Allelic Exclusion in pTalpha -deficient Mice: No Evidence for Cell Surface Expression of Two T Cell Receptor (TCR)-beta Chains, but Less Efficient Inhibition of Endogeneous Vbeta right-arrow (D)Jbeta Rearrangements in the Presence of a Functional TCR-beta Transgene

By Anna Krotkova,* Harald von Boehmer,Dagger and Hans Jörg Fehling*

From the * Basel Institute for Immunology, CH-4005 Basel, Switzerland; and the Dagger  Institut Necker, Institut National de la Santé et de la Recherche Medicale 373, F-75730 Paris Cedex 15, France

Summary
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
References


Summary

Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-beta chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-beta chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-beta rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-beta rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-alpha (pTalpha ) chain, a core component of the pre-TCR. Staining of CD3+ thymocytes and lymph node cells with antibodies specific for Vbeta 6 or Vbeta 8 and a pool of antibodies specific for most other Vbeta elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTalpha -deficient mice expressing a functionally rearranged TCR-beta transgene. Interestingly, although the transgenic TCR-beta chain significantly influenced thymocyte development even in the absence of pTalpha , it was not able to inhibit fully endogeneous TCR-beta rearrangements either in total thymocytes or in sorted CD25+ pre-T cells of pTalpha -/- mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.


Functional TCR genes are assembled by a program of somatic gene rearrangements from variable (Vbeta )1 gene segments, diversity (Dbeta ) genes, and joining (Jbeta ) elements at the TCR-beta loci and from Valpha and Jalpha elements at the TCR-alpha loci. At the TCR-beta locus, Dbeta right-arrow Jbeta rearrangements precede Vbeta right-arrow DJbeta rearrangements. Although this process of V(D)J recombination could theoretically give rise to cells with two in-frame TCR rearrangements at corresponding alleles, and thus two functional alpha  or beta  TCR genes, virtually all T lymphocytes of the alpha beta lineage express only one particular TCR-beta chain, a phenomenon referred to as allelic exclusion. Analysis of an increasing number of T cell clones and hybridomas has revealed that allelic exclusion at the TCR-beta locus is largely due to the fact that alpha beta T cells carry as a rule only one productive TCR-beta rearrangement, whereas the rearrangement on the other allele is either incomplete (DJbeta ) or out of frame (1). These findings are in line with the notion that a productive TCR-beta rearrangement can somehow prevent further rearrangements at the TCR-beta locus. Strong support for this hypothesis has been obtained in mice expressing productively rearranged TCR-beta transgenes (2, 3), which enforce almost complete inhibition of endogeneous Vbeta right-arrow DJbeta rearrangements, whereas Dbeta right-arrow Jbeta rearrangements were essentially unimpaired. In contrast, no inhibition of endogeneous TCR-beta rearrangements could be observed in mice expressing a nonproductive TCR-beta transgene (3).

In mature T cells, the rearrangement status of the TCR-alpha locus differs from that of the TCR-beta locus in that usually both alleles carry Valpha right-arrow Jalpha rearrangements and cells with two functional TCR-alpha alleles are easily detectable (1, 4, 5). In fact, in TCR-alpha transgenic mice there is no or only very inefficient inhibition of endogeneous Valpha right-arrow Jalpha rearrangements (6, 7, 8). Thus, it appears that rearrangements in the TCR-alpha locus continue on both alleles until a receptor is formed that can bind to thymic MHC molecules and induce positive selection, an event that leads to downregulation of RAG expression and complete termination of all TCR gene rearrangements (6, 9, 10, 11).

While, in general, TCR-alpha rearrangements occur relatively late during thymocyte development, primarily at the transition from the double-negative (DN) to the double-positive (DP) stage and during the DP stage itself (12, 13), TCR-beta rearrangements are initiated and completed much earlier, namely at a CD4-8- (DN) stage defined by the expression of the IL-2 receptor alpha  chain (CD25) (12, 14). Any model postulating a negative feedback of functional TCR-beta chains on rearrangement at the second beta  allele therefore presumes a signaling function of TCR-beta in the absence of TCR-alpha . A similar situation is encountered in B cells where IgH chains are thought to inhibit further rearrangements at the IgH locus, well before mature IgL chains become available. The discovery of the pre-B cell receptor (BCR) (15, 16) and pre-TCR (17, 18) provided likely candidates for the signaling machinery mediating allelic exclusion at the corresponding loci in the absence of mature light chains or TCRalpha chains, respectively, because these receptors consist, in the case of the pre-BCR, of a conventional IgH chain paired with surrogate light chains lambda 5 and VpreB (along with signal-transducing Igalpha [mb-1] and Igbeta [B29] proteins) (19) and, in the case of the pre-TCR, of a conventional TCR-beta chain disulfide-linked to the invariant pre-TCR-alpha (pTalpha ) chain (in association with components of the CD3 complex) (20). Surprisingly, however, analysis of the limited number of mature B cells that develop in lambda 5-deficient and therefore pre-BCR-defective mice did not provide any evidence for violation of allelic exclusion (21). On the other hand, more recent experiments seem to indicate that allelic exclusion is not fully operating in the absence of lambda 5 when precursor rather than mature B cells are studied (22).

The role of the pre-TCR in allelic exclusion of the TCR-beta locus has been investigated lately in mouse chimeras that were generated by injecting TCR-beta -transgenic, pTalpha -/- embryonic stem (ES) cells into RAG-/- blastocysts (23). Analysis of ES cell-derived thymocytes in these chimeric mice revealed equivalent inhibition of endogeneous Vbeta right-arrow DJbeta rearrangements in the presence and absence of the pTalpha chain and provided no evidence for the existence of T lymphocytes with more than one TCR-beta chain (23). Here, we report on allelic exclusion in stable lines of pTalpha -/- and TCR-beta -transgenic, pTalpha -/- mice rather than chimeric animals, which permitted a more rigorous analysis. Our results indicate an involvement of the pre-TCR in allelic exclusion at the TCR-beta locus, but they also show that TCR-beta signaling is not completely compromised in the absence of pTalpha , a finding that may explain why it has been difficult so far to demonstrate an effect of the pre-TCR, and by inference of the pre-BCR, on allelic exclusion when analysing pTalpha - (23) or lambda 5-deficient mice (21).


