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. 2003 Jan;108(1):16–23. doi: 10.1046/j.1365-2567.2003.01544.x

Identification of a new cluster of T-cell receptor delta recombining elements

Grzegorz K Przybylski *,†, Jens Wanzeck †, Martie C M Verschuren ‡, Jacques J M Van Dongen ‡, Stephan Serke †, Christian A Schmidt †
PMCID: PMC1782872  PMID: 12519298

Abstract

Within the human T-cell receptor δ (TCRD) gene we have identified a new cluster of seven δ recombining elements (δRec2.1–2.7), located 2·6–5·2 kilobases downstream of the Vδ2 gene segment. The δRec2 elements are isolated recombining signal sequences (RSS), which were shown to rearrange with the Dδ3 and Jδ1 segments of the TCRD gene as well as with the ψJα of the TCRA gene. Rearrangements involving the δRec2 elements were found in all peripheral blood (PB) samples from 10 healthy individuals, although their frequency was about 100-fold lower than that of classical δRec rearrangements. The total frequency of δRec2 rearrangements was lower in PB T lymphocytes, as compared with thymocytes, suggesting that they are deleted during T-cell development. The decrease of the frequency of the δRec2-Dδ3 rearrangements was most prominent: 11 times lower in PB T lymphocytes than in thymocytes. Since the δRec2-Jδ1 rearrangements contained the Dδ3 segment in the junctional region, we assume that they are derived from the δRec2-Dδ3 rearrangements. In contrast, the majority of δRec2-ψJα rearrangements did not contain the Dδ3 segment, indicating that they are single step rearrangements. The δRec2-Jδ1 and δRec2-ψJα rearrangements seem to be T-lineage specific, but the δRec2-Dδ3 rearrangements were also found at very low frequencies in B lymphocytes and natural killer cells. Our results suggest that δRec2 rearrangements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Vα-Jα rearrangements. We hypothesize, that in a similar manner to the classical δRec rearrangements, the δRec2 rearrangements might also contribute to T-cell differentiation towards the TCR-αβ lineage.

Introduction

The T-cell receptor δ gene (TCRD) is located within the TCRA gene and consists of eight variable (V), three diversity (D), four joining (J) and a single constant (C) region.1–5 The V, D and J segments are flanked by specific recombination signal sequences (RSS) consisting of conserved palindromic heptamer and nonamer sequences, separated by less-conserved 12-base pair (bp) or 23-bp spacers. According to the 12/23 rule, rearrangements predominantly occur between gene segments with differently sized spacers.6,7 The TCR gene rearrangements during T-cell differentiation start with VDJ recombination of the TCRD gene, followed by the recombination of the TCRG and the TCRB genes, respectively.8 Productive rearrangements of the TCRD and TCRG genes lead to expression of the TCR-γδ, present on a small population of mature T lymphocytes. Non-productively rearranged TCRD genes remain silent or are deleted by subsequent Vα-Jα recombinations. Besides coding regions, a non-functional TCRD recombining element (δRec) was identified upstream from the Vδ2 gene segment.9 Rearrangements involving δRec (δRec-ψJα, δRec-Jα58, δRec-Jδ1 and δRec-Dδ3) are frequently found in human thymocytes9–12 and are detectable at low levels in peripheral blood (PB) lymphocytes.12 The higher frequency of δRec-ψJα and δRec-Jα58 rearrangements in human thymocytes, as compared to mature T cells, strongly suggests that these rearrangements are deleted during differentiation.9,12 This is supported by the observation that in cell culture conditions, promoting the development of αβ T cells, the δRec rearrangements occur very rapidly, before the productive rearrangement of the TCR-α.13 Therefore, the TCRD deleting recombinations, preceding the Vα-Jα recombinations, are thought to be pivotal steps in the commitment to the TCR-γδ versus TCR-αβ lineage.9,12–14

As a result of the limited number of Vδ, Dδ and Jδ segments, restriction fragment analysis usually allows for precise determination of gene segments involved in TCRD gene rearrangements.15 However, 20–30% of TCRD gene rearrangements in acute leukaemias cannot be precisely identified by restriction fragment length analysis or by polymerase chain reaction (PCR).15–17 Recently, we identified an acute mixed-lineage leukaemia (AMLL) with a gene rearrangement between a new TCRD recombining element (δRec2) and the Jδ1 gene segment.18 In this study, we have analysed rearrangements involving the δRec2 element in normal leucocytes, sorted T lymphocytes, B lymphocytes, natural killer (NK) cells and thymocytes. We identified a new cluster of seven TCRD recombining elements (δRec2.1–δRec2.7), capable of rearranging with the Dδ3, Jδ1 and ψJα gene segments.

