[Show abstract][Hide abstract] ABSTRACT: The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ(-/-) xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.
Proceedings of the National Academy of Sciences 10/2015; DOI:10.1073/pnas.1519118112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Severe combined immunodeficiency (SCID) represents congenital disorders characterized by a deficiency of T cells caused by arrested development in the thymus. Yet the nature of these developmental blocks has remained elusive because of the difficulty of taking thymic biopsy specimens from affected children.
We sought to identify the stages of arrest in human T-cell development caused by various major types of SCID.
We performed transplantation of SCID CD34(+) bone marrow stem/progenitor cells into an optimized NSG xenograft mouse model, followed by detailed phenotypic and molecular characterization using flow cytometry, immunoglobulin and T-cell receptor spectratyping, and deep sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor δ (TRD) loci.
Arrests in T-cell development caused by mutations in IL-7 receptor α (IL7RA) and IL-2 receptor γ (IL2RG) were observed at the most immature thymocytes much earlier than expected based on gene expression profiling of human thymocyte subsets and studies with corresponding mouse mutants. T-cell receptor rearrangements were functionally required at the CD4(-)CD8(-)CD7(+)CD5(+) stage given the developmental block and extent of rearrangements in mice transplanted with Artemis-SCID cells. The xenograft model used is not informative for adenosine deaminase-SCID, whereas hypomorphic mutations lead to less severe arrests in development.
Transplanting CD34(+) stem cells from patients with SCID into a xenograft mouse model provides previously unattainable insight into human T-cell development and functionally identifies the arrest in thymic development caused by several SCID mutations.
The Journal of allergy and clinical immunology 10/2015; DOI:10.1016/j.jaci.2015.08.022 · 11.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clonal CD8(+)/T-cell receptor (TCR)αβ(+) 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-Vβ and TCR-Vα clonotypes in a cohort of 26 CD8(+)/TCRαβ(+) 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 TCRβ (TCRB) complementarity determining region 3 (CDR3) amino-acid LGL clonotypes. We observed a lack of clear TCRA and TCRB CDR3 homology in CD8(+)/TCRαβ(+) 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(+)/TCRαβ(+) T-LGL and TCRγδ(+) T-LGL and thus underlines the idea that the LGL types have different etiopathogenesis. The heterogeneity of clonal CD8(+)/TCRαβ(+) T-LGL proliferations might in fact suggest that multiple pathogens or autoantigens are involved.
Blood Cancer Journal 01/2014; 4(1):e172. DOI:10.1038/bcj.2013.70 · 3.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives:
To address whether successive B-cell lymphomas, diagnosed within a 5- to 15-year interval, are recurrences or unrelated primary lymphomas.
Immunoglobulin heavy and κ light chain gene rearrangements were studied using multiplex polymerase chain reaction fragment assays and sequence analysis in 61 patients.
Clonal patterns of the multiple lymphomas from 36 patients were determined and classified accordingly: 30 recurrences, 2 possible recurrences, 2 different clones with a common origin, and 2 unrelated primary lymphomas.
Regardless of subtype, 89% to 94% of late B-cell lymphoma relapses were recurrences of the primary tumor. Therefore, routinely investigating the possible clonal relationship between successive lymphomas may not be warranted except for specific lymphoma subtypes such as diffuse large B-cell lymphomas.
American Journal of Clinical Pathology 07/2013; 140(1):114-26. DOI:10.1309/AJCPI14GXNWASVUZ · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
To investigate a putative involvement of chronic (super-)antigenic stimulation in driving T-cell expansion in SS.
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.
Sequence analysis of clonally rearranged TCRB genes showed over-representation of Vβ8, Vβ13, Vβ17, Vβ21 and Vβ22, and under-representation of Vβ2 and Jβ1.1 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.
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 Vβ and Jβ gene usage does seem to point to a restricted TCR repertoire, possibly as a result of superantigenic selection prior to neoplastic transformation.
British Journal of Dermatology 03/2011; 165(1):78-84. DOI:10.1111/j.1365-2133.2011.10308.x · 4.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: > To investigate the role of X chromosomal inactivation (XCI) in systemic sclerosis (SSc) and its effects on forkhead box P3 (Foxp3) expression in T regulatory cells (Tregs).
