive (DP) thymocytes expressing low levels of the {alpha}ß TCR-CD3 complexes. At this stage, through receptor/coreceptor interaction with MHC products, a small proportion of DP cells are positively selected (13). Positive selection results in the arrest of V(D)J recombination, an increase in {alpha}ß TCR-CD3 expression, and the modulation of coreceptor expression (14) to yield CD4+ or CD8+ single positive (SP) cells, which eventually migrate to the periphery.

In contrast to {alpha}ß T cells, {gamma}{delta} thymic cell development is far less understood, partly due to their small number (as compared with {alpha}ß+ thymocytes) and lack of phenotypic markers (other than the {gamma}{delta} TCR). Recent studies indicate that a significant proportion of TCR{delta} and TCR{gamma} rearrangements are completed earlier than those for TCRß, at the DN CD44+CD25+ stage (8, 9). Also, analysis of mice deficient for TCRß-chain expression (TCRß-/- mice that produce {gamma}{delta}+ T cells only (15)) has identified thymic cell populations in which in-frame TCR{delta} rearrangements are enriched, suggesting that development in the {gamma}{delta} pathway similarly goes through a selection event(s) (16). However, the precise stage at which this process would take place in normal mice and the composition of the receptor complexes involved are still unclear.

With respect to the aforementioned models of lineage commitment, the question remains as to what are the forces that drive the choice between the TCR {alpha}ß and {gamma}{delta} differentiation pathways. Perhaps productive TCR ß or {gamma}{delta} rearrangements divert differentiation away from the {gamma}{delta} or {alpha}ß lineage, respectively (17, 18, 19). Silencing of the TCR{gamma} gene and/or deletion of the TCR{delta} gene (i.e., by V{alpha}-to-J{alpha} recombination) would be factors which, subsequently, contribute to lock the developing cells into the {alpha}ß cell lineage (4, 20). Lack of pT{alpha} expression and/or inhibition of Vß-to-DJß joining by expressed {gamma}{delta} TCRs would play a similar role in the {gamma}{delta} developmental pathway (5). However, these views have been challenged by the findings that the differentiation of small populations of {alpha}ß and {gamma}{delta} lineage-committed T cells from several transgenic and knock-out mouse models may be independent of the nature of the expressed receptors (18, 21, 22).

Recently, we have described knock-out mice that show reduced levels of TCRß gene recombination and a block in {alpha}ß T cell differentiation after deletion of the only defined transcriptional TCRß gene enhancer (Eß) at the TCRß locus (23). In the present study, we characterize more precisely the TCRß gene expression and T cell developmental defects in the Eß-/- thymus. Our results emphasize the role of Eß in {alpha}ß T cell development and provide additional implications with respect to the processes of {alpha}ß vs {gamma}{delta} lineage commitment and differentiation.


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

Wild-type (WT) C57BL/6J mice, single knockout TCRß-deficient (TCRß-/-), and TCR{delta}-deficient (TCR{delta}-/-) mice (15, 24), heterozygous (Eß+/-) and homozygous (Eß-/-) Eß-deleted (23) mice, and double knock-out TCR{delta}-deficient (TCR{delta}-/-) Eß-/- mice (25) were used in this study. TCRß-/- and TCR{delta}-/- mice, both on a C57BL/6J genetic background, were obtained from The Jackson Laboratory (San Diego, CA). All mice were housed in a specific pathogen-free animal facility in accordance with institutional guidelines. Mice were sacrificed for analysis between 4 and 6 wk of age. Initial analyses were performed using Eß-/- mice bred on a mixed (S129 x BALB/c) genetic background; subsequent studies using Eß-/- animals bred on a C57BL/6J background gave essentially the same results.

Antibodies

Biotinylated, FITC-, PE-, and allophycocyanin-conjugated mAbs against CD8 (53-6.7), CD4 (H129.19), CD44 (Pgp-1), CD25 (7D4), TCRß (H57-597), TCR{gamma}{delta} (GL3), Vß3 TCR (KJ25), CD3-{epsilon} (2C11), and CD24/heat-stable Ag (HSA; M1/69) were purchased form PharMingen (San Diego, CA). Biotinylated mAbs against the B220 (RA3-6B2; B cell-specific), Mac-1 (M1/170; macrophage-specific), and Gr-1 (RA6-8c5; granulocyte-specific) markers were from Caltag (Tebu, Le Perray en Yvelines, France). Biotinylated mAbs were revealed using streptavidin tricolor (Caltag).

Flow cytometry and cell cycle analysis

Lymphocyte preparation and cell-staining with saturating levels of mAbs were conducted according to published protocols (for example, see Ref. 26). For cell-surface analyses, 5–50 x 105 gated events were acquired using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and were analyzed with the Lysis II software. Cell sorting was performed as described previously (26, 27) using a FACStarPlus (Becton Dickinson). For cell sorting of Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- thymocytes, the sorting windows were defined in such a way that 1) only cells expressing high levels of both CD4 and CD8 were purified, and 2) within the selected CD4+CD8+ cells, the {gamma}{delta}+ and {gamma}{delta}- sorting gates do not overlap.

For propidium iodide staining of DNA, 5 x 105 sorted cells were washed in PBS-0.2% BSA and then fixed in PBS-70% ethanol for 30 min at 4°C. Cells were washed with PBS-0.1% glucose, treated with RNase A (Boehringer Mannheim, Meylan, France; 1 µg/ml) for 15 min at room temperature, and stained with propidium iodide (Sigma-Aldrich, Saint-Quentin Fallavier, France; 25 µg/ml in PBS-0.1% glucose) for 20 min at room temperature and then for 10 min at 4°C. Analysis was performed on a FACScan (Becton Dickinson) with appropriate doublet discrimination.

Nucleic acid extraction, reverse transcription, and long-range PCRs (LR-PCRs)

Total RNA and genomic DNA were simultaneously extracted from purified cell populations (~5 x 105 sorted cells) using TRIzol (Life Technologies, Cergy Pontoise, France) as recommended by the manufacturer. RNA samples were treated with RNase-free DNase I (Pharmacia, Orsay, France) and were converted to cDNA by reverse transcription, using the Ready-to-Go T-primed first strand kit (Pharmacia).

