Detection of terminal deoxynucleotidyl transferase (TdT) by flow cytometry in leukemic disorders

We applied a new technique to the detection of intracellular TdT in 26 leukemic patients, including 16 non-T acute lymphoblastic leukemia (ALL), four T-ALL, one T-lymphoblastic lymphoma in leukemia phase, one undifferentiated leukemia, one de novo lymphoblastic phase of chronic myeloid leukemia, and three acute monocytic leukemias (AMOL). Mononuclear cell suspensions were incubated in saponin to permeabilize the cell membrane. The cells were then stained by indirect immunofluorescence (IF) using anti-human TdT monoclonal antibodies and were analyzed by flow cytometry (FCM). The TdT results were compared with those obtained by biochemical TdT assay (26 cases), immunoperoxidase determination (PAP) (12 cases), and fluorescence microscopy (seven cases). The results obtained by PAP and fluorescence microscopy were 100% concordant with those obtained by FCM and biochemical assay. TdT determination by FCM allows the analysis of large numbers of cells in a fast, objective, and reliable manner, as compared with biochemical assay, PAP, and fluorescence microscopy determinations.     

Terminal deoxynucleotidyl transferase (TdT) enzyme in thymus and bone marrow: I. Age-associated decline of TdT in humans and mice

This study was aimed at characterizing terminal deoxynucleotidyl transferase (TdT) levels in populations of normal human and murine lymphocytes and toward correlating TdT enzyme levels with the biological process of aging. A newly developed method that utilizes a small number of cells was employed to determine TdT levels in bone marrow and thymus cells following cell fractionation at unit gravity sedimentation. By these methods, cell fractions with high TdT activity were found to comprise only 5–10% of the parent cell pools. In the human bone marrow, we show here that TdT-positive cell fractions are largely depleted of HTLA, E-rosette forming, and mitogen-responsive cells, whereas TdT-positive human thymocyte fractions contain a high percentage of HTLA and E-rosette-positive cells. Our observations in the murine model confirm the earlier observations that TdT activity decreases with age. We further show here that the age-associated decline of TdT in the bone marrow preceded that in the thymus. As is true for the mouse, TdT activity in human bone marrow and thymus was also found to decrease with advancing age. The decline in TdT was not associated with a change in cell distribution profiles after unit gravity sedimentation of bone marrow or thymus cells. From these data, the age-associated loss of TdT cannot be attributed to a loss of a particular subpopulation of cells.     

The production of terminal deoxynucleotidyl transferase (TdT) positive cells in cultures of human pluripotent hemopoietic progenitor cells

A transient increase in terminal deoxynucleotidyl transferase positive (TdT+) cells was observed during the early phase of (less than or equal to day 5) cultures supporting the growth of pluripotent myeloid progenitor cells (CFU-mix). T-cell growth-promoting medium and erythropoietin were not required. The rapidity with which TdT+ cells appeared in cultures and the results of cultures where TdT+ cells were high initially (greater than 800 cells/culture) were not consistent with their having been produced by proliferation of pre-existing TdT+ cells from the bone marrow inoculum. The results suggest production of TdT+ cells from a TdT-negative precursor either by altered enzyme expression or by production of TdT+ progeny.     

Cytogenetic heterogeneity in blast crisis (BC) of chronic myelogenous leukemia (CML)

New cytogenetic findings are reported in a patient who entered into an accelerated blastic phase of chronic myelogenous leukemia (CML). The cytogenetic findings of this case can be described as 46, xy, t (5;7) (q 31;q 11), t (9;22) (q 34;q 11), Ph'. The prognostic implications in such patients with rare and unusual cytogenetic findings are discussed.     

