In vivo boosting of lung natural killer and lymphokine-activated killer cell activity by interleukin-2: comparison of systemic, intrapleural and inhalation routes.

Natural killer (NK) cells are thought to play a role in host defence against malignancy and infection, in immunoregulation and as precursor cells in a generation of lymphokine-activated killer (LAK) cells which can lyse NK-resistant tumour cells. As the lung is a major site for malignancy and infection and as there are large numbers of lymphoid cells including NK cells in the interstitial compartment of the lung, we evaluated the capacity of interleukin-2 (IL-2), a lymphokine capable of augmenting NK activity in vitro, to augment lung NK cell activity in vivo, using different routes of IL-2 administration. We compared both systemic (i.v. and i.p.) and local (intrapleural and inhalation) routes of IL-2 administration (50,000 U/daily for 5 days) using CBA mice, assessing NK and LAK cell activity in the spleen (systemic) and in the lung. The target cells used for these studies were the YAC-1 (NK-sensitive) and P815, NO36 and HA56 (NK-resistant, LAK-sensitive) cell lines. Splenic NK activity was increased by 1.4-1.9-fold for i.v./i.p., respectively, compared with controls with both systemic routes of administration, and lung NK activity was increased 3.2-fold and 3.8-fold (i.v./i.p, respectively, P less than 0.05), to levels which were comparable to systemic (splenic) NK activity following the same therapy. Intrapleural IL-2 administration similarly enhanced lung NK activity (3.3-fold) and splenic NK activity (1.3-fold; P less than 0.05 versus controls for both). Surprisingly, inhaled IL-2 suppressed both splenic and lung NK cell activity (84 +/- 8% and 78 +/- 10% suppression, respectively, P less than 0.05). LAK cell activity was also enhanced in the lung by 1.8-8-fold in response to i.v., i.p. and intrapleural IL-2, whereas inhaled IL-2 was ineffective in generating LAK cell activity. These results suggest that the systemic and intrapleural administration of IL-2 effectively boost pulmonary NK and LAK activity whereas inhalation of IL-2 does not. Thus, in clinical situations where boosting of local lung NK or LAK cell activity is desired, these routes of IL-2 administration may be effective.     

Asbestos fibres inhibit the in vitro activity of lymphokine-activated killer (LAK) cells from healthy individuals and patients with malignant mesothelioma.

Asbestos exposure is associated with an increased incidence of several malignancies, including malignant mesothelioma (MM). This study evaluates the relationship between asbestos exposure and the in vitro generation and function of LAK cells, an immune effector cell population with powerful lytic activity against MM cells. Both serpentine (chrysotile) and amphibole (amosite and crocidolite) forms of asbestos fibres suppress LAK cell generation, viability (by 5-11%, P less than 0.02) and cell recovery (by 13-15%, P less than 0.02). However, the LAK cells generated in the presence of the amphiboles were as effective as unexposed cells in lysing both standard tumour cell targets (K562, 56.4% lysis versus 61.5%, respectively, P greater than 0.5; NS; Daudi, 60.5% lysis versus 64.5% P greater than 0.5; NS), and MM tumour cell targets (mean of three MM cell lines 48.3% versus 46.3%, P greater than 0.5; NS), whereas the function of LAK cells generated in the presence of chrysotile was significantly reduced against three out of the five tumour cell targets tested (P less than 0.03). In the presence of asbestos fibres, LAK cell function was reduced against all five tumour cell targets (P less than 0.01), irrespective of whether the cell donors were healthy individuals or patients with MM. NK cell activity was also suppressed (P less than 0.01). The serpentine form of asbestos, chrysotile, was significantly more suppressive of both effector cell functions than either of the amphiboles (P less than 0.01). These findings suggest that asbestos exposure may suppress the function and in some instances the generation of immune effector cell mechanisms, thereby increasing the risk of disease and malignancy.     

Functional activity of natural killer cells and killer T cells in mice after ovarian transplantation

Tashkent Postgraduate Medical Institute, Ministry of Health of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Lopatkin). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 9, pp. 273–275, September, 1991.     

Generation of natural killer cells from hematopoietic stem cells in vitro for immunotherapy

Natural killer (NK) cells are part of the innate immune system and are an alluring option for immunotherapy due to their ability to kill infected cells or cancer cells without prior sensitization. Throughout the past 20 years, different groups have been able to reproduce NK cell development in vitro, and NK cell ontogeny studies have provided the basis for the establishment of protocols to produce NK cells in vitro for immunotherapy. Here, we briefly discuss NK cell development and NK cell immunotherapy approaches. We review the factors needed for NK cell differentiation in vitro, which stem cell sources have been used, published protocols, challenges and future directions for Good Manufacturing Practice protocols.     