Analysis of T cell-specific transcripts and gene rearrangements by RT-PCR and LR-PCR assays, using cDNA or genomic DNA templates and locus-specific primers, were performed essentially as described previously (25, 27). RT-PCR was performed for 22 cycles of 30 s at 94°C, 30 s at 58°C, and 1 min at 72°C; LR-PCR was performed for 25 cycles of 30 s at 94°C, 1 min at 58°C, and 1 min at 72°C. Individual RT-PCR used the following pairs of forward and reverse primers: V{delta}4-C{delta}, 5'-CCGCTTCTCTGTGAACTTCC-3' and 5'-GCTGCTAGGAAAACTCTCCT-3'; V{gamma}4-C{gamma}1, 5'-TGTCCTTGCAACCCCTACCC-3' and 5'-ATTGCCACAGACAGATGTTGT-3'; V{alpha}8-C{alpha}, 5'-ACCCAGACAGAAGGCCTGGTCACT-3' and 5'-GAGGGAGCTGAGTGGGTG-3'; pT{alpha}, 5'-CTGCAACTGGGTCATGCTTC-3' and 5'-GTCCAAATTCTGTGGGTGGG-3'; RAG-2, 5'-CACATCCACAAGCAGGAAGTACAC-3' and 5'-GGTTCAGGGACATCTCCTACTAAG-3'; CD4, 5'-GAGAAGACGCTGGTGCTGGG-3' and 5'-CCCACAACTCCACCTCCTC-3'; CD8, 5'-CAAGCATCTACTGGCTGCGGG-3' and 5'-GTGGGGGAACGGGCATTGCTT-3'; and ß-actin, 5'-GTGGGCCGCTCTAGGCACCAA-3' and 5'-CTCTTTGATGTCACGCACGATTTC-3'. Individual LR-PCR used the following pairs of forward and reverse primers: V{delta}4- or V{delta}5-J{delta}1, 5'-CCGCTTCTCTGTGAACTTCC-3' or 5'-CAGATCCTTCCAGTTCATCC-3' and 5'-CAGTCACTTGGGTTCCTTGTCC-3'; V{gamma}1-J{gamma}4, 5'-CCGGCAAAAAGCAAAAAAGTT-3' and 5'-ACTACGAGCTTTGTCCCTTTG-3'; V{gamma}2-J{gamma}2, 5'-TACCGGCAAAAAACAAATC-3' and 5'-CAGAGGGAATTACTATGAGC-3'; V{gamma}4-J{gamma}1, 5'-TGTCCTTGCAACCCCTACCC-3' and 5'-CAGAGGGAATTACTATGAGC-3'; and Cß2, 5'-TGTGGCAGGCTCTAATTAAAT-3' and 5'-GCTATAATTGCTCTCCTTGATGGCCTG-3'. After amplification, PCR products were electrophoresed on 1% agarose/0.5% NuSieve gels, transferred to Nylon membranes (Gene Screen Plus; NEN Life Science Products, LeBlanc Mesnil, France), and hybridized with [{gamma}-32P]-labeled locus-specific oligonucleotide probes internal to the corresponding primers. Images were generated by use of a phosphoimager (BAS 1000; Fuji, Raytest France S.A.R.L., France) and quantified using MacBAS software.

Ligation-mediated PCR (LM-PCR)

Detection of in vivo-generated signal ends (SEs) by LM-PCR, using genomic DNA ligated to the unidirectional BW linker (28) as template, as well as verification for the presence of the DNA in the BW linker-ligated samples by PCR using primers specific for CD14 (5'-GCTCAAACTTTCAGAATCTACCGAC-3' and 5'-AGTCAGTTCGTGGAGGCCGGAAATC-3'), were conducted as described previously (25, 29). Depending on the TCR gene segment analyzed, the following primers were used in two successive rounds of amplification: J{alpha}50 (round 1, 5'-CCACGTCCAGATGCCAACTTGAAA-3'; round 2, 5'-GAGAGGAGTGCTGAAAACAGCCTT-3'); J{delta}1 (round 1, 5'-TGTTGTTCCCACATGCTGCTCAAAC-3'; round 2, 5'-AACCTCCTGTAAGCTAACCCATCCT-3'); and J{gamma}1 (round 1, 5'-CCAACTGAACTCCTTCTATTTTCTGTTGGTG-3'; round 2, 5'-AACTCCAGGGAGAACAGTGTATGAG-3'). PCR products were analyzed as described in the previous section.

PCR-RFLP

Diverse TCR{delta} and TCR{gamma} gene rearrangements were studied by PCR-RFLP, according to published protocols (11, 30). Briefly, genomic DNA from sorted thymocytes was PCR-amplified using appropriate pairs of TCR{delta} and TCR{gamma} primers as follows: V{delta}4- or V{delta}5-J{delta}1, 5'-CCATCGATGGCCGCTTCTCTGTGAACTTCC-3' or 5'-CCATCGATGGCAGATCCTTCCAGTTCATCC-3' and 5'-CAGTCACTTGGGTTCCTTGTCC-3'; V{gamma}2-J{gamma}2, 5'-CCATCGATGGTACCGGCAAAAAACAAATC-3' and 5'-TGAATTCCTTCTGCAAATACCTTG-3'; and V{gamma}4-J{gamma}1, 5'-CCATCGATGGTGTCCTTGCAACCCCTACCC-3' and 5'-TGAATTCCTTCTGCAAATACCTTG-3'. PCR products were gel purified, digested with the ClaI restriction enzyme (forward primers contain a ClaI site (ATCGAT) on their 5' side) to generate fragments of predicted size that spanned the polymorphic junctions, and then labeled using T4 polynucleotide kinase and [{gamma}-32P]ATP (Amersham, Les Ulis, France). After ethanol precipitation, the labeled products were resolved on 5% denaturing polyacrylamide gels, parallel to calibrated sequencing ladders from DNA fragments of known size. Gels were used to generate images and quantification data, as described above. Lengths of predicted in-frame joints were calculated from published sequences.

Calculation of PCR-RFLP values for TCR{delta} and TCR{gamma} selection

Assuming that rearrangements occur with an equal frequency in all three reading frames, within a population of rearranging TCR{delta} cells, 1/3 will carry an in-frame ({delta}+) allele after the initial attempt and 2/3 will carry an out-of-frame ({delta}-) allele. Given the absence of allelic exclusion at the TCR{delta} locus (31), rearrangement could proceed to the second allele in the former subpopulation, yielding cells that have either an additional {delta}+ allele ({delta}+/{delta}+; 1/3 x 1/3) or a {delta}- allele ({delta}+/{delta}-; 2/3 x 1/3). Among the remaining 2/3 cells, rearrangement on the second allele yields cells with either a {delta}+ allele ({delta}-/{delta}+; 1/3 x 2/3) or an additional {delta}- allele ({delta}-/{delta}-; 2/3 x 2/3). In total, all {delta} alleles in the cell population undergo rearrangement, and 1/3 + (1/3 x 1/3) + (1/3 x 2/3) are {delta}+. Cells carrying a {delta}+ rearrangement(s) (corresponding to a total of 2[1/3 + (1/3 x 2/3)] {delta} rearranged alleles) enter the selected pool, whereas the {delta}-/{delta}- cells are eliminated. Therefore, when scanning TCR{delta} selected cells by PCR-RFLP, the theoretical ratio (N) between the number of {delta}+ alleles (numerator) vs that of total rearranged ({delta}+ and {delta}-) alleles (denominator) is as follows.

