Cytotoxicity Of Cytokine-induced Killer Cells Research Articles

Human interferon-alpha increases the cytotoxic effect of CD56+cord blood-derived cytokine-induced killer cells on human B-acute lymphoblastic leukemia cell lines

Cytokine-induced killer (CIK) cells may represent a promising immunotherapy for the treatment of children with relapsing B-lineage acute lymphoblastic leukemia (B-ALL) following hematopoietic stem cell transplantation (HSCT). Therefore, we investigated the possibility of combining adoptive immunotherapy with CIK cells and human interferon-alpha (hIFN-α) in order to potentiate the cytotoxicity of CIK cells against B-ALL. Cord blood- derived CIK (CB-CIK) cells were differentiated, stimulated with phosphate-buffered saline (PBS) or hIFN-α, and tested for cytotoxic activity. We tested the anti-leukemic and graft-versus-host disease (GvHD) effects of CB-CIK cells in a human xenograft NOD/SCID/γc(-) (NSG) mouse model. Bulk CB-CIK cells showed very moderate cytotoxic activity while the subpopulation of CD56(+) CB-CIK cells showed significant cytotoxic activity against B-ALL cells. hIFN-α significantly augmented the cytotoxicity of CD56(+)CB-CIK cells in vitro and induced signal transducer and activator of transcription-1 (STAT1) phosphorylation. In addition, CD56(+)CB-CIK cells could delay mouse mortality significantly in vivo, and this effect was enhanced significantly by hIFN-α (P = 0.022). Furthermore, unlike CB mononuclear cells or peripheral blood mononuclear cells (PBMC), CD56(+)CB-CIK cells, alone or stimulated with hIFN-α, caused either no GvHD or mild GvHD, respectively, when injected into sublethally irradiated NSG mice. CD56(+)CB-CIK cells are effective cytotoxic agents against human B-ALL cell lines in vitro and possess anti-leukemic activity that is potentiated by hIFN-α in an NSG mouse model in vivo. These pre-clinical data support the testing of this immunotherapeutic approach in the clinic for the treatment of B-ALL.

A phase I/II clinical trial of autologous cytokine-induced killer cells as adjuvant immunotherapy for acute and chronic myeloid leukemia in clinical remission

Cytokine-induced killer (CIK) cells have shown remarkable cytotoxicity against various tumors in vitro and in animal studies. We report on the clinical outcome of autologous CIK cells for patients with acute (AML) and chronic (CML) myeloid leukemia in remission. Eleven of the 13 recruited AML patients undergoing autologous peripheral blood stem cell transplant (autoPBSCT) were given autologous CIK cell infusion upon engraftment post-transplant and followed-up for disease relapse. Eleven CML patients on Imatinib with residual disease detectable by polymerase chain reaction (PCR) were given infusion and monitored by quantitation of the bcr-abl transcript. Despite the presence of interferon (IFN)-γ-secreting T cells against various AML- and CML-associated peptides at sporadic time-points and demonstration of in vitro cytotoxicity of CIK cells against autologous and allogeneic AML targets, there was no survival benefit in AML patients post-autoPBSCT given CIK cells compared with historical controls. For CML patients, all continued to have a detectable bcr-abl transcript fluctuating within a range comparable to their pre-treatment baseline, although two had a transient but non-sustainable disappearance of bcr-abl transcript. There were no adverse reactions except for fever within the first day of infusion. Our small series, while confirming safety, failed to demonstrate a clinical benefit of autologous CIK cells given in its current form for AML and CML. Further manipulation of CIK cells to improve anti-leukemic potency and specificity, together with the preparation of patients to create a more conducive milieu for in vivo expansion and persistence of infused CIK cells, should be explored.

