Posttranscriptional Silencing of VβDJβCβ Genes Contributes to TCRβ Allelic Exclusion in Mammalian Lymphocytes

Natalie C. Steinel, Brenna L. Brady, Andrea C. Carpenter, Katherine S. Yang-Iott and Craig H. Bassing
J Immunol July 15, 2010, 185 (2) 1055-1062; DOI: https://doi.org/10.4049/jimmunol.0903099

Abstract

Feedback inhibition of V(D)J recombination enforces Ag receptor allelic exclusion in mammalian lymphocytes. Yet, in-frame VβDJβ exons can assemble on both alleles in human and mouse αβ T lineage cells. To elucidate mechanisms that enforce TCRβ allelic exclusion in such cells, we analyzed Vβ expression and rearrangement in mice containing a functional Vβ14DJβ1.5Cβ1 gene (Vβ14NT) and/or Vβ8.2DJβ1.1Cβ1 transgene (Vβ8Tg). The majority of Vβ14NT and Vβ8Tg αβ T lineage cells expressed only Vβ14+ or Vβ8+ TCRβ-chains, respectively, and lacked Vβ rearrangements on wild-type TCRβ loci. However, endogenous Vβ rearrangements and αβ T lineage cells expressing endogenous Vβs from wild-type alleles alone or with the prerearranged Vβ in cell surface TCRβ-chains were observed in Vβ14NT and Vβ8Tg mice. Although nearly all Vβ8Tg:Vβ14NT thymocytes and splenic αβ T cells expressed Vβ8+ TCRβ-chains, only half of these lymphocytes expressed Vβ14+ TCRβ-chains, even though similar steady-state levels of Vβ14NT mRNA were expressed in Vβ8+Vβ14+ and Vβ8+Vβ14− populations. Our data demonstrated that posttranscriptional silencing of functionally assembled endogenous VβDJβCβ genes can enforce TCRβ allelic exclusion and reveal another mechanism that contributes to the development of lymphocytes with monospecific Ag receptors.

The adaptive immune systems of jawed vertebrates consist of T and B lymphocytes that express cell surface T cell Ag receptor (TCR) or B cell Ag receptor complexes. TCR and Ig V region exons are assembled in developing lymphocytes through the recombination of germline V(D)J gene segments (1). In mammals, the combination of possible rearrangement events within single genetic loci encoding each TCR and Ig chain contributes to diversification of Ag receptor binding specificities. However, in cartilaginous fish, each individual type of Ig chain is encoded by fully preassembled, partially preassembled, or unassembled germline gene segments located within hundreds of independent genetic loci (2). Most lymphocytes in jawed vertebrates express cell surface Ag receptor chains from a single allele or locus, a phenomenon that is referred to as Ag receptor allelic or haplotypic exclusion. For example, ∼99% of mouse and human αβ T cells express cell surface TCRβ-chains from a single allele (3–5). The majority of lymphocytes in mice and humans assemble a single in-frame exon within TCRβ, IgH, and IgL loci due to feedback inhibition of variable, diversity, and joining [V(D)J] recombination, which is signaled by the expression of functional TCR or Ig chains and enforces Ag receptor allelic exclusion (6). In contrast, restricted expression of functional Ig genes from a single genetic locus seems to be the major mechanism that mediates haplotype exclusion in lymphocytes of cartilaginous fish (7).

In humans and mice, αβ T lymphocytes develop through a differentiation program that involves the assembly, expression, and selection of a functional VβDJβCβ gene from one allele (8). TCRβ genes are assembled through DJβ intermediates in CD4−CD8− (double-negative [DN]) thymocytes (9). Transcription through a functional VβDJβ rearrangement generates TCRβ-chains that can pair with pTα molecules to form pre-TCRs (8). These receptors signal feedback inhibition of further Vβ rearrangement to enforce TCRβ allelic exclusion and select DN cells for differentiation into CD4+CD8+ (double-positive [DP]) thymocytes (8). DN cells that assemble an out-of-frame VβDJβ rearrangement on the first allele can attempt Vβ rearrangement on the second allele (9). In DP cells, TCRα genes are assembled on both alleles from Vα and Jα segments (10). In-frame VαJα rearrangements generate TCRα-chains that can associate with TCRβ molecules to form αβ TCRs (8). Positive selection of αβ TCRs promotes further differentiation of DP cells into CD4+ or CD8+ (single-positive) thymocytes (8). These cells exit the thymus and migrate to the spleen and other peripheral locations as naive mature αβ T cells. However, DP thymocytes expressing autoreactive αβ TCRs are frequently eliminated by apoptosis (8). TCRβ allelic exclusion has been hypothesized to prevent autoimmunity by facilitating the development and selection of cells with αβ TCRs of a single specificity (11).