Enzymatic activity of naturally occurring 1-acylglycerol-3-phosphate-O-acyltransferase 2 mutants associated with congenital generalized lipodystrophy

Mutations in the gene encoding 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) have been reported in patients with congenital generalized lipodystrophy (CGL). AGPAT2, a 278 amino acid protein, belongs to the acyltransferase enzyme family, and has two conserved motifs, NHX(4)D and EGTR, involved in the enzymatic activity. The AGPATs catalyze acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA) during the biosynthesis of glycerophospholipids and triglycerides from glycerol-3-phosphate. The present studies were designed to determine the enzymatic activity of AGPAT2 mutants found in CGL patients to provide a molecular explanation for the phenotype and to obtain additional information about the structure-function relationship of AGPAT2 protein. The enzymatic activities of the wild type AGPAT2 and mutants were determined in cell lysates of overexpressing Chinese hamster ovary cells by measuring the conversion of [(3)H]LPA to [(3)H]PA in the presence of oleoyl-coenzyme A. Whereas, the R68X, 221delGT, 252delMRT, D180fsX251, and V167fsX183 mutants had markedly reduced enzymatic activity (median <15% of the wild type), the mutants, 140delF, G136R, and L228P, retained median activity ranging from 15% to 40% of the wild type enzyme. However, the missense mutant, A239V, had 90% of the wild type activity. We suggest that reduction in AGPAT2 enzymatic activity underlies the loss of adipose tissue in CGL. Our observations reveal an important role of various carboxy-terminal residues in determining the enzymatic activity of AGPAT2.     

Bone marrow necrosis intravitally recognized in four cases of blastic leukaemia.

Bone marrow necrosis (BMN) is a rare intravitally recognized finding in acute leukaemia with an uncertain clinical significance. The clinical events in 4 patients with AML, ALL, AMoL and blastic transformation of CGL in whom bone marrow cytology and histology revealed BMN are reviewed. One patient with BMN at clinical presentation of AML entered complete, long lasting remission with marrow restoration after the standard DAT therapy. In the three remaining patients survival after BMN diagnosis was 6, 11, and 14 weeks. Clinical, haematological, histological and marrow scanning findings and their significance for early diagnosis and means to asses the extent and evaluation of BMN will be discussed. In contrast to the most earlier reports, BMN does not appear to confer a poor prognosis in all patients with blastic leukaemia.     

Localization of terminal deoxynucleotidyl transferase (TdT) in rat thymus using immunoperoxidase methods

Summary Indirect and peroxidase anti-peroxidase (PAP) immunoenzymatic methods were used to detect terminal deoxynucleotidyl transferase (TdT) in imprints and formalin-fixed paraffin sections of normal rat thymus. TdT is found in the nuclei of small lymphocytes in imprint samples from neonatal and adult rat thymus, showing granular or circular patterns of peroxidase reaction products. Diffuse brown reaction products of peroxidase are located in both the nuclei and cytoplasm of medium and large lymphocytes. Indirect measurements show that, as age progresses, the percentage of peroxidase-positive cells decreases in all types of lymphocytes, from 72.4% on the 11th day to 54.8% in the 5th month, whereas that of negative cells increases from 14.4% to 39.4%. In formalin-fixed paraffin sections, peroxidase-positive lymphocytes are found mainly in the cortex and cortico-medullary boundary, and only rarely in the medulla.     

Differences in distribution of ribonuclease isoenzymes in cytosol and granules in normal human granulocytes and in granulocytes of patients with chronic granulocytic leukemia (CGL)

Distribution of ribonuclease (RNAase), acid phosphatase (acid Ph-ase) and beta glucuronidase (BGU) between the granule, cytosol-soluble and post-granule fractions in normal human granulocytes and in granulocytes of chronic granulocytic leukemia (CGL) was studied. CGL granulocytes were found to display relative RNAase activity 1.2 times higher, relative acid Ph-ase activity 2.5 times higher than normal granulocytes. The granule fraction of CGL granulocytes showed 1.4 times higher relative RNAase activity but 0.87 times lower acid Ph-ase activity and the same BGU activity as normal granulocytes. On the other hand, the supernatant soluble fraction of CGL granulocytes showed 4.4 times higher relative RNAase activity, 1.2 times higher relative acid Ph-ase activity and BGU 2.2 times higher than in cytosol soluble fraction of normal granulocytes. Thus, cytosol soluble fraction of CGL granulocytes show a relative activity of the lysosomal enzymes studied which is remarkably higher than in normal granulocytes. The percentage distribution of RNAase, acid Ph-ase and BGU showed that CGL granulocytes contain only 36% of total RNAase activity versus 46% of that in normal ones. On the other hand, CGL granulocytes in cytosol soluble fraction will contain 48% of total RNAase versus 29% of total RNAase in cytosol of normal granulocytes. The isoenzyme profiles of RNAase of granule fractions were similar in normal and CGL granulocytes, while the RNAase isoenzyme profiles of cytosol fractions were different for normal and CGL granulocytes, indicating that some essential part of CGL granulocyte cytosol RNAase differs from RNAase contained in granules and in cytosol of normal granulocytes.     

