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.

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.

Research on stress-induced apoptosis of natural killer cells and the alteration of their killing activity in mouse liver

AIM:To investigate the stress-induced apoptosis of natural killer(NK)cells and the changes in their killing activity in mouse livers.METHODS:A restraint stress model was established in mice.Flow cytometry was employed to measure the percentage of NK cells and the changes in their absolute number in mouse liver.The cytotoxicity of hepatic and splenic NK cells was assessed against YAC-1 target cells via a 4 h 51Cr-release assay.RESULTS:The restraint stress stimulation induced the apoptosis of NK cells in the liver and the spleen,which decreased the cell number.The number and percentage of NK cells in the spleen decreased.However,the number of NK cells in the liver decreased,whereas the percentage of NK cells was significantly increased.The apoptosis of NK cells increased gradually with prolonged stress time,and the macrophage-1(Mac-1)+NK cells were more susceptible to apoptosis than Mac-1-NK cells.Large numbers of Mac-1-NK cells in the liver,which are more resistant to stress-induced apoptosis,were observed than the Mac-1-NK cells in the spleen.The stress stimulation diminished the killing activity of NK cells in the spleen was significantly decreased,but the retention of numerous Mac-1-NK cells in the liver maintained the killing ability.CONCLUSION:Significant stress-induced apoptosis was observed among Mac-1+NK cells,but not Mac-1-NK cells in the mouse liver.Stress stimulation markedly decreased the killing activity of NK cells in the spleen but remained unchanged in the liver.

Effects of Intraoperative Administration of Dexmedetomidine on the Percentage of T-Lymphocyte Subsets and Natural Killer Cells in Patients with Colorectal Cancer

Study Objective: To observe the effect of dexmedetomidine (DEX) on T-lymphocyte subsets and natural killer (NK) cells in the peripheral blood of perioperative patients with colorectal cancer. Design: A random double-blind control clinical study. Setting: A university hospital. Patients: Forty patients with colorectal cancer, ASA I-П. Interventions: All patients were randomly divided into a DEX group (n = 20) and a control group (n = 20). Before induction of anesthesia, epidural catheters were placed in the L1-L2 or T12-L1 intervertebral spaces. The DEX group received 1 μg/kg of DEX (200 μg/50 ml) intravenously for 15 min prior to the surgery, which was then infused at a rate of 0.5 μg/kg/h until 30 min before the end of the surgery. The control group received an intravenous infusion of saline (50 ml) instead of DEX during the same periods as the DEX group. All patients received routine anesthesia and postoperative analgesia. Measurements: Blood samples from all patients were collected at the following time points: before anesthesia (T0), 24 h after surgery (T1), 48 h after surgery (T2) and 72 h after surgery (T3). Changes in T-lymphocyte subsets (CD3+, CD4+, CD8+, CD4+/CD8+) and NK cells were determined by flow cytometry. Main Results: Compared with the control group, the percentages of CD3+ and CD4+ cells and the CD4+/CD8+ ratio in the DEX group increased significantly from T1 to T3

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.

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.

Recovery of natural killer cells is mainly in post-treatment period in chronic hepatitis C patients treated with sofosbuvir plus ledipasvir

AIM To investigate how natural killer(NK) cells are affected in the elimination of hepatitis C virus(HCV) by sofosbuvir/ledipasvir, two highly effective direct-acting antivirals(DAAs). METHODS Thirteen treatment-na?ve and treatment-experienced chronic hepatitis C(CHC) patients were treated with sofosbuvir/ledipasvir, and NK cells were detected at baseline, weeks 2, 4, 8 and 12 during therapy, and week post of treatment(Pt)-12 and 24 after the end of therapy by multicolor flow cytometry and compared with those from 13 healthy controls. RESULTS All patients achieved sustained virological response. There was a significant decline in CD56^(bright) NK cell frequencies at week 8(P = 0.002) and week 12(P = 0.003), which were altered to the level comparable to healthy controls at week Pt-12, but no difference was observed in the frequency of CD56^(dim) NK cells. Compared with healthy controls, the expression levels of NKG2A, NKp30 and CD94 on NK cells from CHC patients at baseline were higher. NKG2A, NKp30 and CD94 started to recover at week 12 and reached the levels similar to those of healthy controls at week Pt-12 or Pt-24. Before treatment, patients have higher interferon(IFN)-γ and perforin levels than healthy controls, and IFN-γ started to recover at week 8 and reached the normalized level at week Pt-12. CONCLUSION NK cells of CHC patients can be affected by DAAs, and phenotypes and function of NK cells recover not at early stage but mainly after the end of sofosbuvir/ledipasvir treatment.