Generation and analysis of mice containing different preassembled VβDJβCβ transgenes demonstrated that expression of a functional TCRβ-chain can inhibit rearrangement and expression of endogenous Vβ segments (12). Enforcement of allelic exclusion by such feedback inhibition predicts that ∼60% of αβ T cells contain DJβ intermediates and ∼40% contain out-of-frame VβDJβ rearrangements on their nonselected alleles (13). Yet, sequence analyses of TCRβ joins or mRNA revealed the presence of two in-frame VβDJβ rearrangements in 5–10% of mouse and human αβ T cells that exhibit allelic exclusion (14, 15). In addition, in-frame endogenous Vβ14Jβ rearrangements were found, but not expressed, in ∼10% of αβ T cell hybridomas generated from mice with a modified TCRβ locus that permits direct Vβ14-to-Jβ rearrangement (16). Moreover, VβDJβCβ genes that were assembled in-frame within a transgenic TCRβ mini-locus were not expressed on αβ T cells of mice containing a preassembled TCRβ transgene (17). Furthermore, although TCRβ-mediated feedback inhibition is blocked in pTα−/− thymocytes, TCRβ allelic exclusion is maintained in the αβ T lineage cells of pTα−/− mice (18, 19). Collectively, these data indicate that additional mechanisms must restrict the cell surface expression of functionally assembled TCRβ genes; however, the absence of an allotypic Cβ marker in humans and mice has prevented definitive conclusions. Thus, to elucidate mechanisms that enforce allelic exclusion in cells with two functional VβDJβCβ genes, we analyzed Vβ expression and rearrangement in αβ T lineage cells of mice containing one allelic copy of a preassembled functional endogenous TCRβ gene and/or classical TCRβ transgene.

Materials and Methods

Mice

Generation and characterization of Vβ8Tg mice and LN2 embryonic stem cells containing the preassembled Vβ14Dβ1Jβ1.5Cβ gene were previously described (20, 21). Vβ8Tg mice were bred onto a 129SvEv (Taconic Farms, Germantown, NY) background. LN2 cells were used to generate mice with the Vβ14NT allele transmitted through the germline. These mice were mated with 129SvEv mice to isolate the Vβ14NT allele from the other rearranged TCRβ and TCRα alleles. These Vβ14NT/+ mice were maintained on a 129SvEv background and mated with one another to generate the Vβ14NT/+, Vβ14NT/NT, and wild-type (WT) mice used in the experiments. All experiments were performed on 4–6-wk-old mice in accordance with relevant institutional and national guidelines and regulations and approved by the Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee. None of the individual or compound mutant mice appeared different or exhibited phenotypes by which they could be distinguished from WT littermates or age-matched controls.

Flow cytometry

Single-cell suspensions of lymphocytes from thymuses and spleens were incubated with RBC lysis buffer (0.7 M NaCl and 17 mM Tris HCl). Cells were washed with FACS staining buffer (PBS containing 0.5% BSA) and stained with the following Abs from BD Pharmingen (San Diego, CA): allophycocyanin–anti-Cβ (553174), allophycocyanin-cy7–anti-B220 (552094), FITC–anti-Vβ14 (553258), PE–anti-Vβ8 (553862), PE–anti-Vβ10 (553285), biotin–anti-Vβ6 (553192), biotin–anti-Vβ5 (553188), and PE-Cyc7-straptavidin (557598). Cells were stained in FACS staining buffer. Live cells were gated on the basis of forward or side scatter and DAPI exclusion (D1306; Invitrogen). Data were collected on an LSR II and were analyzed using FlowJo; 500,000 events were collected for each sample file. All displayed events were gated on single DAPI−B220−TCRβ+ cells.

PCR analysis of Vβ rearrangements

Total thymocytes or splenocytes were lysed in rapid lysis buffer (0.1 M Tris [pH 8.5], 0.2% SDS, 0.005 M EDTA, 0.2 M NaCl, and 250 μg/μl proteinase K). Genomic DNA was isolated by isopropanol precipitation. PCR conditions for a final volume of 25 μl were 10× PCR Buffer (Qiagen, Valencia, CA), 0.2 mM 2′-deoxynucleoside 5′-triphosphate (Applied Biosystems, Foster City, CA), 0.2 mM each primer, 5 U HotStarTaq polymerase (Qiagen), and 500 ng DNA. PCR cycles were 94°C for 3 min; 40 cycles of 94°C for 45 s, 60°C for 1 min, 30 s, and 72°C for 2 min, 30 s; and 72°C for 10 min. The Vβ-specific primers and the 3′Jβ1.2 primer (P2) were described previously (22). The 3′Jβ2.2 primer was 5′-CTCCAACCCTGACTCAGATCCCCACC-3′. The Cβ2 primers were 5′-CAAACAAAAGGCTACCCTCGTG-3′ and 5′-GCAGACAGAACCCCCTGATGATAG-3′.

Generation and analysis of hybridomas

The generation and analysis of TCRβ gene rearrangements in Vβ14NT/+ and Vβ14NT/NT αβ T cell hybridomas were conducted as previously described (16, 23, 24).

Analysis of Vβ14DJβ1.5Cβ1 mRNA expression

The sort purification of Vβ14+Vβ8+ and Vβ14+Vβ8− thymocytes from Vβ8Tg:Vβ14NT/+ mice was conducted on a FACS Aria, with staining and gating strategy identical to that described above for flow cytometry. RNA was isolated using TRIzol, and poly-A cDNA was generated using the NEB Protoscript II cDNA synthesis kit. Expression levels of Vβ14DJβ1.5Cβ1 and GAPDH mRNAs were determined by quantitative PCR (qPCR) on an ABI 7500 Fast Real-Time PCR machine using the following primer pairs: Vβ14F 5′-AGGCCACAATGCTATGTATTGGT-3′ and Vβ14R 5′-TGAGGTTGGAAGCGACTTGA-3′ or GAPDHF 5′-CTTCACCACCATGGAGAAGGC-3′ and GAPDHR 5′-GGCATGGACTGTGGTCATGAG-3′.

