Natural Killer Assays Research Articles

Serum KL-6 concentrations as a novel biomarker of severe COVID-19.

Severe acute respiratory syndrome coronavirus 2–induced direct cytopathic effects against type I and II pneumocytes mediate lung damage. Krebs von den Lungen‐6 (KL‐6) is mainly produced by damaged or regenerating alveolar type II pneumocytes. This preliminary study analyzed serum concentrations of KL‐6 in patients with coronavirus disease (COVID‐19) to verify its potential as a prognostic biomarker of severity. Twenty‐two patients (median age [interquartile range] 63 [59‐68] years, 16 males) with COVID‐19 were enrolled prospectively. Patients were divided into mild‐moderate and severe groups, according to respiratory impairment and clinical management. KL‐6 serum concentrations and lymphocyte subset were obtained. Peripheral natural killer (NK) cells/µL were significantly higher in nonsevere patients than in the severe group (P = .0449) and the best cut‐off value was 119 cells/µL. KL‐6 serum concentrations were significantly higher in severe patients than the nonsevere group (P = .0118). Receiver operating characteristic analysis distinguished severe and nonsevere patients according to KL‐6 serum levels and the best cut‐off value was 406.5 U/mL. NK cell analysis and assay of KL‐6 in serum can help identify severe COVID‐19 patients. Increased KL‐6 serum concentrations were observed in patients with severe pulmonary involvement, revealing a prognostic value and supporting the potential usefulness of KL‐6 measurement to evaluate COVID‐19 patients' prognosis.

Preclinical study of adoptive immunotherapy with natural killer cells in metastatic colorectal cancer.

e13103 Background: Several immune-based approaches for treatment of metastatic colorectal cancer (mCRC) are currently under investigation. Aims of our study were to grow and characterize tumor cells (TC) obtained from mCRC patients, and to investigate their susceptibility to autologous natural killer (NK) cell recognition and killing in vitro. Methods: Tumor samples and peripheral blood mononuclear cells from 10 mCRC patients undergoing surgery were collected. Establishment of TC lines that are representative of the original tumor and could be expanded in vitro for a significant number of passage was performed according with our previously described method (Turin I et al, Cytotherapy 2007). CD56+CD3- NK cells were incubated overnight with medium alone or activated with IL-2 (100U/ml) or IL-15 (20ng/ml) and tested against 51Cr-labeled primary TC in NK assay or in ADCC assay after pre-coating of TC with anti-EGFR monoclonal antibody cetuximab. Results: Eight stable mCRC lines have been obtained and characterized to confirm their neoplastic origin. Patient NK cells display low capacity to lyse autologous TC (median 12%, range 5%-17%). IL-2 and IL-15 incubation significantly potentiate NK cytotoxic capacity (median18%, range 6%-62% and median 35%, range 28%-65%, respectively). These results refer to an effector:target ratio of 25:1. ADCC assay has shown that cetuximab pre-coating of TC results on an increasing of their susceptibility to NK-mediated lysis. Phenotypical and molecular analysis of activating NK receptors and their ligands present on surface of TC are currently under evaluation. Conclusions: NK cells derived from mCRC patients display measurable cytotoxic activity against autologous primary TC. Susceptibility of NK-mediated lysis is strongly enhanced by pre-incubation of NK cell with cytokines and by pre-coating of TC with cetuximab. These findings, presented upon approval by the study’s data monitoring committee and undergoing confirmation in a larger series, may support a pilot study of NK cells pre-activated ex vivo for treatment of mCRC patients.

A longitudinal study of cell-mediated immunity in pigs infected with porcine parvovirus.

