The human T cell receptor genes are targets for chromosomal abnormalities in T cell tumors

T cells express either of the two forms of antigen-specific receptors, the alpha/beta and gamma/delta heterodimers. Their structure closely resembles that of immunoglobulins, and the variable part of the receptor molecule is created by somatic assembly of variable, diversity, and joining regions. The genetic structure of T cell receptor (TCR) genes and their rearrangement in T cell development have been elucidated in great detail in recent years. The human genes for the gamma and beta subunits are located on the short and long arms of chromosome 7, respectively, whereas the delta- and alpha-chain genes are located in tandem on the centromeric half of the long arm of chromosome 14. Expression of either alpha/beta or gamma/delta TCR complexes on T cells in the developing thymus is likely to proceed in an ordered fashion and results in the appearance of distinct T cell subpopulations. The process of DNA rearrangements required for the generation of functional variable region genes also predisposes lymphoid cells to aberrant DNA rearrangements, which can be detected as chromosomal abnormalities such as translocations and inversions. Molecular analysis of such aberrant rearrangements has shown that rearranging loci are fused to loci unrelated to antigen receptor genes. Furthermore, the breakpoint structures represent nonproductive intermediates in the hierarchy of physiological rearrangements. Accordingly, T cell tumors arising early in T cell development often carry chromosomal abnormalities involving the delta-chain locus, whereas tumors generated later in T cell development tend to show aberrations in the alpha-chain gene. This pattern seems to reflect the stage-specific accessibility of TCR loci for rearrangement by the recombinase machinery. This enzyme is guided by specific recombination signals that can sometimes also be found at the site of breakage on the participating locus in chromosomal abnormalities. Although some features of the mechanism of aberrant rearrangements are known, their biological consequences are less well understood. However, molecular analysis of the mechanism of chromosomal aberrations in T cell tumors suggests that their biological consequences may vary. Firm evidence for the pathogenic significance is missing for most of these lesions. This provides a challenge to molecular immunology to determine how chromosomal abnormalities are involved in tumor pathogenesis.     

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.     

Abnormal recombination of Igh D and J gene segments in transformed pre-B cells of scid mice

Studies of Ig and TCR genes in transformed lymphocytes of scid mice have revealed aberrant DNA rearrangements. Here we present a more detailed analysis of the Igh gene recombination in nine scid pre-B cell lines transformed by Abelson murine leukemia virus. We found 85% of the rearranged Igh alleles to contain abnormal Dh-Jh deletions of varying size. All of these deletions encompassed Jh elements and extended into the Igh enhancer region, occasionally involving the switch (S) region of the C mu gene. Some of these rearrangements removed most of the Dh elements, but none appeared to extend to the Vh genes. DNA sequence analysis of the two abnormally rearranged Igh alleles in one pre-B cell line showed that no Dh or Jh coding sequences were retained at the recombination sites though heptamer-like (CACTGTG) recognition signal sequences were present in the absence of nonamer (GGTTTTTGT) recognition signal sequences. These results imply that a deregulated recombinase activity may be responsible for the abnormal Dh-Jh deletions and the absence of Vh-Dh joining in established lines of Abelson murine leukemia virus-transformed scid pre-B cells.     

Characterization of TCR gene rearrangements during adult murine T cell development

Development of the alphabeta and gammadelta T cell lineages is dependent upon the rearrangement and expression of the TCRalpha and beta or gamma and delta genes, respectively. Although the timing and sequence of rearrangements of the TCRalpha and TCRbeta loci in adult murine thymic precursors has been characterized, no similar information is available for the TCRgamma and TCRdelta loci. In this report, we show that approximately half of the total TCRdelta alleles initiate rearrangements at the CD44highCD25+ stage, whereas the TCRbeta locus is mainly in germline configuration. In the subsequent CD44lowCD25+ stage, most TCRdelta alleles are fully recombined, whereas TCRbeta rearrangements are only complete on 10-30% of alleles. These results indicate that rearrangement at the TCRdelta locus can precede that of TCRbeta locus recombination by one developmental stage. In addition, we find a bias toward productive rearrangements of both TCRdelta and TCRgamma genes among CD44highCD25+ thymocytes, suggesting that functional gammadelta TCR complexes can be formed before the rearrangement of TCRbeta. These data support a model of lineage commitment in which sequential TCR gene rearrangements may influence alphabeta/gammadelta lineage decisions. Further, because TCR gene rearrangements are generally limited to T lineage cells, these analyses provide molecular evidence that irreversible commitment to the T lineage can occur as early as the CD44highCD25+ stage of development.     