Materials and Methods

Mice. C57BL6 (B6) mice, which were used in most experiments as wild-type controls, were purchased from IFFA-Credo (France). All other genetically modified mice (pTalpha -/-, TCR-beta -transgenic, RAG-2-/-, and the respective intercross offspring) were bred and maintained in a specific pathogen-free facility of the Basel Institute for Immunology (Switzerland). All animals used were 6-12 wk of age. pTalpha -deficient mice were identified by Southern blotting of PstI-digested tail DNA using as a probe a genomic PstI-XhoI fragment (~610 bp) corresponding to positions 5,432 to 6,046 of the pTalpha gene (numbering according to reference 24). TCR-beta -transgenic mouse lines expressing functionally rearranged, Vbeta 8.2 transgenes have been described previously (2). In our experiments, we have used the line with ~20 copies. Transgenic mice were identified by PCR with 1 µg of genomic tail DNA as template and primers specific for Vbeta 8.2 (5'-GCATGGGCTGAGGCTGATCCATTA-3') and a region located immediately 3' of the Jbeta 2 cluster (5'-TGAGAGCTGTCTCCTACTATCGATT-3'). Cycling conditions were as follows: 1 min at 94°C (denaturation); 30 s at 94°C; 1 min at 55°C; and 1 min 30 s at 72°C; 25 cycles. Amplification of a ~920-bp fragment indicated the presence of TCR-beta transgenes. Breeding stocks of RAG-2-deficient mice have been provided by Dr. F.W. Alt (Boston, MA). RAG-2-/- animals were identified based on the absence of B220+/surface Ig+ B lymphocytes as evidenced by double staining of peripheral blood cells with B220- PE and sheep anti-mouse Ig-FITC antibodies.

Antibodies and Flow Cytometry. The following antibodies have been used (all purchased from PharMingen, San Diego, CA, unless stated otherwise): FITC anti-mouse CD3epsilon (2C11); biotin anti-mouse CD3epsilon (500A2); FITC anti-mouse CD4 (H129.19); R-PE anti-mouse CD4 (H129.19; GIBCO BRL, Gaithersburg, MD); FITC anti-mouse CD8alpha (53-6.7); R613 anti-mouse CD8alpha (53-6.7; GIBCO BRL); FITC anti-mouse CD11b (Mac1alpha ) (M1/70); FITC anti-mouse CD19 (1D3); biotin anti-mouse CD25 (7D4); R-PE anti-mouse CD44 (IM7); PE anti-mouse gamma delta TCR (GL3); FITC anti-mouse Ly-6G (GR1) (RB6-8C5); R-PE anti-mouse CD45R/B220 (RA3-6B2); FITC anti-mouse Vbeta 6 (RR4-7); FITC anti-mouse Vbeta 8.1/8.2 (MR5-2); PE anti- mouse Vbeta 2 (B20.6); PE anti-mouse Vbeta 3 (KJ25); PE anti-mouse Vbeta 4 (KT4); PE anti-mouse Vbeta 5.1/5.2 (MR9-4); PE anti-mouse Vbeta 6 (RR4-7); PE anti-mouse Vbeta 7; PE anti-mouse Vbeta 8.1/8.2 (MR5-2); PE anti-mouse Vbeta 10b (B21.5); PE anti-mouse Vbeta 11 (RR3-15); PE anti-mouse Vbeta 13 (MR12-3); FITC sheep anti- mouse Ig F(ab')2 fragment (Silenus, Australia). Streptavidin conjugates: Streptavidin-Tricolor (Caltag, CA) and streptavidin-APC (Molecular Probes, OR).

For flow cytometry, single cell suspensions from thymi and lymph nodes (axial, mesenteric, inguinal) were prepared in PBS containing 2% calf serum (CS). The number of viable cells was determined using a Coulter counter. Thymocytes were stained at 5 × 106 cells per ml in PBS, 2% CS containing the relevant antibodies at saturating concentrations. When pools of anti-Vbeta reagents were used, it proved necessary to dialyze the antibody mix containing all first-step antibodies immediately before the staining, to eliminate the cytotoxicity of the concentrated reagents. Dialysis was performed at 4°C in ultra thimbles (Schleicher & Schuell UH 100/10; cutoff ~10,000 MM) against two changes of PBS, 2% CS for 2 × 30 min. Phenotypes and proportions of thymocyte subsets were analyzed by three-color flow cytometry using a FACScan® (Beckton Dickinson, Mountain View, CA) and the Lysis II program. The data depicted in Figs. 2 and 3 were analyzed with the program Cellquest (Becton Dickinson). Dead cells were excluded from the analysis by forward- and side-scatter gating, when analyzing numerically small subpopulations also by addition of propidium iodine (PI) and gating on PI- cells. Four-color analyses and cell sorting were performed on a FACStar Plus® (Beckton Dickinson) equipped with a pulse processor for forward-scatter width (FSC-W) in order to gate out cell doublets.
Fig. 2. Vbeta 8.1/8.2 versus Vbeta -pool staining of lymph node cells from TCR-beta -transgenic mice expressing (pTalpha +) or lacking (pTalpha -/-) a functional pTalpha gene (four-color analysis). The transgene-encoded receptor contains the Vbeta 8.2 element. Staining and gating was done as described in Fig. 1.
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Fig. 3. The effects of TCR-beta transgenes on various aspects of thymic T cell development in the absence of pTalpha (the right most panel in A, B, and C). A TCR-beta -transgenic mouse expressing functional pTalpha (TCR-beta -transgenic, pTalpha +), a wild-type C57BL6 (WT [B6]), and a nontransgenic pTalpha -deficient (pTalpha -/-) mouse are included as controls. (A) CD4/CD8 profiles. The figures on top of each panel give the total number of thymocytes found in the particular mouse analyzed in this experiment. These values are very typical for mice with the respective genotype as seen in many similar experiments. (B) Analysis of triple-negative (CD3-CD4-CD8-) thymocytes for differential expression of the developmental markers CD25 and CD44. Thymocytes from three mice of the same genotype were pooled. CD4- and CD8-expressing cells (single positive and DP thymocytes) from B6 and TCR-beta -transgenic, pTalpha + mice were complement depleted (see Materials and Methods) before staining. For cytofluorometric analysis, thymocytes were incubated with an antibody mix containing CD4-FITC, CD8-FITC, CD3-FITC, GR1-FITC, MAC1-FITC, CD19-FITC (the latter three antibodies are specific for granulocytes, macrophages, and B cells, respectively), CD44-PE and CD25-biotin, and in a second step with streptavidin-Tricolor. FITC-positive cells were excluded from the analysis by electronic gating (data not shown). The cells shown in the four panels are thus highly enriched for immature triple-negative thymocytes. (C) The generation of gamma delta -expressing cells is suppressed in TCR-beta -transgenic mice, even in the absence of pTalpha . Thymocytes were stained with a PE-conjugated TCR-gamma delta -specific antibody and CD3-biotin, in a second step with streptavidin-Tricolor. Only thymocytes that are positive for both CD3 and TCR-gamma delta were considered as genuine gamma delta -expressing cells (as defined by the rectangular gate). The data shown in A and C were obtained in the same experiment with the same mice.
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Fig. 1. Vbeta 8.1/8.2 versus Vbeta -pool staining of thymocytes and lymph node cells from a wild-type C57BL6 (WT [B6]) and a pTalpha -deficient (pTalpha -/-) mouse (four-color analysis). Thymocytes or lymph node cells were stained with an antibody mix containing CD3-biotin, Vbeta 8.1/8.2- FITC, and nine distinct PE-conjugated anti-Vbeta antibodies (anti-Vbeta 2, 3, 4, 5.1/5.2, 6, 7, 9, 10b, 13). CD3+ cells were detected with a streptavidin-Tricolor reagent. Before acquisition, PI was added. (Top) Dot blots of cells that have passed three gates: the FSC/SSC gate for live cells, the gate for PI- cells, and the pulse-width gate that eliminates cell doublets (data not shown). (Bottom) The cells depicted have passed in addition the gate for CD3high cells shown in the top.
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Depletion of CD4 and CD8 Expressing Thymocytes. Single-cell suspensions from thymi of three individual mice with the same genotype were pooled, resuspended in 8 ml serum-free DMEM medium, and incubated simultaneously with 1 ml of anti-CD8 (31M) and 1 ml of anti-CD4 (RL172.4) antibody supernatants on ice for 30 min. The cells were then washed in 10 ml DMEM, 2% CS and resuspended in 9 ml of the same medium. After adding 1 ml of freshly dissolved rabbit complement (Low-Tox-M, Cedarlane, Hornby, Canada), the suspension was incubated at 37°C for 40 min. Surviving cells were purified by Ficoll density-gradient centrifugation, washed and resuspended in PBS, 2% CS. The efficiency of CD4/CD8 depletion was usually more than 95% as determined by staining purified thymocytes with R-PE anti-mouse CD4 (H129.19) and R613 anti-mouse CD8alpha (53-6.7) antibodies, which bind epitopes distinct from those recognized by antibodies 31M and RL172.4.