Terminal deoxynucleotidyl transferase, TdT, as a marker for leukemia and lymphoma cells

Terminal deoxynucleotidyl transferase, TdT, was assayed in the mononucleate cells of blood and bone marrow from 121 patients with leukemias at the onset of disease and from 95 subjects with malignant lymphomas at diagnosis. This intracellular marker was also investigated by cytoimmunofluorescent tests in 17 other cases of initial leukemias and in 3 diagnosed lymphoblastic lymphomas. Generally, the TdT levels were significantly enhanced in the blasts of the following: acute undifferentiated leukemias; the more immature types of acute lymphoblastic leukemias i.e., the null, non-T non-B, common, early T and pre-B subgroups; a fraction of blastic crises in chronic myelogenous leukemias; and many lymphoblastic lymphomas. TdT might also be slightly increased in the mononucleate blood cells obtained from the most immature forms of acute myelogenous leukemias. Relapses with changes in cell phenotypes were occasionally observed in previously TdT-positive leukemias as a result of clonal evolution of the disease. The leukemias with blasts containing high levels of TdT were usually responsive to treatment with corticosteroids and vincristine. TdT is an oligoclonal marker characterizing several populations of undifferentiated or poorly differentiated blasts that tend to develop towards or along the lymphoid pathway. Together with specific immunological markers, this enzyme is useful to define the particular type of leukemic cells. It also serves to identify the quasi-lymphoblastic nature of the malignant clone, a helpful indication for the choice of therapy.     

Phenotypic features and proliferative activity of B cell progenitors in X-linked agammaglobulinemia

In this study, we applied mAb and heterologous antisera in double marker combinations to investigate the phenotype and the proliferative activity of immature B lineage cells in XLA. Bone marrow (BM) samples from eight male adult patients with no circulating B lymphocytes were studied. The proportions and the phenotype of the earliest identifiable B cell progenitors, expressing nuclear terminal deoxynucleotidyl transferase (TdT), cytoplasmic CD22, and membrane CD19 and CD10 were identical to those observed in normal BM. In XLA these cells represented 1.2% to 22% of BM mononuclear cells; 5% to 42% and 1% to 45% of such cells weakly expressed CD20 and CD37, respectively, and invariably lacked CD13 and CD33. Cytoplasmic mu+ sIg- pre-B cells were seen in low numbers (0.1% to 0.3%) in four samples and were undetectable in the remaining four. Consequently, the ratio TdT+/c mu+ was greater than 100 in five out of eight samples studied in contrast to the less than 10 values seen in normal individuals. The proliferative activity of B lineage progenitor cells was studied by using Ki67 and anti-bromodeoxyuridine mAb. Although the proliferation of TdT+ cells in XLA was comparable with that seen in normal BM samples (24% to 59% of TdT+ were Ki67+ and 11% to 27% incorporated bromodeoxyuridine), this was dramatically reduced in the c mu+ cells (no c mu+, Ki67+ seen in three samples where pre-B cells were observed). Thus, the abnormalities of B cell differentiation in XLA are first seen at the c mu+ pre-B stage and suggest a maturation block in the transition between TdT+, c mu- pre-pre-B cells and c mu+ pre-B cells. The severity of this block may be variable, allowing the generation of a near normal number of pre-B cells in some patients, which nevertheless have a defective proliferative activity. Finally, our study further supports the concept that the effects of the "XLA gene" are confined within the B lineage by demonstrating that the proportions of T cells bearing TCR-alpha beta and TCR-gamma delta in XLA are similar to those seen in normal individuals.     

