CIK Cells Research Articles

A Positive Role of Cytokine-Induced Killer Cell Therapy on Gastric Cancer Therapy in a Chinese Population: A Systematic Meta-Analysis.

BackgroundChemotherapy and radiation therapy provide limited improvement in survival of gastric cancer patients after tumor resection. It is essential to develop a novel therapeutics for gastric cancer. In the recent years, cytokine-induced killer cells (CIKs)-based adoptive immune therapy has been explored in gastric cancer patients. Due to the small number of patients included in each clinical trial and low-power statistical analysis, the effectiveness of this approach is still unclear. To address this issue, we systemically analyzed the relevant clinical trial data published in recent years by powerful statistical meta-analysis.Material/MethodsClinical data was searched by multiple electronic databases with a term “gastric cancer” and “cytokine-induced killer cells”. Six relevant clinical trials with case-control studies were extracted for our meta-analysis, including 318 patients receiving CIK cell therapy and 369 patients receiving conventional therapy.ResultsOverall survival (OS) and odds ratio (OR) were analyzed for patients at 1, 2, 3, and 5 years post-CIK cell therapy and post-conventional therapy. Heterogeneity and publication bias were analyzed for included data quality and publication bias. Our meta-analysis from 6 clinical trials suggests that CIK cell therapy significantly increased 5-year OS from 27±2.44% to 49±7.62% (p<0.05) and 5-year OR up to 1.77 (p<0.05). The increased 5-year survival rate was also highly correlated with the increased CD3+ T cell number and ratio of CD4+/CD8+ in the CIK treated patients.ConclusionsCIK cell therapy significantly increased 5-year survival rate compared to conventional chemotherapy among gastric cancer patients. The study provides powerful statistical evidence for large-scale clinical trials with CIK cell therapy.

Fish IRF3 up-regulates the transcriptional level of IRF1, IRF2, IRF3 and IRF7 in CIK cells.

Interferon Regulatory Factors (IRFs) belong to a family of transcription factor involved in transcriptional regulation of type I IFN and IFN-stimulated genes (ISG) in cells. In the present study, an IRF3 full-length cDNA (termed CiIRF3, JX999055) and its promoter sequence were cloned by homology cloning strategy and genome walking from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiIRF3 is comprised of a 5'UTR (195 bp), a 3'UTR (269 bp) and a largest open reading frame (ORF) of 1377 bp encoding a polypeptide of 458 amino acids. CiIRF3 has a conservative DNA-binding domain (DBD) at N-terminal and a relatively conserved interferon regulatory factors association domain (IAD). Phylogenetic tree analysis indicated that CiIRF3 gathers together with other IRF-3 from higher vertebrates in the same branch. The promoter sequence of CiIRF3 (596 bp) consists of three IRF-E, a C/EBP beta, a NF-kappa B and a TATA-BOX. CiIRF3 was constitutively expressed at low level in different grass carp tissues but was rapidly up-regulated with Poly I:C stimulation. To study the molecular mechanism of CiIRF3 regulating the transcription of IRFs, CiIRF3 was expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assays revealed the affinity of CiIRF3 protein with promoters of CiIRF1, CiIRF2, CiIRF3 and CiIRF7 respectively. Then, CIK cells were co-transfected with pcDNA3.1-CiIRF3, pGL3-promotor (pGL3-CiIRF1, pGL3-CiIRF2, pGL3-CiIRF3, pGL3-CiIRF7) and luciferase reporter vector respectively. The cotransfection experiment showed that CiIRF3 increased the promoter activity of CiIRF1, CiIRF2, CiIRF3 and CiIRF7. Furthermore, overexpression of CiIRF3 in CIK cells also up-regulated the expressions of CiIRF1, CiIRF2, CiIRF3 and CiIRF7. So, CiIRF3 can improve the transcriptional level of CiIRF1, CiIRF2, CiIRF3 and CiIRF7.