Allelic exclusion at the TCR delta locus and commitment to gamma delta lineage: different modalities apply to distinct human gamma delta subsets

Expression of a beta-chain, as a pre-TCR, in T cell precursors prevents further rearrangements on the alternate beta allele through a strict allelic exclusion process and enables precursors to undergo differentiation. However, whether allelic exclusion applies to the TCR delta locus is unknown and the role of the gamma delta TCR in gamma delta lineage commitment is still unclear. Through the analysis of the rearrangement status of the TCR gamma, delta, and beta loci in human gamma delta T cell clones, expressing either the TCR V delta 1 or V delta 2 variable regions, we show that the rate of partial rearrangements at the delta locus is consistent with an allelic exclusion process. The overrepresentation of clones with two functional TCR gamma chains indicates that a gamma delta TCR selection process is required for the commitment of T cell precursors to the gamma delta lineage. Finally, while complete TCR beta rearrangements were observed in several V delta 2 T cell clones, these were seldom found in V delta 1 cells. This suggests a competitive alpha beta/gamma delta lineage commitment in the former subset and a precommitment to the gamma delta lineage in the latter. We propose that these distinct behaviors are related to the developmental stage at which rearrangements occur, as suggested by the patterns of accessibility to recombination sites that characterize the V delta 1 and V delta 2 subsets.     

A novel type of aberrant T cell receptor alpha-chain gene rearrangement. Implications for allelic exclusion and the V-J recombination process

In the process of analyzing the contribution of nonproductive alpha- and beta-chain gene rearrangements to the allelic exclusion of TCR gene expression, we have found a novel type of aberrant alpha-gene rearrangement. In one alpha-allele of the mouse KB5-C20 T cell clone, a J alpha gene segment has been abutted precisely to a sequence that does not display any homology to known V and D gene segment. The appended sequence originates from within the V alpha locus and is located, in the germ-line, 1 kb upstream of a member of the V alpha 2-gene segment subfamily. No recombination signal sequences have been found contiguous to the recombination point. These observations indicate that in normal T lymphocytes, TCR alpha-genes may be affected by aberrant rearrangements similar to those that predominate in human T cell tumors containing chromosome 14 inversion or translocation. Furthermore, compilation of published data and cloning and sequencing of three additional alpha-alleles has allowed us to examine the status of alpha-loci in nine mouse T cell clones expressing functional alpha beta-heterodimers. Interestingly, in contrast to the situation observed at the beta-locus, only 1 of 18 analyzed alpha-alleles has retained a germ-line unrearranged configuration. In addition, in each T cell clone, alpha-rearrangements on homologous chromosomes were unevenly distributed over the J alpha region and shown to generally involve neighboring J alpha gene segments.     

Mechanisms controlling termination of V-J recombination at the TCRgamma locus: implications for allelic and isotypic exclusion of TCRgamma chains

Analyses of Vgamma-Jgamma rearrangements producing the most commonly expressed TCRgamma chains in over 200 gammadelta TCR(+) thymocytes showed that assembly of TCRgamma V-region genes display properties of allelic exclusion. Moreover, introduction of functionally rearranged TCRgamma and delta transgenes results in a profound inhibition of endogenous TCRgamma rearrangements in progenitor cells. The extent of TCRgamma rearrangements in these cells is best explained by a model in which initiation of TCRgamma rearrangements at both alleles is asymmetric, occurs at different frequencies depending on the V or J segments involved, and is terminated upon production of a functional gammadelta TCR. Approximately 10% of the cells studied contained two functional TCRgamma chains involving different V and Jgamma gene segments, thus defining a certain degree of isotypic inclusion. However, these cells are isotypically excluded at the level of cell surface expression possibly due to pairing restrictions between different TCRgamma and delta chains.     

Unraveling the consecutive recombination events in the human IGK locus

In addition to the classical Vkappa-Jkappa, Vkappa-kappa deleting element (Kde), and intron-Kde gene rearrangements, atypical recombinations involving Jkappa recombination signal sequence (RSS) or intronRSS elements can occur in the Igkappa (IGK) locus, as observed in human B cell malignancies. In-depth analysis revealed that atypical JkappaRSS-intronRSS, Vkappa-intronRSS, and JkappaRSS-Kde recombinations not only occur in B cell malignancies, but rather reflect physiological gene rearrangements present in normal human B cells as well. Excision circle analysis and recombination substrate assays can discriminate between single-step vs multistep rearrangements. Using this combined approach, we unraveled that the atypical Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints most probably result from ongoing recombination following an initial aberrant JkappaRSS-intronRSS signal joint formation. Based on our observations in normal and malignant human B cells, a model is presented to describe the sequential (classical and atypical) recombination events in the human IGK locus and their estimated relative frequencies (0.2-1.0 vs < 0.03). The initial JkappaRSS-intronRSS signal joint formation (except for Jkappa1RSS-intronRSS) might be a side event of an active V(D)J recombination mechanism, but the subsequent formation of Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints can represent an alternative pathway for IGK allele inactivation and allelic exclusion, in addition to classical Ckappa deletions. Although usage of this alternative pathway is limited, it seems essential for inactivation of those IGK alleles that have undergone initial aberrant recombinations, which might otherwise hamper selection of functional Ig L chain proteins.     

