Presentation_1.PPTX

Katharina Bröker ,Evgeny Sinelnikov ,Dirk Gustavus ,Udo Schumacher ,Ralf Pörtner ,Hans Hoffmeister ,Stefan Lüth ,Werner Dammermann

doi:10.3389/fbioe.2019.00194.s001

Abstract

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, IFNγ 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 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 in vitro. Altogether, these characteristics promise a high clinical potency of thus produced NK cells awaiting further evaluation.

Highlights

Natural killer cells are innate effector lymphocytes which mediate potent immune responses against stressed, malignant as well as virus-infected cells
Up to now, according to the World Health Organization cancer is still the second leading cause of death worldwide and around 90% of all cancer-deaths are linked to the formation of distant metastases (Chaffer and Weinberg, 2011)
Since NK cells are known to control circulating tumor cells and by this mechanism prevent metastasis formation, NK cell immunotherapy is a promising approach for cancer treatment (Brodbeck et al, 2014; Krasnova et al, 2017)

Summary

Introduction

Natural killer cells are innate effector lymphocytes which mediate potent immune responses against stressed, malignant as well as virus-infected cells. The exocytosis pathway involves the release of cytotoxic granules containing perforin and granzymes as well as the secretion of pro-inflammatory cytokines including IFNγ which induce target cell lysis (Smyth et al, 2005). In this context, NK cell activation depends on a balance between signals generated from different germline-encoded activating as well as inhibitory receptors. The second pathway is mediated via interaction of death receptors on the target cell and their cognate ligands on the NK cell resulting in apoptosis of the target cell (Zamai et al, 1998; Smyth et al, 2001). The formed so called immunological synapse (IS) ensures the directed release of death-inducing substances and facilitates additional cell-cell interactions

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Results

Conclusion