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.

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.

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.

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.

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.

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.

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.

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.

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.

Tumor microenvironment in primary liver tumors: A challenging role of natural killer cells

In the last years,several studies have been focused on elucidate the role of tumor microenvironment(TME)in cancer development and progression.Within TME,cells from adaptive and innate immune system are one of the main abundant components.The dynamic interactions between immune and cancer cells lead to the activation of complex molecular mechanisms that sustain tumor growth.This important cross-talk has been elucidate for several kind of tumors and occurs also in patients with liver cancer,such as hepatocellular carcinoma(HCC)and intrahepatic cholangiocarcinoma(iCCA).Liver is well-known to be an important immunological organ with unique microenvironment.Here,in normal conditions,the rich immune-infiltrating cells cooperate with non-parenchymal cells,such as liver sinusoidal endothelial cells and Kupffer cells,favoring self-tolerance against gut antigens.The presence of underling liver immunosuppressive microenvironment highlights the importance to dissect the interaction between HCC and iCCA cells with immune infiltrating cells,in order to understand how this cross-talk promotes tumor growth.Deeper attention is,in fact,focused on immune-based therapy for these tumors,as promising approach to counteract the intrinsic anti-tumor activity of this microenvironment.In this review,we will examine the key pathways underlying TME cell-cell communications,with deeper focus on the role of natural killer cells in primary liver tumors,such as HCC and iCCA,as new opportunities for immune-based therapeutic strategies.

Impact of Fusobacterium nucleatum in the gastrointestinal tract on natural killer cells

BACKGROUND Gut microbial dysbiosis contributes to the development and progression of colorectal cancer(CRC).Natural killer(NK)cells are involved in early defense mechanisms to kill infective pathogens and tumor cells by releasing chemokines and cytokines.To better understand the relationship between the gut microbiome and CRC,it was hypothesized here that a high abundance of Fusobacterium nucleatum(F.nucleatum)in the gastrointestinal tract could cause reduced NK cell activity.AIM To identify associations between gastrointestinal tract F.nucleatum levels and NK cell activity.METHODS In vitro experiments were performed on NK cells treated with F.nucleatum,Peptostreptococcus anaerobius,and Parvimonas micra to identify the effects of gut microbiome species on NK cells.Following 24 and 48 h of treatment,NK cell counts were measured.In parallel studies,C57BL/6 mice were given broadspectrum antibiotics in their drinking water to reduce resident gut flora.After 3 wk,the mice received the various bacterial species or phosphate-buffered saline(PBS)via oral gavage every 2 d for 6 wk.At the study end,blood samples were acquired to perform NK cell activity assessment and cytokine analysis.Intestinal tissues were collected and analyzed via immunohistochemistry(IHC).RESULTS The data show that after 3 wk of broad-spectrum antibiotic treatment,levels of total bacteria and F.nucleatum were markedly decreased in mice.Gavage of F.nucleatum significantly decreased NK cell activity relative to the activities of cells from mice treated with antibiotics only and PBS.The administration of F.nucleatum decreased the proportion of NK46+cells based on IHC staining and increased the production of interleukin-1βand tumor necrosis factor-α.CONCLUSION High levels of F.nucleatum in the gastrointestinal tract reduced NK cell activity in mice,and the decrease in NK cell activity might be affected by increased proinflammatory cytokines after F.nucleatum treatment.

Fasudil prevents liver fibrosis via activating natural killer cells and suppressing hepatic stellate cells

