TCRβ chain repertoire of peripheral αβ T cells is generated through the stepwise assembly and subsequent selection of TCRβ V region exons during thymocyte development. To evaluate the influence of a two-step recombination process on Vβ rearrangement and selection, we generated mice with a preassembled Dβ1Jβ1.1 complex on the Jβ1ω allele, an endogenous TCRβ allele that lacks the Dβ2-Jβ2 cluster, creating the Jβ1DJβ allele. As compared with Jβ1ω/ω mice, both Jβ1DJβ/ω and Jβ1DJβ/DJβ mice exhibited grossly normal thymocyte development and TCRβ allelic exclusion. In addition, Vβ rearrangements on Jβ1DJβ and Jβ1ω alleles were similarly regulated by TCRβ-mediated feedback regulation. However, in-frame VβDJβ rearrangements were present at a higher level on the Jβ1DJβ alleles of Jβ1DJβ/ω αβ T cell hybridomas, as compared with on the Jβ1ω alleles. This bias was most likely due to both an increased frequency of Vβ-to-DJβ rearrangements on Jβ1DJβ alleles and a preferential selection of cells with in-frame VβDJβ exons assembled on Jβ1DJβ alleles during the development of Jβ1DJβ/ω αβ T cells. Consistent with the differential selection of in-frame VβDJβ rearrangements on Jβ1DJβ alleles, the Vβ repertoire of αβ T cells was significantly altered during αβ TCR selection in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice, as compared with in Jβ1ω/ω mice. Our data indicate that the diversity of DJβ complexes assembled during thymocyte development influences TCRβ chain selection and peripheral Vβ repertoire.

Generation and expression of a diverse repertoire of Ag receptors on the cell surface of lymphocytes are essential for adaptive immunity. TCR and Ig chains consist of V regions that bind Ag and C regions. During lymphocyte development, TCR and Ig V region exons (genes) are assembled through the rearrangement of germline V, D, and J gene segments (1). V(D)J recombination is initiated by the lymphocyte-specific RAG1/RAG2 (RAG) endonuclease, which induces DNA double-strand breaks between participating gene segments and their flanking recombination signal sequences (RSSs),3 generating blunt signal ends and hairpin-sealed coding ends (2). Signal ends are repaired by generally expressed core nonhomologous end-joining (NHEJ) proteins to form signal joins, whereas coding ends are opened by additional NHEJ proteins and then repaired by the core NHEJ factors to form V(D)J coding joins (3, 4). This process generates Ag receptor chain diversity through the combination of joining events, the inherent imprecision of NHEJ, and the random addition of nontemplate (N) nucleotides by the lymphocyte-specific TdT protein (1).

In humans and mice, αβ T lymphocytes develop through a differentiation program that involves the assembly, expression, and selection of TCR genes (5). TCRβ genes are assembled through an ordered process in which Dβ-to-Jβ rearrangements are detectable in c-kit (CD117)+CD44+CD25 early T lineage progenitors (ETPs) and CD117+CD44+CD25+ (stage II) CD4/CD8 (double-negative (DN)) thymocytes, and Vβ rearrangements initiate in CD117CD44CD25+ (stage III) DN cells (6, 7). Although Dβ-to-Jβ recombination is not required for Vβ rearrangement, the deletion of sequences between Dβ and Jβ segments, such as the 3′Dβ RSSs, may facilitate Vβ-to-DJβ recombination (8, 9, 10). The assembly and expression of a productive (in-frame) VβDJβ rearrangement on the first allele generate TCRβ chains that pair with pTα molecules to form pre-TCRs that select DNIII cells for further development (5). This β-selection process involves signals that rescue DNIII thymocytes from apoptosis, promote rapid cellular proliferation, direct differentiation into CD117CD44CD25 DNIV cells and then CD4+/CD8+ (double positive (DP)) thymocytes, and prevent Vβ-to-DJβ rearrangements on the second allele to ensure TCRβ chains are expressed from only one allele (5). In DP thymocytes, TCRα genes are assembled on both alleles from Vα and Jα segments (11). Productive VαJα rearrangements generate TCRα chains that can associate with TCRβ chains to form αβ TCR receptors, which are then subject to selection (12). Negative selection of αβ TCRs leads to cell death, whereas positive selection rescues DP cells from apoptosis and promotes their further development to CD4+ or CD8+ (single-positive (SP)) thymocytes, which exit the thymus as αβ T cells (12). Due to the imprecision of V(D)J joining, only one-third of VβDJβ rearrangements are assembled in-frame. Thymocytes that assemble a nonproductive (out-of-frame) VβDJβ rearrangement on the first allele can undergo Vβ rearrangement on the second allele, which drives differentiation if assembled in-frame (13). Thus, in addition to their selected VβDJβ rearrangements, ∼60% of normal αβ T cells contain DJβ rearrangements and ∼40% contain out-of-frame VβDJβ rearrangements on their nonselected alleles, a phenomenon referred to as the 60:40 ratio (13, 14). This pattern of TCRβ rearrangements indicates that Vβ-to-DJβ rearrangements occur on one allele at a time in DNIII cells (13); however, there is no evidence that distinguishes whether Dβ-to-Jβ rearrangements occur asynchronously between alleles or on both alleles simultaneously.

The TCRβ chain repertoire of peripheral αβ T lymphocytes is generated through the assembly and selection of VβDJβ rearrangements during thymocyte development. Generation of primary TCRβ repertoires is not random because rearrangements between both Dβ and Jβ segments and Vβ segments and DJβ complexes occur at varying relative levels in DN thymocytes, as determined, at least in part, by genetic variations of their flanking RSSs (15, 16, 17, 18, 19). The relative frequency at which Vβs are expressed in DN thymocytes before β-selection and in DP cells before αβ TCR selection is similar, suggesting that Vβ repertoire is not substantially altered during β-selection (20). In contrast, the relative frequency at which Vβs are expressed in DP thymocytes vs SP thymocytes and peripheral αβ T cells can be dramatically different, indicating that the Vβ repertoire of TCRβ chains is shaped during αβ TCR selection (21, 22, 23, 24, 25, 26, 27). The length of VβDJβ joins also is modulated during thymocyte development with shorter sequences selected for during DP to SP differentiation (28, 29).

There are many gaps in our understanding of the mechanisms that evolved to control the regulated assembly and selection of TCRβ chains. The tripartite joining of Vβ, Dβ, and Jβ segments is required for generation of TCRβ chains with normal-sized third CDRs, which are involved in Ag binding (30). Accordingly, selective pressure enforced by Ags may have caused TCRβ loci to evolve Vβ, Dβ, and Jβ segments such that TCRβ chains include amino acids encoded by Dβ nucleotides and gain the increased junctional diversity of two joining events. The assembly of Ig H chain genes from VH, DH, and JH segments occurs through DJH intermediates and is subject to allelic exclusion (14), whereas the assembly of TCRδ genes occurs through both DJδ and VDδ intermediates and exhibits allelic inclusion (31). In this context, an ordered two-step recombination process also could have evolved to provide an additional level of regulatory control important for preventing Vβ to DJβ rearrangements on both alleles and enforcing TCRβ allelic exclusion (18).

