Natural Killer Cell Activity Assay Research Articles

Multifunctional Microparticles with Stimulation and Sensing Capabilities for Facile NK Cell Activity Assay.

Natural killer (NK) cells are a subset of innate lymphoid cells playing an important role in immune surveillance and early defense against infection and cancer. They recognize and directly kill infected or transformed cells. At the same time, they produce various cytokines and chemokines to regulate other immune cells. NK cell activity can be a useful marker for health screenings because impaired NK cell functions may indicate a more susceptible environment for infection or tumor development. Currently, most NK cell activity assays are focused on measuring either cytokine secretion, in particular, interferon γ (IFN-γ), or cytotoxicity against target cells such as K562, thus only providing partial information on NK cell activity. In order to develop a comprehensive test for measuring NK cell function, cytotoxicity and cytokine secretion ability should be measured simultaneously. In addition, current NK cell assays are performed by stimulating NK cells with cocktails of cytokines, antibody-coated beads, or live target cells. In this study, we developed multifunctional microparticles for NK cell activity assay (MNAs) that allow simultaneous stimulation and sensing various NK cell activities, including cytokine secretion and cytotoxicity. The surfaces of MNAs are decorated with multiple functional biomolecules, including antibodies that stimulate NK cells by engaging NK cell activating receptors, antibodies that can capture cytokines secreted by NK cells, and a peptide sensor that reacts with granzyme B, a key molecule released by NK cells for cytotoxicity. The performances of MNAs are assessed using flow cytometry and live cell imaging. NK cell activity is measured by simply mixing MNAs with NK cells and performing flow cytometry, and the results are comparable to those measured by standard NK cell activity assays.

Neutral polysaccharide from Panaxnotoginseng enhanced cyclophosphamide antitumor efficacy in hepatoma H22-bearing mice

BackgroundOur previous studies demonstrated that the administration of crude Polysaccharide from Panax notoginseng (CPPN) can effectively prolong the lifespan of tumor-bearing mice via boosting the host immune system as well as weak cytotoxicity against hepatocellular carcinoma (HCC). In the present study, Neutral Polysaccharide (NPPN) were further purified from crude polysaccharide isolated from panax notoginseng. The effects of NPPN on the immune function and hematopoietic function of mice with low immunity and myelosuppression induced by cyclophosphamide (CTX) were investigated. The effect of NPPN combined with CTX on the tumor inhibition rate of the H22 tumor-bearing mice and the impact of NPPN on the proliferation of H22 liver cancer cells in vitro were investigated.MethodsCPPN was obtained by water extraction and alcohol precipitation method, and further purified by DEAE Sepharose Fast Flow ion exchange resin column. NPPN was added to the immunosuppressed with myelosuppression mice induced by CTX. Thymus index, spleen index, lymphocyte proliferation stimulation index by adding of concanavalin A, determination of serum hemolysin, NK cell activity assay, mice carbon clearance experiment, blood count tests were detected. The tumor inhibition rate of the H22 tumor-bearing mice treated with NPPN combined with CTX was recorded.ResultsNPPN and 4 kinds of acid polysaccharide from Panax notoginseng (APPN) were successfully isolated from the CPPN by DEAE Sepharose Fast Flow ion exchange resin column. NPPN inhibited the growth of H22 cells and significantly increase the tumor inhibition rate of the H22 tumor-bearing mice combined with CTX. The elevation of the cellular and humoral immunity levels as well as a variety of blood count tests indicators of immunosuppressive with myelosuppression mice may contribute to the antitumor activity of NPPN.ConclusionNPPN has a potential antitumor activity for the treatment of liver cancer combined with cyclophosphamide.

Assessment of NK Cell Activity Based on NK Cell-Specific Receptor Synergy in Peripheral Blood Mononuclear Cells and Whole Blood.