Porcine parvovirus (PPV) is an ubiquitous pathogen causing reproductive failure in swine. Protection against reproductive failure caused by acute PPV infection has commonly been related to the presence of specific antibodies in the dam. However, the role of cell-mediated immunity during chronic PPV infection remains to be elucidated, and may be relevant to the pathogenesis of novel diseases such as postweaning multisystemic wasting syndrome (PMWS), which may be triggered by coinfection with PPV and porcine circovirus type 2 (PCV2). To investigate whether pigs infected with PPV generate a cell-mediated immune response, a longitudinal infection experiment was performed, using swine leukocyte antigens (SLA) class I characterized growing pigs (haplotype H7/H7). Pigs were intranasally inoculated with PPV at 0, 80, and 136 days. At predetermined time points, peripheral blood mononuclear cells (PBMC) were isolated, and virus-specific lymphoproliferative responses and the cytolytic activities of cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells were examined. Cytolytic assays were performed by the chromium release method, using as targets a syngeneic porcine kidney cell line established for the purpose (CTL assays) and K562 cells (NK assays). A specific proliferative response of PBMC from virus-infected pigs to PPV was observed from day 101 onwards. In contrast, PBMC from mock-infected pigs did not proliferate in response to PPV. Flow cytometric analysis indicated that the CD4+CD8+ T-cell subset of PBMC proliferated in response to virus antigen, in keeping with the assumed role for these cells in immunological memory. This is, to our knowledge, the first indication of a cellular immune response following PPV infection. A weak CTL activity, which peaked on days 80 and 87, was observed in PPV-infected pigs. In vitro restimulation of PBMC with live PPV did not induce further CTL activity. A pronounced NK cell activity was detected in both virus-infected and control pigs throughout the experiment, and may have negatively affected the sensitivity of the CTL assay. In conclusion, the findings of a late lymphoproliferative response together with weak CTL activity are in keeping with an effective control of acute PPV infection by humoral immunity, but open the possibility that cellular immunity may play a role in controlling PPV reinfection. Finally, we find that the established experimental model using SLA characterized pigs may constitute a valuable tool for future studies of CTL activity in pigs.

Measurement of NK activity by the microcytotoxicity assay (MCA): a new application for an old assay

Natural killer (NK) cells are spontaneously cytotoxic immune effector cells with the ability to selectively destroy tumor cells without harming normal cells. To perform this function, NK cells utilize two main cytotoxicity pathways, the well known perforin/granzyme-mediated secretory/necrotic killing and the recently defined TNF family ligand-mediated non-secretory/apoptotic killing. The former mechanism is manifested mainly against a few cultured leukemia cell targets, while the latter mediates killing against a large variety of tumor cell targets. Therefore, the biological role and significance of these mechanisms might be different. The NK cell-mediated necrotic killing has been reliably and selectively measured in humans by the standard 4-h 51Cr release assay (CRA) against K562 myeloid leukemia cell targets. However, no standardized high throughput assay is available for testing the NK cell-mediated apoptotic killing. Here, we introduce the modified MCA as a convenient method for measuring perforin/granzyme-independent NK cell-mediated apoptotic killing. The assay is performed in microwells of Terasaki tissue culture microtest plates, using adherent tumor cell targets, which are selectively susceptible to non-secretory/apoptotic killing and resistant to secretory/necrotic killing mediated by NK cells. Target cells are plated in microwells and incubated overnight to adhere to the plastic surface and to regenerate cell surface-bound TNF family receptors. Following this adherence, target cells are co-incubated with freshly isolated human peripheral blood mononuclear leukocytes (PBMNL) or purified subpopulations of immune cells for 24 h in various effector/target (E/T) ratios. During this incubation, dead target cells become non-adherent and are removed by washing the wells. Remaining adherent (viable) target cells are fixed, stained and optically counted. A notable dose-dependent (peak at 200:1 E/T ratio), time-dependent (peak at 24 h of incubation) and donor-dependent killing of tumor cells was consistently and reproducibly induced by PBMNL of normal donors. Using purified subpopulations of immune cells, it was demonstrated that among PBMNL, CD3 −CD56 +CD16 + mature NK cells are the only mediators of tumor cell killing in MCA, as well as in CRA. Comparative studies of NK activity detected by MCA and CRA, performed with PBMNL from normal individuals and breast cancer patients, showed no significant correlation between the cytotoxicities measured in the two assays. In addition, while NK activity measured in CRA was normal in most breast cancer patients, NK activity assessed in MCA was decreased in a large majority of the patients. Thus, MCA is a sensitive NK assay, which is biologically different from CRA, and may be clinically relevant. MCA has also a higher throughput, and is more practical and economical than CRA.