The mouse TCRβ locus consists of 20 functional Vβ segments and two Dβ-Jβ clusters (Dβ1-Jβ1 and Dβ2-Jβ2) that each contains a single Dβ segment (Dβ1 or Dβ2) and six functional Jβ segments (Jβ1.1-Jβ1.6 or Jβ2.1-Jβ2.7). In a population of DN thymocytes, Dβ2 rearranges to all six functional Jβ2 segments and Dβ1 rearranges to all 12 functional Jβ segments, creating DβJβ complexes of 18 Dβ-Jβ joining combinations. In the DNIII population, all Vβ segments rearrange to these Dβ1Jβ1, Dβ1Jβ2, and Dβ2Jβ2 complexes, with deletion of Dβ1Jβ1 complexes and germline Jβ1 segments upon joining to either Dβ1Jβ2 or Dβ2Jβ2 complexes. Moreover, in DNIII cells with primary Vβ rearrangements to Dβ1Jβ1 complexes, secondary Vβ rearrangements theoretically can occur to Dβ2Jβ2 complexes, resulting in deletion of the VβDβ1Jβ1 complexes. Because the number and complexity of TCRβ rearrangements present obstacles for the investigation of mechanisms that regulate the assembly of TCRβ V region exons, we previously used gene targeting to delete the endogenous Dβ2-Jβ2 cluster and create the Jβ1ω allele on which Vβ rearrangements only can be targeted to DβJβ1 complexes of six Dβ-Jβ joining combinations (18). Heterozygous and homozygous Jβ1ω mice exhibit αβ T cell development, TCRβ rearrangement, Vβ repertoire, and TCRβ allelic exclusion indistinguishable from wild-type mice with unmodified TCRβ loci (18). Thus, to evaluate the influence of Dβ-to-Jβ rearrangement on Vβ rearrangement and TCRβ selection, we generated and analyzed mice with a preassembled Dβ1Jβ1.1 complex on the Jβ1ω allele. Due to practical considerations, our analysis was unfortunately limited to one particular Jβ1 segment and one DβJβ coding join of a defined sequence and length, which could introduce biases in Vβ rearrangement and TCRβ selection. Yet, such potential biases also would indicate unequivocally that DJβ complexes assembled in DN thymocytes influence these downstream processes.

The DJβ targeting vector was constructed in pLNTK (32). The 5′ homology arm is a 2.8-kb KpnI/BamHI fragment containing a preassembled Dβ1Jβ1.1 complex PCR cloned from splenocytes, which was then blunted and ligated into the SalI site of pLNTK. The 3′ homology arm is a 1.8-kb BamHI/SacI genomic fragment containing Jβ1.2 through Jβ1.6, which was blunted and ligated into the XhoI site of pLNTK. This fragment also contained a HindIII site that was inserted just inside the BamHI site before subcloning. The completed targeting vector was sequenced to confirm the integrity of the preassembled Dβ1Jβ1.1 complex. The 5′KO probe is a 300-bp PCR product amplified with primers 5′-GGATCCTGAGAACTGGACATAAGGG-3′ and 5′-TTTAATCACTGTGTACTTCC-3′. The 3′KO probe is a 546-bp EcoRI/KpnI fragment.

The DJβ targeting vector was electroporated into Jβ1ω/ω embryonic stem (ES) cells (18), as previously described (33) to generate Jβ1DJβNeo/ω ES cells. Targeted clones were identified by Southern blot analysis with the 5′Dβ1 probe on EcoRI-digested genomic DNA (5.5-kb Jβ1DJβNeo, 9.3-kb Jβ1ω) and confirmed with the 3′Jβ1 probe on HindIII-digested DNA (5.7-kb Jβ1DJβNeo, 8.9-kb Jβ1ω). Targeted ES cells were infected with recombinant AdenoCre and subcloned. Cre-deleted subclones were identified by Southern blot analysis with the 5′Dβ1 probe on EcoRI-digested genomic DNA (5.5-kb Jβ1DJβNeo, 8.7-kb Jβ1DJβ, 9.3-kb Jβ1ω) and confirmed with the 3′Jβ1 probe on BamHI-digested DNA (8.7-kb Jβ1DJβNeo, 13.5-kb Jβ1DJβ, 8.3-kb Jβ1ω).

Generation of Jβ1ω/ω mice was previously described (18). DO11.10 TCRβ transgenic mice (34) were bred with Jβ1DJβ/DJβ mice to generate Vβ8TgJβ1DJβ/DJβ mice.

Cells from single-cell suspensions of the thymuses and spleens of 4- to 6-wk-old mice were counted and then stained with indicated combinations of FITC-conjugated anti-CD8, anti-Vβ5, anti-Vβ8, anti-Vβ10b, and anti-Vβ14 Abs; PE-conjugated anti-CD4, anti-CD8α, anti-Cβ, and anti-Vβ10b Abs; allophycocyanin-conjugated anti-Cβ and anti-CD4; biotin-conjugated anti-Vβ14 and anti-Vβ12; and streptavidin (SA)-PE-Cy7, SA-FITC, and SA-allophycocyanin reagents (BD Pharmingen). For analysis of DN subsets, thymocytes were stained with a mixture of PE-conjugated Abs for TCRβ, TCRδ, CD8α, CD45R, CD19, CD11c, CD11b, Ter119, and NK1.1, as well as PE-Cy7-conjugated anti-CD25 and allophycocyanin-conjugated anti-CD117 Abs (BD Pharmingen). Data acquisition was conducted on a BD FACSCalibur equipped with BD CellQuest Pro, and data analysis was performed with FlowJo software (Tree Star). Each FACS experiment was done at least three separate times on independent mice of each genotype.

Hybridomas were generated by fusion of the BW-1100.129.237 thymic lymphoma cell line (35) with Con A- and IL-2-stimulated αβ T cells, as previously described (33). Hybridoma genomic DNA was isolated and subjected to Southern blot analysis. The Southern blot analysis of TCRβ rearrangements was conducted with the 5′Dβ1 and 3′Jβ1 probes on either EcoRI- or HindIII-digested genomic DNA isolated from the hybridomas. The 5′Dβ1 probe is a 400-bp NheI fragment. The 3′Jβ1 probe is a 777-bp DrdI fragment. PCR analyses of Vβ rearrangements to Dβ1Jβ1.1 and Dβ1Jβ1.2 complexes in hybridomas and sort-purified DNIII cells were conducted, as previously described (18).

