DC-CIK Cells Research Articles

Effect of interleukin-15 and dendritic cells on the phenotype and cytotoxicity of cytokine-induced killer cells

Objective To investigate the effect of interleukin 15 (IL-15) and dendritic cells (DC) on the phenotype and cytotoxicity of cytokine-induced killer (CIK) cells.Methods Spleen mononuclear cells were harvested from C57BL/6 mice and cultured in anti-CD3 antibody-coated plate with interferon-γ (IFN-γ) and IL-2 containing medium to induce CIK cells.Bone marrow derived-DC was prepared.IL-15,DC or both were added to the CIK culture medium.CIK,CIK,IL-15-CIK,DC-CIK,DC/IL-15-CIK cells were produced,respectively.The percentage of CD3 + CD8 +,CD3 + NK1.1 + cells in these CIK cells were analyzed with FCM.Cytotoxicity of different CIK cells on mouse liver carcinoma hepal-6 cells were determined through detection of lactate dehydrogenase (LDH) release.Results The ratio of CD3 + CD8 + and CD3 + NK1.1 + cells in the spleen mononuclear cells of C57BL/6 mice before stimulation was (12.3 ± 2.1) ％ and (1.1 ± 0.5) ％,respectively.After culturing for 14 days in vitro,the ratio of CD3 + CD8 + cells in CIK,IL-15-CIK,DC-CIK and DC/IL-15-CIK cells was (18.8 ± 2.4)％,(22.1 ± 2.0)％,(28.2 ±2.6)％ and (34.7 ±2.3)％,respectively.The ratio of CD3 +NK1.1 + cells was (17.1±2.6)％,(18.9 ±3.4)％,(22.5±1.5)％ and (25.9±1.8)％.There was significant difference between IL-15 treatment group and control.The cytotoxicity of DC-CIK and DC/IL-15-CIK cells on liver carcinoma Hepal-6 cells were significantly higher than CIK cells as effector-target ration range from 2.5∶1.0 to 20.0∶1.0 (P ＜ 0.05).Cytotoxicity of DC-CIK and DC/IL-15-CIK was (54.9 ± 3.1) ％ and (58.75 ± 4.34) ％ as effector-target ratio was 20.0∶ 1.0.Conclusion Both IL-15 and DC increase the ratio of CD3 + CD8 + and CD3 + NK1.1 + cells in CIK cells,and synergetic effects also present.CIK cells stimulated with DC showed increased anti-tumor effect,and IL-15 can further enhance the antitumor effect of DC-CIK cells. Key words: Cytokine-induced killer; Interleukin-15 ; Dendritic cells ; Cytotoxicity

Effect of DC-CIK cell on the proliferation, apoptosis and differentiation of leukemia cells

ObjectiveTo observe the effect of co-culture cytokine-induced killer cells (CIK) and homologous dendritic cells (DC) on the proliferative activity and phenotype change of the DC-CIK cell and the cell killing activity of leukemia HL-60. Methods50 mL cord blood sample was obtained from infants delivered by full term healthy woman and the cord blood mononuclear cells were isolated by density gradient centrifugation. Non-adherent cells were collectedfor the induction culture of CIK, adherent cells were differentiated into mature DC; cultured mature DC was mixed with and CIK in the proportion of 1:5 for 12 d. Killing activity of DC-CIK co-cultured cell on leukemia HL-60 was detected by MTT assay. ResultsCompared with CIKs, the co-cultured DC-CIKs presented a markedly higher proliferation and killing activity. ConclusionsCo-culture of DC-CIK cells led to a significant increase of the proliferation and cytotoxicity of CIK.

The research of antitumor activities in vitro of DCs loading antigen prouced by radiofrequency ablation of tumor combined with CIK cells