The betulinic acid-induced human CIK cell cytotoxicity to gastric cancer cell line SGC-7901 and its mechanism

Objective To observe the effect of betulinic acid(BetA) on the growth of human cytokine induced killer(CIK) cells and the killing activity of CIK cells on the gastric cancer cells in vitro before and after induced by betulinic acid,explore its mechanism.Methods Peripheral blood mononuclear cell (PBMC) were separated form the healthy and were induced with various of cytokine to become CIK cells in vitro.CIK cells were collected on the tenth day and were induced with betulinic acid in different concentrations,followed by 48 h,the colorimetric methyl thiazolyl tetrazolium(MTT) method assay the proliferation rate of human CIK cells.Flow cytometry (FCM) was used to detect the expression changes of perforin,granzyme B and CD107a of human CIK cells before and after betulinic acid-induced.Lactate dehydrogenase (LDH) release assay was used to measure the influence on cytotoxic activity of CIK cells induced by betulinic acid against gastric cancer cell line SGC-7901 in vitro.Western blot assay was used to measure the extracellular signal-regulated kinase1/2 (ERK1/2),and adapter proteins SH2-domain containing leukocyte protein of 76KD(SLP-76) and linker for activative of T cells(LAT) expression changes of human CIK cells before and after drug-induced.Results Betulinic acid can promote CIK cells growth when the concentration were in 0.08-10 μg/ml,the expression of perforin,granzyme B and CD107a of CIK cells were significantly higher than control group(P＜0.05) when the concentration of betulinic acid were in 0.3 μg/ml.In the meanwhile,the cytotoxic activity of CIK cells in vitro against gastric cancer cell line SGC-7901 were also remarkably higher than the control group (P＜0.05).The expression of SLP-76,LAT and ERK1/2 were significantly increased to a certain extent than the control group( P＜0.05 ),when CIK cells were treated with betulinic acid.Conclusion These results suggest that betulinic acid can promote CIK cells growth in some concentrations and increase the cytotoxic activity of CIK cells against gastric cancer cell line SGC-7901,its mechanism may related with two factors,on the one hand,enhancing the activity of SLP-76,LAT and ERK1/2,on the other hand,increasing the expression of perforin,granzyme B and CD107a on the surface of CIK cells. Key words: Betulinic acid; Gastric cancer cell line; CIK cells

English

This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a characteristic flow cytometric profile where a number of peaks of progressively halving CFSE fluorescence intensity were observed. The test principle of cytotoxicity is based on target cell labeling with CFSE and subsequent DNA-labeling with PI for the identification of target cells with compromised cell membranes. Results of CFSE fluorescence profile of the entire cell population showed that these cells underwent up to 6 divisions after 21 days. The percentage of total dividing cell population was 97.12%, of which 17.28% cells went through 6 rounds and 6.84% cells entered the seventh generation. The cytotoxicity of CIK cells showed a significant and positive correlation with effector: target (E:T) ratio ranging from 50:1 to 6.25:1. CFSE labeling combined with flow cytometry can therefore be used to monitor the proliferation of CIK cells. Moreover, it presents an advantageous method to measure cytotoxicity of CIK cells by avoiding radioactive substance, increasing sensitivity at low E:T ratio, analyzing on a cell-to-cell basis and allowing for long-term incubation.   Key words: Flow cytometry, CFSE, cytokine-induced killer cell, proliferation, cytotoxicity.

Immunomodulation of inducible co‐stimulator (ICOS) in human cytokine‐induced killer cells against cholangiocarcinoma through ICOS/ICOS ligand interaction

To evaluate the immunomodulation of inducible co-stimulator (ICOS) in cytokine-induced killer (CIK) cells against cholangiocarcinoma. CIK cells were generated from normal peripheral blood mononuclear cells. Methyl thiazolyl tetrazolium assay was performed to assess proliferation of CIK-ICOS and controlled CIK cells; ELISA was used to analyze the expression of cytokines. Reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the expression of ICOS ligand (ICOSL) in CIK cells and human cholangiocarcinoma cell line QBC939 cells. The cytotoxicity of CIK cells was determined either by lactate dehydrogenase-releasing assay in vivo or alteration of tumor size prior to and after the treatment of CIK cells in vivo. CIK-ICOS cells proliferated more and expressed higher secretion a level of interferon-γ than the controlled CIK. These cells exhibited higher cytotoxicity against cholangiocarcinoma cell lines at all efficacy: toxicity (E:T) ratios tested than the controlled CIK cells. More importantly, the anti-ICOSL antibody was able to attenuate the elevated cytotoxicity mediated by ICOS overexpression. When injected into cholangiocarcinoma xenografts in severe combined immunodeficiency mice, CIK-ICOS cells survived better than the controlled CIK cells around xenografts and significantly reduced the growth rate of cholangiocarcinoma, with least volume increase and more severe necrosis of the xenografts than controlled mice treated with saline, CIK or CIK-enhanced green fluorescent protein. ICOS can enhance the cytotoxic effect of CIK cells against cholangiocarcinoma both in vitro and in vivo. This effect is mediated by ICOS-augmented cytokine secretion and cell proliferation, and in part through ICOS-ICOSL interaction.