BACKGROUND Fasudil,as a Ras homology family member A(RhoA)kinase inhibitor,is used to improve brain microcirculation and promote nerve regeneration clinically.Increasing evidence shows that Rho-kinase inhibition could improve liver fibrosis.AIM To evaluate the anti-fibrotic effects of Fasudil in a mouse model of liver fibrosis induced by thioacetamide(TAA).METHODS C57BL/6 mice were administered TAA once every 3 d for 12 times.At 1 wk after induction with TAA,Fasudil was intraperitoneally injected once a day for 3 wk,followed by hematoxylin and eosin staining,sirius red staining,western blotting,and quantitative polymerase chain reaction(qPCR),and immune cell activation was assayed by fluorescence-activated cell sorting.Furthermore,the effects of Fasudil on hepatic stellate cells and natural killer(NK)cells were assayed in vitro.RESULTS First,we found that TAA-induced liver injury was protected,and the positive area of sirius red staining and type I collagen deposition were significantly decreased by Fasudil treatment.Furthermore,western blot and qPCR assays showed that the levels of alpha smooth muscle actin(α-SMA),matrix metalloproteinase 2(MMP-2),MMP-9,and transforming growth factor beta 1(TGF-β1)were inhibited by Fasudil.Moreover,flow cytometry analysis revealed that NK cells were activated by Fasudil treatment in vivo and in vitro.Furthermore,Fasudil directly promoted the apoptosis and inhibited the proliferation of hepatic stellate cells by decreasingα-SMA and TGF-β1.CONCLUSION Fasudil inhibits liver fibrosis by activating NK cells and blocking hepatic stellate cell activation,thereby providing a feasible solution for the clinical treatment of liver fibrosis.

Donor liver natural killer cells alleviate liver allograft acute rejection in rats

BACKGROUND:Liver enriched natural killer (NK) cells are of high immune activity.However,the function of donor liver NK cells in allogeneic liver transplantation (LTx) remains unclear.METHODS:Ten Gy of whole body gamma-irradiation (WBI) from a 60 Co source at 0.6 Gy/min was used for depleting donorderived leukocytes,and transfusion of purified liver NK cells isolated from the same type rat as donor (donor type liver NK cells,dtl NKs) through portal vein was performed immediately after grafting the irradiated liver.Post-transplant survival observation on recipients and histopathological detection of liver grafts were adoptive to evaluate the biological impact of donor liver NK cells on recipients’ survival in rat LTx.RESULTS:Transfusion of dtl NKs did not shorten the survival time among the recipients of spontaneous tolerance model (BN to LEW rat) after rat LTx,but prolonged the liver graft survival among the recipients depleted of donor-derived leukocytes in the acute rejection model (LEW to BN rat).Compared to the recipients in the groups which received the graft depleted of donor-derived leukocytes,better survival and less damage in the allografts were also found among the recipients in the two different strain combinations of liver allograft due to transfusion of dtl NKs.CONCLUSIONS:Donor liver NK cells alone do not exacerbate liver allograft acute rejection.Conversely,they can alleviate it,and improve the recipients’ survival.

Advancement of chimeric antigen receptor-natural killer cells targeting hepatocellular carcinoma

With the advance of genome engineering technology,chimeric antigen receptors(CARs)-based immunotherapy has become an emerging therapeutic strategy for tumors.Although initially designed for T cells in tumor immunotherapy,CARs have been exploited to modify the function of natural killer(NK)cells against a variety of tumors,including hepatocellular carcinoma(HCC).CAR-NK cells have the potential to sufficiently kill tumor antigen-expressing HCC cells,independent of major histocompatibility complex matching or prior priming.In this review,we summarize the recent advances in genetic engineering of CAR-NK cells against HCC and discuss the current challenges and prospects of CAR-NK cells as a revolutionary cellular immunotherapy against HCC.

CD4+ T cells and natural killer cells: Biomarkers for hepatic fibrosis in human immunodeficiency virus/hepatitis C virus-coinfected patients

AIM To characterize peripheral blood natural killer(NK) cells phenotypes by flow cytometry as potential biomarker of liver fibrosis in human immunodeficiency virus(HIV)/hepatitis C virus(HCV) coinfected patients.METHODS Peripheral mononuclear cells from 24 HIV/HCV(HBVnegative) coinfected and 5 HIV/HCV/HBV seronegative individuals were evaluated. HIV/HCV coinfected patients were divided in to groups: G1, patients with METAVIR F0-F2 and G2, patients with METAVIR F3-F4. NK surface cell staining was performed with: AntiCD3(APC/Cy7), anti-CD56(PE/Cy5), anti-CD57(APC), anti-CD25(PE), anti-CD69(FITC), anti-NKp30(PE), antiNKp46(PE/Cy7), anti-NKG2D(APC), anti-DNAM(FITC); anti-CD62L(PE/Cy7), anti-CCR7(PE), anti-TRAIL(PE), anti-Fas L(PE), anti CD94(FITC). Flow cytometry data acquisition was performed on BD FACSCanto, analyzed using Flow Jo software. Frequency of fluorescence was analyzed for all single markers. Clinical records were reviewed, and epidemiological and clinical data were obtained.RESULTS Samples from 11 patients were included in G1 and from 13 in G2. All patients were on ARV, with undetectable HIV viral load. Liver fibrosis was evaluated by transient elastography in 90% of the patients and with biopsy in 10% of the patients. Mean HCV viral load was(6.18 ± 0.7 log10). Even though, no major significant differences were observed between G1 and G2 regarding NK surface markers, it was found that patients with higher liver fibrosis presented statistically lower percentage of NK cells than individual with low to mild fibrosis and healthy controls(G2: 5.4% ± 2.3%, G1: 12.6% ± 8.2%, P = 0.002 and healthy controls 12.2% ± 2.7%, P = 0.008). It was also found that individuals with higher liver fibrosis presented lower CD4 LT count than those from G1(G2: 521 ± 312 cells/μL, G1: 770 ± 205 cells/μL; P = 0.035).CONCLUSION Higher levels of liver fibrosis were associated with lower percentage of NK cells and LTCD4+ count; and they may serve as noninvasive biomarkers of liver damage.

