Terminal Deoxynucleotidyl Transferase Gene Research Articles

Targeting TdT gene expression in Molt-4 cells by PNA-octaarginine conjugates

Peptide nucleic acid (PNA) is an amide based structural nucleic acid mimic with potential applications in gene therapeutic drug discovery. In the present study, we evaluated and compared the effects on gene expression, cell viability and apoptosis of two antisense PNA-d-octaarginine conjugates, targeting sequences at the AUG translation start site or the 5′-UTR of the TdT (terminal deoxynucleotidyl transferase) gene, as well as a sense oligomer corresponding to the 5′-UTR-antisense, in Molt-4 cells. The protein level of TdT was determined by flow cytometry, and qPCR was used for mRNA expression analysis. Mismatch PNAs were used as control to address the sequence/target spcifity of the biological effects. The results showed that treatment with the AUG- and to slightly lesser extent with the 5′-UTR-antisense PNAs reduced the TdT mRNA as wel as the protein level, whereas only very low effect was observed for the 5′-UTR-sense PNA. A parallel effect was observed on reduced cell survival and increased rate of apoptosis. Our findings suggest that antisense PNAs can inhibit expression of the TdT gene and induce apoptosis in Molt-4 cells.

The expression of terminal deoxynucleotidyl transferase and paired box gene 5 in Merkel cell carcinomas and its relation to the presence of Merkel cell polyomavirus DNA.

Merkel cell carcinoma (MCC) tumor samples frequently express B-lymphoid lineage markers. However, the reasons for expression of specific B-lymphoid lineage markers are still unclear. We studied the expression of TdT and PAX5 (two B-cell lymphoid lineage markers) in a large pool of MCC tissue microarray samples. Immunoexpression and staining intensities of TdT and Pax-5 were statistically correlated with patient, tumor, Merkel cell polyomavirus (MCV), and disease-specific parameters. In a cohort of 117 MCC patients and their corresponding tumor samples, TdT was expressed in 37 (31.6%) samples and PAX5 in 26 (22.2%). Simultaneous immunostaining for TdT and PAX5 was observed in 13 (11.1%) samples. A statistically significant relationship was observed between MCV virus copy number and positive TdT expression (P = 0.0056). Similarly, a significant relationship was also observed between positive TdT and tumor MCV virus positivity (P = 0.000495). We observed frequent TdT and PAX5 immunoexpression in MCC tumor samples. However, simultaneous immunoexpression of these markers was scarce. TdT expression was statistically significantly associated with MCV positivity. The absence of a statistically significant association between tumor parameters and disease progression markers undermines the systemic use of these markers in clinical practice.

Single Cell Trajectory Detection Orders Hallmarks of Early Human B Cell Development

