Expression and modulation of progesterone induced blocking factor (PIBF) and innate immune factors in human leukemia cell lines by progesterone and mifepristone.

Progesterone (P), required for successful pregnancy, influences autoimmune, infectious, and malignant diseases via adaptive and innate immune effects. P induces NK inhibitor progesterone induced blocking factor (PIBF) in CD8+ T cells. PIBF isoforms could permit solid tumor immune escape. Expression and modulation of PIBF and innate immune proteins by P in leukemia cells and leukocyte subpopulations have not been reported. Ten T, seven myeloid, six B, five epithelial, fibroblast BG9, G-CSF mobilized CD34+ stem cells, and peripheral blood mononuclear cells were screened for PIBF mRNA by RT-PCR, and protein by immunohistochemistry in SRIK-NKL, MOT, U937, HL60, R-CLL, MD-E, 729pH6neo, SRIH-B(ATL), SRIK-B(T-PLL), and MeWo. Cell lines expressing PIBF and exemplifying myeloid/monoblast, natural killer/T, and B lineages were cultured with and without 0.5 - 5 microM P or 0.5 - 0.05 microM mifepristone (RU486) for 24 h. Subsequently they were examined for changes in the expression of mRNA by RT-PCR and protein by immunohistochemistry for PIBF and some innate immune factors. All cells expressed PIBF mRNA; protein only in four (SRIK-NKL, U937, SRIK-B(T-PLL) and HL60) out of 10 cell lines tested. P increased and RU486 decreased PIBF in U937, SRIK-B(T-PLL) and SRIK-NKL. P upregulated TLR-4 in U937, and HNP1 - 3, LL-37, IRAK-2, and IRAK-4 in multiple lines and RU486 down regulated these. PIBF may be used by some leukemias to evade immune surveillance and is a potential therapeutic target. P may impact infection and autoimmunity via effects on LPS receptor, TLR signaling, and antimicrobial peptides.

Inhibition of deoxynucleotide-polymerizing enzyme activities of human leukemia lymphoblasts and simian sarcoma virus by tilorone and thirteen of its analogs.

Tilorone, which is 2,7-bis[2-(diethylamino)ethoxy]-9H-fluoren-9-one dihydrochloride, and 13 of its analogs inhibited human cellular DNA polymerases alpha and beta assayed with activated DNA as template and also cellular DNA polymerase gamma and DNA polymerase from simian sarcoma virus assayed with poly(A) (dT)12-18 as template. Terminal deoxynucleotidyltransferase (TdT), which has no template requirement, was not inhibited by any of the 14 compounds when d(A)12-18 or d(G)12-18 was used as initiator. Three compounds did not inhibit TdT assayed with activated DNA as initiator, but 11 compounds did, and these 11 compounds were generally less inhibitory to TdT than to the other DNA polymerases. The three compounds that did not inhibit TdT assayed with activated DNA but did inhibit the other DNA polymerases will be useful in the characterization of TdT activity. Modifications of the polycyclic ring structure of tilorone and the kinds of substituent groups attached to the ring structures influenced the degree of inhibition of all enzymes.

High terminal deoxynucleotidyl transferase activity in a new T-cell line (RPMI 8402) of acute lymphoblastic leukemia origin.

High activity of terminal deoxynucleotidyl transferase (terminal transferase) was found in a new "thymus-dependent" cell line (RPMI 8402) which is of acute lymphoblastic leukemia origin. This enzyme resembled the terminal transferase from other human cells in all its properties including Km (0.7 x 10(-6) m for dGTP). The high activity of this enzyme in RPMI 8402 and fresh acute leukemia lymphoblasts, in contrast to the low activity of this enzyme reported for "thymus-independent' cells, suggested that this cell line may have originated from leukemia cells. Moreover, the high activity of terminal transferase in RPMI 8402 cells should make feasible large-scale purification of this enzyme for detailed studies.

Deoxynucleotide-polymerizing enzyme activities in T- and B-cells of acute lymphoblastic leukemia origin.

