Kuopio Center for Gene and Cell Therapy, Kuopio, Finland.
A.I. Virtanen Institute, Biotechnology and Molecular Medicine Unit, University of Eastern Finland, Kuopio, Finland.
Front Immunol. 2023 Sep 28;14:1227064. doi: 10.3389/fimmu.2023.1227064. eCollection 2023.
Natural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma .
NK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured from co‑cultures with the glioma cell lines U‑87 MG, U‑87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell‑selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL‑21/15, IL‑27/18/15 and IL-12/18/15).
The expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL‑12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15.
According to our findings the proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to this extent, and these findings could be used for upscaling clinical NK cell manufacturing.
自然杀伤 (NK) 细胞是先天免疫系统的一部分，是抵抗癌症的第一道防线。由于它们具有识别和杀死肿瘤细胞的天然机制，因此 NK 细胞被认为是异体即用型细胞免疫疗法的一种潜在选择。在这里，我们的目标是确定针对胶质母细胞瘤的基于细胞因子的、无饲养细胞的、激活和扩增 NK 细胞的最佳方案。
从人外周血中富集 NK 细胞，并使用不同的激活和细胞因子组合进行 16 天的扩增。根据 NK 细胞活力、功能、扩增率和纯度来评估扩增条件。通过与神经胶质瘤细胞系 U-87 MG、U-87 MG EGFR vIII、LN-229、U-118 和 DK-MG 的共培养来测量扩增的 NK 细胞的细胞毒性和脱颗粒作用。从最终 39 种不同条件中选择最佳的扩增方案：三步磁性细胞分选（去除 CD3+细胞、富集 CD56+细胞、去除 CD3+细胞后再富集 CD56+细胞）；四种激活方案（连续、预激活、再激活和增强）；和四种细胞因子组合（IL-2/15、IL-21/15、IL-27/18/15 和 IL-12/18/15）。
根据供体和扩增条件的不同，扩增率在 2-50 倍之间变化。从起始材料中进行连续的两步磁性细胞分选（去除 CD3+细胞和富集 CD56+细胞），并使用已知能产生细胞因子诱导的记忆样 NK 细胞的 IL-12/18/15 细胞因子进行预激活，可获得最佳的扩增率和纯度。通过再激活增强这些记忆样 NK 细胞的细胞毒性，减少供体差异。当在扩增的最后阶段用 IL-12/18/15 或 IL-21/15 增强时，产生最具细胞毒性的 NK 细胞。
根据我们的发现，NK 细胞的增殖能力和功能受到多种因素的影响，例如供体、起始材料的组成、细胞因子组合和激活方案。细胞因子改变了 NK 细胞的表型和功能，这在它们对神经胶质瘤细胞系的反应中表现明显。据我们所知，这是迄今为止进行的最全面的比较研究，这些发现可用于临床 NK 细胞生产的放大。