AbstractAbstract 1044 Background:Normal B cell development in the bone marrow (BM) is a seemingly well-understood, progressive process and thus represents a suitable test system in which to apply an algorithmic approach to modeling cellular differentiation. In humans, hematopoietic stem cells form lymphoid progenitor cells that develop into pro- then pre- B cells and finally those cells that escape negative selection become immature B cells that leave the BM for the peripheral immune organs. Flow cytometry can track these stages using the expression of immunophenotypic cell surface markers, including those for progenitors (CD34, CD38), early B cell populations (CD10), as well as those of more mature B cells (CD20, IgM). Expression of the B cell transcription factor PAX5, and immune diversity conferring enzymes terminal deoxynucleotidyl transferase (TdT) and recombination-activating gene (RAG) can also be tracked at the single cell level. Regulatory signaling by factors in the BM orchestrates critical checkpoints in the B cell developmental program, such as Interleukin (IL)-7-mediated STAT5 phosphorylation and signaling downstream the preB cell receptor/B cell receptor (BCR) (p-BLNK, p-Syk, p-PLCγ2, p-Erk). Successful coordination of these signals with immunoglobulin gene rearrangement results in a burst of proliferative expansion prior to maturation/exit to the periphery. Failure of any one of these processes results in B cell deletion while certain dysregulations driven by oncogenic processes can result in malignancy.While much of this core understanding has been founded in murine models, the rarity of early B cell progenitors and lack of genetic tools has complicated definition of B cell development in humans. Using 42 parameter mass cytometry in combination with a novel single-cell trajectory finding algorithm, we have now laid a human B cell developmental process in primary human BM to an unprecedented level of detail, mapping out the expression pattern of virtually all relevant B cell immunophenotypic markers as well as intracellular enzyme, transcription factor and regulatory modification simultaneously, at the single cell level. Methods:The mononuclear cell fraction of multiple healthy human marrows was characterized by simultaneously analyzing 42 antibody parameters with mass cytometry targeting a multitude of phenotypic markers, intracellular signaling molecules, hallmarks of cell cycle and apoptosis all in the context of in vitro perturbations relevant to B cell development (including IL-7 and BCR crosslinking). The resulting multidimensional data was modeled using a novel, scalable, robust graph-based trajectory algorithm that iteratively refines a solution trajectory using random landmarks to reduce variability. Populations of interest were prospectively isolated and a novel qPCR assay was created to quantitate immunoglobulin heavy chain (IgH) rearrangement in genomic DNA. Results/Conclusion:Modeling of the resulting data was undertaken using this algorithm (termed Wanderlust) that devised and ordered cellular relationships based on the average phenotypic progression from our defined starting point, in this case, CD34+CD38- hematopoietic stem cells, in order to calculate a developmental trajectory. The predicted trajectory was then used to inform a traditional 'gating' analysis of the data and provide a higher resolution view of human B cell development than previously published. It both confirmed established steps in human B cell progression, and importantly, revealed new populations of early B cell progenitors based on expression of CD34, CD38, CD24 and TdT. These populations were corroborated to be of B-lineage and ordered as predicted based on the progressive rearrangement of the IgH locus by qPCR of extracted genomic DNA. We aligned previously unregistered key developmental checkpoints such as STAT5 activation in response to IL-7 and proliferation in response preBCR expression with traditional immunophenotypic cell populations. While predicted in silico, and then molecularly verified and staged in vitro, these regulatory events all lay within discrete cell subsets that can now be demarcated using conventional cytometric methods. Together, this provides a backbone on which to further examine both healthy regulatory events as well as the corruption of this developmental process such as in malignant or immunodeficient states. Disclosures:No relevant conflicts of interest to declare.

Immune Defects of B- and T-Cell Compartments in Natalizumab-Induced Progressive Multifocal Leukoencephalopathy,

Abstract 3228▪▪This icon denotes a clinically relevant abstract Objective:Progressive multifocal leukoencephalopathy (PML) is a rare, but often fatal demyelinating brain disease caused by the JC virus, which usually occurs in immunosuppressed patients, including those with hematological malignancies or receiving monoclonal antibodies-based immunotherapies. PML is likely the result of a complex combination of several pathogenic mechanisms, such as alterations of peripheral cell-mediated immunity and mobilization of JC virus-carrying CD34+-hematopoetic stem cells and pre-B-cells. Taking advantage of the availability of samples from a multiple sclerosis (MS) patient treated with the anti-α4β1 monoclonal antibody natalizumab who developed PML, which was monitored for 35 months since before therapy initiation, we investigated the role of B and T lymphocytes in PML onset. Methods:Real-Time PCR was used to measure the release of T and B cells from the production sites by means of T-cell receptor excision circles (TRECs) and K-deleting excision circles (KRECs) analysis and to quantify transcripts for immature hematopoietic cells such as terminal deoxynucleotidyl transferase, CD34, and pre-B lymphocyte gene 1. Naïve and mature T- and B-cell subsets were identified by flow cytometry, T-cell heterogeneity was quantified by spectratyping and IgA, IgG and IgM by turbidimetric assay. Data were compared to those of untreated and natalizumab-treated MS patients and healthy donors. Results:After 34 months of natalizumab therapy, a 42 years old female developed PML, diagnosed on the basis of magnetic resonance imaging and JC virus positivity in cerebrospinal fluid. Before therapy, her thymus and bone marrow produced a significant low number of TRECs+ and KRECs+ cells. While TRECs remained low during all therapy period, KRECs and transcripts for pre-B lymphocyte gene 1, which is selectively expressed in pre-B cells, peaked after 6 months of therapy, remained high at 12 and 15 months of treatment, and then decreased at the moment of PML onset. Flow cytometry confirmed a deficient production of CD4+CD45RA+CCR7+CD31+ recent T emigrants, counterbalanced by an increased number of CD8+CCR7–CD45RA+ TEMRA cells for all observation period, but showed a modification of peripheral CD4 and CD8 cell number only at the moment of PML. While the percentage of naïve B cells increased by about 70% after 6 months of therapy, the number of B lymphocytes within each B-cell subpopulations remained low for the entire treatment period. T-cell repertoire and immunoglobulin production were altered. Interpretation:Although performed in a single patient, data all agree in demonstrating that a deficient production of new TRECs+ T lymphocytes, together with an increase of newly produced KRECs+ B cells just few months after therapy beginning may predispose to PML. These findings encourage further researches on the utility of the TRECs/KRECs assay as a potential tool for the identification of patients at risk of developing PML after monoclonal antibodies-based therapies. Disclosures:No relevant conflicts of interest to declare.