Materials and methods

Cell samples

DNA was extracted, according to standard procedures, from the PB leucocytes of 10 healthy donors, and eight thymus samples obtained from four human fetuses (15–20 weeks old) and four infants (1 day to 20 months old) undergoing cardiac surgery. All human cell samples were obtained according to the guidelines for informed consent of the Medical Ethics Committees of the Karol Marcinkowski University Medical School (Poznan, Poland), Charité Clinic, Humboldt University (Berlin, Germany) and the Erasmus University Rotterdam (Rotterdam, the Netherlands).

Purification of leucocyte subsets by cell-sorting

Thirty millilitres of ethylenediaminetetraacetic acid-anticoagulated PB samples was stained with saturating amounts of paired monoclonal antibodies (mAb): (a) CD3-phycoerythrin [PE; Becton Dickinson Immunocytometry System (BDIS); San Jose, CA] plus CD66b-fluorescein isothiocyanate [FITC; Beckman Coulter Immunotech (BCI); Marseille, France] to sort CD3+ T lymphocytes and CD66b+ neutrophils, and (b) CD19-FITC (BCI), CD16-PE (BCI) and CD56-PE (BDIS) to sort CD19+ B lymphocytes and CD16+ and/or CD56+ NK cells. The NK-cell population might contain a small fraction of NK T cells (CD3+ CD56 and/or CD16+). The sorting window was set on sideward light scatter versus specific fluorescence. Sorting was performed on a FACS Vantage sorter (BDIS) equipped with an Enterprise 621 single laser. The purity of the sorted populations exceeded 96% in each sort-run.

Nested-multiplex PCR

A first-round PCR of 50 μl total volume was performed with: 65 ng, 325 ng, 1300 ng, or 3250 ng of genomic DNA, 10 pmol of the 5′ primer δRec2.1a(− 102) and three 3′ primers: Dδ3a(+ 180), Jδ1a(+ 155) and ψJαa(+ 208), 10 nmol each dATP, dCTP, dGTP, dTTP (Perkin Elmer Cetus, Norwalk, CT); 2·5 U Taq polymerase and a PCR buffer (Perkin Elmer Cetus) including 10 mm Tris–HCl, pH 8·3, 50 mm KCl, 1·5 mm MgCl2 and 0·001% (w/v) gelatin. After the initial denaturation at 94° for 5 min, 35 cycles consisting of 94° for 1 min, 60° for 1 min and 72° for 2 min were performed followed by the final extension step at 72° for 5 min. Since δRec2 elements are grouped in a cluster, in the first-round multiplex PCR all possible rearrangements between δRec2 elements and the gene segments Dδ3, Jδ1 and ψα were amplified.

The second-round PCR of 50 μl total volume was performed with: 1 μl of the first-round PCR product, 10 pmol of one of the 5′ primers: δRec2.1b(− 66), δRec2.2(− 59), δRec2.3(− 370), δRec2.4(− 79), δRec2.5(− 483), δRec2.6(− 62) or δRec2.7(− 107) combined with one of the nested 3′ primers: Dδ3b(+ 77), Jδ1b(+ 102) or ψJαb(+ 126). Twenty-one specific nested PCRs were performed from each multiplex first-round PCR. To exclude contamination of PCR reagents non-template controls were included in all reactions. PCR products were analysed by 2·0–3·0% agarose gel electrophoresis with ethidium bromide staining. The sequences of the oligonucleotide primers, synthesized by TIB MOLBIOL (Berlin, Germany), are listed in Table 1. Primers Jδ1a(+ 155) and Dδ3b(+ 77) were published by Yokota et al.19 Other primers were designed by us, based on the nucleotide sequence of the TCRD gene deposited in GenBank.5

Table 1.