Models for antigen receptor gene rearrangement. II. Multiple rearrangement in the TCR: allelic exclusion or inclusion?

This series of papers addresses the effects of continuous Ag receptor gene rearrangement in lymphocytes on allelic exclusion. The previous paper discussed light chain gene rearrangement and receptor editing in B cells, and showed that these processes are ordered on three different levels. This order, combined with the constraints imposed by a strong negative selection, was shown to lead to effective allelic exclusion. In the present paper, we discuss rearrangement of TCR genes. In the TCR alpha-chain, allelic inclusion may be the rule rather than the exception. Several previous models, which attempted to explain experimental observations, such as the fractions of cells containing two productive TCRalpha rearrangements, did not sufficiently account for TCR gene organization, which limits secondary rearrangement, and for the effects of subsequent thymic selection. We present here a detailed, comprehensive computer simulation of TCR gene rearrangement, incorporating the interaction of this process with other aspects of lymphocyte development, including cell division, selection, cell death, and maturation. Our model shows how the observed fraction of T cells containing productive TCRalpha rearrangements on both alleles can be explained by the parameters of thymic selection imposed over a random rearrangement process.     

Inhibition of T-cell receptor beta-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck.

The variable region genes of the T cell receptor (TCR) alpha and beta chains are assembled by somatic recombination of separate germline elements. During thymocyte development, gene rearrangements display both an ordered progression, with beta chain formation preceding alpha chain, and allelic exclusion, with each cell containing a single functional beta chain rearrangement. Although considerable evidence supports the view that the individual loci are regulated independently, signaling molecules that may participate in controlling TCR gene recombination remain unidentified. Here we report that the lymphocyte-specific protein tyrosine kinase p56lck, when overexpressed in developing thymocytes, provokes a reduction in V beta--D beta rearrangement while permitting normal juxtaposition of other TCR gene segments. Our data support a model in which p56lck activity impinges upon a signaling process that ordinarily permits allelic exclusion at the beta-chain locus.     

The Hill-Robertson effect and the evolution of recombination

In finite populations, genetic drift generates interference between selected loci, causing advantageous alleles to be found more often on different chromosomes than on the same chromosome, which reduces the rate of adaptation. This "Hill-Robertson effect" generates indirect selection to increase recombination rates. We present a new method to quantify the strength of this selection. Our model represents a new beneficial allele (A) entering a population as a single copy, while another beneficial allele (B) is sweeping at another locus. A third locus affects the recombination rate between selected loci. Using a branching process model, we calculate the probability distribution of the number of copies of A on the different genetic backgrounds, after it is established but while it is still rare. Then, we use a deterministic model to express the change in frequency of the recombination modifier, due to hitchhiking, as A goes to fixation. We show that this method can give good estimates of selection for recombination. Moreover, it shows that recombination is selected through two different effects: it increases the fixation probability of new alleles, and it accelerates selective sweeps. The relative importance of these two effects depends on the relative times of occurrence of the beneficial alleles.     

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.     

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.     

Intermolecular V(D)J recombination is prohibited specifically at the joining step

V(D)J recombination, normally an intramolecular process, assembles immunoglobulin and T cell receptor genes from V, D, and J coding segments. Oncogenic chromosome translocations can result from aberrant rearrangements, such as occur in intermolecular V(D)J recombination. How this is normally prevented remains unclear; DNA cleavage, joining, or both could be impaired when the recombination signal sequences (RSS) are located in trans, on separate DNA molecules. Here, we show that both trans cleavage and joining of signal ends occur efficiently in vivo. Unexpectedly, trans joining of coding ends is severely impaired (100-to 1000-fold), indicating that protection against intermolecular V(D)J recombination is established at the joining step. These findings suggest a novel surveillance mechanism for eliminating cells containing aberrant V(D)J rearrangements.     