U5 monoclonal antibody identifies a novel lymphocyte surface antigen preferentially expressed in human circulating natural killer cells with high cytotoxic activity.

U5 monoclonal antibody (mAb), developed against Japanese monkey lymphocytes, identified a glycoprotein of 32 000 MW that is expressed in a subset of human circulating natural killer (NK) cells. The distribution of U5 molecules was restricted among CD16+ cells, and U5 antigen was preferentially expressed in the CD38+ subset. U5+ CD16+ CD56+ cells were highly active on NK assay against K562 target cells. Variations in cytolytic activities and mRNA expression of perforin, granzyme B and Fas ligand (FasL) were observed in U5- CD16+ CD56+ cells depending on the donor. We found that in some donors, a phenotypically mature (CD16+ CD56+) but functionally immature subset was present in the peripheral circulation. The U5- CD16+ CD56+ cells of some donors exhibited negligible cytolytic activity with no detectable expression of the above mRNAs, whereas those of the other donors had a significant but lower cytolytic activity with a reduced expression of granzyme B mRNA as compared with those of U5+ CD16+ CD56+ cells. Concanavalin A (Con A) stimulation induced an expression of U5 antigen in U5- CD16+ CD56+ cells accompanied by an up-regulation of granzyme B mRNA expression. These findings suggest that U5 antigen may be a novel molecule involved in the maturation or differentiation of human circulating NK cells.

Induction of transporter associated with antigen processing by interferon gamma confers endothelial cell cytoprotection against natural killer-mediated lysis.

T lymphocytes react minimally with nonactivated endothelial cells (ECs). However, natural killer (NK) lymphocyte interactions with resting ECs are rapid, avid, and result in endothelial activation and/or cytotoxicity. The molecular basis for these interactions and EC sensitivity to NK-mediated lysis is unclear. To address the EC-specific nature of NK sensitivity, we used syngeneic human umbilical vein ECs, dermal microvascular ECs, dermal fibroblasts, and B lymphoblastoid cell lines in calcein-AM retention NK assays with allogeneic NK effector cells and found the EC lines consistently more NK-sensitive. Because NK inhibitory receptors are engaged by membrane major histocompatibility complex (MHC) I molecules and MHC I-deficient targets are NK-sensitive, we investigated the quantitative levels of membrane MHC I on the panel of syngeneic lines. Highly sensitive ECs expressed similar (or higher) levels of membrane MHC I than their syngeneic NK-resistant counterparts. Pretreatment of ECs with gamma interferon (IFN-gamma) conferred protection against NK-mediated lysis, with much more rapid kinetics (2-6 hr) than those required for membrane MHC I hyperinduction (>8 hr). These kinetics are consistent with induction of transporter associated with antigen processing (TAP) expression and function. As opposed to NK-resistant cell lines, TAP-1 was undetectable in resting ECs. Recombinant expression of the TAP inactivator ICP47 by adenoviral-mediated transduction was used to selectively inhibit IFN-gamma-mediated EC TAP function. ICP47 expression abrogated EC cytoprotection conferred by IFN-gamma. We demonstrate a relationship between both basal and induced TAP-1 expression/function and EC sensitivity to NK-mediated cytotoxicity. We discuss the influence of an induced MHC I-associated peptide repertoire on vascular vulnerability to cytotoxic lymphocytes.

