Dataset Information

Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells.

ABSTRACT: Adoptive immunotherapy with Cytokine Induced Killer (CIK) cells has shown antitumor activity against several kinds of cancers 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. Literature provides scanty 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 (day 1, day 14 and day 21) of CIK cells expansion. Mature CIK cells (day 21) produce a great variety of interleukins and secreted proteins that can be divided into 3 groups based on their secretion quantity: high (IL-13, RANTES, MIP-1? and 1?), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-?, VEGF and GMCSF) and low (IL-1?, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, Eotaxin, PDGF-bb, FGF basic, G-CSF and MCP-1) secreted. Moreover, comparing PBMC (day 1) and mature CIK cells (day 14 and 21) secretome, we observed that IL-5, IL-10, IL-13, GM-CSF, VEGF resulted greatly up-regulated, while IL-1?, IL-6, IL-8, IL-15, IL-17, eotaxin, MCP-1, and RANTES were down-regulated. We also performed a gene expression profile analysis of patient-derived CIK cells showing that mRNA for the different cytokines and secreted proteins were modulated during PBMC to CIK differentiation. We highlighted previously unknown secretory properties and provided for the first time a comprehensive molecular characterization of CIK cells. Our findings provide rationale to explore the functional implications and possible therapeutic modulation of CIK secretome.

SUBMITTER: Mesiano G

PROVIDER: S-EPMC5630476 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells.

Mesiano Giulia G Zini Roberta R Montagner Giulia G Bianchi Nicoletta N Manfredini Rossella R Chillemi Antonella A Aglietta Massimo M Grignani Giovanni G Lampronti Ilaria I Fiorino Erika E Malavasi Fabio F Sangiolo Dario D Gambari Roberto R Ferrari Davide D

Molecular medicine (Cambridge, Mass.) 20170809

Adoptive immunotherapy with Cytokine Induced Killer (CIK) cells has shown antitumor activity against several kinds of cancers 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. Literature provides scanty 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 ...[more]

PMID: 28805233

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Project description:Background & aimsCytokine-induced killer (CIK) cell-based immunotherapy is effective as an adjuvant therapy in early stage hepatocellular carcinoma (HCC) but lacks efficacy in advanced HCC. We aimed to investigate immune suppressor mechanisms in HCC, focusing on the role of myeloid-derived suppressor cells (MDSCs) in response to CIK therapy.MethodsMDSCs were quantified by flow cytometry and quantitative real-time PCR. Cytokines were detected by cytokine array. A lactate dehydrogenase cytotoxicity assay was performed in the presence or absence of MDSCs to study CIK function against HCC cells in vitro. An FDA-approved PDE5 inhibitor, tadalafil, was used to target MDSCs in vitro and in vivo. Two different murine HCC cell lines were tested in subcutaneous and orthotopic tumor models in C57BL/6 and BALB/c mice. The antitumor effects of human CIKs and MDSCs were also tested in vitro.ResultsAdoptive cell transfer of CIKs into tumor-bearing mice induced inflammatory mediators (e.g., CX3CL1, IL-13) in the tumor microenvironment and an increase of tumor-infiltrating MDSCs, leading to impaired antitumor activity in 2 different HCC models. MDSCs efficiently suppressed the cytotoxic activity of CIKs in vitro. In contrast, treatment with a PDE5 inhibitor reversed the MDSC suppressor function via ARG1 and iNOS blockade and systemic treatment with a PDE5 inhibitor prevented MDSC accumulation in the tumor microenvironment upon CIK cell therapy and increased its antitumor efficacy. Similar results were observed when human CIKs were tested in vitro in the presence of CD14+HLA-DR-/low MDSCs. Treatment of MDSCs with a PDE5 inhibitor suppressed MDSC suppressor function and enhanced CIK activity against human HCC cell lines in vitro.ConclusionOur results suggest that targeting MDSCs is an efficient strategy to enhance the antitumor efficacy of CIKs for the treatment of patients with HCC.Lay summaryCytokine-induced killer cells are a mixture of immune cells given to eliminate cancer cells. However, not all patients respond to this treatment. Herein, we show in 2 different liver cancer models that myeloid-derived suppressor cells are increased in response to cytokine-induced killer cell therapy. Targeting these myeloid-derived suppressor cells may provide an additional therapeutic benefit alongside cytokine-induced killer cell therapy.

Project description:Cytokine Induced Killer (CIK) cells are heterogeneous ex vivo expanded T lymphocytes with mixed T-NK phenotype and endowed with a wide MHC-unrestricted antitumor activity against both solid and hematologic malignancies. CIK cells can be easily ex vivo expanded up to clinical relevant rates from circulating peripheral blood mononuclear cells (PBMC), according to a standard protocol involving timed stimulation with IFN- γ (day 0), Ab-antiCD3 OKT3 (day 1) and IL-2 (from day 1 to the end). Nowadays literature provides scanty information on cytokines, chemokines and growth factors secreted by CIK cells. A comprehensive molecular profiling of CIK cells is needed to complete the knowledge of their biologic and functional features. In this perspective we performed a gene expression profile analysis of basal PBMC and ex vivo expanded CIK cells generated from 3 patients with histologic confirmed Gastrointestinal Stromal Tumors (GIST). The principal component analysis (PCA) shows that the CIK cell samples clustered together and were clearly separated from PBMC ones. First, we checked the mRNA expression levels of secreted proteins and we found a significant upregulation of GM-CSF, IL-5, IL-13, and IFN-γ in CIK cells. Moreover, IL-1Ra, IL-1β, IL-6, IL-10, IL-15, IL-8, Eotaxin and MCP-1 are downregulated in CIK cells if compared to PBMC. Noteworthy, among differentially expressed genes (DEGs) we identified other secreted molecules that are upregulated in patient-derived CIK cells, which can contribute to their tumor killing activity: GZMA, GZMB, GZMK, PRF1, IL-32, and LTA. Furthermore, to better characterize the CIK cell phenotype, we studied the surface antigen expression. As expected, we found the 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 the upregulation in CIK cells of well-known surface antigens, like CD3D, CD3G, IL2Ra, IL2RG, CD226/DNAM1, ITGAL/LFA-1, KLRK1/NKG2D, NCR3/NKp30, and TRAIL/TNFSF10. Next, in order to identify differentially expressed pathways during CIK cells maturation, we performed a functional analysis by using Ingenuity Pathway Analysis (IPA) software. Among inactivated functions in CIK cells, we found chemotaxis of myeloid cells, phagocytosis, migration of granulocytes and engulfment of leukocytes. Noteworthy, functional gene categories as proliferation of cells, cell death of lymphocytes, cell death of mononuclear leukocytes, apoptosis of B lymphocytes, and quantity of CD4+ T-lymphocytes resulted activated, in agreement with the selection and expansion of CIK cell precursors induced by in vitro treatment of PBMC. Finally, IPA analysis predicted as activated categories cytotoxicity of lymphocytes, cytotoxicity of cells, and cytotoxicity of natural killer cells. In particular, genes involved in cytotoxic mechanism of CIK cells like NCR3/NKp30, KLRK1/NKG2D, TRAIL/TNFSF10, CD226/DNAM1, CD244, CD69, CD96, GZMA, GZMB, PRF1 are differentially expressed. With this work we highlighted previously unknown secretory properties and provided for the first time a comprehensive molecular characterization of CIK cells, opening the possibility to operate on secretory level to improve CIK cell cytotoxic activity against tumors.

Dataset Information

Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells.

Publications

Analytic and Dynamic Secretory Profile of Patient-Derived Cytokine-Induced Killer Cells.

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