Natural killer (NK) cells are cytotoxic innate lymphocytes endowed with a unique ability to kill a broad spectrum of cancer and virus-infected cells. Given their key contribution to diverse diseases, the measurement of NK cell activity (NKA) has been used to estimate disease prognosis or the effect of therapeutic treatment. Currently, NKA assays are primarily based on cumbersome procedures related to careful labeling and handling of target cells and/or NK cells, and they require a rapid isolation of peripheral blood mononuclear cells (PBMCs) which often necessitates a large amount of blood. Here, we developed an ELISA-based whole blood (WB) NKA assay involving engineered target cells (P815-ULBP1+CD48) providing defined and synergistic stimulation for NK cells via NKG2D and 2B4. WB collected from healthy donors (HDs) and patients with multiple myeloma (MM) was stimulated with P815-ULBP1+CD48 cells combined with IL-2. Thereafter, it utilized the serum concentrations of granzyme B and IFN-γ originating in NK cells as independent and complementary indicators of NKA. This WB NKA assay demonstrated that MM patients exhibit a significantly lower NKA than HDs following stimulation with P815-ULBP1+CD48 cells and had a good correlation with the commonly used flow cytometry-based PBMC NKA assay. Moreover, the use of P815-ULBP1+CD48 cells in relation to assessing the levels of NKG2D and 2B4 receptors on NK cells facilitated the mechanistic study and led to the identification of TGF-β1 as a potential mediator of compromised NKA in MM. Thus, our proposed WB NKA assay facilitates the reliable measurement of NKA and holds promise for further development as both a clinical and research tool.

A Unique Enzymatically Hydrolyzed Whey Protein Positively Impacts Measures of Immunity

Abstract Objectives Milk proteins appear to have little direct impact on the immune system when consumed directly. The digestive system breaks them down into absorbable size and they provide nutritive value. However, it has been discovered that enzymatically hydrolyzed peptides can more directly influence the immune system, presumably before digestion completes their hydrolysis during gut immune system exposure. The term ‘bioactive centers’ will be used to describe these unique peptides. Methods After screening several different versions of enzyme hydrolyzed whey, a new bioactive center-containing product was found that had promise for further immunological research. It was compared against transfer factors, 4Life Research's colostrum filtrate, which have previously been studied for their immune activity. Results In a natural killer cell activity assay, the bioactive centers product was significantly (70%) more effective than IL-2, the standard stimulant, and equally as effective as transfer factors. A second experiment confirmed that this effect was consistent across different production batches of the product. A third experiment found that a combination of the bioactive centers and transfer factors enhanced the activity by 10% on average. In a second study, the bioactive centers were evaluated in a mouse model. They were as effective as transfer factors in stimulating NK cell activity, antibody production, TNF-alpha and IL-2 production, and in stimulating phagocytosis. In most cases, results were higher, though not statistically significantly so, for the transfer factor/bioactive centers combination. Conclusions Since the discovery of transfer factors, work both in and outside of 4Life Research has continued to try to identify new approaches to positively impacting the immune system. These results show that bioactive centers from whey can have such activity. Further work is needed to fully understand their impact in human health. Funding Sources 4Life Research.

Assessment of Human Natural Killer Cell Events Driven by FcγRIIIa Engagement in the Presence of Therapeutic Antibodies.

One mechanism of action for clinical efficacy by therapeutic antibodies is the promotion of immune-related functions, such as cytokine secretion and cytotoxicity, driven by FcγRIIIa (CD16) expressed on natural killer (NK) cells. These observations have led to research focusing on methods to increase Fc receptor-mediated events, which include removal of a fucose moiety found on the Fc portion of the antibody. Further studies have elucidated the mechanistic changes in signaling, cellular processes, and cytotoxic characteristics that increase ADCC activity with afucosylated antibodies. Additionally, other studies have shown the potential benefits of these antibodies in combination with small molecule inhibitors. These experiments demonstrated the molecular and cellular mechanisms underlying the benefits of using afucosylated antibodies in combination settings. Many of these observations were based on an artificial in vitro activation assay in which the FcγRIIIa on human NK cells was activated by therapeutic antibodies. This assay provided the flexibility to study downstream effector NK cell functions, such as cytokine production and degranulation. In addition, this assay has been used to interrogate signaling pathways and identify molecules that can be modulated or used as biomarkers. Finally, other therapeutic molecules (i.e., small molecule inhibitors) have been added to the system to provide insights into the combination of these therapeutics with therapeutic antibodies, which is essential in the current clinical space. This manuscript aims to provide a technical foundation for performing this artificial human NK cell activation assay. The protocol demonstrates key steps for cell activation as well as potential downstream applications that range from functional readouts to more mechanistic observations.