Frequency and phenotype of natural killer cells and natural killer cell subsets in bovine lymphoid compartments and blood

Natural killer (NK) cells are widely distributed in lymphoid and non‐lymphoid tissues, but little is known about the recirculation of NK cells between blood and tissues. This is relevant to understanding recirculation in the steady‐state and also for determining the roles for NK cells in vaccine‐induced immunity and responses to infection. Therefore, the percentage of NK cells and their phenotype across peripheral blood, afferent lymph and lymph nodes in steady‐state conditions was investigated in cattle using the pseudo‐afferent lymphatic cannulation model. CD2⁺ CD25^lo NK cells were the predominant subset of NK cells within the blood. In contrast, CD2⁻ CD25^hi NK cells were the main subset present within the skin‐draining afferent lymphatic vessels and lymph nodes, indicating that CD2⁻ NK cells are the principal NK cell subset trafficking to lymph nodes via the afferent lymphatic vessel. Furthermore, a low percentage of NK cells were present in efferent lymph, which were predominantly of the CD2⁻ subset, indicating that NK cells can egress from lymph nodes and return to circulation in steady‐state conditions. These compartmentalization data indicate that NK cells represent a population of recirculating lymphocytes in steady‐state conditions and therefore may be important during immune responses to vaccination or infection.     

人LAK细胞的体外抗肿瘤作用

用重组IL-2(rIL-2)以及部分纯化的IL-2(PPIL-2)体外激活人外周血单个核细胞.(PBM),使之形成LAK细胞,然后借助于~(51)Cr释放实验,研究了正常人和肿瘤病人的LAk细胞对传代的肿瘤细胞系和新鲜实体瘤细胞的杀伤能力。实验结果表明:1.二种来源的LAK细胞均能明显杀伤传代的肿瘤细胞系,包括NK敏感的K562细胞和NK抵抗的Daudi细胞。2.采用数种新鲜实体瘤细胞作靶,二种来源的LAg细胞同样具有明显的广谱杀伤力,这证实该群杀伤细胞确系LAK细胞。3.不同来源的实体瘤细胞对LAK细胞的杀伤敏感性不同,表现为杀伤程度上的差异,这似乎提示某些肿瘤对LAK细胞杀伤存在抗性。     

Signal transduction via phosphorylated adhesion molecule, LFA-1{beta} (CD18), is increased by culture of natural killer cells with IL-2 in the generation of lymphokine-activated killer cells

It is well known that IL-2 stimulates natural killer (NK) cellsto express lymphokine activated killer (LAK) activity and thatthis stimulation prompts the acquisition of the ability to lysepreviously insensitive target cells. The possible role of adhesionmolecules in the IL-2 activation process was probed by focussingon a lymphocyte function-associated antigen (LFA)-1-dependentmodel system. A mAb to the LFA-1ß chain abrogatedLAK activity, but only moderately suppressed NK activity, suggestinga differential role for LFA-1ß In LAK compared withNK mediated lysis. Orthophosphate labeling demonstrated thatthe LFA-1ß chain was strongly phosphorylated in LAKbut not NK cells; in contrast, the chain was phosphorylatedsimilarlyin both effector cell types. At least a portion ofthe phosphorylation of the ß chain was on tyrosineresidues, as shown by Western blotting with anti-phosphotyrosineantibody of LFA-1ß immunoprecipitates. Crosslinkingof the LFA-1ß chain with plastic-adhered antibodystimulated Ca²⁺-dependent release of cytoplasmic lytic granulesand induced phosphatidyl inositol turnover in LAK but not NKcells. We conclude that the IL-2-induced phosphorylation oftheß chain of the LFA-1 adhesion molecule in LAK cellsand associated alteration in signal transduction may be importantin the stimulation of LAK cell activity in NK cells.     

Controlled infection with a therapeutic virus defines the activation kinetics of human natural killer cells in vivo

Human natural killer (NK) cells play an important role in anti‐viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon‐inducible molecules CD69 and tetherin peaked 24–48 h post‐infection, coincident with a peak of interferon‐induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post‐infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus‐based therapy. Furthermore, our results suggest the existence of a post‐infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.     

Alteration of T cells and natural killer cells during chickenpox in infancy

To determine the reasons for the low immune response and the mild morbidity of chickenpox in infancy, we investigated alteration of T cells and natural killer (NK) cells during chickenpox in children <1 year and 2 years old using flow cytometry. The CD4/CD8 ratio decreased only in the <1-year-old group from the acute to the convalescent phase (P<0.05). The CD3–CD16+CD56+ and CD57–CD16+ counts increased in the <1-year-old group, but those in the 2-year-old group did not increase from the acute to the convalescent phase. The CD3–CD16+CD56+ counts and the CD57-CD16+ counts and percentage were larger in the <1-year-old group than those in the 2-year-old group (P=0.001,P=0.002, andP<0.05) in the convalescent phase. These results seem to indicate that the low immune response in infancy after chickenpox are related to the small number of CD8 in contrast with CD4 and that increased subsets of NK cells during chickenpox may correlate to the mild morbidity of chickenpox in infancy.     

