Global transcriptome-wide analysis of CIK cells identify distinct roles of IL-2 and IL-15 in acquisition of cytotoxic capacity against tumor
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Cytokine-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.
CIKIL-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.
The 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.
Through 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.
KeywordsCIK cells Interleukin 2 Interleukin 15 Deep sequencing Transcriptome
Cancer is still a leading cause of diseases related death all over the world. It was estimated that 7.6 million people were dead from various types of cancer in 2008, and the figure will continue to rise to 13.1 million in 2030 . Fortunately, significant progress has been made to develop better approaches to prevent, diagnose and treat cancer in the past several years. These advances have made more people survive with their cancer today. However, these new approaches are not completely effective to all of cancers, and side effects were brought by some of treatments. Among these advances, immunotherapy has shown its large potential in cancer therapy. Cytokine-induced killer (CIK) cells, a subset of T lymphocytes with a natural killer T cell phenotype, have been proven to be effective to most of tumors in vitro and in vivo . CIK cells exhibit potent cytolytic activities against tumor cells with minimal adverse effects. CIK cells are prepared from peripheral blood mononuclear cells (PBMCs) by priming with IFN-γ, and maintained with monoclonal antibody against CD3 (OKT3) and interleukin-2 in the following days . During the generation of CIK cells, monoclonal antibody against CD3 provided mitogenic signals to T lymphocytes. Priming with IFN-γ is to activate the monocytes which provide contact-dependent (CD58/LFA-3) and soluble (IL-12) crucial signals promoting generation of autophagy and antigen cross-presentation . In following bulk culture, IL-2 promotes T cell proliferation, survival and acquisition of cytolytic effector function.
IL-15 is a cytokine which stimulate growth of NK, NKT cells and activated T lymphocytes in peripheral, and it has similar biological properties with IL-2 in innate immunity . Studies have suggested that IL-15 bind to subunits of IL-2 receptor and common gamma chain . Because IL-15 and IL-2 share common signaling components, they mediate a series of similar signaling events. These events include activation of the Janus kinase (Jak) and STAT pathways. The two cytokines both can facilitate the induction of tumor toxic effector T cells and proliferation of NK cells. However, IL-15 and IL-2 are differed in their cDNA/protein sequence and contribute differently to T cell-mediated immune response . Although IL-2 is a growth and survival factor, it plays important role in Fas-mediated activation-induced cell death (AICD) of CD4 T cell. In contrast, IL-15 promotes the survival of T lymphocytes by inhibiting IL-2-mediated CD4+ T cell AICD .
In our previous study, we have shown that CIK cells stimulated with combination of IL-2 and IL-15 exhibited enhanced cytotoxic capacity against lung cancer both in vitro and in vivo. Interestingly, we found that CIK cells activated with IL-2 and IL-15 could up-regulate the expression levels of IFN-γ and TNF-α in vivo compared to CIK cell stimulated with IL-2 alone . In order to identify the roles of IL-2 and IL-15 during induction of tumor toxic function of CIK cells, we performed comparative transcriptome analysis between CIK cells prepared with IL-15 and IL-2 respectively by Ion PI mRNA sequencing (RNA-seq) for the first time. The mRNAs isolated from CIKIL-15 cells and CIKIL-2 cells were transcribed into cDNAs which were applied to deep sequencing. The results of RNA-seq were analyzed by a series of bioinformatic methods including mapping, gene differential expression analysis, gene ontology (GO) and pathway analysis. Our finding will provide evidence for optimizing the CIK cell propagation strategy which produces more effective CIK cells against tumor.