The pattern of gene expression and gene dose profiles of 6-Mercaptopurine- and 6-Thioguanine-resistant human leukemia cells

Exposure of MOLT4 human T-cell leukemia cells to 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) resulted in acquired resistance associated with attenuated expression of the genes encoding concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). To identify other alterations at the RNA and DNA levels associated with 6-MP- and 6-TG resistance, we compared here the patterns of gene expression and DNA copy number profiles of resistant sublines to those of the parental wild-type cells. The mRNA levels for two nucleoside transporters were down-regulated in both of the thiopurine-resistant sublines. Moreover, both of these cell lines expressed genes encoding the enzymes of purine nucleotide composition and synthesis, including adenylate kinase 3-like 1 and guanosine monophosphate synthetase at significantly lower levels than wild-type cells. In addition, expression of the mRNA for a specialized DNA polymerase, human terminal transferase encoded by the terminal deoxynucleotidyl transferase (DNTT) gene, was 122- and 93-fold higher in 6-TG- and 6-MP-resistant cells, respectively. The varying responses to 6-MP- and 6-TG observed here may help identify novel cellular targets and modalities of resistance to thiopurines, as well as indicating new potential approaches to individualization therapy with these drugs.

Regulation of gene expression during thymocyte differentiation by Protein Phosphatase 1 (64.16)

Abstract The goal of our research is to define signal transduction pathways that regulate thymocyte differentiation. We have previously reported that Protein Phosphatase 1 (PP1) directly dephosphorylates and regulates the activity of the Ikaros protein - a master regulator of lymphocyte differentiation (Popescu et al. J Biol Chem 2009 284:13869). We extended our studies to determine whether PP1-mediated dephoshorylation of Ikaros controls Ikaros’ function in regulating the expression of the terminal deoxynucleotidyl transferase (TdT) gene during thymocyte differentiation. An Ikaros mutant that is unable to interact with PP1 (IK 465/7A), and that is subsequently hyperphosphorylated, was compared to the wild type Ikaros for DNA-binding affinity toward the TdT promoter and for ability to repress transcription of the TdT gene using a luciferase reporter assay. Chromatin immunoprecipitation assays showed that the loss of PP1-mediated dephosphorylation of Ikaros leads to decreased Ikaros DNA-binding affinity for the D’ regulatory element of the TdT promoter in vivo. The IK 465/7A mutant also failed to repress transcription of TdT in the luciferase reporter assay, suggesting that Ikaros’ interaction with PP1 and subsequent dephosphorylation is essential for its function as repressor of TdT transcription. Results suggest that the PP1 signal transduction pathway regulates thymocyte differentiation by controlling the function of Ikaros and transcriptional repression of the TdT gene.