Genotype characteristics and immunological indicator evaluation of 311 hemophagocytic lymphohistiocytosis cases in China

BackgroundPrimary hemophagocytic lymphohistiocytosis (pHLH) is a genetic disorder that is classically diagnosed by genetic testing. Secondary HLH (sHLH) is usually caused by infections, malignancies, or autoimmune disorders, but may display some mutations or polymorphisms. Rapid immunological assays examining natural killer (NK) cell activity, degranulation function (CD107a), and protein expression related to genetic deficiencies have been recommended for early pHLH identification.MethodsA retrospective analysis of 311 HLH patients from a Chinese population was performed to evaluate the potential correlations between genetic testing and rapid immunological assays; genotyping characteristics, age of onset, and etiology were examined.ResultsAmong the 128 (128/311) patients who were positive in the genetic screening, the most frequently detected mutant gene was UNC13D (29%), followed by LYST (21%), PRF1 (17%), and STXBP2 (10%). Among pHLH patients (n = 39), the majority (67%) had PRF1 and UNC13D defects. FHL-2 was predominant (12/27, 44%) in patients aged under 18, while FHL-3 was the most common (6/12, 50%) in adults. Differences in genetic variant types and etiological components were noted in HLH patients based on the age of onset. NK cell activity and CD107a were observed to show a consistent trend (Ptrend < 0.001) when grouping patients according to the severity of the genetic variant type. Moreover, NK cell activity was generally consistent within a certain range of ΔCD107a values (Ptrend < 0.001). The PPV for bi-allelic degranulation gene mutations in patients with CD107a < 5% was 38.9% (7/18), while the PPV in patients with CD107a ≤10% was 16.7% (13/78). The PPV for pHLH was 41.4% (29/70) with NK cell activity ≤13%. To further evaluate the diagnostic efficacy of NK cell activity assay in pHLH, a receiver operating characteristic (ROC) curve was generated and showed an area under the curve (AUC) of 0.872, and the optimal cutoff value was determined to be 13.425% with a sensitivity of 84.21% and specificity of 80.67% when the corresponding Youden index was maximized. Flow cytometry screening for deficient proteins, including perforin, SAP, and XIAP, showed a relatively high sensitivity (83.33–93.33%). The positive predictive values (PPVs) of perforin and XIAP were relatively low (20.83–26.92%), but the negative predictive values (NPVs) for all three were excellent (all > 98%).ConclusionsVarious immunological indicators have different clinical prediction and application values for the diagnosis of pHLH. The degree of reduction of immunological indicators also needs attention, and choosing appropriate cutoff value may be of important significance in guiding clinical judgment for pHLH.

Heat-treated Lactobacillus plantarum increases the immune responses through activation of natural killer cells and macrophages on in vivo and in vitro models.

Recently, Lactobacillus plantarum (nF1) has been reported to have immune-enhancing effects in an immunosuppressed-animal model. Natural killer (NK) cells and macrophages play important roles in the immune responses. However, immunomodulatory effects of heat-treated Lactobacillus plantarum-nF1 (hLp-nF1) on the activation of NK cells and macrophages have not been elucidated. We assessed whether hLp-nF1 could elevate the activation of NK cells and macrophages using cyclophosphamide (CP)-induced immunosuppressed BALB/c mice and RAW 264.7 macrophages. A nitric oxide (NO) assay, enzyme-linked immunosorbent assay, Western blot analysis and NK cell activity assay were used to examine the effects of hLp-nF1 on the immune enhancement.Results/Key findings. Administration of hLp-nF1-elevated NK cell activities and serum levels of TNF-α, IL-2, and IL-12 in CP-induced immunosuppressed mice. In RAW 264.7 macrophages, treatment with hLp-nF1 increased the production of NO and expression of inducible NO synthase. Simultaneously, hLp-nF1 increased the production of TNF-α, IL-2, and IL-6 from RAW 264.7 cells. Finally, hLp-nF1 induced activation of nuclear factor-κB and phosphorylation of IκBα. We identified that hLp-nF1 has an immune-enhancing effect through the activation of NK cells and macrophages. Therefore, these findings suggest that hLp-nF1 would be helpful to enhance the immunity.