Inhibition of Natural Killer Cell Activity against Cytomegalovirus-Infected Fibroblasts by Nitric Oxide-Releasing Agents

The addition of nitric oxide (NO)-releasing agents, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), 3-{(±)-(E)-ethyl-2′-[(E)-hydroxyimino]-5-nitro-3-hexenecarbamoyl}-pyridine (NOR 4), significantly inhibited natural killer (NK) cell activity against cytomegalovirus (CMV)-infected cells. Inhibition of NK cell activity was due to NO released in the culture medium because the concentration of nitrite in the culture medium correlated with the inhibition of NK cell activity and the addition of an antagonist of NO, [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide] (carboxy-PTIO), to NK assay restored NK cell activity. The mechanism of inhibition of NK cell activity against CMV-infected cells by NO-releasing agents includes (1) inhibition of the production of interferon (IFN)-α by CD16 (Leu11b)-depleted cells cultured with CMV-infected cells and (2) inhibition of the activation process of NK cell by IFN-α. It is suggested that the production of NO by an inflammatory process may lead to the inhibition of NK cell-mediated cytotoxicity against CMV-infected cells.

Enhancement of Porcine Natural Killer Cell Activity by Recombinant Human and Murine IL-12

These studies describe the effects of recombinant human (rHu) and recombinant murine (rMu) interleukin 12 (IL-12) in the porcine system. To examine the effects of rHu and rMu IL-12, porcine PBL were cultured with rHu and rMu IL-12, and then used for a NK assay. Significant enhancement of porcine NK cell cytotoxicity was observed, and maximal enhancement was reached after 26 hr culture in nanogram quantities (5 ng/ml = 10 units/ml) of rHu and rMu IL-12. Monoclonal antibody (mAb) specific for human IL-12, C8.6, blocked rHu IL-12 enhanced porcine natural killer (NK) cytotoxicity. It was also shown that rHu IL-12 had additive effects on enhancement of NK cell cytotoxicity with PNK-E/G7 mAbs which react with cytolytic trigger molecules on porcine NK cells. By using purified NK cells, porcine tumor necrosis factor-α (TNF-α) secretion was measured on rHu IL-12 and PNK-E/G7 mAb-activated NK cells. rHu IL-12 and PNK-E/G7 mAbs synergize to induce TNF-α secretion. These data indicate that rHu and rMu IL-12 are cross-reactive in the porcine system and suggest the potential use of the porcine system as an important animal model for further study of rHu IL-12.

1235 A protein kinase inhibitor, h-7, induces apoptosis in a drosophila neuronal cell line

To investigate the role of microfilaments in natural killer (NK) cell-mediated cytotoxicity, general microfilament inhibitors, cytochalasins B,D and dihydrocytochalasin B, and a selective inhibitor of myosin light chain kinase (MLCK) which regulates microfilament contraction, i.e. 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9) were examined in an NK assay system. ML-9 inhibited NK cell activity in a dose-dependent manner without affecting target cell binding, whereas cytochalasins suppressed the binding. The dextran suspension method revealed that ML-9 inhibits the programming for the lysis stage of the lytic process. In the single cell assay, the addition of ML-9 after target cell binding had occurred inhibited the lysis of bound target cells, whereas the addition of cytochalasins in a similar manner did not affect it. Thus, these results suggest the possibility that microfilament contraction is involved in the lytic mechanism of NK cell-mediated cytolysis. However, the mechanism whereby cytochalasins inhibit target cell binding remains unclear.

The use of non-radioactive chromium as an alternative to 51Cr in NK assay

A novel method to measure target cell cytolysis based on the use of ‘cold’, non-radioactive chromium and on the determination of metal release by graphite furnace atomic absorption spectroscopy (FAAS) is proposed. Natural killer (NK) assays were performed by labelling target cells with chromium as Na 2CrO 4, and results were compared with those obtained by conventional overnight labelling with 51Cr of targets killed by the same effectors. The cytotoxic capacity of peripheral blood lymphocytes from healthy subjects was evaluated, and NK activity measured with both methods showed a good agreement at each of the tested effector to target cell ratios (between 100:1 and 1:1), with a high and significant coefficient of correlation ( r = 0.931, p < 0.0001). The selection of the appropriate Cr concentrations for labelling target cells took into account both the sensitivity of our instrumentation and the possible toxic effects of the metal. A study of the effects of Cr on the cell line (K562) which is usually employed as a target in NK tests showed that Cr could have a detrimental effect on cellular function, with significant numbers of cells with depolarised mitochondria and reduced DNA synthesis after 24 h incubation using Cr levels higher than 15 μmol/l (780 μg/l). The method proposed here has a number of advantages, including the use of a non-radioactive tracer, limited costs, high sensitivity and reproducibility, and the possibility of storing samples. In addition, the technique uses a fixed Cr concentration which is known to be non toxic.

