SoonHo Kweon

Background: Natural Killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. Here, we report a new effective method to expand human NK cells ex vivo using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in combination with a short exposure to soluble IL-21. In addition, we describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction. Methods: K562-OX40L cells were generated by lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Soluble IL-21 was also added in various concentrations only once at the beginning of the culture. NK cells were expanded for 4 to 5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, CD107a degranulation) of these expanded NK cells were compared to those generated by using K562 feeder cells. Results: The culture of NK cells with K562-OX40L cells in combination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis between the feeder cells and NK cells as well as the homotypic interaction between NK cells through the OX40-OX40L axis were both necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and CD107 degranulation. Conclusion: Our data suggest that OX40 ligand is a potent co-stimulant for the robust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.

Background: Measurement of natural killer (NK) cell function has important clinical utility in several diseases. Although the flow cytometry (FC)-based 4-h NK cytotoxicity assay using peripheral blood mononuclear cells (PBMCs) in the clinical laboratory has been used for this purpose, this assay requires large amounts of blood and a rapid PBMC isolation step. Here, we developed an FC-based overnight NK cytotoxicity assay using whole blood (WB), and applied it to patients with liver diseases. Methods: Peripheral blood of healthy volunteers (n = 28) and patients with liver diseases, including hepatocellular carcinoma (n = 19) and liver cirrhosis (n = 7), was analyzed for complete blood count, absolute NK cell count, and NK cell activity (NKA). NKA was evaluated in three assay types: an FC-based overnight WB NK cytotoxicity assay using carboxyfluorescein diacetate succinimidyl ester-labeled K562 cells in the presence of various cytokine combinations (including interleukin (IL)-2, IL-18, and IL-21), an FC-based 4-h PBMC NK cytotoxicity assay, and an FC-based CD107a degranulation assay using WB and PBMCs. Results: Optimal cytokine combinations for NK cell activation in WB were determined (IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21). A good correlation was observed between WB and PBMC NK cytotoxicity assays; absolute NK cell counts were better correlated with the WB NK cytotoxicity assay than with the PBMC NK cytotoxicity assay. This WB NK cytotoxicity assay showed that patients with liver diseases had significantly lower NK cytotoxicity than healthy volunteers, under stimulation with various cytokines (p < 0.001). Conclusion: The proposed FC-based overnight WB NK cytotoxicity assay correlates well with the conventional 4-h PBMC NK cytotoxicity assay, demonstrating future potential as a supportive assay for clinical laboratory research and observational studies.

Natural killer (NK) cells are a subset of innate lymphoid cells playing an important role in immune surveillance and early defense against infection and cancer. They recognize and directly kill infected or transformed cells. At the same time, they produce various cytokines and chemokines to regulate other immune cells. NK cell activity can be a useful marker for health screenings because impaired NK cell functions may indicate a more susceptible environment for infection or tumor development. Currently, most NK cell activity assays are focused on measuring either cytokine secretion, in particular, interferon γ (IFN-γ), or cytotoxicity against target cells such as K562, thus only providing partial information on NK cell activity. In order to develop a comprehensive test for measuring NK cell function, cytotoxicity and cytokine secretion ability should be measured simultaneously. In addition, current NK cell assays are performed by stimulating NK cells with cocktails of cytokines, antibody-coated beads, or live target cells. In this study, we developed multifunctional microparticles for NK cell activity assay (MNAs) that allow simultaneous stimulation and sensing various NK cell activities, including cytokine secretion and cytotoxicity. The surfaces of MNAs are decorated with multiple functional biomolecules, including antibodies that stimulate NK cells by engaging NK cell activating receptors, antibodies that can capture cytokines secreted by NK cells, and a peptide sensor that reacts with granzyme B, a key molecule released by NK cells for cytotoxicity. The performances of MNAs are assessed using flow cytometry and live cell imaging. NK cell activity is measured by simply mixing MNAs with NK cells and performing flow cytometry, and the results are comparable to those measured by standard NK cell activity assays.

Cellular dynamics under complex topographical microenvironments are important for many biological processes in development and diseases, but systematic investigation has been limited due to the lack of technology. Herein, we developed a new dynamic cell patterning method based on a cell-friendly photoresist polymer that allows in situ control of cell dynamics on nanostructured surfaces. Using this method, we quantitatively compared the spreading dynamics of cells on nanostructured surfaces to those on flat surfaces. Furthermore, we investigated how cells behaved when they simultaneously encountered two topographically distinct surfaces during spreading. This method will allow many exciting opportunities in the fundamental study of cellular dynamics.