T cell receptor gamma and delta gene rearrangements in scid thymocytes. Similarity to those in normal thymocytes.

The nature of TCR gamma and delta gene rearrangements in 4- to 6-week-old scid thymocytes was examined by using the polymerase chain reaction technique, cloning, and DNA sequencing. Analysis of 78 sequences indicates that TCR gamma and delta gene rearrangements in scid mice generally resemble those in thymocytes from normal young adult mice. V gamma 1, V gamma 2, and V gamma 5 rearrangements are heterogeneous, with extensive N region addition and nucleotide excision from the recombining coding segments. In addition, homogeneous and fetal-like V gamma 3, V gamma 4, and V delta 1 rearrangements are observed. These rearrangements are currently difficult to interpret but may be significant with respect to whether certain homogeneous joints in normal mice are due to cellular selection or to the rearrangement process. scid TCR gamma and delta gene nucleotide sequences also reveal direct V-J delta joining, inter-(V-J-C gamma) cluster joining, and the possibility of inversional rearrangement at the gamma locus. Short sequence homologies may contribute to V(D)J recombination and to the rescue of blocked coding joints.     

Biased T-cell receptor delta element recombination in scid thymocytes.

Thymocytes in mutant mice with severe combined immunodeficiency (scid thymocytes) show ongoing recombination of some T-cell receptor delta gene elements, generating signal joints quantitatively and qualitatively indistinguishable from those in wild-type fetal thymocytes. Excised D delta 2-J delta 1 and D delta 1-D delta 2 rearrangements are detectable at levels equivalent to or greater than those in thymocytes from wild-type mice on fetal day 15. Signal junctional modification, shown here to occur frequently in wild-type adult but not newborn excised D delta 2-J delta 1 junctions, can occur normally in adult scid thymocytes. Excised D delta 1-D delta 2 scid junctions, similar to wild-type thymocytes, include pseudonormal coding junctions as well as signal junctions. Inversional D delta 1-D delta 2 rearrangements, generating conventional hybrid junctions, are also reproducibly detectable in scid thymus DNA. These hybrids, unlike those reported for artificial recombination constructs, do not show extensive nucleotide loss. In contrast to the normal or high incidences of D delta 1-, D delta 2-, and J delta 1-associated signal junctions in scid thymocytes, V delta 1, V gamma 3, and V gamma 1.2 signal products are undetectable in scid thymocytes or are detectable at levels at least 10-fold lower than the levels in wild-type fetal thymocytes. These findings confirm biased T-cell receptor element recombination by V(D)J recombinase activity of nontransformed scid thymocytes and indicate that analysis of in vivo-mediated gene rearrangements is important for full understanding of how the scid mutation arrests lymphocyte development.     

T-cell receptor alpha locus V(D)J recombination by-products are abundant in thymocytes and mature T cells.

In addition to the assembled coding regions of immunoglobulin and T-cell receptor (TCR) genes, the V(D)J recombination reaction can in principle generate three types of by-products in normal developing lymphocytes: broken DNA molecules that terminate in a recombination signal sequence or a coding region (termed signal or coding end molecules, respectively) and DNA molecules containing fused recombination signal sequences (termed reciprocal products). Using a quantitative Southern blot analysis of the murine TCR alpha locus, we demonstrate that substantial amounts of signal end molecules and reciprocal products, but not coding end molecules, exist in thymocytes, while peripheral T cells contain substantial amounts of reciprocal products. At the 5' end of the J alpha locus, 20% of thymus DNA exists as signal end molecules. An additional 30 to 40% of the TCR alpha/delta locus exists as remarkably stable reciprocal products throughout T-cell development, with the consequence that the TCR C delta region is substantially retained in alpha beta committed T cells. The disappearance of the broken DNA molecules occurs in the same developmental transition as termination of expression of the recombination activating genes, RAG-1 and RAG-2. These findings raise important questions concerning the mechanism of V(D)J recombination and the maintenance of genome integrity during lymphoid development.     

Characterization of excised DNA intermediates associated with V(D)J recombination at the T-cell receptor delta locus.