Allelic exclusion may not apply to TCR{gamma} gene recombination either (32). Moreover, each of the two TCR {gamma}1, {gamma}2, and {gamma}4 alleles (33) may attempt rearrangement in any one cell. Therefore, cells carrying a {gamma}- joint on both alleles at a given {gamma} locus (for example, the {gamma}1 locus) may theoretically be rescued into the selected pool after a {gamma}+ rearrangement at any one of the four remaining alleles (i.e., the two {gamma}2 and {gamma}4 alleles). Because PCR-RFLP analyzes only one locus at a time, the N ratio when scanning TCR {gamma}1 selected cells, for example, is decreased according to the following revised formula, in which the number of {gamma}+ alleles (the numerator) remains unchanged, whereas that of the {gamma}- alleles (the denominator) is augmented in proportion to the number of {gamma}1-/{gamma}1- cells rescued in the sampled population after an in-frame {gamma}2 or {gamma}4 rearrangement.

Note that, in this situation in which up to six TCR{gamma} alleles could be rearranged, the predicted value of in-frame junctions in the case of TCR{gamma} selection is close to that associated with random recombination (0.33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of TCRß chain expression in Eß-/- T cells

The Eß-/- mouse presents a severe defect in TCRß gene recombination and a block in {alpha}ß T cell development (23). Impaired {alpha}ß T cell development is best characterized by reduced cellularity and altered cellular profiles in thymuses from the Eß-/- animals compared with those from WT or heterozygous (Eß+/-) littermates (Tables I and II, Fig. 1Go, and data not shown). On average, total thymocytes in the Eß-/- mutants are decreased by >15-fold (Table IGo), even though we noted important variations (from 3.5 x 106 to 42 x 106) among Eß-/- individuals. When stained for CD4 and CD8 surface expression, Eß-/- thymocytes contain an abnormally high proportion of DN cells and, conversely, a reduced proportion of DP cells; genuine CD4high and CD8high SP cells are missing (Fig. 1GoA, upper middle and upper left panels). Reported to the absolute cell numbers, Eß-/- DN thymocytes are present in normal or slightly diminished numbers, whereas both Eß-/- DP and SP cells are severely reduced, accounting for the collapse in thymus cellularity (Table IGo).



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  		FIGURE 1. Flow cytofluorometric analysis of T lineage cells from 4-wk-old Eß+/-, Eß-/-, TCR{delta}-/- Eß-/-, and TCRß-/- mice. A, Total thymocytes were analyzed by flow cytometry for the expression of CD4 vs CD8 (upper panels), and DN thymocytes were analyzed for the expression of CD44 vs CD25 (lower panels). For the CD44/CD25 studies, DN T lineage cells were analyzed after staining of thymocytes with anti-CD44-PE, anti-CD25-FITC, and anti-CD3, -CD4, -CD8, -B220, -Mac-1, and -Gr-1 (all biotinylated and revealed with streptavidin tricolor) mAbs and gating on the CD3-, CD4-, CD8-, B220-, -Mac-1, and -Gr-1 negative window. The absolute number of thymocytes found in each type of mouse is shown on the top; quadrant percentages are indicated in the lower right quadrant (CD4/CD8 analysis) or the upper right quadrant (CD44/CD25 analysis). B, Thymocytes (left panel) and lymph node cells (right panel) were analyzed for the cell surface expression of TCRß-chains using the Cß-specific H57.597 mAb. Note that H57.597 staining was slightly lower in the case of the Eß-/- mouse compared with that of the TCRß-/- mouse.

 

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  		Table I. Absolute numbers and percentage of thymocytes in Eß+/-, Eß-/-, and TCR{delta}-/- Eß-/- mice: anti-CD4/-CD8 staining of total thymocytes1

 
The cellular profiles described above, notably the severe reduction but not disappearance of DP cells, are similar to those reported for TCRß-chain-deficient (TCRß-/-) mice which, after gene targeting, carry a large (>15-kb) deletion within the TCRß locus (15). In TCRß-/- mice, it has been shown that the production of DP thymocytes is highly, if not entirely, dependent on TCR{delta}-chain expression, because these cells were almost completely absent when the mutation was introduced onto a TCR{delta}-deficient (TCR{delta}-/-) background (15, 16, 34). To test whether a similar effect occurs in the Eß-/- mice, these were crossed with TCR{delta}-/- mice (24), and thymuses from double mutant (TCR{delta}-/- Eß-/-) animals were analyzed as above (Table IGo and Fig. 1GoA, upper right panel). Strikingly, thymus cellularity was further reduced in the TCR{delta}-/- Eß-/- mice, showing little interindividual variation. Moreover, TCR{delta}-/- Eß-/- thymuses essentially lacked DP and SP cells, which is indicative of a drastic block in early T cell differentiation. This was confirmed by analysis of the CD44/CD25 profiles from DN thymocytes (Table IIGo and Fig. 1GoA, lower panels). Thus, although CD44+CD25+ and CD44-CD25+ DN cells are proportionally increased in the TCR{delta}-/- Eß-/- thymus, these cells fail to efficiently populate the CD44-CD25- compartment. A similar, but less marked, block of T cell differentiation was observed for Eß-/- DN cells. We conclude that, as in the TCRß-/- mice, deletion of Eß leads to a block in early T cell development that can be bypassed, albeit inefficiently, by expression of TCR{delta}-chains.


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  		Table II. Absolute numbers and percentage of thymocytes in Eß+/-, Eß-/-, and TCR{delta}-/- Eß-/- mice: anti-CD44/-CD25 and anti-{gamma}{delta} stainings of CD4-/CD8- DN thymocytes1

 
The above results strongly argue that the Eß mutation is not permissive for the residual expression of TCRß-chains (otherwise DP and SP cells would presumably be found in the TCR{delta}-/- Eß-/- thymus). To confirm this, thymus and lymph node cells from an Eß-/- mice were analyzed using the H57-597 mAb, which reacts against pan-TCRß-chains; Eß+/- and TCRß-/- cells were used as positive and negative controls, respectively (Fig. 1GoB). As expected, surface H57-597 staining was observed for cells from the Eß+/- mouse (both H57-597low and H57-597high cells were found in the Eß+/- thymus, whereas only H57-597high cells were found in the lymph nodes) but not for those from the TCRß-/- mouse. Importantly, thymus and lymph node cells from the Eß-/- mouse were also H57-597-negative. These results were reproduced upon testing several individual animals (I.L., unpublished data), establishing the Eß-/- mouse as an additional model of TCRß-chain (hence pre-TCR) deficiency. It is noteworthy that {gamma}{delta}+ cells are found in increased numbers and at a higher percentage among the Eß-/- DN thymocytes (Table IIGo), implying that the Eß deletion does not block {gamma}{delta} T cell development.

