Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-lnduced Killer Cells
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Adoptive immunotherapy with cytokine induced killer (CIK) cells has shown antitumor activity against several kinds of cancer in preclinical models and clinical trials. CIK cells are a subset of ex vivo expanded T lymphocytes with T-NK phenotype and MHC-unrestricted antitumor activity. The literature provides scant information on cytokines, chemokines and growth factors secreted by CIK cells. Therefore, we investigated the secretory profile of CIK cells generated from tumor patients. The secretome analysis was performed at specific time points (d 1, d 14 and d 21) of CIK cell expansion. Mature CIK cells (d 21) produce a great variety of interleukins and secreted proteins that can be divided into three groups based on their secretion quantity: high (interleukin [IL]-13, regulated on activation normal T cell expressed and secreted [RANTES] chemokine, MIP-1α and 1β), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-γ, vascular endothelial growth factor [VEGF] and granulocyte-macrophage colony-stimulating factor [GM-CSF]) and low (IL-1β, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, eotaxin, platelet-derived growth factor-bb, basic fibroblast growth factor, G-CSF and monocyte chemoattractant protein [MCP]-1). Moreover, comparing peripheral blood mononuclear cells (PBMCs) (d 1) and mature CIK cells (d 14 and 21) secretomes, we observed that IL-5, IL-10, IL-13, GM-CSF and VEGF were greatly upregulated, while IL-1β, IL-6, IL-8, IL-15, IL-17, eotaxin, MCP-1 and RANTES were downregulated. We also performed a gene expression profile analysis of patient-derived CIK cells, showing that mRNA for the different cytokines and secreted proteins was modulated during PBMC-to-CIK differentiation. We highlight previously unknown secretory properties and provide, for the first time, a comprehensive molecular characterization of CIK cells. Our findings provide a rationale to explore the functional implications and possible therapeutic modulation of CIK secretome.
Adoptive immunotherapy with cytokine-induced cells holds promise as a new therapeutic approach in the setting of metastatic solid tumors refractory to standard treatments. Cytokine-induced killer (CIK) cells are heterogeneous ex vivo expanded T lymphocytes with mixed T-NK phenotype and endowed with wide MHC-unrestricted antitumor activity against both solid and hematologic malignancies (1, 2, 3, 4, 5, 6, 7). CIK cells can be easily expanded ex vivo up to clinical relevant rates from circulating peripheral blood mononuclear cells (PBMCs), according to a standard protocol involving timed stimulation with interferon (IFN)-γ (d 0), anti-CD3 moAb OKT3 (d 1) and interleukin (IL)-2 (from d 1 to the end) (8, 9, 10). The MHC-independent tumor-killing ability of CIK cells favors their possible clinical translation, as, in theory, they could be applied to all patients regardless their human leukocyte antigen haplotype. CIK cells have a T-NK mixed phenotype with variable rates of CD3+CD56+ cells, considered mainly responsible for the antitumor activity (1,11,12). CIK cells express some activating receptors shared with natural killer (NK) cells such as NKG2D, DNAX accessory molecule-1 (DNAM-1) and low levels of NKp30, while they do not express NKp44 and NKp46, inhibitory killer immunoglobulin-like receptors NKG2A and CD94 (13). The antitumor activity of CIK cells is mainly due to the NKG2D receptor intensely expressed on the membrane of CIK cells. The main ligands recognized by NKG2D are MHC class I-related molecules A and B (MIC A/B) and members of the unique long 16-binding proteins, stress-inducible proteins expressed by tumor cells of various origin (3,4,14, 15, 16, 17, 18). Recent clinical trials support their initial activity and excellent safety profile in challenging settings such as lung, renal, liver, breast and gastrointestinal cancers (19).
It is known that CIK cells have a predominant Th1 phenotype, with reported secretion of IFN-γ and tumor necrosis factor (TNF)-α (20,21), which are involved in regulating innate and adaptive immunities. The other positive regulatory cytokines that are secreted by CIK cells are IL-2 and IL-4 (20,21).
