T-cell receptor V delta-J alpha rearrangements in human thymocytes: the role of V delta-J alpha rearrangements in T-cell receptor-delta gene deletion.

The differentiation mechanisms that force thymocytes into the T-cell receptor (TCR)-alpha beta or TCR-gamma delta lineage are poorly understood, but rearrangement processes in the TCR-alpha/delta locus are likely to play an important role. It is assumed that the TCR-delta gene is deleted prior to V alpha-J alpha rearrangements by rearrangement of the so-called TCR-delta-deleting elements delta Rec and psi J alpha. However, the TCR-delta gene can also be deleted via V delta-J alpha rearrangements. We studied the different TCR-delta-deleting rearrangements of V delta 1, delta Rec, V delta 2 and V delta 3 to J alpha gene segments in human thymocytes and peripheral blood using polymerase chain reaction analysis. The V delta 1 gene segment is the most upstream V delta gene segment tested and appears to rearrange to almost all J alpha gene segments. In contrast, the delta Rec and V delta 2 gene segments only rearrange to the 5'-located J alpha gene segments, thereby preserving an extensive TCR-alpha combinatorial diversity, because most J alpha gene segments are kept available for subsequent V alpha-J alpha rearrangements. Based on our combined data we hypothesize that the different V delta gene segments and the delta Rec gene segment play different roles in T-cell development with regard to TCR-delta deletion.     

Early activation of TCR alpha gene rearrangement in fetal thymocytes.

We have previously demonstrated that the onset of TCR alpha gene rearrangement is mainly restricted to the J alpha50 gene in fetal day 1delta thymocyte hybridomas. Now, J alpha50 rearrangements from fetal thymocyte hybridomas and from day 15.5 fetal thymus have been isolated and sequenced. We demonstrate that J alpha50 is rearranged to the rearranged Vdelta1 Ddelta2 gene segment. This indicates that the TCR alpha rearrangement process is initiated in fetal thymocytes far earlier than previously thought. These thymocytes have their delta genes still accessible for rearrangement and therefore, are controlled by the TCR delta enhancer (Edelta) (and/or another TCR delta specific cis-acting element). Our results further suggest that both Edelta and the TCR alpha enhancer (Ealpha) are active at the onset of alpha rearrangements or alternatively, the initial activation of the J alpha locus is controlled by Edelta. In addition, Vdelta1 Ddelta2 J alpha50 gene segments are replaced by secondary alpha rearrangements, indicating that thymocytes with the early alpha rearrangement are committed to the alphabeta lineage.     

Diversity of novel recombining elements suggests developmentally programmed expression of the T cell receptor alpha/delta locus.

During fetal ontogeny, the first wave of gamma delta T lymphocytes appears in the thymus at day 14 of gestation assembling predominantly T cell receptors (TcR) with V gamma 3 and V delta 1. To identify V delta gene segments that are transcribed at day 16, subsequent to the first wave of V delta 1 expression, delta chain cDNA was amplified by the anchored polymerase chain reaction with single-sided specificity for C delta. Unexpectedly, most of the cDNA clones do not contain V gene segments. In some cDNA clones an alternative splice from the leader exon to the C delta exon has deleted the whole variable region exon. In other cDNA clones, multiple non-V-like elements are juxtaposed to the D delta 2 and J delta 1 gene segments. A large number of these diverse elements appear to be rearranged in fetal thymocytes, bringing V alpha gene segments located upstream of the recombining element into proximity to the J alpha locus. It is proposed that these rearrangements make irreversible the commitment to the TcR alpha beta lineage and determine a programmed read out of different clusters of V alpha gene segments.     