A versatile flow cytometry-based assay for the determination of short- and long-term natural killer cell activity

A flow cytometry based method has been developed to assess natural killer (NK) cell activity in both short-term (4 h) and long-term (18 h) NK assays. Target cells were either labeled with PKH-2, c′FDA or D275. Simultaneously, dead cells were identified by counter-staining with the nuclear dye propidium iodide. Using flow cytometry, only D275 in combination with propidium iodide permits the differentiation of four cell populations: live target cells, dead target cells, live effector cells, and dead effector cells. Even after the extended incubation periods (18 h) necessary for the determination of NK activity in some domestic animals these four populations remain clearly distinguishable. Comparison of results with cells of normal human individuals obtained using this D27S/propidium iodide flow cytometry assay with data derived from fluorescence microscopy or an endogenous lactate dehydrogenase release assay shows a strong correlation. Since in long-term NK assays a high proportion of dead effector cells is constantly observed this cell population frequently limits the use of the lactate dehydrogenase release assay but does not interfere with the flow cytometry assay presented here. Using this novel assay, we have demonstrated the suppressive effects of defined glycosaminoglycans on long-term porcine NK activity.

Measurement of NK activity in effector cells purified from canine peripheral lymphocytes

Natural killer (NK) cells spontaneously lyse a variety of tumor cells in vitro, and are believed to play an important role in host resistance to tumor growth and metastasis in vivo. As part of our work in comparative oncology, we have designed and validated a canine NK cell assay. Of several lymphocyte isolation techniques evaluated, sedimentation of whole blood through a two-step Ficoll/Hypaque gradient ( sp. gr. 1.066 1.119 ) followed by plastic adherence of monocytes resulted in the most pure lymphocyte population (> 95% lymphocytes). Of four cell lines evaluated as targets in the NK assay, a canine thyroid adenocarcinoma (CTAC) cell line was determined to be most sensitive, and a lymphoblastoid (CT45-S) cell line was determined to be most resistant to NK lysis. A 15 h effector-target incubation period using these targets resulted in reproducible measurement of cell specific lytic activity. Passage of canine lymphocytes through nylon wool columns did not result in a significant increase in NK activity. A final sedimentation of purified lymphocytes through a 45 50% Percoll gradient concentrated NK activity into a single band of lymphocytes. Lymphocytes forming conjugates with CTAC target cells were 5.5–6.5 μm in diameter, and were characterized by a reniform nucleus and varying numbers of electron-dense cytoplasmic granules.

Interferon-alpha-dependent and -independent participation of accessory cells in natural killer cell-mediated lysis of HSV-1-infected fibroblasts.

Human natural killer (NK) cells require an HLA-DR+ accessory cell (AC) population to lyse herpes simplex virus-type 1 (HSV-1)-infected fibroblasts (HSV-Fs) but not K562 target cells. It has been postulated that ACs may function by producing interferon-alpha (IFN-alpha), which stimulates NK cells. Using a sequential enrichment protocol, ACs were found to coenrich with the interferon-producing cells (IPCs). Treatment of the ACs with a protein synthesis inhibitor, emetine, inhibited both their IFN production and AC function, results that support a central role for IFN in AC activity. In contrast, when the arginine analogue canavanine was added to NK assays, no IFN-alpha was produced and NK(HSV-Fs) activity was only partially inhibited. Consistent with IFN-independent AC function, treatment with either polyclonal sheep or bovine anti-IFN-alpha neutralized all the IFN-alpha produced during the NK assays but caused either no or partial reduction of NK(HSV-Fs) activity, respectively. However, when limiting numbers of ACs were used, the bovine antiserum neutralized both IFN-alpha and NK(HSV-Fs) activity. We further found that HLA-DR+ cells are required for cell clustering, suggesting a role for cell contact. Finally, fixation of activated ACs prevented the accessory function. Together, these results demonstrate that ACs can provide help to NK cells in both IFN-alpha-dependent and -independent manners.