Our previous gene-targeted modification of the endogenous Dβ1Jβ1 cluster demonstrated that a loxP site and a novel BamHI site inserted just 3′ of Jβ1.2 on one allele could be used to distinguish between VβDJβ1.1 and VβDJβ1.2 rearrangements and had no discernable effects on thymocyte development, Vβ rearrangement, or VβDJβ1.1 and VβDJβ1.2 expression (18). Thus, we used gene-targeted mutation to replace the germline sequences spanning the Dβ1 and Jβ1.1 segments with a preassembled Dβ1Jβ1.1 complex (Fig. 1) on a single allele of Jβ1ω/ω ES cells. This Dβ1Jβ1.1 complex was isolated by PCR amplification and subcloning of Dβ1Jβ1.1 joins on DJβ rearranged alleles of wild-type splenic αβ T cells. The isolated Dβ1Jβ1.1 complex does not contain N or P nucleotides, and compared with the full sequences of Dβ1 and Jβ1.1 is missing one C nucleotide. The initial targeting event resulted in the replacement of the endogenous sequences with the Dβ1Jβ1.1 complex and insertion of a neomycin-resistant gene (Neor) flanked by loxP sites just 3′ of the endogenous Jβ1.2 segment, creating the Jβ1DJβNeo allele (Fig. 1,A). Next, we deleted the Neor gene through transient expression of the Cre recombinase in Jβ1DJβNeo/ω ES cells to leave a single loxP site inserted just 3′ of the endogenous Jβ1.2 segment, creating the Jβ1DJβ allele (Fig. 1,A). We also inserted a novel HindIII site next to the loxP site to distinguish between TCRβ rearrangements on the Jβ1DJβ and Jβ1ω alleles (Fig. 1). Finally, we used Jβ1DJβ/ω ES cells to generate germline Jβ1DJβ/ω mice and then bred these with Jβ1ω/ω mice and also with each other to generate Jβ1DJβ/ω and Jβ1DJβ/DJβ mice, respectively. The comparative analysis of Jβ1ω/ω and Jβ1DJβ/DJβ mice will allow us to evaluate whether complete subversion of the tripartite TCRβ recombination process influences αβ T cell development, TCRβ rearrangement, Vβ repertoire, and/or TCRβ allelic exclusion. In addition, the analysis of TCRβ rearrangements in Jβ1DJβ/ω will reveal whether the assembly of a DJβ complex could affect Vβ rearrangement or influence TCRβ selection.

FIGURE 1.

Gene-targeted generation of Jβ1DJβ/ω ES cells. A, This schematic diagram illustrates the Jβ1ω allele that lacks the Dβ2-Jβ2.7 segments, positioning of the DJβ targeting construct, and the resulting Jβ1DJβNeo and Jβ1DJβ alleles. Open boxes depict the Dβ1 and Jβ1 segments; their adjacent RSSs are triangles. Upon gene targeting, the preassembled Dβ1Jβ1.1 complex replaced the germline Dβ1 and Jβ1.1 segments, and a neomycin resistance gene (Neor, rectangle) flanked by loxP sites (filled ovals) was inserted between the Jβ1.2 and Jβ1.3 segments. Following Cre-mediated deletion of the neor gene, the remaining segments align approximately to the position of an endogenous Dβ1Jβ1.1 complex. The solid horizontal bars indicate the relative locations of the 5′KO, 3′KO, 5′Dβ1, and 3′Jβ1 probes. Restriction site designations are as follows: B, BamHI; H, HindIII; R, EcoRI; S, SacI. B, Shown are the sequences of the germline Dβ1 segment with the 5′Dβ1 RSS and the heptamer of the 3′Dβ1 RSS (top sequence), the preassembled Dβ1Jβ1.1 complex (middle sequence), and the germline Jβ1.1 segment with the Jβ1.1 RSS (bottom sequence).

FIGURE 1.

Gene-targeted generation of Jβ1DJβ/ω ES cells. A, This schematic diagram illustrates the Jβ1ω allele that lacks the Dβ2-Jβ2.7 segments, positioning of the DJβ targeting construct, and the resulting Jβ1DJβNeo and Jβ1DJβ alleles. Open boxes depict the Dβ1 and Jβ1 segments; their adjacent RSSs are triangles. Upon gene targeting, the preassembled Dβ1Jβ1.1 complex replaced the germline Dβ1 and Jβ1.1 segments, and a neomycin resistance gene (Neor, rectangle) flanked by loxP sites (filled ovals) was inserted between the Jβ1.2 and Jβ1.3 segments. Following Cre-mediated deletion of the neor gene, the remaining segments align approximately to the position of an endogenous Dβ1Jβ1.1 complex. The solid horizontal bars indicate the relative locations of the 5′KO, 3′KO, 5′Dβ1, and 3′Jβ1 probes. Restriction site designations are as follows: B, BamHI; H, HindIII; R, EcoRI; S, SacI. B, Shown are the sequences of the germline Dβ1 segment with the 5′Dβ1 RSS and the heptamer of the 3′Dβ1 RSS (top sequence), the preassembled Dβ1Jβ1.1 complex (middle sequence), and the germline Jβ1.1 segment with the Jβ1.1 RSS (bottom sequence).

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To evaluate the potential influence of Dβ-to-Jβ rearrangement on αβ T cell development, we analyzed thymocytes and splenocytes isolated from Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice. Both Jβ1DJβ/ω and Jβ1DJβ/DJβ mice exhibited similar numbers of thymocytes and splenocytes as Jβ1ω/ω mice (data not shown). FACS analysis of Jβ1DJβ/ω and Jβ1DJβ/DJβ thymocytes with anti-CD4 and anti-CD8 Abs revealed a distribution of DN, DP, and SP populations similar to those in Jβ1ω/ω thymocytes (Fig. 2, A, D, and E). In addition, FACS analysis of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ splenocytes with anti-CD4 and anti-CD8 Abs revealed similar percentages of CD4+ and CD8+ αβ T cells in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice, as compared with in Jβ1ω/ω mice (Fig. 2, B and E). FACS analysis of Jβ1DJβ/ω and Jβ1DJβ/DJβ thymocytes with anti-CD117 and anti-CD25 Abs showed a similar distribution of ETPs, stage II, stage III, and stage IV DN cells as Jβ1ω/ω thymocytes (Fig. 2, C and D). Despite these similarities, there were detectable differences in the DNIII and DNIV populations and statistically significant differences in SP thymocytes and CD4+ and CD8+ splenic αβ T cell populations among Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice (Fig. 2, D and E). These data demonstrate that, although neither a single fixed DJβ rearrangement nor the sequence of the particular DJβ complex used has any substantial effect on thymocyte development, the inability to assemble a diverse DJβ repertoire has subtle or significant influences on different stages of αβ T cell differentiation. The decreased ratios of DNIII to DNIV cell populations that correlate with increased copy number of the Jβ1DJβ allele may reflect that Vβ rearrangements occur at a higher frequency on the Jβ1DJβ allele in DNIII thymocytes and/or DNIII cells expressing VβDJβ chains from Jβ1DJβ alleles are preferentially selected. The statistically significant differences in SP thymocytes and CD4+ and CD8+ splenic αβ T cell populations among Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice suggest that selection of αβ TCR containing VβDJβ chains with the preassembled Dβ1Jβ1.1 complex may be altered.

FIGURE 2.

Normal αβ T cell development in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice. A and B, Shown are representative anti-CD4 and anti-CD8 FACS analysis of cells isolated from the thymuses (A) and spleens (B) of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice. The percentage of DN, DP, CD4+ SP, and CD8+ SP thymocytes (A) and CD4+ and CD8+ αβ T cells (B) is indicated. C, Shown are representative anti-CD117 and anti-CD25 FACS analysis of thymocytes negative for mature cell markers (TCRβ, TCRδ, CD4, CD8α, CD19, CD11c, CD11b, B220, and NK1.1). D and E, Bar graphs showing the average frequency of cells within each thymocyte developmental stage and peripheral T cell population from at least five mice of each genotype. The error bars are SEM. Significant differences have been calculated using two-tailed Student’s t test.

