Designing a risk prognosis model based on natural killer cell-linked genes to accurately evaluate the prognosis of gastric cancer

Background:This study was aimed at identifying natural killer(NK)cell-related genes to design a risk prognosis model for the accurate evaluation of gastric cancer(GC)prognosis.Methods:We obtained NK cell-related genes from various databases,followed by Cox regression analysis and molecular typing to identify prognostic genes.Various immune algorithms and enrichment analyses were used to investigate the mutations,immune status,and pathway variations among different genotypes.The key prognostic genes were assessed using the least absolute shrinkage and selection operator(Lasso)regression analysis and univariate Cox regression analysis.Thereafter,the risk score(RS)prognosis model was constructed based on the selected important prognostic genes.A Receiver Operating Characteristics(ROC)curve was plotted for analyzing the robustness of the model.Subsequently,the decision and calibration curves were used for assessing the reliability and prediction accuracy of the proposed model.The‘pRRophetic’R software package was utilized for predicting the half-maximal inhibitory concentration(IC50)of immunotherapy and chemotherapy drugs.Results:We screened 21 prognostic genes and three molecular subtypes and found that the C1 subtype had the worst prognosis.Further,the pathways promoting tumor proliferation,such as epithelial-mesenchymal transition were significantly up-regulated.The results also showed that the macrophages in the M2 stage were significantly infiltrated in the C1 subtype,and there was significant overexpression in the C1 subtype,accompanied by a severe inflammatory reaction.The C1 was highly sensitive to drugs like 5-fluorouracil and paclitaxel.The ROC,calibration curve,and decision curve showed that the risk model was robust and strongly reliable.Conclusion:Overall,our proposed NK cell-related RS model can be used as a more accurate prediction index for GC patients,providing a valuable contribution to personalized medicine.

Targeted Therapy of CEA-CAR-NK Cells Against Colorectal Cancer Cells

Objective:Investigate the cytotoxic effect of CAR-NK cells targeting CEA on colorectal cancer cells with positive CEA expression.Methods:The mRNA and protein levels of CEA in different CRC cell lines were detected by qRT-PCR and Western blot analysis.Lentiviral transduction was used to construct CAR-NK cells and empty vector CON-NK cells targeting CEA.Fluorescence microscopy and WB were used to determine whether the cells successfully constructed and expressed CAR structures.The effector NK cells were co-cultured with target cells,and the levels of LDH,IFN-γ,and GM-CSF were detected.The killing rate of effector cells was calculated,and the release of cytokines during the killing of target cells by different effector cells was compared.Results:The expression level of CEA in colorectal cancer patients was significantly higher than that in normal samples and other tumor samples,and the prognosis survival time of patients with high CEA expression was lower than that of CRC patients with low or no CEA expression(P<0.05).The CEA expression of the HT29 cell line was significantly higher than that of the SW1116 cell line at both the mRNA and protein levels.CEA-CAR-NK92 cells and CON-NK92 cells expressed green fluorescence under a microscope,and WB results showed that CEA-CAR-NK92 cells successfully expressed the CAR structure.Compared with CON-NK92 cells and NK92 cells,CEA-CAR-NK92 cells effectively killed HT29 cells(P<0.05).CEA-CAR-NK92 cells secreted a large amount of IFN-γand GM-CSF during the killing of HT29 cells,while the cytokine secretion of CON-NK92 cells and NK92 cells was not significant(P<0.05).Conclusion:CAR-NK92 cells targeting CEA can effectively kill CEA-positive colorectal cancer cells.

