Antigenic relationships in calf thymus and human leukemic cell terminal deoxynucleotidyl transferase.

Antibody to purified terminal deoxynucleotidyl transferase (nucleosidetriphosphate : DNA deoxy-nucleotidylexotransferase, E.C. 2.7.7.31) from calf thymus was prepared in rabbits using terminal deoxynucleotidyl transferase crosslinked to bovine serum albumin. These antibodies, partially purified by 60% ammonium sulfate precipitation and Sephadex G-200 column chromatography, produced one precipitation band with calf thymus terminal deoxynucleotidyl transferase on immunodiffusion. This antibody preparation also inhibited the in vitro activity of terminal deoxynucleotidyl transferase from calf thymus, acute leukemic lymphoblasts and Molt-4 cells but not that of DNA polymerases alpha, beta and psi from these cells     

High-level expression of murine terminal deoxynucleotidyl transferase inEscherichia coli grown at low temperature and overexpressingargU tRNA

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5′-triphosphates onto the 3′-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production inEscherichia coli of murine TdT with minimal proteolysis and high specific activity.     

Cyclic AMP induces terminal deoxynucleotidyl transferase in immature B cell leukemia lines

Terminal deoxynucleotidyl transferase (Tdt), a unique DNA polymerase found only in lymphoid cells, may be involved in the generation of immunoglobulin-combining site diversity. To study the actual metabolic function of the enzyme, we developed a system in which Tdt expression is induced under defined culture conditions. We found that pharmacologic agents that raise intracellular cyclic AMP levels, such as caffeine, induce a three- to 10-fold increase in enzyme biosynthesis rate and activity. This phenomenon is observed only in pre-B cell lines of human or murine origin.     

Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector.

The role of terminal deoxynucleotidyl transferase (TdT) in the insertion of N regions into the junctional sites of immunoglobulin genes was investigated. Pre-B-cell lines capable of continuous rearrangement of immunoglobulin light-chain genes and differing only in the presence or apparent absence of TdT were derived by infecting cells with a TdT retroviral expression vector or a control vector. The cell lines were then superinfected with a retrovirus-based artificial immunoglobulin gene rearrangement substrate. The substrate was allowed to rearrange in the cell lines and the rearranged proviruses were rescued from the cell lines. Nucleotide sequence analysis of the V-J junctions of the proviral rearranged genes showed a fivefold greater frequency of N-region insertion in proviruses rescued from the TdT+ cell lines than in those rescued from the TdT- cell lines, so that at least 50% of the rearrangements that occurred in the presence of TdT had N regions. It is thus evident that TdT can stimulate N-region insertion, and the enzyme is presumably directly responsible for adding nucleotides at V-J and other immunoglobulin and T-cell receptor gene junctions.     

Expression of terminal deoxynucleotidyl transferase in human thymus during ontogeny and development

Expression of the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in human thymus during ontogeny and development. In five fetal thymus samples, the enzyme activity was barely detectable. At birth, the terminal transferase activity remained low. Maximum expression of the enzyme activity occurred between 10 and 40 mo of age. Analysis of six other enzyme activities, adenosine kinase, deoxyadenosine kinase, AMP deaminase, dAMP deaminase, 5' nucleotidase, and adenosine deaminase confirmed the normal status of the thymic tissue. A careful analysis of thymic architecture revealed that involution did not occur as a result of the disease process that necessitated cardiac surgery. By immunofluorescence, the TdT antigen was localized exclusively in the nucleus of cortical thymocytes. Protein immunoblotting studies indicated that human thymic terminal transferase exists as a single high m.w. species in individuals under 30 mo of age. Thereafter, a variant m.w. species is detectable. The increase in expression of this enzyme coincides with the increase observed in serum immunoglobulin levels during maturation and precedes the maximum development of the human thymus.     

Polyamines and terminal deoxynucleotidyl transferase expression In KM 3 pre-B cell line during phorbol ester induced differentiation.

The aliphatic polyamines, putrescine, spermine and spermidine belong to a category of molecules implicated in DNA replication. Their synthesis is strongly activated during the G₁ period and they have been implicated in the regulation of cell proliferation and differentiation. Terminal transferase is a DNA polymerase present in pre-T and pre-B cells and its expression can be modulated by phorbol ester treatment. In this study we have monitored the relationship of intracellular polyamine levels with terminal deoxynucleotidyl transferase down-regulation induced by 12-0-tetradecanoyl phorbol myristate 13-acetate treatment in the human pre-B KM-3 cell line. Phorbol myristate acetate can cause an increase, at 4 and 8 hours of differentiation, of intracellular levels of putrescine as well as a decrease in terminal deoxynucleotidyl transferase synthesis showing the probable involvement that polyamines have in the differentiation process.     