Lymphocyte development requires the assembly of antigen receptor genes through the specialized process of V(D)J recombination. This process is initiated by cleavage at the junction between coding segments (V, D, and J) and the recombination signal sequences that border these segments, resulting in generation of double-strand break intermediates. We have used a two-dimensional gel system to characterize broken molecules arising from V(D)J recombination at the T-cell receptor (TCR) delta locus and have identified linear species excised by Ddelta1-Ddelta2 and V-Ddelta2 rearrangement in thymus DNA. Relatively few (approximately 10) V-Ddelta2-excised linear species were detected in DNA from fetal thymocytes. The sizes of these species corresponded to the estimated distances between Ddelta2 and the V gene segments utilized by gammadelta T cells and indicated that both Ddelta2-proximal and -distal V gene segments are targeted for V-Ddelta2 rearrangement. Similar-sized species were observed in DNA from thymocytes of scid mice in which T-cell development is arrested prior to TCR expression. Since previous studies suggest that the TCR alpha/delta locus encodes more than 100 V gene segments, our results indicate that a few select V gene segments are predominantly targeted for rearrangement to Ddelta2, and this primarily accounts for the restricted Vdelta gene repertoire of gammadelta T cells.     

A delta T-cell receptor deleting element transgenic reporter construct is rearranged in alpha beta but not gamma delta T-cell lineages.

T cells can be divided into two groups on the basis of the expression of either alpha beta or gamma delta T-cell receptors (TCRs). Because the TCR delta chain locus lies within the larger TCR alpha chain locus, control of the utilization of these two receptors is important in T-cell development, specifically for determination of T-cell type: rearrangement of the alpha locus results in deletion of the delta coding segments and commitment to the alpha beta lineage. In the developing thymus, a relative site-specific recombination occurs by which the TCR delta chain gene segments are deleted. This deletion removes all D delta, J delta, and C delta genes and occurs on both alleles. This delta deletional mechanism is evolutionarily conserved between mice and humans. Transgenic mice which contain the human delta deleting elements and as much internal TCR delta chain coding sequence as possible without allowing the formation of a complete delta chain gene were developed. Several transgenic lines showing recombinations between deleting elements within the transgene were developed. These lines demonstrate that utilization of the delta deleting elements occurs in alpha beta T cells of the spleen and thymus. These recombinations are rare in the gamma delta population, indicating that the machinery for utilization of delta deleting elements is functional in alpha beta T cells but absent in gamma delta T cells. Furthermore, a discrete population of early thymocytes containing delta deleting element recombinations but not V alpha-to-J alpha rearrangements has been identified. These data are consistent with a model in which delta deletion contributes to the implementation of a signal by which the TCR alpha chain locus is rearranged and expressed and thus becomes an alpha beta T cell.     

Junctional sequences of T cell receptor gamma delta genes: implications for gamma delta T cell lineages and for a novel intermediate of V-(D)-J joining

Nucleotide sequences of a large number of V-(D)-J junctions of T cell receptor (TCR) gamma and delta genes show that most fetal thymocytes express on their surface one of just two gamma delta TCRs known to be expressed by epidermal gamma delta T cells (s-IEL) or intraepithelial gamma delta T cells associated with female reproductive organs (r-IEL). In contrast, gamma delta TCRs expressed on adult thymocytes are highly diverse as a result of multiple combinations of gene segments as well as junctional deletions and insertions, indicating that developmental time-and cell lineage-dependent mechanisms exist that control the extent of gamma delta TCR diversity. In addition, this study revealed a new type of junctional insertion (P nucleotides), which led to a new model of V-(D)-J joining generally applicable to immunoglobulin and TCR genes.     

Transrearrangements between antigen receptor genes in normal human lymphoid tissues and in ataxia telangiectasia.