Enhancement of Cytotoxicity against IL-15 Up-regulate NKG2D Expression on Cytokine-induced Killer Cells

Objective To analyse the effects of IL-15 on the expression of NKG2D and the cytotoxicity of cytokine-induced killer(CIK) cells against human esophagus carcinoma cell EC9706 in vitro.Methods Peripheral blood mononuclear cells were isolated from healthy donors then divided into two groups: the control group(cells were cultured in the presence of IFN-γ,anti-CD3 antibody and IL-2) and IL-15 group(cells were cultured in the presence of IFN-γ,anti-CD3 antibody,IL-2 and 10 ng/ml IL-15).Phenotypic characteristics of CIK cells and the expression of NKG2D were analyzed by flow cytometry.After 14 days culture,cytotoxicity of CIK cells against EC9706 cells was measured by using a standard LDH releasing assay.Effector cells were added to target cells at E∶T radios of 20∶1,30∶1.In blocking experiments,NKG2D monoclonal antibody was added to CIK cells for 15 min before plating at 30∶1 E∶T ratio.Results NKG2D molecules were significantly up-regulated during the culture period on CD3+ cells(the CIK cells population) and the CD56+cells.The differences of NKG2D expressions were significant between IL-15 group and the control group(P0.05).Cytotoxicity of CIK cells treated by the IL-15 group was higher than that of the control group,both at 20∶1 E∶T ratios and at 30∶1 E∶T ratios(P0.05).When the NKG2D molecules on CIK cell ular membrance were blocked by the NKG2D monoclonal antibody,the cytolytic activity in the control group cells and IL-15 group cells was significantly inhibited.Conclusion IL-15 up-regulated the expression of NKG2D on CIK cells,which enhanced the NKG2D mediated cytotoxicity against EC9706 cells.CIK cells played a role through the NKG2D molecules.

Cytotoxic effect of trans-cinnamaldehyde on human leukemia K562 cells

To investigate the effects of trans-cinnamaldehyde (TCA) on the human leukemia K562 cell line and the cytotoxicity of cytokine-induced killer (CIK) cells against K562 cells. Apoptosis, Fas expression, and mitochondrial transmembrane potential in K652 cells were analyzed using flow cytometry. K562 cells were labeled with CFSE. The cytotoxic effect of expanded CIK cells on CFSE-labeled K562 cells was determined by FACS flow cytometry. Treatment with TCA 180 micromol/L for 9 h induced apoptosis in 8.9%+/-1.23% of K562 cells. Treatment with 120 or 180 micromol/L TCA for 24 h significantly increased the apoptotic cells to 18.63%+/-1.42 % and 38.98%+/-2.74%, respectively. TCA significantly upregulates Fas expression and decreases mitochondrial transmembrane potential in K562 cells. TCA treatment at 120 and 180 micromol/L for 9 h enhanced the percentage of lysis of K562 cells by expanded CIK cells from 34.84%+/-2.13% to 48.21%+/-2.22 % and 64.81%+/-3.22% at the E:F ratio of 25:1 and from 49.26%+/-3.22% to 57.81%+/-5.13% and 73.36%+/-5.98% at E:F ratio of 50:1. TCA exerts cytotoxic effects on human leukemia K562 cells by inducing apoptosis and synergizing the cytotoxicity of CIK cells against K562 cells. These properties of TCA are beneficial to the treatment of leukemia, even in the patients who have received hematopoietic stem cells transplantation (HSCT).