FIGURE 2.

Normal αβ T cell development in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice. A and B, Shown are representative anti-CD4 and anti-CD8 FACS analysis of cells isolated from the thymuses (A) and spleens (B) of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice. The percentage of DN, DP, CD4+ SP, and CD8+ SP thymocytes (A) and CD4+ and CD8+ αβ T cells (B) is indicated. C, Shown are representative anti-CD117 and anti-CD25 FACS analysis of thymocytes negative for mature cell markers (TCRβ, TCRδ, CD4, CD8α, CD19, CD11c, CD11b, B220, and NK1.1). D and E, Bar graphs showing the average frequency of cells within each thymocyte developmental stage and peripheral T cell population from at least five mice of each genotype. The error bars are SEM. Significant differences have been calculated using two-tailed Student’s t test.

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Because an allotypic marker has not been found or created for TCRβ chains, many studies of TCRβ allelic exclusion have been conducted using transgenic mice that express a prerearranged, in-frame VβDJβ rearrangement randomly integrated into the genome (36, 37, 38, 39, 40, 41, 42). Thus, to investigate whether a two-step recombination process is required for enforcement of TCRβ allelic exclusion, we generated and analyzed Vβ expression in Jβ1DJβ/DJβ mice expressing a transgenic in-frame Vβ8DJβ rearrangement (Vβ8Tg). FACS analysis of Vβ8Tg and Vβ8TgJβ1DJβ/DJβ splenocytes with an anti-Cβ Ab and an anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 Ab failed to detect any differences in the presence of αβ T cells expressing these Vβs on the cell surface with Vβ8 in either mouse (Fig. 3,A). In addition, FACS analysis of Vβ8Tg and Vβ8TgJβ1DJβ/DJβ splenocytes with an anti-Vβ8 Ab and either an anti-Vβ12 or anti-Vβ14 Ab did not detect any difference in αβ T cells expressing both Vβ8 and another Vβ on the cell surface (Fig. 3 B). These data indicate that a two-step recombination process is not required for enforcement of TCRβ allelic exclusion in Vβ8Tg mice, at least at the level of detection of FACS using anti-Vβ Abs specific for TCRβ chains with different Vβs. We also conducted FACS analysis with different combinations of anti-Vβ-specific Abs on thymocytes and splenocytes of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice (data not shown), but the technical limitations of this approach did not have sufficient specificity or sensitivity of staining to determine whether the expected small populations of allelically included cells were present.

FIGURE 3.

Normal allelic exclusion in Vβ8Tg:Jβ1DJβ/DJβ αβ T cells. A, Shown are representative anti-Cβ and anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 FACS analysis of cells isolated from the spleens of wild-type, Jβ1DJβ/DJβ, Vβ8Tg, and Vβ8TgJβ1DJβ/DJβ mice. The percentage of Cβ+ cells that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 is indicated. B, Shown are representative anti-Vβ12 and anti-Vβ8 or anti-Vβ14 and anti-Vβ8 FACS analysis of Cβ+ cells isolated from the spleens of wild-type, Jβ1DJβ/DJβ, Vβ8Tg, and Vβ8TgJβ1DJβ/DJβ mice. The percentage of Vβ12+, Vβ8+, and Vβ12+Vβ8+ or Vβ14+, Vβ8+, and Vβ14+Vβ8+ cells is indicated.

FIGURE 3.

Normal allelic exclusion in Vβ8Tg:Jβ1DJβ/DJβ αβ T cells. A, Shown are representative anti-Cβ and anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 FACS analysis of cells isolated from the spleens of wild-type, Jβ1DJβ/DJβ, Vβ8Tg, and Vβ8TgJβ1DJβ/DJβ mice. The percentage of Cβ+ cells that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 is indicated. B, Shown are representative anti-Vβ12 and anti-Vβ8 or anti-Vβ14 and anti-Vβ8 FACS analysis of Cβ+ cells isolated from the spleens of wild-type, Jβ1DJβ/DJβ, Vβ8Tg, and Vβ8TgJβ1DJβ/DJβ mice. The percentage of Vβ12+, Vβ8+, and Vβ12+Vβ8+ or Vβ14+, Vβ8+, and Vβ14+Vβ8+ cells is indicated.

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The observed enforcement of TCRβ allelic exclusion in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice suggests that Vβ-to-DJβ rearrangements on Jβ1DJβ alleles are subject to TCRβ-mediated feedback inhibition. However, despite impaired TCRβ-mediated feedback inhibition in pTα-deficient thymocytes, dual Vβ-expressing αβ T cells are not observed in either pTα-deficient mice or pTα-deficient mice containing a TCRβ transgene, indicating that TCRβ allelic exclusion also can be enforced by post Vβ-to-DJβ recombination mechanisms (38, 42). The 60:40 ratio of αβ T cells with VβDJβ/DJβ and VβDJβ/VβDJβ rearrangements reflects the regulation of Vβ rearrangement by TCRβ-mediated feedback inhibition (13). Therefore, to evaluate the influence of a two-step recombination process on TCRβ-mediated feedback regulation, we generated a panel of 88 Jβ1DJβ/DJβ αβ T cell hybridomas and analyzed TCRβ rearrangements by Southern blot analysis. We first assayed TCRβ rearrangements on EcoRI-digested genomic DNA using the 5′Dβ1 and 3′Jβ1 probes, each of which hybridizes to the same 5.5-kb EcoRI germline fragment from the Jβ1DJβ allele (Fig. 1,A). We found that 55 of 88 (62%) Jβ1DJβ/DJβ αβ T cell hybridomas retained 5.5-kb 5′Dβ1 and 3′Jβ1 bands, indicating they contained only one VβDJβ rearrangement (Table I,A). The remaining 33 of 88 (38%) lacked the 5.5-kb 5′Dβ1 and 3′Jβ1 bands, but contained two novel-sized 3′Jβ1 bands, revealing they carried VβDJβ rearrangements on both Jβ1DJβ alleles (Table I A). These data demonstrate that Jβ1DJβ/DJβ αβ T cells exhibit the normal 60:40 ratio of VβDJβ/DJβ and VβDJβ/VβDJβ rearrangements. Thus, Vβ rearrangements on Jβ1DJβ alleles are subject to normal TCRβ feedback regulation, consistent with the observed enforcement of TCRβ allelic exclusion in Jβ1DJβ/DJβ and Vβ8TgJβ1DJβ/DJβ αβ T cells.

Table I.

