Activated γδ T Cells Promote Dendritic Cell Maturation and Exacerbate the Development of Experimental Autoimmune Uveitis (EAU) in Mice.

Our previous study reveals that gamma delta (γδ) T cells were activated and dendritic cells (DCs) underwent maturation during the inflammation phase in experimental autoimmune uveitis (EAU) mice, and the interaction between DCs and γδ T cells may significantly exacerbate the development of EAU. However, the interactions between DCs and γδ T cells that can affect DCs maturation to influence EAU development must be further addressed. In this study we showed that mature DC numbers in TCR-δ-/- (KO) EAU mice were lower than those in wild-type (WT) C57BL/6 (B6) mice. The γδ T cells harvested from WT EAU mice secreted more interferon-γ (IFN-γ), however, after blocking IFN-γ, the maturation of DCs was significantly downregulated. By contrast, the percentage of IFN-γ- and IL-17-producing CD4+ T cells in KO EAU mice decreased to a greater extent than that in WT EAU mice during the inflammatory phase. Additionally, the levels of IFN-γ/IL-17 in serum were in agreement with those of CD4+ T cells. Furthermore, after activated γδ T cells injection, the inflammatory symptoms of EAU mice were more aggravated. In vitro co-cultures of both cell types showed that activated γδ T cells may induce DCs to generate higher levels of intracellular cell adhesion molecule-1 (ICAM-1/CD54), CD80, CD83, and CD86. Moreover, co-culture of the two cells may induce the activation of CD4+ T cells. Taken together, our results demonstrated that activated γδ T cells may promote DCs maturation and further enhance the generation of Th1/Th17 cells in EAU mice, resulting in exacerbated EAU.

IMGT® Biocuration and Comparative Analysis of Bos taurus and Ovis aries TRA/TRD Loci.

The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens to generate immunity, and mount stronger attacks each time the pathogen is encountered. T cell receptors are the antigen receptors of the adaptive immune response expressed by T cells, which specifically recognize processed antigens, presented as peptides by the highly polymorphic major histocompatibility (MH) proteins. T cell receptors (TR) are divided into two groups, αß and γδ, which express distinct TR containing either α and ß, or γ and δ chains, respectively. The TRα locus (TRA) and TRδ locus (TRD) of bovine (Bos taurus) and the sheep (Ovis aries) have recently been described and annotated by IMGT® biocurators. The aim of the present study is to present the results of the biocuration and to compare the genes of the TRA/TRD loci among these ruminant species based on the Homo sapiens repertoire. The comparative analysis shows similarities but also differences, including the fact that these two species have a TRA/TRD locus about three times larger than that of humans and therefore have many more genes which may demonstrate duplications and/or deletions during evolution.

γδ T Cell-Secreted XCL1 Mediates Anti-CD3-Induced Oral Tolerance.

Oral tolerance is defined as the specific suppression of cellular and/or humoral immune responses to an Ag by prior administration of the Ag through the oral route. Although the investigation of oral tolerance has classically involved Ag feeding, we have found that oral administration of anti-CD3 mAb induced tolerance through regulatory T (Treg) cell generation. However, the mechanisms underlying this effect remain unknown. In this study, we show that conventional but not plasmacytoid dendritic cells (DCs) are required for anti-CD3-induced oral tolerance. Moreover, oral anti-CD3 promotes XCL1 secretion by small intestine lamina propria γδ T cells that, in turn, induces tolerogenic XCR1+ DC migration to the mesenteric lymph node, where Treg cells are induced and oral tolerance is established. Consistent with this, TCRδ-/- mice did not develop oral tolerance upon oral administration of anti-CD3. However, XCL1 was not required for oral tolerance induced by fed Ags, indicating that a different mechanism underlies this effect. Accordingly, oral administration of anti-CD3 enhanced oral tolerance induced by fed MOG35-55 peptide, resulting in less severe experimental autoimmune encephalomyelitis, which was associated with decreased inflammatory immune cell infiltration in the CNS and increased Treg cells in the spleen. Thus, Treg cell induction by oral anti-CD3 is a consequence of the cross-talk between γδ T cells and tolerogenic DCs in the gut. Furthermore, anti-CD3 may serve as an adjuvant to enhance oral tolerance to fed Ags.