Effect of dendritic cells transfected with total RNA of human colon cancer Lovo cells on specific cytotoxicity of cytokine-induced killer cells in vitro

Objective To explore the effect of dendritic cells (DC) primed by total RNA extracted from human colon cancer Lovo cell on specific cytotoxicity of cytokine-induced killer cells (CIK) in vitro.Methods Cord blood mononuclear cells extracted by Ficoll density gradient centrifuge were induced into CIK and DC cells separately, and their Immunophenotype was detected by Flow cytometer. Trizol harvested total RNA from colon cancer cell Lovo and the RNAs were loaded to DCs obtained from cord blood as tumor anti gens. Effectors were grouped accordingly as CIK cells co-cultured with DCs transfected with Lovo RNA, CIK cells co- cultured with unloaded DCs and CIK cells. Targets was Lovo cells. In vitro cytotoxicity of CHK cells was extured with DCs loaded with Lovo RNA(76.49%±4.21%), DC + CIK group was lower(53.84% ± 2.15%),and CIK cells group possessed the lowest cytotoxicity(32.20% ± 3.07%), showing statistic significance( P ＜0.05). Conclusion Extraction of total RNA from tumor cells is simple and easy for clinical implementation.Total RNAs acted as antigen to pulse DCs can strengthen the specific cytotoxicity of CIK cells, which will have good prospects for clinical application. Key words: Dendritic cell; Killer cells; Antigens

Dendritic cell-activated cytokine-induced killer cells enhance the anti-tumor effect of chemotherapy on non-small cell lung cancer in patients after surgery

Cytokine-induced killer (CIK) cells have shown cytolytic activity against several tumor cells in vitro and in animal tumor models. Furthermore, CIK cells activated by dendritic cell (DC) stimulation show increased anti-tumor activity. This study aimed to evaluate the clinical efficacy of DC-activated CIK cell treatment following regular chemotherapy and the effects of this therapy on immune responses in patients with non-small cell lung cancer (NSCLC) after surgery. A paired study, with 42 patients in each group with stage I-IIIa NSCLC after surgery, was performed. Patients received chemotherapy alone (CT) or chemotherapy and DC-activated CIK cell treatment (immuno-CT). Disease-free survival (DFS) and overall survival were evaluated. CIK cell cytotoxicity against tumor cells was detected using a lactate dehydrogenase-based method. Serum cytokine levels in the immuno-CT group were detected using cytokine antibody arrays. The cytotoxicity of CIK cells was significantly enhanced by DC activation. The 2-year overall survival rate in the immuno-CT group was significantly improved compared with the CT group (94.7 +/- 3.6% versus 78.8 +/- 7.0%, P < 0.05). The 2-year DFS of these two groups showed no significant difference. DC-activated CIK cell treatment increased production of cytokines that have known anti-tumor effects, including IFN-gamma, MIG, TNF-alpha and TNF-beta, in patients who had no progression, but they were not found in patients who developed recurrence/metastasis. This study suggests that the role of DC-activated CIK cells in improvement of chemotherapy for malignant tumor treatment is associated with up-regulation of the production of cytokines involved in the anti-tumor effect.

Effects of inducible co-stimulator gene on the cytotoxic activity of cytokine-induced killer cells against cholangiocarcinoma cells

Objective To explore the effects of inducible co-stimulator (ICOS) gene on the cytotoxic activity of cytokine-induced killer (CIK) cells against cholangiocarcinoma cells. Methods CIK-ICOS cells were obtained by stable transfecting ICOS genes into CIK cells through the adenovirus vector whereas untransfected and EGFP-transfected CIK cells were treated as controls. The proliferation and apoptosis of different CIK cells, as well as their cytotoxicity against cholangiocarcinoma cells in the three groups were detected. The expressions of IFN-T, IL-2 and TNF-α in the supernatant of different CIK cells were measured by ELISA. SCID mice with cholangiocarcinoma were randomly divided into CIK group, CIK-EGFP group, CIK-ICOS group and normal saline group. The cytotoxic activity of CIK-ICOS cells against cholangiocarcinoma cells in vivo was observed. Results CIK-ICOS cells displayed better proliferation than CIK cells and CIK-EGFP cells. At day 20 and 23 of culture, the apoptosis rate of CIK-ICOS cells was 0.69% and 0.89%, respectively, while that of the CIK cells was 2.90% and 4.92%. The cytotoxic effect of CIK-ICOS cells at different E: T ratio against cholangiocarcinoma cells was significantly stronger than that of CIK cells and CIK-EGFP cells (F=13.37, 6.46, 25.51, P<0.05). The concentration of IFN-γ in CIK-ICOS cultured supernatant was (49.50±4.73)μg/L, which was significantly higher than that in the cultured supernatant of CIK cells [(30.53±3.73)μg/L] and CIK-EGFP cells [(30.12±2.64)μg/L](F=38.89, P<0.05). The growth of cholangiocarcinoma was significantly slower in CIK-ICOS group than that in CIK group and CIK-EGFP group, whereas the necrosis area of tumor was larger and the CIK cells in CIK-ICOS group was more than those in the other two groups. Conclusions CIK cells had the function of killing cholangiocarcinoma cells in vitro and in vivo. After ICOS genes were transfected into CIK cells, the survival time of CIK cells in vitro was prolonged and the proliferation of CIK cells was enhanced, as well as the secretion of IFN-γ was increased so that the cytotoxicity of CIK cells against cholangiocarcinoma cells in vitro and in vivo was enhanced. Key words: Cholangiocarcinoma; Inducible co-stimulator; Cytokine-induced killer cells