Optimal culture conditions for the generation of natural killer cell-induced dendritic cells for cancer immunotherapy

Dendritic cell (DC)-based vaccines continue to be considered an attractive tool for cancer immunotherapy. DCs require an additional signal from the environment or other immune cells to polarize the development of immune responses toward T helper 1 (Th1) or Th2 responses. DCs play a role in natural killer (NK) cell activation, and NK cells are also able to activate and induce the maturation of DCs. We investigated the types of NK cells that can induce the maturation and enhanced function of DCs and the conditions under which these interactions occur. DCs that were activated by resting NK cells in the presence of inflammatory cytokines exhibited increased expression of several costimulatory molecules and an enhanced ability to produce IL-12p70. NK cell-stimulated DCs potently induced Th1 polarization and exhibited the ability to generate tumor antigen-specific cytotoxic T lymphocyte responses. Our data demonstrate that functional DCs can be generated by coculturing immature DCs with freshly isolated resting NK cells in the presence of Toll-like receptor agonists and proinflammatory cytokines and that the resulting DCs effectively present antigens to induce tumor-specific T-cell responses, which suggests that these cells may be useful for cancer immunotherapy.     

Soluble HLA-G molecules induce apoptosis in natural killer cells

Membrane-bound human leukocyte antigen-G (HLA-G) molecules are primarily expressed by cytotrophoblasts of the fetus. They are thought to protect the fetus from immunologic attack by the maternal immune system and have recently been associated with transplantation graft acceptance. In addition, soluble HLA-G molecules (sHLA-G) have been shown to play a role in the success of pregnancies, but are upregulated in certain cancers. However, the exact mechanism for this regulation has remained elusive. The aim of this study was to examine the mechanism by which sHLA-G interact with natural killer (NK) cells in vitro. sHLA-G effectively blocked NK lysis of target cells via fracticide killing of NK cells by apoptosis. These studies support the protective role of sHLA-G in immunologic reactions by interacting with NK cells, thus providing a regulatory function.     

Liaison between natural killer cells and dendritic cells in human gestation

A successful pregnancy relies on immunological adaptations that allow the fetus to grow and develop in the uterus, despite being recognized by maternal immune cells. Among several immunocompetent cell types present within the human maternal/fetal interface, DC-SIGN~ dendritic cells （DCs） and CD56＋ natural killer （NK） cells are of major importance for early pregnancy maintenance, not only generating maternal immunological tolerance but also regulating stromal cell differentiation. Previous reports show the presence of NK-DC cell conjugates in first trimester human decidua, suggesting that these cells may play a role in the modulation of the local immune response within the uterus. While effective immunity is necessary to protect the mother from harmful pathogens, some form of tolerance must be activated to avoid an immune response against fetal antigens. This review article discusses current evidence concerning the functions of DC and NK cells in pregnancy and their liaison in human decidua.     

In vivo kinetics of human natural killer cells: the effects of ageing and acute and chronic viral infection

Human natural killer (NK) cells form a circulating population in a state of dynamic homeostasis. We investigated NK cell homeostasis by labelling dividing cells in vivo using deuterium-enriched glucose in young and elderly healthy subjects and patients with viral infection. Following a 24-hr intravenous infusion of 6,6-D(2)-glucose, CD3(-) CD16(+) NK cells sorted from peripheral blood mononuclear cells (PBMC) by fluorescence-activated cell sorter (FACS) were analysed for DNA deuterium content by gas chromatography mass spectrometry to yield minimum estimates for proliferation rate (p). In healthy young adults (n=5), deuterium enrichment was maximal approximately 10 days after labelling, consistent with postmitotic maturation preceding circulation. The mean (+/- standard deviation) proliferation rate was 4 x 3 +/- 2 x 4%/day (equivalent to a doubling time of 16 days) and the total production rate was 15 +/- (7 x 6) x 10(6) cells/l/day. Labelled cells disappeared from the circulation at a similar rate [6 x 9 +/- 4 x 0%/day; half-life (T((1/2))) < 10 days]. Healthy elderly subjects (n=8) had lower proliferation and production rates (P=2 x 5 +/- 1 x 0%/day and 7 x 3 +/- (3 x 7) x 10(6) cells/l/day, respectively; P=0 x 04). Similar rates were seen in patients chronically infected with human T-cell lymphotropic virus type I (HTLV-I) (P=3 x 2 +/- 1 x 9%/day). In acute infectious mononucleosis (n=5), NK cell numbers were increased but kinetics were unaffected (P=2 x 8 +/- 1 x 0%/day) a mean of 12 days after symptom onset. Human NK cells have a turnover time in blood of about 2 weeks. Proliferation rates appear to fall with ageing, remain unperturbed by chronic HTLV-I infection and normalize rapidly following acute Epstein-Barr virus infection.     