Recognition of Listeria Infection by Germline Elements of the Vγ1.1 Vδ6.3 TCR.

γδNKT cells are an abundant γδT cell population with restricted Vγ1.1 Vδ6.3 gene usage and phenotypic and functional similarity to conventional αß-invariant NKT cells. The γδNKT population responds to Listeria infections, but specific ligands are not known. In this work, we studied the CDR3 requirements of the γδNKT TCR, Vγ1.1Vδ6.3 for recognizing naive macrophages, and macrophages infected with Listeria We expressed four different variants of the Vγ1.1Vδ6.3 TCR in TCR-deficient hybridomas, one with germline-encoded sequences and three with nongermline-encoded sequences. All of the hybridomas were activated when cultured in the presence of macrophages, and the activation was increased when the macrophages were infected with Listeria This indicates that these TCRs can recognize a self-ligand present in macrophages and suggests that the ligand is modified or upregulated when the cells are infected with Listeria One of the three nongermline-encoded Vγ1.1 variants induced a lower activation level compared with the other variants tested in this study, suggesting that recognition of the Listeria-induced ligand involves the CDR3γ region of the TCR.

The Armadillo (Dasypus novemcinctus): A Witness but Not a Functional Example for the Emergence of the Butyrophilin 3/Vγ9Vδ2 System in Placental Mammals.

1-5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain a TRGV9/TRGJP rearrangement and a TRDV2 comprising Vδ2-chain. They respond to phosphoantigens (PAgs) like isopentenyl pyrophosphate or (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) in a butyrophilin 3 (BTN3)-dependent manner and may contribute to the control of mycobacterial infections. These cells were thought to be restricted to primates, but we demonstrated by analysis of genomic databases that TRGV9, TRDV2, and BTN3 genes coevolved and emerged together with placental mammals. Furthermore, we identified alpaca (Vicugna pacos) as species with typical Vγ9Vδ2 TCR rearrangements and currently aim to directly identify Vγ9Vδ2 T cells and BTN3. Other candidates to study this coevolution are the bottlenose dolphin (Tursiops truncatus) and the nine-banded armadillo (Dasypus novemcinctus) with genomic sequences encoding open reading frames for TRGV9, TRDV2, and the extracellular part of BTN3. Dolphins have been shown to express Vγ9- and Vδ2-like TCR chains and possess a predicted BTN3-like gene homologous to human BTN3A3. The other candidate, the armadillo, is of medical interest since it serves as a natural reservoir for Mycobacterium leprae. In this study, we analyzed the armadillo genome and found evidence for multiple non-functional BTN3 genes including genomic context which closely resembles the organization of the human, alpaca, and dolphin BTN3A3 loci. However, no BTN3 transcript could be detected in armadillo cDNA. Additionally, attempts to identify a functional TRGV9/TRGJP rearrangement via PCR failed. In contrast, complete TRDV2 gene segments preferentially rearranged with a TRDJ4 homolog were cloned and co-expressed with a human Vγ9-chain in murine hybridoma cells. These cells could be stimulated by immobilized anti-mouse CD3 antibody but not with human RAJI-RT1Bl cells and HMBPP. So far, the lack of expression of TRGV9 rearrangements and BTN3 renders the armadillo an unlikely candidate species for PAg-reactive Vγ9Vδ2 T cells. This is in line with the postulated coevolution of the three genes, where occurrence of Vγ9Vδ2 TCRs coincides with a functional BTN3 molecule.

Insight into normal thymic activity by assessment of peripheral blood samples.

The thymus is a highly specialized organ for T cell receptor (TCR) rearrangement and selection mechanisms that ensure the formation of functional and self-tolerant cells. Little is known about how peripheral blood assessment of thymic function reflects thymus activity during infancy. We compared thymic function-related markers in the thymus with those in peripheral blood in order to check their correlations. We concomitantly blood samples from immunocompetent infants who underwent cardiac surgery that involved thymectomy. The studied thymic markers included TCR excision circles (TRECs), four different TCRD (TCR delta chain) gene rearrangements, the TCR repertoire, regulatory T cells (Tregs, defined as the CD4+CD25+FOXP3+ cell population) and real-time quantitative polymerase chain reaction (RQ-PCR) mRNA expression of forkhead box P3 (FOXP3). Twenty patients were enrolled in this study. Their mean age at the time of the surgery was 3 months/5 days ± 3 months/18 days. There was a significant correlation between thymic and peripheral blood levels of TREC, all four TCRD gene rearrangements and the amount of Tregs. The levels of these parameters were significantly higher in the thymus than those detected in the peripheral blood. The TCR repertoire distribution in both samples was similar. FOXP3 mRNA levels in the thymus and peripheral blood correlated well. Our findings demonstrated a strong and significant correlation between peripheral blood and intra-thymic activity parameters during infancy. Assessment of these parameters in peripheral blood can be used to accurately estimate different intra-thymic capacities for assessing T cell function in health and disease.