Clinical utility of a novel natural killer cell activity assay for diagnosing non-small cell lung cancer: a prospective pilot study.

PurposeAlthough decreased natural killer cell activity (NKA) has been observed in many solid cancers, clinical implication of NKA has been scarcely investigated in lung cancer. The objective of this study was to evaluate the potential of using NKA to support diagnosis of non-small cell lung cancer (NSCLC).Materials and methodsWe prospectively evaluated and compared peripheral blood NKA using a novel interferon-gamma releasing assay in healthy population (n=40), patients with benign lung disease (n=40), and those with NSCLC (n=71). We explored the correlation between NKA and clinical parameters and assessed diagnostic performance of NKA for NSCLC using receiver operating characteristic curve analysis.ResultsMedian NKA values in healthy population, patients with benign lung disease, and those with NSCLC were 1,364.2, 1,438.2, and 406.3 pg/mL, respectively. NKA in NSCLC patients was significantly lower than that in the other two control groups (both P<0.001). At a cutoff value of NKA at 391.0 pg/mL, the area under the curve was 0.762 (95% CI: 0.685–0.838, P<0.001), with a sensitivity of 52.3%, a specificity of 91.0%, a positive predictive value of 85.3%, and a negative predictive value of 65.4% for the diagnosis of NSCLC. Multivariate analysis demonstrated that diagnosis of NSCLC is the only clinical parameter that was significantly associated with NKA (P<0.001).ConclusionThis pilot study showed that patients with low NKA were more likely to have lung cancer. Further studies are warranted in order to establish the clinical utility of NKA test for diagnosing lung cancer.

A Case of Type 2 Hypersensitivity to Rasburicase Diagnosed with a Natural Killer Cell Activation Assay

Drug hypersensitivity reactions can lead to different clinical pictures depending on the underlying immunological mechanism. Diagnosis tests are already available to assess the most frequent drugs hypersensitivity reactions, which are mediated by specific IgE or T cells. However, it remains challenging to diagnose type 2 hypersensitivity reactions (T2HR), which can lead to severe cytopenia and liver failure. Here, we describe a case of T2HR to rasburicase, an uricolytic agent used to prevent tumor lysis syndrome. In this patient, sensitization was associated with the production of specific IgG able to bind to leukocytes. We found that patient NK cells were specifically activated in the presence of rasburicase and autologous serum, which led to exocytosis of lytic granules. This antibody-dependent cell cytotoxicity mechanism may lead to cytopenia observed in the patient. Moreover, this NK cell activation assay could be used to improve the diagnosis of a T2HR to rasburicase and, by extent, to other drugs. These data also suggest that NK cells could play an important role in the pathophysiological mechanism of T2HR.

Application of an improved flow cytometry-based NK cell activity assay in adult hemophagocytic lymphohistiocytosis.

Low or absent natural killer (NK) cell activity is included as one of the HLH-2004 diagnostic criteria. To improve the diagnosis of HLH, we aimed to establish a rapid and reliable NK cell activity assay that avoids the use of radioactivity. The K562 cell line, as standard NK target cells, was engineered to stably express enhanced green fluorescent protein (EGFP), which can be quantified by flow cytometry. The EGFP-flow cytometry method for measuring NK cell activity was improved by double staining of early and late apoptotic target cells. Whole-blood samples from healthy volunteers were assessed with this method, which demonstrated that optimal conditions were effector-target ratio of 10:1 and incubation time of 4 h. This method was further evaluated for samples from 113 HLH patients and 64 healthy volunteers. Mean NK cell activity in either primary or secondary HLH patients was significantly lower (P<0.001) than in healthy individuals (20.23±4.12%). Furthermore, primary HLH patients (10.76±2.54%) exhibited even lower (P<0.001) NK cell activity compared with secondary HLH patients (15.01±3.62%). We have optimized and implemented this method in clinically relevant samples.

