Lack of common TCRA and TCRB clonotypes in CD8(+)/TCRαß(+) T-cell large granular lymphocyte leukemia: a review on the role of antigenic selection in the immunopathogenesis of CD8(+) T-LGL.

Clonal CD8(+)/T-cell receptor (TCR)αß(+) T-cell large granular lymphocyte (T-LGL) proliferations constitute the most common subtype of T-LGL leukemia. Although the etiology of T-LGL leukemia is largely unknown, it has been hypothesized that chronic antigenic stimulation contributes to the pathogenesis of this disorder. In the present study, we explored the association between expanded TCR-Vß and TCR-Vα clonotypes in a cohort of 26 CD8(+)/TCRαß(+) T-LGL leukemia patients, in conjunction with the HLA-ABC genotype, to find indications for common antigenic stimuli. In addition, we applied purpose-built sophisticated computational tools for an in-depth evaluation of clustering of TCRß (TCRB) complementarity determining region 3 (CDR3) amino-acid LGL clonotypes. We observed a lack of clear TCRA and TCRB CDR3 homology in CD8(+)/TCRαß(+) T-LGL, with only low level similarity between small numbers of cases. This is in strong contrast to the homology that is seen in CD4(+)/TCRαß(+) T-LGL and TCRγδ(+) T-LGL and thus underlines the idea that the LGL types have different etiopathogenesis. The heterogeneity of clonal CD8(+)/TCRαß(+) T-LGL proliferations might in fact suggest that multiple pathogens or autoantigens are involved.

A restricted clonal T-cell receptor αß repertoire in Sézary syndrome is indicative of superantigenic stimulation.

BACKGROUND: Sézary syndrome (SS) is a cutaneous T-cell lymphoma characterized by erythroderma, lymphadenopathy and malignant clonal T cells in the skin, lymph nodes and peripheral blood. A role for superantigens in the pathogenesis of SS has been postulated before. OBJECTIVES: To investigate a putative involvement of chronic (super-)antigenic stimulation in driving T-cell expansion in SS. METHODS: Antigenic specificity of the T-cell receptor (TCR) was assayed by molecular analysis of the TCRA (n=11) and TCRB (n=28) genes, followed by detailed in silico analysis. RESULTS: Sequence analysis of clonally rearranged TCRB genes showed over-representation of Vß8, Vß13, Vß17, Vß21 and Vß22, and under-representation of Vß2 and Jß1.1 when compared with healthy controls. No similarity was detected in amino acid motifs of the complementarity determining region 3 (CDR3). Analysis of TCRA rearrangements showed that there was no common Vα or Jα gene usage, and that TCRA CDR3 amino acid motifs were not highly similar. CONCLUSIONS: The lack of clear stereotypic TCRA and TCRB CDR3 amino acid motifs would argue against involvement of a single common antigen in the pathogenesis of SS. Nevertheless, the skewing of Vß and Jß gene usage does seem to point to a restricted TCR repertoire, possibly as a result of superantigenic selection prior to neoplastic transformation.

Human T-cell lines with well-defined T-cell receptor gene rearrangements as controls for the BIOMED-2 multiplex polymerase chain reaction tubes.

The BIOMED-2 multiplex polymerase chain reaction (PCR) tubes for analysis of immunoglobulin and T-cell receptor (TCR) gene rearrangements have recently been introduced as a reliable and easy tool for clonality diagnostics in suspected lymphoproliferations. Quality and performance assessment of PCR-based clonality diagnostics is generally performed using human leukemia/lymphoma cell lines as controls. We evaluated the utility of 30 well-defined human T-cell lines for quality performance testing of the BIOMED-2 PCR primers and protocols. The PCR analyses of the TCR loci were backed up by Southern blot analysis. The clonal TCRB, TCRG and TCRD gene rearrangements were analyzed for gene segment usage and for the size and composition of their junctional regions. In 29 out of 30 cell lines, unique clonal TCR gene rearrangements could be easily detected. Besides their usefulness in molecular clonality diagnostics, these cell lines can now be authenticated based on their TCR gene rearrangement profile. This enables their correct use in molecular clonality diagnostics and in other cancer research studies.

Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies.

