Wagner Juliane, Pfannenstiel Viktoria, Waldmann Anja, Bergs Judith W J, Brill Boris, Huenecke Sabine, Klingebiel Thomas, Rödel Franz, Buchholz Christian J, Wels Winfried S, Bader Peter, Ullrich Evelyn
Children's Hospital, Goethe University, Frankfurt am Main, Germany.
Division for Stem Cell Transplantation and Immunology, Department for Children and Adolescents Medicine, Hospital of the Goethe University Frankfurt, Frankfurt am Main, Germany.
Front Immunol. 2017 Jun 12;8:676. doi: 10.3389/fimmu.2017.00676. eCollection 2017.
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, 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 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 culturing protocol combined efficient expansion and high cytolytic functionality of NK cells for treatment of radiation-resistant RMS.
横纹肌肉瘤（RMS）是儿童最常见的软组织恶性肿瘤。尽管近几十年来进行了深入研究，但转移性或复发性疾病患者的预后几乎没有改善。抗肿瘤治疗的新治疗理念旨在调节患者的免疫系统，以增强其对肿瘤细胞的攻击性或靶向作用。除了手术、放疗和化疗外，直接应用细胞因子、抗体或过继性细胞疗法激活免疫是很有前景的方法。近年来，自然杀伤（NK）细胞的过继性转移成为转化医学的焦点，因为它们对转化的恶性细胞具有高细胞毒性潜力。NK细胞治疗的一个主要挑战是它需要大量功能性NK细胞。因此，目前正在开发NK细胞扩增方案。许多培养策略基于添加饲养细胞或辅助细胞，在最终NK细胞产品临床应用前需要将其去除。在本研究中，我们探讨了使用共同γ链细胞因子，特别是IL-15和IL-21的无饲养细胞扩增方法。我们的结果表明IL-15在NK细胞扩增方面具有很高的潜力，而IL-21可触发NK细胞成熟和功能。因此，我们建立了一个两阶段扩增方案，先用IL-15诱导早期NK细胞扩增，然后短期暴露于IL-21以增强NK细胞对RMS细胞的细胞毒性活性。进一步的功能分析显示脱颗粒增强以及促炎细胞因子如干扰素-γ和肿瘤坏死因子-α的分泌增加。在一项概念验证研究中，我们还观察到过继性转移的经IL-15扩增和IL-21增强的NK细胞与使用荧光素酶转导的RMS异种移植模型的图像引导高精度放射治疗联合应用时的治疗效果。总之，这种两阶段无饲养细胞培养方案结合了NK细胞的高效扩增和高细胞溶解功能，用于治疗抗辐射的RMS。