Cell lines and reagents
Human lung adenocarcinoma (SPC-A-1 cells) and gastric tumor cells (BGC823) were obtained from Chinese Type Culture Collection (Shanghai, PR China). FITC conjugated anti-CD56 antibody and R-phycoerythrin conjugated anti-CD3 antibody used in identifying CIK phenotypic markers were purchased from BD Biosciences. The cell viability assay kit (Cell Counting Kit-8) was purchased from Dojindo, Molecular Technologies. Reagents for CIK cells generation including OKT3, IFN-γ, IL-2 and IL-15 were from Miltenyi Biotec. Experiments involving human peripheral blood were reviewed and approved by Bioethics Committee of Yan’an Affiliated Hospital of Kunming Medical University. Written informed consents have been given from all volunteers participated in this study.
Generation of CIKIL-2 and CIKIL-15(Standard protocols)
The Bioethics Committee of Yan’an Affiliated Hospital of Kunming Medical University has approved the investigation protocols to draw blood from healthy volunteers after written informed consent for the purposes of preparation CIK cells against tumor and deep sequencing. CIK cells were prepared from PBMCs which were isolated by standard Ficoll separation. PBMCs were cultured in RPMI 1640 growth medium at a density of 5 × 106 cells/mL. The RPMI 1640 growth medium for CIK contained 10% FBS, 2% L-glutamine and antibiotics. The generation of CIK cells was primed by adding 1000 U/mL IFN-γ on day 0 and 100 ng/mL anti-CD3 antibody and 500 U/mL IL-2 or 10 ng/mL IL-15 within the following 15 days of culture. The CIK cells were propagated every 5 days with RPMI 1640 growth medium supplemented with anti-CD3 antibody and IL-2 or IL-15 respectively . The CIK cells were expanded for 15 days and analyzed every 5 days.
Cytotoxicity assay based on CCK-8
After co-culture with CIK cells for 48 hours, the cell viabilities of two tumor cells were determined by CCK-8 based methods. Briefly, 10uL of CCK-8 solution was added in each well, and the plates were incubated at 37°C for 2–4 hours. After incubation, the absorbance of each well was read by a spectrophotometer at 450 nm. Each sample for one treatment was calculated by values from 5 independent samples.
RNA extraction and quality control
Total RNA was extracted from each sample using TRIzol Reagent (Life technologies, USA) according to the protocol from manufacturer. The concentration of each sample was measured by NanoDrop 2000 (Thermo Scientific, USA). The quality was assessed by the Agilent2200 (Agilent, USA).
Whole transcriptome libraries preparation and deep sequencing
The sequencing library of each RNA sample was prepared by using Ion Total RNA-Seq Kit v2 according to the protocol provided by manufacturer (Life technologies, USA). Briefly, poly(A)-containing mRNA was purified from 5 ug total RNA with Dynabeads (Life technologies, USA). The mRNA was fragmented using RNaseIII and purified. The fragmented RNA was hybrized and ligated with Ion adaptor. The RNA fragments were reverse-transcribed and amplified to double-stranded cDNA. Then, the amplified cDNA was purified by magnetic bead based method, and the molar concentration was determined for each cDNA library. Emulsion PCR was performed using template of cDNA library. The Template-Positive Ion PITM Ion SphereTM Particles were enriched and loaded on the Ion PITM chip for sequencing.
Filtering raw reads and mapping
The raw reads ≥50 bp which passed filtering were used for mapping. We used the Masplicing as our RNA-seq data mapping analysis tool whose core program is Bowtie that can identify the exon-exon splicing immediately and accurately .
Identification of differentially expressed genes
We applied the DEseq to filter the differentially expressed genes for the CIKIL-15 and CIKIL-2 groups. After the statistical analysis, we selected the differentially expressed genes according to the FDR threshold (FDR < 0.05) .
GO analysis was applied to analyze the main function of the differential expression genes according to the Gene Ontology which is the key functional classification of NCBI [12, 13]. Generally, Fisher’s exact test and χ2 test were used to classify the GO category, and the false discovery rate (FDR) was calculated to correct the P-value, the smaller the FDR, the small the error in judging the p-value [14, 15]. The FDR was defined as , where N k refers to the number of Fisher’s test P-values less than χ2 test P-values. We computed P-values for the GOs of all the differential genes. The significant GO terms were defined as P value <0.05 and FDR <0.05. Concerning on the treatment of GO term redundancy, we have adopted strategy of filtering out terms by picking only one from each leaf-to-root path.
