Trav15-dv6 family Tcrd rearrangements diversify the Tcra repertoire

TCR and BCR repertoires rely on combinatorial diversity imparted by V(D)J recombination of variable (V), diversity (D), and joining (J) gene segments at their respective antigen receptor (AgR) loci (Schatz and Ji, 2011). The lymphocyte-specific recombination-activating gene (RAG) proteins mediate double-stranded DNA breaks at recognition sites, called “recombination signal sequences” (RSSs), flanking the V, D, and J segments of the AgR loci. These breaks are then modified, and the segments are ligated via nonhomologous end joining and spliced to a constant gene segment (C) to make AgR chain transcripts (Schatz and Ji, 2011; Helmink and Sleckman, 2012). RAG binding at AgR loci is primarily restricted to recombination centers (RCs), chromatin regions characterized by highly accessible clusters of J (and sometimes D) segments (Ji et al., 2010; Schatz and Ji, 2011). In order for V-to-(D)J recombination to occur, V segments must be brought into proximity of the RC (Jhunjhunwala et al., 2009; Lin et al., 2018). This process is tightly regulated, and ordered recombinations are integral to successful generation of a diverse TCR repertoire. Tcrd, Tcrg, and Tcrb rearrangements occur at the CD4⁻CD8⁻ (double-negative [DN]) stage of thymocyte development (Capone et al., 1998). Subsequently, Tcra undergoes V_α-to-J_α recombination at the CD4⁺CD8⁺ (double-positive [DP]) stage (Petrie et al., 1995).

Because Tcra recombinations involve only V_α and J_α segments, Tcra is capable of undergoing multiple rounds of rearrangement to generate a functional, in-frame TCRα chain (Petrie et al., 1993; Wang et al., 1998). The first V-to-J_α rearrangement is referred to as the “primary rearrangement,” and all subsequent Tcra recombination events are referred to as “secondary rearrangements” (Carico and Krangel, 2015). Primary Tcra rearrangements typically involve the most V_α-proximal J_α segments (Thompson et al., 1990), which constitute the initial Tcra RC. High-throughput sequencing (HTS) of the Tcra repertoire demonstrates that early V_α rearrangements are broadly distributed, including the proximal half of the V_α array as well as substantial central and distal V_α contributions. Secondary rearrangements proceed to more distal V_α and J_α segments over time, with the Tcra RC continually retargeted in stepwise fashion to more distal J_α segments following each round of V_α-J_α rearrangement (Hawwari and Krangel, 2007; Ji et al., 2010; Schatz and Ji, 2011; Carico et al., 2017). Notably, in a mouse in which all thymocytes have a knock-in of the rearrangement Trav17-Traj57 (Buch et al., 2002), the progression of secondary rearrangements was found to be tightly constrained, with each round of secondary recombinations limited to the most proximal of the remaining V_α and J_α segments (Carico et al., 2017). Because the combinatorial space occupied by secondary rearrangements downstream of a single primary rearrangement is narrow, repertoire diversity must depend on broad use of V_α segments in primary rearrangements.

The Tcra-Tcrd locus displays a unique structure among AgR loci, wherein D_δ and J_δ gene segments are nested between the V_α and J_α gene segments, and V_δ segments are interspersed among the V_α segments in both mouse and man (Fig. 1 A; Glusman et al., 2001; Carico and Krangel, 2015). Intriguingly, many of the central and distal V_α segments used in primary and early secondary rearrangements were found to be immediately upstream of segments that can be used as Tcrd recombination substrates (Carico et al., 2017). Most cells that express αβTCRs first undergo Tcrd rearrangement on at least one allele (Livak et al., 1995; Nakajima et al., 1995; Sleckman et al., 1998; Shih et al., 2012). These truncating recombinations delete V_α segments located between the recombined V_δ and D_δ segments before Tcra rearrangement begins. Therefore, Tcrd rearrangement could bias Tcra recombination toward the use of more distal V_α segments for primary rearrangements than would be observed on alleles that do not recombine Tcrd.