Cellular characteristics of the Eß-/- DP thymocytes

Examples of pre-TCR-independent DP cell development in several models of natural or engineered genetic mouse mutants have fueled speculation about the underlying developmental mechanisms and their relevance to the physiology of thymic cell differentiation. Issues of particular concern are 1) the actual lineage ({alpha}ß or {gamma}{delta}) of the resulting TCRß- DP thymocytes, 2) the extent to which the DN to DP cell transition depends upon a cis-autonomous or, alternatively, trans-induction mechanism, and 3) the nature of the specific receptors that drive this stage of thymocyte differentiation (4, 5, 6). Because we wished to understand how our Eß-/- mice fit into the present picture, we undertook a series of cytofluorometric analyses focusing on the DP cells present in the mutant thymus; thymocytes from heterozygous Eß+/- mice were also analyzed. The results are reported in Fig. 2Go and Table IIIGo. Tricolor staining using anti-CD4, -CD8, and -{gamma}{delta} mAbs indicated the presence of a minor population of anti-{gamma}{delta} reactive cells (hereafter referred to as {gamma}{delta}+) within the DP compartment from the Eß-/- thymuses (Fig. 2Go). On average, the {gamma}{delta}+ cells account for 16.6% of Eß-/- DP thymocytes, which is lower than the proportion of {gamma}{delta}+ cells in the Eß-/- DN compartment (mean value of 24%) but significantly higher than that of Eß-/- DP thymocytes which were nonspecifically stained by an anti-Vß3 TCR mAb (mean value of 5.2%; Tables I–III, Fig. 2Go, and data not shown). Within the Eß-/- thymus, {gamma}{delta}+ high expressors were mainly found in the DN cell compartment, whereas the Eß-/- DP {gamma}{delta}+ thymocytes consisted mostly of low to intermediate with a few high {gamma}{delta}+ expressors (as judged by cytofluorograph profiles and comparison of the mean values of rate fluorescence intensity). Under these experimental conditions, only a few percent of specific, anti-{gamma}{delta}-reacting cells was seen among the DP thymocytes from either heterozygous Eß+/- or WT mice (0.5%–<2%) as well as the DP thymocytes from TCRß-/- mice (<4%) (Fig. 2Go and data not shown).



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  		FIGURE 2. A minor population of {gamma}{delta}+ thymocytes is present within the Eß-/- DP cell compartment. Total thymocytes from the indicated mice were triple-stained with anti-CD4, -CD8 and -{gamma}{delta}, or -Vß3 TCR mAbs and, using flow cytofluorometry, CD4-CD8-/DN and CD4+CD8+/DP thymocytes were analyzed for {gamma}{delta} cell surface expression (left and middle panels, respectively), and CD4+CD8+/DP thymocytes were analyzed for Vß3 cell surface expression (right panels; the anti-Vß3 TCR mAb, which has been produced in the same species and possesses the same biochemical properties as the anti-{gamma}{delta} mAb, is used here as control for nonspecific staining of the Eß-/- DP thymocytes). Eß-/- #1 and #2 correspond to two mutant individuals. The percentages of {gamma}{delta}+ or Vß3+ staining are indicated. A subpopulation of Vß3+ cells was detected within the SP compartments from both Eß+/- and TCR{delta}-/- thymi (e.g., accounting for 4.8 and 3.6% of their CD4-CD8+/SP thymocytes, respectively; data not shown).

 

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  		Table III. Flow cytometric analysis of T cell-specific cell surface markers and extent of cell cycling within the DP thymocytes from the Eß+/- and Eß-/- mice

 
Four-color cytofluorometric analyses using additional mAbs against the T cell-specific surface markers CD24/HSA, CD3-{epsilon}, or CD25 allowed us to further compare the {gamma}{delta}+ and {gamma}{delta}-unreactive (hereafter referred to as {gamma}{delta}-) subsets within the Eß-/- DP compartment (Table IIIGo). Both HSA and CD25 staining are indicators of thymocyte immaturity, the latter often being associated with reduced cell proliferation at the DN-to-DP transition (35). CD3-{epsilon} staining usually correlates with high levels of TCR expression. High percentages of HSA-expressing cells were found in all DP subsets, whether Eß+/- or Eß-/-, with equally prominent values in both the Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- subsets (mean rates >=94%) and a slightly lower value in the Eß+/- DP subset (mean rate = 86.3%). On the other hand, mean percentages of cells expressing high levels of CD3-{epsilon} were lower in the Eß-/- DP subpopulations compared with Eß+/- DP thymocytes, the {gamma}{delta}- subset being predominantly affected (0.5 vs 20.3%, a 40-fold difference). Notably, a similar comparison for CD25 staining demonstrated moderately higher levels of expression within the Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- subsets (15.7 and 6 vs <2%, respectively).

Finally, after cell sorting and propidium iodide staining, we analyzed the Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- subsets with respect to their level of >2N DNA content (reflecting the extent of cell cycling; Table IIIGo). Compared with the Eß+/- total DP controls, the Eß-/- DP cells displayed lower percentages of cells with greater than a diploid DNA content. The lowest value (3.7%) was found for the {gamma}{delta}+ population, which is consistent with this subset containing mostly nondividing cells. However, the {gamma}{delta}- population exhibited a higher level of dividing cells (9.5%).

In summary, our cytofluorometric analyses demonstrate that Eß-/- DP thymocytes consist primarily of moderately dividing cells that are TCR- CD3-{epsilon}-/low, although a sizable population of slowly dividing TCR {gamma}{delta}+ cells is also present. Both populations include a significant percentage of cells that exhibit a relatively immature phenotype, as defined from anti-HSA and -CD25 stainings.

