TY - JOUR A1 - Bröker, Katharine A1 - Sinelnikov, Evgeny A1 - Gustavus, Dirk A1 - Schumacher, Udo A1 - Pörtner, Ralf A1 - Hoffmeister, Hans A1 - Lüth, Stefan A1 - Dammermann, Werner T1 - Mass Production of Highly Active NK Cells for Cancer Immunotherapy in a GMP Conform Perfusion Bioreactor JF - Frontiers in Bioengineering and Biotechnology N2 - 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 ex vivo, 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, IFN gamma production, and cytotoxicity in vitro. 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 anticancer 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 IFN gamma expression upon cultivation with K562 tumor cells and demonstrate a high cytotoxicity toward leukemic as well as solid tumor cell lines in vitro. Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation. KW - natural killer cells (NK cells) KW - cytotoxicity KW - tumor immunity KW - immunotherapy KW - perfusion bioreactor KW - GMP KW - mass production process Y1 - 2019 U6 - https://doi.org/10.3389/fbioe.2019.00194 SN - 2296-4185 VL - 7 PB - Frontiers Research Foundation CY - Lausanne ER - TY - JOUR A1 - Gutbier, Birgitt A1 - Schönrock, Stefanie M. A1 - Ehrler, Carolin A1 - Haberberger, Rainer A1 - Dietert, Kristina A1 - Gruber, Achim D. A1 - Kummer, Wolfgang A1 - Michalick, Laura A1 - Kuebler, Wolfgang M. A1 - Hocke, Andreas C. A1 - Szymanski, Kolja A1 - Letsiou, Eleftheria A1 - Lüth, Anja A1 - Schumacher, Fabian A1 - Kleuser, Burkhard A1 - Mitchell, Timothy J. A1 - Bertrams, Wilhelm A1 - Schmeck, Bernd A1 - Treue, Denise A1 - Klauschen, Frederick A1 - Bauer, Torsten T. A1 - Tönnies, Mario A1 - Weissmann, Norbert A1 - Hippenstiel, Stefan A1 - Suttorp, Norbert A1 - Witzenrath, Martin T1 - Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2 JF - Critical care medicine N2 - Objectives: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. Design: Controlled, in vitro, ex vivo, and in vivo laboratory study. Subjects: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. Interventions: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. Measurements and Main Results: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1(-/-) mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. Conclusions: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury. KW - acute lung injury KW - pneumococcal pneumonia KW - sphingosine kinase 1 KW - sphingosine-1-phosphate KW - sphingosine-1-phosphate receptor 2 Y1 - 2018 U6 - https://doi.org/10.1097/CCM.0000000000002916 SN - 0090-3493 SN - 1530-0293 VL - 46 IS - 3 SP - e258 EP - e267 PB - Lippincott Williams & Wilkins CY - Philadelphia ER -