Suppressive effects of pentoxifylline on natural killer cell activity.

The methylxanthine derivative pentoxifylline, widely used as a hemorrheologic agent in the treatment of peripheral vascular disease, is now being evaluated for potential applications in patients with cancer. Recent studies have shown that pentoxifylline can modulate a number of neutrophil functions at in vitro concentrations of at least 50 micrograms/ml. Using a standard51chromium-release assay, we studied the suppressive effects of pentoxifylline on natural killer (NK) cell activity and found that pentoxifylline, at concentrations of 50 and 100 micrograms/ml, suppressed the in vitro NK cell activity of healthy volunteers by 25% and 75%, respectively. Postreaction supernatants from chromium-release studies were then assayed for prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) by using enzyme-linked immunosorbent assay. Concentrations of both PGE2 and TNF-alpha were increased by more than five times in those assay wells containing pentoxifylline. Moreover, the addition of 1 microgram/ml indomethacin to the NK assay system containing pentoxifylline, completely inhibited PGE2 production and abrogated the pentoxifylline-induced NK suppression. The addition of PGE2 (1 x 10(-6) mol/L) to the assay system suppressed NK activity, whereas addition of 1 or 10 ng/ml TNF-alpha did not. Theophylline, another methylxanthine, failed to suppress NK activity like pentoxifylline at equimolar concentrations. Our studies provide the first evidence that concentrations of pentoxifylline of at least 50 micrograms/ml suppressed NK cell function by inducing PGE2 synthesis from effector peripheral-blood mononuclear cells.

Negative modulation of human NK cell activity by purinoceptors: 1. Effect of exogenous adenosine triphosphate

A variety of receptors, including purinergic receptors (for adenosine and adenosine nucleotides), are present on lymphoid cells. We have investigated the negative modulatory action of ATP, a high-energy purinergic compound, on the human peripheral blood natural killer (NK) cells. Micromolar doses of exogenous ATP produced a strong, direct, reversible, and dose-dependent (2.5 × 10 −5 to 8 × 10 −4 M) inhibition of NK cell-mediated cytotoxic activity in 98% of the 131 healthy donors tested. The inhibitory effect of ATP could be demonstrated in unseparated total lymphocytes and enriched NK cells (Percoll density gradient or negative selection by complement lysis) in 2-or 16-hr NK assays, at various effectontarget ratios, at suboptimal and saturating concentrations of the K562 targets. The inhibitory constants ID 20 and ID 50max for ATP (2 hr), which were 5.6 × 10 −5 and 8 × 10 −5 M, respectively, appear to be a relatively stable feature of an individual. The mechanism of action of ATP was also explored. It is not due to a simple chelation by ATP of the divalent cations Mg 2+ and Ca 2+ which play a role in the binding/lytic reactions during NK cell-mediated oncolysis. ATP treatment of lymphocytes neither nonspecifically decreased the number of viable cells nor specifically eliminated the NK cells, including their subsets, CD57 +16 −, CD57 +16 +, and CD57 − 16 + as revealed by immunofluorescence staining. Use of an enzyme-like kinetic approach to define the nature of ATP-induced inhibition suggests that it may be an “uncompetitive” type (Lineweaver-Burke reciprocal plot), because both V max (maximal velocity of oncolysis) and K m (Michaelis constant) were reduced simultaneously from 1.53 × 10 4 and 2.64 × 10 4 to 0.44 × 10 4and 0.73 × 10 4, respectively, in presence of ATP. Other methods (Eadie-Hofstee's and Hane's equations) confirmed that ATP does elicit a qualitatively and quantitatively similar shift in these kinetic parameters. Finally, an 18-hr preactivation of effectors by interleukin 2 (IL2) resulted in partial protection from the negative modulation of NK activity by ATP, while the presence of ATP during preincubation did not interfere with the desensitizing effect of IL2. The biological relevance of these findings are discussed and it is concluded that by interacting with appropriate (non-FcRIII?) cell-surface binding sites such as P 2 purinergic receptors, ATP is capable of negatively modulating the antitumor cytotoxic activity of human NK cells. This downregulation can be partially overcome by IL2 that up-regulates NK cells. Such a model system could be helpful in elucidating the mechanism of action of purinergic agonists such as ATP in human NK cells.