Comprehensive information on the secretory activity of CIK cells is limited and needs to be more deeply explored to improve our knowledge of CIK cell biology and possible clinical applications. Investigation of CIK cell secretome can provide novel insights into its physiological mechanisms as well as a better understanding of immunological processes in this context. CIK cell performance is positively or negatively modulated by both cell-to-cell interactions and soluble factors secreted by CIK cells themselves or other cells. T regulatory lymphocytes (Tregs) have been shown to impair CIK cell activity. It has been demonstrated that depletion of Tregs before starting the ex vivo culture improved CIK cell proliferation and tumor-killing activity (22). These effects were at least in part attributed to TGF-beta1 and glucocorticoid-induced tumor necrosis factor receptor (22). We hypothesize that other plasma membrane molecules or soluble factors have a role in modulation of CIK cell performance. It has been reported that IL-10 suppresses CIK cell activity and the co-culture of CIK cells with DC can reverse its effect (23).
In this study we explored the comprehensive secretory activity of patient-derived CIK cells, at both the protein and mRNA level. Furthermore, we conducted a dynamic analysis to highlight possible variations of different factors (cytokines, chemokines and growth factors) during the ex vivo expansion of CIK cells.
Materials and Methods
Ex Vivo Expansion and Phenotype Characterization of CIK Cells
CIK cells were expanded from peripheral blood collected from five patients with histologically confirmed gastrointestinal stromal tumors (GISTs) at the Candiolo Cancer Institute, Fondazione del Piemonte per L’Oncologia-IRCCS. All individuals provided informed consent for blood donation according to a protocol approved by the internal review board and ethics committee. As control for the cytokines secretion analysis, we also generated CIK cells from five healthy donors. Cryopreserved PBMCs were seeded at a concentration of 2 × 106 cells/mL according to the standard protocol (8,9), including 21 d of culture in RPMI-1640 medium (Gibco BRL Life Technologies Italia, Monza, Italy) supplemented with 10% fetal bovine serum (Sigma Aldrich, Milan, Italy), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco BRL Life Technologies Italia) at 37°C and 5% CO2, with the timed addition of IFN-γ (1000 U/mL on d 0), Ab anti-CD3 OKT3 (50 ng/mL on d 1) and IL-2 (300 U/mL on d 1 up to the end, refreshing the medium every 2–3 d) (all factors are from Miltenyi Biotec, Calderara di Reno, Bologna, Italy). At d 0, a small amount of each PBMC culture (7 × 106) was seeded at the same concentration and culture conditions (2 × 106 cells/mL in RPMI-1640 medium with 10% fetal bovine serum, 100 U/mL penicillin and streptomycin at 37°C and 5% CO2) but without the addition of INF-γ. Aliquots of culture medium (4.5 mL) and 3 × 106 of PBMCs were collected at d 1 for quantification of cytokines and RNA extraction, respectively. Culture medium was centrifuged to precipitate floating cells and stored at −30°C, while PBMCs were collected in Invitrogen™ TRIzol™ (Thermo Fisher Scientific, Milan, Italy) and stored at −80°C. The same procedure was repeated for each CIK cell culture at d 14 and 21.
The final expansion rate calculated on both bulk CIK cells and the CD3+CD56+ subset was determined. Phenotype of CIK cells was weekly analyzed starting from d 0 by standard flow cytometric assay. The following monoclonal antibodies (mAbs) were used: CD3-FITC, CD4−PE, CD56-APC, CD8−PE and CD314-APC (anti-NKG2D) (all mAbs were from Miltenyi Biotec). The Miltenyi Biotec Treg Detection kit (CD4/CD25/Foxp3) (APC) was used to detect T regulatory cells within CIK cultures at d 14 and 21. Labeled cells were read on FACS CyAn ADP (Beckman Coulter, Cassina De’ Pecchi, Milan, Italy) and analyzed using Summit software.