A hypothermal storage solution for cytokine-induced killer cells derived from cord blood

We measured the killing activities of cytokine-induced killer cells (CIK) cells after hypothermal storage of such cells to identify an effective freezing solution. Fourteen days after induction of CIK cells from cultured umbilical cord blood, cells were frozen and stored in a new storage solution (10% [v/v] dimethyl sulphoxide [DMSO]; 10% [w/v] Dextran; and lymphocytes, but without serum) and traditional cell freezing solution (15% [v/v] DMSO; 10% [w/v] human albumin; and lymphocytes, also without serum). Cells were thawed and cultured after 180 days, and cell morphology and proliferation were investigated. Flow cytometric measurement (FCM) was used to define the immune phenotype. The lactate dehydrogenas ( LDH) method was employed to test the ability of stored and freshly induced CIK cells to kill The myelogenous leukemia cell line K562. The morphologies and immune phenotypes of freshly induced and stored (in either solution) cells were similar. The proliferative activity and killing capacity of fresh CIK cells and those stored in the new solution were similar, and higher than those of cells stored in the traditional solution.

Modification of cytokine-induced killer cells with chimeric antigen receptors (CARs) enhances antitumor immunity to epidermal growth factor receptor (EGFR)-positive malignancies.

Epidermal growth factor receptor (EGFR, ErbB1, Her-1) is a cell surface molecule overexpressing in a variety of human malignancies and, thus, is an excellent target for immunotherapy. Immunotherapy targeting EGFR-overexpressing malignancies using genetically modified immune effector cells is a novel and promising approach. In the present study, we have developed an adoptive cellular immunotherapy strategy based on the chimeric antigen receptor (CAR)-modified cytokine-induced killer (CAR-CIK) cells specific for the tumor cells expressing EGFR. To generate CAR-CIK cells, a lentiviral vector coding the EGFR-specific CAR was constructed and transduced into the CIK cells. The CAR-CIK cells showed significantly enhanced cytotoxicity and increased production of cytokines IFN-γ and IL-2 when co-cultured with EGFR-positive cancer cells. In tumor xenografts, adoptive immunotherapy of CAR-CIK cells could inhibit tumor growth and prolong the survival of EGFR-overexpressing human tumor xenografts. Moreover, tumor growth inhibition and prolonged survival in mice with EGFR(+) human cancer were associated with the increased persistence of CAR-CIK cells in vivo. Our study indicates that modification with EGFR-specific CAR strongly enhances the antitumor activity of the CIK cells against EGFR-positive malignancies.

Identification and characterization of IKKε gene from grass carp Ctenopharyngodon idella.

IKKε is an IKK-related kinase implicated in antiviral immune response in higher vertebrates. To elucidate the function of IKKε in teleost fish, grass carp IKKε (gcIKKε) has been cloned and characterized in this paper. The full-length cDNA of gcIKKε is composed of 2529 nucleotides and encodes a polypeptide of 723 amino acids. The mRNA transcription of gcIKKε was constitutively detected in all the selected tissues and the gcIKKε mRNA level increased at 36 h after GCRV infection. Western blot data of both HEK293T cells and EPC cells demonstrated that gcIKKε was around 80 KDa; and immunofluorescence staining data of both NIH3T3 cells and EPC cells determined gcIKKε was a cytosolic protein. The mRNA level of gcIKKε in CIK cells was increased more than 150 times right after poly(I:C) treatment and PMA treatment triggered gcIKKε mRNA transcription in CIK cells more than 100 times. Over-expression of gcIKKε in EPC cells activated the promoter activity of both zebrafish IFN and fathead minnow IFN. gcIKKε mRNA transcription level in CIK cells was increased from 48 h post GCRV infection with different MOIs. All the data support the idea that gcIKKε is a novel teleost IκB kinase recruited in the IFN-mediated antiviral immunity of grass carp.