PJA-BP expression and TCR delta deletion during human T cell differentiation

Recombination of deltaRec to psiJalpha will delete the TCR delta gene, which is thought to play an important role in the bifurcation of the TCR alphabeta versus TCR gammadelta differentiation lineages. We recently detected a DNA-binding protein in human thymocytes, the so- called PJA-BP, which recognizes the psiJalpha gene segment and might be one of the factors involved in the regulation of preferential deltaRec- psiJalpha rearrangements. We now investigate PJA-BP expression and its correlation with TCR delta gene deletion in thymocytes. Our electrophoretic mobility shift assay experiments showed that the PJA-BP is evolutionary conserved in human, murine and simian thymocytes. Using a large series of human hematopoietic malignancies (n = 30), we conclude that PJA-BP expression is thymocyte specific and seems to be restricted to thymocytes committed to the TCR alphabeta lineage. Analysis of seven well-defined human thymocyte subpopulations showed that preferential deltaRec-psiJalpha rearrangements as well as PJA-BP expression can be detected from the immature CD34-/CD1+/CD3- /CD4+/CD8alpha+beta- thymocyte differentiation stage onwards. These experiments indicate that expression of PJA-BP in human thymocytes starts simultaneously with preferential deltaRec-psiJalpha rearrangements, which supports our hypothesis that PJA-BP is one of the factors involved in the preferential recombination of deltaRec to psiJalpha.      

A complex genetic rearrangement in a t(10;14)(q24;q11) associated with T-cell acute lymphoblastic leukemia.

The t(10;14)(q24;q11) is observed in the leukemia cells of 5-10% of cases of T-cell acute lymphoblastic leukemia (T-ALL). Recently, molecular analyses of a number of these translocations revealed simple reciprocal translocations between the T-cell receptor delta chain gene (TCRD) and a region of 10q24. We have characterized, at the molecular level, a t(10;14)(q24;q11) in a patient with T-ALL. The translocation in this case, in contrast to the previous cases, is part of a complex genetic rearrangement. In addition to a reciprocal translocation between the D delta 3 gene segment of TCRD and a region of 10q24, a local inversion occurred within TCRD, involving the D delta 2 and V delta 2 gene segments. As a consequence, the entire joining and constant regions and most of the diversity regions of TCRD are located on the derivative 14 chromosome, whereas the joining and constant regions of TCRA are positioned on the derivative 10 chromosome. The chromosome 10 breakpoint in our patient, as in other t(10;14), clusters within a 9 kb breakpoint region. The occurrence of seven breakpoints within a localized region of chromosome 10 implies the existence of a nearby gene whose activation may have conferred a selective advantage on the leukemia cells. Moreover, as in the previous cases, the translocation in the present study exhibits recombination signal sequences or signal-like sequences adjacent to the breakpoint junction. The presence of such motifs suggests the involvement of the recombinase enzyme system in the generation of this genetic alteration.     

Dominant T-cell receptor beta-chain gene rearrangements indicate clonal expansion in the rheumatoid joint

T-cell receptor (TcR) beta and delta gene rearrangements were studied in anti-CD3 expanded T-cell populations cultured from the synovial membrane (SM) (n = 5) or synovial fluid (SF) (n = 2) of rheumatoid arthritis (RA) patients. Dominant TcR beta-chain gene rearrangements to C beta 1 were demonstrated in all the patients tested and 1-3 expanded clones per patient were found. Clonal rearrangements to C beta 2 were detected in one SM sample (two clones) and one SF sample (one clone). The TcR delta gene was deleted in all the samples tested. We conclude that clonal dominance may be found in expanded T-cell populations from SM and SF of RA patients. Multiple clones may be present, either using the C beta 1 or C beta 2 gene segment.     

A conserved sequence in the mouse variable T cell receptor alpha recombination signal sequence 23-bp spacer can affect recombination

Although the V-gene segments coding for the TCR alpha and delta chains are mixed together in the alpha delta locus and are recombined by the same processes, some gene segments (TRAV) are rearranged only with TCR Jalpha gene segments, some (TRDV) only with TCR Ddelta gene segments and some (TRADV) with both. To date, no molecular signal is known that can characterize these three different types of gene segments. Studying the recombination signal sequences (RSS) of all mouse TCR V-gene segments we observed that 80% of the TRAV contain a palindrome sequence (CTGCAG) or its related variant CTGTAG in their 23-bp spacer. Using gel-shift assays we show that these sequences are specifically recognized by some nuclear proteins that are expressed by fresh thymocytes, fresh lymphocytes and tumor cells. Recombination assays on plasmid substrates in a pre-B cell line showed that RSS containing the CTGCAG sequence can impair recombination. From the protein fractions containing the CTGCAG-binding activity, three proteins were identified: G3BP1 (a nucleic-acid-binding protein with a proposed helicase activity) and two proteins from the high-mobility group (HMG) family--HMGB2 and HMGB3. We hypothesize that these proteins can affect recombination at the TCR alpha delta locus.     