Oligonucleotide primers used in PCR and sequencing analysis

5′ primers:
δRec2.1a(−102) 5′ – ATG AGA ACC CCA AGG CAC AG
δRec2.1b(−66) 5′ – GCC CAA AGT AGC ATA GCT AG
δRec2.2(−59) 5′ – CCC AAA TGA CCT CTC AAG TG
δRec2.3(−370) 5′ – AAG CCT GAT TGT CCT GGC TG
δRec2.4(−79) 5′ – CTG ACA ACC ACA ATT CTC TG
δRec2.5(−483) 5′ – GTT AGG GTC TTA GTC CAC TG
δRec2.6(−62) 5′ – AAT TTC GCA ACG CTT CTC TCC
δRec2.7(−107) 5′ – GGT AAC AGC GTA CAT TCC AGT G
M13(−40) 5′ – GTA AAA CGA CGG CCA G
3′ primers:
Jδ1a(+155) 5′ – AAA TGC TAG CTA TTT CAC CCA
Jδ1b(+102) 5′ – GAT GGA GGA TGC CTT AAC CT
Dδ3a(+180) 5′ – AGT TTC ACC CAA GGA AGA AC
Dδ3b(+77) 5′ – AGG GAA ATG GCA CTT TTG CC
ψJαa(+208) 5′ – CAT GGG AAT AAC TGT AGG CTC
ψJαb(+126) 5′ – GGC ACA TTA GAA TCT CTC ACT G
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The position of the first nucleotide of the primers is indicated upstream (–) or downstream (+) relative to the heptamer RSS.

Nucleotide sequencing of PCR products

Selected PCR products were either cloned into the TA-EASY cloning vector (Promega, Madison, WI) or directly subjected to nucleotide sequencing. The sequencing reaction was performed with primers: M13(− 40), Dδ3b(+ 77), Jδ1b(+ 102), or ψJαb(+ 126) using the ALF sequencer (Pharmacia, Uppsala, Sweden), as recommended by the manufacturer.

Results

Identification of the δRec21−7 cluster

To study gene rearrangements involving the recently identified δRec2 element,18 PCR was performed with DNA extracted from PB of 10 healthy individuals. In a 35-cycle PCR, using the δRec2.1a(− 102) primer in combination with Dδ3a(+ 180), Jδ1a(+ 155), or ψJα(+ 208) primer, no amplification products were observed. Surprisingly, in the second-round PCR, using the nested δRec2.1b(− 66) primer in combination with the nested Dδ3b(+ 77), Jδ1b(+ 102), or ψJαb(+ 126) primers, in addition to the bands representing rearrangements of the δRec2 also several larger bands were observed (Fig. 1). The obtained PCR products were cloned and sequenced. Sequence analysis using the blastn program (National Center for Biotechnology Information, http://http://www.ncbi.nlm.nih.gov/BLAST/) revealed, that the atypical PCR products represented rearrangements of the Dδ3, Jδ1, or ψJα gene segments to regions of the TCRD gene, located 2·6–5·2 kilobases (kb) downstream from the Vδ2 gene segment (GenBank accession no. HUAE000661).5 So far these regions have not yet been reported to be involved in gene rearrangements. These rearrangements had junctional regions with N-nucleotide insertions and nucleotide deletions, indicating that they were mediated by the VDJ recombinase machinery (Fig. 2). Analysis of the germline sequences downstream from the rearrangement breakpoints revealed the presence of palindromic heptamer sequences with an average homology of 75% to the consensus heptamer sequence (CACAGTG). Similarly to the published RSS,20 the first three nucleotides of the heptamers were most conserved showing 100% homology to the consensus sequence. Only two of seven δRec2 elements (2.3 and 2.4) possessed a meaningful nonamer with a 67% and 56% homology to the consensus sequence (ACAAAAACC), separated from the heptamer by a 21-bp and a 23-bp spacer (Fig. 3). Based on the nucleotide sequence of the TCRAD locus (accession no. HUAE000661),5 and our sequence analysis we prepared the first continuous restriction map of the Vδ2-Jδ3 region of the TCRD gene, including the novel δRec2 gene cluster (Fig. 4).

Figure 1.