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.

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.

Why natural killer cells in triple negative breast cancer?

The triple-negative subtype of breast cancer(TNBC)has the bleakest prognosis,owing to its lack of either hormone receptor as well as human epidermal growth factor receptor 2.Henceforth,immunotherapy has emerged as the front-runner for TNBC treatment,which avoids potentially damaging chemotherapeutics.However,despite its documented association with aggressive side effects and developed resistance,immune checkpoint blockade continues to dominate the TNBC immunotherapy scene.These immune checkpoint blockade drawbacks necessitate the exploration of other immunotherapeutic methods that would expand options for TNBC patients.One such method is the exploitation and recruitment of natural killer cells,which by harnessing the innate rather than adaptive immune system could potentially circumvent the downsides of immune checkpoint blockade.In this review,the authors will elucidate the advantageousness of natural killer cell-based immuno-oncology in TNBC as well as demonstrate the need to more extensively research such therapies in the future.

Perioperative Cimetidine Application Modulates Natural Killer Cells in Patients with Colorectal Cancer: A Randomized Clinical Study

Thirty-eight colorectal cancer patients were randomly assigned to treatment group, which took cimetidine in the perioperative period, and control group to which no drug was given. Twenty healthy volunteers served as normal controls. NK cells were measured by immunocytochemical technique.The results showed that NK percentages before treatment in both groups of patients were sig-nificantly lower than those in normal controls (P<0. 05). NK cell percentages at admission, before operation, on the 2nd and the 10th postoperative days were 14. 84± 4. 41, 15. 74 ± 3. 75, 17. 21 ± 3. 69, 21. 05 ± 4. 54, respectively, for the treatment group, and 15. 00±2. 77, 13.05± 2. 46, 14. 21± 2. 19, 15. 58± 1. 68,respectively, for control group. The difference was statistically significant (P<0. 01 ), suggesting that the perioperative administration of cimetidine could help restore NK cells in colorectal cancer patients.

EZH2 identifies the precursors of human natural killer cells with trained immunity

Objective:Trained immunity of natural killer(NK)cells has shown great potential in the treatment of cancers by eliciting enhanced effector responses to restimulation by cytokines or cancer cells for long time periods after preactivation.However,the human NK cells responsible for the generation and maintenance of trained immunity are largely unknown.We hypothesized that heterogeneous human NK cells would respond differentially to stimulation with a combination of IL-12,IL-15,and IL-18,and that an NK cell subset might exist that is mainly responsible for the induction of trained immunity.On the basis of our hypothesis,we aimed to identify the subset from which cytokine-trained human NK cells originate and to explore possible regulatory targets for drug intervention.Methods:Flow cytometry assays were performed to analyze the functions of cytokine-trained NK cells and examine cell division and protein expression in NK cell subsets.Single-cell RNA sequencing(scRNA-seq)plus TotalSeq™technology was used to track the heterogeneity of NK cells during the induction of trained immunity.Results:Traditional developmental markers for peripheral NK cells were unable to identify the precursors of human NK cells with trained immunity.Therefore,we used scRNA-seq plus TotalSeq™technology to track the heterogeneity of NK cells during the induction of trained immunity and identified a unique cluster of CD57−NKG2A+EZH2+IFNG+MKI67+IL12R+IL15R+IL18R+NK cells.Enrichment and pseudotime trajectory analyses suggested that this cluster of NK cells contained the precursor of trained NK cells.We then used flow cytometry to further investigate the role of EZH2 in trained NK precursors and found that CD57−NKG2A+EZH2+NK cells had faster cell cycles and an enhanced trained phenotype,and EZH2 inhibition significantly impaired the induction of trained immunity in NK cells.These results suggested that EZH2 is a unique epigenetic marker of precursors of human NK cells with trained immunity.Conclusions:Our work revealed human NK heterogeneity in the induction of trained immunity,identified the precursor subset for trained NK cells,and demonstrated the critical role of EZH2 in the induction of trained immunity in human NK cells.