Investigation of the Cytotoxicity of CAR-NK-92 Cells Targeting CEA Against Gastric Cancer Cells

Objective:To construct CAR-NK-92 cells targeting carcinoembryonic antigen(CEA)and study their killing effect on gastric cancer cells.Methods:CAR-NK-92 cells targeting CEA were constructed.After co-culturing CAR-NK-92 cells with MKN-45 gastric cancer cells,the killing effect of CAR-NK-92 cells was detected by a lactate dehydrogenase release assay.The secretion levels of gamma interferon and granulocyte-macrophage colony-stimulating factor were measured using an ELISA assay.Results:The lactate dehydrogenase release assay showed that CAR-NK-92 cells had a significant killing effect on MKN-45 cells compared to CON-NK-92 cells,and the difference was statistically significant(P<0.001).ELISA results indicated that the levels of gamma interferon and granulocyte-macrophage colony-stimulating factor secreted by CAR-NK-92 cells and MKN-45 target cells were significantly increased after co-culture(P<0.001).Conclusion:CAR-NK-92 cells targeting CEA exhibit a significant killing effect on CEA-positive gastric cancer cells.

Immune evasion and therapeutic opportunities based on natural killer cells

Natural killer(NK)cells can elicit an immune response against malignantly transformed cells without recognizing antigens,and they also exhibit cytotoxic effects and immune surveillance functions in tumor immunotherapy.Although several studies have shown the promising antitumor effects of NK cells in immunotherapy,their function is often limited in the tumor microenvironment because tumor cells can easily escape NK cell-induced death.Thus,for efficient tumor immunotherapy,the mechanism by which tumor cells escape NK cell-induced cytotoxicity must be fully understood.Various novel molecules and checkpoint receptors that mediate the disruption of NK cells in the tumor microenvironment have been discovered.In this review,we analyze and detail the major activating and inhibitory receptors on the surface of NK cells to delineate the mechanism by which tumor cells suppress NKG2D ligand expression and increase tumor receptor and inhibitory receptor expression[NKG2A,programmed cell death1(PD-1),and T-cell immunoglobulin and immunoreceptor tyrosine inhibitory motif(TIGIT)]on the NK cell surface,and thus inhibit NK cell activity.We also reviewed the current status of treatments based on these surface molecules.By comparing the therapeutic effects related to the treatment status and bypass mechanisms,we attempt to identify optimal single or combined treatments to suggest new treatment strategies for tumor immunotherapy.

Establishment of a prognostic model related to tregs and natural killer cells infiltration in bladder cancer

BACKGROUND Regulatory T cells(Tregs)and natural killer(NK)cells play an essential role in the development of bladder urothelial carcinoma(BUC).AIM To construct a prognosis-related model to judge the prognosis of patients with bladder cancer,meanwhile,predict the sensitivity of patients to chemotherapy and immunotherapy.METHODS Bladder cancer information data was obtained from The Cancer Genome Atlas and GSE32894.The CIBERSORT was used to calculate the immune score of each sample.Weighted gene co-expression network analysis was used to find genes that will have the same or similar expression patterns.Subsequently,multivariate cox regression and lasso regression was used to further screen prognosis-related genes.The prrophetic package was used to predict phenotype from gene expression data,drug sensitivity of external cell line and predict clinical data.RESULTS The stage and risk scores are independent prognostic factors in patients with BUC.Mutations in FGFR3 lead to an increase in Tregs percolation and affect the prognosis of the tumor,and additionally,EMP1,TCHH and CNTNAP3B in the model are mainly positively correlated with the expression of immune checkpoints,while CMTM8,SORT1 and IQSEC1 are negatively correlated with immune checkpoints and the high-risk group had higher sensitivity to chemotherapy drugs.CONCLUSION Prognosis-related models of bladder tumor patients,based on Treg and NK cell percolation in tumor tissue.In addition to judging the prognosis of patients with bladder cancer,it can also predict the sensitivity of patients to chemotherapy and immunotherapy.At the same time,patients were divided into high and low risk groups based on this model,and differences in genetic mutations were found between the high and low risk groups.