The BIOMED-2 Concerted Action BMH4-CT98-3936 on 'Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders' developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB). As no comparable TCRB PCR method pre-existed and only a limited number of samples was tested within the BIOMED-2 study, we initiated this study for further validation of the newly developed TCRB PCR approach by comparing PCR data with previously generated Southern blot (SB) data in a series of 66 immature (ALL) and 36 mature T-cell malignancies. In 91% of cases, concordant PCR and SB results were found. Discrepancies consisted of either failure to detect SB-detected TCRB rearrangements by PCR (6.5%) or detection of an additional non-SB defined rearrangement (2.5%). In 99% of cases (99/100), at least one clonal TCRB rearrangement was detected by PCR in the SB-positive cases. A predominance of complete Vbeta-Jbeta rearrangements was seen in TCRalphabeta(+) T-cell malignancies and CD3-negative T-ALL (100 and 90%, respectively), whereas in TCRgammadelta(+) T-ALL, more incomplete Dbeta-Jbeta TCRB rearrangements were detected (73%). Our results underline the reliability of this new TCRB PCR method and its strategic applicability in clonality diagnostics of lymphoproliferative disorders and MRD studies.

Basic helix-loop-helix proteins E2A and HEB induce immature T-cell receptor rearrangements in nonlymphoid cells.

T-cell receptor (TCR) gene rearrangements are mediated via V(D)J recombination, which is strictly regulated during lymphoid differentiation, most probably through the action of specific transcription factors. Investigated was whether cotransfection of RAG1 and RAG2 genes in combination with lymphoid transcription factors can induce TCR gene rearrangements in nonlymphoid human cells. Transfection experiments showed that basic helix-loop-helix transcription factors E2A and HEB induce rearrangements in the TCRD locus (Ddelta2-Ddelta3 and Vdelta2-Ddelta3) and TCRG locus (psi Vgamma7-Jgamma2.3 and Vgamma8-Jgamma2.3). Analysis of these rearrangements and their circular excision products revealed some peculiar characteristics. The Vdelta2-Ddelta3 rearrangements were formed by direct coupling without intermediate Ddelta2 gene segment usage, and most Ddelta2-Ddelta3 recombinations occurred via direct coupling of the respective upstream and downstream recombination signal sequences (RSSs) with deletion of the Ddelta2 and Ddelta3 coding sequences. Subsequently, the E2A/HEB-induced TCR gene recombination patterns were compared with those in early thymocytes and acute lymphoblastic leukemias of T- and B-lineage origin, and it was found that the TCR rearrangements in the transfectants were early (immature) and not necessarily T-lineage specific. Apparently, some parts of the TCRD (Vdelta2-Ddelta region) and TCRG genes are accessible for recombination not only in T cells, but also in early B-cells and even in nonlymphoid cells if the appropriate transcription factors are present. The transfection system described here appeared to be useful for studying the accessibility of immunoglobulin and TCR genes for V(D)J recombination, but might also be applied to study the induction of RSS-mediated chromosome aberrations.

The role of molecular analysis of immunoglobulin and T cell receptor gene rearrangements in the diagnosis of lymphoproliferative disorders.

AIMS: To investigate whether the analysis of immunoglobulin (Ig)/T cell receptor (TCR) rearrangements is useful in the diagnosis of lymphoproliferative disorders. METHODS: In a series of 107 consecutive cases with initial suspicion of non-Hodgkin's lymphoma (NHL), Southern blot (SB) analysis of Ig/TCR rearrangements was performed. RESULTS: In 98 of 100 histopathologically conclusive cases, Ig/TCR gene results were concordant. In one presumed diffuse large B cell lymphoma (DLCL) and one follicular lymphoma (FL) case no clonality could be detected by SB analysis, or by polymerase chain reaction (PCR) at second stage. In the DLCL, sampling error might have occurred; the FL was revised after an initial diagnosis of reactivity. In many of the histopathologically inconclusive cases Ig/TCR gene SB analysis was helpful, giving support for the histopathological suspicion. However, because of a lack of (clinical) follow up data this could not be confirmed in a few cases. CONCLUSIONS: Experienced haematopathologists or a pathologist panel can diagnose malignant versus reactive lesions in most cases without the need for Ig/TCR gene analysis and can select the 5-10% of cases that might benefit from molecular clonality studies.

Molecular and flow cytometric analysis of the Vbeta repertoire for clonality assessment in mature TCRalphabeta T-cell proliferations.

Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)-PCR analysis of Vbeta-Cbeta transcripts and flow cytometry with a Vbeta antibody panel covering more than 65% of Vbeta domains was validated using 28 SB-defined clonal T-cell receptor (TCR)alphabeta(+) T-ALL samples and T-cell lines. Next, the diagnostic applicability of the V(beta) RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vbeta-Cbeta RT-PCR products were detected in all 47 samples, whereas single Vbeta domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vbeta monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vbeta gene segments not covered by the applied Vbeta monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the "clonal" character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vbeta-Cbeta products and single Vbeta domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular Vbeta analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vbeta antibody studies can be used as a powerful screening method for the detection of single Vbeta domain expression, followed by molecular studies in patients with more than 20% single Vbeta domain expression or large suspect T-cell populations (more than 50%-60%) without Vbeta reactivity.

Flow cytometric analysis of the Vbeta repertoire in healthy controls.

BACKGROUND: Analysis of the T-cell receptor (TCR)-Vbeta repertoire has been used for studying selective T-cell responses in autoimmune disease, alloreactivity in transplantation, and protective immunity against microbial and tumor antigens. For the interpretation of these studies, we need information about the Vbeta repertoire usage in healthy individuals. METHODS: We analyzed blood T-lymphocyte (sub)populations of 36 healthy controls (age range: from neonates to 86 years) with a carefully selected most complete panel of 22 Vbeta monoclonal antibodies, which together recognized 70-75% of all blood TCRalphabeta(+) T lymphocytes. Subsequently, we developed a six-tube test kit with selected Vbeta antibody combinations for easy and rapid detection of single ("clonal") Vbeta domain usage in large T-cell expansions. RESULTS: The mean values of the Vbeta repertoire usage were stable during aging in blood TCRalphabeta(+) T lymphocytes as well as in the CD4(+) and CD8(+) T-cell subsets, although the standard deviations increased in the elderly. The increased standard deviations were caused by the occurrence of oligoclonal T-cell expansions in the elderly, mainly consisting of CD8(+) T lymphocytes. The 15 detected T-cell expansions did not reach 40% of total TCRalphabeta(+) T lymphocytes and represented less than 0.4 x 10(9) cells per liter in our study. Vbeta usage of the CD4(+) and CD8(+) subsets was comparable for most tested Vbeta domains, but significant differences (P < 0.01) between the two subsets were found for Vbeta2, Vbeta5.1, Vbeta6.7, Vbeta9.1, and Vbeta22 (higher in CD4(+)), as well as for Vbeta1, Vbeta7.1, Vbeta14, and Vbeta23 (higher in CD8(+)). Finally, single Vbeta domain expression in large T-cell expansions can indeed be detected by the six-tube test kit. CONCLUSIONS: The results of our study can now be used as reference values in studies on distortions of the Vbeta repertoire in disease states. The six-tube test kit can be used for detection of single Vbeta domain expression in large T-cell expansions (>2.0 x 10(9)/l), which are clinically suspicious of T-cell leukemia.

T cell receptor gamma (TCRG) gene rearrangements in T cell acute lymphoblastic leukemia refelct 'end-stage' recombinations: implications for minimal residual disease monitoring.

The T cell receptor gamma (TCRG) gene configuration was established in a large series of 126 T cell acute lymphoblastic leukemia (T-ALL) patients using combined Southern blotting (SB) and heteroduplex PCR analyses. The vast majority of TALL (96%) displayed clonal TCRG gene rearrangements, with biallelic recombination in 91% of patients. A small immature subgroup of CD3- T-ALL (n = 5) had both TCRG genes in germline configuration, three of them having also germline TCRD genes. In five patients (4%) combined SB and PCR results indicated oligoclonality. In five rearrangements detected by SB, the Vgamma gene segment could not be identified suggesting illegitimate recombination. Altogether, 83% of TCRG gene rearrangements involved either the most upstream Vgamma2 gene (including four cases with interstitial deletion of 170 bp in Vgamma2) and/or the most downstream Jgamma2.3 segment, which can be perceived as 'end-stage' recombinations. Comparative analysis of the TCRG gene configuration in the major immunophenotypic subgroups indicated that TCRgammadelta+ T-ALL display a less mature immunogenotype as compared to TCRalphabeta+ and most CD3- cases. This was reflected by a significantly increased usage of the more downstream Vgamma genes and the upstream Jgamma1 segments. Comparison between adult and pediatric T-ALL patients did not show any obvious differences in TCRG gene configuration. The high frequency, easy detectability, rare oligoclonality, and frequent 'end-stage' recombinations make TCRG gene rearrangements principal targets for PCR-based detection of minimal residual disease (MRD) in T-ALL. We propose a simple heteroduplex PCR strategy, applying five primer combinations, which results in the detection of approximately 95% of all clonal TCRG gene rearrangements in T-ALL. This approach enables identification of at least one TCRG target for MRD monitoring in 95% of patients, and even two targets in 84% of T-ALL.

