T-cell Receptor Delta Gene Research Articles

Enhancer control of local accessibility to V(D)J recombinase.

We have studied the role of transcriptional enhancers in providing recombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) delta gene minilocus. This transgene is composed of unrearranged variable (Vdelta and Vdelta2), diversity (Ddelta3), joining (Jdelta1 and Jdelta3), and constant (Cdelta) gene segments. Previous data indicated that with the TCR delta enhancer (Edelta) present in the Jdelta3-Cdelta intron, V(D)J recombination proceeds stepwise, first V to D and then VD to J. With the enhancer deleted or mutated, V-to-D rearrangement is intact, but VD-to-J rearrangement is inhibited. We proposed that Edelta is necessary for J segment but not D segment accessibility and that J segment inaccessibility in the enhancerless minilocus resulted in the observed V(D)J recombination phenotype. In this study, we tested this notion by using ligation-mediated PCR to assess the formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3' of Ddelta3 and 5' of Jdelta1. In five lines of mice carrying multicopy integrants of constructs that either lacked Edelta or carried an inactivated Edelta, the frequency of DSBs 5' of Jdelta1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of Ddelta3 was unaffected. We interpret these results to indicate that Edelta is required for Jdelta1 but not Ddelta3 accessibility within the minilocus, and we conclude that enhancers regulate V(D)J recombination by providing local accessibility to the recombinase. cis-acting elements other than Edelta must maintain Ddelta3 in an accessible state in the absence of Edelta. The analysis of DSB formation in a single-copy minilocus integrant indicates that efficient DSB formation at the accessible RSS 3' of Ddelta3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.

Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells.

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.

An enhancer-blocking element between alpha and delta gene segments within the human T cell receptor alpha/delta locus.

T cell receptor (TCR) alpha and delta gene segments are organized within a single genetic locus but are differentially regulated during T cell development. An enhancer-blocking element (BEAD-1, for blocking element alpha/delta 1) was localized to a 2.0-kb region 3' of TCR delta gene segments and 5' of TCR alpha joining gene segments within this locus. BEAD-1 blocked the ability of the TCR delta enhancer (Edelta) to activate a promoter when located between the two in a chromatin-integrated construct. We propose that BEAD-1 functions as a boundary that separates the TCR alpha/delta locus into distinct regulatory domains controlled by Edelta and the TCR alpha enhancer, and that it prevents Edelta from opening the chromatin of the TCR alpha joining gene segments for VDJ recombination at an early stage of T cell development.

T cell receptor alpha gene rearrangement and transcription in adult thymic gamma delta cells.

T cells belong to two separate lineages based on surface expression of alpha beta or gamma delta T cell receptors (TCR). Since during thymus development TCR beta, gamma, and delta genes rearrange before alpha genes, and gamma delta cells appear earlier than alpha beta cells, it has been assumed that gamma delta cells are devoid of TCR alpha rearrangements. We show here that this is not the case, since mature adult, but not fetal, thymic gamma delta cells undergo VJ alpha rearrangements more frequently than immature alpha beta lineage thymic precursors. Sequence analysis shows VJ alpha rearrangements in gamma delta cells to be mostly (70%) nonproductive. Furthermore, VJ alpha rearrangements in gamma delta cells are transcribed normally and, as shown by analysis of TCR beta-/- mice, occur independently of productive VDJ beta rearrangements. These data are interpreted in the context of a model in which precursors of alpha beta and gamma delta cells differ in their ability to express a functional pre-TCR complex.

Junctional diversity in signal joints from T cell receptor beta and delta loci via terminal deoxynucleotidyl transferase and exonucleolytic activity.

The site-specific V(D)J recombination reaction necessary to assemble the genes coding for immunoglobulin (Ig) and T cell receptor (TCR) variable regions is initiated by a precise double strand cut at the border of the recombination signals flanking the genes. Extensive processing of the coding ends before their ligation accounts for most of the Ig and TCR repertoire diversity. This processing includes both base additions to and loss from the coding ends. On the other hand, it has generally been thought that signal ends are not modified before they are fused, and that signal joints consist of a perfect head-to-head ligation of the recombination signals. In this study, we analyzed signal joints created during the rearrangement of different TCR-beta and TCR-delta genes in thymocytes. We show that a significant fraction (up to 24%) of these signal joints exhibits junctional diversity. This diversity results from N nucleotide additions for TCR-beta signal joints, and from N additions and exonucleolytic digestion for TCR-delta joints. Altogether, our findings suggest that: (a) signal ends can undergo some of the same modifications as coding ends, (b) inversional rearrangement generates more diversity than deletional events, and (c) fine differences exist in the recombinase/DNA complexes formed at each rearranging locus.