Long-distance regulation of fetal V(δ) gene segment TRDV4 by the Tcrd enhancer.

Murine Tcra and Tcrd gene segments are organized into a single genetic locus (Tcra/Tcrd locus) that undergoes V(D)J recombination in CD4(-)CD8(-) double-negative (DN) thymocytes to assemble Tcrd genes and in CD4(+)CD8(+) double-positive thymocytes to assemble Tcra genes. Recombination events are regulated by two developmental stage-specific enhancers, E(δ) and E(α). Effects of E(α) on Trca/Tcrd locus chromatin have been well documented, but effects of E(δ) have not. In this regard, E(α) acts over long distances to activate many V(α) and J(α) segments for recombination in double-positive thymocytes. However, in DN thymocytes, it is unclear whether E(δ) functions over long distances to regulate V(δ) gene segments or functions only locally to regulate D(δ) and J(δ) gene segments. In this study, we analyzed germline transcription, histone modifications, and recombination on wild-type and E(δ)-deficient alleles in adult and fetal thymocytes. We found that E(δ) functions as a local enhancer whose influence is limited to no more than ∼10 kb in either direction (including D(δ), J(δ), and TRDV5 gene segments) in adult DN thymocytes. However, we identified a unique long-distance role for E(δ) promoting accessibility and recombination of fetal V(δ) gene segment TRDV4, over a distance of 55 kb, in fetal thymocytes. TRDV4 recombination is specifically repressed in adult thymocytes. We found that this repression is enforced by a developmentally regulated loss of histone acetylation. Constitutively high levels of a suppressive modification, histone H3 lysine 9 dimethylation, may contribute to repression as well.

[ γδ T lymphocyte function and the polymorphism of T cell receptor V δ chain in lungs of asthmatic patients].

OBJECTIVE: To observe the function of gamma delta T lymphocytes and the polymorphism of T cell receptor V delta chain in the lungs of asthmatic patients and explore the role of gamma delta T cells in airway inflammation. METHODS: Bronchoalveolar lavage fluid BALF was obtained from 7 asthmatic patients and 7 healthy control individuals. The percentage of gamma delta T cell in BALF was measured by flow cytometry. The gamma delta T cell in BALF was purified by magnetic labeled beads. Proliferous activity was examined by MTT assay. Cytokines secreted by gamma delta T cells in medium was assessed by enzyme-linked immunosorbent assay. Polymorphism of T cell receptor V delta chain was detected by RT-PCR and gene scan analysis. RESULTS: The proportion of gamma delta T cell in the BALF of asthmatic patients [(6.39+/-0.71)%] was significantly higher than that in control subjects [(2.62+/-0.37)%] (P<0.01). The proportion of macrophage in the BALF of asthmatic patients [(81+/-4)] was significantly lower than that in control subjects [(86+/-2)] (P<0.05). The proliferation rate of asthmatic patients [(284.2+/-43.6)%] was significantly higher than that of control subjects [(217.5+/-59.5)%] (P<0.05). Interleukin-4 secreted by gamma delta T cells of asthmatic patients [(18.9+/-3.1) pg/ml)] significantly increased when compared with the control subjects [(14.1+/-3.0) pg/ml] (P<0.05). The polymorphism of T cell receptor V delta chain was not significantly different between these two groups. CONCLUSIONS: The increase of gamma delta T cells in the lung of asthmatic patients further exacerbates Th1/Th2 disturbance and airway inflammation. Antigen recognition by gamma delta T cells is non-specific.

Annotation and classification of the bovine T cell receptor delta genes.

