Phenotypic and functional analysis of lymphokine-activated killer (LAK) cell clones. Ability of CD3+, LAK cell clones to produce interferon-gamma and tumor necrosis factor upon stimulation with tumor targets.

Lymphokine-activated killer (LAK) cells are generated by the culture of peripheral blood lymphocytes with interleukin-2 (IL-2). A variety of cells, including T-lymphocytes and natural killer (NK) cells, can be activated by IL-2 to exhibit the ability to kill multiple tumor and "modified-self" targets. Recent reports indicate that culture conditions can determine the phenotype of cells expressing LAK activity. Using limiting dilution techniques, we first generated cloned LAK cells with three culture conditions: autologous human serum (AHS) + IL-2; AHS + IL-2 + 0.1 micrograms/ml phytohemagglutinin and fetal bovine serum and IL-2. We determined that all but one of the 47 LAK cell clones generated with the three culture conditions were CD3+ and T-cell like; one NK-like clone was observed. Clones that were cytotoxic for one target could generally kill multiple targets, and the absence of phytohemagglutinin did not significantly affect the ability of the LAK cell clones to kill multiple targets. The presence of phytohemagglutinin was, however, necessary for the long-term maintenance of proliferation and cytotoxic activity of the LAK cell clones. The mechanism by which LAK cells kill tumor targets is not known. We here demonstrate that LAK cells and LAK cell clones can produce interferon-gamma and tumor necrosis factor (TNF) when stimulated with an erythroleukemia cell, K562. Five of the six CD3+, LAK cell clones tested could be stimulated by K562 cells to produce both interferon-gamma and TNF. However, the ability of the cloned LAK cells to kill K562 cells, as measured in a 4-h 51Cr-release assay, did not correlate with their ability to produce these cytokines. Furthermore, specific antibodies that neutralize the cytotoxic activity of interferon-gamma and TNF did not inhibit killing of K562 cells by LAK cells as measured with a 4-h cytotoxic assay. The cytostatic and cytotoxic activities of interferon-gamma and TNF for tumor cells are well documented, but these cytolytic activities are slower acting and exhibit their maximum effect after 48-96 h. We here propose that LAK cells kill tumor targets by a combination of cell-to-cell-mediated killing and by the release of slower acting cytostatic/cytotoxic cytokines that can inhibit the growth of tumors some distance from the effector cells.

Elevated serum levels of soluble interleukin-2 receptors in HIV infection: no correlation with activated T cells.

Serum levels of soluble interleukin-2 receptors (sIL-2R) were measured in 105 HIV-seropositive individuals simultaneously with T cell subsets and activated T cells (CD3+ and HLA-DR+). Significantly elevated levels of serum sIL-2R were found (564 +/- 259 U/ml versus 258 +/- 87 U/ml in 70 controls, p less than 0.001), as well as increased numbers of activated T cells (mean numbers, 579/microliters in the patients versus 113/microliters in 26 controls, p less than 0.0001). Correlation analysis did not disclose any significant association between elevated sIL-2R and increased activated T cells, nor with decreased CD4+ lymphocytes. These data suggest that sIL-2R in HIV infection do not emanate from activated T cells and are not linked to CD4+ cell loss.

Cyclic AMP and cyclic GMP phosphodiesterase activities in Hodgkin's disease lymphocytes.

Cyclic nucleotide phosphodiesterase (PDE) activities were studied in peripheral blood monocyte-depleted lymphocytes and enriched T-lymphocyte suspensions from thirteen patients with previously untreated Hodgkin's disease (HD) and fourteen age and sex matched healthy volunteers. Monocyte-depleted lymphocytes from HD patients showed PDE-activities which were two times higher than in their normal counterpart cells. The mean cAMP-PDE activity present in enriched HD T-lymphocyte suspensions was four times higher than in control T-lymphocytes, and the mean cGMP-PDE associated with HD T-lymphocytes was three times higher than in the controls. The hydrolytic activities present in both monocyte-depleted and T-lymphocyte enriched cells suspensions remained unchanged in absence or in the presence of calmodulin and calcium. Since depressed cAMP and cGMP resting levels have been observed in HD lymphocytes and lymphocyte subpopulations, our results suggest that the elevated PDE activities are, at least in part, responsible for the alterations in lymphocyte cyclic nucleotide levels.