Failure of bone marrow cryopreservation in chronic granulocytic leukemia: relation to excessive granulo-macrophagic progenitor pool

Autologous bone marrow transplantation (ABMT) in chronic granulocytic leukemia (CGL) aims at reversing the acute or acceleration phases by injection of stem cells collected during the chronic phase. This study was designed to explain an unusual rate of delayed engraftment (50%) in our experience of ABMT in CGL patients. We investigated all the factors possibly responsible for abnormal perpetuation of aplasia following infusion of cryopreserved marrow stem cells. The study of CFU-gm recovery in 41 bags of frozen marrow from 25 patients revealed an overall deficiency with a mean CFU-gm recovery of 55 +/- 38% in CGL patients versus 73 +/- 15% in the control group (p less than 0.001). Our data also showed an inverse linear relation (r = -0.40, p less than 0.05) between CFU-gm concentration and recovery after freezing. A good CFU-gm recovery (greater than or equal to = 50%) was observed in 70% of cases when the concentration was less than 3700 CFU-gm/ml as compared to 30% of cases when the concentration was over 3700 CFU-gm/ml (p less than 0.001). The lack of improvement by diluting rich CFU-gm marrows to reduce CFU-gm concentration/ml, as well as the absence of relationship between CFU-gm recovery after freezing and nucleated cells concentration, suggest a particular fragility of CGL stem cells to freezing, probably related to their excessive amplification. At the present time, we strongly recommend that the highest possible dose of progenitor cells be cryopreserved, preferably at a low concentration, in patients with CGL, and particular attention devoted to the freezing procedure in each individual patient, with numerous appropriate efficiency tests.     

Primitive progenitor cells in the blood of patients with chronic granulocytic leukemia

We measured the number of blast colony-forming cells (Bl-CFC) in the blood of 11 patients with untreated chronic granulocytic leukemia (CGL). The culture system used detects three types of Bl-CFC (Types I, II and III) in normal marrow, of which Bl-CFC (I) are the most primitive and might represent the putative hemopoietic stem cell. The mean numbers of Bl-CFC (I) in CGL blood, normal bone marrow and normal blood were 134 +/- 29 (+/- SEM), 127 +/- 21 and 1.5 +/- 0 respectively per 1 X 10(6) mononuclear cells. These findings are consistent with the concept that CGL is due to a primary increase in stem cell numbers with secondary increases in committed progenitor and leukocyte numbers.     

Inhibition of terminal deoxynucleotidyltransferase by (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate

(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-triphosphate (BVdUTP), known as a specific inhibitor of herpes simplex virus (type 1)-DNA polymerase, was found to be a potent inhibitor of the activity of terminal deoxynucleotidyltransferase (TdT) from calf thymus. BVdUTP was not an efficient substrate of TdT, but it inhibited the incorporation of normal deoxynucleotide substrates in competitive fashion at the nucleotide binding site of TdT molecule. The Ki value for BVdUTP (5 microM) was much less than the Km value for dGTP (83 microM), indicating stronger affinity of the inhibitor to TdT than that of the substrate. These results indicate the usefulness of BVdUTP as a potent inhibitor of TdT for elucidation of the reaction mechanism of this enzyme.     

Mutational analysis of terminal deoxynucleotidyltransferase-mediated N-nucleotide addition in V(D)J recombination

The addition of nontemplated (N) nucleotides to coding ends in V(D)J recombination is the result of the action of a unique DNA polymerase, TdT. Although N-nucleotide addition by TdT plays a critical role in the generation of a diverse repertoire of Ag receptor genes, the mechanism by which TdT acts remains unclear. We conducted a structure-function analysis of the murine TdT protein to determine the roles of individual structural motifs that have been implicated in protein-protein and protein-DNA interactions important for TdT function in vivo. This analysis demonstrates that the N-terminal portion of TdT, including the BRCA-1 C-terminal (BRCT) domain, is not required for TdT activity, although the BRCT domain clearly contributes quantitatively to N-nucleotide addition activity. The second helix-hairpin-helix domain of TdT, but not the first, is required for activity. Deletional analysis also suggested that the entire C-terminal region of TdT is necessary for N-nucleotide addition in vivo. The long isoform of TdT was found to reduce N-nucleotide addition by the short form of TdT, but did not increase nucleotide deletion from coding ends in either human or rodent nonlymphoid cells. We consider these results in light of the recently reported structure of the catalytic region of TdT.     