Results

Expression of endogenous Vβ segments in αβ T lineage cells of mice containing a preassembled functional VβDJβCβ gene

Most investigations of TCRβ allelic exclusion have been conducted through analyses of mice expressing preassembled functional VβDJβCβ transgenes. The physiologic relevance of such studies has been questioned because of the varying extents to which transgenes enforce allelic exclusion, the high copy number of transgenes often required for allelic exclusion, and other potential transgenic artifacts (19, 25, 26). Thus, we sought to study TCRβ allelic exclusion in mice containing a single allelic copy of a preassembled functional endogenous VβDJβCβ gene. Chimeric mice containing preassembled in-frame endogenous TCR genes have been generated through the transfer of αβ T cell nuclei into embryonic stem cells (20). We used stem cells reconstituted with the nucleus of a Vβ14+ αβ T cell to establish mice containing a preassembled functional endogenous Vβ14DJβ1.5Cβ1 gene (Vβ14NT) within their germline. These mice were bred with WT mice to separate the Vβ14NT allele from the other prerearranged TCR alleles, and their offspring were intercrossed to establish mice containing the Vβ14NT gene on one (Vβ14NT/+) or two (Vβ14NT/NT) alleles (Fig. 1A). FACS analysis of Vβ14NT/+ and Vβ14NT/NT thymocytes and splenocytes with anti-Vβ14 and anti-Cβ Abs revealed that most Vβ14NT/+ cells and all Vβ14NT/NT cells expressed Vβ14 within surface TCRβ-chains (Fig. 1B). Notably, cell populations lacking Vβ14 within surface TCRβ-chains (Vβ14−Cβ+) were detectable in Vβ14NT/+, but not Vβ14NT/NT, mice (Fig. 1B). These data indicated that expression of the preassembled functional Vβ14DJβ1.5Cβ1 gene within cell-surface TCRβ-chains can be silenced in Vβ14NT/+ mice.

FIGURE 1.
FIGURE 1.

Mice with a preassembled functional Vβ14DJβ1.4Cβ1 gene develop αβ T cells expressing Vβ14 and Vβ8 segments. A, Schematic representations of the genomic organization of the WT and Vβ14NT TCRβ loci. The relative locations of germline Vβ, Dβ, Jβ, and Cβ gene segments and the preassembled Vβ14DJβ1.4 rearrangement are indicated. B, FACS analysis of Vβ14 expression in Vβ14NT/+ mice. This analysis was conducted on mice of each genotype five independent times. Shown are representative plots of anti-Cβ and anti-Vβ14 stains conducted on thymocytes and splenocytes isolated from Vβ14NT/+ and Vβ14NT/NT mice. C, FACS analysis of Vβ8 expression in Vβ14NT/+ mice. This analysis was conducted on mice of each genotype three independent times. Shown are representative plots of anti-Vβ14 and anti-Vβ8 stains conducted on thymocytes and splenocytes isolated from Vβ14NT/+, Vβ14NT/NT, and WT mice. The percentages of cells within each quadrant are indicated.

In mice, Vβ8 is the most highly represented Vβ within cell-surface αβ TCR because three individual Vβ segments (Vβ8.1, Vβ8.2, and Vβ8.3) exist (27). The presence of Vβ8+Vβ3− and Vβ8+Vβ3+ splenic T cells was observed in mice containing a Vβ3+ TCRβ transgene that prevents the expression of other endogenous Vβ segments (5). Thus, in an initial attempt to characterize the Vβ14−Cβ+ αβ T cell populations in Vβ14NT/+ mice, we conducted FACS analysis of Vβ14NT/+ and Vβ14NT/NT thymocytes and splenocytes with combinations of anti-Vβ8, anti-Vβ14, and anti-Cβ Abs. In Vβ14NT/+ mice, we detected populations expressing only Vβ14, only Vβ8, or Vβ14 and Vβ8 within surface TCRβ-chains (Fig. 1C). The frequencies of Vβ8+ αβ T lineage cells were significantly lower in Vβ14NT/+ mice compared with WT mice (107-fold lower in thymocytes and 57-fold lower in splenocytes) (Fig. 2B, 2C). Yet, we found only Vβ14+Vβ8−Cβ+ populations in Vβ14NT/NT mice (Fig. 1C), indicating that the Vβ14−Cβ+ populations in Vβ14NT/+ mice represent bona fide αβ T cells rather than staining artifacts. These data demonstrated that Vβ8+ TCRβ-chains from the WT allele can be expressed on the surface of Vβ14NT/+ αβ T lineage cells with or without Vβ14+ chains from the Vβ14NT allele, the former of which results in TCRβ allelic inclusion.

FIGURE 2.
FIGURE 2.

Vβ14NT/+ mice develop αβ T cells expressing a limited repertoire of endogenous Vβ segments. A, FACS analysis of Vβ expression in Vβ14NT/+ mice. Shown are representative plots of anti-Vβ14 and anti-Vβ5, anti-Vβ6, or anti-Vβ10 stains conducted on thymocytes and splenocytes isolated from Vβ14NT/+, Vβ14NT/NT, and WT mice. The percentages of cells within each quadrant are indicated. Bar graphs depicting Vβ usage in thymocytes (B) or splenocytes (C) isolated from Vβ14NT/+, Vβ14NT/NT, and WT mice. These analyses were conducted on mice of each genotype three independent times. The numbers above the bars represent fold differences in expression of the particular Vβ segment between the indicated genotypes. Error bars are SEM, and two-tailed Student t tests were performed. **p = 0.001–0.01; ***p < 0.001.