BACKGROUND: gammadelta T cells differ from alphabeta T cells with regard to the types of antigen with which their T cell receptors interact; gammadelta T cell antigens are not necessarily peptides nor are they presented on MHC. Cattle are considered a "gammadelta T cell high" species indicating they have an increased proportion of gammadelta T cells in circulation relative to that in "gammadelta T cell low" species such as humans and mice. Prior to the onset of the studies described here, there was limited information regarding the genes that code for the T cell receptor delta chains of this gammadelta T cell high species. RESULTS: By annotating the bovine (Bos taurus) genome Btau_3.1 assembly the presence of 56 distinct T cell receptor delta (TRD) variable (V) genes were found, 52 of which belong to the TRDV1 subgroup and were co-mingled with the T cell receptor alpha variable (TRAV) genes. In addition, two genes belonging to the TRDV2 subgroup and single TRDV3 and TRDV4 genes were found. We confirmed the presence of five diversity (D) genes, three junctional (J) genes and a single constant (C) gene and describe the organization of the TRD locus. The TRDV4 gene is found downstream of the C gene and in an inverted orientation of transcription, consistent with its orthologs in humans and mice. cDNA evidence was assessed to validate expression of the variable genes and showed that one to five D genes could be incorporated into a single transcript. Finally, we grouped the bovine and ovine TRDV1 genes into sets based on their relatedness. CONCLUSIONS: The bovine genome contains a large and diverse repertoire of TRD genes when compared to the genomes of "gammadelta T cell low" species. This suggests that in cattle gammadelta T cells play a more important role in immune function since they would be predicted to bind a greater variety of antigens.

Differential utilization of T cell receptor TCR alpha/TCR delta locus variable region gene segments is mediated by accessibility.

T cell receptor (TCR) variable region exons are assembled from germline V, (D), and J gene segments, each of which is flanked by recombination signal (RS) sequences that are composed of a conserved heptamer, a spacer of 12 or 23 bp, and a characteristic nonamer. V(D)J recombination only occurs between V, D, and J segments flanked by RS sequences that contain, respectively, 12(12-RS)- and 23(23-RS)-bp spacers (12/23 rule). Additional mechanisms can restrict joining of 12/23 RS matched segments beyond the 12/23 rule (B12/23). The TCRdelta locus is contained within the TCRalpha locus; TCRalpha variable region exons are encoded by TRAV and TRAJ segments and those of TCRdelta by TRDV, TRDD, and TRDJ segments. On the basis of the 12/23 rule, both TRAV and TRDV gene segments are compatible to rearrange with TRDD gene segments; however, TRAV-to-TRDD joins are not observed in vivo. Absence of TRAV-to-TRDD rearrangement might be explained either by B12/23 restriction or by differential accessibility of the TRDV versus TRAV gene segments for rearrangement to TRDD. We used in vitro substrate analysis to reveal that both TRAV and TRDV 23-RSs mediate rearrangements to the 5'TRDD1 12-RS, demonstrating that B12/23 restriction does not explain these rearrangement biases. However, targeted replacement of TRDD1 and its 12-RSs with TRAJ38 and its 12-RS showed that TRDV gene segments rearrange with the ectopic TRAJ38, whereas TRAV segments do not. Our results demonstrate that sorting of TRAV and TRDV gene segments is determined by differential locus accessibility during T cell development.

Striking antigen recognition diversity in the Atlantic salmon T-cell receptor alpha/delta locus.

The complete TCR alpha/delta locus of Atlantic salmon (Salmo salar) has been characterized and annotated. In the 900 kb TCR alpha/delta locus, 292 Valpha/delta segments and 123 Jalpha/delta segments were identified. Of these, 128 Valpha/delta, 113 Jalpha, and a Jdelta segment appeared to be functional as they lacked frame shifts or stop codons. This represents the largest repertoire of Valpha/delta and Jalpha segments of any organism to date. The 128 functional Valpha/delta segments could be grouped into 29 subgroups based upon 70% nucleotide similarity. Expression data confirmed the usage of the diverse repertoire found at the genomic level. At least 99 Valpha, 13 Vdelta 86 Jalpha, 1 Jdelta, and 2 Ddelta segments were used in TCR alpha or delta transcription, and 652 unique genes were identified from a sample of 759 TCRalpha cDNA clones. Cumulatively, the genomic and expression data suggest that the Atlantic salmon T-cell receptor has enormous capacity to recognize a wide diversity of antigens.