Most deletional V-to-DJ_δ recombination events in mice involve D_δ-proximal V_δ segments, thereby leaving most of the V_α array intact (Chen et al., 2015; Zhao et al., 2016). One V_δ family, Trav15-dv6, accounts for most of the distal Tcrd rearrangements that do occur. As Trav15-dv6 rearrangements would truncate the V_α array and change the starting point for primary Tcra rearrangements, we hypothesized that Trav15-dv6 rearrangement may be fundamental to combinatorial diversity of the Tcra repertoire.

In a mouse model of altered chromatin looping at the Tcra-Tcrd locus, V-to-DJ_δ recombination was found to be skewed toward rearrangement of the proximal Trdv2-2 and Trdv3 gene segments, with concomitant reductions in rearrangements to distal V_δ segments (Chen et al., 2015; Zhao et al., 2016). Tcra rearrangements were also found to be less diverse, consistent with a role for Tcrd recombination in Tcra repertoire formation. However, because the primary effect of mutation in this model is on chromatin looping, it remains unclear whether the observed Tcra repertoire phenotype may be a direct consequence of this structural change as opposed to an indirect consequence of altered Tcrd rearrangements.

Here, we employed an HTS approach (Carico et al., 2017) to determine the impact of Tcrd recombination on the Tcra repertoire in DP thymocytes using several novel mouse models. In primary mouse thymocytes incapable of Tcrd rearrangement, we observed a dramatic contraction in Tcra repertoire diversity, directly implicating Tcrd recombination in Tcra repertoire formation. Moreover, deletion of Trav15d-1-dv6d-1 or Trav15-1-dv6-1 revealed the rearrangement of these V_δ segments to be particularly important in targeting primary Tcra rearrangements to the distal and central portions of the V_α array, respectively. Finally, we provide evidence that Trav15-dv6 or flanking elements may also have a direct effect on the Tcra repertoire by facilitating secondary V_α-to-J_α rearrangement events.

To directly assess the impact of Tcrd recombination on the Tcra repertoire, we sought a mouse strain incapable of Tcrd recombination. In this regard, the C_δ-KO mouse undergoes normal Tcrd rearrangement (Itohara et al., 1993), and the E_δ-KO mouse has only a partial defect (Monroe et al., 1999). Therefore, we generated a novel line of mice lacking a 24-kb region containing the D_δ and J_δ segments (Figs. 1 A and S1 A). The resulting mouse, D_δ and J_δ deficient (DJD), ablates V-to-DJ_δ rearrangement and γδT cells, with otherwise normal thymocyte development (Fig. S1, B–D).

Preselection DP thymocytes from DJD mice and their WT littermates were subjected to 5′ rapid amplification of cDNA ends (RACE) and Tcra sequencing. Unique clones were analyzed to determine the frequencies of V-to-J_α rearrangements. As previously reported (Carico et al., 2017), in WT thymocytes, we found the Tcra repertoire to be diverse (Fig. 1 B, left panel; and Fig. S2 A). Proximal J_α segments frequently rearranged with proximal, central, and distal V_α segments. Secondary rearrangements occurred mostly along two distinct diagonals (Fig. S2 B). The major diagonal started with proximal and central V-to-proximal J_α primary rearrangements, followed by secondary rearrangements through the central and distal V_α and J_α segments. The minor diagonal arose from distal V-to-proximal J_α primary rearrangements. In DJD thymocytes, absent Tcrd recombination, we observed restricted V_α use; proximal J_α segments recombined almost exclusively with the most proximal V_α segments (Fig. 1 B, right panel). This reduction in diversity of V_α use in early rearrangements with proximal J_α segments depressed the diversity of secondary rearrangements, with a notable loss in the use of distal V_α segments, even in recombinations with central and distal J_α segments. The major diagonal was constrained and shifted toward more proximal V_α segments along its entire length; the minor diagonal was largely depleted of unique clones.