All 5 thymus-dependent cell (T-cell) lines (Molt-3; Molt-4; RPMI-8402; CCRF-CEM; CCRF-HSB-2) and 7 thymus-independent cell (B-cell) lines (RPMI-8382, RPMI-8392, RPMI-8412, RPMI-8422, RPMI-8432, RPMI-8442, CCRF-SB) established so far from acute lymphoblastic leukemia patients were examined for deoxynucleotide polymerizing enzymes. All T- and B-cells had DNA polymerase gamma, DNA polymerase beta, and terminal deoxynucleotidyl transferase both in the soluble (the latter 2 enzymes only in small amounts) and chromatin fraction, whereas DNA polymerase alpha was found only in the soluble fraction. With respect to their sedimentation and chromatographic behavior, template-primer requirements, Km for deoxythymidine triphosphate or deoxyguanosine triphosphate divalent cation preference, effect of NaCI and inhibitors, the enzymes from T- and B-cells resembled each other and those from other mammalian cells. DNA polymerase alpha, beta, and gamma from T-cells like those from "fresh" acute lymphoblastic leukemia cells, were more thermolabile than those from B-cells or phytohemagglutinin-stimulated normal lymphocytes. In addition, the terminal deoxynucleotidyl transferase from the above cells was completely inactivated in 5 to 6 min at 50 degrees, whereas the DNA polymerase alpha, beta, and gamma retained considerable activity even after heating for 25 min at 50 degrees. DNA polymerase activity of the soluble fraction from T-cells was of the same magnitude as in B-cells when expressed on a DNA basis but twice that of B-cells when expressed on a protein basis. High terminal deoxynucleotidyl transferase activity, equivalent to that observed in acute lymphoblastic leukemia cells, was found in all T-cell lines that, when expressed on a DNA basis, was 30 to 100 times higher than the B-cell lines tested. These results support the suggestion of earlier investigators that T-cell lines examined here may have originated from leukemic cells.

Effect of bleomycin on deoxynucleotide-polymerizing enzymes from human cells.

DNA polymerases alpha and beta from Molt-4 cells are inhibited by bleomycin, whereas DNA polymerase gamma assayed with poly-(A)-(dT)12-18 as the template primer or terminal deoxynucleotidyl transferase assayed with activated DNA, poly(dA), (dG)12-18 or (dA)12-18 as the initiator are not inhibited by this antibiotic. Inhibition by bleomycin increased the Km for template DNA but not that for dTTP. Increasing amounts of bleomycin did not affect the Vmax for DNA polymerase alpha or beta when the amount of template DNA was varied but it reduced the Vmax for these enzymes when dTTP was varied. Moreover, the addition of extra template reversed the bleomycin inhibition but the addition of extra enzyme did not. Although dithiothreitol was required for bleomycin inhibition of DNA polymerase activity, bleomycin preincubated with dithiothreitol (or beta-mercaptoethanol) at pH 6.5 to 9.0 lost its inhibitory activity. This was not the case when DNA was also included in the preincubation mixture. The results obtained in this study indicate that bleomycin inhibits DNA polymerases alpha and beta by a thiol reagent-dependent interaction with the template. Thus, the antitumor activity of bleomycin may be greatly influenced by the concentration of sulfhydryl compounds and their proximity to DNA in the target cells.

Human T-cell leukemia virus I provirus and antibodies in a captive gorilla with non-Hodgkin's lymphoma.

Antibodies reactive against human T-cell leukemia virus I (HTLV-I) were detected by indirect immunofluorescence assay using MT-2 as target cells, enzyme linked immunosorbent assay screen and competition assay, and Western blot analysis in three sera (one collected in 1979) from a captive gorilla which developed diffuse histiocytic lymphoma in 1983. The sera from four other healthy gorillas housed separately were HTLV-I antibody negative. All sera were negative for HTLV-III antibodies by enzyme linked immunosorbent assay. Southern blot analysis of DNA from lymphoma tissue after digestion with BamHI and using complete HTLV-I genome probe gave one 10-kilobase fragment and a characteristic 1.05-kilobase internal fragment detected in all known HTLV-I isolates. These results indicate that the gorilla was infected with HTLV-I or a closely related simian virus several years before the development of lymphoma.

Human T-cell leukemia virus type I or a related retrovirus in patients with mycosis fungoides/Sézary syndrome and Kaposi's sarcoma.