Stage-specific induction of terminal deoxynucleotidyl transferase in a T-lymphoid line upon coculture with a thymic stromal line

We previously reported an in vitro T-cell differentiation system in which the L4 lymphoid clone was cocultured with the St3 stromal line derived from the same murine thymic tumor, 15#4T. L4 cells in L4—St3 cocultures sequentially express Thy-1 and CD4 in a manner typical of normal thymocytes. In contrast, L4 cells grown in medium alone retain their Thy-1⁻CD4⁻ phenotype. We also isolated L4 subclones from the coculture with increasingly differentiated phenotypes with respect to Thy-1 and CD4. We now report induction of an additional thymocyte differentiation marker, terminal deoxynucleotidyl transferase (TdT) in 15#4T cells (and to a lesser extent subcloned L4 cells) upon coculture with St3 stroma. Coculture of 15#4T cells with St3 stroma resulted in expression of TdT as measured by ribonuclease protection for TdT RNA and Western immunoblotting for TdT protein. Cocultured L4 cells were induced for TdT expression to a lesser degree and for a shorter period of time. The magnitude of TdT RNA induction was maximal for cell lines with the least mature differentiation phenotype (15#4T and L4: Thy-1⁻CD4⁻) and decreased proportionally for subclones with increasingly mature phenotype, e.g., L4E cells (Thy-1⁺CD4⁺). TdT protein was undetectable by Western immunoblotting and immunofluorescent staining of the L4E subclone on or off stroma. Recombination-activating gene-1 (RAG-1), which is expressed in immature thymocytes during T-cell receptor rearrangement, but suppressed in mature thymocytes, was also examined using the ribonuclease protection assay. In contrast to TdT, RAG-1 expression was suppressed by coculture with St3 cells for 15#4T and also more mature subclones, indicating regulation by a mechanism independent from TdT. The ordered induction of TdT, Thy-1, and CD4, as well as regulation of RAG-1 in the 15#4T-St3 system, supports the conclusion that this in vitro system is a valuable model for characterizing regulation of these markers in normal thymocytes.     

Distribution of terminal deoxynucleotidyl transferase and purine degradative and synthetic enzymes in subpopulations of human thymocytes

Terminal deoxynucleotidyl transferase (TdT) and purine metabolic enzymes were examined in subsets of human infant thymocytes (defined by surface cell antigens) and normal peripheral T lymphocytes. Putative prothymocytes (RFB-1+, HTA-1+/- large blast-like cells), medium and high density cortical thymocytes (RFB-1+, HTA-1+), and medullary thymocytes (RFB-1-, HTA-1-, OKT3+) were isolated by density gradient centrifugation, monoclonal antibody and complement-mediated cytolysis, and cell-antibody affinity chromatography. Peripheral T lymphocytes were isolated from normal adult mononuclear cells using nylon fiber technique. Adenosine deaminase (ADA) and TdT were highest in prothymocytes 48.8 +/- 14.7 mumol/hr/10(8) cells (mean +/- SE) and 22.9 +/- 1.4 U/10(8) cells, respectively. Both enzymes decreased progressively down the maturation pathway. In peripheral T lymphocytes, ADA was 3.9 +/- 1.5 mumol/hr/10(8) cells, and TdT was undetectable. Purine nucleoside phosphorylase (PNP) and ecto-5'nucleotidase (5'NT) were lowest in cortical thymocytes (27.5 +/- 11.0 nmol/hr/10(6) cells and 2.8 +/- 1.3 nmol/hr/10(6) cells, respectively) and increased with T cell maturation. The PNP level was 124.9 +/- 17.2 nmol/hr/10(6) cells and 5'NT was 30.1 +/- 3.9 nmol/hr/10(6) cells in peripheral T lymphocytes. The deoxynucleoside kinases (deoxyguanosine, deoxyadenosine, and deoxycytidine kinases) paralleled the changes in ADA and TdT activity among the different T subsets. The proliferative activity (labeling index) was highest in the prothymocyte fraction and lowest in peripheral T cells. Variation in the distribution of these enzymes in T cell subsets may explain their different sensitivities to deoxyadenosine and deoxyguanosine toxicity and the different effects on T cell development of ADA or PNP deficiency.     

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.     

Cyclic AMP modulates interleukin-1 action in a cytotoxic T-cell hybridoma

The induction of cytolytic activity in PC60, a murine T-cell hybridoma, is paralleled by a rise in the level of BLT-esterase (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase), a serine esterase specific for activated T-cells. Both interleukin-1 (IL-1) and dibutyryl cAMP were albe to increase the esterase activity in a dose-dependent and saturable manner. When added in combination the two activators showed a strong synergism: BLT-esterase levels were up to three times higher than the sum of the levels due to dibutyryl cAMP and IL-1 added separately. Stimulators of the adenylate cyclase, such as forskolin and cholera toxin, induced a similar enhancement of the BLT-esterase response to IL-1. PC60 cells did not produce any cAMP in response to IL-1. When the two stimuli were added sequentially a second effect for cAMP emerged: preincubation with dibutyryl cAMP or activators of the adenylate cyclase for 4 h or longer completely blocked the action of subsequently added IL-1. Taken together, the data demonstrate a dual modulatory role for cAMP in T-lymphocytes activated by IL-1.