Human bone marrow cells positive for terminal deoxynucleotidyl transferase (TdT), HLA-DR, and a T cell marker may represent prothymocytes

Recent evidence suggests that prothymocytes, which occur in a low frequency in murine bone marrow (BM), are already committed to thymocyte differentiation and discrete from precursor B cells as well as pluripotent hematopoietic stem cells. Furthermore, it was suggested that, in rodents, prothymocytes are positive for the nuclear enzyme terminal deoxynucleotidyl transferase (TdT) and a T cell surface antigen. The human prothymocyte has not been identified as yet. We analyzed human BM cells by double immunofluorescence staining for TdT and the T cell surface markers Tp41 (recognized by the monoclonal antibodies WT1 and 3A1), T11, T1, and T6. In the BM samples tested, neither T1+/TdT+ nor T6+/TdT+ cells were detected, but Tp41+/TdT+ and T11+/TdT+ cells were present in low frequencies. In childhood BM, the frequency was about two to five in 10,000, whereas in adult BM and regenerating BM, these cells were not always detectable, but if detected, their frequency was five- to 10-fold lower. In a triple staining, using fluorescein, rhodamine, and colloidal gold particles as labels, it appeared that all Tp41+/TdT+ cells were also positive for HLA-DR. These Tp41+/HLA-DR+/TdT+ cells were also detectable in low frequencies in the thymus, and occasionally Tp41+/TdT+ and T11+/TdT+ cells were detected in the peripheral blood (PB), suggesting a migration from the BM to the thymus via the PB. The malignant counterpart of the Tp41+/HLA-DR+/TdT+ cell was detected in a patient with acute lymphoblastic leukemia with the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- phenotype and germ-line immunoglobulin heavy chain genes. We postulate that the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- cell represents a human prothymocyte.     

Cellular immunity in insulin-dependent diabetes mellitus (type 1)

Cell-mediated immunity was investigated with T-cell blastic transformation stimulated by phytohaemagglutinin and/or insulin in patients with diabetes mellitus type 1. T-cell blastic transformation was determined in the whole blood by the intake of labelled thymidine intake by the lymphocytic DNA. Healthy individuals and patients with diabetes mellitus type 2 served as control groups. It was found that T-cell blastic transformation stimulated with phytohaemagglutinin is markedly diminished in patients with diabetes mellitus type 1 and to a lesser degree in patients with diabetes mellitus type 2. Insulin increased T-cell blastic transformation in insulin-dependent diabetic patients but has no effect in diabetes mellitus type 2. The obtained results suggest that induction and central phases of the cell-mediated immunological response are diminished in diabetes mellitus independently on its type. Such disorders may have different etiology depending on the type of diabetes mellitus.     

Decreased bone marrow iron in chronic granulocytic leukaemia: a consistent finding not reflecting iron deficiency

The iron status of 50 patients with Ph'-positive chronic granulocytic leukaemia (CGL) was evaluated at diagnosis by means of bone marrow and blood studies. A decreased or absent iron in semiquantitative estimation on bone marrow smears was observed in 92% of patients, and 88% had a low sideroblast score. In contrast, normal Hb and serum iron concentrations were found in the majority of cases, and only two out of the 50 patients displayed a decreased serum ferritin. To ascertain whether the bone marrow pattern of iron depletion could be due to an expansion of the red cell mass, the latter parameter was measured by isotopic methods in a subgroup of 11 patients. Normal or slightly increased values were obtained in all cases. We conclude that absent or decreased marrow iron is a common feature in the chronic phase of CGL, that generally does not reflect true iron deficiency. Since such a finding is also usual in polycythaemia vera and idiopathic myelofibrosis, it should be included among the features shared by the chronic myeloproliferative disorders.     

