Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro.

Objective: 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 in vitro. Methods: 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 in vitro from co­cultures with the glioma cell lines U­87 MG, U­87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell­selection 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, IL­21/15, IL­27/18/15 and IL-12/18/15). Results: 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 IL­12/18/15 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. Conclusion: According to our findings the ex vivo 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.

Transcriptomics uncovers substantial variability associated with alterations in manufacturing processes of macrophage cell therapy products.

Gene expression plasticity is central for macrophages' timely responses to cues from the microenvironment permitting phenotypic adaptation from pro-inflammatory (M1) to wound healing and tissue-regenerative (M2, with several subclasses). Regulatory macrophages are a distinct macrophage type, possessing immunoregulatory, anti-inflammatory, and angiogenic properties. Due to these features, regulatory macrophages are considered as a potential cell therapy product to treat clinical conditions, e.g., non-healing diabetic foot ulcers. In this study we characterized two differently manufactured clinically relevant regulatory macrophages, programmable cells of monocytic origin and comparator macrophages (M1, M2a and M0) using flow-cytometry, RT-qPCR, phagocytosis and secretome measurements, and RNA-Seq. We demonstrate that conventional phenotyping had a limited potential to discriminate different types of macrophages which was ameliorated when global transcriptome characterization by RNA-Seq was employed. Using this approach we confirmed that macrophage manufacturing processes can result in a highly reproducible cell phenotype. At the same time, minor changes introduced in manufacturing resulted in phenotypically and functionally distinct regulatory macrophage types. Additionally, we have identified a novel constellation of process specific biomarkers, which will support further clinical product development.