Diffuse large B-cell lymphoma successfully treated with amplified natural killer therapy alone: A case report

BACKGROUND The prognosis of patients with advanced diffuse large B-cell lymphoma(DLBCL)is poor,with a 5-year survival rate of approximately 50%.The mainstay of treatment is multidrug combination chemotherapy,which has been associated with serious side effects.Amplified natural killer(ANK)cell therapy amplifies and activates natural killer(NK)cells to attack only malignant tumors.As ANK cells attack programmed death ligand 1(PD-L1)-positive tumor cells,ANK therapy is considered effective against adult T-cell lymphoma and malignant lymphoma.CASE SUMMARY Herein,we report a case of an older patient with advanced DLBCL who was successfully treated with ANK immunotherapy.A 91-year-old female visited our hospital with sudden swelling of the right axillary lymph node in April 2022.The patient was diagnosed with stage II disease,given the absence of splenic involvement or contralateral lymphadenopathy.ANK therapy was administered.Six rounds of lymphocyte sampling were performed on July 28,2022.To reduce the occurrence of side effects,the six samples were diluted by half to obtain 12 samples.Cultured NK cells were administered twice weekly.The treatment efficacy was evaluated by performing computed tomography and serological tests every 1 or 2 mo.The treatment suppressed lesion growth,and the antitumor effect persisted for several months.The patient experienced mild side effects.PD-L1 immunostaining was positive,indicating that the treatment was highly effective.CONCLUSION ANK therapy can be used as a first-line treatment for malignant lymphoma;the PD-L1 positivity rate can predict treatment efficacy.

HBsAg stimulates NKG2D receptor expression on natural killer cellsand inhibits hepatitis C virus replication

Background： Higher hepatitis B surface antigen （HBsAg） facilitates hepatitis C virus （HCV） clearance inpatients with hepatitis B virus （HBV）/HCV co-infection. We investigated the effect of exogenous HBsAgon the inhibition of HCV replication mediated by natural killer （NK） cells.

The effects of stereotactic body radiotherapy on peripheral natural killer and CD3~+CD56~+ NKT-like cells in patients with hepatocellular carcinoma

Background: Both natural killer(NK) and CD3+CD56+ natural killer T(NKT)-like cells play critical roles in the antitumor response. This study aimed to explore the effects of stereotactic body radiotherapy(SBRT) on peripheral NK and NKT-like cells in patients with hepatocellular carcinoma(HCC), and to identify possible surface markers on these cells that correlate with the prognosis. Methods: Twenty-five HCC patients were prospectively enrolled in our study, and 10 healthy individuals were served as healthy controls. Flow cytometry was used to determine the counts and the percentages of peripheral NK and NKT-like cells, cells with certain receptors, and cells with intracellular interferon-γand TNF-α secretion at different time points, including time points of prior to SBRT, at post-SBRT, and 3-month and 6-month after treatment. The Kaplan-Meier method with the log-rank test was applied for survival analysis. Results: The peripheral NKT-like cells was increased at post-SBRT. Meanwhile, elevated levels of inhibitory receptors and reduced levels of activating receptors of NK cells were also observed in NK cells at post-SBRT, but the levels was not significantly different at 3-month and 6-month as compared with the baseline levels. Lower percentage of NKp30+ NK cells before SBRT and higher percentage of CD158b + NK cells after SBRT were associated with poor progression-free survival. In addition, higher percentage of CD3+CD56+ NKT-like cells was associated with a higher overall survival rate in HCC patients. Conclusions: SBRT has an apparent effect on both peripheral NK and CD3+ CD56+ NKT-like cells. Lower percentage of NKp30 + NK cells before SBRT and higher percentage of CD158b + NK cells after SBRT are correlated with poor patients' PFS. Higher percentage of CD3+ CD56+ NKT-like cells is associated with higher OS in HCC patients.

The Natural Killer Cell: A Historical Perspective and the Use of Supplements to Enhance NKC Activity

The Natural Killer Cell (NKC) is the cell-mediated cornerstone of innate immunity. The purpose of this reviewis to give a historical perspective of the discovery of the Natural Killer Cell (NKC)and to apply the use of supplements in the enhancement of NKC in human cancers for the developmentof human health and well-being.Since the discovery of the NKC, as observed by Nomarski optics, scanning (SEM)/transmission electron microscopy (TEM) with cellular numeration and enrichment using bovine serum albumin (BSA) continuous gradients, there have been significant research and clinical studies to increase the effectiveness of NKC in the destruction of cancer cells. Based on significant research and clinical studies, at least 16 components have been identified that enhance or may enhance, based on their immune modulator activity, the NKC. These supplements include Alpha LipoicAcid, Arabinoxylin, Curcumin, Garlic, Genistein, Ginseng, Lentinan, Mistletoe, N-Acetylcysteine, Resveratrol, Selenium, Vitamin B, Vitamin C, Vitamin D3, Vitamin E and zinc.