Similarly, pathway analysis was used to find out the significant pathway of the differential genes according to KEGG, Biocarta and Reactome [10, 16, 17]. Still, we turn to the Fisher’s exact test and χ2 test to select the significant pathway, and the threshold of significance was defined by P-value and FDR. The significant pathway was identified by P value <0.05 and FDR < 0.05. The enrichment was calculated like the equation above [18, 19, 20].
KEGG database has included metabolism, membrane transport, signal transduction, cell cycle pathways and information about interactions among them. The genes we have selected may involved in two or more signaling pathways. Because of the same genes in different pathways, overlappings between pathways were obvious. We picked the genes in enriched biological pathway and used Cytoscape for graphical representations of pathways .
Co-expression network analysis
For each pair of genes, we calculate the Pearson Correlation and choose the significant correlation pairs (FDR < 0.01) to construct the network . Within the network analysis, degree centrality is the most simplest and important measures of the centrality of a gene within a network that determine the relative importance. Degree centrality is defined as the link numbers one node has to the other . Moreover, to study some properties of the networks, k-cores in graph theory were introduced as a method of simplifying graph topology analysis .
Quantitative reverse-transcription PCR
All the qRT-PCR involved in this study was performed on the CFX96 Touch™ (BIORAD, USA). The first strand of cDNA was synthesized with adjusted concentration of RNA, and corresponding genes were amplified by employing EVA Green Supermix. All the primers used for qRT-PCR were obtained from GeneCopoeia (USA).
Enhanced cell proliferation capacity of CIKIL-15 and superior tumor toxic effect of CIK IL-2
Overview of sequencing data of RNA-seq analysis
Statistics of raw and mapped reads from RNA-seq analysis of CIK cells stimulated by IL-15 and IL-2 respectively
Mapped reads (Rate)
Unique mapping (Rate)
Differential gene expression profiles of CIKIL-15 and CIKIL-2and GO analysis
Up-regulated genes related with functions and phenotypes of CIK IL-15
3.61 × 10−4
7.28 × 10−3
Regulation of cell-cell adhesion; Wnt signaling pathway; immature T cell proliferation in thymus; positive regulation of focal adhesion assembly; T cell differentiation in thymus; cell differentiation; cell-cell signaling; negative regulation of apoptotic process; positive regulation of transcription, DNA-templated
Interleukin 21 receptor
2.53 × 10−4
5.53 × 10−3
Interleukin-21-mediated signaling pathway; natural killer cell activation; cytokine-mediated signaling pathway
E3 ubiquitin-protein ligase
2.02 × 10−3
2.82 × 10−2
Regulation of type I interferon production; positive regulation of type I interferon production; Notch signaling pathway; innate immune response; protein ubiquitination
Intercellular adhesion molecule 4
1.46 × 10−4
3.51 × 10−3
Cell adhesion; cell-cell adhesion; regulation of immune response
Platelet-derived growth factor D
2.38 × 10−5
8.11 × 10−4
Platelet-derived growth factor receptor signaling pathway; cellular response to amino acid stimulus; multicellular organismal development; regulation of peptidyl-tyrosine phosphorylation; positive regulation of cell division
Up-regulated genes related with functions and phenotypes of CIK IL-2
Cytotoxic T-lymphocyte-associated protein 4
1.64 × 10−3
2.38 × 10−2
Immune response; negative regulation of regulatory T cell differentiation; negative regulation of B cell proliferation; T cell costimulation; B cell receptor signaling pathway; cellular response to DNA damage stimulus; positive regulation of apoptotic process
1.11 × 10−3
1.75 × 10−2
Innate immune response; positive regulation of GMCSF biosynthetic process; positive regulation of T-helper 1 cell differentiation; T cell activation; regulation of interleukin-2 biosynthetic process; T cell costimulation