Figure 1

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PCR analysis of δRec2-Jδ1 rearrangements in peripheral blood leucocytes (PB). Lane M, 123-bp molecular weight marker; lanes 1–10, PB samples; lane N, negative control. Nested PCR was performed with the δRec2.1(− 66) and the Jδ1(+ 102) primers. In addition to the δRec2.1-Jδ1 rearrangement, detected in cases 1, 6 and 8, the δRec2.2-Jδ1 rearrangement was detected in case 7 and the δRec2.3-Jδ1 was found in cases 3, 4 and 9.

Figure 2.

Figure 2

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Junctional regions of the rearrangements between the δRec2.1–2.7 elements and the Dδ3, Jδ1 and ψJα segments. N, nucleotides added by the terminal deoxynucleotidyl transferase. The Dδ1–3, Jδ1 and ψJα gene segments are underlined.

Figure 3.

Figure 3

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Sequence homology of the recombining signal sequences (RSS) of the δRec2.1–2.7 elements and the δRec element to the consensus RSS. Regions homologous to the consensus sequence are underlined.

Figure 4.

Figure 4

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Restriction map of the Vδ2-Jδ3 region of the TCRD gene including the δRec2.1–2.7 gene cluster. Open boxes, exons; filled boxes, recombining signal sequences (RSS); E, EcoRI; H, HindIII; B, BamHI; Bg, BglII.

Rearrangements of δRec21−7 elements in peripheral blood leucocytes

To determine the frequency of rearrangements between the seven δRec2 elements and the Dδ3, Jδ1 and ψJα segments, we first performed real-time PCR (TaqMan; Applied Biosystems, Foster City, CA). Since the δRec2.1–2.7 elements are located very close to each other, the individual δRec2 primers also co-amplified other rearrangements of the downstream δRec2 elements. Furthermore, very low numbers of δRec2 rearrangements were measured, which were outside the reproducible range of the real-time PCR. For these reasons we decided to estimate the frequency of rearrangements by our multiplex nested PCR approach using different amounts of PB DNA from 10 healthy individuals: 3250 ng, 1300 ng, or 325 ng, equivalent to 500 000, 200 000 and 50 000 cells, respectively. In the first round of multiplex PCR, with the 5′ primer δRec2.1a(− 102) and three 3′ primers: Dδ3a(+ 180), Jδ1a(+ 155) and ψJαa(+ 208), all possible rearrangements between δRec2.1–2.7 elements and gene segments: Dδ3, Jδ1 and ψJα were amplified. In the second round each particular rearrangement was amplified with one of the seven 5′ δRec2.1–2.7 primers and one of the three 3′ primers (Dδ3b(+ 77), Jδ1b(+ 102), or ψJαb(+ 126) in 21 separate PCR analyses. With this sensitive (full nested) PCR approach we were able to visualize the amplification product of a single copy of rearranged TCRD gene.

The percentage of samples found to be positive for a particular rearrangement decreased with the diminishing amounts of DNA used for PCR, implying that in the majority of samples single copies of the rearranged allele were present (Fig. 5). Subsequent direct sequencing of the PCR products revealed a readable single sequence of the joining region, confirming that a single copy of the rearranged allele was detected in the analysed samples. Furthermore, a second analysis of the same DNA sample showed different δRec2 rearrangements. Even, when rearrangements of the same δRec2 element were amplified again, different joining regions were found in most cases, indicating the presence of a single cell with the δRec2 rearrangement in the analysed DNA sample, rather than monoclonal cell expansion.

Figure 5.

Figure 5

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Analysis of δRec2.1–2.7 rearrangements in peripheral blood leucocytes (PB). Ten DNA samples, equivalent to 500 000, 200 000 and 50 000 cells, were analysed by nested PCR. Numbers of samples with particular rearrangements are shown.

Quantification of δRec2.1–2.7 rearrangements

When DNA amounts corresponding to a given number of cells (n) are analysed, based on the portion (P) of samples positive for a given rearrangement the frequency of the rearrangement (f) can be determined using the following formula: f = 1 − (1 − P)1/n. This formula is a transformation of the binomial distribution formula and was used for calculations of the frequency of each rearrangement. The δRec2.5-Jδ1 was the most frequent rearrangement in PB (f = 24 × 10−6) followed by the δRec2.4-Jδ1 and δRec2.3-Jδ1 (f = 6 × 10−6) (Fig. 5). The δRec2.3–2.5 elements, located in the middle of the cluster, were more frequently rearranged than the upstream or downstream δRec2 elements. Overall, in PB the δRec2.1–2.7 elements were most frequently rearranged to the Jδ1 gene segment (38 × 10−6), less frequently to the Dδ3 segment (11 × 10−6) and rarely to the ψJα segment (3 × 10−6).