頭頚部癌患者におけるＮＫ，ＬＡＫ活性の動態

Natural Killer (NK) and lymphokine-activated killer (LAK) activity as well as surface markers of peripheral blood lymphocytes (PBL) in head and neck cancer patients are investigated with regard to the prognosis of patients.Patients (n=31) were grouped as follows:(1) untreated (UT) group (n=29), (2) treated, disease-free (TDF) group (n=21), and (3) group with recurrent disease (RD;n=6).NK and LAK activities were assayed by 51 Cr release method using K-562 cells in NK assay or Daudi cells in LAK assay as targets. Also LAK induction rate from PBL by interleukin-2 (IL-2) was measured.Analysis of surface markers of PBL (Leu 2, Leu 3, Leu 4, Leu 7, Leu 11, HLA-DR, and M1) including two color analysis (Leu 4×HLA-DR, Leu 2×Leu 15, Leu 3×Leu 8, and Leu 7×Leu 11) were performed using flow cytometry system.NK activity (P<0.01) or LAK induction rate (P<0.01) in the TDF group was significantly increased as compared with the UT group.LAK activity in PBL without IL-2 treatment were not statistically different among TDF, RD, and UT groups.In the study of surface markers of PBL, a significant decrease in the percentage of Leu3+ (P<0.01) or Leu 3+8+ (P<0.01) cells, and significant increase in the percentage of HLA-DR+ (P<0.01), Leu 4+HLA-DR+ (P<0.05), or Leu 3+8- (P<0.05) cells in TDF group were observed as compared with each corresponding immunologial parameter in the UT group.In the RD group, the percentage of Leu 3+ (P<0.001) or Leu 4+ (P<0.05) cells was significantly decreased and the percentage of HLA-DR+ (P<0.01), Leu 7+ (P<0.05), Leu 11+ (P<0.05), or Leu 2+15+ (P<0.05) cells was significantly increased, when compared with the UT group.However, the difference between each pair of UT, TDF and RD groups with regard to the percentage of M1+, Leu 7+11-, Leu 7+11+, or Leu 7-11+cells in PBL was not significant.

Resistance of chronic lymphocytic leukaemia cells to interferon-alpha generated lymphokine activated killer cells.

Recent studies have shown that, when used in early stage disease, interferon-alpha (IFN-alpha) can produce a fall in the number of malignant cells in the peripheral blood of patients with B-CLL. In this study, we investigated the effect of IFN-alpha on natural killer (NK) cell and lymphokine-activated cell (LAK) activity in patients with B-CLL. In vitro, IFN-alpha (500 U/ml for 18 hours) induced LAK activity in patients with B-CLL (27.7 +/- 9.9%, n = 20), and IL-2 (500 U/ml for 5 days) produced similar activity (35.9 +/- 8.8%, n = 7). Despite the induction of LAK activity by IFN-alpha and IL2 in patients with B-CLL, the malignant cells remained resistant to both allogeneic and autologous LAK effectors. NK activity in patients with B-CLL is also low (23.1 +/- 7.2%, n = 20), and B-CLL cells were resistant to NK cell activity. In cold target competition assays, CLL cells did not compete with labelled K562 or Daudi targets in the NK and LAK assays, suggesting that the malignant cells are not recognised by the effector cells, and this may be related to low level of expression of the adhesion receptors, LFA-1 and ICAM-1. Finally, CLL cells were also resistant to antibody dependent cell mediated cytotoxicity, but were susceptible to antibody dependent complement mediated lysis. These results suggest that it is unlikely that the effects of IFN-alpha in B-CLL are due to the enhancement of NK or LAK activity.