The polymerase chain reaction (PCR) was used on DNA obtained from various normal lymphoid tissues to amplify chimeric TCR gene rearrangements involving J segments of the beta gene and V segments of the gamma or delta genes. As found previously for the transrearrangements between the gamma and delta genes, transrearrangements involving the beta gene were more abundant in DNA of the thymus than in DNA of the spleen, lymph node, bone marrow, or PBL. In addition, transrearrangements between Ig H chain V region segment and J segment of TCR delta chain were also found in DNA of normal thymus. Sequence analysis of the trans-rearrangement PCR products revealed structures closely resembling normal intragenic rearrangements, with N insertions and often D segments at the junctions between segments. The sequences analyzed suggest that transrearrangements arise through the action of normal lymphocyte recombinase, involve trans recognition of heptamer/nonamer recombination signals, and follow the 12 + 23 spacer rule. To test whether transrearrangements result from chromosomal rearrangements with breakpoints at the sites of Ag receptor genes, PCR was performed on the DNA of PBL from patients with ataxia telangiectasia, a disorder in which circulating lymphocytes often have numerous karyotypic abnormalities with breakpoints at the cytogenetic positions of these genes. Comparison of the results of PCR on this DNA and that of normal tissues demonstrated a substantially increased frequency for most types of transrearrangements investigated. These results support the interpretation that transrearrangement among TCR genes may occur by chromosomal rearrangement.     

V(D)J recombination intermediates and non-standard products in XRCC4-deficient cells.

V(D)J recombination assembles immunoglobulin (Ig) and T cell receptor (TCR) gene segments during lymphocyte development. Recombination is initiated by the RAG-1 and RAG-2 proteins, which introduce double-stranded DNA breaks (DSB) adjacent to the Ig and TCR gene segments. The broken ends are joined by the DSB repair machinery, which includes the XRCC4 protein. While XRCC4 is essential for both DSB repair and V(D)J recombination, the functions of this protein remain enigmatic. Because the rare V(D)J recombination products isolated from XRCC4-deficient cells generally show evidence of excessive nucleotide loss, it was hypothesized that XRCC4 may function to protect broken DNA ends. Here we report the first examination of V(D)J recombination intermediates in XRCC4-deficient cells. We found that both types of intermediates, signal ends and coding ends, are abundant in the absence of XRCC4. Furthermore, the signal ends are full length. We also showed that alternative V(D)J recombination products, hybrid joints, form with normal efficiency and without excessive deletion in XRCC4-deficient cells. These data indicate that impaired formation of V(D)J recombination products in XRCC4-deficient cells does not result from excessive degradation of recombination intermediates. Potential roles of XRCC4 in the joining reaction are discussed.     

Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus.

We have cloned extrachromosomal circular DNAs containing T cell receptor (TCR) delta gene segments in adult mouse thymocytes and splenocytes. We find that the frequency of circular DNA clones carrying germline delta sequences is lower than that of J alpha probe-positive clones, possibly related to increasing 5' distance from the most upstream J alpha segment. This suggests that the TCR alpha/delta locus is successively rearranged from within and that the delta-containing excision products are progressively diluted out by the subsequent cell division which includes further alpha gene rearrangements. In addition, examination of delta gene excision products revealed newly identified V delta subfamilies, the reciprocal joining of two D delta elements, J delta 2 usage in thymocytes and novel sequences homologous to the human delta-gene deleting elements.     

Gamma-irradiation directly affects the formation of coding joints in SCID cell lines.

SCID mice have a defect in the catalytic subunit of the DNA-dependent protein kinase, causing increased sensitivity to ionizing radiation in all tissues and severely limiting the development of B and T cell lineages. SCID T and B cell precursors are unable to undergo normal V(D)J recombination: coding joint and signal joint products are less frequently formed and often will exhibit abnormal structural features. Paradoxically, irradiation of newborn SCID mice effects a limited rescue of T cell development. It is not known whether irradiation has a direct impact on the process of V(D)J joining, or whether irradiation of the thymus allows the outgrowth of rare recombinants. To investigate this issue, we sought to demonstrate an irradiation effect ex vivo. Here we have been able to reproducibly detect low-frequency coding joint products with V(D)J recombination reporter plasmids introduced into SCID cell lines. Exposure of B and T lineage cells to 100 cGy of gamma irradiation made no significant difference with respect to the number of coding joint and signal joint recombination products. However, in the absence of irradiation, the coding joints produced in SCID cells had high levels of P nucleotide insertion. With irradiation, markedly fewer P insertions were seen. The effect on coding joint structure is evident in a transient assay, in cultured cells, establishing that irradiation has an immediate impact on the process of V(D)J recombination. A specific proposal for how the DNA-dependent protein kinase catalytic subunit influences the opening of hairpin DNA intermediates during coding joint formation in V(D)J recombination is presented.     

