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.

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.

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.

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.

Intraperitoneal perfusion of cytokine-induced killer cells with local hyperthermia for advanced hepatocellular carcinoma

AIM:To study the effect and tolerance of intraperitoneal perfusion of cytokine-induced killer(CIK) cells in combination with local radio frequency(RF) hyperthermia in patients with advanced primary hepatocellular carcinoma(HCC).METHODS:Patients with advanced primary HCC were included in this study.CIK cells were perfused intraperitoneal twice a week,using 3.2 × 10 9 to 3.6 × 10 9 cells each session.Local RF hyperthermia was performed 2 h after intraperitoneal perfusion.Following an interval of one month,the next course of treatment was administered.Patients received treatment until disease progression.Tumor size,immune indices(CD3 +,CD4 +,CD3 + CD8 +,CD3 + CD56 +),alpha-fetoprotein(AFP) level,abdominal circumference and adverse events were recorded.Time to progression and overall survival(OS) were calculated.RESULTS:From June 2010 to July 2011,31 patients diagnosed with advanced primary HCC received intraperitoneal perfusion of CIK cells in combination with local RF hyperthermia in our study.Patients received an average of 4.2 ± 0.6 treatment courses(range,1-8 courses).Patients were followed up for 8.3 ± 0.7 mo(range,2-12 mo).Following combination treatment,CD4 +,CD3 + CD8 + and CD3 + CD56 + cells increased from 35.78% ± 3.51%,24.61% ± 4.19% and 5.94% ± 0.87% to 45.83% ± 2.48%(P = 0.016),39.67% ± 3.38%(P = 0.008) and 10.72% ± 0.67%(P = 0.001),respectively.AFP decreased from 167.67 ± 22.44 to 99.89 ± 22.05 ng/mL(P = 0.001) and abdominal circumference decreased from 97.50 ± 3.45 cm to 87.17 ± 4.40 cm(P = 0.002).The disease control rate was 67.7%.The most common adverse events were low fever and slight abdominal erubescence,which resolved without treatment.The median time to progression was 6.1 mo.The 3-,6-and 9-mo and 1-year survival rates were 93.5%,77.4%,41.9% and 17.4%,respectively.The median OS was 8.5 mo.CONCLUSION:Intraperitoneal perfusion of CIK cells in combination with local RF hyperthermia is safe,can efficiently improve immunological status,and may prolong survival in HCC patients.

Immunotherapy with dendritic cells and cytokine-induced killer cells for hepatocellular carcinoma: A meta-analysis

BACKGROUND Hepatocellular carcinoma(HCC) has been revealed as the second most common cause of cancer-related deaths worldwide. The introduction of cell-based immunotherapy, including dendritic cells(DCs) and cytokine-induced killer cells(CIKs), has brought HCC patients an effective benefit. However, the efficacy and necessity of cellular immunotherapy after different interventional therapy remains to be further explored.AIM To investigate the efficacy of cellular immunotherapy, involving DCs and CIKs,combined with different conventional treatments of HCC.METHODS We performed a literature search on PubMed and Web of Science up to February15, 2019. Long-term efficacy(overall survival and recurrence) and short-term adverse effects were investigated to assess the effectiveness of immunotherapy with DCs and/or CIKs. Review Manager 5.3 was used to perform the analysis.RESULTS A total of 22 studies involving 3756 patients selected by eligibility inclusion criteria were forwarded for meta-analysis. Combined with the conventional clinical treatment, immunotherapy with DCs and/or CIKs was demonstrated to significantly improve overall survival at 6 mo [risk ratio(RR) = 1.07;95%confidence interval(CI): 1.01-1.13, P = 0.02], 1 year(RR = 1.12;95%CI: 1.07-1.17, P< 0.00001), 3 years(RR = 1.23;95%CI: 1.15-1.31, P < 0.00001) and 5 years(RR =1.26;95%CI: 1.15-1.37, P < 0.00001). Recurrence rate was significantly reduced by cellular immunotherapy at 6 mo(RR = 0.50;95%CI: 0.36-0.69, P < 0.0001) and 1 year(RR = 0.82;95%CI: 0.75-0.89, P < 0.00001). Adverse effect assessment addressed that immunotherapy with DCs and/or CIKs was accepted as a safe,feasible treatment.CONCLUSION Combination immunotherapy with DCs, CIKs and DC/CIK with various routine treatments for HCC was evidently suggested to improve patients’ prognosis by increasing overall survival and reducing cancer recurrence.

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.

