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

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.

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.

Effect of anti-asthma Chinese medicine Chuankezhi on the anti-tumor activity of cytokine-induced killer cells

Chuankezhi(CKZ),a new Chinese medicine,plays an important role in immunoregulation.Cytokineinduced killer(CIK)cells have been commonly used for immunotherapy in recent years.In this study,we aimed to investigate the immunoregulatory effect of CKZ on CIK cells.Peripheral blood monocytes were isolated from healthy donors,and CIK cells were generated by culturing monocytes with interferon-gamma(IFN-γ)and interleukin 2.Different concentrations of CKZ were added on day 2.After incubation for 14days in culture,the antitumor effects of CIK cells were measured by cytotoxicity assay.Flow cytometry was used to explore the effect of CKZ on CIK cell immunophenotype,intracellular cytokine production,and apoptosis.The effect of CKZ on the antitumor activity of CIK cells in nude mice was also investigated.CKZ increased the percentage of CD3+CD56+CIK cells but did not significantly change the percentage of CD4+,CD8+,or CD4+CD25+CIK cells.CKZ-conditioned CIK cells showed a greater ability to kill tumor cells,as well as a higher frequency of IFN-γand TNF-αproduction,compared with the CIK cells in the control group.CKZ also suppressed the apoptosis of CIK cells in vitro.Furthermore,CKZ combined with CIK cells had a stronger suppressive effect on tumor growth in vivo than the CIK,CKZ,or normal saline control groups.Our results indicate that CKZ enhances the antitumor activity of CIK cells and is a potential medicine for tumor immunotherapy.

Feeder-free differentiation of human iPSCs into natural killer cells with cytotoxic potential against malignant brain rhabdoid tumor cells

Natural killer(NK)cells are cytotoxic immune cells that can eliminate target cells without prior stimulation.Human induced pluripotent stem cells(iPSCs)provide a robust source of NK cells for safe and effective cell-based immunotherapy against aggressive cancers.In this in vitro study,a feeder-free iPSC differentiation was performed to obtain iPSC-NK cells,and distinct maturational stages of iPSC-NK were characterized.Mature cells of CD56^(bright)CD16^(bright)phenotype showed upregulation of CD56,CD16,and NK cell activation markers NKG2D and NKp46 upon IL-15 exposure,while exposure to aggressive atypical teratoid/rhabdoid tumor(ATRT)cell lines enhanced NKG2D and NKp46 expression.Malignant cell exposure also increased CD107a degranulation markers and stimulated IFN-γsecretion in activated NK cells.CD56^(bright)CD16^(bright)iPSC-NK cells showed a ratio-dependent killing of ATRT cells,and the percentage lysis of CHLA-05-ATRT was higher than that of CHLA-02-ATRT.The iPSC-NK cells were also cytotoxic against other brain,kidney,and lung cancer cell lines.Further NK maturation yielded CD56^(-ve) CD16^(bright)cells,which lacked activation markers even after exposure to interleukins or ATRT cells-indicating diminished cytotoxicity.Generation and characterization of different NK phenotypes from iPSCs,coupled with their promising anti-tumor activity against ATRT in vitro,offer valuable insights into potential immunotherapeutic strategies for brain tumors.

Cytokine-induced killer cells showing multidrug resistance and remaining cytotoxic activity to tumor cells after transfected with mdr1 cDNA

Background Routine treatment of cancer such as surgery, radiation or chemotherapy is sometimes unable to erdiacate metastatic malignant cells. So we tried a new method and increased the adoptive immunotherapy of Cytokine-induced killer (CIK) cells in tumor patients and the multidrug resistance (mdr1) cDNA was transfected into CIK cells. Methods CIK cells were obtained from peripheral blood and induced by IFN-γ, anti-CD3 monoclonal antibody, IL-2 and IL-1. CIK cells were transfected with plasmid PHaMDR containing human mdr1 cDNA by electroporation. RT-PCR was used to detect mdr1 mRNA in transfected CIK cells. P-glycoprotein (P-gp) expressed on surface of CIK cells was assayed by FITC-conjugated anti-P-gp monoclonal antibody and flow cytometry. Multidrug resistance to doxorubicin and colchicine and cytotoxic activity to human breast cancer cell line MCF7 were performed using MTT method. Results mdr1 mRNA was detected in transfected CIK cells. P-gp was expressed on the surface of the transfected CIK cells, and the P-gp positive cells reached 21%-37% of the total CIK cells after transfection. The IC50 to doxorubicin increased to 22.3-45.8 times, and that to colchicines to 6.7-11.35 times, as compared to those of untransfected CIK cells. However, the cytotoxic activity to MCF7 cell line remained unaltered.Conclusions CIK cells were successfully transfected with mdr1 cDNA by using electroporation. The transfected CIK cells had the characteristics of multidrug resistance without change in their cytotoxic activity to tumor cells.