Highly diverse TCR delta chain repertoire in bovine tissues due to the use of up to four D segments per delta chain.

Tissue-specific distribution of gammadelta TCRs with limited TCR diversity is a common phenomenon in species with a low percentage of gammadelta T cells like humans and mice. We set out to investigate whether this is also the case in cattle (Bos taurus), a species with high percentages of gammadelta T cells. Using a method that was independent of variable (V) segment-specific primers, we generated 65 unique TCR delta chain sequences. We found no evidence for preferential use of certain Vdelta segments in lymph node, skin, spleen, small intestine, large intestine, and blood. The delta chain CDR3 length distribution was very wide in each tissue, which was confirmed by spectratyping. The highly variable CDR3 length was due to the use of up to four diversity (D) segments by one bovine delta chain. Human and murine delta chains contain only one or two D segments. The five functional Ddelta segments that we describe here were identified at cDNA and genomic level, and are the first ruminant D segments described. Fourteen TCR delta chain sequences used novel Vdelta1 segments, and one expressed a novel member of the Vdelta3 family. The number of known functional Vdelta segments in cattle including these new ones is 42 now, but the total number may be much higher. A high number of Vdelta segments in combination with the use of up to four out of five D segments, and the possibility of using non-template encoded (N) nucleotides on either side of these, makes the potential bovine delta chain repertoire much bigger than any known TCR chain. This situation is quite different from the situation in humans and mice, and suggests that the differences between gammadelta high and gammadelta low species in distribution, diversity, and function of gammadelta T cells may be substantial.

T-cell receptor gamma and delta gene rearrangements and junctional region characteristics in south Indian patients with T-cell acute lymphoblastic leukemia.

Clonal T-cell receptor (TCR) gamma and delta gene rearrangements were studied in 40 T-ALL cases (pediatrics, 29; adults, 11) using PCR with homo-heteroduplex analysis. At least one clonal TCRG or TCRD rearrangement was detected in 34 (85%) cases. TCR gamma (TCRG) rearrangement was detected in 25 (62.5%) cases that included 16 (55%) pediatrics and 9 (81.8%) adults. TCR delta (TCRD) rearrangement was detected in 14/40 (35%) cases, which included 12 (41%) pediatrics and 2 (18%) adults. The frequency of VgammaI-Jgamma1.3/2.3 was significantly more in adults than pediatrics (81.8% vs. 41.3%, P=0.02). In TCRD, Vdelta1-Jdelta1 was rearranged in 10 (25%) cases. The surface membrane CD3 positive cases are significantly associated with absence of TCRD rearrangements (surface membrane CD3+ TCRdelta- 84% vs. surface membrane CD3- TCRdelta- 48%, P value=0.03). Junctional region sequence analyzed with 10 cases each, of TCRG and TCRD, revealed an average junctional region of 7.4 nucleotides (range 2-18 nucleotides) in TCRG and 27 nucleotides (range 14-42 nucleotides) in TCRD-complete rearrangements. In TCRG, trimming at the ends of Vgamma and Jgamma germline nucleotides resulted in deletion, on an average of 9.2 nucleotides. In TCRD, deletion of nucleotides of the Vdelta and Jdelta gene segments on an average was 3.5 nucleotides. The junctional region of TCRD is more diverse than TCRG; nevertheless, the frequency of TCRG was more than that of TCRD and hence we rely more on TCRG clonal markers to quantitate the minimal residual disease in T-ALL.

Molecular cytogenetic study of 126 unselected T-ALL cases reveals high incidence of TCRbeta locus rearrangements and putative new T-cell oncogenes.