Chimeric gamma-delta signal joints. Implications for the mechanism and regulation of T cell receptor gene rearrangement.

Rearrangement of Ag receptor genes requires recognition by the lymphocyte recombinase of heptamer-nonamer signal sequences followed by two endonucleolytic cleavages and two DNA ligations to form the coding and signal joints. The phenomenon of trans-rearrangement, in which Ag receptor gene segments located on different chromosomes recombine to yield chimeric products, provides an in vivo system in which to investigate the ability of the recombinase to carry out each of these functions in trans. Trans-rearrangements between TCRG and TCRD loci, similar in structure and frequency to those observed previously in human lymphoid tissues, were demonstrated in normal mouse thymus by PCR with crossed V gamma/J delta and V delta/J gamma primer pairs. A simple mechanistic model for trans-rearrangement was then tested. This model posits an ability of the recombinase to catalyze the formation of both coding and signal joints in trans and therefore predicts that trans-rearrangements will generate chimeric signal joints. In adult thymus, chimeric D delta 2-J gamma 1 and D delta 2-J gamma 2 signal joints, containing fused heptamer-nonamer sequences, could be detected by PCR and were each present at frequencies sufficient to account for a large proportion of the corresponding TCRG/TCRD trans-rearrangements. In agreement with the predictions of the model, chimeric signal joints were found as both linear chromosomal and circular episomal DNA. The results provide a framework for understanding the formation of chromosomal translocations in normal and neoplastic lymphoid cells and support the possibility of a looping mechanism for standard gene rearrangement. To test the form of regulation of TCRG rearrangement, the frequencies of specific signal joints from standard and trans-rearrangements were compared. Although J gamma 1 and J gamma 2 segments participated with equal frequency in trans-rearrangement with D delta 2, only the J gamma 1 segment participated in standard rearrangement with V gamma 5. The results suggest that V-J recombination in the TCRG locus is regulated directly at the DNA level by cis-acting constraints which do not affect the accessibility of individual TCRG gene segments to recombination in trans.     

Characterization of an avian (Gallus gallus domesticus) TCR alpha delta gene locus.

Mammalian TCR delta genes are located in the midst of the TCR alpha gene locus. In the chicken, one large V delta gene family, two D delta gene segments, two J delta gene segments, and one C delta gene have been identified. The TCR delta genes were deleted on both alleles in alpha beta T cell lines, thereby indicating conservation of the combined TCR alpha delta locus in birds. V alpha and V delta gene segments were found to rearrange with one, both or neither of the D delta segments and either of the two J delta segments. Exonuclease activity, P-addition, and N-addition during VDJ delta rearrangement contributed to TCR delta repertoire diversification in the first embryonic wave of T cells. An unbiased V delta 1 repertoire was observed at all ages, but an acquired J delta 1 usage bias occurred in the TCR delta repertoire. The unrestricted combinatorial diversity of relatively complex TCR gamma and delta loci may contribute to the remarkable abundance of gamma delta T cells in this avian representative.     