T cell receptor excision circle assessment of thymopoiesis in aging mice

Signal joint T cell receptor delta (TCRD) excision circles (TRECs) are episomal DNA circles generated by the DNA recombination process that is used by T lymphocytes to produce antigen-specific alpha/beta T cell receptors. Measurement of TRECs in thymocytes and peripheral blood T cells has been used to study thymus output in chickens and humans. We have developed a real-time quantitative-PCR assay for the specific detection and quantification of mouse TCRD episomal DNA circles excised from the TCRA locus during TCRA gene rearrangement (mTRECs). We found that the mouse TCRD TRECs detected with this assay were predominantly in na?ve phenotype CD4(+) and CD8(+) T cells. In a series of aged mice (range 6-90-week-old) we determined the absolute number of thymocytes and the number of molecules of mTRECs/100,000 thymocytes. We found that the absolute number of thymocytes dramatically decreased with age (P<0.05) and that molecules of mTREC/100,000 thymocytes also declined with mouse age (P<0.05). Splenocytes were isolated from aging mice and the frequency of na?ve phenotype CD4 and CD8 cells determined. There was a significant drop in both CD4 and CD8 na?ve peripheral T cells in the aged mice over time. mTREC analysis in purified CD4(+) and CD8(+) splenocytes demonstrated a constant level of mTRECs in the CD4 compartment until age 90 weeks, while the mTRECs in the CD8 compartment fell with age (P<0.05). By combining the mouse TREC assay with T cell phenotypic analysis, we demonstrated that IL-7 administration to young mice induced both increased thymopoiesis and peripheral T cell proliferation. In contrast, IL-7 treatment of aged mice did not augment thymopoiesis, nor induce expansion of splenic T cells. Thus, thymus output continues throughout murine adult life, and the thymic atrophy of aging in mice is not reversed by administration of IL-7.     

Flow cytometric and immunohistochemical characterization of the gamma/delta T-lymphocyte population in normal human lymphoid tissue and peripheral blood.

We determined the quantitative and topographic distribution of gamma/delta lymphocytes in normal human lymphoid tissue and peripheral blood using a monoclonal antibody that detects a framework determinant on delta molecules and delineated the immunophenotypic characteristics of the gamma/delta lymphocyte population by one- and/or two-color immunohistochemical and two- and/or three-color flow cytometric analysis. Variable, but generally small, numbers of gamma/delta lymphocytes are present in peripheral blood and in all lymphoid tissues. The vast majority, greater than or equal to 90%, of lymphoid tissue delta lymphocytes reside in interfollicular (T-cell) zones. Approximately 90% of delta thymocytes are present in the thymic medulla. The percentage of CD3-positive T cells that express delta are: spleen 12.5 +/- 8.1%, peripheral blood 4.0 +/- 3.1%, appendix 2.9 +/- 1%, lymph node 2.2 +/- 1%, thymus 1.4 +/- 0.5%, and tonsil 0.7 +/- 0.5%. We further demonstrated that 1) gamma/delta-thymocytes and gamma/delta peripheral lymphocytes express T-cell lineage restricted antigens CD3 and CD2 but only a variable subset, 30% to 90%, express T-cell lineage associated antigens CD5 and/or CD8; (2) approximately 60% of gamma/delta thymocytes express low-density CD4 while all gamma/delta peripheral lymphocytes lack detectable CD4; 3) gamma/delta lymphocytes lack natural killer (NK), macrophage, and B-cell associated antigens CD16, CD14, and CD20, respectively, but greater than or equal to 70% of gamma/delta T lymphocytes express CD11b, Leu7, and NKH-1, antigens, which are also expressed by suppressor/cytotoxic and NK cells; and 4) a large subpopulation, approximately 25%, of gamma/delta thymocytes are in S1-G2 phase, while greater than or equal to 98% of gamma/delta peripheral lymphocytes are small lymphocytes in G0-G1 phase and lack activation/proliferation markers. Together these results indicate that gamma/delta lymphocytes are resting, mature T cells that probably play a primary role in suppressor/cytotoxic phenomena. They also indicate that gamma/delta lymphocytes variably express multiple-cell surface antigens associated with various cell lineages, suggesting that gamma/delta lymphocytes represent a considerably more heterogeneous cell population than previously appreciated and that they may actually subserve multiple functions.     