The murine TCRβ locus contains 20 functional Vβ segments that can be expressed as part of cell-surface αβ TCRs (28). Thus, to evaluate whether endogenous Vβ segments other than Vβ8 are expressed within TCRβ-chains on αβ T cells of Vβ14NT/+ mice, we conducted FACS analysis of Vβ14NT/+ and Vβ14NT/NT thymocytes and splenocytes with combinations of anti-Vβ5, anti-Vβ6, anti-Vβ10, anti-Vβ12, anti-Vβ14, and anti-Cβ Abs. We found cell populations expressing Vβ5 or Vβ6 segments within surface TCRβ-chains on Vβ14+ and Vβ14− cells in Vβ14NT/+ mice (Fig. 2A). Yet, we observed only Vβ14+Cβ+ populations in Vβ14NT/NT mice (Fig. 2A), indicating that the Vβ5+Cβ+ and Vβ6+Cβ+ cells in Vβ14NT/+ mice also represent bona fide αβ T cells rather than staining artifacts. We were unable to detect bona fide αβ T lineage cells expressing Vβ10 or Vβ12 within TCRβ-chains on Vβ14NT/+ or Vβ14NT/NT mice (Fig. 2C, data not shown). The frequency of αβ T lineage cells expressing Vβ5 and Vβ6 were significantly lower in Vβ14NT/+ mice compared with WT mice (Vβ5 was 85-fold lower in thymocytes and 43-fold lower in splenocytes; Vβ6 was 19-fold lower in thymocytes and 21-fold lower in splenocytes) (Fig. 2C). These data demonstrated that a limited repertoire of endogenous Vβ segments is expressed within TCRβ-chains on Vβ14NT/+ αβ T lymphocytes, which results in TCRβ allelic inclusion or occurs in association with silenced cell-surface expression of the preassembled functional Vβ14DJβ1.5Cβ1 gene.

For purposes of comparison between Vβ14NT and TCRβ transgenic mice, we conducted the same FACS analyses on thymocytes and splenocytes of mice expressing a preassembled functional Vβ8.2DJβ1.1Cβ1 transgene from one allele (Vβ8Tg) (21). Vβ8Tg mice were shown to exhibit feedback inhibition of Vβ rearrangement and TCRβ allelic exclusion (25, 29). Consistent with these published findings, we observed that almost all Vβ8Tg αβ T cells expressed Vβ8 within cell-surface TCRβ-chains (Fig. 3A). We failed to detect significant populations of αβ T cells expressing Vβ10 in Vβ8Tg mice (Fig. 3B). Although we observed cell populations expressing Vβ5, Vβ6, and Vβ14 segments within surface TCRβ-chains of Vβ8Tg αβ T lineage cells (Fig. 3B), the frequencies of cells expressing these Vβ segments in Vβ8Tg mice were significantly lower than those in WT mice (Vβ5 was 23-fold lower in thymocytes and 47-fold lower in splenocytes; Vβ6 was 17-fold lower in thymocytes and 19-fold lower in splenocytes; Vβ14 was 8-fold lower in thymocytes and 4-fold lower in splenocytes) (Fig. 3C, 3D). TCRβ-chains with these Vβ segments are observed predominantly on cells that also express surface Vβ8+ chains, resulting in TCRβ allelic inclusion (Fig. 3B). Because similar Vβ5+Cβ+ and Vβ6+Cβ+ populations were observed in Vβ14NT/+ mice, these findings demonstrated that a limited repertoire of endogenous Vβ segments also is expressed within surface TCRβ-chains on Vβ8Tg αβ T lymphocytes. Collectively, our data and published observations (5) indicate that the incomplete downregulation of endogenous Vβ expression and silenced cell-surface expression of functional TCRβ-chains are not unique to TCRβ transgenic mice but rather are general phenomena of mice containing preassembled functional VβDJβCβ genes.

FIGURE 3.
FIGURE 3.

Vβ8Tg mice develop αβ T cells expressing a limited repertoire of endogenous Vβ segments. A, FACS analysis of Vβ8 expression in Vβ8Tg mice. Shown are representative plots of anti-Cβ and anti-Vβ8 stains conducted on thymocytes and splenocytes isolated from Vβ8Tg mice. B, Shown are representative plots of anti-Vβ8 and anti-Vβ14, anti-Vβ5, anti-Vβ6, or anti-Vβ10 stains conducted on thymocytes and splenocytes isolated from Vβ8Tg and WT mice. The percentages of cells within each quadrant are indicated. Bar graphs depicting Vβ usage in thymocytes (C) or splenocytes (D) isolated from Vβ8Tg and WT mice. These analyses were conducted on mice of each genotype three independent times. The numbers above the bars represent fold differences in expression of the particular Vβ segment between the two genotypes. Error bars represent SEM.

A larger repertoire of endogenous Vβ segments rearranges in thymocytes containing preassembled functional VβDJβCβ genes