Molecular features responsible for the absence of immunoglobulin heavy chain protein synthesis in an IgH(-) subgroup of multiple myeloma.

This study involved 12 patients with multiple myeloma (MM), in whom malignant plasma cells did not contain immunoglobulin heavy chain (IgH) protein chains. Southern blot analysis revealed monoallelic J(H) gene rearrangements in 10 patients, biallelic rearrangement in 1 patient, and biallelic deletion of the J(H) and C(micro) regions in 1 patient. Heteroduplex polymerase chain reaction analysis enabled the identification and sequencing of 9 clonal J(H) gene rearrangements. Only 4 of the joinings were complete V(H)-(D)-J(H) rearrangements, including 3 in-frame rearrangements with evidence of somatic hypermutation. Five rearrangements concerned incomplete D(H)-J(H) joinings, mainly associated with deletion of the other allele. Curiously, in at least 1 of these 5 cases the second allele seemed to be in germline configuration, whereas the in-frame V(kappa)-J(kappa) gene rearrangements contained somatic mutations. The configuration of the IGH genes was further investigated by use of C(H) probes. In 5 patients the rearrangements in the J(H) and C(H) regions were not concordant, probably caused by illegitimate IGH class switch recombination (chromosomal translocations to 14q32. 3). These data indicate that in many IgH(-) MM patients illegitimate IGH class switch rearrangement or illegitimate deletion of the functional V(H)-(D(H))-J(H) allele are responsible for IgH negativity. For example, the exclusive presence of D(H)-J(H) rearrangements in combination with mutated IGK genes can only be explained in terms of normal B-cell development, if the second (functional) IGH allele is deleted, which was probably the case in most patients. Therefore, defects at the DNA level are responsible for the lack of IgH protein production in most IgH(-) MM patients.

E2A and EBF act in synergy with the V(D)J recombinase to generate a diverse immunoglobulin repertoire in nonlymphoid cells.

Immunoglobulin (Ig) and T cell receptor (TCR) genes are assembled during lymphocyte maturation through site-specific V(D)J recombination events. Here we show that E2A proteins act in concert with RAG1 and RAG2 to activate Ig VK1J but not Iglambda VlambdaIII-Jlambda1 rearrangement in an embryonic kidney cell line. In contrast, EBF, but not E2A, promotes VlambdaIII-Jlambda1 recombination. Either E2A or EBF activate IgH DH4J recombination but not V(D)J rearrangement. The Ig coding joints are diverse, contain nucleotide deletions, and lack N nucleotide additions. IgK VJ recombination requires the presence of the E2A transactivation domains. These observations indicate that in nonlymphoid cells a diverse Ig repertoire can be generated by the mere expression of the V(D)J recombinase and a transcriptional regulator.

Easy detection of all T cell receptor gamma (TCRG) gene rearrangements by Southern blot analysis: recommendations for optimal results.

Southern blot analysis of T cell receptor (TCR) gene rearrangements has proven to be a helpful tool to establish clonality in T cell leukemias and lymphomas. To improve the detection of clonal TCR gamma (TCRG) gene rearrangements by Southern blot analysis, we designed four new Jgamma probes and determined the most optimal restriction enzymes to be used with these probes. Based on detailed analysis of the sequences as well as on hybridization experiments with the TCRGJ21 probe, the Jgamma1.2 and Jgamma2.1 downstream areas were found to be highly homologous, suggesting that during evolution the duplication of the Jgamma region was followed by deletion of the tentative Jgamma2.2 gene segment. Southern blot analysis of 51 T cell acute lymphoblastic leukemias (T-ALL) revealed that all TCRG gene rearrangements can be detected by use of the TCRGJ13 probe in EcoRI digests and the TCRGJ21 probe in PstI digests. Additional probes and digests allow a more precise identification of the exact type of TCRG gene rearrangements in the majority of cases. Almost 90% of the TCRG gene rearrangements in T-ALL involved the Jgamma2 region (16% Jgamma2.1 and 72% Jgamma2.3), whereas Jgamma1 region rearrangements were particularly found in TCRgammadelta+ T-ALL. This information has implications for design of primer sets for PCR analysis at diagnosis and for PCR target choice in detection of minimal residual disease during follow-up of T-ALL patients.

Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia.

Several frequently applied polymerase chain reaction strategies for analysis of immunoglobulin heavy-chain gene rearrangements were compared by analyzing 70 B-cell lymphoproliferative disorders and 24 reactive lymphoid lesions. Southern blot analysis was used as the "gold standard" for clonality assessment. For polymerase chain reaction analysis, primers directed against framework (FR) 3 (FR3-A and FR3-B), FR2, and FR1 of the variable gene segments and against joining gene segments of the immunoglobulin heavy-chain gene were used. Polymerase chain reaction products were analyzed by high-resolution fingerprinting analysis using radiolabeled nucleotides, gene scanning using fluorochrome-labeled primers, and heteroduplex analysis. All of the assays generated polyclonal patterns in the reactive tissues. The sensitivity in detecting monoclonality as defined by Southern blotting varied between 60% (heteroduplex analysis with FR3 primers) and 77% (high-resolution fingerprinting analysis with FR2 primers). Comparison of the three FR3 assays revealed that gene scanning had the highest sensitivity (69%), probably because it could detect small aberrant monoclonal amplicons. False-negative results were especially frequent in follicular center lymphoma (n = 20), but also in diffuse large B-cell lymphoma (n = 18), both renowned for having mutated variable gene segments, potentially leading to primer mismatching. For example, in follicular center lymphoma, the FR3, FR2, and FR1 region primer sets detected clonality in only 35 to 40, 65, and 50%, respectively. Combining these techniques, 17 (85%) of 20 follicular center lymphoma samples showed monoclonality. Therefore, especially in follicular center lymphoma, diffuse large B-cell lymphoma, and, to a lesser extent, in other lymphomas, multiple variable gene segment primer sets must be used for a reliable assessment of clonality. Our results also suggest that gene scanning is somewhat more sensitive than other read-out systems. Heteroduplex analysis, however, is a reliable alternative within a diagnostic setting, avoiding the use of radioactivity or expensive automated sequencing equipment and fluorochrome-labeled (oligo)nucleotides.

Detection of T cell receptor beta (TCRB) gene rearrangement patterns in T cell malignancies by Southern blot analysis.

Reliable detection of clonal T cell receptor beta (TCRB) gene rearrangements is essential for clonality assessment of suspect T cell proliferations. Since no appropriate Southern blot probes were available, we developed a new set of optimized TCRB gene probes. The TCRBJ1 and TCRBJ2 probes are positioned just downstream of the Jbeta1 and Jbeta2 gene segments, respectively, and can be used for detection of both incomplete Dbeta-Jbeta and complete Vbeta-Jbeta rearrangements, whereas the TCRBD1U and TCRBD2U probes upstream of the Dbeta segments can be used to confirm incomplete Dbeta-Jbeta rearrangements. Less frequently occurring Vbeta-Dbeta rearrangements can easily be detected via the downstream TCRBD1 and TCRBD2 probes. Although both EcoRI and HindIII are appropriate restriction enzymes to be used in combination with all these probes, false-positivity due to partial digestion of the EcoRI site in the Jbeta2-Cbeta intron has to be excluded via the TCRBC probe. Application of the TCRB probes in a large series of nearly 200 T cell malignancies revealed clonal rearrangements in all immature and mature TCR alphabeta+ T cell malignancies, in the vast majority of TCR gammadelta+ T cell acute lymphoblastic leukemias (T-ALL) (approximately 95%) and even in most CD3- T-ALL (approximately 80%). TCRB gene rearrangement patterns differed between the various categories of T cell malignancies. An increased frequency of complete Vbeta-Jbeta1 rearrangements was observed in TCR alphabeta+ T-ALL as compared to CD3- and TCR gammadelta+ T-ALL (33% vs 16% and 11%, respectively), and also incompletely rearranged V-D, D-D, or D-J alleles in the beta2 region occurred more frequently in TCR alphabeta+ cases than in CD3- and TCR gammadelta+ T-ALL (27% vs 15% and 18%, respectively). Furthermore, in comparison to TCR alphabeta+ T-ALL, less Vbeta-Jbeta1 and more Vbeta-Jbeta2 rearrangements were detected in mature TCR alphabeta+ T cell malignancies. The occurrence of the different types of TCRB rearrangement patterns has implications for PCR-based clonality assessment and for PCR-based detection of minimal residual disease via TCRB gene analysis. For instance, focussing on the beta2 region of T-ALL will allow detection of rearrangements in 70%, 75%, and 90% of CD3-, TCR gammadelta+, and TCR alphabeta+ cases, respectively. Therefore the here-described results will facilitate the design of the most appropriate primer sets for PCR analysis of TCRB gene rearrangements at the DNA level.

TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation.

Recent studies have identified several populations of progenitor cells in the human thymus. The hematopoietic precursor activity of these populations has been determined. The most primitive human thymocytes express high levels of CD34 and lack CD1a. These cells acquire CD1a and differentiate into CD4(+)CD8(+) through CD3(-)CD4(+)CD8(-) and CD3(-)CD4(+) CD8alpha+beta- intermediate populations. The status of gene rearrangements in the various TCR loci, in particular of TCRdelta and TCRgamma, has not been analyzed in detail. In the present study we have determined the status of TCR gene rearrangements of early human postnatal thymocyte subpopulations by Southern blot analysis. Our results indicate that TCRdelta rearrangements initiate in CD34(+)CD1a- cells preceding those in the TCRgamma and TCRbeta loci that commence in CD34(+)CD1a+ cells. Furthermore, we have examined at which cellular stage TCRbeta selection occurs in humans. We analyzed expression of cytoplasmic TCRbeta and cell-surface CD3 on thymocytes that lack a mature TCRalphabeta. In addition, we overexpressed a constitutive-active mutant of p56(lckF505) by retrovirus-mediated gene transfer in sequential stages of T-cell development and analyzed the effect in a fetal thymic organ culture system. Evidence is presented that TCRbeta selection in humans is initiated at the transition of the CD3(-)CD4(+)CD8(-) into the CD4(+)CD8alpha+beta- stage.

Immunophenotypic and immunogenotypic characteristics of TCRgammadelta+ T cell acute lymphoblastic leukemia.

Thirty T cell receptor (TCR)gammadelta+ T cell acute lymphoblastic leukemias (T-ALL) were analyzed for their immunophenotype, as well as for the rearrangements and junctional regions of the TCRG and TCRD genes. In 15 cases membrane expression of TCRgammadelta proteins could be studied extensively by flow cytometry with a new Vgamma/Vdelta antibody panel. Virtually all TCRgammadelta+ T-ALLs expressed TdT, CD2, CD3, CD5, CD6, and CD7, but they were heterogeneous in their CD1/CD4/CD8 immunophenotype. The majority expressed either CD4+/CD8- or CD4+/CD8+, whereas only 7/30 TCRgammadelta+ T-ALLs lacked both antigens. Despite heterogeneity in the rearranged TCRG and TCRD genes, we found preferential protein expression of VgammaI (21/30), Jgamma2.3 (19/30) and Cgamma2 (21/30) gene products in the TCRgammadelta+ T-ALL. Expressed TCRD genes were largely limited to Vdelta1-Jdelta1, except for six patients who expressed non-Vdelta1 TCRdelta chains (Vdelta2-Jdelta1, Vdelta2-Jdelta3, Vdelta3-Jdelta1, Vdelta6-Jdelta2, and two Valpha-Jdelta1). In spite of the relatively limited combinatorial repertoire of the TCRG and TCRD genes, the junctional region diversity of the expressed genes was extensive. The Vgamma/Vdelta antibody panel confirmed the predominant, but not exclusive, expression of VgammaI and Vdelta1 proteins. Importantly, not a single T-ALL expressed the common peripheral blood Vgamma9+/Vdelta2+ phenotype. These immunogenotypic and immunophenotypic characteristics represent excellent targets for flow cytometric and PCR-based detection of 'minimal residual disease' in all TCRgammadelta+ T-ALL. Comparison of non-Vdelta1+ TCRgammadelta T-ALLs with the more common Vdelta1+ type showed a trend towards a more mature immunogenotype in the former. Firstly, more complete TCRD rearrangements were identified on the non-expressed allele in the non-Vdelta1+ group (83% vs 43%); secondly, a higher frequency of 'end-stage' Jgamma2.3 gene rearrangements was found in non-Vdelta1 cases on both TCRG alleles (83% vs 66%); thirdly, a higher frequency of complete TCRB rearrangements was found in non-Vdelta1 cases (79% vs 50%).