Previous work described the progression of secondary rearrangements emanating from a single primary recombination as proceeding ∼1.33 V_α segments per J_α (Carico et al., 2017). According to our hypothesis, DJD thymocytes should be depleted of V-J_α combinations resulting from primary rearrangements distal to the most proximal Trav15-dv6 family member, Trav15-2-dv6-2, as well as any secondary rearrangements that occur as a consequence of those primary rearrangements. To assess this, we overlaid a step-function diagonal (4V_α:3J_α, corresponding to 1.33 V_α per J_α) over the DJD−WT difference map, with its origin just distal to Trav15-2-dv6-2 (Fig. 1 C). The step-function diagonal predicts almost exactly the region of depletion of the combinatorial Tcra repertoire in DJD thymocytes. In WT thymocytes, the region above the diagonal accounts for 43% of the repertoire; in DJD thymocytes, it accounts for 22% (Fig. 1, B and C; P < 0.0001 by χ² test with Yates’s correction). However, this is a substantial underestimate of the impact on some distal V_α rearrangements. Prior work demonstrated that the SD of the V_α distribution used with any J_α gradually increases from proximal to distal across the J_α array (Carico et al., 2017). Because this increase is not accounted for by the step-function diagonal, we would expect representation of the most distal V_α-distal J_α combinations to persist, although at reduced frequencies, in the region above the diagonal in DJD thymocytes (compare Fig. 1 B, right panel, with Fig. 1 C). This effect should not impact quantification along the minor diagonal; accordingly, rearrangements in this region account for 7.7% of the repertoire in WT but only 2% in DJD thymocytes, a reduction of 74%. We conclude that Tcrd rearrangement expands the combinatorial diversity of the Tcra repertoire by diversifying V_α use.

Primary Tcra rearrangements are poorly represented in the steady-state Tcra repertoire. To more robustly investigate the impact of Tcrd rearrangement on primary Tcra rearrangement, we directly visualized Tcra rearrangements in the earliest DP thymocytes. We previously analyzed these cells in mice carrying a tamoxifen-inducible Tcrd^CreER allele together with a Rosa26^{fl-STOP-fl-ZsGreen} (hereafter Rosa26^ZsG) reporter allele (Carico et al., 2017). In these mice, tamoxifen injection causes ZsGreen expression in cells with transcriptionally active Trdc, including DN thymocytes, most of which, failing to diverge to the γδT cell fate, progress to rearrange Tcra at the DP stage (Madisen et al., 2010; Zhang et al., 2015). To analyze rearrangement on the DJD allele, we mutated Trac on the Tcrd^CreER allele to prevent annealing of Trac 5′ RACE primers (Fig. S1 A). In mice containing Rosa26^ZsG and a Trac-mutated Tcrd^CreER (Tcrd^CreERTrac^M) allele, in combination with either a WT or DJD allele, we analyzed Tcra recombination in ZsGreen⁺ DP thymocytes at 12 h after tamoxifen injection. These primary and early secondary recombinations, in cells that compose <20% of the DP population (Carico et al., 2017), are typically overshadowed by secondary recombinations in the steady-state repertoire. As previously reported (Carico et al., 2017), on WT alleles, we found these recombinations to be restricted to the first half of the J_α array (Fig. 2 A, left panel), with the majority of rearrangements focused on the most proximal J_α segments, Traj58-Traj48. V_α use involved the first half of the V_α array, along with a cluster of relatively distal V_α segments. Save for an unexpected reduction in use of Traj58, DJD alleles displayed minimal change in overall J_α use (Fig. 2 A, right panel; and Fig. 2 B). However, V_α use on DJD alleles was restricted compared with WT thymocytes, with overall use of segments distal to Trav15-2-dv6-2, the most proximal Trav15-dv6 segment, reduced from 59.4% to 32.2% on DJD alleles (P < 0.0001 by χ² test with Yates’s correction). We conclude that Tcrd recombination diversifies primary V_α use, leading to a more robust repertoire of secondary rearrangements and ultimately imparting combinatorial diversity upon the Tcra repertoire.