Our observations that endogenous Vβ segments are expressed within TCRβ-chains on the surface of Vβ14NT/+ and Vβ8Tg αβ T cells indicate that Vβ rearrangements must have occurred on WT alleles in developing Vβ14NT/+ and Vβ8Tg thymocytes. Because surface expression of preassembled functional VβDJβCβ-chains can be silenced, FACS analysis with anti-Vβ–specific Abs cannot be used as an accurate readout of Vβ-to-DJβ rearrangements. Thus, to ascertain the repertoire of Vβ rearrangements in αβ T lineage cells of Vβ14NT/+ and Vβ8Tg mice, we conducted PCR-based analysis of VβDJβ joins in WT, Vβ8Tg, Vβ14NT/+, and Vβ14NT/NT thymocytes. The murine TCRβ locus contains 20 functional Vβ segments and two Dβ-Jβ clusters (Dβ1-Jβ1 and Dβ2-Jβ2), each with one Dβ segment and six functional Jβ segments (Fig. 4A). We used combinations of Vβ- and Jβ-specific primers to amplify potential rearrangements of each functional endogenous Vβ segment to DJβ complexes involving Jβ1.1/Jβ1.2 or Jβ2.1/Jβ2.2 segments (Fig. 4A). We found that the levels of rearrangements involving many Vβ segments to DJβ1.1/DJβ1.2 and DJβ2.1/DJβ2.2 complexes were undetectable or substantially reduced in Vβ8Tg and Vβ14NT/+ cells compared with in WT cells (Fig. 4B). In contrast, we found that the levels of rearrangements involving Vβ5, Vβ6, Vβ7, Vβ8, Vβ14, Vβ15, Vβ16, and Vβ17 segments to DJβ1.1/DJβ1.2 complexes were unchanged or slightly reduced in Vβ8Tg and Vβ14NT/+ cells compared with in WT cells (Fig. 4B). These data demonstrated that a limited repertoire of endogenous Vβ segments can rearrange at appreciable levels in Vβ14NT/+ and Vβ8Tg thymocytes, despite the presence of a preassembled functional VβDJβCβ gene/transgene.

FIGURE 4.
FIGURE 4.

Endogenous Vβ segments rearrange in Vβ14 NT/+ and Vβ8Tg thymocytes. A, PCR strategy for amplification of Vβ rearrangements to DJβ1.1/DJβ1.2 and DJβ2.1/DJβ2.2 complexes. Shown is a schematic representation of the WT TCRβ locus depicting the relative locations of representative Vβ, Dβ, Jβ, and Cβ gene segments, as well as the primers located just downstream of Jβ1.2 or Jβ2.2 and the Cβ2 primers. B, PCR analysis of potential Vβ-to-DJβ1 and Vβ-to-DJβ2 rearrangements. Shown are representative PCR amplifications of Vβ rearrangements to DJβ1.1/DJβ1.2 (top panels) and DJβ2.1/DJβ2.2 (bottom panels) complexes for the indicated Vβ segments performed on DNA isolated from Vβ8Tg, Vβ14NT/+, or WT thymocytes. The amounts of DNA and numbers of PCR cycles used were previously demonstrated to amplify rearrangements within the linear range for the WT sample. C, qPCR analysis of Vβ-to-DJβ1 and Vβ-to-DJβ2 rearrangements. Shown are representative PCR amplifications of Vβ rearrangements to DJβ1.1/DJβ1.2 and DJβ2.1/DJβ2.2 complexes for the indicated Vβ segments using serial 1:5 dilutions of DNA isolated from WT, Vβ8Tg, Vβ14NT/+, or Vβ14NT/NT thymocytes. Also shown is a representative PCR amplification of Cβ2 as a control for DNA content.

Our PCR data seem to be in conflict with previous studies concluding that the levels of rearrangements involving all endogenous Vβ segments are substantially reduced in αβ T lineage cells of Vβ8Tg mice (25, 29). These previous experiments quantified Vβ rearrangements only by PCR amplification of VβDJβ2 joins because, theoretically, VβDJβ1 joins can form on extrachromosomal excision circles that might not be subject to feedback inhibition. To our knowledge, the direct quantification of chromosomal VβDJβ rearrangements in αβ T cells of mice containing preassembled TCRβ transgenes/genes has not been reported. Therefore, we generated panels of Vβ14NT/+ and Vβ8Tg αβ T cell hybridomas and quantified chromosomal TCRβ rearrangements by Southern blot analysis on EcoRI-digested genomic DNA using a series of TCRβ locus probes. Of the 82 Vβ14NT/+ hybridomas analyzed, 66 (81%) contained DJβ rearrangements and 10 (12%) contained VβDJβ rearrangements on the WT TCRβ allele. Similarly, of the 129 Vβ8Tg hybridomas analyzed, 102 (79%) contained DJβ rearrangements on one or both WT alleles, and 12 (9.3%) contained VβDJβ rearrangements on one or both TCRβ alleles (Table I). The remaining 6 (7%) Vβ14NT/+ and 15 (11%) Vβ8Tg αβ T cell hybridomas contained germline TCRβ loci, Vβ-to-Dβ rearrangement, or rearranged loci with Southern blot patterns suggesting aberrant Dβ-to-Jβ rearrangements that deleted Jβ-coding sequences (Table I). In addition to VβDJβ joins on selected alleles, ∼60% of normal αβ T cells contained DJβ joins, and ∼40% contained VβDJβ joins on nonselected alleles. Accordingly, the overall level of chromosomal VβDJβ rearrangements was reduced only ∼4-fold in Vβ14NT/+ and Vβ8Tg αβ T lineage cells compared with in WT cells.

