Ontogeny of terminal deoxynucleotidyl transferase-positive cells in lymphohemopoietic tissues of rat and mouse.

The ontogeny of hemopoietic cells which contain the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in rats and mice. During fetal life, TdT-positive cells were first detected in the thymus, where they appeared on or about day 17 of gestation. TdT-positive cells were not found in fetal liver, spleen, or bone marrow, but appeared in bone marrow and spleen on the day after birth. In the rat, peak levels of TdT-positive cells were attained at 3 to 4 weeks of age in thymus, bone marrow, and spleen, accounting for 67, 3.9, and 2.3% of nucleated cells, respectively. The percentages of TdT-positive cells in thymus and bone marrow decreased gradually thereafter, whereas, TdT-positive cells in spleen were no longer detectable by 7 weeks of age. Normal percentages of TdT-positive cells were found in bone marrow and spleen from neonatally thymectomized rats and congenitally athymic (nu/nu) mice. Dexamethasone treatment resulted in a marked decrease in TdT-positive cells. The results are discussed with respect to the putative role of TdT-positive hemopoietic cells as thymocyte progenitors.     

Distribution of terminal deoxynucleotidyl transferase in bovine serum albumin gradient-fractionated thymocytes and bone marrow cells of normal and leukemic mice.

The distribution of terminal deoxynucleotidyl transferase (TdT) peaks I and II, in single cell suspensions of thymuses, bone marrow, and peripheral lymphoid organs fractionated in discontinuous bovine serum albumin gradients, was examined in a variety of mouse strains and Fischer 344 rats to relate the normal patterns of thymocyte differentiation to the leukemic process. TdT peaks I and II were found in fractions A (10 to 23%), B (23 to 26%), and C (26 to 29%) of the thymus of both normal and leukemic C57BL/6 mice, whereas only peak I was found in the same fractions of AKR mice. TdT in bone marrow was found mainly in fraction A in both normal and leukemic mice. The specific activity of TdT in this fraction, which comprises only 1 to 5% of the total bone marrow cell population, was similar to that of the thymus. The cell population of fraction A of the bone marrow was found to increase (10 to 15-fold) in leukemic mice. Only low levels of TdT activity were found in either whole or fractionated bone marrow of athymic NIH Swiss (nu/nu) mice.     

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.     

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.     

The distribution of terminal deoxynucleotidyl transferase (TdT) among subsets of thymocytes in the rat.

Terminal deoxynucleotidyl transferase (TdT), a unique DNA-polymerizing enzyme,has been shown to be present in a moderately dense subpopulation of rat thymocytes separated on discontinuous Ficoll density gradients. This subpopulation has been characterized by using antigenic and functional markers to identify directly and quantify cortical and medullary thymocytes. The TdT-positive thymocytes are depleted by cortisone administration, lack responsiveness to phytohemagglutinin, concanavalin-A, and histocompatibility alloantigens, bear surface antigens characteristic of cortical thymocytes (bone marrow lymphocyte antigen) and lack surface antigens characteristic of medullary thymocytes (rat-masked thymocyte antigen and histocompatibility antigens). The results indicate that TdT is present exclusively (or in markedly higher concentrations) in a subset of cells which comprised about 65% of cortical thymocytes. Two other major subsets of cortical thymocytes were identified which appeared to be TdT-negative. A minor subset of very low density cortical thymocytes was also defined. These observations have provided insight into the possible pathways of thymocyte ontogeny.     

In vitro studies on lymphocyte differentiation. II. Generation of terminal deoxynucleotidyl transferase-positive cells in long-term cultures of mouse bone marrow.

The enzyme TdT is the earliest known marker of lymphocytic differentiation in rodents. Cells containing this enzyme were demonstrated in suspension cultures of mouse bone marrow cells that had been maintained in vitro for periods of 7 to 45 days. The cells were detected by immunofluorescence using purified antibodies to homogeneous TdT. Between 0.04 and 2.0% of cultured bone marrow cells from a variety of mouse strains were positive. More than 40% of the TdT-positive cells incorporated 3H-thymidine during a 20-min pulse. Surface Ig and Thy-1 antigens were not detected on the TdT-positive cells. The prevalence of TdT-positive cells was decreased 10-fold in cultures that had been treated with 10(-6) M hydrocortisone 24 hr before harvesting. The results indicate that lymphoid progenitor cells can be generated in vitro.     

