The formation of immunological synapse (IS) is essential for natural killer (NK) cells to eliminate target cells. Although elucidation of the overall characteristics and formation processes of IS is ongoing, the mechanisms for regulating the stability of IS by cytoskeleton remain to be understood. The current study reports that Nogo receptor 1 (NgR1) plays a key role in modulating NK cell-mediated killing by destabilization of IS formation. NgR1 deficiency or blockade results in improved tumor control of NK cells by enhancing NK-totarget cell contact stability and regulating F-actin dynamics during IS formation. Clinically, patients with a tumor expressing abundant NgR1 ligand showed poor prognosis despite high levels of NK cell infiltration. Thus, our study identifies NgR1 as an immune checkpoint involved in IS formation and reveals a potential approach to improve the killing effect of NK cells in cancer immunotherapy.

Background: Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient availability of NK cells and limited UCB volume, more effective culture methods are required. NK cell expansion and functionality are largely affected by the culture medium. While human serum is a major affecting component in culture media, the way it regulates NK cell functionality remains elusive. We elucidated the effects of different culture media and human serum supplementation on UCB NK cell expansion and functionality.Methods: UCB NK cells were cultured under stimulation with K562-OX40L-mbIL-18/21 feeder cells and IL-2 and IL-15 in serum-containing and serum-free culture media. The effects of the culture media and human serum supplementation on NK cell expansion and cytotoxicity were evaluated by analyzing the expansion rate, activating and inhibitory receptor levels, and the cytotoxicity of the UCB NK cells. Results:The optimal medium for NK cell expansion was Dulbecco's modified Eagle's medium/Ham's F12 with supplements and that for cytotoxicity was AIM V supplemented with Immune Cell Serum Replacement. Shifting media is an advantageous strategy for obtaining several highly functional UCB NK cells. Live cell imaging and killing time measurement revealed that human serum enhanced NK cell proliferation but delayed target recognition, resulting in reduced cytotoxicity.Conclusions: Culture medium supplementation with human serum strongly affects UCB NK cell expansion and functionality. Thus, culture media should be carefully selected to ensure both NK cell quantity and quality for adoptive cell therapy.

Image-based cell sorting is essential in biological and biomedical research. The sorted cells can be used for downstream analysis to expand our knowledge of cell-to-cell differences. We previously demonstrated a user-friendly image-activated microfluidic cell sorting technique using an optimized and fast deep learning algorithm. Real-time isolation of cells was carried out using this technique with an inverted microscope. In this study, we devised a recently upgraded sorting system. The cell sorting techniques shown on the microscope were implemented as a real system. Several new features were added to make it easier for the users to conduct the real-time sorting of cells or particles. The newly added features are as follows: (1) a high-resolution linear piezo-stage is used to obtain in-focus images of the fast-flowing cells; (2) an LED strobe light was incorporated to minimize the motion blur of fast-flowing cells; and (3) a vertical syringe pump setup was used to prevent the cell sedimentation. The sorting performance of the upgraded system was demonstrated through the real-time sorting of fluorescent polystyrene beads. The sorter achieved a 99.4% sorting purity for 15 μm and 10 μm beads with an average throughput of 22.1 events per second (eps).