A high-throughput assay of NK cell activity in whole blood and its clinical application

Natural killer (NK) cells are lymphocytes of the innate immune system and have the ability to kill tumor cells and virus-infected cells without prior sensitization. Malignant tumors and viruses have developed, however, strategies to suppress NK cells to escape from their responses. Thus, the evaluation of NK cell activity (NKA) could be invaluable to estimate the status and the outcome of cancers, viral infections, and immune-mediated diseases. Established methods that measure NKA, such as 51Cr release assay and CD107a degranulation assay, may be used to determine NK cell function, but they are complicated and time-consuming because they require isolation of peripheral blood mononuclear cells (PBMC) or NK cells. In some cases these assays require hazardous material such as radioactive isotopes. To overcome these difficulties, we developed a simple assay that uses whole blood instead of PBMC or isolated NK cells. This novel assay is suitable for high-throughput screening and the monitoring of diseases, because it employs serum of ex vivo stimulated whole blood to detect interferon (IFN)-γ secreted from NK cells as an indicator of NKA. After the stimulation of NK cells, the determination of IFNγ concentration in serum samples by enzyme-linked immunosorbent assay (ELISA) provided a swift, uncomplicated, and high-throughput assay of NKA ex vivo. The NKA results microsatellite stable (MSS) colorectal cancer patients was showed significantly lower NKA, 263.6±54.5pg/mL compared with healthy subjects, 867.5±50.2pg/mL (p value <0.0001). Therefore, the NKA could be utilized as a supportive diagnostic marker for microsatellite stable (MSS) colorectal cancer.

Specific antibody‐dependent cellular cytotoxicity responses associated with slow progression of HIV infection

Antibody-dependent cellular cytotoxicity (ADCC) is potentially an effective adaptive immune response to HIV infection. However, little is understood about the role of ADCC in controlling chronic infection in the small number of long-term slow-progressors (LTSP) who maintain a relatively normal immunological state for prolonged periods of time. We analysed HIV-specific ADCC responses in sera from 139 HIV(+) subjects not on antiretroviral therapy. Sixty-five subjects were LTSP, who maintained a CD4 T-cell count > 500/μl for over 8 years after infection without antiretroviral therapy and 74 were non-LTSP individuals. The ADCC responses were measured using an natural killer cell activation assay to overlapping HIV peptides that allowed us to map ADCC epitopes. We found that although the magnitude of ADCC responses in the LTSP cohort were not higher and did not correlate with CD4 T-cell depletion rates, the LTSP cohort had significantly broader ADCC responses compared with the non-LTSP cohort. Specifically, regulatory/accessory HIV-1 proteins were targeted more frequently by LTSP. Indeed, three particular ADCC epitopes within the Vpu protein of HIV were recognized only by LTSP individuals. Our study provides evidence that broader ADCC responses may play a role in long-term control of HIV progression and suggests novel vaccine targets.

Ultrasound complex enzymes assisted extraction and biochemical activities of polysaccharides from Epimedium leaves

Ultrasound complex enzymes (UCE) were used to extract crude polysaccharides (CEP) from Epimedium leaves. The response surface methodology (RSM) based on Box–Behnken Design (BBD) was employed to further optimize the extraction conditions. The optimum conditions were: complex enzyme dose of 50, 250, 200, 100U/g (papain, pectase, cellulase and α-amylase), extraction temperature 46.8°C, ultrasound time 42.3min, pH 4.28, and ultrasound power 311W. Under these conditions, the experimental yield of CEP was 5.98%, which was well matched with the value predicted by the model. Three major polysaccharide fractions (EP-1, EP-2 and EP-3) were obtained by ultrafiltration, chromatography on DEAE-Sepharose Fast Flow and Sephadex G-100 column. Their chemical–physical characteristics were investigated using chemical methods, gas chromatography (GC) and high-performance size-exclusion chromatography (HPSEC). Biochemical characterization of the polysaccharide fractions was evaluated on the basis of free radical scavenging, ferric-reducing antioxidant power (FRAP), lymphocyte proliferation, macrophage phagocytosis and NK cell activity assay in vitro. The results showed that all fractions contained higher galacturonic acid (GalA) and galactose (Gal), possessed considerable antioxidant activity, and could promote proliferation of splenic lymphocytes, activate macrophages and enhance NK cells’ activity. UCE as a more effective and environment-friendly technique could be employed for extracting active ingredients from plant materials, extensively applied in pharmaceutical and food industries.