Developmental abnormalities of terminal deoxynucleotidyl transferase positive bone marrow cells and thymocytes in New Zealand mice: effects of prostaglandin E1

The enzyme TdT was used as a marker with which to study the ontogeny of primitive lymphopoietic cells in NZ strain mice. A marked accumulation of abnormally large, rapidly proliferating TdT+ cells was seen in the subcapsular region of the thymus cortex in the NZB and NZB/W mice. This abnormal accumulation of TdT+ thymocytes was most pronounced in the NZB/W hybrid and persisted for at least the first 16 wk of life. In addition, significantly elevated percentages of TdT+ bone marrow cells (presumptive prothymocytes) were present in NZB, NZW, and NZB/W mice between 1 and 4 wk of age, with the highest mean peak levels occurring in the NZB strain. Treatment of both normal and adrenalectomized BALB/c and NZB/W mice with pharmacologic doses (7 to 10 mg/kg) of PGE1 caused a marked, dose-dependent decrease in thymus weight and thymus cell number within 12 to 18 hr. Histologic and cell separation studies showed that this was due to the selective depletion of PNA+ TdT+ cortical thymocytes. Similarly, PGE1 caused a reversible, dose-dependent decrease in the percentage of TdT+ bone marrow cells. In contrast, PGF2 alpha, which is not therapeutically active against autoimmunity in NZB/W mice, had no detectable effect on TdT+ bone marrow cells or thymocytes in BALB/c or NZB/W mice. These results directly document the existence of abnormalities in the development of lymphopoietic precursor cells in the bone marrow and thymus cortex of NZ strain mice prior to the onset of autoimmune phenomena. The results also raise the possibility that the therapeutic efficacy of exogenous PGE1 in autoimmune NZ strain mice may be related, at least in part, to its ability to rectify the abnormal development of these early lymphoid cells.     

Non-germ-line elements (NGE) are present in the T cell receptor beta-chain genes isolated from the mutant mouse, motheaten (me/me)

Extra nucleotides (which we call NGE, for non-germ-line elements) are inserted at the junctions of rearranged V, D, and J segments in the immunoglobulin heavy and T cell receptor beta-chain V region genes. NGE addition helps diversity HV3 regions of these genes. It is believed that NGE are added enzymatically and without template during the joining process. Terminal deoxynucleotidyl transferase (TdT) is thought to be the cause of NGE formation. TdT is normally detected in murine thymus and bone marrow cells, but its presence in the immunodeficient mutant mouse, Motheaten (me/me), is extremely reduced in these tissues. To determine whether this TdT deficiency could affect NGE formation during the V-D-J joining of antigen receptor genes, we cloned several rearranged T cell receptor beta-chain genes from thymocytes of me/me mice. Our sequence analysis revealed that Motheaten thymus beta-chain genes have approximately 4 base pair NGE, which are comparable in size to the NGE of wild-type genes. These results do not support the idea that TdT is the NGE-forming enzyme, although it is still possible that a low but residual level of TdT is capable of NGE formation in Motheaten. Alternatively, our data may suggest that the TdT activity in Motheaten T cells is normal; however, the number of TdT+ cells is greatly reduced in the Motheaten thymus, due to a severe defect in T cell development.     

Modulation of in vitro myelopoiesis by LGL: different effects on early and late progenitor cells

We describe here the modulatory activity of human peripheral blood natural killer (NK) cells on the growth and differentiation of myeloid progenitor cells at different stages of maturation. NK-enriched cell fractions containing 54 to 75% large granular lymphocytes (LGL) and displaying high levels of NK activity significantly inhibited the growth of late (7 day) granulocyte-macrophage colony-forming cells (CFU-GM) from about 50% of normal human bone marrow samples. However, the same fractions strongly enhanced the growth of early (14 day) stem cells from peripheral blood. Enhancing activity on early CFU-GM from blood was greater in highly purified NK cell preparations containing 96% LGL than in NK-depleted T cell preparations from the same donors. Analogous to the results when using the NK-enriched fractions, the NK-purified preparations inhibited late CFU-GM and stimulated the early ones. We conclude from these observations that human LGL have a modulatory effect on myelopoiesis depending on the maturation stage of the progenitor cell.     

Response of lympho-hemopoietic tissue to Corynebacterium parvum by study of DNA enzymes and H3-TdR metabolism.

The effects of Corynebacterium parvum on the lymphohemopoietic tissues of mice was investigated by H3-TdR metabolism, organ weights, DNA-polymerase-alpha activity and terminal deoxynucleotidyl transferase (TdT) activity. Marked increase in spleen size occurred. The increase in size was accompanied by increases in DNA-P-alpha activity and H3TdR uptake. This indicated that the spleen was a major site of cell proliferation and of increase in population size after C. parvum stimulation. Thymus and bone marrow changes are also described. The thymus showed a marked decrease in size which was maximal in 10 days and showed recovery thereafter. Thymus TdT activity fell immediately and may reflect either a release or lysis of thymocytes which contain TdT activity. However, no change in TdT activity was measurable in the bone marrow or spleen. The principal cell population increase had phagocytic activity and was presumably monocytes-macrophages, and an increase in such cells was found in the spleen.     

A critical comparison of commonly used procedures for the assay of terminal deoxynucleotidyl transferase in crude tissue extracts.