Trav15-dv6 segments comprise the most frequent of the distal and central V_δ contributions to the Tcrd repertoire; the only other similarly located V_δ gene segments, Trav14d-3-dv8 and Trav16d-dv11 (Fig. S2), are rarely used (Chen et al., 2015; Zhao et al., 2016). We observed that in primary recombinations of the WT Tcra locus, most central and distal V_α use occurs immediately upstream of Trav15-dv6 family members, particularly Trav15-1-dv6-1 and Trav15d-1-dv6d-1 (left panels of Figs. 1 B and 2 A). These early central and distal recombinations are dramatically reduced in DJD mice (Fig. 1 B, right; Fig. 1 C; Fig. 2 A, right; and Fig. 2 B), suggesting that Trav15-dv6 recombinations in DN cells may contribute to diversifying the Tcra repertoire. To analyze the impact of Trav15-dv6 rearrangements on Tcra recombination, we analyzed two lines of mice, one with a deletion of ∼2 kb spanning Trav15d-1-dv6d-1 and a second with a similar deletion of Trav15-1-dv6-1 (Fig. S1 A) for their Tcra clonal repertoires. We hypothesized that the loss of either would result in a substantial reduction in recombination events emanating in a diagonal pattern from the most proximal upstream V_α and moving distally through the remaining V_α segments.

Primary and secondary Tcra recombinations in the minor diagonal, predicted to occur as a consequence of Tcrd rearrangements involving Trav15d-1-dv6d-1, represented 3.1% of the Tcra repertoire in WT DP thymocytes but only 0.8% of the repertoire in cells lacking Trav15d-1-dv6d-1 (Fig. 3, A and B, region 1; P < 0.0001 by χ² test with Yates’s correction). Similarly, major diagonal primary and secondary Tcra recombinations, predicted to occur as a consequence of Tcrd rearrangements involving Trav15-1-dv6-1, accounted for 21.2% of the repertoire in WT thymocytes but only 16.6% of the repertoire in cells lacking Trav15-1-dv6-1 (Fig. 4, A and B, region 2; P < 0.0001 by χ² test with Yates’s correction). In contrast, the representation of minor diagonal Tcra recombinations predicted to occur as a consequence of rearrangements involving more distal Trav15-dv6 family members increased slightly in Trav15-1-dv6-1–deleted DP cells (Fig. 4, A and B, region 1). In both lines of mice, rearrangements involving V_α segments downstream of the deleted Trav15-dv6 family member increased proportionally (Fig. 3 B, region 3; and Fig. 4 B, region 4). Because reduced minor diagonal Tcra rearrangements were observed in both DJD mice and Trav15d-1-dv6d-1–deleted mice and reduced major diagonal Tcra rearrangements were observed in both DJD mice and Trav15-1-dv6-1–deleted mice, we conclude that central and distal Tcrd rearrangements involving Trav15-dv6 family members diversify the Tcra repertoire.

In thymocytes lacking either Trav15d-1-dv6d-1 or Trav15-1-dv6-1, we observed changes in the Tcra repertoire that were not predicted to arise due to loss of Tcrd recombination to either segment. In the absence of Trav15d-1-dv6d-1, rearrangements between distal V_α and distal J_α segments outside the minor diagonal were reduced to 13.1% of the repertoire from 19.1% in WT; these secondary rearrangements lie on the major diagonal and are predicted to arise from primary rearrangements involving central V_α segments (Fig. 3, A and B, region 2; P < 0.0001 by χ² test with Yates’s correction). Similarly, in Trav15-1-dv6-1–deleted mice, major diagonal secondary rearrangements involving central and distal V_α segments were unexpectedly reduced to 37.4% of the repertoire from 43.1% in WT (Fig. 4, A and B, region 3; P < 0.0001 by χ² test with Yates’s correction); these secondary rearrangements are predicted to arise from primary rearrangements involving proximal V_α segments. Moreover, particularly in Trav15-1-dv6-1–deleted mice, reduced secondary rearrangements were associated with increased use of V_α segments immediately J_α-proximal to the deleted region (Fig. 4). None of these changes were observed in the DJD repertoire (Fig. 2). This implies that Trav15-dv6 family members impact Tcra repertoire diversification by an additional mechanism that is separate from their roles as Tcrd recombination substrates. We propose that the Trav15-1-dv6-1 and Trav15d-1-dv6d-1 deletions have these additional effects on Tcra repertoire diversity because they impair the propagation of secondary Tcra rearrangements to V_α segments beyond the deleted region.