Quantification of Cytokines and CIK Cell Secretome
Cytokines released in culture medium by PBMCs (d 1, absence of INF-γ) and CIK cells (d 14 and 21) were measured by Bio-Plex cytokine assay (Bio-Rad Laboratories, Hercules, CA, USA) as described elsewhere (24). The Bio-Plex cytokine assay is designed for multiplexed quantitative measurement of different cytokines in a single well using as little as 50 µL of supernatants collected at three time points. To perform the experiments, we used premixed multiplex beads of the Bio-Plex human cytokine Human 27-Plex Panel (Bio-Rad Laboratories), which included 27 secreted proteins: cytokines, chemokines and growth factors (IL-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 p70, IL-13, IL-15, IL-17, fibroblast growth factor [FGF]-basic, eotaxin, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], IFN-γ, IP-10, monocyte chemoattractant protein [MCP]-1 [MCAF], macrophage inflammatory protein [MIP]-1α, MIP-1β, platelet-derived growth factor [PDGF]-bb, regulated on activation normal T cell expressed and secreted [RANTES] chemokine, tumor necrosis factor [TNF]-α and vascular endothelial growth factor [VEGF]). IL-2 detection was included to have an internal positive control. Briefly, 50 µL of cytokine standards or samples (supernatants from seeded cells) were incubated with 50 µL of anti-cytokine conjugated beads in 96-well plates for 30 min at room temperature (RT) with shaking. Plates were then washed three times with 100 µL of Bio-Plex wash buffer using the Bio-Plex Pro Wash Station (Bio-Rad Laboratories), then 25 µL of diluted detection antibody was added and plates were incubated for 30 min at RT with shaking. After three washes, 50 µL of streptavidinphycoerythrin was added, and the plates were incubated for 10 min at RT with shaking. Finally, plates were washed three times, beads were suspended in Bio-Plex assay buffer and samples were analyzed on Bio-Rad 96-plate reader using the Bio-Plex suspension array system and Bio-Plex manager software (Bio-Rad Laboratories) (24). Secretome of patient-derived CIK cells was compared with those of healthy donors to evaluate possible differences in the secretory pathways.
RNA Extraction and Secretome Gene Expression Profile
Total cellular RNA was isolated from 3 million PBMCs (d 1, absence of INF-γ) and CIK cells (d 14) obtained from 5 GIST patients, using Invitrogen TRIzol (Thermo Fisher Scientific). We performed a microarray analysis of CIK cells at d 14 instead of d 21 (mature CIK cells) to study gene expression changes during CIK cell maturation. Disposable RNA chips (Agilent RNA 6000 Nano LabChip kit; Agilent Technologies) were used to determine the purity/integrity of RNA samples with an Agilent 2100 Bioanalyzer. NanoDrop ND-1000 spectrophotometer was used to evaluate the RNA sample concentration, and 260/280 nm and 260/230 nm ratios were considered to verify the purity of total RNA. For microarray analysis, we selected the three RNA sample pairs with the best RNA integrity number. Briefly, cDNA synthesis and biotin-labeled target synthesis were performed using the Gene Atlas 3′ IVT Plus Reagent Kit according to the standard protocol supplied by Affymetrix. The HG-U219 Array Strip (Affymetrix, Santa Clara, CA, USA) hybridization, staining and scanning were performed using the GeneAtlas platform.
Gene expression profile data were analyzed with the Partek GS 6.6 software package and normalized using the robust multiarray average procedure (25). Differentially expressed genes (DEGs) were then selected using a supervised approach with the analysis of variance module included in the Partek GS package. In particular, we selected all the probe sets with a fold change contrast ≥2 in the pairwise comparison of CIK cells with PBMCs, and a false discovery rate (q-value) <0.05. Ingenuity Pathway Analysis (IPA) (https://doi.org/www.ingenuity.com) was used to study cellular processes and pathways modulated in CIK cells.
All microarray data were submitted to the Gene Expression Omnibus repository (https://doi.org/www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc = GSE97581).
Results are expressed as mean ± standard deviation. Comparisons between samples of d 14 and d 1 and between d 21 and d 1 were made by using nonparametric unpaired t test (Mann-Whitney). Differences between groups were considered significant when *p < 0.05 and highly significant when **p < 0.01.
All supplementary materials are available online at https://doi.org/www.molmed.org.
Ex Vivo Expansion and Phenotype of CIK Cells
Characteristics of patients and CIK cells.