Rearrangements in JβDJβ αβ T cell hybridomas

A. TCRβ rearrangement phenotypes in Jβ1DJβ/DJβ and Jβ1DJβ/ω αβ T cell hybridomas
Genotype Total VβDJβ/DJβ VβDJβ/VβDJβ 
Jβ1DJβ/DJβ 88 55 (62.5%) 33 (37.5%) 
Jβ1DJβ/ω 247 144 (58.0%) 103 (42.0%) 
A. TCRβ rearrangement phenotypes in Jβ1DJβ/DJβ and Jβ1DJβ/ω αβ T cell hybridomas
Genotype Total VβDJβ/DJβ VβDJβ/VβDJβ 
Jβ1DJβ/DJβ 88 55 (62.5%) 33 (37.5%) 
Jβ1DJβ/ω 247 144 (58.0%) 103 (42.0%) 
B. Frequency of complete VβDJβ exon rearrangements on the DJβ allele versus the ω allele in heterozygous VβDJβ/DJβ Jβ1DJβ/ω αβ T cell hybridomas (144 total)
Genotype Allele Vβ to DJβ 
Jβ1DJβ/ω DJβ 99 (69.0%) 
 ω 45 (31.0%) 
B. Frequency of complete VβDJβ exon rearrangements on the DJβ allele versus the ω allele in heterozygous VβDJβ/DJβ Jβ1DJβ/ω αβ T cell hybridomas (144 total)
Genotype Allele Vβ to DJβ 
Jβ1DJβ/ω DJβ 99 (69.0%) 
 ω 45 (31.0%) 

To evaluate whether the presence of the preassembled Dβ1Jβ1.1 complex might either enhance or impair Vβ-to-DJβ rearrangement, we first sought to determine whether overall Vβ rearrangements occur at a similar level on the Jβ1DJβ and Jβ1ω alleles. Unfortunately, due to the inherent biases of amplifying overall VβDβJβ1 rearrangements of one fixed size on Jβ1DJβ alleles vs six different sizes on Jβ1ω alleles, solid conclusions are not possible from PCR-based analysis of Vβ-to-DJβ rearrangements in nonselected DNIII thymocytes. The novel HindIII site inserted just downstream of the preassembled DJβ1.1 complex enables us to distinguish between TCRβ rearrangements on the Jβ1DJβ and Jβ1ω alleles in Jβ1DJβ/ω αβ T cells. Thus, we generated a panel of 247 Jβ1DJβ/ω αβ T cell hybridomas and analyzed VβDJβ rearrangements in these cells by Southern blot analysis and by PCR amplification and sequencing. We first assayed TCRβ rearrangements on EcoRI-digested genomic DNA using the 5′Dβ1 and 3′Jβ1 probes, each of which hybridizes to the same 9.4-kb germline EcoRI fragment on the Jβ1ω allele (Fig. 1 A). We found that all 247 hybridomas lacked the 9.4-kb 3′Jβ1 band, but contained a novel-sized 3′Jβ1 band, revealing they had DJβ or VβDJβ rearrangements on the Jβ1ω allele. In addition, we found that 144 (58%) contained a single 5′Dβ1 band and 103 (42%) lacked any 5′Dβ1 bands. These data indicate that Jβ1DJβ/ω αβ T cells also exhibit the normal ratio of VβDJβ/DJβ and VβDJβ/VβDJβ rearrangements.

If Vβ-to-DJβ rearrangements occur at equal frequency on the Jβ1DJβ and Jβ1ω alleles and VβDJβ rearrangements involving the single preassembled Dβ1Jβ1.1 complex and the normal repertoire of rearranged Dβ1Jβ1 complexes are similarly selected, Jβ1DJβ/ω αβ T cells of the VβDJβ/DJβ configuration should contain an equal frequency of VβDJβ rearrangements on the Jβ1DJβ and Jβ1ω alleles. Thus, we next assayed TCRβ rearrangements using the 5′Dβ1 probe on HindIII-digested genomic DNA of the 144 Jβ1DJβ/ω αβ T cell hybridomas with VβDJβ rearrangements on a single allele. The 5′Dβ1 probe hybridizes to an 8.9-kb germline HindIII fragment on the Jβ1ω allele and to a 2.5-kb germline fragment on the Jβ1DJβ allele (Fig. 1,A). We found that 45 of 144 (31%) retained and 99 of 144 (69%) lost the 2.5-kb 5′Dβ1 band (Table I B), revealing that the majority of Jβ1DJβ/ω αβ T cells contain VβDJβ rearrangements on the Jβ1DJβ allele. This observation indicates that either Vβ rearrangements occur at a higher frequency on the Jβ1DJβ allele in DN cells or thymocytes expressing VβDJβ chains from Jβ1DJβ alleles are preferentially selected during development.

In addition to their in-frame and selected VβDJβ rearrangements, ∼60% of normal αβ T cells contain DJβ rearrangements and ∼40% contain out-of-frame VβDJβ rearrangements on their nonselected alleles (13, 14). Because Jβ1DJβ/ω αβ T cell hybridomas exhibit this normal 60:40 ratio, the frequency at which out-of-frame VβDJβ rearrangements occur on the Jβ1DJβ allele of Jβ1DJβ/ω αβ T cells with VβDJβ/VβDJβ rearrangements can be used to distinguish between increased recombination vs preferential selection. In this regard, if Vβ-to-DJβ rearrangements occurred at an increased frequency on Jβ1DJβ alleles, but were selected equally as those on Jβ1ω alleles, greater than one-half of the VβDJβ joins on the Jβ1DJβ alleles in these cells would be out-of-frame. In contrast, if Vβ-to-DJβ rearrangements occurred at the same frequency on Jβ1DJβ and Jβ1ω alleles, but were selected preferentially on the Jβ1DJβ allele, greater than one-half of the VβDJβ joins on the Jβ1DJβ alleles in these cells would be in-frame. We conducted PCR on the genomic DNA isolated from 25 of these hybridomas using primers that hybridize to each specific Vβ and a primer that hybridizes 3′ of the HindIII site on the Jβ1DJβ allele. PCR products were digested with either HindIII or BamHI to distinguish between amplified Vβ rearrangements to the preassembled Dβ1Jβ1.1 complex on the Jβ1DJβ allele vs to Dβ1Jβ1.1 or Dβ1Jβ1.2 complexes on the Jβ1ω allele. Sequence analysis of 25 PCR products representing Vβ rearrangements to the preassembled DJβ1.1 complex revealed that 13 (52%) were out-of-frame and 12 (48%) were in-frame (Table II). These data are not compatible with either of the simple scenarios outlined above. Consequently, we conclude that both an increased frequency of Vβ-to-DJβ rearrangements on Jβ1DJβ alleles and a preferential selection of cells with in-frame VβDJβ rearrangements on Jβ1DJβ alleles during the development of Jβ1DJβ/ω αβ T cells contribute to the bias for in-frame VβDJβ rearrangements on the Jβ1DJβ alleles of Jβ1DJβ/ω αβ T cells.

Table II.