Detection of Endogeneous TCR Rearrangements. High molecular weight thymocyte DNA was prepared as follows: cells from two thymi (pTalpha + mice) or three thymi (pTalpha -/- mice) of the same genotype were pooled and digested in proteinase K solution (50 µg/ml proteinase K, 0.5% SDS) at 50°C for 24 h. The DNA was phenol/chloroform extracted, precipitated with ethanol, recovered, and dissolved in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) at a concentration of 100 µg/ml. PCR-based analysis of TCR-beta chain rearrangements was performed using an assay originally described by van Meerwijk et al. (25) and modified by Anderson et al. (26). Vgamma 4-Jgamma 1 rearrangements (nomenclature according to reference 27) were detected in a similar fashion. Primers specific for Vbeta 4 (5'-CCTGATATGCGAACAGTATCTAGGC-3'), Vbeta 6 (5'-GAAGGCTATGATGCGTATCG-3'), Vbeta 11 (5'-TGCTGGTGTCATCCAAACACCTAG-3'), Vbeta 12 (5'-AGTTACCCAGACACCCAGACATGA-3'), and a region immediately downstream of the last Jbeta segment in the Jbeta 2 gene cluster (5'-TGAGAGCTTGTCTCCTACTATCGATT-3') were used to detect Vbeta right-arrow (D)Jbeta rearrangements, and primers specific for Vgamma 4 (5'-TGTCCTTGCAACCCCTACCC-3') and Jgamma 1 (5'-CAGAGGGAATTACTATGAGC-3') were used to detect the respective rearrangement in the TCR-gamma locus. Template DNA was used at three or more different concentrations in each experiment to ensure linearity of the PCR signal. As a control for the amount of template DNA, aliquots of a PCR mastermix were amplified with oligonucleotides specific for the IgM constant region (5'-CACTAGCCACACCCTTAGCAC-3' and 5'-TGGCCATGGGCTGCCTAGCCCGGGACTT-3'). PCR amplifications were performed in 50 µl reaction buffer containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.01% gelatin, 2.5 mM MgCl2, 0.2 mM of each dNTP (Pharmacia, Piscataway, NJ), 1 pmol of each primer, 0.5U Perfect Match PCR enhancer (Stratagene, La Jolla, CA), 1 U AmpliTaq DNA polymerase (Hoffmann-La Roche, Basel, Switzerland) and the indicated amount of template DNA. The amplification cycle consisted of 45 s at 94°C, 1 min 30 s at 62°C (Vbeta right-arrow [D]Jbeta ) or 50°C (Vgamma 4right-arrow Jgamma 4), and 2 min at 72°C. The cycle was repeated 26 times. PCR products were separated on 1.2% agarose gels, blotted with a vacuum blotter (Bio-Rad; Richmond, CA) onto Hybond N+ nylon membrane (Amersham, Arlington Heights, IL) and detected by hybridization with end-labeled Jbeta 2.7-(5'-TATGAACAGTACTTCGGTCCC-3'), Jgamma 1-(5'-TGAATTCCTTCTGCAAATACCTTG-3'), or Cµ-specific (5'-CCTGCCCAGCACCATTTCCTTC-3') probes, as appropriate.


Results

No Evidence for Cell Surface Expression of Two TCR-beta Chains in pTalpha -/- Mice.

Assuming sufficient time for TCR-beta rearrangements on both alleles, no feedback inhibition after a functional TCR-beta chain has been generated and no selective disadvantage of cells with two functional TCR-beta chains, one would expect that up to 1/5 (20%) of all TCR-beta + cells express two types of beta  chains, indicating the complete absence of allelic exclusion (see reference 4 for calculations). Any, even partial violation of these assumptions would reduce the percentage of double-expressing cells accordingly, albeit unpredictably. Given the low frequency of cells that express a particular Vbeta element, it is clear that evidence for incomplete allelic exclusion is not easy to obtain by simple cell surface staining with antibodies specific for just two particular Vbeta elements.