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.

Thiopurine use associated with reduced B and natural killer cells in inflammatory bowel disease

To identify which blood and mucosal lymphocyte populations are specifically depleted by thiopurine use in vivo.METHODSThe thiopurines azathioprine and 6-mercaptopurine have been a mainstay of inflammatory bowel disease (IBD) therapy for decades, but their mechanism of action in vivo remains obscure. Although thiopurines are lymphotoxic at high doses, and have been reported to cause T cell apoptosis in vitro, their ability to control IBD at lower doses suggests that they may selectively deplete particular lymphocyte populations. Blood cells from 19 IBD patients on a thiopurine, 19 IBD patients not on a thiopurine, and 38 matched healthy control subjects were analyzed by multiple multi-color flow cytometry panels to quantify the immune cell subsets contained therein, both as a percent of cells, and as an absolute cell count. Similar analyses were performed on colon biopsies from 17 IBD patients on a thiopurine, 17 IBD patients not on a thiopurine, and 49 healthy screening colonoscopy recipients.RESULTSComplete blood counts revealed lower lymphocyte, but not monocyte or granulocyte, counts in IBD patients who were taking thiopurines at the time of sampling. This reduction was restricted to CD3-negative lymphocytes, wherein both natural killer (NK) and B cells were significantly reduced among thiopurine recipients. Among CD19+ B cells, the transitional B cells were particularly depleted, being nearly absent in both blood and colon biopsies of thiopurine recipients. No differences were associated with thiopurine use in CD8+ T cells, mucosa-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, gamma/delta T cells, Th1, Th17, regulatory T cells (Tregs) or naïve CD4+ T cells. However, patients with IBD had significantly more circulating FOXP3+, Helios+ Tregs and fewer iNKT and MAIT cells than healthy controls.CONCLUSIONThiopurine use is associated with reduced B and NK cell, but not T cell, subpopulations in the blood of IBD patients.

EFFECTS OF PERIOPERATIVE CIMETIDINE ADMINISTRATIONON NATURAL KILLER CELLS IN PATIENTS WITHGASTROINTESTINAL CANCER

Objective: To study the effects of perioperative use of cimetidine on natural killer (NK) cells in gastrointestinal (GI) cancer patients. Methods: 49 GI cancer patients were randomized into treatment group which took cimetidine in the perioperative period, and control group which did not take the drug. NK cells were measured by immunocytochemical method, using mouse-anti-human CD57 monoclonal antibody as the primary antibody. Blood samples from 20 healthy volunteers were treated in the same way as normal control. Comparisons were made within and between groups. Results: The NK cell percentage of normal control was 18.50±2.31. Both groups of patients had significantly lower than normal NK percentages before treatment (P<0.05). NK cell percentages at admission, before operation, on the 2nd and the 10th postoperative days were 14.60±3.91, 15.64±3.61, 17.40±3.28, 20.68±4.13, respectively, for the treatment group, and 14.88±2.76, 13.17±2.93, 14.50±2.77, 15.67±2.55, respectively, for control group. The difference between the two groups was statistically significant. Conclusion: Perioperative cimetidine application can help restore NK cells. The drug may be useful to reverse postoperative immuno-depression in GI cancer patients.