Breast cancer stem-like cells can promote metastasis by activating platelets and down-regulating antitumor activity of natural killer cells

OBJECTIVE:To investigate whether cancer stem cells(CSCs) more efficiently activating platelets and evading immune surveillance than non-CSCs thus promoting metastasis.METHODS:We enriched and identified sphereforming cells(SFCs) and coincubated washed platelets with several platelet activators including collagen,4T1 and SFCs.Platelet-coating tumor cells,platelet activation and TGF-β1 release were analyzed.Then natural kell cells(NK) were incubated with supernatants of different activated platelet samples what we called sample release(SR).The degranulation assay and NKG2 D expression on NK cells were conducted by flow cytometry.Finally tissue factor(TF) expression of SFCs or 4T1 were evaluated by western blot.RESULTS:Breast cancer cell line 4T1 could form spheres in serum-free medium at low adherence.Sphere-forming cells expressed high levels of the CD24-/lowCD44 + stem cell phenotype.Both sphere-forming cells or 4T1 were coated with abundant platelets while sphere-forming cells induced significantly higher expression of platelet activating receptor CD62 p than 4T1 did(P < 0.01).And sphere-forming cells induced platelets to produce more TGF-β1 than 4T1 did(P < 0.01).Furthermore,sample releases induced by sphere-forming cells caused more vigorous inhibition of NK cells antitumor reactivity(P < 0.05) and reduced NKG2 D expression(P < 0.01).The final results showed that sphere-forming cells expressed higher levels of TF than 4T1(P < 0.05).CONCLUSION:Our findings indicate that CSCs could efficiently activate platelets,induce platelets to secrete more TGF-β1,decrease NKG2 D expression and inhibit antitumor activity of NK cell,compared with 4T1.And higher levels of TF expression of CSCs may account for this correlation of CSCs and platelets.

Research on the biological activity and anti-tumor effect against lymphoma cells of DC-CIK cells

Objective： To investigate the proliferation capabilities, immunophenotype changes, level of secreted cytokines and activities against lymphoma cells under the condition that cytokine-induced killer （CIK） cells co-cultured with dendritic cells （DC） in vitro. Methods： DC and CIK cells were induced from peripheral blood mononuclear cells of healthy volunteers. They were co-cultured meanwhile CIK cells were cultured alone as controls. Increased number of cells were counted by tapan-blue staining, killing activities were detected by MTT assay, immunophenotype changes were analyzed by flow cytometry, the IL-12 and INF-y levels of the cultured supernatants were detected by ELISA kits. Results： The proliferation capabilities of DC-CIK cells were significantly higher than that of CIK cells （P 〈 0.05）. Under the same condition, the ratio of double positive cells such as CD3^＋ CD8^＋, CD3^＋ CD56^＋ in CIK cells was significantly enhanced by co-cultured with DC cells （P 〈 0.05）. The level of IL-12 and INF-y secreted in supernatants was increased noticeably by co-cultured DC-CIK cells on day 3 compared to CIK cells which were cultured alone （P 〈 0.01 and P 〈 0.05）. Within the effector-target ratio range between 5：1 to 40：1, the activities against lymphoma cells of DC-CIK cells were much higher than that of CIK cells （P 〈 0.05）, and this effect was showed a positive correlation with the effector-target ratio. Conclusion： The proliferation capabilities, the level of secreted cytokines and the activities against lymphoma cells of DC-CIK cells were significantly higher than those of CIK cells. The research might provides theoretical and experimental basis for clinical immunotherapy of DC-CIK cells.

Double potentialities of tumoricide and hematopoiesis of human bone marrow cells activated by IL-2 and IL-3

The biomodulative and hematopoietic potentialities of IL-2 and IL-3 activatedbone marrow(ABM)cells from patients with lung adenocarcinoma were studied in vitro.Human bone marrow(BM)cells could be activated by IL-2 in culture for 7d.TheseIL-2 ABM cells had higher cytolytic activities against cells of H 7402 cell line and freshautologous adenocarcinoma cells and maintained the cytotoxicities longer than IL-2 acti-vated peripheral blood lymphocytes(APBLs),a point of possible importance in adoptiveimmunotherapy for cancer patients.The IL-2 ABM cells also had similar number ofBFU-E and CFU-GM to that had fresh BM cells if 1L-3 was added 48h alter IL-2 inculture.The IL-2 and IL-3 ABM cells might be used to eliminate tumor cells and tosupply reconstitutive elements of BM for autologous bone marrow transplantation.