Novel excision products of T cell receptor gamma gene rearrangements and developmental stage specificity implied by the frequency of nucleotide insertions at signal joints.

We have cloned circular DNA excised by T cell receptor (TcR) gamma 1, gamma 2 and gamma 3 gene rearrangements in fetal and adult mouse thymocytes. Circular DNA contained a signal joint reciprocal to the genomic V-J coding joint. Although signal joints without nucleotide insertions are common in immunoglobulin (Ig) and TcR gene rearrangements, the signal joint of gamma found in adult thymocytes contained non-germ-line element (N) insertions at high frequency, while no insertions were found in fetal thymocytes. Thus developmental stage specificity of TcR gamma gene rearrangements is faithfully reflected on the signal joint of excision products. In addition, examination of gamma gene excision products revealed circular DNA products of TcR gamma-alpha transrearrangements, but no evidence of V gamma gene replacement in a rearranged segment.     

Hepatosplenic gammadelta T-cell lymphoma following seven malaria infections

Hepatosplenic gammadelta T-cell lymphoma (HSTL) is a clinicopathological entity associated with an immunocompromised status in approximately 25% of patients. Herein is described a case of HSTL in a 53-year-old Brazilian man with seven previous malaria infections, initially misdiagnosed as a hyperreactive splenomegaly due to chronic malaria. A characteristic lymphoid infiltrate was observed in spleen, liver and bone marrow sinusoids/sinuses. Neoplastic cells had a CD45RO+, CD2+, CD7+, CD3+, CD5-, CD8+, CD56+, perforin+, FasL-negative, T-cell receptor (TCR)alphabeta-negative, TCRgammadelta+ profile. Analyses of gamma and delta TCR rearrangements confirmed diagnosis of gammadelta T-cell lymphoma by detecting VgammaI/Vdelta1-Jdelta1 clonal rearrangements. Sensitive polymerase chain reaction (PCR) for Plasmodium falciparum, Epstein-Barr virus and herpesvirus-8 failed to demonstrate infection. The disease progressed to a fatal outcome following cutaneous infiltration and leukemic proliferation. The authors also comment on the association of lymphoma and infection, focusing on PCR diagnosis of TCRgamma and delta clonal rearrangements and the presumed pathogenic events leading to HSTL in the context of chronic malaria infection. Initial lymphomagenic stages might not be direct consequences of antigenic stimulation of Vdelta1 T-cells, but might depend on interactions between gammadelta T and B cells during cooperative or regulatory responses to Plasmodium sp.     

A new mechanism for generating TCR diversity: A TCR Jalpha-like gene that inserts partial nucleotide sequences in a D-gene manner

delta-TCR genes of two gammadelta-T cell hybridomas were found to contain an identical 19-nt sequence in their non-germline N-regions. To determine if this sequence represented a third murine TCR Ddelta gene, genomic PCR was performed by using it as a primer together with primers for interspersed repetitive elements (IRE). Sequencing revealed that the 19-nt segment is part of a 61-nt gene with flanking 5' and 3' recombination signaling sequences (RSS). Southern blot analysis confirmed the presence of this 61-nt gene in the genome of several mouse strains. The gene is unusual in that the distal 24 nucleotides of its 3' RSS region are contributed by the 5' portion of a B2 IRE sequence that includes an apparently non-functional RNA splice site within the 3' nonamer sequence. It has sequence similarities with the Ddelta1 gene (81%) at its 5' end and with Jalpha genes (73%) overall. Tyramide-FISH analysis identified the gene to exist within or adjacent to the TCR alpha/delta locus on chromosome 14. Surveys of available TCR sequences reveal possible partial insertions of the 61-nt gene in other delta-TCR and in alpha-TCR gene sequences. Thus, the unique 61-nt gene is Jalpha gene-like in structure but D gene-like in function.     

Developmental T cell receptor gene rearrangements: relatedness of the alpha/beta and gamma/delta T cell precursor.