The Trav15d-1-dv6d-1 and Trav15-1-dv6-1 deletions encompass regions spanning ∼500 bp upstream of the transcription start site to ∼1 kb downstream of the gene body. These deleted regions are highly conserved between Trav15-1-dv6 family members and include the promoter, the RSS, and downstream E-box sites that are highly accessible and, by chromatin immunoprecipitation followed by sequencing (ChIP-seq), are occupied by E2A at levels surpassed within the Tcra-Tcrd locus only by Tcra enhancer E-box sites (Fig. S3; Heng et al., 2008; Roy et al., 2018; Yoshida et al., 2019). Loss of the Trav15-dv6 RSS could inhibit more distal secondary rearrangements if the propagation of sequential rearrangements requires closely spaced RSSs. However, Trav15-dv6 family members were not found to be frequently recombined in the WT Tcra repertoire, suggesting that Trav15-dv6 RSSs are often skipped during secondary rearrangements (Fig. 1 B). Moreover, the distance between the nearest functional V_α segments flanking Trav15-dv6 is reduced from ∼37 kb to ∼35 kb on the deleted alleles. We considered that Trav15-dv6 deletion might influence secondary rearrangements by disrupting the CCCTC binding factor (CTCF)–mediated chromatin loop organization. However, binding sites for CTCF were not disrupted on the Trav15d-1-dv6d-1– or Trav15-1-dv6-1–deleted alleles (Shih et al., 2012), and the deletions do not obviously create de novo CTCF binding sites (Martin et al., 2011).

It remains possible that Trav15-dv6 segments influence secondary Tcra recombinations via effects on chromatin accessibility mediated by flanking cis-acting elements. The Trav15-1-dv6-1 promoter drives accessibility, transcription, and recombination of Trav15-1-dv6-1 in DN thymocytes (Naik et al., 2015) but displays only modest accessibility and is not expected to influence neighboring V segments (Heng et al., 2008; Yoshida et al., 2019). Perhaps a better candidate is the highly accessible downstream E2A-bound element (Fig. S3). E2A is a known regulator of Tcrd recombination and γδT cell fate (Bain et al., 1999) and may influence Trav15-dv6 rearrangement in DN thymocytes. As the E2A-bound region remains highly accessible in DP thymocytes, it could have extended effects on accessibility and RAG binding at RCs that form downstream of Trav15-dv6 family members during secondary rearrangement or on V_α substrates upstream of Trav15-dv6 family members. The cis region may alternatively serve as a mediator of locus structure to facilitate recombination; such features have been observed at other AgR loci (Barajas-Mora et al., 2019). Further exploration of this phenomenon is warranted to better understand these perturbations of secondary Tcra rearrangements on Trav15d-1-dv6d-1– and Trav15-1-dv6-1–deleted alleles.

Our repertoire studies emphasize that capture of V gene segments within the Tcra-Tcrd locus occurs via distinct mechanisms during Tcrd and Tcra rearrangement. Capture of V_δ gene segments by the Tcrd RC in DN thymocytes can occur over very long distances. By contrast, capture of V_α gene segments by Tcra RCs appears to occur predominantly as a result of short-range interactions once those segments are brought into proximity of the RC by prior rounds of rearrangement. One exception is the residual rearrangement of central and distal V_α segments in early rearrangements on DJD alleles (Fig. 2 A, right panel); the cluster of rearrangements between V segments upstream of Trav15d-2-dv6d-2 and J segments Traj58-Traj48 is not ablated, but rather reduced from 11.4% to 3.5% (P < 0.0001 by χ² test with Yates’s correction). We think that these rearrangements, which include Trav15d-1-dv6d-1, may result from residual chromatin marks carried over from DN thymocytes in the absence of Tcrd recombination, facilitating DN-like V capture in early DP thymocytes.