Lymphoid gene expression in Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- thymocytes

The presence of {gamma}{delta}+ cells within the DP compartment of TCRß-/- mice has been questioned (15, 22). Indeed, these studies lead to the conclusion that TCRß-/- DP thymocytes correspond to "{alpha}ß -like" T cells, based on the expression of TCR{alpha} transcripts (15) and the presence of V(D)J recombinase-mediated DNA double strand breaks (DSBs) flanking the upstream J{alpha} gene segments (22). To verify that the Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- thymocytes represent truly distinct entities and further clarify their lineage, we conducted analyses of selected gene activities within the two subsets. First, we used RT-PCR to analyze expression of several T cell-specific genes in total RNA from sorted Eß-/- thymocytes, including rearranged TCR{delta}, {gamma}, and {alpha} genes and the pT{alpha} gene. As controls, RNA from purified Eß+/- thymocytes and cultured B cells were included in the analyses. Representative data are shown, which emphasize the distinction between the Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- cell subsets (Fig. 3GoA, lanes 4 and 5). Thus, as predicted, high levels of V{delta}4-C{delta} and V{gamma}4-C{gamma}1 transcripts were detected within the Eß-/- DP {gamma}{delta}+ subset, as opposed to the Eß-/- DP {gamma}{delta}- subset, where these transcripts were barely detectable. In the latter subset, V{delta}4-C{delta} transcripts slightly predominated over V{gamma}4-C{gamma}1 transcripts. However, conversely, mature-sized V{alpha}8-C{alpha} transcripts and pT{alpha} transcripts of both the pT{alpha}a and pT{alpha}b isotypes (35) were found in the Eß-/- DP {gamma}{delta}- subset but not in the Eß-/- DP {gamma}{delta}+ subset. As expected for the Eß+/- control, TCR{delta} and TCR{gamma} as well as pT{alpha} transcripts were detected within the DN population whereas, conversely, mature TCR{alpha} transcripts were found to predominate in the DP cells (lanes 1 and 2; note that the pattern of pT{alpha} gene expression in the Eß+/- thymus, showing high and low levels in DN and DP thymocytes, respectively, reproduces that described for WT thymus (36)). Predictably, B cells lacked TCR{delta}, {gamma}, {alpha}, and pT{alpha} transcripts (lane 6). Consistent with the above results, V{delta}5-C{delta}, V{gamma}7-C{gamma}1, V{gamma}2-C{gamma}2, and V{gamma}1-C{gamma}4 transcripts were more prevalent within the Eß-/- DP {gamma}{delta}+ subset, whereas V{alpha}2-C{alpha} and V{alpha}5-C{alpha} transcripts strongly predominated within the Eß-/- DP {gamma}{delta}- subset (I.L., unpublished data). cDNA products derived from Eß-/- DP {gamma}{delta}- cells and amplified using V{alpha} and C{alpha} primers did not hybridize to a J{delta}1-specific probe (not shown), suggesting that they correspond to VJ{alpha}-C{alpha} messengers rather than to alternatively spliced VDJ{delta}-C{alpha} hybrids (37), an assumption also consistent with the finding of V{alpha}-J{alpha} rearrangements in genomic DNA from Eß-/- DP {gamma}{delta}- cells (I.L., unpublished data).



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  		FIGURE 3. T cell-specific gene expression in Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- thymocytes. A, Thymocytes from 4-wk-old Eß+/- or Eß-/- mice were sorted by flow cytofluorometry into the indicated DN, DP, and DP {gamma}{delta}+ or {gamma}{delta}- subpopulations, respectively. Total RNA was isolated from the sorted cells and from the 7A.4 B cell line used here as a negative control (B cells) and was analyzed by RT-PCR using standard protocols. The amplified gene transcripts are indicated to the right of the panels. B, Same as A, except that RNA from WT thymus and kidney, as well as that from sorted Eß-/- DN {gamma}{delta}+ thymocytes, was also used. Lanes 1-3 show a titration of the indicated transcripts in the WT background. Input material was kept constant (50 ng of cDNA) by using decreasing amounts of thymus and increasing amounts of kidney samples, respectively. Lane 1, undiluted thymus; lanes 2 and 3, 1:3 and 1:9 dilutions, respectively. In both A and B, the bottom panel shows the amplified products from ß-actin to control for the amount and quality of RNA.

 
Additional RT-PCR analyses demonstrated the production of high levels of CD4 and CD8 transcripts in both the Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- subsets but, as expected, not in purified Eß-/- DN {gamma}{delta}+ cells (Fig. 3GoB, lanes 5–7), a finding that strengthens our cytofluorometric results and shows that the Eß-/- DP {gamma}{delta}+ cells are not merely contaminants from the DN compartment. Finally, recombinase-activating gene 1 (RAG1) and RAG2 transcripts were found in cells from the {gamma}{delta}- subset only (Fig. 3GoA and data not shown; the RAG1 and RAG2 transcripts code for the core components of the V(D)J recombinase (3)). The latter findings strongly suggested the possibility of ongoing V(D)J recombination in the Eß-/- DP {gamma}{delta}- but not the {gamma}{delta}+ subset. This was confirmed by using LM-PCR to detect the blunt-ended intermediates (SEs) produced by RAG-mediated DNA DSB cleavage (29). DSBs were detected at the TCR-J{alpha}50, -J{delta}1, and -J{gamma}1 segments in genomic DNA from the DP {gamma}{delta}- but not the DP {gamma}{delta}+ subset (Fig. 4Go). DSB profiles within the Eß-/- DP {gamma}{delta}- subset, including prominent TCR-J{alpha}50 SEs and less intense J{delta}1 and J{gamma}1 SEs, appear related to those in Eß+/- DP cells and distinct from those in both the Eß+/- and Eß-/- DN subsets in which J{delta} and J{gamma} cleaved products predominate (compare lanes 1-3 and 5). Altogether, these results confirm, at the molecular level, the existence of two distinct subpopulations of Eß-/- DP {gamma}{delta}+ and TCR- thymocytes. TCR{delta} and {gamma} transcription and the absence of ongoing V(D)J recombination within the former subset are consistent with its {gamma}{delta}+ cellular phenotype. Conversely, the preferential expression of pT{alpha} and VJ{alpha}-C{alpha} products together with the presence of J{alpha} SE products of V(D)J recombination within the Eß-/- DP {gamma}{delta}- subset indicate that it consists mostly of "{alpha}ß -like" differentiating T cells. However, the production of pT{alpha} transcripts within these cells at levels close to those in DN thymocytes underscores the relatively immature phenotype of this population with respect to that of conventional DP thymocytes.