Intrahepatic and peripheral blood phenotypes of natural killer and T cells: differential surface expression of killer cell immunoglobulin‐like receptors

Deep characterization of the frequencies, phenotypes and functionalities of liver and peripheral blood natural killer (NK), natural killer T (NKT) and T cells from healthy individuals is an essential step to further interpret changes in liver diseases. These data indicate that CCR7, a chemokine essential for cell migration through lymphoid organs, is almost absent in liver NK and T cells. CD56^bright NK cells, which represent half of liver NK cells, showed lower expression of the inhibitory molecule NKG2A and an increased frequency of the activation marker NKp44. By contrast, a decrease of CD16 expression with a potential decreased capacity to perform antibody‐dependent cellular cytotoxicity was the main difference between liver and peripheral blood CD56^dim NK cells. Liver T cells with an effector memory or terminally differentiated phenotype showed an increased frequency of MAIT cells,T‐cell receptor‐γδ (TCR‐γδ) T cells and TCR‐αβ CD8⁺ cells, with few naive T cells. Most liver NK and T cells expressed the homing markers CD161 and CD244. Liver T cells revealed a unique expression pattern of killer cell immunoglobulin‐like receptors (KIR) receptors, with increased degranulation ability and higher secretion of interferon‐γ. Hence, the liver possesses a large amount of memory and terminally differentiated CD8⁺ cells with a unique expression pattern of KIR activating receptors that have a potent functional capacity as well as a reduced amount of CCR7, which are unable to migrate to regional lymph nodes. These results are consistent with previous studies showing that liver T (and also NK) cells likely remain and die in the liver.     

Subsets of human natural killer cells and their regulatory effects

Human natural killer (NK) cells have distinct functions as NK^tolerant, NK^cytotoxic and NK^regulatory cells and can be divided into different subsets based on the relative expression of the surface markers CD27 and CD11b. CD27⁺ NK cells, which are abundant cytokine producers, are numerically in the minority in human peripheral blood but constitute the large population of NK cells in cord blood, spleen, tonsil and decidua tissues. Recent data suggest that these NK cells may have immunoregulatory properties under certain conditions. In this review, we will focus on these new NK cell subsets and discuss how regulatory NK cells may serve as rheostats or sentinels in controlling inflammation and maintaining immune homeostasis in various organs.     

Activity of human natural killer cells under different experimental conditions

Laboratory of Immunochemistry, N. F. Gamaleya Research Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow. (Presented by Academician of the Russian Academy of Medical Sciences S. V. Prozorovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol 114, No. 8, pp. 187–189, August, 1992.     

P-glycoprotein-mediated multidrug resistance and lymphokine-activated killer cell susceptibility in ovarian carcinoma

The sensitivity of tumor cells to lysis by natural killer (NK) and interleukin-2 (IL-2)-activated killer (LAK) cells was studied in three ovarian carcinoma cell lines (2780.9S, SKOV-3, and CHOAUXB1), four multidrug-resistant (MDR) variants, and a melphalan-resistant line. The antitumor activity of LAK cells was evaluated both by⁵¹Cr release and by conjugate formation assays. Four of four P-glycoprotein-positive (P-gp⁺) MDR ovarian carcinoma cell line variants were lysed by human LAK cells to a greater extent than were their drug-sensitive counterparts. In contrast, a melphalan-resistant ovarian carcinoma cell line that does not overexpress P-gp (P-gp^–) did not exhibit an increased susceptibility to LAK cells relative to its parental cell line. Two of the four P-gp⁺ MDR ovarian carcinoma cell line variants were tested for human NK cell susceptibility and this was found to be unchanged or decreased. The P-gp⁺ MDR ovarian carcinoma cell line 2780.AD645 showed a higher frequency of tumor cell binding to LAK cells than did the drug-sensitive parental line. A monoclonal antibody (mAb) against a cell surface epitope of P-gp, MRK16, used at 1 g/ml, enhanced the LAK susceptibility of P-gp⁺ MDR ovarian carcinoma cell lines. However, when incubation with 10 g/ml MRK-16 antibody (Ab) was followed by 12.5 g/ml F(ab)₂ goat anti-mouse (GAM) immunoglobulin (Ig), the increased LAK susceptibility of P-gp⁺ MDR cell lines was inhibited. These data strongly suggest that P-glycoprotein-positive MDR ovarian carcinoma cells not only are targets for LAK cells, but are more sensitive than their drug-sensitive parental lines. This is in contrast to their susceptibility to NK cells, which is low to start with and remains unchanged or even decreased in MDR cells. It is postulated here that P-gp or associated changes result in a greater frequency of effector-target cell binding, leading to increased LAK cell cytotoxicity.