Detection of T-cell receptor delta gene rearrangement in T-cell malignancies by clonal specific polymerase chain reaction and its application to detect minimal residual disease.

A clonal-specific polymerase chain reaction technique to detect T-cell receptor delta gene rearrangement in acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) was evaluated. It was applied to detect minimal residual disease. A sensitive and specific technique to detect minimal residual disease for T-cell malignancies was explored. Southern analysis and polymerase chain reaction (PCR) were used to detect the rearranged V-D-J segment of T-cell receptor delta (TCR delta) gene from malignant cell specimens of patients with leukemia and lymphoma of T-cell lineage. The PCR product was sequenced and from the DNA sequences of the V-D-J region, a 3' anti-sense primer was designed and synthesized for clonal specific PCR (CS-PCR). T-cell receptor delta (TCR-delta) gene rearrangement was studied in 40 cases of acute leukaemia and lymphoma of T-cell lineage at diagnosis. Using Southern analysis, the positive rates were 28 and 32% for the 18 T-lymphoma and 22 T-ALL, respectively. A one stage Polymerase Chain Reaction (PCR) technique was used to detect the rearrangement in Southern positive cases and the PCR positive rates were 80 and 86%, respectively. The PCR technique had a sensitivity of 0.1%. Serial follow-up marrow specimens were available from 4 T-ALL patients following chemotherapy for monitoring of minimal residual disease. Their PCR products were DNA sequenced. A 3' primer was designed for each case for a clonal specific (CS) PCR. The technique had a sensitivity of 0.003%. It was applied to detect minimal residual disease in serial follow-up marrow samples. The first patient had persistent negative CS-PCR results and enjoyed continuous remission for more than 3 years. The second patient with negative one stage PCR but positive CS-PCR results had eventual relapse of leukaemia. The other two patients never achieved a morphological remission. These preliminary results appeared to support the usefulness of these PCR techniques in detecting minimal residual disease and predicting relapses for ALL. However, further clinical correlation in larger populations of patients is necessary.

Pattern of T-cell receptor delta gene rearrangement by Southern blotting and polymerase chain reaction technique in Hong Kong Chinese patients with non-T-cell hematological malignancies.

It has been recognized that clonal T-cell receptor delta (TCR delta) gene rearrangement is present in both T-and B-cell malignancies. The highly sensitive polymerase chain reaction (PCR) technique may be applicable to cases of leukemia and lymphoma of non-T-cell origin for detection of minimal residual disease (MRD). A PCR technique was used in this study to investigate the pattern of clonal TCR delta gene rearrangement in Hong Kong Chinese patients with non-T-cell hematological malignancies. Seventy-three patients with the diagnosis of acute leukemia and non-Hodgkin's lymphoma of non-T-cell origin were included in this study. There were 20 patients with common ALL (cALL), seven precursal B-cell ALL (PreB-ALL), 23 acute myeloid leukemia (AML), and 23 non-Hodgkin's lymphoma of B-lineage (B-NHL). Clonal rearrangement was detectable by Southern analysis using a J delta 1 probe in 41 per cent of ALL of B-lineage but in none of the B-NHL or AML. The samples were also studied further by monoclonal PCR amplification for TCR delta gene rearrangement. Three different sets of primers were employed to detect clonal rearrangement of the TCR delta gene. The V delta 1(D)J delta 1 recombination typically seen in T-cell malignancies were not seen in any of the of the non-T-cell malignancies. The V delta 2 (D) D delta 3 recombination was found exclusively in ALL of B-lineage and was seen in 73 per cent of the Southern positive cases. Although clonal TCR delta gene rearrangement was undetectable by Southern analysis in our AML cases, 26 per cent had a V delta 2(D)J delta 1 recombination found by the PCR technique. Sensitivity of the PCR technique was determined by serial mixing and was up to 5-10 leukemic cells per 10(4) nucleated cells. It was apparent from this study that it was feasible to detect clonal TCR delta gene rearrangement by the PCR technique in a proportion of the cases of non-T-cell hematological malignancies. The PCR technique can be applied to detect residual leukemic cells in marrow of patients in an apparent morphological complete remission. The value of this application requires further clinical evaluation and correlation.

A DNA binding protein in human thymocytes recognizes the T cell receptor-delta-deleting element psi J alpha.