CD3+CD56+ Day 0 (%)
CD3+CD56+ Day 14 (%)
CD3+CD56+ Day 21 (%)
CD3+ Day 21 (%)
CD3+CD8+ Day 21 (%)
CD3+NKG2D+ Day 21 (%)
Secretome of CIK Cells
Secretome Gene Expression Profile of CIK Cells
We investigated the molecular networks activated during the ex vivo expansion of CIK cells. We performed gene expression profiling of RNA obtained from CIK cells collected at specific time points: at d 1 (basal PBMCs) and at d 14 (CIK cells) (see Materials and Methods). PTs 2, 3 and 4 were selected for microarray analysis based on the best RNA integrity number.
Of note, among DEGs we identified other secreted molecules that were upregulated in mature patient-derived CIK cells, which could contribute to their tumor-killing activity: GZMA, GZMB, GZMK, PRF1, IL-32 and LTA (Supplementary Table S1). Furthermore, as shown in Supplementary Table S2, to better characterize the CIK cell phenotype, we studied the surface antigen expression. As expected, we found downregulation of several myeloid differentiation markers (CD14, CD9, CD93, CSF2RA, CSF2RB, EPB41L3, receptors for Fc fragments of immunoglobulins, ITGAX, MCEMP1 and TREM1) and B cell antigens (CD19, CD24, CD79A, CXCR5 and MS4A1). Moreover, microarray data confirmed upregulation in CIK cells of well-known surface antigens like CD3D, CD3G, IL-2Ra, IL-2RG, CD226/DNAM1, ITGAL/LFA-1, KLRK1/NKG2D, NCR3/NKp30 and TRAIL/TNFSF10.
Next, to identify differentially expressed pathways during CIK cell maturation, we performed a functional analysis by using IPA software (December 2016 release). Among inactivated functions in CIK cells, we found “chemotaxis of myeloid cells,” “phagocytosis,” “migration of granulocytes” and “engulfment of leukocytes” (Supplementary Table S3).
Immune cells play a fundamental role against cancer; however, in many cases tumor cells become able to circumvent the activity of innate and adoptive immune response (26). Cancer immunotherapy holds great potential against various forms of cancer (27,28), exploiting either antibody or cell-mediated responses. Among adoptive immunotherapy approaches, CIK cells represent an intriguing therapeutic option, since they are endowed with intense MHC-independent antitumor activity against both hematopoietic and solid tumors (9,29, 30, 31, 32).
Identification of cytokines secreted by CIK cells during their differentiation sheds light on mediators that could positively or negatively modulate tumor growth by acting at different levels. An obvious impact of the study is that it provides a rationale to explore the functional implications and possible therapeutic modulation of CIK secretome.
To unravel CIK secretory properties, we undertook a thorough investigation, starting from the first stages of differentiation and ending at d 21, which is the time point of infusion of CIK cells in patients with cancer. We found that IL-5, IL-10, IL-13, GM-CSF and VEGF production greatly increased during differentiation, whereas IL-4, IL-9 and IL-12 cytokine secretion levels changed minimally. On the contrary, IL-1β, IL-6, IL-8, IL-15, IL-17, eotaxin, MCP-1, IL-1Ra, G-CSF, FGF-basic and PDGF-bb were downregulated over the same time.
GM-CSF mediates a wide variety of effects on cells of the immune system (37). Although GM-CSF is among the growth factors known to promote the expansion of immunosuppressive myeloid-derived suppressor cells (MDSCs), thus promoting tumor growth and metastatic dissemination (38), many reports have also shown relevant positive effects of GM-CSF for immunotherapy (39,40). These include T cell activation, maturation of dendritic cells and stimulation of humoral and cell-mediated responses (41). Data obtained in mice, using herpes simplex virus armed with GM-CSF, have shown its efficacy against pancreatic cancer (42). Preclinical and clinical studies have shown GM-CSF antitumor activity against melanoma, either alone or in association with antimelanoma vaccines (43,44). GM-CSF represents a successful choice in association with anti-GD2 monoclonal antibodies, also for patients with high-risk neuroblastoma (39).