Antibodies reactive with human T-cell leukemia virus type I (HTLV-I) proteins p19, p24, gp46, p56, and gp68 were detected in four of 27 patients with mycosis fungoides/Sézary syndrome (MF/SS) and one patient with Kaposi's sarcoma using radioimmunoprecipitation and Western blot analysis. Seroreactivity patterns to HTLV-I proteins of MF/SS sera were indeterminate or limited in comparison with sera of patients with adult T-cell leukemia/lymphoma. HTLV-I gag- and tax/rex-specific DNA was demonstrated in peripheral blood from three of the MF/SS patients and from the patient with Kaposi's sarcoma by the polymerase chain reaction. HTLV-I-specific DNA sequences were not detected in a cohort of seven seronegative MF/SS patients. The frequency of HTLV-I infection was four of 27 or 14.8% among the MF/SS patients, which is several hundredfold higher than in normal blood donors. The present data suggest a possible association of HTLV-I or a related retrovirus with mycosis fungoides/Sézary syndrome and Kaposi's sarcoma.

Phorbol ester-induced differentiation of a non-T/non-B human leukemic cell line (REH) to macrophage-like cells.

Treatment of non-T/non-B human leukemic cell line REH with 5 X 10(-9) M 12-O-tetradecanoylphorbol-13-acetate (TPA) at 37 degrees resulted in their adherence to culture flasks by 24 to 36 hr and, after 72 hr, the entire surface of the flask/coverslips became covered with macrophage-like cells containing pseudopodia. Wright-Giemsa-stained untreated cells had blast morphology, whereas TPA-treated cells (adherent or excess cells remaining in suspension) had characteristic morphology of macrophages and phagocytized large numbers of latex beads. Untreated REH cells were negative for nitroblue tetrazolium reduction, Sudan Black B, and peroxidase, and they were weakly positive for periodic acid-Schiff, acid phosphatase, chloroacetate esterase (pH 6.8), and nonspecific (naphthol AS-D acetate, pH 6.8) esterase, whereas TPA-treated cells (adherent or in suspension) gave strong reaction for these stains except for peroxidase and chloroacetate esterase which showed moderate reaction. Furthermore, the nonspecific esterase activity of TPA-treated cells and weak activity in 10% of untreated cells was strongly inhibited by NaF, a characteristic of monocytic series of cells. Lysozyme activity was not detected in culture supernatant from control or TPA-treated cells. No cytoplasmic immunoglobulin was detected in untreated or TPA-treated cells, and the monocyte/granulocyte antigen (detected by MCS-2 monoclonal antibody) which was absent from untreated REH cells was expressed in TPA-treated cells. TPA-treated cells lost common acute lymphoblastic leukemia antigen but showed significantly elevated expression of histocompatibility locus DR antigen. Terminal transferase estimated by immunofluorescence and biochemical assay was high in untreated REH cells, whereas TPA-treated cells were negative in terminal transferase immunofluorescence and had only negligible terminal transferase activity in biochemical assay. All these changes in REH cells observed on TPA treatment represent the differentiation of a human leukemic non-T/non-B-cell line to macrophage-like cells for the first time which indicates that some non-T/non-B acute lymphoblastic leukemia cells may have latent monocyte-like phenotype.

High terminal deoxynucleotidyl transferase activity in acute myelogenous leukemia.

Although leukocytes from all 13 acute lymphoblastic leukemia patients examined had high terminal deoxynucleotidyl transferase (terminal transferase) activity (20 to 100 units/mg of cellular DNA, where 1 unit equals 1 nmole of nucleotide polymerized in 1 hr) and those from 21 acute myelocytic leukemia patients had low terminal transferase activity (0.2 to 2 units/mg of cellular DNA), the bone marrow and peripheral blood leukocytes from 2 patients with acute myelocytic leukemia, diagnosed on the basis of clinical features and the morphology, cytochemistry, and cytogenetics of the leukemic cells, had terminal transferase activity (39 to 52 units/mg of cellular DNA) equivalent to that found in leukemic lymphoblasts. These results bring under question the specificity of high terminal transferase activity outside of the thymus as a marker for leukemic lymphoblasts and, secondarily, the derivation of acute lymphoblastic leukemia cells in all cases from thymocytes. Perhaps malignant transformation in a pleuripotent stem cell with derepression of the genome for terminal transferase could account for high terminal transferase activity observed in certain leukemic cells.

Modified nucleotide polymers as inhibitors of DNA polymerases.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.

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

Expression of mRNA and proteins for toll-like receptors, associated molecules, defensins and LL-37 by SRIK-NKL, a CD8+ NK/T cell line.