CD4 +CD25 + Regulatory T Cells Decreased the Antitumor Activity of Cytokine-Induced Killer (CIK) Cells of Lung Cancer Patients

: CD(4) (+)CD(25) (+) regulatory T cells (Tregs) have been shown to inhibit cytotoxic lymphocytes-mediated immune responses. Cytokine-induced killer (CIK) cells exert high impact on adoptive immunotherapeutic approaches. Therefore, the purpose of this report was to determine the effect of Tregs on CIK cell growth and CIK-induced cytotoxicity for inhibition of tumor growth in vivo as well as in vitro. After depletion of CD(4) (+)CD(25) (+) cells before culture, the proliferation and cytotoxicity of CIK cells, which indicated in bromodeoxyuridine (BrdU) and lactic dehydrogenase (LDH) assays, were significantly increased. Depletion of CD(4) (+)CD(25) (+) cells preculture also enhanced the suppression effect on the lung cancer cells inoculated in experimental animals. Blockage of glucocorticoid-induced tumor necrosis factor receptor (GITR) and transforming growth factor beta1 (TGF-beta1) by antibodies partially abrogated the suppressive effect of CD(4) (+)CD(25) (+) cells on CIK. These results indicated that Tregs could inhibit the antitumor activity of CIK cells. The molecules TGF-beta and GITR may contribute to the suppressive function of CD(4) (+)CD(25) (+) cells.

The apoptotic and proliferative fate of cytokine-induced killer cells after redirection to tumor cells with bispecific Ab

Cytokine-induced killer (CIK) cells are ex vivo expanded T cells with co-expression of CD3 and CD56 and NK activity. They have recently been evaluated in a phase I/II clinical trial against malignant lymphoma. Bispecific Ab (bsAb) redirect CIK cells to tumor targets, thus enhancing their cytotoxicity. While bsAb may improve T-cell mediated anti-tumor activity, little is known about the fate of effector cells upon redirection to tumor targets using a bsAb. Using ex vivo-activated CIK cells, Her2/neu expressing breast and ovarian cell lines and a F(ab')2 Her2/neu x CD3 bsAb, we investigated the anti-tumor activity and the proliferative and apoptotic outcome of CIK cells. When redirected to tumor targets with bsAb, there was a significant increase in anti-tumor activity as well as an increase in both CIK cell proliferation and apoptosis. The addition of agonistic Ab against CD28 did not significantly increase proliferation or apoptosis of CIK cells redirected to CD80- and CD86- tumor targets. To attempt to reduce T-cell apoptosis, we incubated CIK cells in the presence of the pan-caspase inhibitor z-VAD-fmk, which led to a partial reduction in T-cell apoptosis without increasing cellular cytotoxicity. bsAb are effective in redirecting activated T cells to tumor targets and such redirection leads to both T-cell proliferation and apoptosis that are not altered by co-stimulation through CD28. Effector cell apoptosis can be reduced by using a caspase inhibitor but this does not increase CIK cell cytotoxicity.

Immunotherapy of Primary Ovarian Cancer Using Autologous Cytokine Induced Killer Cells Retargeted with Bispecific Antibodies: A Preclinical Study.