The TCR is a heterodimeric molecule composed of either gamma delta or alpha beta chains. The differentiation mechanisms that force thymocytes into the gamma delta alpha beta lineage are poorly understood, but rearrangement processes in the TCR-delta alpha locus are likely to play an important role. The TCR-delta gene complex is flanked by the delta-deleting elements delta Rec and psi J alpha, which are assumed to delete the TCR-delta gene before V alpha-J alpha rearrangement. The nonproductive delta Rec-psi J alpha recombination occurs at high frequency in both fetal and postnatal immature thymocytes. To find DNA binding proteins involved in the delta Rec-psi J alpha preferential rearrangement, we performed electrophoretic mobility shift assays using the recombination signal sequence of psi J alpha with additional upstream and downstream sequences. We observed a 180-kDa DNA binding protein in nuclear extracts from human thymocytes that recognized a 46-bp binding site on the psi J alpha gene segment, containing the core motif GTTAATAGG. The psi J alpha binding protein, which we call PJA-BP, was also detected in immature CD3-T cell lines with TCR-delta genes deleted on both alleles, in a TCR-alpha beta+ cell line, and in two of four myeloid cell lines. This protein was absent in a TCR-gamma delta+ T cell line, in nonhemopoietic cell lines, and in all but one B cell lines tested. Although we could detect binding activity of the PJA-BP to some other TCR-J alpha gene segments, we postulate that binding of PJA-BP to the psi J alpha gene segment is one of the factors involved in the preferential delta Rec-psi J alpha gene rearrangement process.

Role of T cell receptor delta gene in susceptibility to celiac disease.

There is a strong genetic influence on the susceptibility to celiac disease. Although in the vast majority of patients with celiac disease, the HLA-DQ(alpha1*0501, beta1*0201) heterodimer encoded by the alleles HLA-DQA1*0501 and HLA-DQB1*0201 seems to confer the primary disease susceptibility, it cannot be excluded that other genes contribute to disease susceptibility, as indicated by the difference in concordance rates between monozygotic twins and HLA identical siblings (70% vs. 30%). Obviously other genes involved in the genetic control of T cell mediated immune response could potentially influence susceptibility to celiac disease. The density of T cells using the gammadelta T cell receptor (TCR) is considerably increased in the jejunal epithelium of patients with celiac disease, an abnormality considered to be specific for celiac disease. This suggests an involvement of gammadelta T cells in the pathogenesis of the disease. To ascertain whether the TCR delta (TCRD) gene contributes to celiac disease susceptibility we carried out an association study and genetic linkage analysis using a highly polymorphic microsatellite marker at the TCRD locus on chromosome 14q11.2. The association study demonstrated no significant difference in allele frequencies of the TCRD gene marker between celiac disease patients and controls; accordingly, the relative risk estimates did not reach the level of statistical significance. In the linkage analysis, performed in 23 families, the logarithm of the odds (LOD) scores calculated for celiac disease versus the TCRD gene marker excluded linkage, suggesting that there is no determinant contributing to celiac disease status at or 5 cM distant to the analyzed TCRD gene marker. In conclusion, the results of the present study provide no evidence that the analyzed TCRD gene contributes substantially to celiac disease susceptibility.

Regulation of T cell receptor delta gene rearrangement by c-Myb.

Developmental activation of VDJ recombination at the T cell receptor (TCR) delta locus is controlled by an intronic transcriptional enhancer (E delta). Transcriptional activation by E delta is dependent on c-Myb. To determine whether c-Myb plays a role in the activation of TCR-delta gene rearrangement, we compared VDJ recombination in transgenic mice carrying two versions of a human TCR-delta gene minilocus recombination substrate. One includes a wild-type E delta, whereas the other carries an E delta with a mutation that abrogates c-Myb binding. We demonstrate that an intact Myb binding site is necessary for efficient rearrangement of the minilocus substrate, suggesting that c-Myb plays a crucial role in activating VDJ recombination at the endogenous TCR-delta locus.

Alpha beta and gamma delta T cells can share a late common precursor.