THE EFFECT OF PHENYLACETATE ON THE EXPANSION AND CYTOTOXIC ACTIVITY OF ADHERENT LAK CELLS FROM PATIENTS WITH HEPATOCELLULAR CARCINOMA

Objective: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and study the synergistic anti-tumor effect of phenylacetate (PA) and A-LAK cells. Methods: A-LAK cells were obtained from peripheral blood mononuclear cells (PBMC) of patients with hepatocellular carcinoma (HCC) by using L-phenylalanine methyl ester (PME) to deplete immunosuppressive monocytes. The proliferation of SMMC7721 cell line treated with PA was studied. A-LAK cells were treated with the supernatant of SMMC7721 cells which had been pretreated with PA and the changes of the proliferation and anti-tumor activity of A-LAK cells were investigated. Results: The expansion of A-LAK cells was significantly higher than that of non-adherent LAK (NA-LAK) cells as well as regular LAK cells. The growth of SMMC7721 cells was significantly suppressed by PA. The supernatant of cultured tumor cells intensively suppressed the proliferation and cytotoxicity of A-LAK cells, but the suppressive effect of supernatant treated with PA previously was decreased. Conclusion: A-LAK cells could be simply prepared by using PME, and showed a synergistic anti-tumor effect with the combination of PA.

Natural killer cells in liver diseases

The liver has been characterized as a frontline lymphoid organ with complex immunological features such as liver immunity and liver tolerance. Liver tolerance plays an important role in liver diseases including acute inflammation, chronic infection, autoimmune disease, and tumors. The liver contains a large proportion of natural killer （NK） cells, which exhibit heterogeneity in phenotypic and functional characteristics. NK cell activation, well known for its role in the immune surveillance against tumor and pathogen-infected cells, depends on the balance between numerous activating and inhibitory signals. In addition to the innate direct ＂killer＂ functions, NK cell activity contributes to regulate innate and adaptive immunity （helper or regulator）. Under the setting of liver diseases, NK cells are of great importance for stimulating or inhibiting immune responses, leading to either immune activation or immune tolerance. Here, we focus on the relationship between NK cell biology, such as their phenotypic features and functional diversity, and liver diseases.

Inositol hexaphosphate-induced enhancement of natural killer cell activity correlates with suppression of colon carcinogenesis in rats

AIM： To investigate the anti-neoplastic effect of inositol hexaphosphate （InsP6 or phytic acid） on dimethylhydrazine （DMH）-induced colon tumor in rats and its effect on blood natural killer （NK） cell activity. METHODS： Healthy Wistar rats, 4 wk old, were divided into control group （fed with common food） and InsP6 group （fed with common food＋2% sodium inositol hexaphosphate in the drinking water）, 15 rats in each group. Both groups were injected with 1,2-dimethylhydrazine subcutaneously （20 mg/kg body weight） once a week for 20 wk. Rats were killed after 21 wk. The whole large intestine was isolated to determine the general condition of tumors and to test blood NK cell activity by lactate-dehydrogenase-release assay. RESULTS： Administration of InsP6 significantly increased blood NK cell activity in DMH-induced colorectal tumor in rats. InsP6 group had a smaller tumor size on average and a smaller number of tumors than the control group. Its mortality was also higher than that in control. However, the variables of body weight and tumor incidence were not significantly different between the two groups. CONCLUSION： InsP6 can increase blood NK cell activity in DMH-induced colon tumor in rats and inhibit tumor growth and metastasis in rats.

Tretinoin or retinol enhancement of lymphokine-activated killer cell proliferation and cytotoxicity against human bladder cancer cells in vitro

目的：研究维Ａ酸（Ｔｒｅ）或维生素Ａ（Ｒｅｔ）对膀胱肿瘤患者淋巴因子激活的杀伤细胞（ＬＡＫ细胞）的增殖和对膀胱肿瘤细胞的细胞毒作用．方法：分别用细胞计数和ＭＴＴ法测定ＬＡＫ细胞的增殖和细胞毒作用．结果：Ｔｒｅ或Ｒｅｔ１０－１００ｎｍｏｌ·Ｌ－１加强由白细胞介素２（ＩＬ２）诱导的ＬＡＫ细胞的增殖和对膀胱肿瘤细胞的杀伤作用．结论：Ｔｒｅ或Ｒｅｔ增强膀胱肿瘤患者ＬＡＫ细胞增殖及对膀胱肿瘤的细胞毒作用．

Clinical Detection of Peripheral Blood Natural Killer Cell Activity

Natural Killer (NK) cells are specific immune cells in human immune system. They have a quick effect and can exert a cytotoxic function without prior sensitization, and they show great application potential in cell-based immunotherapy, anti-infection in vivo. NK cell activity in peripheral blood can be used as one of the biomarkers of immune function response. It has a great positive guiding significance for the clinical prognosis of tumor patients, the prevention of cancer and anti-aging. The clinical detection strategies of NK cell activity in circulation mainly grouped into five types: methyl thiazolyl tetrazolium colorimetric, lactate dehydrogenase release, radionuclide labeling, flow cytometry and NK Vue cytokine release method. It has played an important role in different stages of clinical application development. This paper will make a comparative review of the above-mentioned detection strategies for the NK cell activity.