217 women with SSc and 107 healthy women (controls) were included in the study. From these subjects, DNA was isolated from total peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes after magnetic bead separation. All samples were assessed for skewed XCI patterns with the Human Androgen Receptor Assay. The outcome was assessed by linear regression. CD4+ CD25+ cells were then isolated and intracellular Foxp3 expression was assessed by flow cytometry.
Skewing was not associated with increased age in patients with SSc, in contrast to the control population (r = 0.45, p < 0.0001). Taking this into account, a significantly higher frequency of skewed XCI was found in patients with SSc compared with controls (p = 0.001). No difference in skewing was observed between the immune cell subsets. In addition, a higher concentration of Foxp3+ cells exhibiting a lower Foxp3 mean fluorescence intensity was found in the patients with SSc, with profound XCI skewing (both p < 0.001) associated with less efficient suppressive activity (p=0.012).
Skewed XCI plays a role in susceptibility to SSc, is not restricted and influences Foxp3 expression and the suppressive capacity of Tregs.
Annals of the rheumatic diseases 12/2010; 69(12):2213-6. DOI:10.1136/ard.2010.129999 · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Leukemia is one of the leading journals in hematology and oncology. It is published monthly and covers all aspects of the research and treatment of leukemia and allied diseases. Studies of normal hemopoiesis are covered because of their comparative relevance.
[Show abstract][Hide abstract] ABSTRACT: In addition to the classical Vkappa-Jkappa, Vkappa-kappa deleting element (Kde), and intron-Kde gene rearrangements, atypical recombinations involving Jkappa recombination signal sequence (RSS) or intronRSS elements can occur in the Igkappa (IGK) locus, as observed in human B cell malignancies. In-depth analysis revealed that atypical JkappaRSS-intronRSS, Vkappa-intronRSS, and JkappaRSS-Kde recombinations not only occur in B cell malignancies, but rather reflect physiological gene rearrangements present in normal human B cells as well. Excision circle analysis and recombination substrate assays can discriminate between single-step vs multistep rearrangements. Using this combined approach, we unraveled that the atypical Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints most probably result from ongoing recombination following an initial aberrant JkappaRSS-intronRSS signal joint formation. Based on our observations in normal and malignant human B cells, a model is presented to describe the sequential (classical and atypical) recombination events in the human IGK locus and their estimated relative frequencies (0.2-1.0 vs < 0.03). The initial JkappaRSS-intronRSS signal joint formation (except for Jkappa1RSS-intronRSS) might be a side event of an active V(D)J recombination mechanism, but the subsequent formation of Vkappa-intronRSS and JkappaRSS-Kde pseudohybrid joints can represent an alternative pathway for IGK allele inactivation and allelic exclusion, in addition to classical Ckappa deletions. Although usage of this alternative pathway is limited, it seems essential for inactivation of those IGK alleles that have undergone initial aberrant recombinations, which might otherwise hamper selection of functional Ig L chain proteins.
The Journal of Immunology 10/2004; 173(6):3878-88. DOI:10.4049/jimmunol.173.6.3878 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The t(5;14)(q35;q32) is a novel cryptic translocation in pediatric T-cell acute lymphoblastic leukemia (T-ALL), involving HOX11L2 or CSX on 5q35. The 14q32 breakpoints are heterogeneous. Because the t(5;14)(q35;q32) is hard to detect using conventional karyotyping, it is easily missed in routine diagnostics. Here we describe the development and application of split signal fluorescence in situ hybridization (FISH) assays for both HOX11L2 and CSX, for detection of t(5;14) possibly present in T-ALL patients.
We developed and validated two split signal FISH assays for metaphase and interphase detection of t(5;14) in T-ALL patients. We also investigated the involvement of IGH on 14q32. In addition, HOX11L2 and SIL-TAL1 expression was studied using reverse transcription polymerase chain reaction (RT-PCR).
