Bröker Katharina, Sinelnikov Evgeny, Gustavus Dirk, Schumacher Udo, Pörtner Ralf, Hoffmeister Hans, Lüth Stefan, Dammermann Werner
Center of Internal Medicine II, Brandenburg Medical School, University Hospital Brandenburg, Brandenburg, Germany.
Department of Anatomy and Experimental Morphology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Front Bioeng Biotechnol. 2019 Aug 13;7:194. doi: 10.3389/fbioe.2019.00194. eCollection 2019.
NK cells have emerged as promising candidates for cancer immunotherapy, especially due to their ability to fight circulating tumor cells thereby preventing metastases formation. Hence several studies have been performed to generate and expand highly cytotoxic NK cells , e.g., by using specific cytokines to upregulate both their proliferation and surface expression of distinct activating receptors. Apart from an enhanced activity, application of NK cells as immunotherapeutic agent further requires sufficient cell numbers and a high purity. All these parameters depend on a variety of different factors including the starting material, additives like cytokines as well as the culture system. Here we analyzed PBMC-derived NK cells of five anonymized healthy donors expanded under specific conditions in an innovative perfusion bioreactor system with respect to their phenotype, IFNγ production, and cytotoxicity . Important features of the meander type bioreactors used here are a directed laminar flow of medium and control of relevant process parameters. Cells are cultivated under "steady state" conditions in perfusion mode. Our data demonstrate that expansion of CD3 T cell depleted PBMCs in our standardized system generates massive amounts of highly pure (>85%) and potent anti-cancer active NK cells. These cells express a variety of important receptors driving NK cell recruitment, adhesion as well as activation. More specifically, they express the chemokine receptors CXCR3, CXCR4, and CCR7, the adhesion molecules L-selectin, LFA-1, and VLA-4, the activating receptors NKp30, NKp44, NKp46, NKG2D, DNAM1, and CD16 as well as the death ligands TRAIL and Fas-L. Moreover, the generated NK cells show a strong IFNγ expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines . Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation.
自然杀伤（NK）细胞已成为癌症免疫治疗的有前景的候选者，特别是由于它们具有对抗循环肿瘤细胞从而防止转移形成的能力。因此，已经进行了多项研究来生成和扩增高细胞毒性的NK细胞，例如通过使用特定的细胞因子来上调它们的增殖以及不同激活受体的表面表达。除了增强的活性外，将NK细胞用作免疫治疗剂还进一步需要足够的细胞数量和高纯度。所有这些参数取决于多种不同因素，包括起始材料、细胞因子等添加剂以及培养系统。在这里，我们分析了来自五名匿名健康供体的外周血单个核细胞（PBMC）衍生的NK细胞，这些细胞在创新的灌注生物反应器系统中在特定条件下进行了扩增，分析了它们的表型、γ干扰素（IFNγ）产生和细胞毒性。这里使用的曲折型生物反应器的重要特征是培养基的定向层流和相关工艺参数的控制。细胞在灌注模式下的“稳态”条件下培养。我们的数据表明，在我们的标准化系统中扩增CD3 T细胞耗尽的PBMC可产生大量高纯度（>85%）且具有强效抗癌活性的NK细胞。这些细胞表达多种驱动NK细胞募集、黏附以及激活的重要受体。更具体地说，它们表达趋化因子受体CXCR3、CXCR4和CCR7，黏附分子L-选择素、淋巴细胞功能相关抗原-1（LFA-1）和极晚期抗原-4（VLA-4），激活受体自然杀伤细胞蛋白30（NKp30）、自然杀伤细胞蛋白44（NKp44）、自然杀伤细胞蛋白46（NKp46）、自然杀伤细胞激活受体2D（NKG2D）、DNAX辅助分子-1（DNAM1）和CD16，以及死亡配体肿瘤坏死因子相关凋亡诱导配体（TRAIL）和Fas配体（Fas-L）。此外，所产生的NK细胞在用K562肿瘤细胞培养时显示出强烈的IFNγ表达，并对白血病以及实体瘤细胞系表现出高细胞毒性。总之，这些特性预示着如此产生的NK细胞具有很高的临床效力，有待进一步评估。