Human CD4- CD8- Invariant Natural Killer T Cells Promote IgG Secretion from B Cells Stimulated by Cross-Linking of Their Antigen Receptors

Immunoglobulin (Ig) M production can be induced by the interaction of thymus-independent type-2 (TI-2) antigen (Ag) with B cell Ag receptors (BCRs) without the involvement of conventional T cells;for IgG production through the same process, however, a second signal is required. Previous studies have reported that invariant natural killer T (iNKT) cells may be responsible for the second signal involved in IgG production. In the present study, we addressed whether human iNKT cells could participate in the production of Ig against TI-2 Ag in vitro. Two major distinct subsets of human iNKT cells, CD4+ CD8β- (CD4) and CD4- CD8β- [double negative (DN)] cells, were generated from peripheral blood monocytes from a healthy volunteer. BCR engagement, triggered by anti-IgM antibody stimulation, examined here as a model of BCR engagement triggered by TI-2 Ag, induced abundant IgM production by B cells. Both CD4 and DN iNKT cells reduced IgM production and conversely enhanced IgG production in a dose-dependent manner. In addition, IgG production by CD19+CD27- (naïve) and CD19+CD27+ (memory) B cells was predominantly promoted by DNiNKT cells rather than CD4 iNKT cells;nevertheless, IgM production by both B cell subsets was similarly reduced by either subset of iNKT cells. These results suggest that the DN iNKT subsets may preferentially promote Ig class switching by B cells upon stimulation with TI-2 Ag.

Subgroups of peripheral immune effector cells in cervical cancer patients are more sensitive to radiation therapy than chemotherapy

Background:CD8 positive T lymphocytes and natural killer(NK)cells in the peripheral blood of cervical cancer patients exhibit varying sensitivities to radiotherapy and chemotherapy.Methods:A total of 50 healthy peoples and 60 cervical cancer patients were recruited.The patients with cervical cancer were separated into two groups:radiation and chemotherapy,and blood sample were collected before and after treatment.Data on the proportion of CD8 positive T lymphocytes and NK cells were gathered for analytical evaluation.Results:Compared to healthy individuals,patients with cervical cancer exhibit a reduced proportion of CD8 positive T cells within their peripheral blood.And for patients with cervical cancer,radiation therapy has been found to be more effective than chemotherapy in increasing the proportion of CD8 positive T lymphocytes and NK cells.Conclusions:These results suggest that radiation therapy increases the levels of CD8 positive T lymphocytes and NK cells within the peripheral blood of patients with cervical cancer.The study hypothesis that the changes in the percentage of CD8 positive T lymphocytes may serve as a potential indicator for predicting treatment efficacy.

Natural Killer Cell-Based Immunotherapy for Cancer: Advances and Prospects

Natural killer (NK) cells are key innate immune cells that provide the first line of defense against viral infection and cancer. Although NK cells can discriminate between "self" and "non-self," recognize abnormal cells, and eliminate transformed cells and malignancies in real time, tumors develop several strategies to escape from NK cell attack. These strategies include upregulating ligands for the inhibitory receptors of NK cells and producing soluble molecules or immunosuppressive factors. Various types of NK cells are currently being applied in clinical trials, including autologous or allogeneic NK cells, umbilical cord blood (UCB) or induced pluripotent stem cell (iPSC)-derived NK cells, memory-like NK cells, and NK cell line NK-92 cells, for the treatment of different types of tumors. Chimeric antigen receptors (CARs)-NK cells have recently shown great potential due to their redirect specificity and effective antitumor activity. In this review, we summarize the mechanisms of tumor escape from NK cell recognition, the current status and advanced progress of NK cell-based immunotherapy, ways of enhancing the antitumor capacity of NK cells in vivo, and major challenges for clinical practice in this field.