Functional characterizations of IPS-1 in CIK cells: Potential roles in regulating IFN-I response dependent on IRF7 but not IRF3

IPS-1, as the sole adaptor of RIG-I and MDA5, plays a central role in innate antiviral immunity. In this study, we investigated potential roles of IPS-1 in innate immunity and the domain-requirement of IPS-1 for its signaling in grass carp (Ctenopharyngodon idella). Overexpression experiment showed that CiIPS-1 mediated IFN-I signal possibly dependent on CiIRF7 but not CiIRF3. Post GCRV challenge, CiIPS-1 could enhance antiviral immune responses. CARD and TM domains were crucial for antiviral function of CiIPS-1, and TRAF motif played an assistant role. PRO domain seemed as a negative regulator but was pivotal for the initiation of CiIFN-I and CiMx1. Post viral/bacterial PAMPs stimulation, CiIPS-1-mediated signaling was tightly controlled. CARD domain of CiIPS-1 could significantly elicit poly I:C/LPS/PGN-mediated signaling. PRO domain negatively regulated CiIRF7 and CiIFN-I but was indispensable for inductions of CiMx1 and CiIL-1β. TRAF motif and TM domain regulated the signaling presumably in a cooperative fashion. Post poly I:C stimulation, TRAF motif negatively regulated CiIRF7, CiIFN-I and CiIL-1β at a relative early time while TM domain functioned at a relative late time. TRAF motif was indispensable for the production of CiMx1, while TM domain slightly negatively regulated the expression. Post LPS and PGN stimulation, TRAF motif excited an assistant and persistent negative role on CiIFN-I, CiIRF7 and CiIL-1β induction, but was crucial for induction of CiMx1. TM domain slightly negatively regulated LPS- and PGN-triggered signaling. Taken together, CiIPS-1 not only exerted important functions in antiviral immune response but also participated in viral/bacterial PAMPs-triggered immune response which was tightly controlled to prevent harmful effects resulting from excessive activation. This study provided novel insights into the pivotal role of IPS-1 in innate immunity.

Increased survival time of a patient with metastatic malignant melanoma following immunotherapy: A case report and literature review.

Metastatic malignant melanoma is treated with chemotherapy and radiotherapy. A number of previous studies have indicated that cytokine-induced killer cells (CIK cells) are a heterogeneous cell population that express cluster of differentiation (CD)3 and CD56, in addition to the natural killer cell NKG2D activating receptor. CIK cells possess major histocompatibility complex-unrestricted cytotoxicity towards cancer, but not towards normal targets. The present study investigated whether the addition of CIK cells resulted in an improved therapeutic response in a patient with metastatic malignant melanoma. In the current case, a patient with metastatic malignant melanoma received CIK therapy, which resulted in a relatively long survival time of 28 months. To the best of our knowledge, there have been no previous studies reporting such positive effects in a patient who received CIK cell immunotherapy. Based on the findings of the present study, CIK cell therapy may be an option that results in a good prognosis in certain patients with metastatic malignant melanoma.

Screening and analysis on the protein interaction of the protein VP7 in grass carp reovirus.

Grass carp reovirus (GCRV) has caused serious economic losses for several decades in China. The protein VP7 is one of the important structural proteins in GCRV. Recent studies indicated that the protein VP7 had the commendable antigenicity and immunogenicity. The protein VP7 cooperated with VP5 could change the conformation of the cell membrane and facilitate entry of GCRV into host cells. We speculated that the protein VP7 should play an important role in the pathogenesis of GCRV. In order to explore the function of the protein VP7, the bait protein expression plasmid pGBKT7-vp7 and the cDNA library of CIK cells were constructed. By yeast two-hybrid system, after multiple screening with the high screening rate medium, rotary verification, sequencing and bioinformatics analysis, the interactions of the protein VP7 with ribosomal protein S20 (RPS20) and eukaryotic translation initiation factor 3 subunit b (eIF3b) in CIK cells were identified. RPS20 played the important roles in the generation of influenza B virus and a variety of diseases. eIF3b was relative to the infection of some viruses. This study suggested that the protein VP7 played the role in viral replication and most likely interacted with host proteins by RPS20 and eIF3b. The interaction mechanisms of the protein VP7 with RPS20 and eIF3b, and the subsequent effector mechanisms needed to be further studied. The corresponding protein interaction of the protein VP7 was not acquired in bioinformatics. The protein VP7 and its untranslated region may have the unknown special function. This study laid the foundation for deeply exploring the function of the protein VP7 in GCRV and had the important scientific significance for exploring the pathogenic mechanism of GCRV.