Frequency of δRec2.1–2.7 rearrangements in thymocytes and PB cell populations

To gain insight into the biological meaning of δRec2 rearrangements, we compared the frequency of δRec2.1–2.7 rearrangements in thymocytes (four fetal and four postnatal samples) (Fig. 6), sorted PB T lymphocytes (CD3+) (eight samples), B lymphocytes (CD19+) (six samples), NK cells (CD56+ and/or CD16+) (six samples) and neutrophils (CD66b+) (five samples). For each cell population DNA samples of 65 ng, equivalent of 10 000 cells, were analysed. In fetal thymocytes the δRec2-Dδ3 rearrangements were more frequent than the δRec2-Jδ1 rearrangements, fDδ3 = 930 × 10−6 and fJδ1 = 306 × 10−6, respectively (Fig. 7). In the more mature postnatal thymocytes the frequency of δRec2-Dδ3 rearrangements decreased (fDδ3 = 306 × 10−6), while the frequency of δRec2-Jδ1 rearrangements increased (fDδ3 = 571 × 10−6) (P = 0·016 in Fisher exact test calculated for the number of Dδ3 and Jδ1 rearrangements detected in fetal and postnatal thymocytes: 21/8 and 8/13, respectively). In mature PB T lymphocytes the ratio of δRec2-Dδ3 to δRec2-Jδ1 decreased dramatically (4/15; P = 0·001); fDδ3 = 55 × 10−6 and fJδ1 = 326 × 10−6. Particularly, the δRec2.4-Dδ3 and δRec2.7-Dδ3 rearrangements, observed in all fetal thymocyte samples, were virtually absent in T lymphocytes; 1/8 and 0/8, respectively (P = 0·0000). Interestingly, we also detected the δRec2 rearrangements in B lymphocytes and in NK cells. Since both cell populations showed predominantly the δRec2-Dδ3 rearrangements (Fig. 7), T-lymphocyte contamination could virtually be excluded. All but one of the neutrophil samples were negative for δRec2 rearrangements; the single δRec2-Jδ1 rearrangement might be derived from a minor T-lymphocyte contamination.

Figure 6.

Figure 6

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PCR analysis of δRec2-Dδ3 rearrangements in human thymocytes. Lane M, 123 bp molecular weight marker; lane 1 and lanes 6–8, fetal thymocytes; lanes 2–5 postnatal thymocytes; lane N, negative control. Nested PCR was performed with the δRec2.1(− 66) and the Dδ3(+ 77) primers. The δRec2.1-Dδ3 rearrangement was detected in case 6, δRec2.2-Dδ3 in cases 3, 7 and 8, δRec2.3-Jδ1 in case 1 and the δRec2.4 in cases 1 and 5.

Figure 7.

Figure 7

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Frequency of δRec2.1–7 rearrangements in thymocytes, mature T lymphocytes, NK cells, B lymphocytes, neutrophils and peripheral blood leucocytes (PB).

Usage of the Dδ3 gene segment in the δRec2-Jδ1 and the δRec2-ψJα rearrangements

Sequence analysis of the junctional regions revealed that all but one of the δRec2-Jδ1 rearrangements (12/13) contained the Dδ3 segment and therefore resembled the Vδ-Jδ rearrangements (Fig. 2). In contrast, the majority of the δRec2-ψJα rearrangements (7/8) did not contained the Dδ3 segment and therefore were of the Vα-Jα type (P = 0·001).