Effects of acupuncture treatment on natural killer cell activity,pulse rate,and pain reduction for older adults:an uncontrolled,observational study

OBJECTIVE： The aim of this study was to examine the changes in natural killer （NK） cell activity, pulse rate, and pain intensity among older adults before and after acupuncture treatment. METHODS： Fifty-six individuals （16 males and 40 females）, aged 60 to 82 years （mean age 72.4 ＋ 5.0）, who were experiencing pain in the shoulder, low back, or knee, participated in the study. NK cell activity, leukocyte differentiation （granulocytes and lymphocytes）, pulse rate, and blood pressure values were obtained. Pain intensity was evaluated by using the visual analog scale （VAS）. The Wilcoxon test was used to analyze NK cell activity, leukocytes （granulocyte counts and granulocyte-to-lymphocyte ratio）, and the VAS score in accordance with the location of pain complaints before and after acupuncture treatment. RESULTS： NK cell activity decreased after acupuncture treatment for pain in the shoulder-pain and knee-pain groups. Further, the lymphocyte and granulocyte counts increased after acupuncture treatment for the shoulder-pain group. Pulse rate decreased for the shoulder-pain, low-back- pain, and knee-pain groups after acupuncture treatment. The VAS score decreased after acupuncture treatment for the shoulder-pain, low-back-pain, and knee-pain groups. CONCLUSION： This study showed that in older adults, acupuncture treatment decreases pulse rate, relieves pain in the shoulder, low back, and knee, and reduces NK-cell activity.

Tumor immunotherapy: New aspects of natural killer cells

A group of impressive immunotherapies for cancer treatment, including immune checkpoint-blocking antibodies,gene therapy and immune cell adoptive cellular immunotherapy, have been established, providing new weapons to fight cancer. Natural killer（NK） cells are a component of the first line of defense against tumors and virus infections. Studies have shown dysfunctional NK cells in patients with cancer. Thus, restoring NK cell antitumor functionality could be a promising therapeutic strategy. NK cells that are activated and expanded ex vivo can supplement malfunctional NK cells in tumor patients. Therapeutic antibodies, chimeric antigen receptor（CAR）, or bispecific proteins can all retarget NK cells precisely to tumor cells. Therapeutic antibody blockade of the immune checkpoints of NK cells has been suggested to overcome the immunosuppressive signals delivered to NK cells.Oncolytic virotherapy provokes antitumor activity of NK cells by triggering antiviral immune responses. Herein,we review the current immunotherapeutic approaches employed to restore NK cell antitumor functionality for the treatment of cancer.

Intrahepatic natural killer T cell populations are increased in human hepatic steatosis

AIM: To determine if natural killer T cell (NKT) populations are affected in nonalcoholic fatty liver disease (NAFLD). METHODS: Patients undergoing bariatric surgery underwent liver biopsy and blood sampling during surgery. The biopsy was assessed for steatosis and immunocyte infiltration. Intrahepatic lymphocytes (IHLs) were isolated from the remainder of the liver biopsy, and peripheral blood mononuclear cells (PBMCs) were isolated from the blood. Expression of surface proteins on both IHLs and PBMCs were quantified using flow cytometry. RESULTS: Twenty-seven subjects participated in thisstudy. Subjects with moderate or severe steatosis had a higher percentage of intrahepatic CD3+/CD56+ NKT cells (38.6%) than did patients with mild steatosis (24.1%, P = 0.05) or those without steatosis (21.5%, P = 0.03). Patients with moderate to severe steatosis also had a higher percentage of NKT cells in the blood (12.3%) as compared to patients with mild steatosis (2.5% P = 0.02) and those without steatosis (5.1%, P = 0.05). CONCLUSION: NKT cells are significantly increased in the liver and blood of patients with moderate to severe steatosis and support the role of NKT cells in NAFLD.