REGULATORY EFFECTS OF HUMAN RECOMBINANT IL-6 ON NATURAL KILLER CELL ACTIVITY OF HUMAN FETAL SPLEENS

In the present study it was proved first that human recombinant interleukin-6(HrIL-6) significantly augmented natural killer(NK) cell activity derived from human fetal spleens against K562 target cells in a 4 hours 51Cr release assay. The enhancement of NK activity with 24 hours preincubation in HrlL-6 was dose-dependent, and significantly higher than that of fresh NK cells and controls cultured with RPMI-1640 medium alone (P<0.001). We also found that IL-6 was able to augment NK activity from different fetal spleens at 20 to 40 weeks of gestation (up to 2.24 to 2.78 times), and no difference of NK activity of fetal splenocytes treated by HrIL-6 was observed between different fetal age (32.3% to 45.4%, P>0.05). Furthermore, IL-6-augmented NK activity of fetal splenocytes was very similar to adult levels (P>0.05). These finding strongly indicated that IL-6 plays an important role in the development of NK cell function during the gestational period, suggesting that IL-6 may be of importance in the regulation of host defense mechanisms against malignancies and viral diseases.

Modulating effect of mitomycin or cisplatin on lymphokine-activated killer cell proliferation and antitumor activity to bladder cancer cell lines in vitro

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THE ULTRASTRUCTURAL OBSERVATION OF TARGET CELLS ATTACKED BY HUMAN LAK

In order to explore the killing mechanism of LAK, we observed the morphological change of K562 and Raji attacked by human LAK with transmission electron microscope, The result showed that 1 hour after coculture of LAK and target cell, target cell was significantly damaged.Part of target cells died via necrosis, and part via apoptosis. Our findings show that human LAK can kill target cells via necrosis and apoptosis simultaneously

Autologous Natural Killer Cell/Natural Killer T Cell Immunotherapy of Malignant Diseases

Adoptive immunotherapy, the therapeutic infusion of ex vivo activated cancer-fighting white blood cells that was pioneered by Dr. Steven Rosenberg over 30 years ago, has become more widespread due to outstanding published research documenting the clinical efficacy of this strategy. Based on the well-established in vivo functions of NK and NKT cells, their integral role in the efficacy of certain chemotherapeutic and immunomodulatory agents, and their direct therapeutic action as displayed in clinical trials, the use of autologous natural killer cell infusions is an appropriate and warranted therapeutic option for the treatment of malignant diseases, especially in patients whose disease is refractory to standard treatments such as chemotherapy and radiation.

TREATMENT OF PATIENTS WITH MALIGNANT PLEURAL EFFUSIONS DUE TO ADVANCED LUNG CANCER BY TRANSFER OF AUTOLOGOUS LAK CELLS COMBINED WITH rIL-2 OR rIL-2 ALONE

Experimental study both in vitro and in vivotogether with clinical trials showed that LAKcells have antitumor and antimetastatic effects(1-5)and that these effects are closely related tothe number of LAK cells transferred and the ad-ministration of rIL-2(1,6-8).Usually,autologousPBL’s are used as the source of LAK precursorsin the adoptive immunotherapy of cancer patients.But this not only puts an added burden on thecancer patient,it can cause serious side effectsas well(9).Although TIL’s may provide a solu-tion to this problem(10,11),their isolation fromsolid tumors is complex and consumes many rea-gents.We have reported that the isolation oflymphocytes from malignant ascites or from ma-lignant pleural effusions is not only simple

RESEARCHES ON THE ANTI-AUTOLOGOUS TUMOR EFFECTS OF HUMAN CANCER-KILLING CELLS IN NUDE MICE

An investigation of human splenic LAK cells killing autologous tumor in vivo has been performed in this study. Briefly, an ovary embryo carcinoma (OEC) removed surgically from patient was transplanted Into nude mice (s. c. ) and cultured in vitro, that would be used as targets. Lymphokine-activated killer (LAK) cells were generated from splenic lymphocytes of the OEC patient, who died two and half months after operation, by co- culture with recombinant human interleukin- 2 ( rIL- 2 ) in vitro. The results from Winn' s test in nude mice suggested that these LAK cells could effectively inhibit the tumorlgenicity of autologous tumor m vitro.