Sequence analysis of VβDJβ coding joins on the Jβ1DJβ alleles of Jβ1DJβ/ω αβ T cell hybridomas with biallelic VβDJβ rearrrangements

Sequence (Codons)PNPDJβ Sequence (Codons)Frame
Vβ16 AGCTTAGCC    AG GGGGCA Out 
Vβ11 AGCAGCCTC    AG GGGGCA Out 
Vβ8.2 AGCGGT GA  ATA  A CAGGGGGCA In 
Vβ10 GCCAGCAGC  TAT G  G GGGGCA Out 
Vβ13 AGCAGTTTC    CCGGGACCA Out 
Vβ12 GCCAGCAGG    CAGGGGGCA In 
Vβ9 AGCAGTAGA    G GGGGCA Out 
Vβ15 TGTGGTGCT  CC TC GA CAGGGGGCA In 
Vβ8.2 GCCAGAGGT  GAA A  GA CAGGGGGCA In 
Vβ7 AGCAGTTTA  TAC  CAGGGGGCA In 
Vβ6 GCCAGCAGT AC  CAGGGGGCA In 
Vβ3 AGCAGTCTC  CTA  A CAGGGGGCA Out 
Vβ8.2 AGCGGTGAT  G GGGGCA In 
Vβ1 AGCAGCCAA    GA CAGGGGGCA Out 
Vβ1 AGCAGC  CA  A CAGGGGGCA In 
Vβ11 AGCAGC CT    A CAGGGGGCA In 
Vβ11 AGCAGC  TTA GG  GGGGCA Out 
Vβ12 AGCAGT CCC G GGACAGGGGGCA In 
Vβ10 AGCAGC  TA  GA CAGGGGGCA Out 
Vβ11 AGCAGC CT  T CAA CCC T  GA CAGGGGGCA In 
Vβ9 AGCAGC CA  GA CAGGGGGCA Out 
Vβ16 AGCTTA TG G GGGGCA Out 
Vβ1 AGCAGC CA    GA CAGGGGGCA Out 
Vβ11 AGCAGC  TT  G GGACAGGGGGCA In 
Vβ11 AGCAGC CA  GA CAGGGGGCA Out 
Sequence (Codons)PNPDJβ Sequence (Codons)Frame
Vβ16 AGCTTAGCC    AG GGGGCA Out 
Vβ11 AGCAGCCTC    AG GGGGCA Out 
Vβ8.2 AGCGGT GA  ATA  A CAGGGGGCA In 
Vβ10 GCCAGCAGC  TAT G  G GGGGCA Out 
Vβ13 AGCAGTTTC    CCGGGACCA Out 
Vβ12 GCCAGCAGG    CAGGGGGCA In 
Vβ9 AGCAGTAGA    G GGGGCA Out 
Vβ15 TGTGGTGCT  CC TC GA CAGGGGGCA In 
Vβ8.2 GCCAGAGGT  GAA A  GA CAGGGGGCA In 
Vβ7 AGCAGTTTA  TAC  CAGGGGGCA In 
Vβ6 GCCAGCAGT AC  CAGGGGGCA In 
Vβ3 AGCAGTCTC  CTA  A CAGGGGGCA Out 
Vβ8.2 AGCGGTGAT  G GGGGCA In 
Vβ1 AGCAGCCAA    GA CAGGGGGCA Out 
Vβ1 AGCAGC  CA  A CAGGGGGCA In 
Vβ11 AGCAGC CT    A CAGGGGGCA In 
Vβ11 AGCAGC  TTA GG  GGGGCA Out 
Vβ12 AGCAGT CCC G GGACAGGGGGCA In 
Vβ10 AGCAGC  TA  GA CAGGGGGCA Out 
Vβ11 AGCAGC CT  T CAA CCC T  GA CAGGGGGCA In 
Vβ9 AGCAGC CA  GA CAGGGGGCA Out 
Vβ16 AGCTTA TG G GGGGCA Out 
Vβ1 AGCAGC CA    GA CAGGGGGCA Out 
Vβ11 AGCAGC  TT  G GGACAGGGGGCA In 
Vβ11 AGCAGC CA  GA CAGGGGGCA Out 

Although the Vβ repertoire is not substantially altered during β-selection (20), the relative frequency at which Vβs are expressed in DP vs SP thymocytes can be dramatically different, indicating that the Vβ repertoire of TCRβ chains is shaped during αβ TCR selection (21, 22, 23, 24, 25, 26, 27). The statistically significant differences in SP cells among Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice suggest that incorporation of the preassembled Dβ1Jβ1.1 complex in productive VβDJβ rearrangements may influence αβ TCR selection of DP thymocytes. To investigate this issue, we sought to evaluate whether the repertoire of TCRβ chains is altered upon DP to SP differentiation in Jβ1DJβ/ω or Jβ1DJβ/DJβ mice, as compared with Jβ1ω/ω mice. TCRβ chains are expressed at low/intermediate levels on DP thymocytes undergoing αβ TCR selection and at high levels on positively selected SP cells (12). Thus, we conducted FACS analysis of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ thymocytes with an anti-Cβ Ab and an anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 Ab. As we have done previously to evaluate αβ TCR selection (16), we quantified the percentages of cells expressing particular Vβ segments in TCRβ low/intermediate (DP) and TCRβ high (SP) thymocytes. We found similar percentages of Vβ10+ and Vβ14+ TCRβ intermediate/low and high thymocytes in Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice (Fig. 4, A and B). In contrast, we detected similar percentages of Vβ8+ and Vβ5+ TCRβ low/intermediate thymocytes in Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice, but significant decreases in the percentages of Vβ8+ and Vβ5+ TCRβ high thymocytes in Jβ1DJβ/+ and Jβ1DJβ/DJβ mice, as compared with Jβ1ω/ω mice (Fig. 4, A and B). We also observed a similar percentage of Vβ12+ TCRβ low/intermediate thymocytes in Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice, and a subtle decrease in the percentage of Vβ12+ TCRβ high thymocytes in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice, as compared with Jβ1ω/ω mice (Fig. 4, A and B). These data reveal that incorporation of the preassembled Dβ1Jβ1.1 complex in productive VβDJβ rearrangements influences αβ TCR selection of DP thymocytes expressing particular Vβs. To further address this issue, we also conducted PCR-based analysis of Vβ10 and Vβ12 rearrangements in sort-purified DNIII cells and total thymocytes of Jβ1DJβ/ω mice. We found that the ratio of Vβ10 rearrangements to the preassembled Dβ1Jβ1.1 complex on the Jβ1DJβ allele vs to newly assembled Dβ1Jβ1.1 and Dβ1Jβ1.2 on the Jβ1ω allele was the same in both cellular populations (Fig. 4,C). In contrast, the ratio of Vβ12 rearrangements to the preassembled Dβ1Jβ1.1 complex on the Jβ1DJβ allele vs to newly assembled Dβ1Jβ1.1 and Dβ1Jβ1.2 on the Jβ1ω allele was higher in total thymocytes compared with in sort-purified DNIII cells (Fig. 4 C). These PCR data are consistent with the FACS data and further demonstrate that incorporation of the preassembled Dβ1Jβ1.1 complex in productive VβDJβ rearrangements influences selection of thymocytes expressing particular Vβs.

FIGURE 4.

Altered αβ TCR selection and Vβ repertoire in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice. A, Shown are representative anti-Cβ and anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 FACS analysis of cells isolated from the thymuses of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice. The percentage of Cβ+ low/intermediate and high cells that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 is indicated. B, Bar graphs showing the average percentage of Cβ+ low/intermediate and high thymoctyes that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 are indicated. These values were obtained from three mice of each genotype. The error bars are SEM. Significant differences have been calculated using two-tailed Student’s t test. C, Shown is an image of an ethidium bromide-stained agarose gel resolving Vβ10 or Vβ12 rearrangements in sort-purified DNIII cells and total thymocytes of Jβ1DJβ/ω mice. The identities of the bands are indicated. D, Shown are representative anti-Cβ and anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 FACS analysis of cells isolated from the spleens of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice. The percentage of Cβ+ cells that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 is indicated. E, Bar graphs showing the average percentage of Cβ+ splenic αβ T cells that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 are indicated. These values were obtained from three mice of each genotype. The error bars are SEM. Significant differences have been calculated using two-tailed Student’s t test.