The DND41 T-cell line contains the δRec2.5-Jδ1 rearrangement

The T-ALL-derived DND41 cell line, contains a functional Vδ1-Jδ1 rearrangement the protein products of which pair with the TCR-β chain to form an unusual TCR-βδ heterodimer.21 In addition, the DND41 T-cell line also possesses an unidentified rearrangement to the Jδ1 gene segment on the second allele.22 Based on Southern blot analysis using the Jδ1- and the Vδ2-specific probes,22 we predicted that the DND41 T-cell line might contain one of the δRec2-Jδ1 rearrangements. We indeed identified in a single round PCR a δRec2.5-(Dδ3)-Jδ1 rearrangement (Fig. 2), which was the most common δRec2 rearrangement in T lymphocytes. Since each cell of the DND 41 contains the δRec2.5-Jδ1 rearrangement, we used this cell line to determine the actual sensitivity of the nested-multiplex PCR. We performed the PCR with serial dilutions of the DND 41 T-cell line DNA, corresponding to the number of cells given in the parenthesis: 0·65 ng (100), 195 pg (30), 65 pg (10), 19·5 pg (3), 6·5 pg (1) and 1·95 pg (0·3). We were able to detect the δRec2.5-Jδ1 in the samples containing 100, 30, 10 and 3 cells (data not shown). No amplification product was obtained in the dilutions of 1 and 0·3 cells/sample. Unsuccessful amplification of the single cell sample, assumed to contain one copy of the rearrangement, might be a result of a stochastic phenomenon observed in serial dilutions at this concentration, or because a fraction of target DNA fragments was not amplifiable. The results confirmed the very high sensitivity of the nested-multiplex PCR method and validated its usage for frequency estimation of the δRec2 rearrangements; although the frequencies, calculated on the theoretical single copy sensitivity and 100% efficiency of the nested-multiplex PCR, might be slightly (two- to three-fold) underestimated.

Discussion

The mechanism of thymocyte commitment to the αβ, or γδ lineage is not completely understood. TCRD gene deletion by recombinations of the δRec element was shown to play an important role in cells making the decision to rearrange the TCRA gene and differentiate towards the αβ pathway.9,12–14 We identified a cluster of seven new recombining elements (δRec2.1–2.7), which are located 2·6–5·2 kb downstream of the Vδ2 gene segment. These δRec2 elements are similar to the classical δRec, consist of isolated RSS without an upstream meaningful open reading frame, and are capable of rearranging with the Dδ3, Jδ1 and ψJα gene segments. Rearrangements involving the δRec2.1–2.7 gene cluster were found in PB of all healthy individuals, at an overall frequency in T cells of 0·4 × 10−3, which is 100-fold lower than the classical δRec rearrangements (2–5% in PBL).12

To obtain insight into the consecutive events during T-cell differentiation we compared the frequency of δRec2.1–2.7 rearrangements in thymocytes and in mature PB T-lymphocytes. The ratio of δRec2-Dδ3δ to δRec2-Jδ1 rearrangements was decreasing from 2·6 in fetal thymocytes to 0·6 in postnatal thymocytes and 0·27 in T lymphocytes. Furthermore, virtually all analysed δRec2-Jδ1 rearrangements contained the Dδ3 segment in the junctional region, indicating that during T-cell differentiation the δRec2-Dδ3 complex further recombines to the Jδ1 segment. In contrast, the majority of δRec2-ψJα rearrangements did not contain the Dδ3 segment, suggesting that they are Vα-Jα-like, single-step rearrangements.

Interestingly, the δRec2 rearrangements were not specific for the T-cell lineage. We also found the δRec2-Dδ3 rearrangements in NK cells and in B lymphocytes. This resembles TCRD gene rearrangements in ALL, where the Jδ1 rearrangements (Vδ1-Jδ1, Vδ2-Jδ1, Vδ3-Jδ1 and Dδ2Jδ1) occur almost exclusively in T-ALL while the Dδ3 rearrangements (Vδ2-Dδ3 and Dδ2-Dδ3) are typical for precursor-B-ALL.15,23,24 In vitro experiments showed that the Dδ3 rearrangements are not as tightly regulated as the Jδ1 rearrangements and do not require activation of a TCRD enhancer.25 They can even be induced in non-lymphoid cells via introduction of lymphoid transcription factors. Therefore it might be that the δRec2-Dδ3 rearrangements presented here occur in early lymphoid progenitor cells, which are not yet lineage-committed. The δRec2-Dδ3 rearrangements might proceed to the δRec2-Jδ1 rearrangements, only if the cells differentiate towards the T-cell lineage with activation of the T-cell specific TCRD enhancer.

