Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1×10⁻⁷ M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1×10⁻⁷ M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear³H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of Il-2 through its ability to enhance the expression of intermediate affinity Il-2R on these cells.     

Mesenchymal stem cells alter the frequency and cytokine profile of natural killer cells in abortion-prone mice

Natural killer cells, which play a pivotal role in the establishment and maintenance of normal pregnancy, are the most abundant leukocytes at the fetomaternal interface that their subsets frequencies and cytokine profile are influential factors in the preservation of the decidual tolerogenic microenvironment. Any imbalance in NK cells' frequency and functions could be associated with pregnancy failure. Mesenchymal stem cells (MSCs) are shown to have immunomodulatory effects on NK cells and their cytokine profile. The purpose of this study is to evaluate the impact of MSCs therapy on the cytokine profiles and subpopulations of NK cells in a murine model of recurrent pregnancy loss. Adipose-derived MSCs were injected intraperitoneally to the abortion-prone mice on Day 4.5 of gestation. The abortion rate was determined after MSCs administration and the frequency and cytokine profiles of the different subsets of NK cells were determined using the flow cytometry. Our results showed that, in abortion-prone mice, the frequency of CD49b⁺ NK cells was significantly higher than normal pregnant mice that decreased after therapy. We also demonstrated that MSCs downregulated the production of IFN-γ and upregulated IL-4 and IL-10 production by uNK cells. These findings indicate that MSCs can decrease the infiltration of CD49b⁺ NK cells to the fetomaternal interface and modulate the cytokine profile of NK cells from inflammatory to tolerogenic profile and thereby improve the tolerogenic microenvironment at the fetomaternal interface in benefit of pregnancy maintenance.     

Studies of serum-free medium for the generation of lymphokine-activated killer cells

We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (³H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.Abbreviations IMDM Iscove's Modification of Dulbecco's Medium - rIL-2 recombinant Interleukin - LAK Lymphokine-Activated Killer - RLNL Regional Lymph Node Lymphocytes - PBL Pheripheral Blood Lymphocytes - PBS Phosphate-Buffered Saline - RBC Red Blood Cells - RPMI-AB RPMI 1640 medium supplemented with 10% human AB-type serum Address for offprints: Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., 25 Miyukigaoka, Tsukuba-shi, Ibaraki, 305 Japan     

Target-cell-induced anergy in natural killer cells: suppression of cytotoxic function

 Our earlier studies have demonstrated that natural killer (NK) cells are the effectors that participate during the spontaneous regression of AK-5 tumour in syngeneic hosts. We have shown that the tumour cells are killed by necrosis and apoptosis. In this study, we have examined the induction of functional anergy in NK cells following coculture with fixed AK-5 tumour cells at high ratio. NK cells, upon coculture with fixed AK-5 cells (1:1 ratio), showed loss of cytotoxic function against both AK-5 (antibody-dependent cell cytotoxicity) as well as YAC-1 targets. The response of these cells to the activation by recombinant interleukin-2 and recombinant interferon γ was poor. Induction of tumour necrosis factor α (TNFα) secretion was observed after coculture of NK cells with fixed AK-5 cells. The cocultured cell supernatant inhibited the cytotoxic activity of NK cells, which was partially restored with anti-TNFα antibody. In addition, NK cells, after treatment with fixed tumour cells showed overexpression of the Fas receptor. We have also observed induction of apoptosis in cocultured NK cells. These studies suggest that the fixed tumour cells (antigen) at high ratio are able to suppress NK cell function as well as induce death in NK cells. Received: 16 September 1999 / Accepted: 13 January 2000     

Kinetics of interleukin-2 induced changes in rigidity of human natural killer cells

The success of adoptive immunotherapy is dependent in part on the successful delivery of effector cells to the tumor and the expression of cellular activities, such as adhesion, extravasation, and cytotoxic activity of the effector cells in the tumor. The structural rigidity of the effector cell is an important determinant of these functions. The present study was designed to quantify the changes in cellular rigidity and cytotoxic activity of human natural killer (NK) cells in the presence or absence of interleukin-2 (IL-2). Micropipet aspiration was used to measure the resistance of NK cells to an imposed external deformation. Homogeneous suspensions of NK cells were activated with 1000 U/mL of recombinant IL-2 in vitro and tested for cellular rigidity from 0 to 96 h post stimulation. The IL-2 activated cells increased their rigidity within 24 h and maintained it at this level for 96 h. Prolonged incubation of cells in IL-2 (14 d) resulted in a consistently high rigidity, which was further increased on starvation of the cells from IL-2. The increased rigidity of these cells was maintained throughout 96 h of IL-2 deprivation, although significant relaxation of rigidity was observed between 48 and 96 h. The relaxation of rigidity was associated with an increase in the number of nonviable cells. Reintroduction of IL-2 for 24 h to a culture of NK cells depleted of IL-2 for 48 h did not restore the cells to the predepletion level of rigidity. Cytotoxic activity of the activated NK cells following removal of IL-2 decreased to about 60% of the control activity within 24 h and continued through 72 h postdeprivation. These findings suggest that the initial activation of human NK cells by IL-2 will produce a relatively rapid increase in rigidity that may cause entrapment of these cells in small capillaries in vivo and that removal of IL-2 will produce an additional increase in rigidity, which is associated with decreased functional activity.     