View this table:
Table I. Analysis of TCRβ rearrangements in Vβ14NT/+ and Vβ8Tg αβ T cell hybridomas

This modest reduction in the overall level of chromosomal Vβ rearrangements, compared with the substantial decrease in the numbers of cells expressing endogenous Vβ segments, indicates that not all VβDJβCβ genes assembled in-frame on WT alleles are expressed within TCRβ-chains on Vβ14NT/+ and Vβ8Tg αβ T lineage cells. To further demonstrate this point, we conducted PCR analysis on serially diluted thymocyte DNA to quantify the levels of endogenous Vβ5, Vβ6, Vβ8, Vβ10, and Vβ14 rearrangements to DJβ1.1/DJβ1.2 and DJβ2.1/DJβ2.2 complexes in Vβ8Tg and Vβ14NT/+ cells compared with in WT cells. We found that the levels of Vβ6 and Vβ14 rearrangements to DJβ1.1/DJβ1.2 complexes were comparable among Vβ8Tg, Vβ14NT/+, and WT cells, whereas Vβ6 and Vβ14 rearrangements to DJβ2.1/DJβ2.2 complexes were reduced ∼5-fold in Vβ8Tg cells and ∼25-fold in Vβ14NT/+ cells (Fig. 4C). The levels of Vβ5 rearrangements to DJβ1.1/DJβ1.2 complexes were reduced ∼5-fold in Vβ8Tg cells and ∼25-fold in Vβ14NT/+ cells, and the levels of Vβ5 rearrangements to DJβ2.1/DJβ2.2 complexes were reduced >25-fold in Vβ8Tg and Vβ14NT/+ cells (Fig. 4C). Vβ8 rearrangements to DJβ1.1/DJβ1.2 and DJβ2.1/DJβ2.2 complexes were reduced ∼25-fold and ∼100-fold, respectively, in Vβ14NT/+ cells (Fig. 4C). Because of the genomic organization of Vβ8Tg, we were unable to amplify endogenous Vβ8 rearrangements to DJβ1.1/DJβ1.2 complexes in Vβ8Tg cells; Vβ8 rearrangements to DJβ2.1/DJβ2.2 complexes were reduced >25-fold (Fig. 4C). Consistent with our ability to detect only Vβ14+Vβ8−Cβ+ populations in Vβ14NT/NT mice, we observed no PCR amplicons of Vβ-to-DJβ rearrangements in Vβ14NT/NT cells (Fig. 4C). Our data indicated that the levels of chromosomal Vβ6 and Vβ14 rearrangements in Vβ14NT/+ and Vβ8Tg αβ T lineage cells are reduced to a lesser extent than are the numbers of cells expressing Vβ6 and Vβ14 within surface TCRβ-chains.

Posttranscriptional silencing of functionally assembled endogenous VβDJβCβ genes contributes to TCRβ allelic exclusion

Because αβ T lineage cells expressing two functional TCRβ genes are not selected against (30), our observations are consistent with the notion that not all VβDJβCβ genes assembled in-frame on WT alleles are expressed on Vβ14NT/+ and Vβ8Tg αβ T cells. The inability of VHDJHCH chains to form functional pre-BCRs and promote differentiation can result in their lack of expression on the surface of B cells, ensuring IgH allelic exclusion (31). Yet, the silencing of Vβ14 and Vβ8 expression on Vβ14NT/+ and Vβ8Tg cells, respectively, cannot be due to defects in pairing with pTα because the Vβ14DJβ1.5Cβ1 and Vβ8.2DJβ1.1Cβ1 genes were isolated from selected TCRβ alleles. Mature αβ T lineage cells frequently express intracellular TCRα-chains from both alleles but exhibit TCRα allelic exclusion through posttranslational mechanisms that seem to include competition between TCRα-chains for a single TCRβ-chain or inability of one TCRα-chain to pair with the expressed TCRβ-chain (13, 32). Thus, we next conducted intracellular FACS analysis of Vβ14NT/+, Vβ8Tg, and Vβ14NT/NT αβ T lineage cells with anti-Vβ14 and anti-Vβ8 Abs to evaluate whether analogous mechanisms may restrict cell-surface expression of TCRβ-chains. We found Vβ8+ TCRβ-chains inside Vβ14NT/+, but not Vβ14NT/NT, thymocytes and Vβ14+ TCRβ-chains inside Vβ8Tg thymocytes (Fig. 5A). The percentages of Vβ14NT/+ and Vβ8Tg thymocytes with intracellular and extracellular Vβ8+ and Vβ14+ TCRβ-chains, respectively, were equivalent (compare Figs. 1C, 5A). These data suggested that not all Vβ8DJβCβ and Vβ14DJβCβ genes assembled in-frame on WT alleles are expressed as TCRβ-chains within or on the surface of Vβ14NT/+ and Vβ8Tg cells, respectively.

FIGURE 5.
FIGURE 5.

Silencing of functional VβDJβCβ genes in αβ T lineage cells. A, Intracellular FACS analysis of Vβ14 and Vβ8 expression. This analysis was conducted on mice of each genotype three independent times. Shown are representative plots of anti-Vβ14 and anti-Vβ8 intracellular stains conducted on thymocytes isolated from Vβ14NT/+, Vβ8Tg, Vβ14NT/NT, and Vβ8Tg:Vβ14NT/+ mice. The percentages of cells within each quadrant are indicated. B, Extracellular FACS analysis of Vβ14 and Vβ8 expression. This analysis was conducted on mice of each genotype three independent times. Shown are representative plots of anti-Vβ14 and anti-Vβ8 stains conducted on thymocytes and splenocytes isolated from Vβ14NT/+, Vβ8Tg, and Vβ8Tg:Vβ14NT/+ mice. The percentages of cells within each quadrant are indicated. C, qPCR analysis of Vβ14NT mRNA expression in Vβ8Tg:Vβ14NT/+ αβ T cells. The gating strategy for sorting of Vβ14+Vβ8+ and Vβ14+Vβ8− αβ T cells is shown in the left panel. A bar graph depicting the Vβ14NT mRNA levels relative to GAPDH mRNA levels in Vβ14+Vβ8+ and Vβ14+Vβ8− αβ T cells sort-purified from Vβ8Tg:Vβ14NT/+ mice is shown in the right panel. This experimental analysis was conducted three independent times. Error bars represent SEM.