Terminal deoxynucleotidyl transferase (TdT) is a non-template directed DNA polymerase normally found in vertebrate thymus and bone marrow. Quantitative assay of TdT activity is being widely used as a tool in the differential diagnosis of acute leukemias in man. Clinical specimens of blood and bone marrow often contain 10(7) or fewer cells and require a specific and rapid assay for transferase which can be carried out in crude cell extracts. Commonly used assay methods do not meet these requirements, but can be easily modified to do so.     

The terminal deoxynucleotidyltransferase gene is located on human chromosome 10 (10q23----q24) and on mouse chromosome 19

Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase expressed in immature lymphocytes of the thymus and bone marrow, as well as certain leukemic cells. Chromosomal assignment of the gene coding for human TdT was accomplished by in situ hybridization of a 3H-labeled cDNA probe to human chromosome preparations and by Southern blot analysis of somatic cell hybrid DNAs. The human TdT gene was mapped to the region q23----q24 of chromosome 10. Breaks at this site have been reported in different translocations in human leukemias. The mouse TdT gene was assigned to chromosome 19 by Southern blot analysis of mouse X Chinese hamster somatic cell hybrids. This result adds a fourth locus to the conserved syntenic group on mouse chromosome 19 and human chromosome 10.     

Release of immature cells from the thymus during solid tumor growth: identification by assay of TdT activity.

In vivo anti-tumor activity of spleen cells from C3H/eb mice bearing a syngeneic fibrosarcoma was shown previously to decline to an undetectable level and be replaced by tumor-enhancing activity as tumor growth proceeds. In the light of our findings that thymocytes in the early stages of thymic processing can bring about tumor enhancement, we postulated that premature release of thymocytes and their accumulation in the spleen might account for the loss of the anti-tumor response. In the present experiments an injection of thymocytes did in fact cancel the anti-tumor response of reactive splenocytes from tumor-bearing mice. In order to determine whether premature thymocyte release occurs naturally in the tumor-bearing animals, we assayed activity of the enzyme TdT (as a marker for thymus cells) in the spleens of these mice during progressive tumor growth. Cells with TdT activity were clearly evident in the spleens of the tumor-bearing animals, were derived from the thymus, and accumulated in parallel to the loss of anti-tumour reactivity.     

The kinetics of terminal deoxynucleotidyl transferase-positive cells in the mixed colony assay

Human bone marrow was treated with cytolytic monoclonal antibodies BA-1, BA-2 and BA-3 and examined for terminal deoxynucleotidyl transferase-positive (TdT+) cells during culture in the mixed colony assay. The kinetics of TdT+ cells was compared with untreated bone marrow controls over a 14-day culture period. In control cultures a progressive decline in the number of TdT+ cells occurred during the first three days and by day 4 no TdT+ cells were observed. In antibody-treated cell cultures in which all or the majority of TdT+ cells were removed, no TdT+ cells were detectable after 24 h. Both control and antibody-treated cultures showed comparable colony formation from myeloid, erythroid and multipotential progenitor cells by day 14. The results therefore suggest that the mixed colony assay does not support the growth of TdT+ cells or the production of TdT+ cells from TdT- precursors.     

Post-irradiation thymocyte regeneration after bone marrow transplantation. II. Physical characteristics of thymocyte progenitor cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H x AKR)F1 bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, the thymocyte progenitor cells show the same distribution as CFUs. The relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

Defective lymphopoiesis in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mutant mice. II. Description of a microenvironmental defect for the generation of terminal deoxynucleotidyltransferase-positive bone marrow cells in vitro

We have presented evidence in a previous paper that the development of prothymocytes, pre-B cells, and TdT+ lymphoid precursor cells in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mice is defective. In the present study, we have used a selective culture system that supports the generation of rat- and mouse-origin TdT+ bone marrow lymphoid cells in vitro to further investigate the early stages of lymphopoiesis in me/me and mev/mev mice. The results demonstrate that bone marrow stromal cell feeder layers derived from me/me and mev/mev mice do not support the growth of rat TdT+ cells in vitro, whereas stromal cell feeder layers from heterozygous (+/-) littermates and wild type (+/+) control mice do. Moreover, composite feeder layers formed by mixing as few as one part me/me and mev/mev bone marrow cells with 7 to 9 parts +/- littermate bone marrow cells also fail to effectively support the generation of TdT+ cells in vitro. In contrast to me/me and mev/mev mice, other mutant mouse models of autoimmune (NZB, NZB/W), immunodeficient (nu/nu), and hemopoietic (W/Wv, Sl/Sld) disorders form feeder layers that support normal to elevated levels of TdT+ cell growth in vitro. Thus, to date, only the me/me and mev/mev mutant mice have been found to lack the appropriate microenvironment for the generation of TdT+ bone marrow cells. Histologic analysis of the stromal cell feeder layers that are formed in our culture system shows that multilayered cellular patches, which normally are the most active sites of TdT+ cell development in vitro, are absent in feeder layers of me/me and mev/mev cells. Moreover, feeder layers from mev/mev mice contain a population of MAC 1+, basophilic, nonvacuolated, macrophage-like cells; whereas feeder layers from control mice contain MAC 1+, eosinophilic, vacuolated macrophage-like cells. Stromal cell feeder layers formed by mixtures of me/me or mev/mev and control mouse bone marrow cells contain numerous multilayered cellular patches and vacuolated mononuclear cells, but also contain large numbers of basophilic mononuclear cells. These composite feeder layers have a disproportionately reduced capacity to support the generation of TdT+ cells in vitro. Although the stromal microenvironment of me/me and mev/mev bone marrow does not support the growth of TdT+ cells in vivo or in vitro, the bone marrow from these mutant mice contains detectable numbers of pre-TdT+ cells. Thus, when cultured on normal mouse feeder layers, mutant mouse bone marrow rapidly generates TdT+ cells in vitro, albeit at significantly reduced levels as compared to +/- littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