MicroRNA profiling of CD3+ CD56+ cytokine-induced killer cells.

Studies have proven that IL-2 and IL-15 showed contrasting roles during CIK cells preparation. By employing microarray, we analyzed miRNA expression profiles of PBMC, CIKIL-2 and CIKIL-15. Advanced bioinformatic analyses were performed to explore the key miRNAs which may regulate cell proliferation and anti-tumor activity of CIK. We identified 261 differentially expressed miRNAs (DEMs) between PBMC and CIKIL-2, and 249 DEMs between PBMC and CIKIL-15. MiR-143-3p/miR-145-5p was miRNA cluster which may positively regulate cell proliferation. In contrast, miR-340-5p/miR-340-3p cluster may negatively regulate cell proliferation via induction apoptosis, which may cause decreased cell proliferation capacity of CIKIL-2. MiRNA-target interaction analysis indicated that 10 co-downregulated miRNAs may synergistically turn on the expression of a pool of tumor cytotoxic genes in CIK cells. The DEMs between CIKIL-2 and CIKIL-15 may contribute to enhanced tumor cytotoxic capacity of CIKIL-2. Importantly, we found that repressed miR-193a-5p may regulate the expressions of inhibitory receptor KLRD1. The results of the validation assay have shown that KLRD1 were upregulated in CIK cells. Our findings have provided new insights into mechanisms of CIK cells production and tumor cytotoxic function, and shed light on their safety for clinical trial.

Effect of adoptive immunotherapy with CIK cells combined with interleukin-2 on immunologic function and life qualities of patients with non-small cell lung cancer

Objective To investigate the changes of immunologic functions and life qualities after the auto-cytokine induced killer (CIK) cells combined with interleukin-2(IL-2) being transfused back to the patients with non-small cell lung cancer (NSCLC). Methods Thirty-eight NSCLC patients were enrolled, and 19 patients received not only chemotherapy and radiotherapy but also adoptive immunotherapy with CIK cells combined with IL-2 compared with the other 19 patients who only accepted radiotherapy and chemotherapy. The changes of immunologic functions and life qualities after the CIK cells combined with IL-2 being transfused back to the patients with NSCLC were observed. Immune targets of the treatment group and control group, such as peripheral blood CD3+, CD4+, CD8+ T cell percentage, ratio of CD4+ /CD8+ and Th1/Th2, were observed at the end and after 3 month of the radiation and chemotherapy. Results Both of the two groups were neither having obvious adverse reactions. After CIK cells combined with IL-2 treatment, the life qualities of patients improved, such as 18 cases of mental improvement, 15 cases of appetite improvement, 12 cases of sleep improvement, and 3 cases of fatigue symptoms improvement. The percentage of CD3+, CD4+ cells in the treatment group increased [(66.39±9.22)% vs (42.98±7.23)%, t=5.45, P=0.00; (32.27±3.75)% vs (26.38±2.51)%, t=5.73, P=0.00], the percentage of CD8+ cells declined [(17.51±1.85)% vs (20.90±2.31)%, t=5.21, P=0.00], the rate of CD4+ /CD8+ increased and the rate of Th1/Th2 obversed [(1.86±0.32) vs (1.27±0.19), t=7.13, P=0.00; (1.15±0.48) vs (0.91±0.30), t=2.42, P=0.02]. There were significant differences between the two groups. Conclusion Adoptive immunotherapy with CIK cells combined with IL-2 is safe in clinical, which can improve the immunologic functions and life qualities of the patients with NSCLC. Key words: Carcinoma, non-small-cell lung; Immunotherapy; Cytokine induced killer cells

Proteomic identification and characterization of Ctenopharyngodon idella tumor necrosis factor receptor-associated protein 1 (CiTrap1): An anti-apoptosis factor upregulated by grass carp reovirus infection