Immunoglobulin and T cell receptor gene rearrangement patterns in acute lymphoblastic leukemia are less mature in adults than in children: implications for selection of PCR targets for detection of minimal residual disease.

In order to gain insight into immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements in adult acute lymphoblastic leukemia (ALL), we studied 48 adult patients: 26 with precursor-B-ALL and 22 with T-ALL. Southern blotting (SB) with multiple DNA probes for the IGH, IGK, TCRB, TCRG, TCRD and TAL1 loci revealed rearrangement patterns largely comparable to pediatric ALL, but several differences were found for precursor-B-ALL patients. Firstly, adult patients showed a lower level of oligoclonality in the IGH gene locus (five out of 26 patients; 19%) despite a comparable incidence of IGH gene rearrangements (24 out of 26 patients; 92%). Secondly, all detected IGK gene deletions (n = 12) concerned rearrangements of the kappa deleting element (Kde) to Vkappa gene segments, which represent two-thirds of the Kde rearrangements in pediatric precursor-B-ALL and only half of the Kde rearrangements in mature B cell leukemias. Thirdly, a striking predominance of immature Ddelta2-Ddelta3 cross-lineage recombinations was observed (seven out of 16 TCRD rearrangements; 44%), whereas more mature Vdelta2-Ddelta3 gene rearrangements occurred less frequently (six out of 16 TCRD rearrangements; 38% vs >70% in pediatric precursor-B-ALL). Together these data suggest that the Ig/TCR genotype of precursor-B-ALL is more immature and more stable in adults than in children. We also evaluated whether heteroduplex analysis of polymerase chain reaction (PCR) products of rearranged Ig and TCR genes can be used for identification of molecular targets for minimal residual disease (MRD) detection. Using five of the major gene targets (IGH, IGK, TCRG, TCRD and TAL1 deletion), we compared the SB data and heteroduplex PCR results. High concordance between the two methods ranging from 96 to 100% was found for IGK, TCRG and TAL1 genes. The concordance was lower for IGH (70%) and TCRD genes (90%), which may be explained by incomplete or 'atypical' rearrangements or by translocations detectable only by SB. Finally, the heteroduplex PCR data indicate, that MRD monitoring is possible in almost 90% of adult precursor-B-ALL and >95% of adult T-ALL patients.

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.

Human T cell leukemias with continuous V(D)J recombinase activity for TCR-delta gene deletion.

The so-called TCR-delta-deleting elements, deltaRec and psiJ alpha, flank the major part of the TCR-delta gene complex. By rearranging to each other, the deltaRec and psiJ alpha gene segments delete the TCR-delta gene complex and prepare the allele for subsequent TCR-alpha rearrangement. This intermediate rearrangement is thought to be a specific rearrangement event. In our studies on TCR-delta deletion mechanisms, we identified several T cell acute lymphoblastic leukemias (T-ALL) with continuous activity of the deltaRec-psiJ alpha rearrangement process. Extensive Southern blot, PCR, and sequencing analyses on the coding joints as well as the signal joints of the deltaRec-psiJ alpha rearrangements in these patients allowed us to prove that this continuous rearrangement activity occurred in the leukemic cells and that these cells, therefore, represent a polyclonal subpopulation within the otherwise monoclonal T-ALL. In additional studies, we also identified a T cell line (DND41) with continuous activity of the deltaRec-psiJ alpha rearrangement process. Our data suggest that the ongoing deltaRec-psiJ alpha gene rearrangements predominantly occur in T cells that cannot express a functional TCR-gammadelta, due to biallelic out-of-frame TCR-delta and/or TCR-gamma gene rearrangements. The described T-ALL and the T cell line can serve as an experimental model in further studies on the regulatory elements involved in the specific deletion of the TCR-delta gene complex.