The joining of germ-line V alpha to J alpha genes replaces the preexisting V alpha-J alpha complexes in a T cell receptor alpha, beta positive T cell line

To determine whether T cell receptor genes follow the same principle of allelic exclusion as B lymphocytes, we have analyzed the rearrangements and expression of TCR alpha and beta genes in the progeny of the CD3+, CD4-/CD8- M14T line. Here, we show that this line can undergo secondary rearrangements that replace the pre-existing V alpha-J alpha rearrangements by joining an upstream V alpha gene to a downstream J alpha segment. Both the productively and nonproductively rearranged alleles in the M14T line can undergo secondary rearrangements while its TCR beta genes are stable. These secondary recombinations are usually productive, and new forms of TCR alpha polypeptides are expressed in these cells in association with the original C beta chain. Developmental control of this V alpha-J alpha replacement phenomenon could play a pivotal role in the thymic selection of the T cell repertoire.     

Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease

Gaucher disease results from an autosomal recessive deficiency of the lysosomal enzyme glucocerebrosidase. The glucocerebrosidase gene is located in a gene-rich region of 1q21 that contains six genes and two pseudogenes within 75 kb. The presence of contiguous, highly homologous pseudogenes for both glucocerebrosidase and metaxin at the locus increases the likelihood of DNA rearrangements in this region. These recombinations can complicate genotyping in patients with Gaucher disease and contribute to the difficulty in interpreting genotype-phenotype correlations in this disorder. In the present study, DNA samples from 240 patients with Gaucher disease were examined using several complementary approaches to identify and characterize recombinant alleles, including direct sequencing, long-template polymerase chain reaction, polymorphic microsatellite repeats, and Southern blots. Among the 480 alleles studied, 59 recombinant alleles were identified, including 34 gene conversions, 18 fusions, and 7 downstream duplications. Twenty-two percent of the patients evaluated had at least one recombinant allele. Twenty-six recombinant alleles were found among 310 alleles from patients with type 1 disease, 18 among 74 alleles from patients with type 2 disease, and 15 among 96 alleles from patients with type 3 disease. Several patients carried two recombinations or mutations on the same allele. Generally, alleles resulting from nonreciprocal recombination (gene conversion) could be distinguished from those arising by reciprocal recombination (crossover and exchange), and the length of the converted sequence was determined. Homozygosity for a recombinant allele was associated with early lethality. Ten different sites of crossover and a shared pentamer motif sequence (CACCA) that could be a hotspot for recombination were identified. These findings contribute to a better understanding of genotype-phenotype relationships in Gaucher disease and may provide insights into the mechanisms of DNA rearrangement in other disorders.     

The kinetics of early T and B cell immune recovery after bone marrow transplantation in RAG-2-deficient SCID patients

The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT.     

儿童急性淋巴细胞白血病特异性标记物的精准检测方法研究

目的：T细胞和免疫球蛋白重链基因重排是微小残留病灶水平的特异性标记物,而微小残留病灶的水平与儿童急性淋巴细胞白血病的复发强烈相关。应用传统的聚合酶链式反应方法来监测IgH／TCR基因重排不仅耗时、耗人力,而且敏感度较低。本研究旨在探索一种更为高效和敏感与实用的监测IgH／TCR基因重排的精准检测方法。方法：应用多重PCR技术检测26个患有急性淋巴细胞白血病的儿童的外周血样品中的标记物,这些儿童是在中国哈尔滨市最近两年内被诊断的患者。分别应用基因扫描和毛细血管电泳方法检测IgH（FRI,FRII,FRⅢ）／TCR（TCRB,TCRγ）基因重排和分析PCR产物的片段。结果：IgH／TCR基因重排和对IgH基因重排的阳性率分别为92．3％和75％,在26个病例中,4个复发病人的IgH的三个片段（FRI,FRII,FRⅢ）基因重排显示阳性。进一步分析显示复发与ign基因重排呈线性相关。结论：实验与临床应用表明,基因扫描这种方法对于IgH／TCR基因重排的检测是可靠的、实用的,因而可用于儿童急性淋巴细胞白血病的诊断和随访。     

Accumulating postzygotic isolation genes in parapatry: a new twist on chromosomal speciation

Chromosomal rearrangements can promote reproductive isolation by reducing recombination along a large section of the genome. We model the effects of the genetic barrier to gene flow caused by a chromosomal rearrangement on the rate of accumulation of postzygotic isolation genes in parapatry. We find that, if reproductive isolation is produced by the accumulation in parapatry of sets of alleles compatible within but incompatible across species, chromosomal rearrangements are far more likely to favor it than classical genetic barriers without chromosomal changes. New evidence of the role of chromosomal rearrangements in parapatric speciation suggests that postzygotic isolation is often due to the accumulation of such incompatibilities. The model makes testable qualitative predictions about the genetic signature of speciation.     

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.