Objective To study the in vitro anti-tumor activity of dendritic cells (DCs) loading with antigen produced by radiofrequency ablation of tumor lysate in situ combined with cytokine-induced killer cells (CIK).Methods CIK ceils derived from BALB/C mouse spleen and DCs derived from bone marrow were prepared,and experimental model of murine colon carcinoma were established for radiofrequency ablation.The supernatant of tumor tissue in situ lysis after repeated freezing and thawing were tested by lowry protein quantitative statutory,amounting to a final concentration of 5 μg/ml,then load to the first 5 days of culture DCs (Ag-DC),2 days later,co-cultured with CIK cells after the first seven days of culture 48 h (Ag-DC-CIK).Flow cytometry was used to analyze costimulatory molecules on the surface of the cells,and CCK-8 assay to detect in vitro cytotoxic activity.Results The DCs loading with antigen resulted in an increase in the proportion of CD86 + CD11 c +,MHC Ⅱ + CD11 c + and MHC Ⅱ + CD80 + cells.The main effector cells of CIK cells were CD3 + NK1.1 + cells.The percentage of CD3 + NK1.1 + cells was 1.45％ on the first day of the culture ; while when they had been cultured for 7 days,the percentage CD3 + NK1.1 + significantly increased to 36.9％.The cytotoxicity of Ag-DC-CIK cells toward C26 cells was much more efficient than that of DC-CIK,CIK cells.The cytotoxic activity of the former was significantly lower than the latter and the same target ratio.When the ratios of effector cells to target cells were 5 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (74.9 ± 3.5) ％,; while the DC-CIK was (71.2 ± 2.1) ％ and the CIK cells was (68.7 ± 2.9) ％.The difference was statistically significant(F =7.007,P =0.007).When the ratios of effector cells to target cells were 10 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (82.3 ± 4.5) ％,while the DC-CIK cells was (77.1 ± 5.1) ％,and the CIK cells was (72.7 ± 2.8) ％.The difference was statistically significant (F =7.727,P =0.005).When the ratios of effector cells to target cells were 20 ∶ 1,the cytotoxic activity of Ag-DC-CIK cells against C26 cells was (83.2 ± 1.9) ％,while the DC-CIK cells was (77.2 ± 4.2) ％,and the CIK cells was (73.0 ± 2.6) ％.The difference was statistically significant (F =16.594,P =0.000).Conclusion DCs loading with antigen produced by radiofrequency ablation of tumor in situ pyrolysis products can improve in vitro cytotoxic activity combined with CIK cells,which can provide a new comprehensive cancer treatment strategy. Key words: Dendritic cells; Colonic neoplasms; Radiofrequency ablation

Adoptive cellular immunotherapy for the treatment of patients with breast cancer: A meta-analysis

BackgroundTo evaluate the therapeutic efficacy of dendritic cells (DC) alone, cytokine-induced killer (CIK) cells alone and the combination of DC and CIK cells in the treatment of breast cancer, we performed a systemic review of the relevant published clinical studies, collectively referred to as DC-CIK cell therapy. MethodsSix hundred thirty-three patients with breast cancer were assigned to cohorts, and a meta-analysis was conducted. ResultsThe treatment of breast cancer with DC-CIK cells was associated with a significantly improved 1-year survival (P = 0.0001). The Karnofsky performance status scale of the patients treated with DC-CIK cells was significantly improved compared with that of the non-DC-CIK group (P < 0.0001). The percentage of T cells (CD3+, CD4+ and CD4+CD8+), CD16+ monocytes, and CD3+CD56+ natural killer T cells in the peripheral blood of cancer patients was significantly increased (P ≤ 0.05), whereas the percentage of CD4+CD25+ regulatory T cells was not significantly decreased (P = 0.32) in the DC-CIK treatment group compared with the non-DC-CIK group. The levels of interleukin-2, interleukin-12, tumor necrosis factor-α, interferon-γ, and nucleolar organizer region protein in the peripheral blood of cancer patients, which reflect immune function, were significantly increased (P < 0.001) after DC-CIK cell treatment. Furthermore, after DC-CIK treatment, the average levels of the alpha-fetoprotein, cancer antigen embryonic antigen and carbohydrate antigen tumor markers were decreased (P < 0.00001). ConclusionsDC-CIK cell therapy markedly prolongs survival time, enhances immune function, and improves the efficacy of the treatment of breast cancer patients.

DC-CIK cell therapy combined with transcatheter arterial chemoembolization and radiofrequency ablation for treatment of small hepatocellular carcinoma

DC-CIK cell therapy combined with transcatheter arterial chemoembolization and radiofrequency ablation for treatment of small hepatocellular carcinoma

Clinical Study Of DC-CIK (dendritic cells and cytokine-induced killer cells ) Eliminate Minimal Residual Leukemia