The δRec2.1–2.7 elements, similarly to the close-by upstream Vδ2 region, were much more frequently rearranged to the Dδ3 and Jδ1 segments than to the ψJα segment. On the other hand the regions further upstream from Vδ2 gene segments (δRec, Vδ1 and Vα) segments, more easily recombine to the ψJα and Jα segments.12,26 Based on these findings, and on the published data, the following model of the recombinatorial process of the TCRAD locus can be deduced. The recombination of the TCRD gene starts from the middle of the gene with the Dδ2-Dδ3 rearrangement.8 Subsequently, the proximal upstream (δRec2.1–2.7 or Vδ2) and/or downstream (Jδ1–4) segments are joined to the Dδ2-Dδ3 complex. If a functional Vδ2(Dδ)Jδ rearrangement takes place the rearranged gene will be expressed and the recombinase machinery will be switched off. Should a non-functional rearrangement occur, e.g. δRec2-Jδ or out-of-frame Vδ2(Dδ)Jδ, the complex can be deleted by the δRec-ψJα rearrangement, which, finally, will be replaced by the Vα-Jα recombination.

In spite of extensive studies on TCRD gene rearrangements in ALL, a substantial number of rearrangements involving the Jδ1 segment (30% in T-ALL and 20% in pre-B-ALL) and also several rearrangements in the Vδ2 region remain unidentified. Potentially, a portion of these ALLs may possess δRec2 rearrangements. In our collectives, nine of 29 acute leukaemias with TCRD gene rearrangements exhibited atypical TCRD gene rearrangements.27 In one of those cases (AMLL) we identified the δRec2.1-Jδ1 rearrangement,18 and in this study we identified the δRec2.5-Jδ1 rearrangement in a T-ALL-derived cell line DND41. Apparently, a portion of acute leukaemias with atypical TCRD gene rearrangements contains δRec2 rearrangements. Since the δRec2 rearrangements may be useful for monitoring minimal residual disease in leukaemia patients,28 further studies are needed to determine the frequency of δRec2 rearrangements in T-ALL, precursor-B-ALL and AMLL.

In mice, three δRec elements have been found.29 The murine δRec1 is a homologue of the human classical δRec, and is involved in the TCRD deleting rearrangements, although less frequently than in humans.10 Human homologues of the murine δRec2 and δRec3 have not been described so far. Comparison of the nucleotide sequence of the human δRec2.1–2.7 gene cluster (GenBank accession no. HUAE000661)5 with the analogous region of the murine TCRD gene (GenBank accession no. AF019412) revealed an overall homology of 55% between the two species. The highest homology (77%) was found within a 102-bp region (HUAE000661; 180 744–180 845), located 143 bp 5′ from the δRec2.4 heptamer and within a 313-bp region (HUAE000661; 180 121–180433), located 122 bp 5′ from the δRec2.3 heptamer (70%). Although the overall homology between the two species was similar for the classical δRec region (58%) and the δRec2 region (55%), the δRec2.1–2.7 RSS were less conserved (54–72% homology) than the RSS of the classical δRec (79%). Particularly, the heptamers, which were highly conserved between the human δRec and murine δRec1, were absent in all of the murine regions analogous to human δRec2.1–2.7. Therefore, it seems unlikely that murine regions analogous to human δRec2.1–2.7 might be involved in gene rearrangements.

We hypothesize that rearrangements involving the newly identified δRec2 elements are transient steps in the recombinatorial process of the TCRAD locus and are probably deleted by subsequent Vα-Jα rearrangements. Therefore, similarly to the more frequent classical δRec rearrangements, the δRec2 rearrangements may also play a role in T-cell differentiation towards the αβ pathway in a minor portion of T-lymphoid progenitor cells.

Acknowledgments

We gratefully acknowledge Mrs A. van Lessen for excellent cell sorting. This work was supported in part by the German Josê Carreras Leukemia Foundation (C.A.S. and G.K.P.), the Committee for Scientific Research, Poland, grant KBN 3.P05A.102·22 (G.K.P.), and the Dutch Cancer Society, grant EUR 95–1015 (M.C.M.V and J.J.M.v.D.). Sadly, Stephan Serke died after the completion of this paper.

Abbreviations

δRec

δ recombining element

PB

peripheral blood

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