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.

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.

TCRgammadelta+ large granular lymphocyte leukemias reflect the spectrum of normal antigen-selected TCRgammadelta+ T-cells.

T-cell large granular lymphocytes (LGL) proliferations range from reactive expansions of activated T cells to T-cell leukemias and show variable clinical presentation and disease course. The vast majority of T-LGL proliferations express TCRalphabeta. Much less is known about the characteristics and pathogenesis of TCRgammadelta+ cases. We evaluated 44 patients with clonal TCRgammadelta+ T-LGL proliferations with respect to clinical data, immunophenotype and TCR gene rearrangement pattern. TCRgammadelta+ T-LGL leukemia patients had similar clinical presentations as TCRalphabeta+ T-LGL leukemia patients. Their course was indolent and 61% of patients were symptomatic. The most common clinical manifestations were chronic cytopenias - neutropenia (48%), anemia (23%), thrombocytopenia (9%), pancytopenia (2%) - and to a lesser extent splenomegaly (18%). Also multiple associated autoimmune (34%) and hematological (14%) disorders were found. Leukemic LGLs were predominantly positive for CD2, CD5, CD7, CD8, and CD57, whereas variable expression was seen for CD16, CD56, CD11b, and CD11c. The Vgamma9/Vdelta2 immunophenotype was found in 48% of cases and 43% of cases was positive for Vdelta1, reflecting the TCR-spectrum of normal TCRgammadelta+ T-cells in adult PB. Identification of the well-defined post-thymic Vdelta2-Jdelta1 selection determinant in all evaluable Vgamma9+/Vdelta2+ patients, is suggestive of common (super)antigen involvement in the pathogenesis of these TCRgammadelta+ T-LGL leukemia patients.

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.

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.

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.

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.

Detection of minimal residual disease in acute leukemia patients.

Diagnostic techniques, routinely used in clinical practice for monitoring acute leukemia patients, are able to detect only 1-5% of malignant cells. At present, two main techniques are being introduced for detection of minimal residual disease (MRD) in leukemia, namely immunological marker analysis and the polymerase chain reaction (PCR) technique with general sensitivity of 10(-4)-10(-5). Immunological marker analysis allows detection of unusual and aberrant immunophenotypes, and is usually performed by flow cytometry. PCR analysis allows detection of leukemia-specific DNA sequences, such as fusion regions of chromosome aberrations and junctional regions of rearranged immunoglogulin (Ig) genes and T-cell receptor (TcR) genes. The applicability of the immunophenotyping and PCR-mediated MRD techniques is dependent on the type of leukemia. In virtually all acute lymphoblastic leukemias, PCR analysis of Ig and TcR genes can be used, and immunophenotypic MRD detection is also possible in 70-80% of cases. In AML, immunophenotypic MRD detection can be applied in approximately 80% of cases and PCR analysis of chromosome aberrations in 25-40%. Each MRD technique has its advantages and limitations, which have to be weighed carefully to make an appropriate choice. Furthermore, standardization of the MRD techniques is needed before they are used for stratification or adaptation of treatment protocols. Finally, the clinical impact of MRD detection for the various subtypes of acute leukemias has to be established.

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.