The change in the mode of V segment capture between the DN and DP stages of thymocyte development correlates with a conformational change in the Tcra-Tcrd locus, with the V array being contracted in DN and extended in DP (Shih and Krangel, 2010). Prior work has shown that long-range capture of V_H segments by the Igh RC in pro-B cells occurs via long-range RAG scanning and cohesin-mediated loop extrusion, facilitated by reduced expression of the cohesin unloader Wapl (Hu et al., 2015; Lin et al., 2018; Ba et al., 2020; Hill et al., 2020; Dai et al., 2021). However, RAG scanning from the Tcrd RC appears to be effectively contained within an 80-kb chromatin loop domain formed by the INT1-2 and T early α CTCF binding elements in DN thymocytes (Chen et al., 2015; Zhao et al., 2016). This suggests that long-range capture of V_δ gene segments may occur via diffusion. In contrast, short-range V_α capture by the Tcra RC in DP thymocytes can be envisioned to occur either by relatively short-range RAG scanning or by diffusive interactions. Regardless, our data suggest that Tcra repertoire diversity in mice results from synergy between two different modes of V segment capture. We expect that our conclusions on Tcra repertoire diversity in mice likely extend to humans, since the human Tcra-Tcrd locus is similarly organized with V_δ gene segments distributed among central and distal V_α gene segments.

To generate DJD mice, female C57BL/6J mice were mated to male strain 129 mice. The resulting F1 embryos were subjected to pro-nuclear injection of reagents for two guide–mediated CRISPR/Cas9 (Cong et al., 2013; Singh et al., 2015) to delete an ∼24.3-kb region spanning Trdd1-Trdj2 at the Tcra-Tcrd locus. The upstream guide sequence was 5′-GAC​TCA​CCT​GTT​ATC​AAA​CT-3′, and the downstream guide sequence was 5′-ATA​ATG​CTA​AAA​TTA​CCT​GC-3′. Offspring were screened for deletion on the 129 Tcra-Tcrd allele using PCR and Sanger sequencing (Duke University DNA Analysis Facility). Appropriately targeted mice of mixed C57BL/6J and 129 genetic background were crossed once to strain 129, and DJD heterozygotes were then intercrossed to obtain homozygous WT and DJD littermates for analysis.

Tcrd^CreER/CreERRosa26^ZsG/ZsG mice, with modified strain 129 Tcra-Tcrd alleles on a C57BL/6 genetic background (backcrossed >10 generations), were described previously (Zhang et al., 2015). These mice were further modified by deletion of 138 bp spanning the 5′ portion of Trac exon 1 to generate Tcrd^CreER/CreER Trac^M/+Rosa26^ZsG/ZsG mice. Two guide–mediated CRISPR/Cas9 targeting was accomplished by electroporation using upstream guide 5′-CAG​GCA​AGA​GCG​GAA​CTC​TC-3′ and downstream guide 5′-GAG​ACC​GAG​GAT​CTT​TTA​AC-3′. The founder mouse was backcrossed once to Tcrd^CreER/CreERRosa26^ZsG/ZsG, and mice heterozygous for the Trac mutation were then intercrossed to obtain Tcrd^CreER/CreERTrac^M/MRosa26^ZsG/ZsG mice. These were then crossed to DJD heterozygotes to obtain littermates containing a Rosa26^ZsG allele and Tcrd^CreERTrac^M allele paired with either a WT 129 or DJD 129 Tcra-Tcrd allele on a mixed C57BL/6 and 129 genetic background.