Tumor necrosis factor ligand superfamily member 10
7.02 × 10−18
4.45 × 10−15
Immune response; activation of cysteine-type endopeptidase activity involved in apoptotic process regulation of extrinsic apoptotic; signaling pathway in absence of ligand; apoptotic process; positive regulation of extrinsic apoptotic signaling pathway; positive regulation of release of cytochrome c from mitochondria; apoptotic signaling pathway; positive regulation of cysteine-type endopeptidase activity involved in apoptotic process; positive regulation of apoptotic process
2.32 × 10−6
1.18 × 10−4
Immune response; inflammatory response; immunoglobulin secretion; positive regulation of endothelial cell apoptotic process; B cell proliferation; positive regulation of interleukin-12 production; leukocyte cell-cell adhesion
Interferon regulatory factor 7
3.12 × 10−5
1.02 × 10−3
Innate immune response; inflammatory response; positive regulation of type I interferon-mediated signaling pathway; positive regulation of type I interferon production; toll-like receptor signaling pathway
Pathways analysis of CIKIL-15 and CIKIL-2
Differentially expressed genes act network
Gene co-expression network
Validation of representative genes by qRT-PCR
Although clinical trials of CIK cells in cancer therapy were widely performed in China, fewer studies on molecular mechanism of their anti-tumor function were observed [33, 34]. The pioneering work of CIK cells was performed by Schmidt-Wolf from Stanford. The authors indicated that CIK cells were a subset of non-MHC-restricted T cells expressing both CD3 and CD56, and CIK cells showed potent cytotoxicity against a variety of tumor cells . The efficiency of CIK cells preparation is dependent on T cell proliferation and cytolytic activity against tumor. To generate CIK cells with high quality, cytokines such as IL-1, IL-7, IL-15 and IL-12 have been employed instead of IL-2 or in combination with IL-2 . Of these cytokines, IL-15 is widely tested in CIK cells preparation against several tumor cells. In this study, the results have indicated that CIKIL-15 exhibit enhanced proliferation capacity than CIKIL-2, whereas, CIKIL-2 showed more efficient cytotoxic effect against tumor cells than CIKIL-15. Consistently, the results from transcriptome analysis have shown corelationship with their functional characteristics.
IL-15 is a pleiotropic cytokine which promote T cells and NK cells proliferation and survival [35, 36]. To better elucidate the mechanism of increased proliferation capacity induced by IL-15, we have found that Wnt 4 and PDGFD which were correlated with cell proliferation were up-regulated in CIKIL-15. Wnt signaling pathway is widely involved in cell proliferation and differentiation . It has been reported that Wnt agonist promoted mouse muscle cell proliferation, and specific silencing RNA knockdown of Wnt 4 significantly reduced muscle cell proliferation . Moreover, study showed that the expression of Wnt 4 was required for proliferation of cells in mouse coelomic epithelium . By pathway interaction analysis, we have found that Wnt signaling pathway is located at the center of the network, which got the most interactions with other pathways. Therefore, we suggested that Wnt signaling be one of the most important pathways which contributed to the improved proliferation capacity of CIKIL-15. Except for Wnt 4, PDGFD is also a proliferation promoting factor which regulates several cellular processes including cell proliferation, apoptosis and transformation . Over-expression of PDGFD in mouse or human breast cancer cell significantly increased cell proliferation while silencing PDGFD expression decreased proliferation and increased apoptosis . Studies have indicated that PDGFD promoted cell proliferation by increasing DNA binding capacity of NF-κB and down-regulation of PDGFD inhibit tumor invasion through inactivation of Notch-1 and NF-κB signaling . Therefore, the up-regulation of Wnt 4 and PDGFD may be responsible for enhanced cell proliferation of CIKIL-15.