Human tumor necrosis factor receptor-associated protein 1 (Trap1) is a mitochondrial protein identical to heat shock protein 75 (HSP75) that plays an important role in protecting cells from oxidative stress and apoptosis. In this study, grass carp (Ctenopharyngodon idella) tumor necrosis factor receptor-associated protein 1 (designated as CiTrap1) was identified through two-dimensional electrophoresis (2-DE) analysis and its pattern of expression was investigated in grass carp kidney (CIK) cells infected with grass carp reovirus (GCRV). The full length cDNA of CiTrap1 contained an opening reading frame of 2157 bp that encoded a peptide of 718 amino acids. Phylogenetic analyses indicated that the CiTrap1 shared 87% identity with its homologue from zebrafish (Danio rerio). The transcriptional level of CiTrap1 in CIK cells was upregulated post virus infection as well as poly (I: C) stimulation. Following virus infection, grass carp PTEN-induced putative kinase 1 (PINK1) and Sorcin, whose coding proteins interact with Trap1 in human, were simultaneously upregulated with CiTrap1. Typical characteristics of apoptosis were observed in CIK cells infected with GCRV by DAPI staining, DNA ladder electrophoresis, TUNEL assay and Annexin Ⅴ labeling. RNAi-mediated silencing of CiTrap1 in CIK cells resulted in the increased rate of virus-induced apoptotic cells. The results of this study suggest that CiTrap1 is involved in the host's innate immune response to viral infection possibly through protecting infected cells from apoptosis.

Tumor mica status predicts the efficacy of immunotherapy with cytokine-induced killer cells for patients with gastric cancer

In this study, we determine the relationship between the expression of major histocompatibility complex class I chain-related gene A (MICA) in gastric cancer tumors after D2 gastrectomy and the clinical outcome of a CIK-containing adjuvant therapy. Ninety-five consecutive patients with gastric cancer after D2 gastrectomy who received adjuvant chemotherapy combined with CIK cell therapy were enrolled. The MICA expression of their tumors was determined by immunohistochemistry (IHC). High expression of MICA protein was documented by IHC in 38 of 95 tumor samples (40.0 %). The MICA status was significantly associated with the age and stage, p = 0.008 and 0.023, respectively. Analysis of NKG2D on in vitro expanded CIK cells showed that the percentages of NKG2D+ in CD3+/CD56+, CD3−/CD56+, and CD3+/CD8+ cells populations were 97.2 ± 1.4, 97.9 ± 1.8, and 95.6 ± 2.1 %, respectively. For patient with high MICA-expressing tumors, the median DFS and OS were longer than for the patients with tumors with low expression of MICA; 46.0 versus 41.0 months (p = 0.027), and 48.0 versus 42.0 months (p = 0.031), respectively. In a multivariate analysis, stage and MICA expression were independent prognostic factors for DFS and OS. Our findings show that adjuvant chemotherapy plus CIK therapy treatment is a promising modality for treating gastric cancer patients after D2 gastrectomy. Especially, those who have tumors with high expression of MICA were more likely to benefit from such a treatment strategy. Subsequent studies in clinical trial cohorts will be required to confirm the clinical utility of these markers.

IL-12 induced CIK cells combined with chemotherapy improve immune function and efficacy in patients with esophageal carcinoma

IL-12 induced CIK cells combined with chemotherapy improve immune function and efficacy in patients with esophageal carcinoma

CMV-Specific Cytokine-Induced Killer Cells Comprise Concomitant Cytotoxicity Against Leukemia and Cytomegalovirus

