NK cells recognize cancer and viral cells by binding their activating receptors to antigens presenting on the membrane of target cells. Although the activation mechanism of NK cells is a subject of extensive research today, the role of the composition and spatial distribution of activating ligands in NK cell cytotoxicity is barely understood. In this work, we engineered a nanochip whose surface was patterned with matrices of antigens for NKG2D activating receptors. These matrices mimicked the spatial order of the surface of antigen presenting cells with molecular resolution. Using this chip, we elucidated the effect of the antigen spatial distribution on the NK cell spreading and immune activation. We found that the spatial distribution of the ligand within the 100 nm length-scale provides the minimal conditions for NKG2D regulated cell spreading. Furthermore, we found that the immune activation of NK cells requires the same minimal spatial distribution of activating ligands. Above this threshold, both spreading and activation plateaued, confirming that these two cell functions work hand in hand. Our study provides an important insight on the spatial mechanism of the cytotoxic activity of NK cells. This insight opens the way to rationally designed antitumor therapies that harness NK cytotoxicity.

Studying how different signaling pathways spatially integrate in cells requires selective manipulation and control of different transmembrane ligand-receptor pairs at the same time. This work explores a novel method for precisely arranging two arbitrarily chosen ligands on a micron-scale two-dimensional pattern. The approach is based on lithographic patterning of Au and TiO films, followed by their selective functionalization with Ni-nitrilotriacetic acid-histidine and biotin-avidin chemistries, respectively. The selectivity of chemical and biological functionalizations is demonstrated by X-ray photoelectron spectroscopy and immunofluorescence imaging, respectively. This approach is applied to produce the first type of bifunctional surfaces with controllably positioned ligands for activating the receptors of natural killer (NK) immune cells. NK cells were used as a model system to demonstrate the potency of the surface in guiding site-selective cell attachment and activation. Upon applying the suitable ligand or ligand combination, the surfaces guided the appropriate single- or bifunctional attachment and activation. These encouraging results demonstrate the effectiveness of the system as an experimental platform aimed at the comprehensive understanding of the immunological synapse. The great simplicity, modularity, and specificity of this approach make it applicable for a myriad of combinations of other biomolecules and applications, turning it into the "Swiss knife" of biointerfaces.