Natural killer (NK) cells, innate lymphocyte effectors, kill virus-infected host and tumor cells in non-MHC, non-TCR restricted fashion, unlike T cells. The role of NK cells in recognition and killing of pathogens remains unknown. Expression of the ten human pathogen associated molecular pattern or toll-like receptors (TLR's), associated molecules, and antimicrobial peptides alpha, beta defensins, and LL-37 by NK cells has not been investigated previously. We report our CD8+ NK/T cell line SRIK-NKL, derived from leukemic phase of acute lymphoblastic lymphoma, expresses mRNA and protein for 8/10 TLR's, associated proteins for signaling, defensins, cathelicidin/LL-37, and responds to live bacteria by cell proliferation and increased IFN-gamma, TNF-alpha, TNF-beta, and MIP-1alpha production.

Differences in chromatin from normal and leukemic human cells as shown by digestion with restriction nucleases.

Restriction endonuclease MspI digested significantly more than HpaII the DNA and chromatin from normal and leukemic human cells, although both enzymes digested DNA more than chromatin. Moreover, DNA and chromatin from normal cells showed higher digestion by HpaII compared to DNA and chromatin from leukemic cells indicating higher frequency of Cm5CCG in latter DNA. EcoRII and BstNI, which have the recognition sequence CCATGG but cut at different points, digested all DNAs significantly, as did BstNI for chromatin from all sources. However, chromatin from normal cells showed only limited digestion by EcoRII which significantly digested chromatin from leukemic cells. This could result from subtle differences in the conformation of normal and leukemic cell chromatin involving recognition sequences for EcoRII.

Alterations in enzyme expression on 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of chronic lymphocytic leukemia cells.

Several enzyme activities were examined to establish a relationship between their expression and terminal differentiation of B-chronic lymphocytic leukemia (CLL) cells to plasma cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). Although adenosine deaminase activity did not change significantly, thymidine phosphorylase and purine nucleoside phosphorylase increased 2-3-fold on TPA-induced differentiation of CLL cells. In addition, cytochemical reactions for non-specific esterase and acid phosphatase changed from very weak to intense on differentiation of CLL cells to plasma cells. The above markers, particularly cytochemical, could be useful for the classification of B-cell malignancies and for studying B-cell differentiation.

Polyamine changes during senescence and tumorogenesis in plants.

Putrescine, spermidine, spermine and two unknowns designated as A and B were detected in first seedling leaves of barley (Hordeum vulgare L. var. Wolfe). The levels of these polyamines in first seedling leaves from 4-day-old barley plants grown in darkness or in light were comparable and did not change significantly after exposure of dark grown plants to light for 24 h. No significant consistent changes in the amounts of above polyamines, except perhaps decline in spermidine, were noted during senescence of intact or excised first seedling leaves of barley and this spermidine decline was suppressed during retardation of senescence of excised leaves by 10 mg/l kinetin in the dark. In addition, putrescine, spermidine, spermine, cadaverine and diaminopropane (0.2 mM, 1 mM, 10 mM) had no effect on senescence of excised barley leaves in the dark and both spermine and spermidine induced bleaching of the leaves in the light. Both spermine and spermidine (approx. 10 mM) inhibited RNase and DNase activities but stimulated phosphodiesterase activity (assayed with bis-p-nitrophenyl phosphate as substrate) in crude soluble extracts from barley leaves. Purified snake venom phosphodiesterase activity assayed with RNA as substrate was, however, stimulated by 300-400% by 7-14 mM spermine or spermidine indicating similar possibilities for barley phosphodiesterase. These results together with the presence of multiple species of these enzymes and a decline in net soluble RNase and DNase activities during senescence in barley leaves reported previously, make it unlikely that inhibition of RNase activity in vitro by polyamines could be correlated with their effect on senescence. Putrescine, spermidine and spermine were detected in normal and crown gall tumor tissue cultures of tobacco (Nicotiana tabacum var Wisconsin 38) and in tobacco mosaic virus (TMV)-infected freshly excised pith tissue from tobacco which represented non-proliferating tissue. The level of all three polyamines was several-fold higher in cultured tissues compared to the non-dividing freshly excised pith tissue and the tumor cultures had several-fold higher spermidine and putrescine respectively compared to normal tissue cultures. These results indicate high levels of polyamines in growing tissues but no consistent pivotal changes in polyamines during senescence. The results also do not support polyamines being natural anti-senescent compounds in plants or that their anti-senescent compounds effect could result from inhibition of RNase activity.