Decreased membrane "fluidity" of T lymphocytes from untreated patients with Hodgkin's disease.

Plasma membrane "fluidity" of peripheral blood T lymphocytes from untreated patients with Hodgkin's disease (HD) and healthy controls was studied using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4(trimethylamino)phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). In 13 consecutive patients a significant increase of T lymphocyte plasma membrane microviscosity was observed with both DPH and TMA-DPH. These alterations seemed unrelated to the cholesterol (Chol) and phospholipid (PL) content of HD T lymphocytes since the Chol/PL ratio was comparable in both HD and control cells. Since prostaglandin E2 (PGE2) from monocytic origin has been claimed to be responsible for the impairment of cell-mediated immunity (CMI) associated with HD, we studied the effect of exogeneously added PGE2 (0.1 microM) on control subjects T lymphocyte membrane "fluidity". Using the fluorescent probe DPH and the spin labelled fatty acid probe 16 NMS for electron paramagnetic resonance study, we observed a PGE2-induced fluidization of control T lymphocyte membranes which is specifically located in the inner part of the plasma membrane, whereas the plasma membrane surface seemed unaffected by PGE2 as judged by the TMA-DPH probe. Thus, PGE2 does not appear to be responsible for the alterations of T lymphocyte membranes observed in HD. Intrinsic alterations and/or other mediators might be involved.

Interferon enhanced natural killer function in Hodgkin's disease.

Natural killer (NK) cell activity of peripheral blood mononuclear cells from 19 untreated patients with active Hodgkin's disease and 22 age- and sex-matched healthy individuals was evaluated using classical K 562 cells as targets in a 4-h assay. A significant decrease was demonstrated in the patients (P less than 0.01) in accordance with previous data from the literature. Preincubation of effector cells with alpha leucocyte recombinant interferon (500 units/10(6) cells/ml) led to a significant increase in NK function in the patients (P less than 0.001). However, 4 patients still remained below the normal range and some patients were poor responders to interferon indicating that the mechanisms of depressed NK cell activity associated with Hodgkin's disease could not be overcome by interferon at least in some patients.

Depressed NK cell activity of peripheral blood mononuclear cells in untreated hodgkin's disease: enhancing effect of interferon in vitro.

Natural killer (NK) cell activity of unseparated peripheral blood mononuclear cells from 30 untreated patients with Hodgkin's disease and 22 age- and sex-matched normal controls was evaluated using the classical K 562 cells as targets. A significant defect was demonstrated in the patients with stage I-II and seemed to be more profound in patients with advanced disease (stage III-IV) and in those with B symptoms. The differences between subgroups of patients, however, were not statistically significant, mostly because of the wide dispersion of individual data. Pre-incubation of effector cells with alpha A leucocyte recombinant interferon led to a clear increase in NK cell activity in 4 of 6 patients tested, showing that depressed NK activity in Hodgkin's disease is still susceptible to the enhancing effect of interferon, at least in some patients.

Stage-related decrease in natural killer cell activity in untreated patients with mycosis fungoides.

Natural killer activity of peripheral blood mononuclear cells against the human cell line K 562 was evaluated in 11 patients with mycosis fungoides and simultaneously in 10 age- and sex-matched controls. In the patient group, nine had no previous treatment and in two topical therapy had been discontinued more than 3 months before. None had any associated disease or concurrent therapy that could interfere with the immune system. Patients with early disease showed a mean specific lysis and a range of individual data similar to the controls whereas patients with advanced disease had a significant defect of natural killer activity at effector: target ratios of 100 : 1, 50 : 1, and 25 : 1, as shown by the Mann-Whitney test. Preincubation of effector cells with alpha-interferon for 1 h in a single patient with low natural killing capacity led to a clear increase of the specific lysis, suggesting reduced functional activity rather than depletion of effector cells.

Alterations of peripheral blood lymphocyte cyclic AMP and cyclic GMP in untreated patients with hodgkin's disease.