Distribution of natural killer cells and T-lymphocyte subsets in peripheral blood,gallbladder cancer and surrounding tissue

BACKGROUND: The patient with malignant tumor always show immunologic function drawback and ingravescent with tumor development, especially in the aspect of cell-mediated immunity. This study was undertaken to define the relationship between the immune function of local cells and cancer development by investigating the distribution of natural killer (NK) cells and T-lymphocyte subsets in peripheral blood, the cancer tissue and the tissue surrounding gallbladder carcinoma. METHODS: The numbers of CD4(+) and CD8(+) T-lymphocytes and NK cells were measured by flow cytometry in samples taken from gallbladder cancer tissue, the surrounding tissues and peripheral blood of 38 patients, and compared with the numbers in the peripheral blood and gallbladder tissue of 30 patients with cholecystitis as controls. RESULTS: The numbers of CD4(+) and CD8(+) T-cells and NK cells in gallbladder cancer tissues were significantly higher than those in the surrounding tissue and gallbladder with gallstone. However, the ratio of CD4(+)/CD8(+) was lower in the cancer tissue than that in the surrounding tissue and tissue from gallbladders with gallstones. The distribution of CD4(+) and CD8(+) T-cells and NK cells in mucous membrane of cholecystitis gallbladder and that in the tissue surrounding gallbladder cancer were significantly different. CONCLUSIONS: Disproportionate and imbalanced distribution of NK cells and subsets of T-lymphocytes occurs in the mucous membrane proper of gallbladder cancer and surrounding tissue. Although gallbladder cancer tissue has higher expressions of CD4(+), CD8(+) and NK cells, the immune function is low or in an inhibited state. In gallbladder cancer immunization therapy, local cellular immunological function should be enhanced and the protective barrier improved.

Human natural killer cells for targeting delivery of gold nanostars and bimodal imaging directed photothermal/photodynamic therapy and immunotherapy

Objective:To construct a novel nanoplatform GNS@CaCO3/Ce6-NK by loading the CaCO3-coated gold nanostars(GNSs)with Chlorin e6 molecules(Ce6)into human peripheral blood mononuclear cells(PBMCs)-derived NK cells for tumor targeted therapy.Methods:GNS@CaCO3/Ce6 nanoparticles were prepared and characterized by TEM and UV-vis.The cell surface markers and cytokines secretion of NK cells before and after loading the GNS@CaCO3/Ce6 nanoparticles were detected by Flow Cytometry(FCM)and ELISA.Effects of the GNS@CaCO3/Ce6-NK cells on A549 cancer cells was determined by FCM and CCK-8.Intracellular fluorescent signals of GNS@CaCO3/Ce6-NK cells were detected via Confocal laser scanning microscopic(CLSM)and FCM at different time points.Intracellular ROS generation of GNS@CaCO3/Ce6-NK cells under laser irradiation were examined by FCM.The distribution of GNS@CaCO3/Ce6-NK in A549 tumor-bearing mice were observed by fluorescence imaging and PA imaging.The combination therapy of GNS@CaCO3/Ce6-NK under laser irradiation were investigated on tumor-bearing mice.Results:The coated CaC03 shell on the surface of GNSs exhibited prominent delivery and protection effect of Ce6 during the cellular uptake process.The as-prepared multifunctional GNS@CaCO3/Ce6-NK cells possessed bimodal functions of fluorescence imaging and photoacoustic imaging.The as-prepared multifunctional GNS@CaCO3/Ce6-NK cells could actively target tumor tissues with the enhanced photothermal/photodynamic therapy and immunotherapy.Conclusions:The GNS@CaCO3/Ce6-NK shows effective tumor-targeting ability and prominent therapeutic efficacy toward lung cancer A549 tumor-bearing mice.Through fully utilizing the features of GNSs and NK cells,this new nanoplatform provides a new synergistic strategy for enhanced photothermal/photodynamic therapy and immunotherapy in the field of anticancer development in the near future.