The subdivision of T cells into alpha beta and gamma delta subtypes is conserved throughout vertebrate development. The respective alpha beta and gamma delta T-cell receptors (TCRs) are encoded by somatically rearranged genes. There has been broad speculation as to whether an individual thymocyte can become either a gamma delta T cell or an alpha beta T cell as a result of stochastic gene rearrangement processes, or whether the two types of T cell are derived from separate lineages. Many of the experimental findings are apparently conflicting, however, and the issue--a basic one in immunology and development--remains unresolved. To address this issue, we have used the recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, which allows us to examine quantitatively the status of TCR gamma and delta genes in postnatal alpha beta T cells and their progenitors. Interestingly, such cells are depleted of productively rearranged delta and gamma genes, which can encode delta and gamma TCR polypeptide chains. However, in mice that can rearrange TCR delta gene segments, but in which the TCR delta gene is non-functional in other respects, no such depletion of productive rearrangements is seen. The quantitative data that we have obtained fulfill the predictions of the stochastic hypothesis: that is, a progenitor T cell first attempts to become a gamma delta T cell and, if unsuccessful, then attempts to become an alpha beta T cell. Thus, alpha beta and gamma delta T cells can derive from a common precursor thymocyte. In the simplest case, therefore, lineage-determining factors are the successful rearrangement of both gamma and delta genes before TCR alpha gene rearrangements occur, which lead to deletion of the TCR delta locus and thereby preclude further gamma delta T-cell differentiation. In contrast, successful rearrangement of the TCR beta locus remains compatible with cells becoming either gamma delta or alpha beta T cells.

Germ-line transcription of the T cell receptor delta gene in mouse hematopoietic cell lines.

T cell receptor (TCR) genes are initially activated in hematopoietic stem cells that have been committed to the T cell lineage during ontogeny. We have established cell lines derived from hematopoietic organs which express truncated TCR delta mRNA (1.3 kbp). In this study, cDNA clones of this mRNA were analyzed to characterize TCR delta gene activation in early T cell development. Transcription started from an intron between D delta 2 and J delta 1, 72 bp upstream from J delta 1, and continued to J delta 1 and C delta exons, which were directly spliced to J delta 1. Thus, the truncated mRNA expressed by the cell lines was determined to be a germ-line transcript. The promoter activity of the DNA fragment between D delta 2 and J delta was assessed by its ability to drive the transcription of a reporter gene linked to it in the cell line expressing the TCR delta germ-line transcript. TCR delta germ-line transcription was found not only in these cell lines but also in fetal liver and thymus cells. These findings indicate that the TCR delta germ-line transcription is a naturally occurring event in developing T cells. The significance of germ-line transcription of TCR delta genes is unknown, but it may be an event that follows T-lineage commitment or, at least, may be closely associated with this commitment.

C-Myb and core-binding factor/PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor delta enhancer.

A T-cell-specific transcriptional enhancer lies within the J delta 3-C delta intron of the human T-cell receptor delta gene. We have previously shown that a 30-bp element, denoted delta E3, acts as the minimal TCR delta enhancer and that within delta E3, adjacent and precisely spaced binding sites for core-binding factor (CBF/PEBP2) and c-Myb are essential for transcriptional activity. These data suggested that CBF/PEBP2 and c-Myb synergize to mediate transcriptional activity but did not establish the molecular basis for synergy. In this study, we have examined in detail the binding of CBF/PEBP2 and c-Myb to delta E3. We found that CBF/PEBP2 and c-Myb could simultaneously occupy the core site and one of two overlapping Myb sites within delta E3. However, equilibrium binding and kinetic dissociation experiments suggest that the two factors bind to delta E3 independently, rather than cooperatively. This was found to be true by using isoforms of these factors present in extracts of transfected COS-7 cells, as well as the natural factors present in nuclear extracts of the Jurkat T-cell line. We further showed that CBF/PEBP2 and c-Myb provide unique transactivation functions, since the core-Myb combination cannot be substituted by dimerized core or Myb sites. We propose that spatially precise synergy between CBF/PEBP2 and c-Myb may result from the ability of the two factors to form a composite surface that makes unique and stereospecific contacts with one or more additional components of the transcriptional machinery.

Heterogeneity of the T-Cell Receptor 8 Gene Indicating Subclone Formation in Acute Precursor B-Cell Leukemias