To alleviate this problem, we used a combination of antibodies directed against Vbeta 6 or Vbeta 8.1/8.2 elements on one hand and a pool of nine antibodies recognizing TCRs that contain most of the remaining Vbeta elements on the other. Vbeta 6- or Vbeta 8.1/8.2-specific antibodies were chosen as individual antibodies, because they detect the two most frequent Vbeta T cell populations on the B6 background, representing ~5% and 15% of all TCR-alpha beta + cells, respectively. To increase further the sensitivity of our analysis, we included as a third color a CD3epsilon -specific antibody, which allowed us to gate on CD3+ thymocytes/lymphocytes and exclude all other cells that do not express a TCR or only at low levels. Dead cells, which are known to absorb antibodies nonspecifically and which could therefore give rise to apparently double-expressing cells, were excluded not only by gating on live cells in a forward scatter/side scatter (FSC/SSC) analysis, but also by including PI staining as a fourth color and subsequent gating on PI- cells. By using the FSC pulse-width program on our flow cytometer, we could also efficiently exclude cell doublets, which otherwise would have raised the background of false TCR double-expressing cells.

Fig. 1 shows the result of a representative analysis using the Vbeta 8.1/8.2-specific antibody as the individual antibody and anti-Vbeta 2, 3, 4, 5.1/5.2, 6, 7, 9, 10b, and 13 as pooled antibodies. In the B6 control mouse, the frequency of CD3+ thymocytes and lymph node cells scoring positive for Vbeta 8.1/8.2 and one of the Vbeta s represented in the pool is extremely low (~0.1-0.2%). This value may reflect the low proportion of T cells that genuinely express two beta  chains, violating allelic exclusion, as suggested by studies of human T lymphocytes (28, 29), or it could just indicate the level of nonspecific background staining in our experiment. More important in this context, however, no significant increase in the percentage of potentially double-expressing cells could be detected in the pTalpha -/- mouse, despite this very sensitive staining technique. These data clearly show that T cells and thymocytes expressing intermediate to high levels of the alpha beta TCR are allelically excluded even in pTalpha -/- mice, at least at the level of cell surface expression. Equivalent results were obtained in a similar staining with Vbeta 6 as the individual antibody versus a pool of antibodies including anti-Vbeta 8.1/8.2 (data not shown).

Lack of Cell Surface Expression of Endogeneous TCR-beta Chains in TCR-beta -transgenic, pTalpha -deficient Mice.

To increase further the sensitivity of our anti-Vbeta staining, we included TCR-beta -transgenic mice in our analysis. As transgenic line, we used mice expressing a functional Vbeta 8.2 TCR-beta transgene, previously shown to prevent expression of endogeneous TCR-beta chains (2). As a consequence, essentially all mature T cells in these mice stain positive for Vbeta 8.2, while no other Vbeta elements can be detected on the cell surface. To assess the efficiency of allelic exclusion in the absence of pTalpha , we crossed mice of the TCR-beta -transgenic line with pTalpha -deficient animals and performed a four-color cytofluorometric analysis as described above, using Vbeta 8.1/8.2 to detect the transgenic TCR-beta chain and all other available anti-Vbeta reagents in a pool to detect expression of endogeneous TCR-beta chains. Although the presence of the transgene allowed us to screen essentially all CD3+ cells for expression of two TCR-beta chains rather than a fraction of cells expressing a particular Vbeta element (i.e., Vbeta 8.1/8.2 or Vbeta 6) as above, we still found no evidence for cell surface expression of two distinct TCR-beta chains, as endogeneous TCR-beta chains could not be detected either on thymocytes (data not shown) or on mature T cells (Fig. 2) even in the absence of pTalpha . These data, in conjunction with those from nontransgenic pTalpha -/- mice, strongly suggest that functional TCR-beta chains can regulate the expression of other TCR-beta genes even in the absence of an intact pre-TCR.

A TCR-beta Transgene Can Mediate Effects in Early T Cell Development Despite the Absence of pTalpha .

pTalpha -deficient mice exhibit a severe defect in early T cell development, which leads to a dramatic decrease in the relative proportion and absolute number of DP thymocytes and a concomitant, more than 90% reduction in total thymic cellularity (30). While analyzing Vbeta surface expression in TCR-beta -transgenic mice, we noticed that the presence of the TCR-beta transgene on a pTalpha -/- background had a mild, but reproducible, effect on thymic cellularity, causing an up to threefold increase in the total number of thymocytes as compared with nontransgenic pTalpha -/- controls (Fig. 3; data not shown). This was usually accompanied by a reversion in the DP/DN thymocyte ratio (Fig. 3, compare the pTalpha -/- and the TCR-beta -transgenic, pTalpha -/- panels), although the relative increase in the proportion of DP versus DN thymocytes varied considerably between individual TCR-beta -transgenic, pTalpha -/- mice, possibly reflecting the weakness of the signal involved in this phenomenon.

The observation that a TCR-beta transgene could influence thymic cellularity even in the absence of pTalpha , prompted us to analyze the effect of the beta  transgene on early T cell development in pTalpha -deficient mice. Early T cell development within the CD4-8- population can be subdivided into four discrete stages that are defined by the differential expression of CD44 and CD25 surface markers (31). In normal mice, TCR-beta rearrangements take place at or shortly before the CD25+44-/low stage and only those thymocytes that manage to express a functional TCRbeta chain can form a complete pre-TCR, which is required to trigger expansion and progression to the next developmental stage, defined as CD25-CD44-/low. In pTalpha -deficient mice, which cannot assemble an intact pre-TCR, this transition is severely impaired and relatively few CD25-CD44-/low cells are generated (Fig. 3 B, compare the two left panels). On the other hand, in pTalpha + mice expressing a TCRbeta transgene, essentially all CD25+ thymocytes can form a pre-TCR, resulting in a strong increase in the proportion of CD25-44-/low cells and a concomitant reduction in the percentage of CD25+CD44-/low thymocytes (Fig. 3 B, third panel from the left). Interestingly, analysis of TCR-beta -transgenic mice lacking pTalpha revealed a marked down regulation of CD25 in comparison with nontransgenic pTalpha -/- mice and the appearance of a significant population of CD25-44-/low cells (compare second and fourth panel in Fig. 3 B). However, this effect of the TCR-beta transgene was not as prominent as in the pTalpha + background. These data indicate that the TCR-beta transgene can actually influence the CD25+ compartment of CD4-8- thymocytes in the absence of pTalpha in a similar way as in pTalpha + mice, although to a much lesser extent.