Cyclic AMP and cyclic GMP are important regulatory agents of lymphocyte functions. Depressed T-lymphocyte functions are frequently associated with Hodgkin's disease and suppressor monocytes have been implicated in the pathogenesis of this defect. In the present study cAMP and cGMP resting levels were measured in lymphocytes from 18 untreated patients with Hodgkin's disease using a sensitive radioimmunoassay. A significant decrease of cAMP (P less than 0.001) and, to a lesser degree, of cGMP (P less than 0.01) was found in monocyte-depleted lymphocyte suspensions from the patients compared to controls. Studies of patient and control lymphocyte subpopulations showed in patients a clear deficit of cAMP in T-depleted lymphocytes, rather than in T cells, with a low cAMP/cGMP molar ratio in both subpopulations. From this data it is clear that factors other than prostaglandin-mediated suppression of monocyte origin are involved in the pathogenesis of the T-lymphocyte depression associated with Hodgkin's disease.

Immunomodulating effects of a short-term oral treatment with C 1821 in untreated cancer patients: a controlled study.

C 1821 is a purified glycoprotein extract from Klebsiella pneumoniae serotype 2 with a molecular weight of about 350,000. It enhances immune responses in animals when given orally and the oral route of administration is devoided of any toxicity even in humans. The present controlled trial showed that C 1821 given per os at the single daily dose of 4 mg for 14 days in untreated cancer patients (mostly lymphomas) significantly enhanced delayed cutaneous hypersensitivity to recall antigens using the Multitest system (7 antigens). It also increased basal levels of lymphocyte cAMP and particularly of cGMP which were decreased in these patients. When incubated in vitro with lymphocytes from either normal controls or patients, C 1821 showed a dose-dependent stimulation of cAMP synthesis which was more pronounced in patients than in controls.

Enhancement of delayed cutaneous hypersensitivity in untreated cancer patients given a short-term oral treatment with C 1821.

C 1821 is a purified glycoprotein extract of Klebsiella pneumoniae serotype 2 with immunomodulating properties in animals (in vivo and in vitro) and in humans (in vitro). The compound is devoid of any apparent toxicity when given orally. The aim of the present work was to evaluate the effects of a short term oral administration of C 1821 on delayed cutaneous hypersensitivity to recall antigens in untreated cancer patients (mostly lymphomas). Consecutive patients were alternately allocated to receive C 1821 or placebo for 14 days. C 1821 restored and significantly (P less than 0.02) enhanced skin reactions, as shown using the Multitest system.

Comparative role of polyamines in division and plastid differentiation of Euglena gracilis.

Regulation of polyamine biosynthesis during growth and differentiation of Euglena gracilis was investigated. Increased activity of L-ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyzes the initial step in polyamine synthesis in Euglena, and accumulation of polyamines were observed prior to DNA replication in synchronous cultures of heterotrophically or photoautotrophically grown cells. In photoautotrophic cells three maxima of polyamine synthesis were observed during the light period of the cell cycle. The transition form quiescence to active growth was accompanied in heterotrophic Euglena by a very large stimulation of ornithine decarboxylase activity and polyamine synthesis; the decrease in growth potential of these cells was correlated with a decrease in polyamine levels. In contrast, differentiation of Euglena i.e. a shift from heterotrophic to photoautotrophic mode of living in the absence of division, led only to a minor stimulation of polyamine biosynthesis. Alpha-Methylornithine, an inhibitor of ornithine decarboxylase, blocked the growth of heterotrophic Euglena, and depletion of intracellular polyamines decreased the differentiation rate. Both events could be reversed by addition of putrescine to the growth medium. This study suggests that Euglena requires a minimal intracellular level of polyamines to grow and differentiate under optimal conditions. This requirement seems to be more stringent for cell division.

Studies on polyamine biosynthesis in Euglena gracilis.

Euglene gracilis (strain Z) was found to contain five polyamines which could be separated by high-pressure cation-exchange chromatography. 1,3-Diaminopropane, putrescine, norspermidine (N-(3-aminopropyl)-1,3-diaminopropane), spermidine and norspermine (N,N'-bis(aminopropyl)-1,3-diaminopropane) were identified. Biosynthesis of putrescine in E. gracilis proceeds through decarboxylation of L-ornithine, no arginine decarboxylase (EC 4.1.1.19) activity could be detected. The properties of the enzymes ornithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1.50) in this alga were found to be similar to those of the enzymes isolated from animal tissues or yeast cells. A bioxynthetic scheme is proposed which relates the different polyamines occurring in E. gracilis.