Abstract
Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children. Despite intensive research in recent decades the prognosis for patients with metastatic or relapsed diseases has hardly improved. New therapeutic concepts in anti-tumor therapy aim to modulate the patient’s immune system to increase its aggressiveness or targeted effects toward tumor cells. Besides surgery, radiotherapy and chemotherapy, immune activation by direct application of cytokines, antibodies or adoptive cell therapy are promising approaches. In the last years, adoptive transfer of natural killer (NK) cells came into the focus of translational medicine, because of their high cytotoxic potential against transformed malignant cells. A main challenge of NK cell therapy is that it requires a high amount of functional NK cells. Therefore, ex vivo NK cell expansion protocols are currently being developed. Many culturing strategies are based on the addition of feeder or accessory cells, which need to be removed prior to the clinical application of the final NK cell product. In this study, we addressed feeder cell-free expansion methods using common γ-chain cytokines, especially IL-15 and IL-21. Our results demonstrated high potential of IL-15 for NK cell expansion, while IL-21 triggered NK cell maturation and functionality. Hence, we established a two-phase expansion protocol with IL-15 to induce an early NK cell expansion, followed by short exposure to IL-21 that boosted the cytotoxic activity of NK cells against RMS cells. Further functional analyses revealed enhanced degranulation and secretion of pro-inflammatory cytokines such as interferon-γ and tumor necrosis factor-α. In a proof of concept in vivo study, we also observed a therapeutic effect of adoptively transferred IL-15 expanded and IL-21 boosted NK cells in combination with image guided high precision radiation therapy using a luciferase-transduced RMS xenograft model. In summary, this two-phased feeder cell-free ex vivo culturing protocol combined efficient expansion and high cytolytic functionality of NK cells for treatment of radiation-resistant RMS.
Highlights
With their ability to detect and directly destroy virally infected or malignant cells, natural killer (NK) cells form an important part of the first line defense of the immune system
IL-15-Driven NK Cell Expansion Is Not Impaired by a Short-term Boost with IL-21
NK cells were isolated by an immunomagnetic negative selection and expanded ex vivo under feeder-cell free cultivation conditions addressing the impact of different cytokines
Summary
With their ability to detect and directly destroy virally infected or malignant cells, natural killer (NK) cells form an important part of the first line defense of the immune system. They can be activated rapidly via germ-line encoded receptors that recognize the presence of stress ligands or absence of self-antigens on target cells [1,2,3,4,5]. In vivo development and survival of NK cells require cytokines [6,7,8]. Direct injection of IL-2 has been shown to be accompanied by severe side effects, such as vascular leak syndrome, activation-induced cell death, and strong induction of regulatory CD4pos T cells, which did not occur after IL-15 administration [15, 16]
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