Cytokine induced killer (CIK) cells are ex-vivo activated and expanded CD8+ natural killer T cells that have been shown to have anti-tumor activity. This is the first study exploring cell killing of primary ovarian carcinoma with and without bispecific antibodies (BSAbs). Primary cancer cells and autologous CIK cells were collected from women with epithelial ovarian cancer. BSAbs against CA125 (BSAbxCA125) and Her2 (BSAbxHer2) were developed using chemical heteroconjugation. On FACS analysis, the expansion and stimulation of CIK cells resulted in a significant increase of CD3+CD8+ and CD3+CD56+ T cells. With enhancement by BSAbs, the mean percent lysis in a 51Cr release assay of fresh ovarian cancer cells exposed to autologous CIK cells increased from 22% (±0.3) to 89% (±2.1) at an effector to target ratio (E:T) of 100:1. Anti-NKG2D antibodies significantly attenuated the CIK activity by 57% on primary cells. In a xenograft SCID mouse model, real-time tumor regression and progression was visualized using a non-invasive in vivo bioluminescence imaging system. Four hours after CIK cell injection, we were able to visualize CD8+NKG2D+ CIK cells infiltrating Her2-expressing cancer cells on fluorescence microscopy. Mice that underwent adoptive transfer of CIK cells redirected with BSAbxCA125 (p=0.0002) and BSAbxHer2 (p=0.0002) had a significant reduction in tumor burden and improvement in survival versus those treated with CIK cells alone (p=0.03). BSAbs significantly enhanced the cytotoxicity of CIK cells in primary ovarian cancer cells and in our in vivo mouse model. The mechanism of cytolysis appears to be mediated in part by the NKG2D receptor.

Effects of Ganoderma lucidum polysaccharides on proliferation and cytotoxicity of cytokine-induced killer cells

To study the effects (and the mechanisms thereof) of Ganoderma lucidum polysaccharides (Gl-PS) on the proliferation and the anti-tumor activity of cytokine-induced killer (CIK) cells, and to make use of CIK cells as a means to investigate the interactions between Gl-PS and cytokines. CIK cells were prepared by using the standard protocol as a positive control. Experimental groups also underwent the standard protocol, except that Gl-PS (400 mg/L or 100 mg/L) was added and the dose of anti-CD3 and interleukin-2 they received was reduced by 50% and 75%, respectively. For negative controls, Gl-PS in the experimental protocol was replaced with soluble starch or methylcellulose (400 mg/L or 100 mg/L). CIK cell proliferation, cytotoxicity, and phenotype were determined by using the Trypan blue exclusion method, MTT assay, and flow cytometry. By synergizing cytokines, Gl-PS (400 mg/L or 100 mg/L) could decrease the amount of cytokine in lymphokine activated killer (LAK) cells and CIK cells culture, but had no significant effect on the proliferation, cytotoxicity, or phenotype of LAK cells, or CIK cells induced by cytokines at higher doses alone, in which CIK cells expanded about 80-fold and the main effectors, CD3+NK1.1+ cells, expanded by more than 15%. The cytotoxicity of CIK cells in experimental groups was 79.3%+/-4.7%, 76.9%+/-6.8% versus the positive control 80.7%+/-6.8% against P815 (P>0.05) and 88.9%+/-5.5%, 84.7%+/-7.9% versus the positive control 89.8%+/-4.5% against YAC-1 (P>0.05). The activity of Gl-PS could mostly be blocked by anti-CR3. Gl-PS was shown to be a promising biological response modifier and immune potentiator. The effect of Gl-PS on CIK cells is possibly mediated primarily through complement receptor type 3.

Generation of CD3+ CD56+ cytokine-induced killer cells and their in vitro cytotoxicity against pediatric cancer cells.