Kinetics and Regulation of NK Activity by Interleukin-2 and Interferon in Acute Toxoplasmosis

Natural killer (NK) activity against Toxoplasma gondii tachyzoites and tumour cells during acute toxoplasmosis was investigated using a single-cell NK assay. During the course of infection the percentage of lymphocytes binding tachyzoites and tumour cells did not vary significantly and NK activity was enhanced due to an increase in the specific cytolytic capacity per cell. To determine whether regulatory mechanisms mediated by cytokines might explain the increased NK activity, the kinetics of interleukin-2 (IL-2) and interferon (IFN) production and the correlation between their concentrations and the level of NK activity were analysed at the same time. As the infection progressed NK activity increased in spite of the fact that IL-2 production decreased (except for a small increase during the first day of infection). However, IFN production increased gradually in close temporal and quantitative association with the overall increase in NK activity. These results suggest that T. gondii, via its ability to produce interferon, enhances NK activity against itself and other cells.

Immunomodulation by 9-amino-1,2,3,4-tetrahydroacridine (THA): 1. Down-regulation of natural cell-mediated cytotoxicity in vitro

THA (Tacrine), a drug used in the experimental therapy of dementia of Alzheimer's disease type, and whose biochemical site of action is believed to be the neural cholinesterase, is shown, for the first time, to be an immunosuppressant in vitro on normal human peripheral blood lymphocytes in microgram quantities. THA down-regulates non-MHC restricted natural killer (NK) cell activity without affecting the general viability of cells. This down-regulation can be demonstrated at all effector and target (K562) concentrations, in purified resting NK cells as well as in lymphokine (interleukin 2) activated killer cells in 3- or 16-h NK assays and in all the blood samples tested. Kinetic analysis shows that the Vmax (maximal cytotoxic potential) and Km of NK cell-mediated cytolysis are also attenuated. Single cell assays using agarose matrix reveal that THA moderately interferes with tumor target binding/recognition events and strongly abrogates the delivery of lethal hit, thus lowering the frequency of active killer cells among THA-treated lymphocytes. THA down-regulates NK cells upon direct interaction and does not require the help of non-NK cells. The THA sensitive site(s) on NK cells does not appear to be perturbed significantly either by their proliferative status or by membrane modulations that may be normally induced by interleukin 2. The in vitro immunomodulatory pharmacological properties of THA reveal that the biological site of action of THA extends to non-neural cells also. Such non-neural models may be helpful in exploring the pathophysiological neuroimmunomodulatory properties of THA at cellular and molecular levels.

Induction of natural killer activity by Xanthenone analogues of flavone acetic acid: Relation with antitumour activity

Flavone-8-acetic acid (FAA) induces haemorrhagic necrosis and tumour regression in experimental tumours and induces natural killer (NK) activity. Xanthenone-4-acetic acid (XAA) forms the basis of a series of analogues of FAA which vary in antitumour potency. FAA, XAA and 15 XAA derivatives were tested for their ability to induce either NK activity in mouse spleens or haemorrhagic necrosis in mouse colon 38 tumours. Some derivatives were active in both assays (one at a dose 8-fold lower than that of FAA). When both assays were quantitated, a significant correlation ( r = 0.85; P < 0.001) was found. NK assays could be useful in screening compounds such as FAA and XAA analogues which appear to mediate their antitumour activity by biological response modification. Since tumour necrosis may not be mediated directly by NK cells, FAA and active XAA derivatives may exert pleiotropic effects that include NK induction and tumour necrosis by acting on host cells to release cytokines.