Fragmentation of DNA and chromatin during senescence in barley leaves.

DNA or chromatin from young (7 day) first seedling leaves of barley showed only one component which migrated little into 1% agarose gels on electrophoresis. However, DNA or chromatin from senescent (17-, 19-, and 23-day-old) leaves showed additional dispersed components migrating throughout the length of the gel which increased with age. These low molecular weight components increased even more on autodigestion of chromatin from senescent leaves by its associated DNase or by digestion of DNA from senescent leaves with partially purified chromatin DNase. DNA or chromatin from young leaves also produced gel pattern similar to old leaves on digestion with partially purified chromatin DNase from old barley leaves. Thus, fragmentation of DNA and chromatin by chromatin associated DNase, previously shown to increase 4000% on aging, occurs during senescence in barley leaves.

Examination of HTLV-I ELISA-positive leukemia/lymphoma patients by western blotting gave mostly negative or indeterminate reaction.

We used enzyme-linked immunosorbant assay (ELISA) and Western blotting, with "purified" human T-cell leukemia virus I (HTLV-I), to test for HTLV-I antibodies in 2583 plasma samples from 1053 leukemia/lymphoma patients treated at Roswell Park Memorial Institute, mostly between 1972 and 1984, and in 110 sera samples from normal healthy persons. The results demonstrate that ELISA and Western blot assay have limitations for HTLV-I antibody detection in an adult T-cell leukemia/lymphoma (ATL) nonendemic population. This conclusion is based on the many false reactives obtained by ELISA, and weak and indeterminate reaction (mostly p19 band) on Western blotting. All moderate to strongly HTLV-I ELISA-positive samples tested were negative for human immunodeficiency virus (HIV) antibodies. Although 6/27 mycosis fungoides (MF) patients tested gave mostly a weak reaction on HTLV-I ELISA, 3/6 MF patients gave multiple bands (p19, p31, p36, gp46) on Western blotting and three samples from one patient gave the same p31, p36, and gp46 bands. This may suggest involvement of some HTLV-I-related virus in MF. These results also indicate that prevalence of HTLV-I infection in leukemia/lymphoma patients was rare, if it exists at all, since, despite the reactivity of some sera with HTLV-I-suspected antigens, none of the samples satisfy the USPHS criteria for positivity which is based on the detection of antibodies to gag protein p24 and to an env gene product gp46 or gp61/68.

Differential selectivity of 5-fluorouracil and 5'-deoxy-5-fluorouridine in cultured human B lymphocytes and mouse L1210 leukemia.

The role of differential metabolic activation of a 5-fluorouracil (FU) prodrug, 5'-deoxy-5-fluorouridine (dFUR), in achieving selective cytotoxicity was investigated in cultured human (dFUR), in achieving selective cytotoxocity was investigated in cultured human B lymphocytes and murine leukemia L1210 cells. B cells were cross-sensitive to FU and dFUR. On the other hand, leukemia L1210 cells were sensitive to FU but resistant to dFUR. The difference in the biological activities of FU and dFUR in B and L1210 cells correlated with (a) the metabolism of dFUR to FU by intact B (60% conversion) and L1210 (no conversion) cells, and (b) the phosphorylase activity of B (660 nmoles converted in 2 hr per mg protein) and L1210 (undetectable) cells. The intracellular metabolism of FU and dFUR was studied using a reversed-phase ion-pair high pressure liquid chromatographic assay. FU and dFUR shared similar metabolic pathways in B cells; their anabolites included FU ribose and deoxyribose nucleosides and nucleotides. In L1210 cells, FU was anabolized to 5-fluorouridine triphosphate and 5-fluorodeoxyuridine monophosphate, whereas dFUR was present mainly as the unchanged drug. Further metabolism studies using dFUR with tritium label in either the FU moiety or the altered sugar moiety established that the metabolic pathway of dFUR to cytotoxic FU anabolites in the B cells was via phosphorolysis to FU. These data indicate that, on a cellular level, an FU prodrug such as dFUR, which is activated by cytosolic enzyme, has a different selectivity from that of FU, and that the basis of differential selectivity is the initial phosphorolysis to FU.