A certain number of pediatric cancer patients still succumb to relapse following conventional treatment of their malignancies. One of the mechanisms of relapse is escape from immunity. Adoptive cellular immunotherapy with effector cells has the potential to overcome this escape. In adults, the CD3+ CD56+ cell, a cytokine-induced killer (CIK) cell, appears to be a promising effector cell type with the greatest cytotoxicity. This effector cell type may work in children as well. No similar studies with children have been published. We speculated that expanded CD3+ CD56+ cells obtained from pediatric cancer patients during remission would act similarly against various pediatric tumor cell lines; therefore, we undertook the present study to find support for our speculation. This study was undertaken to generate and expand CD3+ CD56+ CIK cells from normal peripheral blood mononuclear cells (PBL) obtained from 6 children with cancer (2 with acute lymphoblastic leukemia, 2 with large cell lymphoma, and 2 with osteosarcoma) in remission after intensive chemotherapy and to study the cytotoxic activities of these cells against chronic myeloid leukemia cell line K562 t(9;22), 4 pediatric tumor cell lines [infant acute lymphoblastic leukemia RS4 t(4;11), TEL/AML acute lymphoblastic leukemia REH t(12;21), alveolar rhabdomyosarcoma Rh-Cr t(2;13), and Ewing sarcoma EW-Le t(11;22)], and 2 pediatric glioblastoma multiforme cultured cell lines (G74 and G77). CIK cells were generated and expanded in culture medium to which interferon gamma, monoclonal antibody against CD3, and interleukin 2 were added at appropriate times. Cells were counted by flow cytometry. Net lactate dehydrogenase release from target cells incubated with CIK cells was used as an index of CIK cell cytotoxicity against various pediatric tumor cell lines. The results show that after 21 days in culture CD3+ CD56+ CIK cells derived from the 6 pediatric patients accounted for a median of 28.3% of the entire culture (range, 10.7%-36.4%). Before expansion no such cells were found in any of the 6 children. Median lytic activity rates of CIK cells were 45.5% to 64.5%, rates that contrasted drastically to the lytic activity rates of PBL, which were only 8% to 12%. The findings of the present study are encouraging. They provide information for developing adoptive immunotherapy for future clinical trials with pediatric cancer patients, particularly those patients with minimal residual disease after intensive chemotherapy or stem cell transplantation (especially nonmyeloablative transplantation procedures).

Antitumor activities of human autologous cytokine-induced killer (CIK) cells against hepatocellular carcinoma cells in vitro and in vivo.

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. 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. 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). 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.

Generation of cytokine‐induced killer cells from leukaemic samples with in vitro cytotoxicity against autologous and allogeneic leukaemic blasts

Cytokine-induced killer (CIK) cells are CD3(+)CD56(+) non-major histocompatibility complex (MHC)-restricted immune effector cells. The present report demonstrates that it was possible to expand CIK cells obtained at diagnosis from patients with acute leukaemia. The percentage of CD3(+)CD56(+) CIK cells generated following culture ranged between 7.6% and 65% (median of 35.3%) and these cells were able to kill the human natural killer target K562 cells. Although the same effector cells were able to lyse autologous acute myeloid leukaemia (AML) target cells, they were not able to lyse autologous acute lymphoblastic leukaemia target cells. Pre-absorption of the CIK effector cells by K562 cells did not completely abrogate the cytotoxicity of CIK cells against autologous blasts in 9 out of 12 samples tested. Moreover, it was observed that the cytotoxicity generated by the CIK effector cells against allogeneic leukaemic blasts was similar to that against autologous blasts. The present study suggests the potential application of CIK cells in the immunotherapy of AML, either in minimal disease state, as donor lymphocyte infusion in relapse post allogeneic transplant, or in cases of chemotherapy refractory leukaemia.

Two Pathways of Exocytosis of Cytoplasmic Granule Contents and Target Cell Killing by Cytokine-Induced CD3+CD56+ Killer Cells

Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell-induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3-mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP-sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be-identified target cell surface molecules.

Cytotoxicity of cytokine-induced killer cells coated with bispecific antibody against acute myeloid leukemia cells.

Various types of cytokines have been used in in vitro experiments to generate cytokine-induced killer (CIK) cells that are reactive to patient acute myeloid leukemia (AML) cells. Of these CIK cells, interleukin-2 (IL-2)-activated peripheral blood mononuclear cells, i.e., lymphokine-activated killer (LAK) cells, with the initial addition of the anti-CD3 monoclonal antibody (T3 LAK cells), are the most potent cytotoxic lymphocytes, and have marked proliferative capacity. The cytotoxicity of such T3 LAK cells against CD13+ AML cells is further enhanced by the addition of anti-CD3 x anti-CD13 bispecific antibody (BsAb) during the cytotoxicity assay. The combined use of T3 LAK cells and the BsAb can be used for ex vivo purging of CD13+ AML cells in autologous bone marrow transplantation. Other cytokines, such as IL-7 or IL-7 in combination with IL-2, or newly identified cytokines, will also be tested in attempts to obtain more specific and more potent effector cells. Studies of methods to increase the susceptibility of AML cells to CIK are also required.