Natural killer cells in hepatitis C:Current progress

Patients infected with the hepatitis C virus(HCV) are characterized by a high incidence of chronic infection, which results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The functional impairment of HCV-specific T cells is associated with the evolution of an acute infection to chronic hepatitis. While T cells are the important effector cells in adaptive immunity, natural killer(NK) cells are the critical effector cells in innate immunity to virus infections. The findings of recent studies on NK cells in hepatitis C suggest that NK cell responses are indeed important in each phase of HCV infection. In the early phase, NK cells are involved in protective immunity to HCV. The immune evasion strategies used by HCV may target NK cells and might contribute to the progression to chronic hepatitis C. NK cells may control HCV replication and modulate hepatic fibrosis in the chronic phase. Further investigations are, however, needed, because a considerable number of studies observed functional impairment of NK cells in chronic HCV infection. Interestingly, the enhanced NK cell responses during interferon-α-based therapy of chronic hepatitis C indicate successful treatment. In spite of the advances in research on NK cells in hepatitis C, establishment of more physiological HCV infection model systems is needed to settle unsolved controversies over the role and functional status of NK cells in HCV infection.

Functional distinction of rat liver natural killer cells from spleen natural killer cells under normal and acidic conditions in vitro

BACKGROUND: The microenvironment within solid tumors has often been shown to exhibit an acidic extracellular pH. Although the morphologic and functional differences in natural killer (NK) cells of the liver and spleen have been reported previously under physiological conditions, the difference under acidic conditions is still unclear. This study was to investigate the differences in the morphological and functional characteristics between rat liver and spleen NK cells under normal and acidic conditions in vitro. METHODS: Liver and spleen NK cells were isolated and purified from Sprague-Dawley rats by density gradient centrifugation and the Dynabeads FlowComp TM Flexi system, and stimulated for 4 days with or without IL-2 or treated with low pH or control for different times. Morphology was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cell death and proliferation assays were performed by flow cytometry, IFN-γ production was tested by ELISA, and cytotoxic activity was evaluated by lactate dehydrogenase (LDH) release assay. RESULTS: Liver NK cells had significantly higher levels of cytotoxic activity than spleen NK cells under normal and acidic conditions, and the maximum difference was observed at pH 5.6. Further analysis revealed that the cytotoxic activity of NK cells was correlated with morphology, cell death, proliferative activity and IFN-γ production. By TEM, liver NK cells contained a greater number of electron-dense granules per cell at pH 5.6.Moreover, a modest elevation of cell death and reduction of proliferation of liver NK cells occurred within a range of 5.6-7.2. Interestingly, an acidic extracellular pH only marginally, and not significantly, suppressed IFN-γ production by liver NK cells. CONCLUSION: The sharp morphological and functional differences shown by the two types of NK cells in vitro indicate that liver NK cells are unexpectedly resistant to pH shock.

Association of the frequency of peripheral natural killer T cells with nonalcoholic fatty liver disease

AIM: To investigate whether changes in the frequencyof peripheral natural killer T (NKT) cells were correlatedwith liver disease in patients who had metabolicpredispositions to nonalcoholic fatty liver disease(NAFLD).METHODS: Peripheral blood samples were obtainedfrom 60 Chinese NAFLD patients and 60 age and gendermatched healthy controls. The frequency of peripheralNKT cells was detected by flow cytometry. Clinical andlaboratory data were collected for further analysis. RESULTS: NAFLD patients had a lower frequencyof peripheral NKT cells than healthy controls (1.21%± 0.06% vs 1.62% ± 0.07%, P < 0.001). Furtheranalysis revealed that the frequency of peripheralNKT cells was negatively correlated with body massindex, waist circumference and serum levels of alanineaminotransferase. Logistic regression analysis revealedthat elevated body mass index [hazard ratio (HR):2.991], aspartate aminotransferase levels (HR: 1.148)and fasting blood sugar (HR: 3.133) increased the riskof NAFLD, whereas an elevated frequency of peripheralNKT cells (HR: 0.107) decreased the risk. CONCLUSION: Changes in the frequency of peripheralNKT cells were correlated with NAFLD and a decreasedfrequency of peripheral NKT cells was a risk factor forNAFLD.