Abstract Objective To evaluate the clinical efficacy and safety of allogenic dendritic cells (DCs) and cytokine-induced killer (CIK) cells in the eliminating minimal residual leukemia (MRL). Methods 48 acute leukemia patients with hematological complete remission (CR) but without molecular biological remission (CRM), or patients with minimal residual Leukemia (MRL) were selected from Ping’an Hospital of Shijiazhuang during Jan. 2009 to Jun. 2011. According to the patients’ will, 48 patients divided into combined treatment group and chemotherapy group 24 each. All the patients were in the same general information and disease level. The combined treatment group was treated with DC-CIK and consolidation chemotherapy, and the chemotherapy group was treated with consolidation chemotherapy. PBMCs were collected from healthy donors (the patient's parents or children) to prepare DC-CIK cells. DC-CIK cells were intravenous injected into patients once every 15 days, a total of 4-6 times infusion. The blood routine, bone marrow cells, leukemia related genes, urine and stool routine, liver and kidney biochemistry function, and ECG were observed. Changes of peripheral lymphocyte subsets in patients were detected by flow cytometry. Adverse reactions were examined. Results (1)Eleven cases in the combined treatment group achieved CRM, and the CRM rate was 45.8%; whereas only 2 cases in the chemotherapy group achieved CRM and the CRM rate was 8.3%,the difference was statistically significant(χ2=8.55, P&lt;0.01).(2) Compared with the chemotherapy group, the CFIM (four-color combination flow cytometric immunophenotype of minimal residual leukemia) negative conversion rate of patients in the combined treatment group was significantly raised (66.7% vs 25.0%,χ2=8.39, P&lt;0.01). (3)The negative conversion rate of MRL was higher in the combined treatment group than the chemotherapy group (66.7% vs25.0%, χ2=8.39, P&lt;0.01). (4) After treatment the ratio of CD4+/CD8+ cells was significant increased than before treatment in the combined treatment group (1.3±0.4 vs 0.8±0.4, P&lt;0.05). (5)The complete remission rate (CCR) of patients in the combined treatment group after 3 years was 79.2%, while that in the chemotherapy group was 45.8% (χ2=5.69, P&lt;0.05).(6)No dysfunction of critical organs such as heart, liver and kidney and serious adverse reactions were observed while DC-CIK cells infusion. Conclusion DC-CIK combined with chemotherapy can inhibit leukemia gene, promote the negative conversion rate of CFIM, facilitate the clear of MRL, improve immune function and prolong remission of the patients.No serious adverse reactions were found in patients with DC-CIK infusion. Disclosures: No relevant conflicts of interest to declare.

A Clinical Study of HBsAg‐Activated Dendritic Cells and Cytokine‐Induced Killer Cells During the Treatment for Chronic Hepatitis B

We aim to study the therapeutic effects of HBsAg-activated DCs and cytokine-induced killer (CIK) cells as adoptive immunotherapy in patients with Chronic Hepatitis B (CHB). Autologous HBsAg-activated DC-CIK cells were infused into patients with CHB to evaluate their effect on HBV-DNA, HBsAg, ALT, etc. The viral load in the treatment group decreased significantly (P<0.001), while that in the control group did not decrease (P>0.05). Twenty-one patients (63.6% efficiency) in the treatment group had a viral response (≥2 log decrease in viral load), while four patients (14.8% efficiency) from the control group had a viral response. There were significant differences in the viral responses of the two groups (the control group 63.6% versus the control group 14.8%, P<0.001). We concluded that the immunity was enhanced after HBsAg activation in DCs and CIK cells. Reinfusion of autologous HBsAg-activated DC-CIK cells inhibited HBV proliferation in 21 of 33 (63.6%) patients.

Effects of two different immunotherapies on triple negative breast cancer in animal model

The ability of immune system to react specifically against tumors inspirited the study of triple negative breast cancer (TNBC) immunotherapies. Sixty spontaneous breast cancer TA2 mice were randomly divided into three groups: GM-CSF group, with therapy of granulocyte–macrophage colony-stimulating factor (GM-CSF) combined with breast cancer stem cells associated antigens and cytosine–phosphorothioate–guanine oligodeoxynucleotides (CpG-ODNs); DC–CIK group, with infusions of dendritic cells/cytokine-induced killer (DC/CIK) cells; and PBS group as controls. After therapy, the cellular immunity of mice in GM-CSF group and DC–CIK group was obviously increased, especially for GM-CSF group (P<0.05), tumor regression was obviously observed in GM-CSF group. The survival rate of mice in GM-CSF group was significantly higher compared to DC–CIK group and PBS group. These results indicated that tumor immunotherapy manifested strong killing activity against TNBC. The therapeutic effect of GM-CSF combined with antigens and CpG was better than DC–CIK cells.