Myeloid-derived Suppressor Cells Activate Liver Natural Killer Cells in a Murine Model in Uveal Melanoma

Objective Elevated myeloid-derived suppressor cells(MDSCs)in many malignancies are associated with the increased risk for metastases and poor prognosis.Therefore,a mouse model of intraocular melanoma was established to explore how MDSCs influence liver metastases.Methods In this study,murine B16LS melanoma cells were transplanted into the posterior compartment(PC)of the eye of C57BL/6 mice.Leucocytes from the liver of naive mice and mice bearing melanoma liver metastasis were isolated using isotonic Percoll centrifugation,examined by flow cytometry for their expression of Gr1,CD11b,F4/80,RAE-1,and Mult-1,and further isolated for MDSCs and natural killer(NK)cells.The effects of MDSCs on NK cells were tested by coculturing and assessing the ability of NK cells to produce interferon-gamma(IFN-γ)by ELISA and NK cell cytotoxicity by 3H-thymidine incorporation assay.The impact of IFN-γon liver metastases was examined via selectively depleting IFN-γin vivo.Results The results showed that mice with liver metastases had increased levels of CD11b+Gr1+F4/80+as well as CD11b+Gr1+F4/80−MDSCs.MDSCs significantly enhanced the generation of IFN-γtogether with the cytotoxicity of the NK cells.Furthermore,these effects were cell-cell contact-dependent.Although IFN-γwas not of a toxic nature to the melanoma cells,it profoundly inhibited B16LS cell proliferation.Depleting IFN-γin vivo led to increased liver metastases.Conclusion All these findings first revealed that MDSCs accumulated in liver metastasis of intraocular melanoma could activate the NK cells to produce an effective anti-tumor immune response.Thus,the MDSCs’performance in different tumor models would need more investigation to boost current immunotherapy modalities.

Modulation of the Culture Supernatant of Decidual Cells with Exogenous Cytokines on Killing Activity of Natural Killer Cells in Early Pregnancy

To investigate the important function of cytokines in early pregnancy and to provide basic and experimental evidence for understanding the mechanism of their action. Methods Add interferon γ(IFN γ),interleukin 2(IL 2), interleukin 6(IL 6) and epidermal growth factor(EGF) to the confluent culturing decidual cells with three different concentrations and harvest the culture supernatant after 12, 24 and 48 h separately. Observe the effect of the supernatant on killing activity of NK cells with radioimmunological assay of 51 Cr immersion. Results The culture supernatant of decidual cells can promote the killing activity of NK cells in various degrees, and the effect is independent of the type, concentration and acting time of cytokines. Conclusion In normal pregnancy, decidual cytokine network is in a dynamic equilibrium. Exogenous cytokines would be harm to normal pregnancy by interfering the equilibrium state, but the exact mechanism needs further study.

Transient Receptor Potential Melastatin 3 and Intracellular Calcium in Natural Killer Cells in Multiple Sclerosis

Background: Natural killer (NK) cell phenotypes have reported to be implicated in the pathomechanism of Multiple Sclerosis (MS). Several investigators have observed reduced peripheral numbers, reduced cytotoxic activity, and altered CD56Dim and CD56Bright NK cell phenotypes. This current project, for the first time, investigates the NK cell cytotoxicity, calcium mobilisation and transient receptor potential melastatin 3 (TRPM3) surface expression. Methods: NK cell cytotoxic activity and calcium signaling were examined in CD56Dim and CD56Bright NK cells before and after stimulation using Ionomycin, Pregnenolone sulphate, 2-Aminoethoxydiphenyl borate and Thapsigargin. Purified NK cells were labelled with antibodies to determine TRPM3, CD69 and CD107a surface expression using flow cytometry. Results: Twenty-two MS patients and 22 healthy controls were recruited for this project. Twelve of the 22 previously received Alemtuzumab (Lemtrada&reg;) and the remaining ten reported nil medication. We report TRPM3 was significantly increased in untreated MS patients compared with healthy controls and treated MS patients (p-value 0.034). There was a significant decrease in CD69 surface expression on CD56Dim NK cell phenotype for untreated MS patients (p-value 0.031) and treated MS patients (p-value 0.036). We report altered calcium mobilisation in CD56Bright NK cells and to a lesser extent CD56Dim NK cells between healthy controls, treated and untreated MS patients. Conclusion: This investigation suggests variations in TRPM3 expression and calcium mobilisation of NK cells may be implicated in the pathogenesis of MS. Further investigation is required to determine the mechanism by which alemtuzumab alters calcium signaling in NK cells.