Trav15-1-dv6-1 and Trav15d-1-dv6d-1 deletions were generated in Tcrd^CreER/CreERRosa26^ZsG/ZsGId3^f/f mice (Madisen et al., 2010; Guo et al., 2011; Zhang et al., 2015) containing modified strain 129 Tcra-Tcrd alleles on a C57BL/6 genetic background (backcrossed >10 generations). Two-guide mediated CRISPR/Cas9 targeting was accomplished by electroporation using upstream guide 5′-TCT​TCC​CTT​AAA​GAG​TGA​TA-3′, and downstream guide 5′-GAC​ATT​AGA​GTC​CCT​TAA​AG-3′. Offspring were screened by PCR and Sanger sequencing. Deletions of Trav15-1-dv6-1 and Trav15d-1-dv6d-1 were detected in different founders and maintained separately. Appropriately targeted mice were crossed to Tcrd^CreER/CreERRosa26^ZsG/ZsGId3^f/f mice to obtain Rosa26^ZsG/ZsGId3^f/f mice containing a Trav15-1-dv6-1 KO Tcrd^CreER or a Trav15d-1-dv6d-1 KO Tcrd^CreER allele paired with a Tcrd^CreER allele on a C57BL/6 genetic background. Intercrossing of Trav15-1-dv6-1 KO heterozygous mice generated littermates containing either homozygous Trav15-1-dv6-1 KO Tcrd^CreER or Tcrd^CreER alleles for analysis. Intercrossing of Trav15d-1-dv6d-1 KO heterozygous mice generated littermates containing either homozygous Trav15d-1-dv6d-1 KO Tcrd^CreER or Tcrd^CreER alleles for analysis.

All CRISPR/Cas9-mediated deletions were performed by the Duke University Transgenic and Knockout Mouse Shared Resource. Mice were sacrificed at 4–5 wk of age. Both male and female mice were used; no differences were observed on the basis of sex. All mice were handled under protocols approved by the Duke University Institutional Animal Care and Use Committee and maintained in specific pathogen–free conditions.

For analysis of thymocyte subpopulations, thymi were collected from mice at 3–4 wk of age. To sort DP thymocytes for repertoire analysis, thymi were collected from mice at 4–5 wk of age. To label developing thymocytes with ZsGreen, mice were injected i.p. with a single 100-µl dose of 10 mg/ml tamoxifen (Sigma-Aldrich) in corn oil (Sigma-Aldrich) 12 h before sacrifice.

To obtain preselection DP thymocytes (defined as CD4⁺CD8⁺Lin⁻7AAD⁻CD3ε^lo), total thymocytes were stained with anti-CD4 (GK1.5), anti-CD8α (53-6.7), anti-CD3_ε (145-2C11), 7AAD, and PE-Cy5–conjugated lineage (Lin) markers anti-B220 (RA3-6B2), anti-CD11b (M1/70), anti-CD11c (N418), anti-F4/80 (BM8), anti–Gr-1 (RB6-8C5; Invitrogen), and anti–Ter-119 (TER-119). Preselection DP thymocytes from tamoxifen-injected mice were additionally sorted for ZsGreen⁺. For analysis of thymocyte subpopulations, total thymocytes were additionally stained with Pacific Blue anti-CD25 (PC61), allophycocyanin-Cy7 anti-cKit (2B8), and allophycocyanin anti-γδTCR (GL3). All antibodies were purchased from BioLegend, unless otherwise specified.

Tcra sequencing libraries were prepared as previously described (Carico et al., 2017). Briefly, RNA was extracted using TRIzol (Life Technologies) from sorted preselection DP thymocytes. 5′ RACE was performed on total RNA as previously described (Pinto and Lindblad, 2010; Quigley et al., 2011; Carico et al., 2017), with minor modifications. 700 ng RNA was used as input for template-switch 5′ RACE and cDNA synthesis using SuperScript II (Thermo Fisher Scientific). Kapa HiFi polymerase in 1× Kapa HiFi buffer (Kapa Biosystems) was used for all PCRs in 50 µl total volume per reaction; eight reactions were performed per sample for each round of PCR. PCR products were pooled and purified using the QIAquick PCR purification kit (Qiagen) per the manufacturer’s specifications. Purified products of the first PCR were subjected to a second round of PCR amplification as described (Kozich et al., 2013; Carico et al., 2017) to ligate barcodes and Illumina adapter sequences. Libraries were then pooled and purified using the QIAquick PCR purification kit and resuspended in nuclease-free water.