To demonstrate that the expression of functionally assembled endogenous VβDJβCβ genes within TCRβ-chains can be silenced, we bred Vβ14NT/+ and Vβ8Tg mice to generate Vβ8Tg:Vβ14NT/+ mice. Extracellular FACS analysis of Vβ8Tg:Vβ14NT/+ thymocytes and splenocytes with anti-Vβ14, anti-Vβ8, and anti-Cβ Abs revealed substantial populations of Vβ8+Vβ14+Cβ+ and Vβ8+Vβ14−Cβ+ cells and a minor population of Vβ8−Vβ14+Cβ+ cells (Fig. 5B). Nearly all Vβ8Tg:Vβ14NT/+ cells expressed Vβ8, but only half expressed Vβ14, within cell-surface TCRβ-chains. Intracellular FACS analysis of Vβ8Tg:Vβ14NT/+ thymocytes and splenocytes with anti-Vβ14, anti-Vβ8, and anti-Cβ Abs showed populations of Vβ8+Vβ14+Cβ+, Vβ8+Vβ14−Cβ+, and Vβ8−Vβ14+Cβ+ cells (Fig. 5A), which were present at similar numbers as those observed with extracellular FACS analyses (compare Fig. 5A, 5B). Notably, almost all Vβ8Tg:Vβ14NT/+ cells expressed Vβ8, but only half expressed Vβ14, as part of intracellular TCRβ-chains. These data indicated that the expression of functionally assembled endogenous VβDJβCβ genes within TCRβ-chains can be silenced in mouse lymphocytes.

The silenced expression of functionally assembled VβDJβCβ genes within TCRβ-chains could occur at the level of mRNA or protein expression. To determine the level at which the preassembled functional endogenous Vβ14DJβ1.5Cβ1 gene is silenced, we used qPCR to quantify the steady-state levels of mature Vβ14+ mRNA in sort-purified Vβ8+Vβ14+ and Vβ8+Vβ14− splenic αβ T cells of Vβ8Tg:Vβ14NT/+ mice. We found that the steady-state levels of Vβ14+ transcripts were comparable between each population of cells (Fig. 5C). These data demonstrated that expression of the Vβ14NT gene can be silenced at the level of protein. Thus, we conclude that posttranscriptional silencing of functionally assembled endogenous VβDJβCβ genes can contribute to the enforcement of TCRβ allelic exclusion in mammalian lymphocytes.

Discussion

In this study, we investigated VβDJβCβ gene/transgene expression and the rearrangement and expression of endogenous Vβ segments in αβ T lineage cells of mice containing the Vβ14NT gene and/or Vβ8Tg transgene. We found that most Vβ14NT/+ αβ T lineage cells isolated from 4–6-wk-old mice express only Vβ14 within cell-surface TCRβ-chains. These data provide direct evidence that expression of a functionally assembled VβDJβCβ gene from one allele can enforce TCRβ allelic exclusion, as would be expected from prior analyses of TCRβ transgenic mice. Yet, despite TCRβ feedback inhibition of Vβ rearrangements, a limited repertoire of endogenous Vβ segments is expressed within TCRβ-chains on Vβ14NT/+ αβ T lymphocytes. Expression of these Vβ segments can result in TCRβ allelic inclusion or correlate with silenced expression of the functional Vβ14NT gene within cell-surface TCRβ-chains. We obtained analogous data through the analysis of Vβ8Tg mice. Similar observations have been published for mice containing a Vβ3+ TCRβ transgene (5). Thus, our current study indicated that incomplete downregulation of endogenous Vβ expression and silenced cell-surface expression of preassembled TCRβ-chains are general phenomena in αβ T lineage cells containing functional VβDJβCβ genes/transgenes. However, as discussed below, the expression of endogenous Vβ segments within such mice might be attributable to a common nonphysiologic aspect of V(D)J recombination in DN thymocytes with preassembled functional TCRβ genes/transgenes.

Consistent with the incomplete downregulation of endogenous Vβ expression, we found chromosomal VβDJβ1 joins on WT TCRβ alleles in ∼10% of Vβ14NT/+ and Vβ8Tg αβ T lineage cells. Because ∼40% of normal αβ T cells contain VβDJβ joins on nonselected TCRβ alleles, our data revealed that the overall level of endogenous Vβ rearrangements may be only 4-fold lower in Vβ14NT/+ and Vβ8Tg αβ T lineage cells compared with WT cells. A precise quantification cannot be made because VβDJβ2 joins in WT cells could arise through primary or secondary Vβ rearrangements. This modest reduction seems at odds with published studies demonstrating that TCRβ transgenes, such as Vβ8Tg, inhibit endogenous Vβ rearrangements to an apparently greater extent (25, 29). However, analyses of Vβ rearrangements in TCRβ transgenic mice predominantly have been conducted by PCR amplification of VβDJβ2 joins because, theoretically, VβDJβ1 joins can assemble on extrachromosomal circles that might not be subject to feedback inhibition. Considering the results of our analysis of TCRβ rearrangements in αβ T cell hybridomas generated from mice containing a preassembled TCRβ gene or transgene, reappraisals of conclusions gained from some previous studies of TCRβ-mediated feedback inhibition may be warranted. Still, the frequency of Vβ rearrangements on WT alleles in Vβ14NT/+ and Vβ8Tg cells is greater than we expected, considering the accepted model of TCRβ-mediated feedback inhibition. DNA cleavage during V(D)J recombination activates Ataxia Telangiectasia Mutated-dependent responses that may regulate lymphocyte differentiation and Ag receptor gene rearrangements (33, 34). We recently found that Atm−/− αβ T lineage cells exhibit a greater frequency of TCRβ allelic inclusion than WT cells (N. Steinel and C.H. Bassing, unpublished observations). In this context, the ability of TCRβ genes/transgenes to bypass the necessity of assembling VβDJβCβ genes through DNA cleavage might prevent the activation of Ataxia Telangiectasia Mutated-dependent signals that inhibit endogenous Vβ rearrangements.