To examine the relationships between T cell populations at various stages of development, T cell receptor (TcR) gene rearrangements were compared between the four murine populations of (a) early thymocytes, (b) early splenocytes, (c) adult thymocytes and (d) adult splenocytes. TcR alpha gene rearrangements were shown to progress from 5' to 3' regions of the J alpha locus and from 3' to 5' regions of the V alpha locus during the development of T cells in both the thymus and spleen. Thus, the gene rearrangement potentials of proximal genes varied with age, yielding a biased repertoire in the young vs. adult animal. As evidence that gamma/delta and alpha/beta gene rearrangements appeared concomitantly in individual precursors, it was found that: (a) multiple adult thymocytes bore alpha gene rearrangements on one chromosome and delta gene rearrangements on the homologous chromosome, and (b) V gamma 3-J gamma 1 rearrangements, prominent joins in the early gamma/delta T cell population, were also prominent in the early alpha/beta T cell subset. These data illustrate the non-random nature of the developmental TcR gene rearrangement and suggest that alpha/beta and gamma/delta T cell populations derive from related, if not identical, T cell precursor populations.     

Oligoclonal immunoglobulin heavy-chain and T-cell receptor delta rearrangements persist in a recurrent acute lymphoblastic leukemia with one immunoglobulin kappa rearrangement as a clonal marker.

Acute lymphoblastic leukemias (ALLs) represent the clonal expansion of a lymphoid precursor cell. Therefore, all cells of an ALL should have identical antigen receptor gene rearrangements. In a patient with diploid ALL of the B-cell precursor immunophenotype, seven different clonal rearrangements of the immunoglobulin heavy-chain genes (IgH) were identified, implying the presence of oligoclonal populations. All of these rearrangements were only detectable after a modification of the polymerase chain reaction for the complementarity determining region of the IgH genes using V(H) gene framework 3 and (H) consensus primers. Sequence analysis showed that these rearrangements were completely unrelated to each other. Only two of these rearrangements were detectable by Southern blot analysis. Quantification and single-cell analysis confirmed the high frequency of these latter two rearrangements, as well as their presence in the same clonal population. The other rearrangements characterized less than 5% of the leukemic population. In addition, two T-cell receptor Vdelta2-Ddelta3 (TCRdelta) rearrangements were identified, both at a similar frequency. However, they were derived from different cells. An Igkappa rearrangement represented the only clonal marker in this leukemia. All of the Ig and TCRdelta rearrangements, with the exception of one IgH rearrangement, remained stable throughout the course of the disease. The persistence of such a great number of distinct IgH rearrangements at different quantities within the leukemic population and of the two biclonal TCRdelta rearrangements is compatible with the presence of a clonal disease that is defined by the Igkappa rearrangement.     

Rearrangements of T-cell antigen receptor gamma and delta chain genes are detected in the long-term cultured bone marrow cells of athymic nude mice but not in those of euthymic mice.

We have previously shown that extrathymic rearrangements of T-cell receptor (TcR) gamma and delta chain genes occur in the peripheral lymphoid tissues of athymic nude mice. To further determine where the TcR gene rearrangements occur in nude mice, we investigated the rearrangement and expression of the TcR genes in the long-term cultured bone marrow (LTBM) cells which were homogenous in developments without mature T cells as assessed by FACS analysis. The LTBM derived from euthymic mice contained TcR gamma and delta chain genes in germline configuration, while gene rearrangements of both locus were detected in the LTBM cells from nude mice. These results suggested that gamma and delta gene rearrangements do occur in the bone marrow cells of nude mice and that the T-cell precursors in bone marrow may be increased in frequency in such animals.     

Immunohistochemical detection of CD79a expression in precursor T cell lymphoblastic lymphoma/leukaemias

Twenty-three cases of precursor T cell lymphoid malignancies were examined with respect to CD79a expression. Immunohistochemical staining was performed on frozen tissue sections using a broad panel of antibodies and Southern blot analysis was undertaken using DNA probes encoding immunoglobulin heavy chain (IgH) gene and T-cell receptor (TCR) beta, gamma and delta genes. Twelve (52%) of the 23 cases examined demonstrated CD79a expression. IgH and TCRbeta, gamma and delta gene rearrangements were found in 5, 9, 12 and 20 cases, respectively. CD79a-positive neoplastic cells exhibited a phenotype and genotype characteristic of an early stage of T cell differentiation. Immunohistochemical staining was also performed on human thymus and thymoma to investigate the normality of CD79a expression, to discover that low-level expression of CD79a is common in thymocytes and thymoma-associated lymphocytes. These results suggest that CD79a is expressed weakly and transiently in immature T-lineage cells.     