Precursor B-cell acute lymphoblastic leukemias (B-ALLs) have been shown to be oligoclonal at the Ig heavy-chain (IgH) gene level in up to 40% of cases by Southern blot hybridization. In contrast, oligoclonality as deduced from diversity of T-cell receptor (TcR)-delta gene rearrangements of the immature types (ie, V delta 2-D delta 3, D delta 2-D delta 3) has not been reported, so far. We detected oligoclonality characterized by the coexistence of different junctional regions of identical V delta 2-D delta 3 rearrangements in four childhood precursor B-ALLs. No variation was found in the IgH gene status. Therefore, we define these populations as subclones. Two leukemias displayed the variants in an unequal proportion. In the other two leukemias, for which similar quantities of the coexisting rearrangements were detected, single cell-nuclei polymerase chain reaction (PCR) showed two separate leukemic populations. Subclone formation could not be demonstrated by Southern blot hybridization, but was detectable after PCR amplification of the V delta 2-D delta 3 rearrangement and separation by polyacrylamide gel electrophoresis. The variants arose independently from each other, as deduced from their individual sequences. Using subclone-specific oligonucleotides for hybridization to amplified DNA obtained at diagnosis and during follow-up from bone marrow samples, we demonstrate, (1) specificity of all subclone-deduced probes, (2) that one residual leukemic cell can be detected in 10(4) to 10(5) normal mononuclear cells in a semiquantitative assay, and (3) that none of the subclones persisted after induction therapy. We propose that in a leukemic cell population, TcR-delta gene diversity arises after rearrangements of the IgH genes resulting in apparent clonality at the IgH gene level. However, cells are oligoclonal, if the TcR-delta gene rearrangements are considered. As various subclones may respond differently to chemotherapy, they may hamper the detection of minimal residual disease. Therefore, we use all subclone-specific oligonucleotides for hybridization to amplified DNA from follow-up samples.

Alternative Splicing Restricts Translation of Rearrangements at theT-Cell Receptor δ/α Locus

Lymphocytes expressing alpha beta or gamma delta T-cell receptors (TCR) represent distinct T-cell populations. Because TCR delta genes lie within the TCR alpha locus, the rearrangement processes, transcription, and translation of TCR delta or TCR alpha variable domain exons require tight regulation. Human precursor B-cell leukemias (eg, the REH cell line) constitute an interesting model to study TCR delta/alpha recombination because they rearrange TCR delta/alpha loci along a hierarchically ordered pathway in which V delta 2D delta 3 segments are joined to the J alpha cluster. We now show for REH cells that chimeric TCR delta/alpha variable domain exons are posttranscriptionally modified by alternative splicing resulting in truncated V delta 2C alpha transcripts. This process also takes place during thymic differentiation. CD7+/CD3- T-cell precursors exhibit V delta 2D delta 3 rearrangements. Further differentiation into CD7+/CD3+ thymocytes is associated with the expression of a truncated V delta 2C alpha RNA species. In contrast, chimeric TCR delta/alpha rearrangements containing a V delta 1 segment (but no D delta sequences) are predominantly expressed as full-length V delta 1J alpha C alpha transcripts. These data suggest that alternative splicing constitutes a mechanism that restricts the production of distinct chimeric TCR alpha chains.

Association and linkage study of T-cell receptor delta gene in celiac disease

Association and linkage study of T-cell receptor delta gene in celiac disease

TCR delta gene rearrangements in acute myeloid leukemia with T-lymphoid antigen expression.

In this review we present our data concerning T-cell receptor (TCR) delta gene rearrangements in acute myeloid leukemia with coexpression of T-lymphoid features (CD2/CD4/CD7; Ly+ AML). We found a correlation between TCR delta gene rearrangements and coexpression of these T-lymphoid features. Ten of 66 Ly+ AML and only one of 44 AML cases without this coexpression exhibited TCR delta gene rearrangements (p = .028). In contrast, no correlation was observed between terminal deoxynucleotidyl transferase (TdT) expression and the occurrence of TCR delta gene rearrangements in AML. Rearrangements were found in two of 25 AML with and seven of 71 AML cases without TdT expression. Interestingly, nucleotide sequencing of junctional sites revealed up to 36 N-nucleotides in cases without or with only weak TdT expression indicating downregulation of TdT expression after the TCR rearrangement took place. Complete V delta 1J delta 1 and incomplete D delta 2J delta 1 gene rearrangements were observed most frequently in Ly+ AML. These recombination patterns were similar to patterns observed in acute T-lymphoblastic leukemia with coexpression of myeloid features (My+ T-ALL) suggesting transformation of a common myeloid/T-lymphoid progenitor cell in these cases.