Previous experiments in normal (pTalpha +) mice have shown that a TCR-beta transgene inhibits TCR-gamma rearrangements and the generation of gamma delta expressing cells (32). Interestingly, the TCR-beta transgene was able to suppress gamma  rearrangements, as measured by PCR with primers specific for Vgamma 4 and Jgamma 1, even in the absence of pTalpha (Fig. 4), although somewhat less efficiently than in the presence of pTalpha . The TCR-beta transgene also mediated a strong reduction in the number of gamma delta cells in pTalpha -deficient mice (see Fig. 3 C). This effect appears particularly striking, when taking into account that the percentage and absolute number of gamma delta cells is strongly augmented in normal (nontransgenic) pTalpha -/- mice in comparison to wild-type littermates (30) (Fig. 3 C, compare the two left panels). The inhibition of TCR-gamma rearrangements and the strong suppression of gamma delta T cell development illustrates once more that the TCR-beta transgene is not innocuous despite the absence of pTalpha .


Fig. 4. Expression of TCR-beta transgenes inhibits Vgamma -Jgamma rearrangements. Thymocyte DNA from four types of mice (pTalpha + or pTalpha -/- mice with or without TCR-beta transgenes [beta -TG]) was amplified with primers specific for Vgamma 4 and Jgamma 1 (nomenclature according to reference 27). Three different amounts of template DNA (300 ng, 200 ng, 100 ng) were tested to ensure linearity of the signals. An aliquot of the PCR mastermix was amplified with primers specific for the constant region of IgM to provide an internal control for the amount of template used (Ig-C bands). Specific bands were detected with appropriate end-labeled oligonucleotide probes. N denotes a negative control (kidney DNA).
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Introduction of a functionally rearranged TCRbeta transgene into mice lacking one of the recombination activating genes (RAG-1 or RAG-2) leads to the induction of DP cells and the generation of essentially normal numbers of thymocytes (33), a result thought to reflect the physiological function of the pre-TCR. In this context, it was of interest to determine the potential effects of a TCR-beta transgene on thymocyte development in RAG-/- mice lacking pTalpha . To this end, we crossed RAG-2-/- mice carrying the TCR-beta transgene with mice deficient for both RAG-2 and pTalpha , and analyzed thymi from littermates of the F2 intercross. Fig. 5 shows the result of a representative staining experiment. There was no difference between nontransgenic RAG-2-/-, pTalpha + and RAG-2-/-, pTalpha -/- mice with respect to the total number of thymocytes (~1-4 × 106) and the CD4/CD8 profiles (virtually complete absence of CD4+8+ cells) (Fig. 5, top right; data not shown). Introduction of a TCR-beta transgene in RAG-2-deficient, pTalpha + mice resulted in a dramatic increase in total thymocyte numbers and efficient generation of DP thymocytes, as reported previously (33). Surprisingly, expression of the TCR-beta transgene in RAG-2-/- mice lacking pTalpha resulted in the generation of a significant number of DP thymocytes as well, although the total thymic cellularity was raised only modestly (three- to fivefold over RAG-2-/- controls in four independent experiments), which was also reflected by the limited reduction in the percentage of DN thymocytes in these RAG-/-, pTalpha -/-, TCR-beta -transgenic mice (Fig. 5, bottom right). These findings are in full agreement with our observations regarding TCR-beta -transgenic, pTalpha -/- mice with a wild-type RAG backgound, reported above. Taken together, the data indicate that a transgenic TCR-beta chain can signal in the absence of pTalpha , promoting the differentiation of DN thymocytes into DP cells and inhibiting the generation of gamma delta -expressing cells, but that it cannot induce the massive cellular expansion, which normally accompanies the selection of DN CD25+44- thymocytes expressing a productive TCR-beta chain, unless being associated with pTalpha .


Fig. 5. Expression of TCR-beta transgenes in RAG-2-deficient mice in the absence of pTalpha induce differentiation, but no substantial proliferation of immature thymocytes. CD4 and CD8 staining of thymocytes isolated from a TCR-beta -transgenic, RAG-deficient mouse (pTalpha +) and from a TCR-beta -transgenic mouse that is RAG-deficient and in addition lacks pTalpha (pTalpha -/-). A wild-type C57BL6 (WT [B6]) and a RAG-deficient mouse are included as controls. The figures on top of each panel refer to the total number of thymocytes found in the respective animal. Similar results were obtained in four independent experiments. No differences were found between nontransgenic RAG-/- × pTalpha -/- and RAG-/- × pTalpha + mice (data not shown).
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Endogeneous Vbeta right-arrow (D)Jbeta Rearrangements Are Less Efficiently Blocked in TCR-beta -transgenic Mice Lacking pTalpha .

The expression of productively rearranged TCR-beta transgenes in thymocytes of normal (pTalpha +) mice inhibits Vbeta right-arrow (D)Jbeta rearrangements at endogeneous TCR-beta loci almost completely, which is considered as a paradigm for allelic exclusion. To determine the impact of a transgenic TCR-beta chain on TCR-beta rearrangements in the absence of pTalpha , we compared the relative levels of endogeneous Vbeta right-arrow (D)Jbeta rearrangements in thymocytes from TCR-beta -transgenic and nontransgenic pTalpha + and pTalpha -/- mice, using a sensitive DNA-PCR assay (see Materials and Methods; Fig. 6 A, legend). As reported previously (2, 25, 26), TCRbeta -transgenic thymocytes of pTalpha + mice contained nearly undetectable levels of endogeneous Vbeta rearrangements (Fig. 6 A, lanes 2, 6, 10, 14, 18, 22, 26, 30, 34). Although the transgenic TCRbeta chain was still able to inhibit endogeneous TCR-beta rearrangements to a large degree even in the absence of pTalpha , the inhibition was significantly mitigated, as bands specific for endogeneous rearrangements involving all six functional Jbeta 2 segments were apparent at very substantial levels (Fig. 6 A, lanes 4, 8, 12, 16, 20, 24, 28, 32, 36). Similar results were obtained with two additional Vbeta primers (Vbeta 12, Vbeta 14; data not shown), clearly indicating an involvement of pTalpha in the inhibition process.