IntroductionInfection is one of the main causes of mortality and morbidity after allogeneic stem cell transplantation, especially in patients who received T cell depleted haploidentical stem cells. Reactivation or de novo infection of cytomegalovirus (CMV) is amongst the most frequent complications and occur due to a lack of virus-specific T cells post-transplant. Pre-emptive immunotherapy may support both reconstitution of viral specific responses on one hand and may prevent impending leukemic relapse on the other hand. Therefore we established a protocol to generate CMV-specific cytokine-induced killer cells (CIKpp65) with dual cytolytic function against CMV and AML.ProtocolCIK cells were generated in vitro from peripheral blood mononuclear cells (PBMC) of CMV-seropositive healthy donors using IFN-γ, activating monoclonal anti-CD3 antibody (MAb), interleukin (IL)-2 and IL-15. An additional single stimulation with human CMVpp65 protein was adequate to increase the amount of cytotoxic CMV-specificcells within CIK cells up to 23%. In total the CMVpp65 stimulation resulted in up to 11.0-fold increased frequency of CMV-dextramer+CD8+cells after 15 days of expansion (n=12).ResultsCytotoxicityNext we investigated cell-mediated cytotoxicity against leukemic cell lines THP-1 and K562, pp65 loaded cell line T2 and CMV-infected primary fibroblasts. CIK cell cytotoxicity is described as mediated by activating NK-cell receptor NKG2D. This receptor was blocked in order to determine the specific MHC-mediated cytotoxicity in experiments targeting pp65 loaded cells. The lysis of pp65 loaded cells by CIKpp65 cells was significant higher as compared to conventional CIK cells (effector to target cell ratio of 5:1, 39.9±21.6% to 13.6±10.6%, P<0.01). CIKpp65 cells also induced high cytotoxicity in infected fibroblasts (up to 55%, 10:1 E:T ratio). The anti-leukemic effect was retained in CIKpp65 cells. CIKpp65 cells revealed a mean cytotoxicity of 71.5%, 60.7% and 37.8% against THP-1 and 55.0%, 50.0%, 20.5% against K562 in 40:1, 20:1 and 5:1 E:T ratio, respectively. In contrast, the reactivity against allogeneic PBMC remained low (18% lysis, 40:1 E:T ratio) and allogeneic mock-infected fibroblasts were not lysed at all. This clearly indicates towards the low alloreactive potential of CIKpp65 cells.PhenotypeFurthermore we characterized subpopulation and memory phenotype of CIKpp65 cells in detailed flow cytometric analyses and examined the cytokine secretion pattern by cytometric bead array. After expansion the population mainly consisted of a CD3+CD56- T cell (77.6±4.5%) and CD3+CD56+ T-NK cell phenotype (20.0±12.6%). The T-NK cells additionally co-expressed high amounts of CD8 cytotoxic antigen (63.8±16.8%). Interestingly, the T-NK cell compartment contained higher amounts CMV-specific CD8+ cells (mean 5.5%) than the T cell compartment (mean 1.3%). Expression of activating NKG2D and CD25 receptor was strongly positive in both cell fractions. Remarkably, almost 30% of T-NK cells expressed γδ+ T cell receptor, whereas T cells only expressed 4.5% of this receptor type. The cytotoxic T cells within the CIKpp65 cells consisted of a mixed naïve (CD45RA+CD62L+), central memory (CD45RO+CD62L+) and effector memory (CD45RO+CD62L-) phenotype, the cytotoxic T-NK cells mainly of effector memory and EMRA (CD45RA+CD62L-) phenotype. Cytokine secretion (granzyme B, IFN-γ, MIP-1α, TNF-α, Fas-L, IP-10, IL-10, IL-6 and IL-4) were measured during the expansion period and cytotoxic assays and resulting data confirmed the cytotoxic nature of the cells and indicated towards a mainly TH1 cell type character.ConclusionIn conclusion CIKpp65 cells can easily be generated from donor PBMC and might represent advantage to conventional CIK cells. Our pre-clinical data demonstrate the concomitant cytotoxicity of generated cells against leukemia cells and CMV, as well as low alloreactivity and limited risk to induce GvHD. Therefore CIKpp65 cells may represent an effective tool for pre-emptive immunotherapy in patients which have both an apparent risk of CMV reactivation and leukemic relapse after allogeneic stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Pseudomonas aeruginosa injection enhanced antitumor cytotoxicity of cytokine-induced killer cells derived from cord blood