Additionally, we also have found important evidence which may inhibit the proliferation of CIKIL-2. By differentiated expressed genes and gene act network analysis, we found that CTLA4 and CD80 were up-regulated in CIKIL-2. These two proteins can interact with each other to provide inhibitory signal during T cell activation . In the generation of CIKIL-2, OKT3 and IL-2 were sustainedly presented in the culture system. However, IL-2 mediated activation-induced cell death (AICD) occurred during the following culture . Consistently, our previous phenotypic study of CIKIL-2 have showed that the cells subset of CD3+CD28+ was increasing in the first several days while significantly decreased since the 7th day of culture (Data not shown). These results demonstrated that the interaction between CTLA4 and CD80 may lead to inactivation of CD3+CD28+ T cell and inhibit proliferation of CIKIL-2. The inhibitory signal from ligation of CTLA4 to CD80 is the negative feedback to IL-2 stimulation of CIK cells. Comprehensively, not only up-regulated Wnt 4 and PDGFD but also activation inhibitory signal from CTLA4 and CD80 in CIKIL-2 has resulted in the enhanced proliferation capacity of CIKIL-15. We suggest that supplement with cytokines or mAb which down-regulates the inhibitory signal from CTLA4 and CD80 facilitate proliferation of CIKIL-2 production.
The most important of characteristic of CIK cell is cytolytic activity against tumor. In vitro, CIKIL-2 cells have shown more efficient tumor cytotoxicity than CIKIL-15. The expression of CD40LG and IFR7 were up-regulated in CIKIL-2. CD40LG, which is the ligand of CD40, has shown great potentials in cancer therapy . It has been reported that CD40 is expressed in nearly all B cell malignancy and many solid tumors . The ligation of CD40 on the surface of tumor cells inhibits the growth of tumor and induces apoptosis . Besides CD40LG, IFN-β has also been found to play critical role in anti-tumoral immune response . Interestingly, Moschonas has indicated that stimulation of CD40 by its ligand has promoted the expression of IFN-β through the binding IRF7 to its promoter. IRF7 is a transcriptional factor which regulates the expression of type I interferon . Silencing of IRF7 pathways in breast cancer accelerated bone metastasis through immune escape . Thus CD40LG and IFR7 may work synergically to improve the tumor cytotoxic effect of CIKIL-2.
On the other hand, the expression of DTX4 was up-regulated in CIKIL-15 which positively regulated the production of type I interferon through NLRP4 . Moreover, the expression of IL-21R whose ligand is involved in natural killer cell was also increased in CIKIL-15. Paradoxically, the up-regulation of PDGFD in CIKIL-15 not only could promote the proliferation of CIKIL-15 cells but also promote tumor cells survival through cell and cell interaction in tumor cytotoxic assay. PDGFs are composed of four different polypeptide chains (PDGF A-D). It has been reported that PDGFD was deregulated in most of human malignancies with up-regulated expression in solid tumors . The factor interacts with PDGFR-β and activates downstream signaling phosphatidylinositol 3-kinase (PI3K)/AKT, resulted in tumor progression. Moreover, Li et al. reported that PDGFD is a potent transformation growth factor for NIH/3 T3 which increased the cell proliferation rate . We suggested that up-regulated PDGFD is a double-edged sword in CIKIL-15. Because it favored the proliferation of CIKIL-15 cells during preparation, while it may also promoted the survival and proliferation of tumor cells when CIK cells were in contact with tumor cells.
In this study, deep sequencing was performed to analyze the different gene expression profiles of CIKIL-2 and CIKIL-15 for the first time. By advanced bioinformatic analysis of DEGs, we found that cell proliferation promoting function was dominant in CIKIL-15 involving Wnt signaling pathway and focal cell adhesion. In CIKIL-2, type I interferon signaling pathway and cytokine-cytokine receptor interactions were dominant. Through 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.
We thank Dr. Zhu Xiaoyun for his constructive suggestions to this study. This work is supported by grants from National Natural Science Foundation of China (No. 81160267 and 81360245) and grants from the “Special and Joint Program” of Yunnan Province Science and Technology Department & Kunming Medical University.
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