An interesting observation from CIK cell secretome relates to the very low production of IL-6. Indeed, IL-6 is gaining attention for its role in tumor biology. It has recently been found in tissue samples of human prostate obtained by needle biopsy (45), and an integrated gene and miRNA expression analysis of prostate cancer-associated fibroblasts supports a prominent role for IL-6 in fibroblast activation (46). Moreover, IL-6-mediated signaling in hepatocellular carcinoma has been considered crucial for blocking initiation and malignant growth of this neoplastic disease by the anticancer agent icaritin (47). A protective role in hepatocellular carcinoma has been shown for chemerin, also known as retinoic acid receptor responder protein 2, which inhibits IL-6 and GM-CSF expression and MDSC accumulation (48). IL-6 is also among those cytokines recently identified as tumor-derived factors inducing CD38 expression in ex vivo MDSCs. Interestingly, highly expressing CD38 MDSCs have an elevated ability to suppress activated T cells and promote tumor growth (42).
Our analysis shows that human CIK cells secrete another crucial cytokine that has both positive and negative effects depending on tissue context and conditions. IL-10 exerts positive homeostatic effects by downmodulating global immune response, thus preventing tissue damage and chronic inflammation; however, many reports have shown that IL-10 impairs cytotoxic responses of immune cells against tumors (49). Accordingly, elevated IL-10 concentration in serum and cerebrospinal fluid has been linked to poor prognosis in different tumors (50, 51, 52, 53), and inhibition of IL-10-mediated signaling increases T cell infiltration and responses against mouse tumors (54). However, recent findings demonstrated that IL-10 in combination with oncolytic virotherapy can enhance pancreatic cancer rejection (55).
Another cytokine playing a role in tumor biology is IL-13. Besides CIK cells, IL-13 is secreted by a variety of cell types, including T helper type 2 lymphocytes, mast cells, basophils, eosinophils, dendritic cells and CD8+ T lymphocytes (56). It is released upon stimulation by proteases or allergens, thus inducing eosinophilic inflammation and immunoglobulin E class switching in B cells (57). In monocytes and macrophages, IL-13 inhibits the production of prostaglandins, reactive oxygen, nitrogen intermediates and proinflammatory cytokines, among them IL-1, IL-6, IL-8, TNF-α and IL-12 (58). It has been shown that IL-13 exerts multiple effects on tumor cells. Hence, it favors growth of cutaneous T cell lymphoma and its concentration increase correlates with the number of MDSCs in pancreatic, esophageal and gastric cancer. Accordingly, targeting of the IL13Ralpha2 subunit of IL-13R suppresses breast cancer lung metastasis in mice (59,60). Our study shows that IL-13 is highly produced by CIK cells, therefore it would be worthwhile to study in depth the repercussions of CIK-secreted IL-13 on in vitro and in vivo tumor growth. Chemokines play multiple roles in cancer biology and recruitment of cancer responsive immune cells. We also showed that CIK cells produce VEGF during differentiation, giving rise to questions on potential side effects (ie, favor tumor survival and dissemination). The role of VEGF in cancer angiogenic activity has been deeply investigated, showing that it represents a powerful way for tumors to induce formation of new vessels by both autocrine and paracrine stimulation, thus favoring tumor metastatic dissemination (61). Although the effects of anti-VEGF-based therapy can be transitory (62), VEGF still represents a target for pharmacologically controlling and inhibiting neoangiogenesis and metastatic diffusion (63,64), therefore the previously unknown capacity of CIK cells to secrete this pro-angiogenic factor is relevant (65).
In this report we analyzed the secretory panel of human CIK cells laying a foundation for future studies on modulation of CIK secretome. This will hopefully represent a new way to increase their performance against neoplastic cells and/or contribute to the establishment of a micro-environment of unfavorable conditions for cancer growth.
The authors have no competing interests as defined by Molecular Medicine or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported in part by FPRC ONLUS 5 × 1000, Ministero della Salute 2012; Associazione Italiana per la Ricerca sul Cancro (AIRC), project no. 10005 Special Program Molecular Clinical Oncology 5 × 1000 to AIRC-Gruppo Italiano Malattie Mieloproliferative, AIRC project no. 15337. DS is the recipient of a grant from Ricerca Finalizzata-Giovani Ricercatori Ministero della Salute (GR-2011-02349197).
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