Cord blood (CB) is becoming an extensive source of cytokine-induced killer cells. It had been used in several clinical settings and proven to be efficacious and safe. Therefore, we investigated the possibility of combining CIK cells derived from cord blood (CB-CIK) and Pseudomonas aeruginosa injection (PA-MSHA) in order to enhance the cytotoxicity of CB-CIK cells against tumors. Compared with the CB-CIK cells, the PA-MSHA-treated CB-CIK cells demonstrated with increased proliferation rates, higher expression of activated cell surface marker CD28 and lower expression of inhibited cell surface markers PD-1 and CTLA-4. Furthermore, PA-MSHA-treated CB-CIK cells exhibited more effectively for secreting pro-inflammatory cytokine such as IFN-γ and expressing high levels of TLR2, TLR4 and TLR6. The expression of CD107a was higher in the CD3+CD56+ subset of PA-MSHA-treated CB-CIK cells. Our results indicate that the PA-MSHA-treated CB-CIK cells exhibited a more potent in cytotoxic activity against tumor cells. Thus, PA-MSHA enhanced the antitumor ability of CB-CIK cells.

The Role of Mir-155 in the Apoptosis of Human Lymphoma Cell Induced By Cik Cells

ABSTRACT Aim: To observe the effect of cytokine-induced killer cells (CIK) on the apoptosis of human lymphoma cells (Raji and BJAB), and explore the role of microRNA-155(miR-155) in this process. Methods: MiR-155 was determined by real-time quantitative PCR, and the apoptosis was detected by flow cytometry in Raji and BJAB cells. Psi CHECK2-CEBPB 3′-UTR containing the binding site of miR-155 was constructed, and then transfected into Raji and BJAB cells. Luciferase activity of CEBPB (CCAAT/enhancer binding protein beta) was determined with the assistance of a dual luciferase report system. Results: CIK cells could promote Raji and BJAB cells apoptosis (t = 3.634, P Conclusions: CIK cells could enhance human lymphoma Raji and BJAB cells apoptosis by upregulating miR-155, which may provide a new database to elucidate lymphoma cell therapy using CIK cells. Disclosure: All authors have declared no conflicts of interest.

Expression and functional characterization of PGRP6 splice variants in grass carp Ctenopharyngodon idella

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved pattern recognition receptors from insects to mammals, recognize bacterial PGN and function in antibacterial innate immunity. The existence of alternative splicing is a common feature for PGRP family. Here the splicing pattern from the splicing at the 5′ end of PGRP6 gene was identified in a teleost fish, the grass carp (Ctenopharyngodon idella). Four splice variants of grass carp PGRP6 were designated as gcPGRP6a, gcPGRP6b, gcPGRP6c and gcPGRP6d, respectively. Real-time PCR revealed the different expression of these variants in fish individuals and CIK cell line in response to stimulation with different microbial ligands. Immunofluorescence microscopy and Western blotting showed that the splice variants are intracellular protein. Cell lysates from Epithelioma papulosum cyprini (EPC) cells transfected with gcPGRP6 splice variants are able to bind microbial PAMPs including Lys-type PGN from Staphylococcus aureus, DAP-type PGN from Bacillus subtilis, glucan, mannan, and microorganisms including Streptococcus dysgalactiae, Flavobacterium columnare and Saccharomyces cerevisiae. Moreover, overexpression of gcPGRP6 variants inhibited earlier stage growth of intracellular bacteria. The data also identified a specific role for gcPGRP6c variant in the positive regulation of cytolytic molecule perforin, and for gcPGRP6a, gcPGRP6b and gcPGRP6c variants in positive regulation of antimicrobial peptides (AMPs). However, the gcPGRP6d variant, which encoded basically only the PGRP domain, failed to induce the expression of perforin and AMPs. It is suggested that fish PGRP6 splice variants have common and variant-specific function in innate immune response.

Evaluation of tumor response to cytokine-induced killer cells therapy in malignant solid tumors.