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  		FIGURE 4. V(D)J recombination SE intermediates at the TCR{alpha}{delta} and {gamma} gene loci in Eß-/- DP {gamma}{delta}- thymocytes. Genomic DNA was prepared from sorted Eß+/- and Eß-/- thymocytes and analyzed by LM-PCR. Briefly, DNA from the indicated cells was ligated to the BW linker (28 ) and used as a template in PCR reactions with primers to detect broken SEs associated with J{alpha}50-, J{delta}1-, and J{gamma}1-flanking recombination signal sequences (J{alpha}50 SE, J{delta}1 SE, or J{gamma}1 SE, respectively). Negative controls were performed using linker-ligated genomic DNA from a WT kidney (Kd.WT). The bottom panel shows ethidium bromide-stained amplification products from DNA-PCR reactions using CD14-specific primers to control for the amount of sample DNA loaded in the individual reactions.

 
TCR {delta} and {gamma} gene rearrangements in Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- thymocytes

What is the driving force behind pre-TCR-independent thymocyte development in the Eß-/- thymus and is there a developmental relationship between the two subpopulations of DP {gamma}{delta}+ and "{alpha}ß -like" cells? Although distinct according to cytofluorometric and gene expression criteria, both depend on TCR{delta}-chain expression for development (see above). In pre-TCR-deficient animals, development of DN precursors into DP cells was proposed to depend either on the self-expression of TCR{delta}-chains, possibly in association with TCR{gamma}-chains (16, 18, 22) or, alternatively, on interactions with pre-existing {gamma}{delta}+ cells present in the mutant thymus (38). In the first situation, it is predicted that the resulting DP cells carry in-frame TCR{delta} and TCR{gamma} rearrangements despite the fact that they may not express these products as a possible consequence of their commitment to the {alpha}ß lineage. Such cells would thus differ from DP cells of normal mice in which in-frame TCR{delta} and TCR{gamma} joints appear to be counterselected (17, 30). Conversely, in the second situation, rearrangements at the TCR{delta} and TCR{gamma} loci would at least be expected to not be enriched for in-frame junctions. To define which mechanism could be responsible for the generation of the DP {gamma}{delta}- thymocytes in the Eß-/- mice and analyze their developmental relationship with {gamma}{delta}+ cells, we performed two sets of experiments. First, we used semiquantitative LR-PCR assays on genomic DNA prepared from sorted thymocytes to analyze the relative levels of TCR{delta} and {gamma} gene rearrangements within the Eß- DP {gamma}{delta}- vs {gamma}{delta}+ subpopulations. Second, we used PCR-RFLP to qualitatively analyze the same rearrangements. The latter technique permits the determination of the ratio of in-frame vs out-of-frame rearrangements at any V(D)J recombined locus in a given cell population, hence the probability that the population has been selected (or counterselected) based on a particular TCR chain expression profile (11, 30). Considering that PCR-RFLP samples only the fraction of a cell population in which a given rearrangement has occurred, conclusions with respect to TCR chain selection, based on results obtained using this technique, are dictated by the levels of rearranged loci present within this population. Results from these analyses are shown in Figs. 5Go and 6, respectively. Thus, V{delta}4-to-J{delta}1 and V{delta}5-to-J{delta}1 rearrangements were detected at roughly equivalent levels in Eß-/- DP {gamma}{delta}+ and DP {gamma}{delta}- thymocytes, which are close to those found in DN {gamma}{delta}+ thymocytes from the same mice (Fig. 5Go, top panel and legend for details on quantification, by phosphorimager scanning, of 32P emission from the amplified products). Detection of TCR{delta} joints in "{alpha}ß -like" DP cells is in agreement with published results demonstrating the persistence of these products as extrachromosomal circles in cells that have performed V{alpha}-to-J{alpha} rearrangement (17). Similarly, levels of V{gamma}1-to-J{gamma}4, V{gamma}2-to-J{gamma}2, and V{gamma}4-to-J{gamma}1 rearrangements were also found to be equivalent within the Eß-/- {gamma}{delta}+ (either DN or DP) and DP {gamma}{delta}- subsets (Fig. 5Go, middle panels). Parallel PCR-RFLP assays emphasized the rearrangement profile similarities between the two Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- subsets (Fig. 6Go). Overrepresentation of in-frame V{delta}4-to-J{delta}1 and V{delta}5-to-J{delta}1 junctions was obvious for both Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- cells, showing percentages above 63% in all cases, the lowest values being consistently observed in the latter subset (Fig. 6Go, upper panels). Analysis of V{gamma}2-to-J{gamma}2, V{gamma}4-to-J{gamma}1, and V{gamma}1-to-J{gamma}4 junctions gave lower rates of in-frame joints, ranging from 36 to 59% (Fig. 6Go, lower panels, and data not shown). Overall, these values are in general agreement with those predicted in the case of TCR{delta} and TCR{gamma} selection, respectively (see Materials and Methods for calculation), although this may be difficult to conclude for the junctions and cell subsets showing the lowest percentages (e.g., the V{gamma}2-to-J{gamma}2 rearrangements within the {gamma}{delta}- subset) because such values are close to that associated with random recombination. However, after cloning and sequencing of a total of 48 V{gamma}2-to-J{gamma}2 junctions from Eß-/- DP {gamma}{delta}- thymocytes, we found that 43.7% were in-frame (data not shown), a result which tends to further support a role for TCR{gamma} selection in generating the sampled population. Also consistent with this argument is the higher percentage of in-frame junctions observed for the V{gamma}4-to-J{gamma}1 joints (Fig. 6Go, lower right panel). This is most likely because of the fact that the V{gamma}4 gene carries an in-frame STOP codon at its 3' extremity (39) that precludes {gamma}-chain synthesis unless eliminated by the recombination reaction (i.e., being that STOP codons are not recognized by PCR-RFLP, an excess of in-frame joints involving this particular gene segment relative to other types of V{gamma}-J{gamma} junctions is precisely expected in the case of TCR{gamma} selection).



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  		FIGURE 5. Levels of V{delta}-to-J{delta} and V{gamma}-to-J{gamma} joints in Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- thymocytes. LR-PCR assays were used to detect V{delta}4-to-J{delta}1 rearrangements (top panel) and V{gamma}1-to-J{gamma}4 and V{gamma}2-to-J{gamma}1 rearrangements (middle panels) using genomic DNA from sorted DP {gamma}{delta}+ and {gamma}{delta}- thymocytes. Briefly, DNA was amplified using V-specific forward and J-specific reverse primers. At the TCR{delta} and TCR{gamma} loci, the unrearranged V and J segments are too far apart to be amplified within a common germline fragment. To control for the amount of DNA in the individual reaction, the nonrearranging TCRß2 constant region gene (Cß2) was amplified using forward and reverse primers located 5' of and inside exon 1, respectively. The source of DNA is indicated above each lane; PCR products corresponding to specific fragments are indicated to the right. Lanes 1–3 show a titration of sorted DN {gamma}{delta}+ DNA from an Eß-/- mouse. Input material was kept constant (100 ng of genomic DNA) by using decreasing amounts of DN {gamma}{delta}+ DNA and increasing amounts of Eß-/- kidney DNA. Lane 1, undiluted thymus; lanes 2 and 3, 1-to-5 and 1-to-10 dilutions, respectively. Densitometric analysis using a phosphorimager indicated that levels of gene rearrangements in Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- thymocytes relative to those in DN {gamma}{delta}+ thymocytes were, respectively, 71.6 and 78.4% (V{delta}4-to-J{delta}1); 85.8 and 104% (V{gamma}1-to-J{gamma}4); and 77 and 89.7% (V{gamma}2-to-J{gamma}2), after correcting for differences in the amount of loaded DNA as determined by scanning of Cß2 PCR products.