Effects of Korean red ginseng on cyclophosphamide‐induced immunosuppression: comparative effects of 6‐year and 4‐year old Korean red ginseng

In this study, the protective effects of Korean red ginseng (KRG) against cyclophosphamide (CY)‐induced immunosuppression were investigated in animal models. Male BALB/c mice were divided into 6 groups: Normal and CY‐control groups were fed normal diet and the four test groups received modified diets containing 5 or 10% of 4‐year old or 6‐year old KRG powder for 4 weeks. In the subtype and NK cell activity assay, CY was given intraperitoneally in a single dose of 50 mg/kg three days before sacrifice. For the T‐dependent antibody response, CY was given intraperitoneally in a single dose of 50 mg/kg five days before sacrifice and mice were immunized with sheep red blood cells (SRBCs) intraperitoneally in the next day.CY, as reported, showed suppressed effects on body weights, organ weights, splenic lymphocyte subpopulation (total T cells, CD4+ T cells, CD8+ T cells, macrophages and B cells), antibody‐forming cells to SRBC and NK cell activity in these animals. The administration of KRG powder elevated the depressed relative weights of thymus and spleen and increased the number of splenic lymphocyte subpopulation, anti‐SRBC producing cells that were remarkably decreased by the treatment of CY. 6‐year old KRG powder showed more potent effect than 4‐year old KRG in enhancing immune function in the CY‐immunosuppressed mice. These results suggest that 6‐year old KRG is more potent in stimulating immunity in immunosupressed condition than 4‐year old KRG.

Ptaquiloside-induced immunotoxic effects are reversed by selenium supplementation (87.21)

Abstract Pteridium aquilinum (Pa) is a poisonous plant and its ingestion has been closely associated with human and animal cancers. Our earlier studies have shown that Pa reduces white pulp of spleen and NK cell cytotoxicity in mice. In addition, we observed that selenium (Se) reversed Pa-induced spleen damage. In this study the aim was evaluate if ptaquiloside (Ptq), the main toxic principle found in Pa, is responsible for its immunotoxic effects and if Se would reverse these effects. For in vivo study, mice were separated into 4 groups: control, Ptq, Ptq+Se, and Se. Mice were treated by gavage for 14 days with Ptq (5.3mg/kg BW) and/or Se (1.3mg/kg BW). Mice were killed and it was prepared a non-adherent splenic cells suspensions to perform NK cell activity assay against YAC cell line. For in vitro study, cultures of non-adherent splenic cells were performed for each mouse as follows: untreated, Se 0.1mM, RPMI extract of Pa (RE) 4mg/ml, RE 4mg/ml+Se 0.1mM (co-incubation), and RE 4mg/ml+Se 0.1mM (RE/1h wash and then Se/1h). After treatment, the cells were washed to proceed both NK cell activity assay and MTT assay. We observed that Ptq reduced NK cytotoxicity, which permits to suppose that Ptq is the principle responsible by immunotoxic effects. Moreover, NK cell activity was completely recovered after Se supplementation in vivo and in vitro. These results showed for the first time that Pa-induced immunotoxic effects are caused by Ptq and that these effects are fully recovered by Se.

Electro-acupuncture (EA) induced attenuation of immunosuppression appearing after epidural or intrathecal injection of morphine in patients and rats.

