Multifunctional Nanodevices for the Regulation of Cytotoxic Activity of Natural Killer Cells

Esti Toledo 1,2 Guillaume Le Saux 1,2 Avishai Edri 3 Uzi Hadad 2 Angel Porgador 3 Mark Schvartzman 1,2

Innate immune system is based on natural killer (NK) cells – lymphocytes that distinguish between healthy and diseased cells, and attack tumor, virus-infected and stressed cells. The activity of NK cells regulated through a delicate balance between activating and inhibitory signals delivered by a multitude of activating and inhibitory receptors. However, the exact mechanism of how different receptors integrate their signals, and in particular how their signal integration depends on the receptor spatial organization, is unclear. Recently, biomimetic devices that control spatial organization of receptors within the cell membrane have been extensively used to study how the receptor spatial order regulates cell function, including that of immune lymphocytes. These devices comprise of lithographically patterned nanodots functionalized with cognate ligands for the studied receptors. Yet, these devices have been limited to control only receptor of one type, and thus could not been used to study signal integration between different receptors.

Here, we realized biomimetic devices for the spatial control of two types of receptors. For this purpose, we first developed a novel nanofabrication of precisely positioned pairs of two metallic nanodots, using nanoimprint lithography, sequential angle evaporation, and liftoff. Then, we orthogonally functionalized the nano-pattern with two different ligands, and thereby obtained an artificial membrane of antigen presenting cells with unprecedented diversity and complexity of its molecular composition and structure. We verified the selectivity of our functionalization by super-resolution fluorescence microscopy. Furthermore, we demonstrated the applicability of these devices for the regulation and study of the signaling balance in NK cells. This work provides an unique nano-bio toolbox that paves the way to numerous studies aimed at elucidating the molecular mechanism of signal integrating in cells, with the complexity and resolution impossible up to date.

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