 


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  		FIGURE 6. TCR{delta} and TCR{gamma} rearrangement status in Eß-/- DP {gamma}{delta}+ and DP {gamma}{delta}- thymocytes. PCR-RFLP analyses for V{delta}4-to-J{delta}1, V{delta}5-to-J{delta}1, V{gamma}2-to-J{gamma}2, and V{gamma}4-to-J{gamma}1 rearrangements are shown. DNA from sorted thymocytes was PCR amplified, radiolabeled, and electrophoresed through a sequencing gel. The origin of DNA is indicated above each lane ({gamma}{delta}+ or {gamma}{delta}-, DP {gamma}{delta}+ or {gamma}{delta}- thymocytes, respectively). The position of in-frame rearrangements, determined from parallel DNA sequencing ladders (not show), is indicated by bars on the right of each panel. The frequency of such joints as a percent of the total signal, determined by densitometric analysis using a phosphorimager, is shown below each lane.

 
In conclusion, our analyses showing high levels of predominantly in-frame TCR{delta} joints in the Eß-/- DP {gamma}{delta}- thymocytes are consistent with a role for TCR{delta} selection in determining autonomous development of this cell subset. Similarly, LR-PCR, PCR-RFLP, and sequencing data also support TCR{gamma} selection of most, if not all, cells within this subpopulation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have analyzed the effects of TCRß enhancer deletion on thymic cell differentiation, with the goal of evaluating the resulting T cell developmental defects compared with those described in the existing pre-TCR-deficient mouse models. We have shown that, similar to TCRß-/- mice (15), there is a specific lack of TCRß-chain expression in the Eß-/- animals. In addition, we have shown that the DP cells that develop in the Eß-/- thymus are comprised of distinct TCR- and TCR{gamma}{delta}+ subsets, in dissimilar proportions. The former, predominant subset includes relatively immature T cells committed to the {alpha}ß lineage (as evidenced notably by ongoing TCR-J{alpha} recombination), although they carry mostly productive TCR{delta} gene rearrangements and, most likely, productive TCR{gamma} gene rearrangements as well. Eß-/- {gamma}{delta}+ DP thymocytes, on the other hand, exhibit no V(D)J recombination activity and, as discussed further below, may represent either immediate precursors to the {alpha}ß-committed DP thymocytes or, alternatively, a minor branch of terminally rearranged {gamma}{delta} T cells. These results illustrate the critical function of Eß in regulating {alpha}ß T cell differentiation and shed further light on pre-TCR-independent process(es) of {alpha}ß-T cell development.

Essential role of Eß during {alpha}ß T cell differentiation

The defect in TCRß-chain expression in Eß-deleted T cells suffers from no leakiness; otherwise, DP thymocytes would have developed in the TCR{delta}-/- Eß-/- mice. This is a remarkable phenotype considering the limited extent (560 bp in length) of the Eß deletion (23) compared with the large size (>500 kb in the mouse) and complex structure of the TCRß locus (40). As demonstrated by us and by others (23, 25, 41), deletion of Eß severely affects V(D)J recombination of cis-linked gene segments, although the precise mechanism(s) by which this effect is mediated is still under investigation. Strikingly, knock-out deletion of enhancer elements from other TCR and Ig genes generally results in a consistent but less severe defect (i.e., V(D)J recombination is decreased at the targeted locus, but production of the corresponding polypeptide and mature lymphocytes in the relevant lineage is not completely abolished (42, 43)). This particularity could be related to the fact that, whereas the TCRß locus has only one known enhancer, other Ig and TCR loci carry at least two such elements. However, it is equally possible that, depending on the locus considered, other cis-regulatory elements (e.g., different from transcriptional enhancers (44)) could provide on their own and/or complement, at least so some extent, the recombination enhancing function. Introducing cis-linked mutations in Ag receptor gene loci (45) will help to clarify these issues.

Origin, mode of differentiation, and fate of the Eß-/- DP thymocytes

Analysis of Eß-/- mice adds to the general picture that, paradoxically, the lack of a pre-TCR does not impair the development of a few DP cells (2, 5). Our cellular and molecular studies identify distinct {gamma}{delta}+ and {gamma}{delta}- subsets within the Eß-/- DP compartment. The DP {gamma}{delta}+ subset represents a minority of cells in the mutant thymus. It exhibits the highest rate of CD3-{epsilon} staining and lowest rate of cell divisions. Molecular analyses confirm the {gamma}{delta}+ phenotype and detected no sign of V(D)J recombination in this population. It has been reported that {gamma}{delta}+ cells are not found within the DP compartment from TCRß-/- thymi (22), although the presence of a small population of TCR-{delta}-positive cells has occasionally been described (15, 16). However, these cells have not been characterized in detail, although one report does show that they exhibit a high rate of proliferation (16), opposite to the slow dividing rate that we found for the Eß-/- DP {gamma}{delta}+ thymocytes, suggesting that these two types of {delta}+ DP cells may be different. Conversely, in the same study of TCRß-/- mice, populations of slowly dividing CD44-CD25+ and CD44-CD25- thymocytes that develop along the {gamma}{delta} pathway have been identified (16). The Eß-/- DP {gamma}{delta}+ thymocytes may be derived from similar cells and therefore may represent a minor branch of cells in the {gamma}{delta} lineage that for unknown reasons express the CD4 and CD8 coreceptors. The possible basis for the discrepancy between the Eß-/- and TCRß-/- mouse strains in terms of {gamma}{delta} gene expression within the DP cell compartment is currently unclear. It is unlikely to be the result of a difference in the genetic background, because all strains were bred on the C57BL6/J background for several (n > 9) generations. An intriguing possibility would be that it results, by an unknown mechanism(s), from their significant differences in the engineered genomic alterations at the TCRß locus (e.g., differences in the extent of the targeted deletion; presence or not of the selectable Neo gene (15, 23). Finally, it may be noteworthy that the Eß-/- DP {gamma}{delta}+ subset appears roughly equivalent, in terms of absolute cell numbers, to a minute population of CD4+CD8+ DP {delta}+ thymocytes identified in the normal thymus (16), suggesting that both may belong to the same pathway and that the Eß-/- DP {gamma}{delta}+ subset is not peculiar to this mutant strain. Indeed, a significant proportion of late embryonic thymic {gamma}{delta}+ T cells are DP (46), and the promotion of CD4/CD8 surface expression by the {gamma}{delta} TCR alone has been reported (47).