Terminal deoxynucleotidyl transferase in diagnosis of lymphomas

TdT activities were determined on 29 specimens of mononuclear blood, bone marrow or lymph node cells from 18 patients with non Hodgkin's Lymphomas, 2 Hodgkin's patients and 3 patients with non neoplastic lymph nodes. The neoplastic cells were typed using tests detecting membrane markers (E, Em, SIg), and monoclonal antibodies (MoAb). In a group of 15 patients with Low Grade Malignant Lymphoma (L. lymphocytic, centrocytic and lymphoplasmocytic) 14 cases belonged to B cell phenotype lymphoma, with 3 cases among them with a moderate TdT activity. In one case of lymphocytic lymphoma the cells had the non T, non B, TdT+ characteristics. High TdT activity was observed in both examined patients with lymphoblastic lymphoma and in cells obtained from the lymph node of one Hodgkin's lymphoma case. Although our group was of heterogenic character, our investigations confirm the value of TdT as biochemical marker of immature lymphocytes and its usefulness for differential diagnosis of malignant lymphomas.     

Relationships between terminal transferase expression, stem cell colonization, and thymic maturation in the avian embryo: studies in thymic chimeras resulting from homospecific and heterospecific grafts

Terminal deoxynucleotidyl transferase (TdT) can be detected in 11- to 12-day-old embryonic chick thymuses 5 to 6 days after the first influx of lymphoid stem cells into the thymic rudiment. To identify the main factors of TdT induction, grafting experiments were devised in such a way that the age of the grafted thymus and that of the host were different. Uncolonized embryonic chick thymuses were grafted into chick hosts of different ages. Under these conditions, lymphoid differentiation arose from host lymphoid stem cells (LSC) invading the thymic rudiment. TdT immunofluorescent detection in the first wave of thymocytes showed that the percentages of TdT+ cells were related to the total age of the explant and not to the age of the host (11 to 17 days). Similar results were obtained when the chick thymic rudiment was transplanted into quail embryos, showing that quail LSC have TdT inducibility similar to that of chick LSC while developing in a chick thymic environment. Colonized chick thymuses were also grafted into quail embryos to compare the TdT inducibility of the first lymphoid generation (of chick type) and of the second (of quail origin), taking advantage of the different chromatin structure of quail and chick cells. In these experiments, the majority of chick cells remained TdT negative for as long as 10 days, whereas most lymphocytes of the second generation became TdT+ soon after their arrival in the grafted thymus. Therefore, during embryonic life, most TdT+ cells were derived from the second wave of stem cells, but some early stem cells were also able to acquire the enzyme. In a final series of experiments, early thymic rudiments were cultured in vitro with 14- to 16-day-old bone marrow and then grafted into 3-day-old host embryos. Under these conditions, bone marrow LSC contributed to a variable proportion of the first generation of thymocytes. The percentage of TdT+ cells among the progeny of these bone marrow stem cells was found to be two times higher than that of thymocytes derived from host LSC. These results suggest that, in addition to intrathymic environmental factors, the origin of LSC influences the frequency of TdT expression in their progeny.     

Kinetic properties of polymorphic variants and pathogenic mutants in human cystathionine gamma-lyase

Human cystathionine-gamma-lyase (CGL) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme, which functions in the transsulfuration pathway that converts homocysteine to cysteine. In addition, CGL is one of two major enzymes that can catalyze the formation of hydrogen sulfide, an important gaseous signaling molecule. Recently, several mutations in CGL have been described in patients with cystathioninuria, a rare but poorly understood genetic disease. Moreover, a common single nucleotide polymorphism in CGL, c.1364G>T that converts serine at position 403 to isoleucine, has been linked to elevated plasma homocysteine levels. In this study, we have characterized the pathogenic T67I and Q240E missense mutations and the polymorphic variants at amino acid residues 403 using kinetic and spectrophotometric methods. We report that the polymorphism does not influence the cofactor content of the enzyme or its steady-state kinetic properties. In contrast, the T67I mutant exhibits a 3.5-fold decrease in V max compared to that of wild-type CGL, while the Q240E mutant exhibits a 70-fold decrease in V max. The K Ms for cystathionine for both pathogenic mutants are comparable to that of wild type CGL. The PLP content of the T67I and Q240E mutants were about 4-fold and 80-fold lower than that of wild-type enzyme, respectively. Preincubation of the T67I mutant with PLP restored activity to wild-type levels while the same treatment resulted in only partial restoration of activity of the Q240E mutant. These results reveal that both mutations weaken the affinity for PLP and suggest that cystathionuric patients with these mutations should be responsive to pyridoxine therapy.