Terminal transferase immunofluorescence, enzyme markers and immunological profile of human leukemia-lymphoma cell lines representing different levels of differentiation.

We have examined alterations in terminal deoxynucleotidyl transferase (TdT) immunofluorescence (IF) in MOLT-4 cells during changes in growth conditions. Subsequently, we used cells from 48 human hematopoietic cell lines of different cell lineages and maturation stages to compare the IF and biochemical assays for expression of TdT. In addition, we have attempted to correlate the expression of TdT, adenosine deaminase (ADA), thymidine phosphorylase (TP) and immunological markers with maturation stages in these different cell lines. The results indicate that TdT positive cells remain TdT positive when assayed by either the biochemical or IF tests during growth or early plateau phase, but that cells under poor growth conditions, such as in old cultures, may give a negative TdT IF reaction. Otherwise, biochemical and IF assays for TdT gave comparable results in the 48 cell lines tested, testifying to the reliability of the IF test. Based on the comparisons of the various cell lines studied, it appears that both ADA and TdT decrease progressively as maturation of T cells from Blast I to Blast IV to mature T cells increases. TP was deficient in all T-cell lines compared to normal peripheral blood T cells, which in turn had lower activity compared to normal peripheral blood B cells. Pre-B cells, although indistinguishable from each other by immunological markers and all having low TP and ADA activity, showed heterogeneity, with TdT activity high in some and low in others. All non-T, non-B lines had high TdT activity, but low ADA and TP activity. B- and myelocytic cell lines had low ADA and TdT activity, and showed an increase in TP activity as the maturation of cells increased. These results indicate that the TdT IF test is a reliable procedure for detecting TdT positive cells, and that TdT, ADA and TP could be useful markers for studying the differentiation of human hematopoietic cells.

Phenotype, genotype and cytokine production in acute leukemia involving progenitors of dendritic Langerhans' cells.

To date no hematopoietic progenitors of dendritic Langerhans' cells (DLC), which represent an highly efficient class of antigen presenting cells, have been identified or the cytokines they elaborate have been defined. Here we describe an acute leukemia patient whose blasts (90-96% in peripheral blood and bone marrow) had a phenotype consistent with putative progenitors of DLC. The patient was treated with ara-C and VP-16 but did not achieve remission. The blasts had lobulated nuclei, no cytoplasmic vacuolation or Auer rods and were weakly positive for acid phosphatase and non-specific esterase and negative for PAS, granzyme A, dipeptidyl aminopeptidase IV, ATPase/ADPase and lysozyme production. The blasts were positive for CD1a, CD4, CD16, CD35, HLADR, HLADQ, CD11b, CD11c, CD14, CD33, CD34, CD11a, CD71, CD19, CD25, IL-2R beta and negative for CD2, CD7, CD8, CD10, CD22, CD56, CD57, surface or cytoplasmic CD3, TCR delta and TCR beta, HTLV-1p19 and P-glycoprotein. On liquid culture with or without 5 x 10(-9) M 12-O-tetradecanoylphorbol-13-acetate (TPA) for 3 days, the blasts formed aggregates of proliferating and elongating cells on the wall of the flasks with a decline in CD34, numerous dendritic processes appeared on the cells and there was strong positivity for ATPase/ADPase, but no other changes in phenotype. No macrophages were observed, indicating derivation from separate DLCs. Cytogenetic analysis showed chromosomal abnormalities and electron microscopy showed Birbeck granules. Southern blotting of DNA showed rearrangement of one allele for both JH and TCR beta but no HTLV-1 related sequences. Culture supernatants from blasts cultured with or without TPA showed the production of large amounts of IL-8, IL-6, TNF-alpha, MIP-1 alpha, IL-10 and interferon gamma and modest amounts of IL-1 alpha, GM-CSF and stem cell factor. The presence not only of CD1a, HLADR, HLADQ and many other characteristics including Birbeck granules, but also differentiation along the lines of DLC with appearance of dendritic processes on the cells and expression of ATPase/ADPase activity, indicate that the leukemic blasts in our patient represented a leukemic counterpart of normal progenitors of DLC and the leukemia a new entity which could possibly be classified as AML-M8. Lastly, many pro-inflammatory cytokines produced by DLC could contribute to inflammation and IL-10 to immunosuppression.