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.

The Effect of Granulocyte Macrophage-Colony Stimulating Factor upon the Induction of Peripheral Blood Dendritic and Natural Killer Cells When Given Simultaneously with a Slow Continuous Doxorubicin Infusion

It has been demonstrated that it is safe to give Gm-Csf, together with Doxorubicin, by continuous intravenous infusion, thereby substantially increasing the amount of Doxorubicin administered to the average patient, and assuring that each patient receives an individually-determined safe and maximal amount of drug. It is known that Gm-Csf is a potent inducer of components that are major factors in an immunologic attack upon neoplasms. For that reason, we thought it would be worth evaluating in 4 patients’ surface markers of dendritic precursor cells, dendritic cells [DC], and natural killer [NK] cells during the infusion. While there was substantial variation in individual responses, all 4 patients receiving Gm-Csf developed persistent marked increases in cells with each of these markers. The significance of these findings will be discussed.

Study on the activity and phenotype of decidual natural killer cells in patients with unexplained habitual abortions

Objective:To determine the activity and phenotype of decidual natural killer (NK) cells in patients with unexplained habitual abortions (UHA).Methods:A total of 32 patients with UHA were studied, and 20 cases of normal pregnant women were selected as control group. The levels of CD56 +CD3 - NK cells and their CD56 +CD16 -, CD56 +CD16 + subsets in decidua were detected using two color flow cytometric analysis.The NK cells activity was measured by a chromium 51( 51 Cr) release cytotoxicity assay,with K562 human myeloid leukaemia cells as targets.Results:Compared with control group, the proportion of CD56 +CD3 - NK cells in decidual mononuclear cells(DMC) of UHA patients had no difference, but the CD56 +CD16 - NK cell subset decreased and the CD56 +CD16 + subset increased significantly (P<0.05). The decidual NK cells activities of UHA patients were higher than those of normal controls (P<0.05).Conclusions:NK cell is predominant lymphocyte in normal decidua and plays an important role in maintaining successful pregnancy. Abnormally raised activity and disbalanced CD56 +CD16 +, CD56 +CD16 - subsets of decidual NK cell are associated with UHA and may play a role in reproductive failure.

Ex vivo Expansion of Cytokine-Induced Natural Killer Cells from Tumor Patients for Autologous Therapy

To optimize the expansion conditions for cytokine-induced natural killer cells (CINK), including major CD3 - CD56 + NK cells, and by using the selected method to expand the CINK cells from patients with tumors for the application in the autologous immunotherapy, peripheral blood mononuclear cells (PBMC) from healthy donors were cultivated in stem cell growth medium supplemented with 5% human AB serum, and monoclonal anti-CD3 antibody, IL-2 and phytohaemagglutin (PHA) at different concentrations were added to select the optimal cultural conditions for the CINK cells. The optimized method was used to expand the CINK cells from 12 patients with various malignant tumors, the proportions of CD3 + and CD56 + cells in the expanded cells were determined by flow cytometry, and the cytotoxicity of the CINK cells from patients was detected by MTT assay in comparison with those of cytokine-induced killer (CIK) cells or anti-CDC3 antibody-activated killer (CD3AK) cells. It was found that the expansion of CINK cells could be obtained when these cells were activated with 500 ng/ml of monoclonal anti-CD3 antibody and 100?U/ml of IL-2. The highest degree of cell proliferation (51.3 folds) could be attained in the presence of PHA after 14?d cultivation of cells, and the expanded cell populations included CD3 - CD56 + NK cells (49.8%±7.2%) and CD3 + CD56 + T cells (14.7%±3.9%) could lyse 83.7% of K562 cells and 55.8% of Raji cells at a 10∶1 of effector/target ratio. Also the CINK cells of clinical scale could be expanded from tumor patients by using the optimized method with a similar expansion efficiency and cytotoxicity. These cells showed a higher cytotoxic activities against target cell lines (K562 and Raji cell lines) than those of CIK and CD3AK cells. This improved method has several advantages over other methods, such as the easy manipulation, low costs and the production of functionally active NK cells on a clinical scale from cancer patients without the need for special equipments. This might facilitate the realization of further protocol for evaluating the clinical effects of NK cells in autologous immunotherapies of tumor patients.