Localization of genes for V+LDL plasma cholesterol levels on two diets in the opossum Monodelphis domestica

Plasma cholesterol levels among individuals vary considerably in response to diet. However, the genes that influence this response are largely unknown. Non-HDL (V+LDL) cholesterol levels vary dramatically among gray, short-tailed opossums fed an atherogenic diet, and we previously reported that two quantitative trait loci (QTLs) influenced V+LDL cholesterol on two diets. We used hypothesis-free, genome-wide linkage analyses on data from 325 pedigreed opossums and located one QTL for V+LDL cholesterol on the basal diet on opossum chromosome 1q [logarithm of the odds (LOD) = 3.11, genomic P = 0.019] and another QTL for V+LDL on the atherogenic diet (i.e., high levels of cholesterol and fat) on chromosome 8 (LOD = 9.88, genomic P = 5 x 10(-9)). We then employed a novel strategy involving combined analyses of genomic resources, expression analysis, sequencing, and genotyping to identify candidate genes for the chromosome 8 QTL. A polymorphism in ABCB4 was strongly associated (P = 9 x 10(-14)) with the plasma V+LDL cholesterol concentrations on the high-cholesterol, high-fat diet. The results of this study indicate that genetic variation in ABCB4, or closely linked genes, is responsible for the dramatic differences among opossums in their V+LDL cholesterol response to an atherogenic diet.

Dicer Ablation Affects Antibody Diversity and Cell Survival in the B Lymphocyte Lineage

To explore the role of Dicer-dependent control mechanisms in B lymphocyte development, we ablated this enzyme in early B cell progenitors. This resulted in a developmental block at the pro- to pre-B cell transition. Gene-expression profiling revealed a miR-17 approximately 92 signature in the 3'UTRs of genes upregulated in Dicer-deficient pro-B cells; a top miR-17 approximately 92 target, the proapoptotic molecule Bim, was highly upregulated. Accordingly, B cell development could be partially rescued by ablation of Bim or transgenic expression of the prosurvival protein Bcl-2. This allowed us to assess the impact of Dicer deficiency on the V(D)J recombination program in developing B cells. We found intact Ig gene rearrangements in immunoglobulin heavy (IgH) and kappa chain loci, but increased sterile transcription and usage of D(H) elements of the DSP family in IgH, and increased N sequence addition in Igkappa due to deregulated transcription of the terminal deoxynucleotidyl transferase gene.

Recruitment of Ikaros to Pericentromeric Heterochromatin Is Regulated by Phosphorylation

Ikaros encodes a zinc finger protein that is involved in heritable gene silencing. In hematopoietic cells, Ikaros localizes to pericentromeric heterochromatin (PC-HC) where it recruits its target genes, resulting in their activation or repression via chromatin remodeling. The function of Ikaros is controlled by post-translational modifications. CK2 kinase has been shown to phosphorylate Ikaros at its C terminus, affecting cell cycle progression. Using in vivo labeling of murine thymocytes followed by phosphopeptide mapping, we identified four novel Ikaros phosphorylation sites. Functional analysis of phosphomimetic mutants showed that the phosphorylation of individual amino acids determines the affinity of Ikaros toward probes derived from PC-HC. In vivo experiments demonstrated that targeting of Ikaros to PC-HC is regulated by phosphorylation. The ability of Ikaros to bind the upstream regulatory elements of its known target gene terminal deoxynucleotidyltransferase (TdT) was decreased by phosphorylation of two amino acids. In thymocytes, Ikaros acts as a repressor of the TdT gene. Induction of differentiation of thymocytes with phorbol 12-myristate 13-acetate plus ionomycin results in transcriptional repression of TdT expression. This process has been associated with increased binding of Ikaros to the upstream regulatory element of TdT. Phosphopeptide analysis of in vivo-labeled thymocytes revealed that Ikaros undergoes dephosphorylation during induction of thymocyte differentiation and that dephosphorylation is responsible for increased DNA binding affinity of Ikaros toward the TdT promoter. We propose a model whereby reversible phosphorylation of Ikaros at specific amino acids controls the subcellular localization of Ikaros as well as its ability to regulate TdT expression during thymocyte differentiation.

Identification of a new cis-regulatory element of the terminal deoxynucleotidyl transferase gene in the 5′ region of the murine locus