The remaining TCR- thymocytes constitute a majority of the DP cell subset in the Eß-/- thymus. Although comprised of mostly CD3-{epsilon}- cells and consisting of an elevated proportion of CD25+ cells, this subset obviously includes {alpha}ß lineage-committed thymocytes. Despite the fact that our analyses were performed on cell populations rather than on individual cells, the high level of in-frame TCR{delta} rearrangements found in the {gamma}{delta}- cells indicates that most cells within this subset have gone through a process of {delta} selection, in agreement with genetic evidence from the analysis of TCR{delta}-/- Eß-/- double knock-out mice. Most probably, selection for {gamma}-chain expression also occurred for a majority of cells in this subset, as supported by LR-PCR and PCR-RFLP profiles and sequencing analysis. A similar population of "{alpha}ß -like" TCR-negative cells, of which development would be promoted by the {gamma}{delta} TCR, has been postulated to arise within the TCRß-/- thymus (16, 18, 22). It has been argued that such a population follows a developmental pathway distinct from that of {gamma}{delta}+ cells. Our cellular and molecular analyses, notably those showing distinct dividing rates (Table IIIGo), T cell-specific gene expression profiles, and V(D)J recombination activity between the Eß-/- DP {gamma}{delta}+ and {gamma}{delta}- subsets (Figs. 3Go and 4Go), support this view (but also see below). As emphasized by Livak et al. (22), the possibility that cells carrying the same type of receptor adopt distinct developmental {gamma}{delta} or {alpha}ß lineages strongly supports a "stochastic selection" mechanism of early T cell commitment and differentiation, later amplified by lateral cell-cell interactions that may influence the final outcome (48). However, based on the overall low rates of productive V{gamma}-to-J{gamma} joints found in TCRß-/- DP thymocytes, Hayday and colleagues (16) have discussed another possibility that development of some {delta}-selected cells in this population may be TCR{gamma}-independent, leading then to a more complex picture in which "stochastic selection" and "instructive" modes of {alpha}ß/{gamma}{delta} lineage commitment may coexist. Proposed basis for this type of selection included receptors made of the association of the TCR{delta}-chain with another polypeptide such as, for example, pT{alpha} (for discussion, see Ref. 16). In this regard, we found little support for such a hypothesis because introducing the Eß-/- mutation onto a pT{alpha}- or TCR{alpha}-deficient background (hence testing for two factors that are expressed in the Eß-/- DP {gamma}{delta}- subset; Fig. 3Go) had no obvious effect on DP cell development in the double mutants (I.L., unpublished data). Along the same lines, a significant role for TCR{gamma}-pT{alpha} complexes in mediating the DN-to-DP cell transition (34) was also ruled out because DP cells never exceeded 0.5% of total TCR{delta}-/- Eß-/- thymocytes (Table IGo and Fig. 1GoA). The possibility remains that production of DSBs at the TCRß locus in Eß-/- and TCRß-/- animals (Ref. 25 ; W.M.H. and P.F., unpublished data) may trigger an intracellular signaling pathway(s) that facilitates DP development, by analogy with the induction of the DN-to-DP transition in RAG-deficient mice by sublethal gamma-irradiation (a treatment known to induce DNA DSBs) (49, 50). However, it is difficult to explain how such a process could preferentially affect TCR {delta}-selected cells. Finally, it has to be stressed that the utilization of a {gamma}{delta} TCR (or a putative {delta}-based TCR) to commit and/or differentiate along the {alpha}ß pathway may be a feature that is readily observable in genetically engineered TCRß-deficient mice but that is marginal in the normal situation (confined, for example, to development of a few cells that have failed to productively rearrange the TCRß locus). However, in normal mice the pre-TCR would readily bypass these relatively inefficient processes to play an instructive role that actively boosts {alpha}ß development, as recently proposed (19).

Whereas the Eß-/- "{alpha}ß -like" thymocytes must be eliminated at the DP stage because of the absence of a TCR and lack of positive selection, the behavior of DP cells within the {gamma}{delta}+ subset is more uncertain. Elevated levels of Annexin V staining found in this population (as in the "{alpha}ß -like" DP subset) imply that a large proportion is committed to die (I.L., unpublished data). However, some cells may represent the precursors either of the CD4+/CD8+ SP {gamma}{delta}+ cells present in the peripheral lymphoid tissues in the Eß-/- mouse (I.L. and C.V., unpublished data) or, alternatively, of the Eß-/- DP {gamma}{delta}- cells discussed above (a developmental progression that would imply extensive changes in gene expression programs, e.g., the reactivation of rag gene expression and V(D)J recombination activity, of pT{alpha} gene expression, increased cell proliferation, etc.). These possibilities, which could possibly coexist, are currently under investigation.


   Acknowledgments
 
We thank Drs. M. Bonneville, S. Candéias, and P. Naquet for critical reading of this manuscript, C. Beziers La Fosse for preparing the artwork, N. Brun-Roubereau and M. Barrad for helping in the cytofluorometric analyses, and M. Pontier and G. Warcollier for maintaining the mouse colonies.


   Footnotes
 
1 This work was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique and by specific grants from the Association pour la Recherche sur le Cancer, the Commission of the European Communities, the Fondation Princesse Grace de Monaco (to P.F.), and the Ligue Nationale Contre le Cancer (to I.L. and P.F.). Back

2 Current address: Institut de Recherche Jouveinal/Parke-Davis, 3–9 rue de la Loge, 94265 Fresnes Cedex, France. Back

3 Current address: Institut National de la Santé et de la Recherche Médicale U491, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Back

4 Address correspondence and reprint requests to Dr. Pierre Ferrier, Centre d’Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Case 906, 13288 Marseille Cedex 9, France. Back

5 Abbreviations used in this paper: DN, double negative; DP, double positive; SP, single positive; Eß, TCRß gene enhancer; LR-PCR, long-range PCR; LM-PCR, ligation-mediated PCR; SE, signal end; WT, wild type; HSA, heat-stable antigen; DSB, double strand break; RAG, recombinase-activating gene. Back

Received for publication June 17, 1999. Accepted for publication May 22, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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