Immature and advanced patterns of T cell receptor gene rearrangement among lymphocytes in splenic culture

Bulk populations and 39 hybridomas from splenic Con A cultures were analyzed for rearrangements among TCR genes: alpha, beta, gamma, and delta. Patterns were categorized to reveal general rules governing gene rearrangement within the activated adult peripheral population. Many patterns of gene rearrangement were consistent with previous studies of T cell lines. Additional points of interest were the following: 1) A large proportion of Con A-stimulated splenic cells bore no TCR gene rearrangements. 2) One splenic hybridoma exhibited an unusual gene pattern, with rearrangements, at alpha and beta, but not J gamma 1 or J gamma 2 loci. 3) Multiple gamma rearrangements were noted other than V1.2-J2 and V2-J1. 4) One hybridoma exhibited TCR gene rearrangements typical of day 14 to 15 fetal thymocytes, as well as rearrangements at immunoglobulin gene loci. 5) Among hybridomas with J alpha rearrangements, homologous chromosomes exhibited rearrangements at similar positions along the J alpha locus.     

T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining

We have identified circular DNA containing T cell receptor (TCR) beta gene sequences in mouse thymocytes, thereby providing direct evidence for the intramolecular DNA deletion model of V-D-J joining in TCR beta genes. Two types of excision products of V-D-J joining have been identified. Type I, a circular reciprocal recombinant of normal V-D or D-J joining, contains a 7mer-7mer head-to-head structure expected from an excised product of normal V-D or D-J joining. Type II contains a D beta 2-J beta 1 structure on the circular DNA; the recombination event producing this molecule occurs between an upstream J and a downstream D segment, probably leaving the reciprocal 7mer-7mer structure on the chromosome. Some type I molecules seem to represent excision products of secondary joining after formation of the first D-J or V-D-J structure. The recombination mechanism that generates the circular DNA is discussed.     

Cutting edge: TCR delta gene is frequently rearranged in adult B lymphocytes

TCR gene rearrangement generates diversity of T lymphocytes by V(D)J recombination. Ig genes are rearranged in B cells using the same enzyme machinery. Physiologically, TCR gene is postulated to rearrange exclusively in T lineage, but malignant B precursor lymphoblasts contain rearranged TCR genes in most patients. Several mechanisms by which malignant cells break the regulation of V(D)J recombination have been proposed. In this study we show that incomplete TCR delta rearrangements V2-D3 and D2-D3 occur each in up to 16% alleles in B lymphocytes of all healthy donors studied, but complete VDJ rearrangement was negative at the sensitivity limit of 1%. Data are based on real-time quantitative PCR validated by PAGE and sequencing of the cloned products. Therefore, TCR genes rearrange not exclusively in T lineage. This study opens up further questions regarding the exact extent of the "cross-lineage" TCR or Ig rearrangements in normal lymphocytes, specific subsets in which the cross-lineage rearrangements occur, and the physiological importance of these rearrangements.     

In vivo transposition mediated by V(D)J recombinase in human T lymphocytes

The rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non-homologous end-joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non-specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter-chromosomal transposition in humans mediated by V(D)J recombinase. T-cell isolates were shown to contain TCRalpha signal ends from chromosome 14 inserted into the X-linked hypo xanthine-guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase-mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.     

V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair.

V(D)J recombination has been examined in several X-ray-sensitive and double-strand break repair-deficient Chinese hamster cell mutants. Signal joint formation was affected in four mutants (xrs 5, XR-1, V-3, and XR-V9B cells, representing complementation groups 1 through 4, respectively) defective in DNA double-strand break rejoining. Among these four, V-3 and XR-V9B were the most severely affected. Only in V-3 was coding joint formation also affected. Ataxia telangiectasia-like hamster cell mutants (V-E5 and V-G8), which are normal for double-strand break repair but are X ray sensitive, were normal for all aspects of the V(D)J recombination reaction, indicating that X-ray sensitivity is not the common denominator but that the deficiency in double-strand break repair appears to be. The abnormality at the signal joints consisted of an elevated incidence of nucleotide loss from each of the two signal ends. Interestingly, in complementation groups 1 (xrs 5) and 2 (XR-1), signal joint formation was within the normal range under some transfection conditions. This suggests that the affected gene products in these two complementation groups are not catalytic components. Instead, they may be either secondary or stochiometric components involved in the later stages of both the V(D)J recombination reaction and double-strand break repair. The fact that such factors can affect the precision of the signal joint has mechanistic implications for V(D)J recombination.