Terminal deoxynucleotidyl transferase (TdT) expression is controlled at the transcriptional level, however, the TdT core promoter combining D, D′, an initiator (Inr) and downstream basal elements (DBE) does not recapitulate the whole complex regulation of TdT expression. We hypothesized that important cis-regulatory elements of the gene are located outside of the TdT promoter. In an attempt to identify these elements, we performed DNase I hypersensitivity assays over 24 kb including a 10 kb region located upstream of the transcription start site (+1) and a 14 kb region spanning exons and introns I to VI. Hypersensitive sites (HS) HS1 and HS2 were localized 8.5 and 8 kb upstream of the transcription start site, respectively, and were exclusively detected in TdT + cell types. HS3, HS4 and HS5 were mapped at positions −7, −3.4 and −3 kb, respectively, and detected in both TdT negative and positive cells. HS6, HS7 and HS8 were detected immediately upstream of the TdT promoter. HS10 and HS11 were localized in the first and third intron of the gene. Luciferase reporter assays revealed that HS1, HS2 and HS3 synergize with the TdT promoter to activate transcription in a TdT + pre-T cell line but not in a TdT + pro-B cell line. In summary novel cis-regulatory elements have been identified in the 5′ region of the TdT locus that synergize with the promoter to activate gene expression and our results suggest these elements may be more active in T cells.

The Initiator Core Promoter Element Antagonizes Repression of TATA-directed Transcription by Negative Cofactor NC2

Core promoter regions of protein-coding genes in metazoan genomes are structurally highly diverse and can contain several distinct core promoter elements, which direct accurate transcription initiation and determine basal promoter strength. Diversity in core promoter structure is an important aspect of transcription regulation in metazoans as it provides a basis for gene-selective function of activators and repressors. The basal activity of TATA box-containing promoters is dramatically enhanced by the initiator element (INR), which can function in concert with the TATA box in a synergistic manner. Here we report that a functional INR provides resistance to NC2 (Dr1/DRAP1), a general repressor of TATA promoters. INR-mediated resistance to NC2 is established during transcription initiation complex assembly and requires TBP-associated factors (TAFs) and TAF- and INR-dependent cofactor activity. Remarkably, the INR appears to stimulate TATA-dependent transcription similar to activators by strongly enhancing recruitment of TFIIA and TFIIB and, at the same time, by compromising NC2 binding.

Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells.

Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3'-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA-protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1-TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors.

Expression of Activation-Induced Cytidine Deaminase (AID) in Burkitt Lymphoma Cells: Rare AID-Negative Cell Lines with the Unmutated Rearranged VH Gene

Activation-induced cytidine deaminase (AID) is an enzyme that catalyzes somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) gene. The expression of AID was reported to be confined to the germinal center (GC). Burkitt lymphoma (BL) cells are regarded as being derived from GC cells. BL cells have been reported to have SHM in the Ig gene with variety in terms of degree, antigen selection and ongoing mutation characteristic. We investigated the expression of AID in 15 BL cell lines by RT-PCR. In only 2 BL cell lines, Tree92 and Black93, AID expression was not detected, and the IgVH gene of these 2 cell lines was not mutated. Tree92 expresses terminal deoxy-nucleotidyl transferase (TdT) and recombination activating gene (RAG)1, but Black93 does not, as is typical of the BL phenotype. BL cells are generally derived from GC B-cell expressing AID, but are rarely derived from the stage of B-lineage differentiation in which AID is not yet expressed, causing the absence of mutation in the IgVH.

Substantial N diversity is generated in T cell receptor alpha genes at birth despite low levels of terminal deoxynucleotidyl transferase expression in mouse thymus.

N region diversity in antigen receptors is a developmentally regulated process in B and T lymphocytes, which correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). To precisely determine the onset of TdT gene activation during T cell differentiation and thymic ontogeny, TdT expression was directly detected at the cellular level by in situ hybridization and TdT function was assessed by analyzing the distribution of N additions in alpha and beta TCR genes at early stages of development. Even though TdT transcripts were undetectable at birth, substantial N additions were observed in ValphaJalpha junctions and 3 days later in VbetaDbetaJbeta junctions, indicating that TdT expression could be induced in immature thymocytes much earlier than expected. Indeed low TdT expression level was found in TN3/4 and DP from fetal day 17, suggesting that the onset of TdT expression occurs simultaneously in both populations and may depend on microenvironmental cues. Moreover significant increase in the proportion of thymocytes expressing high levels of TdT mRNA during the first week after birth without a similar increase in the level of N diversity suggests that TdT expression and TdT function in the generation of N diversity are not strictly correlated.

The M603 idiotype is lost in the response to phosphocholine in terminal deoxynucleotidyl transferase-deficient mice.