The FISH assays were validated on cell lines and T-ALL patients. We did not identify cases with a t(5;14)(q35;q32) involving CSX, but we did identify 5 cases of t(5;14) involving HOX11L2 out of 32 T-ALL cases studied; in each case the 14q32 breakpoint was found to be centromeric to the IGH region. All 5 positive cases showed HOX11L2 expression, as did 1 case without t(5;14)(q35;q32). Cases with t(5;14)(q35;q32) involving HOX11L2 did not show TAL1 abnormalities, whereas 5 HOX11L2 negative cases did.
Using the newly developed and validated FISH probe sets, we identified 5 new cases of t(5;14) involving HOX11L2 both on metaphases and interphases. The incidence of the t(5;14)(q35;q32) involving CSX is probably low. RT-PCR results suggest that TAL1 and HOX11L2 expression, or TAL1 aberrations and the t(5;14)(q35;q32) involving HOX11L2 are mutually exclusive.
[Show abstract][Hide abstract] ABSTRACT: Molecular clonality analysis of immunoglobulin (Ig) and T-cell receptor (TCR) genes is a widely used diagnostic tool for discrimination between polyclonal, oligoclonal, and monoclonal lymphoproliferative skin lesions. We studied Ig/TCR clonality in a series of 60 patients with an initial suspicion of (primary) cutaneous B- or T-cell lymphoma (CBCL/CTCL). Clonality of Ig/TCR gene rearrangements was assessed by Southern blot (SB) and polymerase chain reaction (PCR) analysis using standardized PCR primers and protocols of the BIOMED-2 Concerted Action BMH4-CT98-3936. The obtained PCR products were subjected to heteroduplex (HD) and GeneScan (GS) analysis. We compared the data of 154 samples with the histopathologic diagnosis, based on the EORTC classification of skin lymphomas.
Molecular results were largely concordant with histopathology. In 12 CBCL patients PCR analysis of Ig gene rearrangements detected clonality in 83% of cases whereas SB did so in 92%. Clonal TCR gene rearrangements were detected by SB in 68% of CTCL patients, whereas TCRG and TCRB PCR analysis detected clonality in 76% and 66% of cases, respectively. PCR GS analysis of TCR rearrangements appeared to be slightly more informative than HD analysis. Clonality assessment was particularly informative for studying involvement of extracutaneous sites, such as regional lymph nodes, peripheral blood, and bone marrow.
Our study shows that the BIOMED-2 multiplex PCR analysis strategy is a reliable and useful technique in the diagnostic process of patients with an initial suspicion of (primary) CBCL/CTCL and for assessment of extracutaneous dissemination, provided that the results are interpreted in the context of clinical, histologic and immunophenotypic data.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: To investigate whether the analysis of immunoglobulin (Ig)/T cell receptor (TCR) rearrangements is useful in the diagnosis of lymphoproliferative disorders.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Analysis of the T-cell receptor (TCR)-Vβ 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 Vβ repertoire usage in healthy individuals. 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 Vβ monoclonal antibodies, which together recognized 70–75% of all blood TCRαβ+ T lymphocytes. Subsequently, we developed a six-tube test kit with selected Vβ antibody combinations for easy and rapid detection of single (“clonal”) Vβ domain usage in large T-cell expansions. The mean values of the Vβ repertoire usage were stable during aging in blood TCRαβ+ 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 TCRαβ+ T lymphocytes and represented less than 0.4 × 109 cells per liter in our study. Vβ usage of the CD4+ and CD8+ subsets was comparable for most tested Vβ domains, but significant differences (P < 0.01) between the two subsets were found for Vβ2, Vβ5.1, Vβ6.7, Vβ9.1, and Vβ22 (higher in CD4+), as well as for Vβ1, Vβ7.1, Vβ14, and Vβ23 (higher in CD8+). Finally, single Vβ domain expression in large T-cell expansions can indeed be detected by the six-tube test kit. The results of our study can now be used as reference values in studies on distortions of the Vβ repertoire in disease states. The six-tube test kit can be used for detection of single Vβ domain expression in large T-cell expansions (>2.0 × 109/l), which are clinically suspicious of T-cell leukemia. Cytometry 40:336–345, 2000