Adjuvant treatment for triple-negative breast cancer: a retrospective study of immunotherapy with autologous cytokine-induced killer cells in 294 patients

Objective: To examine the efficacy and safety of a sequential combination of chemotherapy and autologous cytokine-induced killer(CIK) cell treatment in triple-negative breast cancer(TNBC) patients.Methods: A total of 294 post-surgery TNBC patients participated in the research from January 1, 2009 to January 1, 2015. After adjuvant chemotherapy, autologous CIK cells were introduced in 147 cases(CIK group), while adjuvant chemotherapy alone was used to treat the remaining 147 cases(control group). The major endpoints of the investigation were the disease-free survival(DFS) and overall survival(OS). Additionally, the side effects of the treatment were evaluated.Results: In the CIK group, the DFS and OS intervals of the patients were significantly longer than those of the control group(DFS:P = 0.047;OS: P = 0.007). The multivariate analysis demonstrated that the TNM(tumor-node-metastasis) stage and adjuvant CIK treatment were independent prognostic factors for both DFS [hazard ratio(HR)= 0.520, 95% confidence interval(CI):0.271-0.998, P = 0.049;HR = 1.449, 95% CI:1.118-1.877, P = 0.005, respectively] and OS(HR=0.414, 95% CI:0.190-0.903, P = 0.027;HR= 1.581, 95% CI:1.204-2.077, P = 0.001, respectively) in patients with TNBC. Additionally, longer DFS and OS intervals were associated with increased number of CIK treatment cycles(DFS: P = 0.020;OS: P = 0.040). The majority of the patients who benefitted from CIK cell therapy were relatively early-stage TNBC patients.Conclusion: Chemotherapy in combination with adjuvant CIK could be used to lower the relapse and metastasis rate, thus effectively extending the survival time of TNBC patients, especially those at early stages.

Hepatocellular carcinoma-specific immunotherapy with synthesized α1,3-galactosyl epitope-ulsed dendritic cells and cytokine-induced killer cells

AIM： To evaluate the safety and clinical efficacy of a new immunotherapy using both α-Gal epitope-pulsed dendritic cells （DCs） and cytokine-induced killer cells. METHODS： Freshly collected hepatocellular carcinoma （HCC） tumor tissues were incubated with a mixture of neuraminidase and recombinant αl,3-galactosyltrans- ferase （αI,3GT） to synthesize α-Gal epitopes on car- bohydrate chains of the glycoproteins of tumor mem- branes. The subsequent incubation of the processed membranes in the presence of human natural anti-Gal IgG resulted in the effective phagocytosis to the tumor membrane by DCs. Eighteen patients aged 38-78 years with stage 111 primary HCC were randomly chosen for the study; 9 patients served as controls, and 9 patients were enrolled in the study group.RESULTS： The evaluation demonstrated that the pro- cedure was safe; no serious side effects or autoimmune diseases were observed. The therapy significantly pro- longed the survival of treated patients as compared with the controls （17.1 ± 2.01 mo vs 10.1 ±4.5 mo, P = 0.00121）. After treatment, all patients in the study group had positive delayed hypersensitivity and robust systemic cytotoxicity in response to tumor lysate as measured by interferon-y-expression in peripheral blood mononuclear cells using enzyme-linked immunosorbent spot assay. They also displayed increased numbers of CD8-, CD45RO- and CD56-positive cells in the peripheral blood and decreased α-fetoprotein level in the se- rum. CONCLUSION： This new tumor-specific immunotherapy is safe, effective and has a great potential for the treat- ment of tumors.

Activation of killer cells with soluble gastric cancer antigen combined with anti-CD3 McAb

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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.

Cisplatin pretreatment enhances anti-tumor activity of cytokine-induced killer cells

AIM： To investigate whether cisplatin （DDP） enhances the anti-tumor activity of cytokine- induced killer （CIK） cells in a murine colon adenocarcinoma model. METHODS： Tumor size and weight served as indicators of therapeutic response. Immunohistochemistry was performed to observe intratumoral lymphocyte infiltration and tumor microvessel density. Changes in the percentage of regulatory T （Treg） cells within the spleens of tumor-bearing mice preconditioned with DDP were monitored using flow cytometry. RESULTS： A marked T cell-dependent, synergistic anti- tumor effect of the combined therapy was observed （1968 ± 491 mm3 ys 3872 ＋ 216 mm3; P = 0,003）, Preconditioning chemotherapy with DDP augmented the infiltration of CD3＋ T lymphocytes into the tumor mass and reduced the percentage of both intratumoral and splenic Treg cells. CONCLUSION： Preconditioning with DDP markedly enhances the efficacy of adoptively transferred CIK cells, providing a potential clinical modality for the treatment of patients with colorectal cancer.

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.