Sequencing and analysis were performed as previously described (Carico et al., 2017), with minor modifications. Briefly, barcoded libraries were pooled and sequenced by the Duke University Sequencing and Genomic Technologies Shared Resource using 300-nt paired-end reads on the Illumina MiSeq platform (version 3 chemistry). Agilent Bioanalyzer analysis was used to determine library molarity and quality, and size selection was performed by the Duke University Sequencing and Genomic Technologies Shared Resource for further purification. A PhiX control library and custom primers were added to the standard Illumina primer mix, as previously described (Carico et al., 2017). Libraries were demultiplexed and assessed for quality and yield using Illumina MiSeq Reporter software.

Analysis was performed using MiXCR (version 3.0.7; Bolotin et al., 2015). The reference library was edited to permit alignments only to 129 sequences (Bosc and Lefranc, 2003). The “align” command was used to align sequencing reads to this reference library. The “assemble” command was then used to identify clones with sequences spanning CDR2 through CDR3. Each clone was assigned to the corresponding V and J segments. The “exportClones” command produced a human-readable form of these data; alignment was manually reviewed. Sequences aligning to pseudogenes and other very infrequently uses genes were manually removed, including Traj61, Traj41, Traj25, Trav5d-2, Trav7d-6, Trav7-6, and Trav18. Trav11 and Trav11d are not distinguishable; both were maintained in this analysis, but the computed distribution of reads between the segments should be ignored. The VDJtools (Shugay et al., 2015) command PlotFancyVJUsage was used to calculate clonal frequencies of V-J recombinations. In R (version 3.3.3; R Core Team, 2020), and heatmaps were generated using the gplots (Warnes et al., 2009) and RColorBrewer (Neuwirth, 2014) packages. For difference maps, the WT repertoire was subtracted from the mutant repertoire. Repertoire differences were reported for regions demarcated by step-function diagonals with a slope of 4V_α:3J_α (or 1.33 V_α per J_α), the previously established relationship for the progression of secondary rearrangements across the V and J arrays (Carico et al., 2017). Total numbers of unique sequences in each region were combined from two replicates per genotype, and differences between genotypes were evaluated by two-tailed χ² test with Yates’s correction for continuity, using GraphPad Prism 6 software. When determining the regional changes to the repertoire in Trav15d-1-dv6d-1– and Trav15-1-dv6-1–deleted models, the deleted segment in each case was excluded from analysis.

Tcra repertoire sequencing data are deposited in the Gene Expression Omnibus under accession no. GSE186044.

E2A ChIP-seq from sorted DP thymocytes (CD1dTet⁻CD4⁺CD8⁺) isolated from Id2^f/fId3^f/fLck-Cre C57BL/6 mice was previously reported (Roy et al., 2018). Here, alignment was performed to mm10 using Bowtie2 (version 2.3.4.1), allowing multiple read alignment (k = 3) to account for sequence similarity within the Tcra-Tcrd locus; all other parameters were set to default. Peak calling was performed using MACS2 (version 2.1.1.20160309); default options were used. Data visualization was performed using the Integrative Genomics Viewer (version 2.8.2).

Fig. S1 shows characterization of mouse strain mutations and thymocyte cell populations in DJD mice. Fig. S2 shows the WT strain 129 Tcra combinatorial repertoire with gene segments and secondary Tcra recombination diagonals identified. Fig. S3 shows Tcra-Tcrd locus E2A ChIP tracks.

We thank Lunden Simpson and Abani Naik for comments on the manuscript and Qi-Jing Li for helpful discussions.

This research was supported in part by National Institutes of Health grants R01 GM41052 and R35 GM136284 (to M.S. Krangel) and P01 AI102853 (to Y. Zhuang).

Author contributions: D.J. Dauphars, A. Mihai, Y. Zhuang, and M.S. Krangel designed the study; D.J. Dauphars and A. Mihai performed the experiments; D.J. Dauphars, A. Mihai, and L. Wang analyzed the data; and D.J. Dauphars, A. Mihai, L. Wang, and M.S. Krangel wrote the manuscript.