Diverse multidrug-resistance-modification agents inhibit cytolytic activity of natural killer cells

Multidrug resistance (MDR) is the phenomenon in which cultured tumor cells selected for resistance to one chemotherapeutic agent simultaneously acquire resistance to several apparently unrelated drugs. MDR in tumor cells is associated with the over-expression of P-glycoprotein, an ATP-dependent cell-membrane transport molecule. P-glycoprotein is also expressed in several normal tissues but its physiological role(s) is unknown. We recently observed that a hierarchy of MDR-like activity exists among human peripheral blood lymphocytes in the order CD8>CD4>CD20 (cytoxic/suppressor T cells, helper T cells and B cells respectively). In this study, we report that natural killer (NK) cells also express MDR-like activity. This activity could be inhibited with verapamil or solutol HS-15, two agents that reverse MDR in tumor cells. These, and four additional reversing agents, were used to investigate the possible role of P-glycoprotein in NK cells. We observed that at 10% of their IC₅₀, five of six reversing agents inhibited NK-cell-mediated cytotoxicity; at higher (but non-toxic) doses, all six agents were inhibitory. These data suggest that NK-cell-mediated cytotoxicity may require the functional expression of an efflux molecule similar or identical to P-glycoprotein.This work was supported in part by grants from the National Cancer Institute (CA 57 470), the American Cancer Association — Illinois Division (no. 91-48), The Arthur Andersen Foundation and the Rayman, Freidman, Ditore and Rosenmutter Funds.     

乙酰胆碱对自然杀伤细胞活性的影响

目的:观察乙酰胆碱(ACh)对自然杀伤(NK)细胞活性的影响,并初步探讨其作用的受体机制.方法:根据不同的实验目的,选择ACh、胆碱能受体激动剂和拮抗剂分别作用于NK细胞,以乳酸脱氢酶(lactate dehydrogenase,LDH)自然释放法检测不同实验条件下NK细胞杀伤肿瘤靶细胞(Yac)的活性.结果:ACh、M受体激动剂毛果芸香碱和N受体激动剂烟碱在10-10～10-6mol/L浓度范围内都能显著抑制NK细胞杀伤肿瘤细胞的活性.M受体拮抗剂阿托品(10-8和10-7mol/L)能完全阻断同浓度ACh抑制NK细胞活性的作用;但N受体拮抗剂筒箭毒碱(10-8和10-7mol/L)不能阻断同浓度ACh抑制NK细胞活性的作用.结论:ACh可抑制NK细胞对肿瘤细胞的杀伤作用,此作用主要由淋巴细胞上的M受体和N1受体介导.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

Natural killer cells and innate immunity to protozoan pathogens

Natural killer (NK) cells are lymphoid cells that mediate significant cytotoxic activity and produce high levels of pro-inflammatory cytokines in response to infection. During viral infection, NK cell cytotoxicity and cytokine production is induced principally by monocyte-macrophage- and dendritic cell-derived cytokines but virally encoded ligands for NK cells are also beginning to be described. NK derived interferon-gamma (IFN-gamma) production is also essential for control of several protozoal infections including toxoplasmosis, trypanosomiasis, leishmaniasis and malaria. The activation of NK cells by protozoan pathogens is also believed to be cytokine-mediated although some recent studies suggest that direct recognition of parasites by NK cells also occurs. Both indirect signalling via accessory cell-derived cytokines and direct signalling, presumably through NK receptors, are needed in order for human malaria parasites (Plasmodium falciparum) to optimally stimulate NK activity.     

Natural killer cell–based immunotherapy: From transplantation toward targeting cancer stem cells

Natural killer (NK) cells are key players of the innate immune system. NK cells provide protection against infectious pathogens and malignancies in cell. This characteristic may be attributable to their intrinsic diverse potentialities and also their cooperation with adaptive immune lymphocytes, known as B and T cells. The growth, recurrence, and metastasis of cancer cells, and the failure of cytoreductive therapies against cancer cells are due to the small population of intratumor stem-like cells, called cancer stem cells (CSCs). Furthermore, NK cells can efficiently eradicate heterogeneous tumor cells after a long-term treatment. Therefore, NK cell–based therapy is a promising strategy to target and break CSC-associated resistance to anticancer drugs treatment. In this review, we have presented an overview of the emerging knowledge of the characteristics, diversities, and mechanism-driven immune surveillance of human NK cells and advances in NK cell–based immunotherapies. Finally, we will discuss how these cells can be applied to introduce the next generation of vaccine- and immune-based approaches to prevent drug resistance.     