CIK cells therapy has been evaluated as an adoptive cell immunotherapy for cancer patients, but there still have not been any standardized systems for evaluating the antitumor efficacy yet. The WHO and RECIST criteria have already been established for a few years but not sufficient to fully characterize the activity of immunotherapy. Based on these two criteria, the irRC was proposed for evaluating the efficacy of immunotherapy. A variety of bioassays for immune monitoring including the specific and non-specific methods, have been established. We recommend detect levels of various immunocytes, immune molecules and soluble molecules to find the correlations among them and clinicopathological characteristics to establish criteria for immunological classification. We also recommend a paradigm shift for the oncologists in the evaluation of immune therapies to ensure assessment of activity based on clinically relevant criteria and time points.

Global transcriptome-wide analysis of CIK cells identify distinct roles of IL-2 and IL-15 in acquisition of cytotoxic capacity against tumor.

BackgroundCytokine-induced killer (CIK) cells are an emerging approach of cancer treatment. Our previous study have shown that CIK cells stimulated with combination of IL-2 and IL-15 displayed improved proliferation capacity and tumor cytotoxicity. However, the mechanisms of CIK cell proliferation and acquisition of cytolytic function against tumor induced by IL-2 and IL-15 have not been well elucidated yet.MethodsCIKIL-2 and CIKIL-15 were generated from peripheral blood mononuclear cells primed with IFN-γ, and stimulated with IL-2 and IL-15 in combination with OKT3 respectively. RNA-seq was performed to identify differentially expressed genes, and gene ontology and pathways based analysis were used to identify the distinct roles of IL-2 and IL-15 in CIK preparation.ResultsThe results indicated that CIKIL-15 showed improved cell proliferation capacity compared to CIKIL-2. However, CIKIL-2 has exhibited greater tumor cytotoxic effect than CIKIL-15. Employing deep sequencing, we sequenced mRNA transcripts in CIKIL-2 and CIKIL-15. A total of 374 differentially expressed genes (DEGs) were identified including 175 up-regulated genes in CIKIL-15 and 199 up-regulated genes in CIKIL-2. Among DEGs in CIKIL-15, Wnt signaling and cell adhesion were significant GO terms and pathways which related with their functions. In CIKIL-2, type I interferon signaling and cytokine-cytokine receptor interaction were significant GO terms and pathways. We found that the up-regulation of Wnt 4 and PDGFD may contribute to enhanced cell proliferation capacity of CIKIL-15, while inhibitory signal from interaction between CTLA4 and CD80 may be responsible for the weak proliferation capacity of CIKIL-2. Moreover, up-regulated expressions of CD40LG and IRF7 may make for improved tumor cytolytic function of CIKIL-2 through type I interferon signaling.ConclusionsThrough our findings, we have preliminarily elucidated the cells proliferation and acquisition of tumor cytotoxicity mechanism of CIKIL-15 and CIKIL-2. Better understanding of these mechanisms will help to generate novel CIK cells with greater proliferation potential and improved tumor cytolytic function.

Molecular cloning and functional characterization of the NFIL3/E4BP4 transcription factor of grass carp, Ctenopharyngodon idella

NFIL3 (nuclear factor interleukin 3-regulated) is an important bZIP transcription factor in the immune response and immune cells' development. Here, we identified the NFIL3 gene from grass carp (Ctenopharyngodon idella; gcNFIL3). The deduced amino acid sequence of gcNFIL3 is 468 residues with a typical bZIP domain. Phylogenetics demonstrated that gcNFIL3 clustered closely with NFIL3 of zebrafish. Real-time PCR revealed gcNFIL3 is constitutively expressed in all tissues examined. Its expression was significantly upregulated in head kidney and trunk kidney after stimulation by bacteria. Immunofluorescence microscopy revealed that gcNFIL3 is mainly expressed in the nucleus. Overexpression of gcNFIL3 reduces Aeromonas hydrophila invasion and proliferation. In CIK cells, gcNFIL3 could induce the activation of NF-kappa B and upregulates the expression of IL10 and IFN. These results indicated that gcNFIL3 has immunoregulatory properties and might play a role in the immune response of fish.