The clinical effects of DC-CIK cells combined with chemotherapy in the treatment of advanced NSCLC

The aim of the study was to evaluate the safety and therapeutic effects of autologous dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) combined with chemotherapy in advanced non-small cell lung cancer (NSCLC) patients. Fifty patients with advanced NSCLC (stages III to IV), who had received therapies in our Center (Department of Biotherapy, Affiliated to Cancer Hospital of Shanxi Medical University, Taiyuan, China) from August 2008 to January 2010, were treated by DC-CIK + chemotherapy as the combined treatment group; fifty advanced NSCLC patients treated with chemotherapy at the same time served as controls. The immunologic function, short-term therapeutic effects, the 1-year survival rate, the life quality, the chemotherapy side effects were compared between the two groups, the safety and therapeutic effects of DC-CIK cells therapy were observed too. There was no obvious change of subsets of T cells in peripheral blood before and after therapy in DC-CIK + chemotherapy group, and IFN-γ was improved after therapy in this group (P < 0.05); in chemotherapy alone group, the ratios of CD3+CD4+, CD3+CD8+, CD3−CD56+ cells and the secretion of IL-2, TNF-α decreased significantly after therapy (P < 0.05); the ratios of CD3+CD8+, CD3+CD56+ were improved after cell culture (P < 0.05). The disease control rate (DCR) of DC-CIK + chemotherapy group was higher than that in the chemotherapy alone group (78.0% vs 56.0%, P < 0.05); the 1-year survival rates of DC-CIK + chemotherapy group and chemotherapy alone group were 50% and 44% respectively, had no significant difference. Compared with chemotherapy alone group, the occurrence of chemotherapy side effects (including bone marrow suppression, nausea and vomiting, peripheral nerve toxicity) was less in the DC-CIK + chemotherapy group (P < 0.05). The physical and appetite were better in DC-CIK + chemotherapy group after therapy. To compare with simple chemotherapy, DC-CIK + chemotherapy for advanced NSCLC is safe and effective, and it can improve patients’ life quality and remission rate, and prolong their survival time.

Review of Chinese clinical trials on CIK cell treatment for malignancies

China is the country where the most clinical trials on CIK cells have been performed. We aimed to provide definite evidence for using CIK cell treatment and extrapolate a common applicative standard for malignancies. We chose the VIP database of Chinese scientific and technological journals to search the literature. We entered the keywords "CIK" or "xi bao yin zi you dao de sha shang xi bao" (the equivalent Chinese phrase for CIK cells, by Chinese characters) and searched for in vivo human trials. In 24 collected trials, 936 patients were treated with CIK cells, 525 men and 246 women. The cultivation time of CIK cells ranged from 7 to 28 days. In five studies, CIK cells were co-cultured with dendritic cells. The total number of CIK cells used ranged from 6×10(6) to 1.5×10(10). The total number of DC-CIK cells used ranged from 1×10(9) to 1.3×10(10). In all studies, those immune parameters and tumour markers examined increased, but not all increased significantly. Of the reported 563 patients, 40 had a complete response, 126 had a partial response, 125 had a minimal response, 135 had stable disease and 58 had progressive disease. The remaining 76 patients did not reach an objective response. The total response rate was 51.7% (291/563). The toxicities were slight. CIK cell treatment is a promising and safe modality for treating malignancies. We proposed a standard for cultivating CIK cells.

Clinical study of co-treatment with DC-CIK cells for advanced solid carcinomas

Objective The aim of this study was to observe the therapeutic effect of cytokine induced killer (CIK) cells in combination with dendritic cells (DCs) on advanced solid carcinoma patients.

1113 T-Bet Mediated Anti-Neoplastic Effects of Dendritic Cell-Cytokine Induced Killer Cells In-Vitro

Background and aims: To investigate the molecular mechanism underlying T-bet mediated anti-neoplastic effects of cytokine induced killer (CIK) cells.Methods: Lymphocytes isolated from peripheral blood of leukemic children were induced with IFN-γ, CD3McAb and IL-2 and co-cultured with dendritic cells (DCs) to generate DC-CIK cells. The morphology and immunophenotype of these cells were determined by electron microscopy and flow cytometry, respectively. IL-2 and IFN-γ levels released by DC-CIK cells were quantified by ELISA. Cytotoxicity of DC-CIK cells against leukemia cell lines was measured by the MTT assay. FCM was used to detect CD4+CD25+Treg cells, while RT-PCR and Western blot were used to determine mRNA and protein expressions of Foxp3 and GATA3 in DC-CIK cells treated with T-bet monoclonal antibody.Results: Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. The main effector cells in this population were CD3+CD8+ cells and CD3+CD56+ cells. We demonstrated a time dependent increase in IL-2 and IFN-γ levels after induction. DC-CIK cells were cytotoxic to B95 cells, Jhhan cells and M07e cells, with the highest cytotoxicity towards B95 cells. Treatment with mouse anti-human T-bet monoclonal antibody resulted in an increase in the proportion of CD4+CD25+Treg cells and elevation of Foxp3 and GATA3 mRNA and protein levels.Conclusion: DC-CIK cells induced with cytokines were strongly cytotoxic towards a number of cancer cell lines. Foxp3 and GATA3 were implicated in the T-bet mediated anti-neoplastic effects of DCCIK cells via activation of the Th1 pathway and suppression of theTh2 and Treg pathways.