Chromosomal aberrations of T-cell receptor (TCR) gene loci often involve the TCRalphadelta (14q11) locus and affect various known T-cell oncogenes. A systematic fluorescent in situ hybridization (FISH) screening for the detection of chromosomal aberrations involving the TCR loci, TCRalphadelta (14q11), TCRbeta (7q34) and TCRgamma (7p14), has not been conducted so far. Therefore, we initiated a screening of 126 T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma cases and 19 T-ALL cell lines using FISH break-apart assays for the different TCR loci. Genomic rearrangements of the TCRbeta locus were detected in 24/126 cases (19%), most of which (58.3%) were not detected upon banding analysis. Breakpoints in the TCRalphadelta locus were detected in 22/126 cases (17.4%), whereas standard cytogenetics only detected 14 of these 22 cases. Cryptic TCRalphadelta/TCRbeta chromosome aberrations were thus observed in 22 of 126 cases (17.4%). Some of these chromosome aberrations target new putative T-cell oncogenes at chromosome 11q24, 20p12 and 6q22. Five patients and one cell line carried chromosomal rearrangements affecting both TCRbeta and TCRalphadelta loci. In conclusion, this study presents the first inventory of chromosomal rearrangements of TCR loci in T-ALL, revealing an unexpected high number of cryptic chromosomal rearrangements of the TCRbeta locus and further broadening the spectrum of genes putatively implicated in T-cell oncogenesis.

IMGT/GeneInfo: T cell receptor gamma TRG and delta TRD genes in database give access to all TR potential V(D)J recombinations.

BACKGROUND: Adaptative immune repertoire diversity in vertebrate species is generated by recombination of variable (V), diversity (D) and joining (J) genes in the immunoglobulin (IG) loci of B lymphocytes and in the T cell receptor (TR) loci of T lymphocytes. These V-J and V-D-J gene rearrangements at the DNA level involve recombination signal sequences (RSS). Whereas many data exist, they are scattered in non specialized resources with different nomenclatures (eg. flat files) and are difficult to extract. DESCRIPTION: IMGT/GeneInfo is an online information system that provides, through a user-friendly interface, exhaustive information resulting from the complex mechanisms of T cell receptor V-J and V-D-J recombinations. T cells comprise two populations which express the alphabeta and gammadelta TR, respectively. The first version of the system dealt with the Homo sapiens and Mus musculus TRA and TRB loci whose gene rearrangements allow the synthesis of the alphabeta TR chains. In this paper, we present the second version of IMGT/GeneInfo where we complete the database for the Homo sapiens and Mus musculus TRG and TRD loci along with the introduction of a quality control procedure for existing and new data. We also include new functionalities to the four loci analysis, giving, to date, a very informative tool which allows to work on V(D)J genes of all TR loci in both human and mouse species. IMGT/GeneInfo provides more than 59,000 rearrangement combinations with a full gene description which is freely available at http://imgt.cines.fr/GeneInfo. CONCLUSION: IMGT/GeneInfo allows all TR information sequences to be in the same spot, and are now available within two computer-mouse clicks. This is useful for biologists and bioinformaticians for the study of T lymphocyte V(D)J gene rearrangements and their applications in immune response analysis.

The effect of a novel recombination between the homeobox gene NKX2-5 and the TRD locus in T-cell acute lymphoblastic leukemia on activation of the NKX2-5 gene.

BACKGROUND AND OBJECTIVES: The NK-like homeobox gene (NKX2-5/CSX) plays a crucial role in cardiac development but is not normally expressed in hematopoietic cells. Here, we describe for the first time a fusion between NKX2-5 and the T-cell receptor delta locus (TRD) resulting in NKX2-5 activation in a case of T-cell acute lymphoblastic leukemia (T-ALL). DESIGN AND METHODS: Genomic DNA from a T-ALL patient with an atypical rearrangement, detected by Southern blotting, was analyzed by ligation-mediated polymerase chain reaction (PCR) with TRD-specific primers. Expression of NKX2-5 was analyzed by real-time quantitative PCR in the T-ALL case with the NKX2-5-TRD rearrangement, 18 other cases of T-ALL, three T-ALL derived cell lines, two non-hematopoietic cell lines, peripheral blood mononuclear cells from six healthy individuals and sorted thymocyte subsets. RESULTS: Sequence analysis of ligation-mediated PCR products revealed a novel rearrangement between the third diversity segment of the TRD locus (TRDD3) and a region on chromosome 5q35.1 located 32 kb downstream of the NKX2-5/CSX gene. As a result of this recombination NKX2-5 was placed under influence of the TRD enhancer, resulting in strong ectopic NKX2-5 expression. High NKX2-5 expression was also found in the T-cell lines PEER and CCRF-CEM, which harbor an NKX2-5-BCL11B rearrangement, and in the embryonic kidney cell line 293. NKX2-5 was not expressed in any of the major thymocyte subsets, in normal peripheral blood mononuclear cells, or in the majority (17/18) of the other cases of T-ALL. INTERPRETATION AND CONCLUSIONS: Our finding of overexpression of yet another homeobox gene in T-ALL further supports the hypothesis that homeobox genes play an important role in malignant transformation of particular types of T-ALL.

