Molecular Scale Spatio-Chemical Control of the Activating-Inhibitory Signal Integration in NK Cells

The role of the spatial juxtaposition between activating and inhibitory receptors in cytotoxic lymphocytes has been strongly debated in the context of the inhibition of immune signaling. The challenge in addressing this problem was so far a lack of experimental tools which can simultaneously manipulate different signaling molecules. Here, we circumvent this challenge by introducing a nanoengineered multifunctional cell niche, in which activating and inhibitory ligands are positioned with molecular-scale variability and control, and applied it to elucidate the role of the spatial juxtaposition between ligands for NKG2D and KIR2DL1 - activating and inhibitory receptors in Natural Killer (NK) cells - in KIR2DL1-mediated inhibition of NKG2D signaling. We realized the niche by a nanopatterning of nanodots of different metals with molecular scale registry in one lithographic step, followed by a novel ternary functionalization of the fabricated bi-metallic pattern and its background to with three distinct biochemical moieties. We found, that within the probed range, the 40 nm gap between the activating and inhibitory ligands provided an optimal inhibition condition. Supported by theoretical modeling and simulations we interpret these findings as a consequence of the size and conformational flexibility of the ligands in their spatial interaction. Our findings provide an important insight onto the spatial mechanism of the inhibitory immune checkpoints, whose understanding is both fundamentally important, and essential for the rational design of future immunotherapies. Furthermore, our approach is highly versatile and paves the way to numerous complex molecular platforms aimed at revealing molecular mechanisms through which receptors integrate their signals.