Abstract
BackgroundNatural killer (NK) cells are an emerging new tool for cancer immunotherapy. To develop NK cell therapeutics from peripheral blood mononuclear cells (PBMCs) of healthy donors, substantial expansion of primary NK cells is necessary because of the very low number of these cells in peripheral blood. In this study, we aimed to investigate the effect of various cytokine alone or combinations, in expanded NK cells and to analyze the synergetic effect of cytokine combinations.MethodsHuman NK cells were isolated from healthy donor PBMC. Purified NK cells were stimulated with single cytokines or combinations of IL-2, IL-15, IL-18, and IL-27. The expanded NK cells were characterized by flow cytometry, cytotoxicity assay, calcein AM assay and Western blot.ResultsWe investigated the synergistic effects of each cytokine, namely, IL-2, IL-15, IL-18, and IL-27, on human NK cells isolated from PBMCs of healthy donors and cultured for 21 days. We identified that IL-15/IL-18/IL-27-mediated activation of NK cells most potently increased NK cell proliferation, cytotoxicity, and IFN-ɣ secretion compared with the activation observed with other treatments, including IL-2, IL-15, and IL-15/IL-18. Additionally, the expression of DNAM-1, NKG2D, CD69, and natural cytotoxicity receptors (NCRs; NKp30 and NKp44) increased on day 21 compared to that on day 0, demonstrating the activation of NK cells. In vitro, expanded NK cells were highly cytotoxic against cancer cells, displaying increased perforin and granzyme B accumulation.ConclusionsTaken together, these results indicated that IL-27 can synergize on NK cell expansion and activation with IL-15 and IL-18. In addition, we described an improved culture method for ex vivo expansion of human NK cells with IL-15/IL-18/IL-27 stimulation and characterized the response of NK cells to this stimulation.
Highlights
Natural killer (NK) cells are an emerging new tool for cancer immunotherapy
Our results suggest that synergistic interactions between IL-15, IL-18, and IL-27 play a crucial role in CD3-CD56+ NK cell functions by improving NK cell cytotoxicity activity, which increases perforin granule accumulation and Interferon gamma (IFN-γ) production
Characterization of peripheral blood mononuclear cells (PBMCs) and NK cells in healthy donors In this study, we evaluated the characterization of NK cells derived from PBMCs from 7, 14, and 21-day cultures
Summary
Natural killer (NK) cells are an emerging new tool for cancer immunotherapy. To develop NK cell therapeutics from peripheral blood mononuclear cells (PBMCs) of healthy donors, substantial expansion of primary NK cells is necessary because of the very low number of these cells in peripheral blood. NK cells are effectors of the innate immune system involved in the clearance of tumor cells and virus-infected cells [2,3,4,5]. The effector function of NK cells is regulated by the balance between activating and inhibitory receptor signals [8]. NK cell inhibition is regulated by killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A, which recognize major histocompatibility complex (MHC) class I molecules on target cells [8]. MHC class I-deficient cancer or transformed cells are highly sensitive to NK cells. Because of these inherent functions of NK cells, they have attracted attention as promising immunotherapeutics for cancer and viral infections [10]
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