Genomic organization of the sheep TRG1@ locus and comparative analyses of Bovidae and human variable genes.

gammadelta T cells commonly account for 0.5%-5% of human (gammadelta low species) circulating T cells, whereas they are very common in chickens, and they may account for >70% of peripheral cells in ruminants (gammadelta high species). We have previously reported the ovine TRG2@ locus structure, the first complete physical map of any ruminant animal TCR locus. Here we determined the TRG1@ locus organization in sheep, reported all variable (V) gamma gene segments in their germline configuration and included human and cattle sequences in a three species comparison. The TRG1@ locus spans about 140 kb and consists of three clusters named TRG5, TRG3, and TRG1 according to the constant (C) genes. The predicted tertiary structure of cattle and sheep V proteins showed a remarkably high degree of conservation between the experimentally determined human Vgamma9 and the proteins belonging to TRG5 Vgamma subgroup. However systematic comparison of primary and tertiary structure highligthed that in Bovidae the overall conformation of the gammadelta TCR, is more similar to the Fab fragment of an antibody than any TCR heterodimer. Phylogenetic analysis showed that the evolution of cattle and sheep V genes is related to the rearrangement process of V segments with the relevant C, and consequentely to the appartenence of the V genes to a given cluster. The TRG cluster evolution in cattle and sheep pointed out the existence of a TRG5 ancient cluster and the occurrence of duplications of its minimal structural scheme of one V, two joining (J), and one C.

Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure.

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4- CD8- (double negative [DN]) and CD4+ CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of approximately 100 variable gene segments contributes substantially to the adult TCRdelta repertoire. Moreover, although most contribute to the TCRalpha repertoire, variable gene segments that are Jalpha proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRdelta repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

T-cell receptor molecular diagnosis of T-cell lymphoma.

Malignant and reactive lymphoproliferations in most cases can be distinguished by histology and immunohistology alone; however, in T-cell lymphoproliferations and lymphoproliferations of unknown origin, histology often is inconclusive, and there is no reliable protein marker of malignancy. At the genomic level T-cell neoplasms clonally rearrange T-cell receptor (TCR) genes that can serve as clonal markers. In comparison with Southern blot analysis, polymerase chain reaction (PCR) techniques increasingly are being used to detect these rearrangements because PCRs are rapid, easy to perform, and can be used to amplify poor-quality deoxyribonucleic acid (DNA) from paraffin-embedded formalin-fixed biopsies. Nevertheless there are a number of problems associated with the detection of gene rearrangements using PCR. The foremost of these is improper primer annealing that will lead to false-negative or false-positive PCR results that may arise from poor primer design, especially for TCRB genes with an extensive variable (V), diversity (D), and joined (J) gene repertoire. There also can be difficulty in discriminating between clonal and polyclonal PCR products unless specific methods such as heteroduplex analysis or gene scanning are used. In this chapter, we describe methods, derived from a recent European collaborative BIOMED-2 program, for the detection of TCRG, B, and D rearrangements. TCRB VJ and DJ gene rearrangements are detected using 23 VB primers, 13 JB primers, and 2 DB primers in 3 multiplex tubes. TCRG VJ gene rearrangements are detected with four VG and two JG primers in two multiplex tubes, and TCRD VJ, VD, DJ, and DD rearrangements are detected with six VD primers, four JD primers, and two DD primers in one multiplex tube. Gaussian distributions of polyclonal PCR products are seen at specific size ranges for both TCRB and TCRG. Interpretation of TCRD rearrangements is more complex because of the wide range in PCR product size and often low numbers of TCRGD cells in target tissue. For all loci, some indication of gene usage can be ascertained by labeling the primers with different fluorochromes and gene scan analysis of PCR products. Furthermore the complementarity of the TCR loci affords an unprecedented high clonal detection rate. In addition, we also describe a set of control gene primers designed to amplify amplicons of 100, 200, 300, 400, and 600 bp for the assessment of the integrity and amplifiability of DNA.