Post-Irradiation Thymocyte Regeneration After Bone Marrow Transplantation Ii. Physical Characteristics of Thymocyte Progenitor Cells

Bone marrow cells were separated according to buoyant density, velocity sedimentation and cell surface charge. Fractionated (C3H × AKR)F₁ bone marrow cells were transplanted into lethally-irradiated C3H recipients. In all fractions, the CFUs content and the capacity to restore the thymus cell population were determined. For all the physical parameters tested, thymocyte progenitor cells show the same distribution as CFUs. the relationship between number of thymocyte progenitor cells and number of CFUs is dependent on density. Bone marrow progenitors of PHA responsive cells are of low buoyant density and show a distribution which resembles the distribution of the progenitors of Thy 1 positive cells. After transplantation of large numbers of bone marrow cells into irradiated mice, no significant change in the CFUs content of the thymus was observed.     

The cytoplasmic expression of CD3 antigens in normal and malignant cells of the T lymphoid lineage

Anti-CD3 (T3) Ab reacting with different proportions of thymocytes (anti-CD3a: UCHT1, anti-CD3b: T10B9, and anti-CD3c: OKT3) were tested for cytoplasmic (cCD3) and membrane (mCD3) expression in the bone marrow, thymus, and blood in man and selected primates. The expression of cCD3a and cCD3c in the perinuclear and Golgi area of large, BrdU-incorporating, strongly TdT+ thymic blasts probably represents one of the earliest signs of T cell commitment, because these blast cells are CD1-, CD4-, CD8-, and mCD3-. The cCD3+, TdT+ cells are normally restricted to the thymus and are absent among the TdT+ cells of bone marrow. The anti-CD3b Ab used, T10B9, co-caps and co-modulates with the other anti-CD3 Ab and is a T cell-specific reagent at a membrane level but does not bind to perinuclear cCD3. Instead, this reagent cross-reacts with a filamentous cytoplasmic network in non-T cells in man and in primates S. oedipus and M. rhesus despite their T cell negativity for mCD3. The characteristics of all T-ALL cases studied: cCD3+, CD7+ along with nuclear TdT+ suggest lineage fidelity to early thymic blasts. As a marked contrast, cCD3 is absent in common ALL and in AML, including cases that concomitantly express CD7 and myeloid antigens. Thus, the cCD3, TdT combination provides a very sensitive assay for residual T-ALL blasts outside the normal thymus.     

Evidence for the cellular origin of Gross virus-induced leukemia in the rat: description of a unique LDH isozyme sub-band in leukemic lymphoid cells and lymphohemopoietic precursor cells

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross-leukemia virus (RAGV) were analyzed for a variety of differentiation markers to define their differentiation state and possible cellular origin. A majority of thymocytes from leukemic rats had the phenotypic characteristics of subcapsular cortical thymocytes that are the most ancestral of the thymocytes. These cells exhibited readily detectable levels of Thy-1 and histocompatibility antigens on their surfaces, they contained terminal deoxynucleotidyl transferase (TdT) and they contained low adenosine deaminase (ADA) and high purine nucleoside phosphorylase (PNP) specific activity. The leukemic thymocytes also contained a sub-band of the LDH-5 isozyme (LDH-5') that was not detected in normal thymocytes but that was present in lymphocyte-rich fractions of postnatal bone marrow, fetal and prepubertal spleen, and fetal and neonatal liver. The tissue distribution and ontogeny of LDH-5'-containing cells is similar to prethymic TdT+ cells in the rat and both TdT and LDH-5' are enriched in a subset of bone marrow "null" cells. These results suggest that TdT+ thymocyte progenitors or their precursors are the targets of leukemic transformation of RAGV.