Similarities and distinctions between murine natural killer cells and lymphokine-activated killer cells

Pretreatment of mice with rabbit anti-asialo GM1 removes both natural killer (NK) effector cells and NK cells responsive to interleukin 2 (IL-2). Spleen cells from these mice do possess normal lymphokine-activated killer (LAK) activity. Young mice (less than 3 weeks of age) do not have NK activity and do not possess IL-2-inducible NK effector cells. Similarly to anti-asialo GM1-treated mice, LAK cells can be generated from these mice. While these experiments indicate clear distinctions between a certain level of NK and LAK precursors, the distinctions are not as clear when analyzing mice congenitally deficient in NK cells. Beige mice which lack NK effector cells and IL-2-inducible NK cells also lack the ability to generate LAK cells. The relationships and differences between NK- and LAK-cell precursors and effectors are discussed.     

Lymphokine activated killer cells

Various subpopulations of human leukocytes may be induced by lymphokines to exert cytotoxic activity. In man major histocompatibility complex non-restricted tumor cell lysis by interleukin-2 (IL-2) induced peripheral blood lymphocytes is attributed mainly to natural killer cells. These T cell receptor negative large granular lymphocytes are called lymphokine activated killer (LAK) cells. In order to explore the potential of LAK cells in tumor therapy, several clinical studies have been conducted, using IL-2 alone or in combination with ex vivo IL-2-activated peripheral blood lymphocytes. Objective responses have reproducibly been achieved only in renal cell carcinoma and malignant melanoma and were associated with considerable toxicity. In view of restricted efficacy and increasing doubts as to whether LAK cells indeed account for the in vivo observed responses, more recent strategies focus on tumor antigen specific cytotoxic T cells or tumor infiltrating lymphocytes. Successful translation of this approach into clinical practice, however, may be dependent on some basic problems of tumor immunology to be solved which were thought to be by-passed by the LAK cell approach.     

Human nonspecific killer cells

The NK and K-cell activity of human leukocytes was investigated as compared with those cells of the K 562 cell line and murine cells covered by xenoantibodies in Graffi erythroblast leukaemia by means of the 51Cr release test. NK and K-cells could be identified in the blood and bone-marrow. However, they could not be identified in the thymus, lymph-nodes, and tonsils. Attempts of cell fraction with the blood of healthy donors revealed that the K-cells must be attributed to non-T-lymphocytes. NK-cells may be found in the fraction of non-T-lymphocytes as well as in that of T-lymphocytes. Killer cell activity tests in children with acute leukaemia resulted in leukaemia cells having NK and K-cell activity only in very rare cases. ALL patients in remission had strongly lowered NK-cell values under chemotherapy. In comparison to that, chemotherapy had no influence on K-cell activity. On the one hand, NK-cell activities were induced in mixed cultures of allogenous lymphocytes of the blood and, on the other hand, in cells of lymph-nodes. Attempts of fractionation, investigations for determining the influence of chemotherapy and attempts of inducing killer cell activity in vitro lead to the conclusion that NK and K-cells may be regarded as similar cell populations, being, however, not identical.     

Lymphokine-activated killer cells are rejected in vivo by activated natural killer cells

A 4-h in vivo cytotoxicity assay was used to study the fate of implanted IL-2-generated, lymphokine-activated killer (LAK) cells in mice undergoing an activated NK cell response. 125Iododeoxyuridine-labeled LAK cells were rejected from selected organs of C57BL/6 mice infected with lymphocytic choriomeningitis virus or treated with IL-2 or the IFN inducer poly I:C. This rejection was abrogated by the selective depletion of NK cells with antibodies to asialo-GM1 and NK1.1 Ag. Similar results were noted when LAK cells were generated from the spleens of B and T cell-deficient severe combined immunodeficiency mice and when LAK cells were implanted into severe combined immunodeficiency mice. These data indicate that NK cells activated by virus infections or by IL-2 infusions directly or indirectly eliminate implanted LAK cells. Because LAK cells are used in the treatment of certain human cancers, the strategy of accompanying this therapy with IL-2 infusions should be reassessed in light of these results.     

NK细胞和NKT细胞发育的转录调控

自然杀伤（natural killer,NK）细胞和自然杀伤T（natural killer T,NKT）细胞是参与机体抗病毒免疫和肿瘤免疫的两群淋巴细胞亚群,是介导先天性免疫（innate immunity）应答和调节适应性免疫（adaptive immunity）应答的重要效应细胞。近年来,随着对NK细胞和NKT细胞及其转录调控因子研究的不断深入,NK细胞和NKT细胞的发育机制逐步被阐明,这将为提高NK细胞和NKT细胞的抗病毒和肿瘤免疫疗效提供新的策略。