Forced expression of terminal deoxynucleotidyl transferase in fetal thymus resulted in a decrease in gammadelta T cells and random dissemination of Vgamma3Vdelta1 T cells in skin of newborn but not adult mice

The repertoire of lymphocyte receptor genes encoded in a germline is further diversified by a number of processes, including the template-independent addition of nucleotides (N regions) by means of terminal deoxynucleotidyl transferase (TdT). Normally, mouse gammadelta T cells in the early fetal thymus, whose T-cell receptor (TCR) genes lack N regions and are encoded by Vgamma3-Jgamma1 and Vdelta1-Ddelta2-Jdelta2 with canonical junctions (invariant Vgamma3Vdelta1), are thought to be the precursors of dendritic epidermal T cells (DETC). We generated mutant mice whose endogenous TdT promoter was replaced with the lck promoter through homologous recombination. These mutant mice expressed TdT in fetal thymus, had abundant N regions and infrequent canonical junctions in gamma and delta rearrangements, and showed a decreased number of gammadelta T cells. Various Vgamma3Vdelta1 T cells, most of which had N regions in their TCR genes, were found to disseminate in the skin of newborn mutant mice, whereas normal numbers of DETCs with the invariant Vgamma3Vdelta1 rearrangement were observed in adult mutants. These data demonstrate that the regulation of TdT expression during fetal development is important for the generation of gammadelta T cells, and that Vgamma3Vdelta1 T cells, which have various junctional sequences in their TCR genes, randomly disseminate in skin, but invariant Vgamma3Vdelta1 T cells have a great advantage for proliferation in skin.     

Molecular characterization of the t(10;14) translocation breakpoints in T-cell acute lymphoblastic leukemia: further evidence for illegitimate physiological recombination

The t(10;14)(q24;q11) translocation is a non-random chromosome change seen in the leukemic cells of 5-10% of patients with T-cell acute lymphoblastic leukemia (T-ALL). Recent studies support the hypothesis that the translocation occurs in the course of aberrant physiological recombination and results in the juxtaposition of a T-cell receptor (TCR) gene in 14q11 with a putative oncogene, TCL3, in 10q24. We cloned and sequenced the translocation breakpoints on both derivative 10q+ and 14q- chromosomes from a patient with t(10;14)(q24;q11) T-ALL. Two distinct diversity segments of TCRD, D delta 2 and D delta 3, were identified at the two translocation breakpoints on chromosome 14. The 9.5 kb DNA that separates these two subunits in the germline was deleted, possibly in the course of a D-D joining event. The two chromosome 10 breakpoints were 10 nucleotides apart and occurred in the immediate vicinity of a pseudo-heptamer signal motif. N-region addition is also evident at the breakpoint on the derivative chromosome 10. Our observations strongly suggest that the IG/TCR recombinase normally involved in V-(D)-J joining was involved in the process of the t(10;14)(q24;q11) translocation.     

Developmentally regulated and lineage-specific rearrangement of T cell receptor Valpha/delta gene segments

To quantitate the frequency of Valpha/delta gene utilization by TCRgammadelta T cells we have generated a large panel of gammadelta T cell hybridomas and characterized their productive VDJ rearrangements. Using three novel mAb specific for the Vdelta5 chain and for several members of the Vdelta6 subfamily together with previously described Valpha- and Vdelta-specific mAb we have also quantitated the frequency of gammadelta and alphabeta cells expressing those Valpha/delta gene segments and located in different anatomical sites. We have also characterized the members of the Vdelta7/ADV10 subfamily expressed in C57BL/6 mice and analyzed the representation of individual ADV10 gene segments in alphabeta and gammadelta cells, as well as in precursor cells, in a situation in which TCR-dependent selection is negligible. Our results show that (i) although many Valpha/delta gene segments have the potential to rearrange to either Ddelta and Jdelta segments or to Jalpha segments, only a limited number of Valpha/delta gene segments are expressed by a quantitatively important fraction of gammadelta cells; (ii) such restricted usage of a limited number of Vdelta gene segments by gammadelta cells is mainly established at the level of V(D)J rearrangement, and (iii) there is very little overlap between Valpha/delta gene segments expressed by gammadelta and alphabeta cells.