Antitumor activities of human autologous cytokineinduced killer(CIK)cells against hepatocellular carcinoma cells in vitro and in vivo

AIM: To characterize the anticancer function of cytokine-induced killer cells (CIK) and develop an adoptive immunotherapy for the patients with primary hepatocellular carcinoma (HCC), we evaluated the proliferation rate, phenotype and the antitumor activity of human CIK cells from healthy donors and HCC patients in vitro and in vivo. METHODS: Peripheral blood mononuclear cells (PBMC) from healthy donors and patients with primary HCC were incubated in vitro and induced into CIK cells in the presence of various cytokines such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1), IL-2 and monoclonal antibody (mAb) against CD3. The phenotype and characterization of CIK cells were identified by flow cytometric analysis. The cytotoxicity of CIK cells was determined by (51)Cr release assay. RESULTS: The CIK cells were shown to be a heterogeneous population with different cellular phenotypes. The percentage of CD3+/CD56+ positive cells, the dominant effector cells, in total CIK cells from healthy donors and HCC patients, significantly increased from 0.1-0.13% at day 0 to 19.0-20.5% at day 21 incubation, which suggested that the CD3+ CD56+ positive cells proliferated faster than other cell populations of CIK cells in the protocol used in this study. After 28 day in vitro incubation, the CIK cells from patients with HCC and healthy donors increased by more than 300-fold and 500-fold in proliferation cell number, respectively. CIK cells originated from HCC patients possessed a higher in vitro antitumor cytotoxic activity on autologous HCC cells than the autologous lymphokine-activated killer (LAK) cells and PBMC cells. In in vivo animal experiment, CIK cells had stronger effects on the inhibition of tumor growth in Balb/c nude mice bearing BEL-7402-producing tumor than LAK cells (mean inhibitory rate, 84.7% vs 52.8%, P【0.05) or PBMC (mean inhibitory rate, 84.7% vs 37.1%, P【0.01). CONCLUSION: Autologous CIK cells are of highly efficient cytotoxic effector cells against primary hepatocellular carcinoma cells and might serve as an alternative adoptive therapeutic strategy for HCC patients.

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.

BIOLOGICAL FEATURES OF HUMAN T-ACTIVATED KILLER CELLS

Objective: To investigate the immunobiological essence of T-activated killer (T-AK) cells induced by anti-CD3 monoclonal antibody (CD3McAb) and recombinant interleukin-2 (rIL-2) co-stimulation. Methods: The cytomorphology, phenotype and cytotoxicity of T-AK cells generated from human peripheral blood mononuclear cells (PBMC) were determined. Results: T-AK cells were similar to activated lymphoblasts in morphology, more than 90% of T-AK cells expressed the phenotypes of T-lymphocytes (CD3 +, CD8 +, and 20%～50% of the cells were NK-like phenotype (CD16 +, CD56 +, some of them expressed IL-2 receptor (CD25 +), CD38 antigen (CD38 +) and MHC-II antigen (HLA-DR+) characteristic marks for the activated T lymphocytes. T-AK cells attacking targets were morphologically large volumes with granules and mainly contained CD8 + and CD56 + cells. T-AK cells possessed high tumoricidal activities against NK-sensitive K562 cells and NK-resistant Raji cells, the cytotoxicity was composed of mainly CD3McAb-activated CD3AK activity (～50%), IL-2 induced LAK activity (～30%), NK activity (～10%) and the activities of inhibitory factors in T-AK supernatant (～10%). Conclusion: T-AK cells are a heterogeneous cell population consisting of mainly activated T lymphocytes and NK-like cells, the main part of T-AK cytotoxicity is the common activities of CD3AK cells and LAK cells.

PURGING OF BONE MARROWS CONTAMINATED WITH MYELOID LEUKEMIC CELLS BY INTERLEUKIN-2 AND LYMPHOKINE-ACTIVATED KILLER CELLS

The capability of recombinant human interleukin-2 ( rhIL-2) and lymphokine-activated killer (LAK) cells in the purging of normal human bone marrows contaminated with human myeloid leukemic cell lines was evaluated. Mixtures of normal human bone marrow mononuclear cells ( BMC) and K562 cells or HL-60 cells (at the BMCK562 ratio of 200:1, 100:1 or 20:1) were incubated with IL-2 with or without LAK cells at the BMC:LAK ratio of 1:1 for one or three days. The nubmers of residual K562 cells, BFU-E and CFU-GM were examined by clonogenic assays. In 200:1 mixture groups without LAK cells, the number of K562 colonies reduced by 50% with no loss of BFU-E and CFU-GM in one-day cultures, and no K562 colonies formed in three-day cultures with about 20% loss of BFU-E and CFU-GM. If the BMC.K562 ratios were 100:1 or 20:1 in the mktures, the leukemic cells could not be eliminated. When the mixtures were incubated with IL-2 and LAK cells, no leukemic cell colonies were detected in the 20:1 group following one-day