Fig. 6. Inefficient inhibition of Vbeta right-arrow (D)Jbeta rearrangements in TCR-beta -transgenic mice lacking pTalpha . PCR-based analysis of genomic DNA isolated from unfractionated thymocytes of nontransgenic and TCR-beta -transgenic pTalpha + and pTalpha -/- mice, as indicated in the panel below the autoradiographs (beta -TG, beta -transgenic). Primers complementary to one of several Vbeta elements were used in combination with a reverse primer positioned immediately downstream of the Jbeta 2 gene cluster, allowing amplification of rearranged, but not germline Vbeta segments, as shown in the diagram and described previously (25, 26). Specific PCR products corresponding to rearrangements that involve each of the six functional Jbeta 2 elements were identified by Southern blotting with a probe hybridizing to a region immediately upstream of the Jbeta 2 primer-annealing site. The numbers below the panels refers to the amount of template DNA used. Ig-C indicates bands corresponding to the IgM constant region, an internal control to estimate the actual amount of template used.
[View Larger Version of this Image (42K GIF file)]

Because the proportion of various thymocyte subpopulations is markedly altered in pTalpha -deficient mice, owing to the severe block in alpha beta T cell development (30), we were concerned that our results might have been influenced inappropriately by differences in the cellular composition of thymi from TCR-beta -transgenic pTalpha + and pTalpha -/- mice, respectively, although it was difficult to envisage specifically how this could have mimicked enhanced endogeneous rearrangements in the absence of pTalpha . Nevertheless, to exclude such a possibility completely and to allow a comparison of equivalent thymic subpopulations, we verified our PCR analysis of endogeneous Vbeta right-arrow (D)Jbeta rearrangements with DNA from sorted CD25+CD44-/low DN thymocytes, which represent the developmental stage at which TCR-beta rearrangements predominantly occur (12, 14). Fig. 7 shows that the result of this analysis was exactly the same as with unfractionated thymocytes; although the transgenic TCR-beta chain could inhibit endogeneous Vbeta right-arrow (D)Jbeta rearrangements in the absence of pTalpha (Fig. 7, compare lanes 4, 8, 12 with lanes 3, 7, 11), the degree of inhibition was less pronounced than in CD25+ thymocytes expressing pTalpha . Taken together, our data clearly indicate that the pTalpha chain contributes to the inhibition of endogeneous Vbeta rearrangements by productive TCR-beta transgenes.


Fig. 7. Analysis of genomic DNA isolated from sorted CD25+ triple-negative (CD3-4-8-) thymocytes. (A) Gates used to isolate CD25+CD44-/low triple-negative thymocytes. Thymocytes from three mice of the same genotype were pooled and stained as described in Fig. 3 B. FITC-positive cells (macrophages, granulocytes, B cells, CD4-, and CD8-expressing cells) were eliminated by electronic gating (data not shown). The panels representing the B6 and the TCR-beta -transgenic, pTalpha + mice have a much higher density of dots, because CD4- and CD8-expressing thymocytes from these mice (but not from pTalpha -/- animals) had been depleted with complement before staining, so that triple-negative thymocytes were already strongly enriched. The lower panels show the purity of the populations after sorting. (B) PCR-based analysis of genomic DNA from sorted CD25+ triple-negative thymocytes. For details, see legend to Fig. 6. In this particular experiment, Vbeta 11 rearrangements involving all Jbeta 2 elements, except Jbeta 2.6, were not appropriately amplified at the highest template concentration (lane 4). This phenomenon is due to excess template DNA and is not related to the mouse genotype, because a failure to amplify Vbeta right-arrow Jbeta rearrangements that correspond to larger PCR bands than Vbeta right-arrow Jbeta 2.6 rearrangements has also been observed in a few other experiments at the highest DNA concentration when the template DNA was derived from wild-type or nontransgenic pTalpha -/- mice.
[View Larger Version of this Image (36K GIF file)]


Discussion

The data reported here demonstrate that expression of a functional TCR-beta chain efficiently prevents the expression of a second functional chain on the cell surface of thymocytes and lymph node T cells in both nontransgenic and TCR-beta -transgenic mice even in the absence of pTalpha . At first glance, this result seems to suggest that an intact pre-TCR is not required for mediating allelic exclusion. However, impaired cell surface expression of two functional TCR-beta chains in pTalpha -/- mice does not automatically exclude a role for the pre-TCR in implementing allelic exclusion in normal mice. Recent data indicate that TCRalpha chains can, at least in part, substitute for pre-TCR-alpha chains, because they induce the generation of almost normal numbers of thymocytes in pTalpha -deficient mice, when expressed at a relatively early developmental stage (34, 35). Moreover, a recent analysis of pTalpha -/- × TCR-alpha -/- and pTalpha -/- × TCR-delta -/- mice has provided strong evidence that a significant proportion of the alpha beta thymocytes that develop in pTalpha -/- mice are actually derived from precursors that have been selected for further development based on the presence of a functional TCR-beta chain and early expression of a conventional TCR-alpha chain (35). The underlying assumption that some Valpha right-arrow Jalpha rearrangements and TCR-alpha expression can occur already in the CD25+ DN subpopulation is supported by a PCR-based analysis of TCR-alpha rearrangements in sorted CD25+ thymocytes from normal mice (36). If early expression of TCRalpha chains in a few CD25+ pre-T cells was responsible for the generation of most mature alpha beta T cells in pTalpha -/- mice, the early formation of an alpha beta TCR in these cells might as well be accountable for the allelic exclusion of TCR-beta chains that is observed despite the absence of pTalpha .

A priori, it would also be possible that neither the TCR-alpha nor the pTalpha chain are involved in mediating allelic exclusion and that TCR-beta chains can signal independently, even in the absence of these two partner chains. Our analysis of TCR-beta -transgenic, pTalpha -/- mice clearly demonstrates that TCR-beta chains can signal without being associated with a pTalpha chain, resulting in marked effects on early T cell development; for instance, a small increase in the number of DP thymocytes, partial downregulation of CD25 in the pre-T cell population, significant inhibition of Vgamma right-arrow Jgamma and Vbeta right-arrow (D)Jbeta rearrangements, suppression of the generation of gamma delta -expressing cells and, most striking, induction of DP thymocyte formation in RAG-/- × pTalpha -/- mice. These effects of a transgenic TCR-beta chain in the absence of pTalpha could be due to the association of TCR-beta chains with some other proteins, like TCR-alpha , TCR-delta (37), or the hypothetical VpreT subunit (20), giving rise to a signaling complex that can assume part but not all of pre-TCR function. The idea that the transgenic TCR-beta chain mediates its effects in the absence of pTalpha via a signal-transducing complex including CD3 components is consistent with the observation that a functionally rearranged TCR-beta transgene can no longer inhibit Vbeta right-arrow (D)Jbeta rearrangements in CD3-deficient mice (B. Malissen, personal communication).