The majority of anti-phosphocholine (PC) antibodies induced by the PC epitope in Proteus morganii (PM) express the M603 idiotype (id), which is characterized by an invariant Asp to Asn substitution at the V(H):D(H) junction. To elucidate the molecular basis by which M603-like B cells acquire the mutations resulting in this invariant substitution, we analyzed the immune response to PC-PM in terminal deoxynucleotidyl transferase (TdT) gene knockout (KO) mice. In the absence of TdT, T15-id antibodies comprised 80-100% of the primary response to PC-PM. Less than 10% of the response in wild-type mice is T15-id(+). In TdT KO mice, the secondary response to PC-KLH was higher than in wild-type mice and was dominated by the germ-line T15-id. About 10% of this response, in both TdT KO and wild-type mice, comprised M167-id(+) antibodies. Additionally, none of the functionally rearranged V1/DFL16.1/J(H)1 cDNA isolated from PC-PM-immunized TdT KO mice showed the Asp/Asn substitution characteristic of PC-binding, PC-PM-induced M603-like antibodies. These data indicate that production of M603-id antibody is TdT dependent, while generation of M167-id antibody is TdT independent, and that in the absence of competition from M603-like B cells, T15-id B cells can respond to PC-PM.

B-cell precursors differentiated from cord blood CD34+ cells are more immature than those derived from granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells.

Umbilical cord blood (CB) has been widely used instead of bone marrow (BM) and peripheral blood (PB) for stem cell transplantation (SCT). However, problems of sustained immunodeficiency after CB transplantation remain to be resolved. To elucidate the mechanism of immunodeficiency, we compared the characteristics of B cells differentiated in vitro from CD34+ cells of CB with those of PB. Purified CD34+ cells from CB and PB were cultured on murine stroma cell-line MS-5 with stem cell factor and granulocyte colony-stimulating factor for 6 weeks. The B-cell precursors (pre-B cells) that differentiated in this culture system, were analysed as to their immunoglobulin heavy chain (IgH) variable region gene repertoire and the expression of B-cell differentiation-related genes. CD10+ CD19+ pre-B cells were differentiated from both PB and CB. Although the usages of IgH gene segments in pre-B cells differentiated from CB and PB were similar, the N region was significantly shorter in CB-derived than PB-derived cells. Productive rearrangements were significantly fewer in cells of CB than PB in the third week. Among a number of B-cell differentiation-related genes, the terminal deoxynucleotidyl transferase (TdT) gene was not expressed in CB-derived cells during the culture. These results indicated that immature features of pre-B cells from CB, such as lack of TdT expression, and a short N region and few productive rearrangements in the IgH gene, might cause the delay in mature B-cell production.

Most alpha/beta T cell receptor diversity is due to terminal deoxynucleotidyl transferase.

The contribution of template-independent nucleotide addition to antigen receptor diversity is unknown. We therefore determined the size of the T cell receptor (TCR)alpha/beta repertoire in mice bearing a null mutation on both alleles of the terminal deoxynucleotidyl transferase (Tdt) gene. We used a method based upon polymerase chain reaction amplification and exhaustive sequencing of various AV-AJ and BV-BJ combinations. In both wild-type and Tdt degrees / degrees mice, TCRAV diversity is one order of magnitude lower than the TCRBV diversity. In Tdt degrees / degrees animals, TCRBV chain diversity is reduced 10-fold compared with wild-type mice. In addition, in Tdt degrees / degrees mice, one BV chain can associate with three to four AV chains as in wild-type mice. The alpha/beta repertoire size in Tdt degrees / degrees mice is estimated to be 10(5) distinct receptors, approximately 5-10% of that calculated for wild-type mice. Thus, while Tdt activity is not involved in the combinatorial diversity resulting from alpha/beta pairing, it contributes to at least 90% of TCRalpha/beta diversity.