FIGURE 4.

Altered αβ TCR selection and Vβ repertoire in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice. A, Shown are representative anti-Cβ and anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 FACS analysis of cells isolated from the thymuses of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice. The percentage of Cβ+ low/intermediate and high cells that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 is indicated. B, Bar graphs showing the average percentage of Cβ+ low/intermediate and high thymoctyes that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 are indicated. These values were obtained from three mice of each genotype. The error bars are SEM. Significant differences have been calculated using two-tailed Student’s t test. C, Shown is an image of an ethidium bromide-stained agarose gel resolving Vβ10 or Vβ12 rearrangements in sort-purified DNIII cells and total thymocytes of Jβ1DJβ/ω mice. The identities of the bands are indicated. D, Shown are representative anti-Cβ and anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 FACS analysis of cells isolated from the spleens of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice. The percentage of Cβ+ cells that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 is indicated. E, Bar graphs showing the average percentage of Cβ+ splenic αβ T cells that express Vβ5, Vβ8, Vβ10, Vβ12, or Vβ14 are indicated. These values were obtained from three mice of each genotype. The error bars are SEM. Significant differences have been calculated using two-tailed Student’s t test.

Close modal

The relative frequency at which Vβs are expressed in peripheral αβ T cells also can be dramatically different due to αβ TCR selection in DP thymocytes (21, 22, 23, 24, 25, 26, 27). The statistically significant differences in CD4+ and CD8+ splenic αβ T cells among Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ mice suggest that incorporation of the preassembled Dβ1Jβ1.1 complex in productive VβDJβ rearrangements also may influence peripheral Vβ repertoire. To investigate this issue, we also evaluated whether the repertoire of TCRβ chains is altered in Jβ1DJβ/ω or Jβ1DJβ/DJβ splenic αβ T lymphocytes, as compared with in Jβ1ω/ω cells. To this aim, we conducted FACS analysis of Jβ1ω/ω, Jβ1DJβ/ω, and Jβ1DJβ/DJβ splenocytes with anti-Cβ Abs and anti-Vβ5, anti-Vβ8, anti-Vβ10, anti-Vβ12, or anti-Vβ14 Abs. We found a similar percentage of Vβ14+ and Vβ10+ splenic αβ T cells as in Jβ1ω/ω mice (Fig. 4, D and E). In contrast, we detected significant decreases in the percentage of Vβ8+ and Vβ5+ splenic αβ T cells in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice, as compared with in Jβ1ω/ω mice (Fig. 4, D and E). We also observed an increase in the percentage of Vβ12+ splenic αβ T cells in Jβ1DJβ/ω and Jβ1DJβ/DJβ mice, as compared with Jβ1ω/ω mice (Fig. 4, D and E); however, these differences were not statistically significant due to variation in the percentage of Vβ12+ cells in mice among experiments. These data demonstrate that incorporation of the preassembled Dβ1Jβ1.1 complex in productive VβDJβ rearrangements also influences the Vβ repertoire of peripheral αβ T cells.

To evaluate the influence of a two-step recombination process on Vβ rearrangement and selection, we generated mice with a preassembled Dβ1Jβ1.1 complex on an endogenous TCRβ allele that also lacks Dβ2-Jβ2 segments. This genetic manipulation created the Jβ1DJβ allele on which Vβ rearrangements only can be targeted to this one particular Dβ1Jβ1 complex of a defined sequence and length. Our comparative analysis of Jβ1DJβ/DJβ mice and Jβ1ω/ω mice in which Vβ rearrangements can be targeted to Dβ1Jβ1 complexes of six possible Dβ-Jβ joining combinations demonstrated that complete subversion of the tripartite TCRβ recombination process does not detectably alter Vβ rearrangement or TCRβ allelic exclusion.

However, our analysis of TCRβ rearrangements revealed that in-frame VβDJβ rearrangements were present at a higher level on the Jβ1DJβ alleles of Jβ1DJβ/ω αβ T cell hybridomas, as compared with on the Jβ1ω alleles. This bias was most likely due to both an increased frequency of Vβ-to-DJβ rearrangements on Jβ1DJβ alleles and a preferential selection of cells with in-frame VβDJβ exons assembled on Jβ1DJβ alleles during the development of Jβ1DJβ/ω αβ T cells. Consistent with the differential selection of in-frame VβDJβ rearrangements on Jβ1DJβ alleles, we found that incorporation of the preassembled Dβ1Jβ1.1 complex in productive VβDJβ rearrangements influences αβ TCR selection of DP thymocytes expressing particular Vβs and also the Vβ repertoire of peripheral αβ T cells. Collectively, our findings indicate that a two-step recombination process is not essential for normal regulation of Vβ rearrangement, but the sequence of DJβ complexes assembled during thymocyte development can influence TCRβ chain selection and peripheral Vβ repertoire.

TCRβ genes are assembled in an ordered fashion such that Dβ-to-Jβ and Vβ-to-DJβ, but not Vβ-to-Dβ, rearrangements occur during thymocyte development. This ordered assembly of TCRβ genes is controlled in part through the developmental stage-specific initiation of Dβ/Jβ in ETP and DNII cells and Vβ recombinational accessibility in DNIII thymocytes (15, 43, 44), most likely directed by activation of germline Dβ-Jβ and Vβ promoters independent of TCRβ gene recombination events (45, 46, 47). Our findings that the overall level of Vβ-to-DJβ rearrangements on Jβ1DJβ and Jβ1ω alleles is similar in Jβ1DJβ/ω αβ T cell hybridomas suggest that the preassembled Dβ1Jβ1.1 complex does not substantially activate RAG accessibility of the 5′Dβ1 RSS or lead to long-range chromatin changes that promote RAG accessibility of Vβ segments. This interpretation is consistent with our previous data that Dβ-to-Jβ rearrangement is not required for activation of Vβ rearrangement (10, 18). However, the observed bias for in-frame VβDJβ exons on the Jβ1DJβ alleles of Jβ1DJβ/ω αβ T cells might reflect an increased level of Vβ-to-DJβ rearrangement on this allele, possibly due to absence of the 3′Dβ1 RSS (10). If so, our data might indicate that Dβ1-to-Jβ1 rearrangements ordinarily do not proceed to completion on both alleles before the biallelic initiation of Vβ RAG accessibility.