Successful Rescue of Refractory/Recurrent Myelogenous Leukemia by Allogeneic Hematopoietic Cell Transplantation and Prophylactic Immunotherapy.

Abstract 4095Poster Board III-1030The clinical outcomes of refractory/recurrent leukemia that salvaged by allogeneic hematopoietic cell transplantation (HCT) is usually poor. It is crucial for the management of those high-risk patients with appropriate conditioning regimens that balance the efficacy and safety well and with enhanced anti-leukemia effect post-HCT. Our previous study has shown that donor's dendritic cell-primed cytokine-induced killer cells (DC-CIK) is a safe and effective therapy in the management of early leukemia recurrence after allogeneic HCT, which fail to or ineligible for current standard treatment. ObjectiveIn present clinical study, we examine the efficacy of refractory/recurrent myelogenous leukemia salvaged by allogeneic HCT and prophylactic immunotherapy. MethodsFrom September 2006 to May 2008, 30 patients with refractory/recurrent myelogenous leukemia (AML 29, CML-BC 1) were enrolled. The median age was 32 (12 to 55) years old. The median blasts in bone marrow were 36% (20% to 87%) prior to conditioning. The grafts were from HLA identical siblings (5), unrelated donors (7), and haploidentical family members (18). Conditioning regiments were individualized according to patients' status as following. Generally, the regimen with high-dose cytarabine plus BUCY2 was used (13 cases). The patients with impaired organ function received above regimen except with fludarabine instead of cyclophosphamide (11 cases). For the recipients with > 40% blasts in bone marrow, melphalan (2 cases) or aclarubicin (1 case) was added into the regimen or FLAG followed by reduced-intensified BUCY2 (3 cases) was employed in order to reduce leukemia burden. Cyclosporine A, methotrexate and mycophenolate mofetil were administrated for GVHD prophylaxis. To prevent leukemia relapse, immunosuppressants were tapered off early post-HCT. Prophylactic immunotherapy including cellular (DLI, DC-CIK, NK cells), and humoral (IL-2, IFN-a, thymosin) was used selectively in the patients who had no GVHD 120 days after HCT. ResultsThe median mononuclear cells in the graft were 7.36 (3.49 to 11.5) ×108/kg. The median CD34+ cells were 4.06 (1.57 to 11.4) ×106/kg. The median CD3+ cells were 1.42 (0.75 to 3.61) ×108/kg. Twenty-nine patients attained sustained engraftment. One died of multi-organ failure before hematopoietic reconstitution. The median time for white blood cells > 1.0 × 109/L, and platelets > 20 × 109/L was 14 (10 to 21) days, 15 (12 to 26) days, respectively. Thirteen patients developed acute GVHD (grade I in 5, grade II in 7, and grade III in 1). Thirteen patients developed chronic GVHD after immunosuppressants' reduction or withdrawal. In addition, 13 patients received prophylactic immunotherapy due to lack of chronic GVHD 120 days post-HCT, then 7 of 13 developed chronic GVHD. With the median follow-up of 15 (3 to 35) months, two (6.7%) patients with AML had hematological recurrence. One patient attained durable complete remission again after treatment with chemotherapy followed by immunotherapy with DLI, DC-CIK, and NK cells. Five (16.7%) patients died (infections in 2, hematological relapse in 1, chronic GVHD in 1, and multi-organ failure in 1). 25 of 30 (83.3%) patients have been in continuous complete remission since salvaged HCT. ConclusionOur preliminary clinical results have shown that the combination of salvaged allogeneic HCT and prophylactic immunotherapy is a promising modality for the treatment of myelogenous leukemia in refractory/recurrent status, even with high leukemia burden. Disclosures:No relevant conflicts of interest to declare.

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.