We also discovered that TCRβ allelic exclusion in mouse lymphocytes can be enforced through silencing the expression of functionally assembled VβDJβCβ genes within cell-surface TCRβ-chains. In Vβ14NT/+ and Vβ8Tg αβ T lineage cells, TCRβ allelic exclusion of some endogenous Vβs mainly occurs through silencing of assembled VβDJβCβ genes involving these Vβ segments. Differential regulation of Vβ expression by inhibition of rearrangement versus silencing in Vβ14NT/+ and Vβ8Tg αβ T lineage cells reinforces previous conclusions that germline transcription and recombinational accessibility of each Vβ segment is regulated individually (35, 36), and distinct cis-acting elements control Vβ rearrangement and TCRβ allelic exclusion (37). The endogenous Vβ segments that are regulated by feedback inhibition versus silencing in Vβ14NT/+ and Vβ8Tg cells are interspersed throughout the TCRβ locus and reside proximal and distal from Dβ-Jβ segments. Our comparison of Vβ promoter, coding, and recombination signal sequences failed to reveal any similarities within or differences between these two groups of Vβs that could provide insight into the mechanistic basis for their distinct regulation. Yet, we did observe a low density of transposons and repetitive genomic sequences directly upstream or downstream of the Vβs that rearranged in Vβ14NT/+ and Vβ8Tg cells. Because these types of DNA elements promote epigenetic changes that inhibit site-specific genomic recombination events in Schizosaccharomyces pombe and Tetrahymena thermophila (38, 39) and possibly DH-to-JH rearrangements in developing B cells (40), there is reason to speculate that Vβ-to-DJβ rearrangements may be downregulated by similar mechanisms. Considering that VHDJHCH transgenes can enforce IgH allelic exclusion without preventing the rearrangement of proximal VH segments (41, 42), postrecombination silencing of Ag receptor genes may contribute to enforce allelic exclusion of more than TCRβ genes.

Posttranscriptional silencing of the Vβ14NT gene contributes to TCRβ allelic exclusion in approximately half of Vβ14NT:Vβ8Tg αβ T lineage cells. We demonstrated that this silencing occurs at the protein level, indicating that the Vβ14NT mRNA is not translated or the Vβ14NT chain is rapidly degraded in ∼50% of αβ T cells expressing the Vβ8.2DJβ1.1Cβ1 transgene. In Vβ14+Vβ8+ cells, the Vβ8Tg and Vβ14NT proteins must each form stable αβ TCR complexes with the TCRα-chains expressed in Vβ14+Vβ8+ cells. Perhaps intrinsic properties or high expression of the Vβ8Tg protein outcompetes the Vβ14NT protein for association with TCRα-chains in Vβ14−Vβ8+ cells, leading to the rapid degradation of free Vβ14NT chains. Transcripts of in-frame VβDJβCβ genes from both alleles have been isolated from WT αβ T cells that exhibit TCRβ allelic exclusion (15); however, experiments to determine the potential expression of both genes within intracellular TCRβ have not been reported. Consequently, future experiments are needed to evaluate whether the regulation of TCRβ allelic exclusion at the protein level is a general mechanism that extends to other endogenous VβDJβCβ genes and occurs in cells lacking preassembled TCRβ transgenes. Although Vβ14ΝΤ can be silenced at the protein level in Vβ14NT:Vβ8Tg cells, our data cannot exclude contributions of other mechanisms, such as transcriptional or translational silencing, to enforce TCRβ allelic exclusion in αβ T lineage cells that have assembled in-frame VβDJβCβ genes on both alleles. Consistent with this notion, TCRβ allelic exclusion of a Vβ13 gene segment inserted upstream of the endogenous Dβ1 segment can be mediated through transcriptional downregulation postrecombination (37). Thus, our findings suggest that to reach unequivocal conclusions, future studies of TCRβ allelic exclusion and feedback inhibition might need to include assays that quantify chromosomal Vβ-to-DJβ rearrangements, VβDJβCβ mRNA, and intracellular TCRβ protein.

Acknowledgments

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by the Department of Pathology and Laboratory Medicine, the Center for Childhood Cancer Research of the Children’s Hospital of Philadelphia, the Abramson Family Cancer Research Institute (to C.H.B.), and University of Pennsylvania Training Grant TG GM-07229 (to B.L.B.). C.H.B. was a Pew Scholar in the Biomedical Sciences and is a Leukemia and Lymphoma Society Scholar.

  • Abbreviations used in this paper:

    DN
    double-negative
    DP
    double-positive
    qPCR
    quantitative PCR
    TCR
    T cell Ag receptor
    Vβ8Tg
    Vβ8.2DJβ1.1Cβ1 transgene
    WT
    wild-type.

  • Received September 21, 2009.
  • Accepted May 12, 2010.

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