Recent analyses of in vitro and transfected plasmid substrates have shown that c-fos deposits RAG onto 3′Dβ RSSs and blocks Vβ-to-Dβ rearrangement through steric hindrance of 5′Dβ RSSs; and Vβ-to-Dβ rearrangements are detectable by PCR in c-fos−/− thymocytes, indicating that Dβ-to-Jβ rearrangements are not completed on all alleles before initiation of Vβ accessibility (8). These findings are at odds with our current observation that initial Vβ-to-DJβ rearrangements are not exclusively targeted to the preassembled Dβ1Jβ1.1 complex in Jβ1DJβ/ω αβ T cells. One possible explanation is that the substantial block in thymocyte development at the DNII stage observed in c-fos−/− mice (8) is caused by decreased Jβ RSS accessibility and/or gross perturbations in the thymocyte differentiation program, rather than by disordered TCRβ rearrangements. Another explanation is that the genomic deletion associated with the preassembled Dβ1Jβ1.1 complex could alter germline Dβ1-Jβ1 transcription (9) and/or nucleosome positioning over the 5′Dβ1 RSS (48, 49, 50, 51) in a manner that reduces RAG accessibility. We have recently found RAG-mediated cleavage of one Igκ allele activates an ATM-dependent signal that prevents V(D)J recombination of the second Igκ allele (B. Yin and C. Bassing, unpublished observations). Thus, it also is conceivable that Dβ-to-Jβ rearrangements on Jβ1ω alleles in Jβ1DJβ/ω DN thymocytes inhibit in trans Vβ-to-DJβ rearrangements on Jβ1DJβ alleles and thereby diminish any advantage that the preassembled Dβ1Jβ1.1 complex confers in cis upon Vβ rearrangement.

TCRβ genes are also regulated such that in-frame VβDJβ rearrangements form only on a single allele in the majority of developing thymocytes. Despite intense efforts, the manner by which Vβ rearrangements are restricted to one allele at a time is completely unknown, although evidence for both stochastic and directed control mechanisms has been provided (13, 52, 53, 54). Our current observations that Vβ rearrangements occur at a similar level on Jβ1DJβ and Jβ1ω alleles in Jβ1DJβ/ω αβ T cell hybridomas and TCRβ allelic exclusion is maintained in Jβ1DJβ/DJβ mice have implications for the potential mechanisms that restrict Vβ rearrangement to one allele at a time. First, our data formally demonstrate that the process of Dβ-to-Jβ rearrangement is not required for monoallelic assembly and expression of in-frame TCRβ exons, and TCRβ allelic exclusion is achieved exclusively through regulation of the Vβ-to-DJβ rearrangement step in developing αβ T cells. Second, within the context of stochastic models of TCRβ allelic exclusion that invoke low recombination efficiency of the Vβ rearrangement step as the underlying mechanism (54), our data demonstrate that deletion of 3′Dβ RSSs upon Dβ-to-Jβ rearrangement to relieve any potential steric hindrance of 5′Dβ RSSs is not the rate-limiting mechanism that restricts Vβ rearrangements to only one allele at a time.

The assembly and expression of TCRβ genes and the selection of TCRβ chains associated with pTα molecules are required for the differentiation of DNIII thymocytes into DNIV and then DP cells (5). Expression of transgenic in-frame VβDJβ rearrangements leads to a reduction in the percentage of DNIII cells and a concomitant increase in the percentage of DNIV thymocytes, demonstrating that the assembly and expression of TCRβ chains is the rate-limiting step in early thymocyte development (55, 56). In this study, we show that Jβ1DJβ/ω and Jβ1DJβ/DJβ mice exhibit slight reductions in the frequency of DNIII thymocytes and concomitant increases in the frequency of DNIV cells. The magnitude of these changes corresponds to the number of Jβ1DJβ alleles, suggesting that the DNIII to DNIV transition and β-selection are slightly enhanced by the presence of the preassembled DJβ complex. The lack of a detectable increase in the level of overall Vβ-to-DJβ rearrangements on Jβ1DJβ alleles as compared with on Jβ1ω alleles in Jβ1DJβ/ω αβ T cell hybridomas suggests that VβDJβ rearrangements involving the particular DJβ join used in this study are better able to pair and/or signal with pTα chains than VβDJβ rearrangements involving the population of DJβ joins normally assembled on Jβ1ω alleles. However, we cannot rule out the possibility that increased rearrangement frequencies of a particular Vβ segment to the preassembled DJβ1.1 complex on Jβ1DJβ alleles as compared with the Dβ1Jβ1 complexes of six Dβ-Jβ joining possibilities on Jβ1ω alleles in DNIII thymocytes contribute to this slightly accelerated early thymocyte development. In this regard, recombination efficiencies can be influenced by the nucleotide composition of coding sequences flanking participating RSSs (57, 58, 59, 60, 61, 62). Unfortunately, firm conclusions require the accurate quantification of VβDJβ rearrangements in nonselected DNIII thymocytes, which is not possible due to the inherent biases of amplifying VβDβJβ1 rearrangements of one fixed size on Jβ1DJβ alleles vs six different sizes on Jβ1ω alleles.

The generation and expression of a broad repertoire of Ag receptors on the surface of lymphocytes are critical for development and function of an effective adaptive immune system. For example, underrepresentation of a particular Vκ segment (VκA2) in the peripheral Igκ repertoire of humans due to allelic polymorphisms is associated with an increased susceptibility to Haemophilus influenzae type b because VκA2 segments are often used in anti-H. influenzae type b Abs (63). It has been known for quite some time that the Vβ repertoire assembled in DN thymocytes can be substantially shaped during αβ TCR selection in the DP thymocytes of mice expressing superantigens (21, 22, 23, 24, 25, 26, 27). Our current observations that incorporation of the preassembled Dβ1Jβ1.1 complex in productive VβDJβ rearrangements affects αβ TCR selection of DP thymocytes expressing particular Vβs and the Vβ repertoire of peripheral αβ T cells demonstrate that DJβ complexes assembled during thymocyte development can influence TCRβ chain selection and peripheral Vβ repertoire, even in the absence of superantigens. In this context, the number and/or identity of amino acids encoded by DJβ joins may differentially affect the ability of TCRβ chains containing particular Vβ segments from pairing with TCRα chains, associating with CD4/CD8 coreceptors, and/or recognizing peptides presented by MHC during αβ TCR. Restriction of endogenous Vα rearrangements to a single functional Jα segment substantially impairs positive selection of αβ TCR in DP thymocytes and leads to a marked reduction in peripheral αβ T cell numbers (64). Moreover, mice containing a single endogenous DH segment exhibit reduced numbers of bone marrow B cells and defective immune responses to a particular T-independent Ag (65). Furthermore, we previously demonstrated that the frequency of Vβ2 and Vβ14 rearrangements is reduced by the presence of other Vβ segments that compete with Vβ2 and Vβ14 for the productive coupling with DJβ1 complexes (66). Thus, we hypothesize that the two Dβ-Jβ clusters and the six Jβ segments within each cluster may have evolved under selective pressure to ensure the most beneficial representation of Vβ segments expressed in the peripheral TCRβ repertoire of αβ T cells.

We thank Heikyung Suh and Megan Gleason for technical help, and Brenna L. Brady for critical evaluation of the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant AI20047 (to F.W.A.) and the Department of Pathology and Laboratory Medicine and Center for Childhood Cancer Research of the Children’s Hospital of Philadelphia (to C.H.B.). A.C.C. is supported by the Training Program in Immune System Development and Regulation at the University of Pennsylvania. C.H.B. is a Pew Scholar in the Biomedical Sciences. F.W.A. is an Investigator of the Howard Hughes Medical Institute.

3

Abbreviations used in this paper: RSS, recombination signal sequence; DN, double negative; DP, double positive; ES, embryonic stem; ETP, early T lineage progenitor; NHEJ, nonhomologous end joining; SA, streptavidin; SP, single positive.

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