In order to investigate the effect of epidural morphine and electroacupuncture (EA) on immune function of postoperative patients, peripheral blood for NK (natural killer) cell activity assay were obtained from patients before and on the 1st, 3rd, 7th day after cholecystectomy. 1 mg morphine injected into epidural space after surgery significantly suppressed NK cell activities via prolonged inhibition and increased the inhibitory intensity of it. EA of "Zusanli (St. 36), Sanyinjiao(Sp. 6), Hegu(L.I.4) and Neiguan(P.6) points enhanced the NK cell activities after epidural injection of morphine. It was further observed that microinjection of 1 microgram/1 microliter naloxone into periaqueductal gray (PAG) partially antagonized the suppression of NK cell activity induced by intrathecal (ith) injection of 40 micrograms/50 microliters morphine, and so does EA of "Zusanli(St. 36)". When EA applied after naloxone microinjected into PAG, the NK cell activity was further augmented as compared to that using EA or PAG microinjection of naloxone alone. The above results suggest epidural morphine combined with EA is a better method to relieve postoperative pain, and opiate receptor in PAG might partially mediate the suppression of NK cell activity induced by intrathecal injection of morphine. Yet, the mechanisms of the attenuation of EA on morphine induced immunosuppression needs further study.

Influence of elevated temperature on natural killer cell activity, lymphokine-activated killer cell activity and lectin-dependent cytotoxicity of human umbilical cord blood and adult blood cells

Purpose : To determine whether hyperthermia is to the benefit or detriment of host immune function, the effect of hyperthermia was evaluated on various functions of T-lymphocytes from human umbilical cord blood and compared to that of adult blood. Methods and Materials: Nonadherent mononuclear cells from cord blood or adult blood were used as the effector cells. To generate lymphokine activated killer (LAK) cells, effector cells were kept in culture for 5 days in complete medium containing recombinant human interleukin-2. To activate effector cells to become cytotoxic, cells were kept in culture in complete medium containing Con A. Cytotoxicity was determined in a standard 4-h chromium release assay using K-562 human erythroleukemic cells (in the natural killer cell activity assay) or Daudi cells (in the LAK cell activity or Lectin dependent cytotoxicity assay) as targets. For heat effects, cells in complete medium were heated at the desired temperature in a water bath for 1 h. Results: Lymphokine-activated killer cell activity, lectin-dependent cytotoxicity and T-cell proliferative capacity were not deficient in human cord blood. Cytotoxic activities of T-cells from adult blood as well as from cord blood can be enhanced at febrile range (⩽ 40°C), and were significantly decreased by exposure to 1 h at 42°C. Conclusion: The febrile responses (⩽ 40°C) to infection, in the course of malignant disease and with biological response modifiers treatment, may all be related to host defense mechanisms. Based on these observations, whole body hyperthermia (⩽ 40°C), in combination with the appropriate cytokines, may have therapeutic potential in the treatment of neonatal infections and malignancies under certain circumstances. Hyperthermia in febrile range may, therefore, confer an important immunoregulatory advantage to the host. In contrast, tumor killing therapeutic temperature (> 42°C) which inhibits host immunocompetence should probably be used only for local hyperthermia.

Enzyme release assay of human NK cell activity using β-galactosidase-expressing K562 target cell line

In the present report, we established a K562 cell line useful for an enzyme release assay of human natural killer (NK) activity. Human myelogenous leukemia cell line, K562, was transfected with a plasmid carrying Escherichia coli β-galactosidase (β-gal) gene. A colony that permanently expresses the enzyme activity was isolated, and designated K562/Zneo. Incubation of K562/Zneo cells (1 X 10 4) with nonadherent human peripheral blood lymphocytes (PBL) resulted in the release of β-gal activity depending on the incubation time and the number of effector cells. Released β-gal activity was assayed sensitively by using 4-methylumbelliferyl-β-D-galactoside, a fluorescent substrate. The cytolytic activity of PBL was augmented significantly when the cells were preincubated with interleukin-2 for 20 h. This enzyme release assay showed a comparable sensitivity to that of 51Cr release assay. Thus, K562/Zneo cell line is thought to be useful for the nonradioactive assay of human NK and lymphokine-activated killer activities.