Methylation of the promoter region may be involved in tissue-specific expression of the mouse terminal deoxynucleotidyl transferase gene

The terminal deoxynucleotidyl transferase gene (TdT) is expressed in mice only in early B and T lymphoid precursors a few days after birth. Transactivating factors have been shown to contribute to the lymphoid specific expression of TdT, but they do not account entirely for the restriction of its expression to early precursors. Since tissue-specific expression can be modulated by other mechanisms such as DNA methylation and DNA accessibility, we evaluated the methylation pattern of the TdT gene in various expressing and non-expressing tissues and cell lines. Lymphoid and non-lymphoid organs differed significantly in their methylation profiles. In the thymus nearly complete demethylation of aHha I site in the promoter was associated with high levels of TdT transcription. There was similar, but weaker demethylation of the TdT promoter in bone marrow, possibly due to the presence of a few TdT expressing B cell precursors. The same methylation status was also associated with TdT expression in different B and T cell lines. Kinetic studies of TdT gene demethylation and TdT transcription during thymus development showed that changes in methylation status were also involved in the differential expression of TdT in fetal and adult life. Footprinting experiments revealed the existence of three regions specifically protected by nuclear extracts from TdT-expressing cells. Together, these results suggest that promoter demethylation is involved in the control of TdT expression and implicate new promoter regions in this regulation.

Biologic and clinical significance of CD7 expression in acute myeloid leukemia.

CD7 antigen, a T-cell lineage associated antigen, is expressed in a minority of patients with acute myeloid leukemia (AML). The biologic and clinical significance of this finding is not clearly established. In this retrospective study of patients with de novo acute myeloid leukemia, we have identified CD7 expression and analyzed its association with markers expressed early in hemopoietic ontogeny and clinical parameters. Among 60 consecutive AML patients, we found six (10%) expressing CD7 on leukemic cells. There were five males and one female and the mean age was 59.6 years (age range: 32-76 years) with no demographic peculiarities. The FAB subtypes were: M0 (2), M1 (1), M2 (1), and M4 (2). CD7 expression was associated with immature antigens CD34, HLA-DR, and terminal deoxynucleotidyl transferase (TdT) and antigen receptor gene rearrangements (rearrangements of T-cell receptor gamma chain in 6/6 and immunoglobulin heavy chain in 2/6). Hepatomegaly was present in three and this was associated with splenomegaly with lymphadenopathy in one patient. Mediastinal or central nervous system involvement was absent. Complete remission was achieved in two patients with standard chemotherapy; one of these is in remission and alive (5 years later), while one died following relapse 9 months later. Three patients had significantly lower response to standard therapeutic regimen (two died during induction and one died 7 months later without ever achieving complete remission). One patient has been excluded in determining the prognostic significance of CD7 due to early death. Our results suggest origin of CD7+ AML from early hemopoietic precursors and indicate biologic aggressiveness in a significant proportion of patients. We suggest evaluation of CD7 in all patients with AML at the time of diagnosis in view of poor clinical outcome.

CYTOKINE GENE EXPRESSION IN MURINE FETAL INTESTINE: POTENTIAL FOR EXTRATHYMIC T CELL DEVELOPMENT

An increasing body of evidence suggests that certain T cell subsets mature extrathymically in the epithelium of the intestine. In the studies reported here, the authors have analysed cytokine/growth factor gene expression, recombinase-activating gene RAG-1 and RAG-2 gene expression and terminal deoxynucleotidyl transferase (TdT) gene expression in mouse fetal intestine and fetal thymus and liver, two known haematopoietic tissues. Stem cell factor (SCF) and interleukin 7 (IL-7) message was abundant in all three tissues during fetal development. IL-2 and IL-4 were not expressed in fetal gut but IL-4 was weakly detected in fetal liver and thymus. IL-9 and IL-13 mRNA was detected in all fetal tissues and IL-15 mRNA was abundant in fetal intestine but only weakly expressed in fetal liver and thymus. mRNA for SCF, IL-7, IL-13 and IL-15 was also detected in fibroblast-like cell lines derived from fetal intestine. RAG-1 and RAG-2 mRNA was detected in all three fetal tissues. TdT mRNA was not detected in fetal gut or liver but was weakly expressed in (fetal day) fd19–20 fetal thymus. Long-term (>6 weeks) in vitro growth of IEL was achieved by coculturing intraepithelial lymphocytes (IEL) with IL-7-secreting fibroblasts in the presence of SCF and IL-2. The data show that the fetal mouse gut provides a suitable environment for lymphocyte development and receptor rearrangement, similar to fetal thymus and liver, even though expansion of intestinal IEL is delayed until 2–3 weeks after birth.