Response of a Murine Epidermal Vγ1/Vδ6-TCR+Hybridoma to Heat Shock Protein HSP-60

In the epidermis, the major population of T lymphocytes expresses a T-cell receptor (TCR) with V gamma 5 and V delta 1 variable regions, which is unique to this tissue. Roberts et al and Ezquerra et al, also describe a minor population of gamma delta-TCR+ cells in the epidermis that expresses a V gamma 1/V delta 6 TCR. These cells are different from other epidermal T cells in that they "spontaneously" produce cytokines, a result thought to be due to autoreactivity. Over the past 5 years, our laboratory has produced V gamma 1/V delta 6+ T-cell hybridomas from many tissue sources. These spontaneously produce cytokines but also are activated by heat shock protein (HSP-60)-derived peptides. Ezquerra et al report that none of their V gamma 1/V delta 6+ epidermal T-cell lines derived from C3H/HeN mice respond to HSP-60. Of the 99 gamma delta-TCR+ hybridomas we have produced from epidermal T cells of C57BL/6 mice, only one expressed the V gamma 1/V delta 6 TCR. This hybridoma, 70BET-2.12, not only spontaneously produces cytokines, but, unlike the V gamma 1/V delta 6-TCR+ epidermal T cells of Ezquerra et al, it also responds to the whole HSP-60 protein and a 17-mer HSP-60 peptide from M. leprae, producing increased levels of interleukin-2 of up to approximately ten-fold above the spontaneous level. This shows that V gamma 1/V delta 6-TCR+ epidermal T cells can respond to HSP-60. To confirm that 70BET-2.12 expresses TCR genes similar to those of cells that have HSP-60 reactivity, V gamma 1-C gamma 4 and V delta 6-C delta cDNA were produced from RNA isolated from this hybridoma, amplified by the polymerase chain reaction, and sequenced. The gamma and delta TCR gene sequences were similar but not identical to previously published sequences of HSP-60-reactive cells from lymphoid and other organs. No explanation can be found for the discrepancy between our findings and those of others at the level of TCR expression such that other strain-specific factors might be responsible for HSP-60 reactivity.

Detection of clonality by polymerase chain reaction in childhood B-lineage acute lymphoblastic leukemia.

DNA-based PCR with various sets of primers for TCR gamma/delta, and Ig heavy chain (IgH) genes were used to study clonality in childhood B-lineage acute lymphoblastic leukemia. Amplification of the IgH CDR-III was observed in 75 of 120 analyzed cases (62.5%). From all analyzed groups, the IgH gene rearrangement was most often observed in pre-B ALL (85.7%) and was rather rare in null-ALL (34.5%). TCR delta gene rearrangement was the most common, and was observed in 77 patients (64.2%). The typical pattern of rearrangements was defined as an incomplete V delta 2 to D delta 3, V delta 2 to D delta 2, or D delta 3 to D delta 2 recombination product. Rearrangements of TCR gamma gene we observed in 61 cases (50.8%). TCR gamma gene rearrangements were detected predominantly in null-ALL and early B-ALL (55.2% and 60%, respectively) and were rather rare in other groups. Of all eight V segments of V gamma I group, the most frequent gene usage concerns regions V gamma 2, V gamma 4, and psi V gamma 7. We have confirmed that IgH gene amplification, together with TCR gamma and delta gene amplification, provides a rapid, sensitive approach to assessing clonality in ALL almost in 100% of cases.

Allelic differences in TCR gamma-chains alter gamma delta T cell antigen reactivity.

Mouse gamma delta T cell hybridomas from various strains that express a TCR-V gamma 1/V delta 6 respond weakly to an autologous Ag and more strongly to a short segment of the mycobacterial heat shock protein-60 (HSP-60). However, V gamma 1/V delta 6 hybridomas derived from AKR mice show greatly reduced or absent responses to these stimuli. To determine whether the lack of response in these AKR hybridomas is caused by polymorphisms found in the expressed AKR gamma and TCR-delta genes or, instead, stems from other genes in AKR, we crossed an AKR mouse with a responder mouse, C57BL/10 (B10), and prepared hybridomas from F1 progeny. Expression of an AKR V gamma 1-J gamma 4-C gamma 4 gene correlated with nonresponsiveness, whereas expression of a B10 V gamma 1-J gamma 4-C gamma 4 gene in most hybridomas ensured responses to both self Ag and the HSP-60 peptide. An allelic difference in the expressed V gamma 6 gene was irrelevant to these responses. Moreover, transfection of a functional B10 V gamma 1-J gamma 4-C gamma 4 gene into an F1 hybridoma variant that had lost the AKR version of this gene restored responses. The allelic gamma gene products differ at nine amino acids in the V region, and three amino acids in the C region. In addition, the AKR C gamma 4 region contains a 16-amino acid insertion. We propose that amino acid differences among those encoded by the AKR V gamma 1-J gamma 4-C gamma 4 gene are responsible for the lack of response, and reduce the ability of the TCR-gamma delta to bind the relevant Ag.