The question arises whether the observed effects of the transgenic TCR-beta chain in the absence of pTalpha have some physiological relevance or whether they are solely due to a peculiarity of the transgenic system; for instance, expression of the TCR-beta transgene at inappropriately high levels. In this context, it should be mentioned that the TCR-beta chains in our TCR-beta -transgenic mice can be expressed on thymocytes in gpi-linked form, a feature not found in nontransgenic animals (38). If the significant inhibition of endogeneous Vbeta right-arrow (D)Jbeta rearrangements in pTalpha -deficient mice was due to a transgenic artifact, one might anticipate a variation in the degree of inhibition when analyzing different TCR-beta -transgenic lines. This may explain, why Xu et al. (23) have seen an equally strong inhibition of endogeneous TCR-beta rearrangements in both pTalpha + and pTalpha -/- thymocytes, while in our experiments the potentially artifactual, transgene-specific effects of the TCR-beta chain may be fortuitously less pronounced, allowing us to detect significant differences in the absence and presence of pTalpha . Moreover, the use of a stable line of transgenic mice allowed us to compare endogeneous rearrangements within the same transgenic background, which was not possible in the chimeric mice of Xu et al. (23), because they were generated from pTalpha + and pTalpha -/- ES cell clones that had been transfected individually with the respective TCR-beta transgene, almost certainly giving rise to pTalpha + and pTalpha -/- animals with distinct transgene copy numbers and/or insertion sites. Whatever the reason for the discrepant results may be, our observation that the inhibition of endogeneous rearrangements at the TCR-beta locus is less profound in the absence of pTalpha , despite the capacity of the transgenic beta  chain to signal in part independently of pTalpha , clearly indicates a role of pTalpha and the pre-TCR in the regulation of Vbeta right-arrow (D)Jbeta rearrangements. The importance of an intact pre-TCR for allelic exclusion at the level of TCR gene rearrangements therefore may be even more obvious in normal (nontransgenic) mice, in which a newly formed, functional TCR-beta chain is by definition expressed at physiological levels and therefore possibly less prone to pTalpha -independent signal transduction. This will be assessed by comparing the rearrangement status at the TCR-beta locus in a statistically significant number of sorted DN CD25+ thymocytes from pTalpha -deficient mice and wild-type littermates at the single cell level.


Footnotes

Address correspondence to H.J. Fehling, Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland. Phone: 41-61-605-1245; FAX: 41-61-605-1364; E-mail: fehling{at}bii.ch

Received for publication 28 April 1997.

1   Abbreviations used in this paper: BCR, B cell receptor; CS, calf serum; D, diversity; DN, double-negative; DP, double-positive; ES, embryonic stem; FSC, forward scatter; J, joining; PI, propidium iodine; pTalpha , pre-TCRalpha ; SP, single-positive; SSC, side scatter; V, variable.

We thank C. Laplace for skillful and efficient technical support; M. Dessing and A. Pickert for expert assistance with cell sorting and four-color cytofluorometric analysis; W. Metzger, E. Wagner, and B. Aschwanden for their care in maintaining the mouse colonies; J. Hatton and R. Schulze for oligonucleotide synthesis; B. Pfeiffer and H. Spalinger for photography, and T. Rolink and S. Gilfillan for critical reading of the manuscript. A. Krotkova is affiliated with the Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow. H. von Boehmer is supported by the Human Frontier Science Foundation. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche, Basel.


References

1. Malissen, M., J. Trucy, E. Jouvin-Marche, P.-A. Cazenave, R. Scollay, and B. Malissen. 1992. Regulation of TCRalpha and beta  gene allelic exclusion during T-cell development. Immunol. Today. 13: 315-322 [Medline].
2. Uematsu, Y., S. Ryser, Z. Dembiç, P. Borgulya, P. Krimpenfort, A. Berns, H. von Boehmer, and M. Steinmetz. 1988. In transgenic mice the introduced functional T cell receptor beta  gene prevents expression of endogeneous beta  genes. Cell. 52: 831-841 [Medline].
3. Krimpenfort, P., F. Ossendorp, J. Borst, C. Melief, and A. Berns. 1989. T cell depletion in transgenic mice carrying a mutant gene for TCRbeta . Nature (Lond.). 341: 742-746 [Medline].
4. Casanova, J.L., P. Romero, C. Widman, P. Kourilsky, and J.L. Maryanski. 1991. T cell receptor genes in a series of class I major histocompatibility complex-restricted cytotoxic T lymphocyte clones specific for a Plasmodium berghei nonapeptide: implications for T cell allelic exclusion and antigen-specific repertoire. J. Exp. Med. 174: 1371-1383 [Abstract].
5. Padovan, E., G. Casorati, P. Dellabona, S. Meyer, M. Brockhaus, and A. Lanzavecchia. 1993. Expression of two T cell receptor alpha  chains: dual receptor T cells. Science (Wash. DC). 262: 422-424 [Medline].
6. Borgulya, P., H. Kishi, Y. Uematsu, and H. von Boehmer. 1992. Exclusion and inclusion of alpha  and beta  T cell receptor alleles. Cell. 69: 529-537 [Medline].
7. Hardardottir, F., J.L. Baron, and C.A. Janeway Jr.. 1995. T cells with two functional antigen-specific receptors. Proc. Natl. Acad. Sci. USA. 92: 354-358 [Abstract].
8. Heath, W.R., and J.F.A.P. Miller. 1995. Expression of two alpha  chains on the surface of T cells in T cell receptor transgenic mice. J. Exp. Med. 178: 1807-1811 [Abstract].
9. Brändle, D., C. Müller, T. Rülicke, H. Hengartner, and H. Pircher. 1992. Engagement of the T cell receptor during positive selection in the thymus down-regulates RAG-1 expression. Proc. Natl. Acad. Sci. USA. 89: 9529-9533 [Abstract].
10. Petrie, H.T., F. Livak, D.G. Schatz, A. Strasser, I.N. Crisp, and K. Shortman. 1993. Multiple rearrangements in T cell receptor alpha  chain genes maximize the production of useful thymocytes. J. Exp. Med. 178: 615-622 [Abstract].
11. Kouskoff, V., J.-L. Vonesch, C. Benoist, and D. Mathis. 1995. The influence of positive selection on RAG expression in thymocytes. Eur. J. Immunol. 25: 54-58 [Medline].
12. Petrie, H.T., F. Livak, D. Burtrum, and S. Mazel. 1995. T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production. J. Exp. Med. 182: 121-127 [Abstract].
13. Wilson, A., J.-P. de Villartay, and H.R. MacDonald. 1996. T cell receptor delta  gene rearrangement and T early alpha  (TEA) expression in immature alpha beta lineage thymocytes: implications for alpha beta /gamma delta lineage commitment. Immunity. 4: 37-45 [Medline].
14. Godfrey, D.I., J. Kennedy, P. Mombaerts, S. Tonegawa, and A. Zlotnik. 1994. Onset of TCR-beta gene rearrangement and role of TCR-beta expression during CD3-CD4-CD8- thymocyte differentiation. J. Immunol. 152: 4783-4792 [Abstract/Free Full Text].
15. Sakaguchi, N., C.N. Berger, and F. Melchers. 1986. Isolation of a cDNA copy of an RNA species expressed in murine pre-B cells. EMBO (Eur. Mol. Biol. Organ.) J. 5: 2139-2147 [Abstract].
16. Kudo, A., and F. Melchers. 1987. A second gene, VpreB in the lambda 5 locus of the mouse which appears to be selectively expressed in pre-B lymphocytes. EMBO (Eur. Mol. Biol. Organ.) J. 6: 2267-2272 [Abstract].
17. Groettrup, M., K. Ungewiss, O. Azogui, R. Palacios, M.J. Owen, A.C. Hayday, and H. von Boehmer. 1993. A novel disulfide-linked heterodimer on pre-T cells consists of the T cell receptor beta  chain and a 33 kD glycoprotein. Cell. 75: 283-294 [Medline].
18. Saint-Ruf, C., K. Ungewiss, M. Groettrup, L. Bruno, H.J. Fehling, and H. von Boehmer. 1994. Analysis and expression of a cloned pre-T cell receptor gene. Science (Wash. DC). 266: 1208-1212 [Medline].
19. Melchers, F., H. Karasuyama, D. Haasner, S. Bauer, A. Kudo, N. Sakaguchi, B. Jameson, and A. Rolink. 1993. The surrogate light chain in B cell development. Immunol. Today. 14: 60-68 [Medline].
20. von Boehmer, H., and H.J. Fehling. 1997. Structure and function of the pre-T cell receptor. Annu. Rev. Immunol. 15: 433-452 [Medline].
21. Kitamura, D., A. Kudo, S. Schaal, W. Müller, F. Melchers, and K. Rajewsky. 1992. A critical role for the lambda 5 protein in B cell development. Cell. 69: 823-831 [Medline].
22. Löffert, D., A. Ehlich, W. Müller, and K. Rajewsky. 1996. Surrogate light chain expression is required to establish immunoglobulin heavy chain allelic exclusion during early B cell development. Immunity. 4: 133-144 [Medline].
23. Xu, Y., L. Davidson, F.W. Alt, and D. Baltimore. 1996. Function of the pre-T-cell receptor alpha  chain in T-cell development and allelic exclusion at the T-cell receptor beta  locus. Proc. Natl. Acad. Sci. USA. 93: 2169-2173 [Abstract/Free Full Text].
24. Fehling, H.J., C. Laplace, M.-G. Mattei, C. Saint-Ruf, and H. von Boehmer. 1995. Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene. Immunogenetics. 42: 275-281 [Medline].
25. van Meerwijk, J.P.M., H. Blüthmann, and H. Steinmetz. 1990. T-cell specific rearrangement of T-cell receptor beta  transgenes in mice. EMBO (Eur. Mol. Biol. Organ.) J. 9: 1057-1062 [Abstract].
26. Anderson, S.J., K.M. Abraham, T. Nakayama, A. Singer, and R.M. Perlmutter. 1992. Inhibition of T-cell receptor beta -chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck. EMBO (Eur. Mol. Biol. Organ.) J. 11:4877-4886.
27. Itohara, S., P. Mombaerts, J. Lafaille, J. Iacomini, A. Nelson, A.K. Clarke, M.L. Hooper, A. Farr, and S. Tonegawa. 1993. T cell receptor delta  gene mutant mice: independent generation of alpha beta T cells and programmed rearrangements of gamma delta TCR genes. Cell. 72: 337-348 [Medline].
28. Davodeau, F., M.A. Peyrat, F. Romagné, A. Necker, M.M. Hallet, H. Vié, and M. Bonneville. 1995. Dual T cell receptor beta  chain expression on human T lymphocytes. J. Exp. Med. 181: 1391-1398 [Abstract].
29. Padovan, E., C. Giachino, M. Cella, S. Valitutti, O. Acuto, and A. Lanzavecchia. 1995. Normal T lymphocytes can express two different T cell receptor beta  chains: implications for the mechanism of allelic exclusion. J. Exp. Med. 181: 1587-1591 [Abstract].
30. Fehling, H.J., A. Krotkova, C. Saint-Ruf, and H. von Boehmer. 1995. Crucial role of the pre-T-cell receptor alpha  gene in development of alpha beta but not gamma delta T cells. Nature (Lond.). 375: 795-798 [Medline].
31. Fehling, H.J., and H. von Boehmer. 1997. Early alpha beta T cell development in the thymus of normal and genetically altered mice. Curr. Opin. Immunol. 9: 263-275 [Medline].
32. von Boehmer, H., M. Bonneville, I. Ishida, S. Ryser, G. Lincoln, R.T. Smith, H. Kishi, B. Scott, P. Kisielow, and S. Tonegawa. 1988. Early expression of a T-cell receptor beta -chain transgene suppresses rearrangement of the Vgamma 4 gene segment. Proc. Natl. Acad. Sci. USA. 85: 9729-9732 [Abstract].
33. Shinkai, Y., S. Koyasu, K. Nakayama, K.M. Murphy, D.Y. Loh, E.L. Reinherz, and F.W. Alt. 1993. Restoration of T cell development in RAG-2-deficient mice by functional TCR transgenes. Science (Wash. DC). 259: 822-825 [Medline].
34. Bruno, L., H.J. Fehling, and H. von Boehmer. 1996. The alpha beta T cell receptor can replace the gamma delta receptor in the development of gamma delta lineage cells. Immunity. 5: 343-352 [Medline].
35. Buer, J., I. Aifantis, J.P. DiSanto, H.J. Fehling, and H. von Boehmer. 1997. Role of different T cell receptors in the development of pre-T cells. J. Exp. Med. 185: 1541-1547 [Abstract/Free Full Text].
36. Mertsching, E., A. Wilson, H.R. MacDonald, and R. Ceredig. 1997. T cell receptor alpha  gene rearrangement and transcription in adult thymic gamma delta cells. Eur. J. Immunol. 27: 389-396 [Medline].
37. Hochstenbach, F., and M.B. Brenner. 1989. T-cell receptor delta -chain can substitute for alpha  to form a beta delta heterodimer. Nature (Lond.). 340: 562-565 [Medline].
38. Groettrup, M., and H. von Boehmer. 1993. T cell receptor beta  chain dimers on immature thymocytes from normal mice. Eur. J. Immunol. 23: 1393-1396 [Medline].

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