Controlling Cytotoxic Activity of Natural Killer (NK) Cells by Nanolithographic Molecular-scale Devices

Rapidly emerging research and clinical studies show promising anti-tumor effects of immunotherapy based on Natural-Killer (NK) cell ­–lymphocytes that belong to innate immune system. NK cell cytotoxicity is regulated by activating receptors that bind their cognate ligands on the surface of target cell. Activating ligands are upregulated in cancer and viral cells, yet, they can be also expressed in certain amount by healthy tissues. Given various levels of expression of NK activating ligands on infected, transformed, and activated immune cells, as well as on several healthy cells, it can be hypothesized that such level of expression regulates the cytotoxicity of NK cells, and that there are minimal requirements for the expression of activation ligands to stimulate the cytotoxic response of NK cells.

To elucidate how the distribution of activating ligands regulates NK cell cytotoxicity, we engineered a nanochip for the controlled activation of human NK cells. The chip contained multiple arrays of controllably positioned Au nanodots functionalized with MHC class I (MICA) – which ligands for NKG2D activating receptors. We fabricated the arrays by nanoimprint, immobilized on them (i)thiol terminated with Ni-chelated Nitrilotriacetic acid and (ii)histidine-conjugated MICA. We verified the selectivity of our functionalization by immunofluorescent staining of immobilized MICA with fluorophore-conjugated antibody.

We produced arrays with different clustering and arrangement of ligated nanodots and monitored their stimulation of NK cells. In particular, we assessed the effect of ligand distribution onto the cell spreading, and onto the cell cytotoxicity by imaging lysosomal-associated membrane protein CD107a. Our study provides an important insight into the spatial mechanism of the cytotoxic activity of NK cells, by establishing the ligand distribution within sub-nanometric length-scale as a critical barrier for the formation of immune synapse and degranulation. This understanding paves the way to rationally designed immunotherapeutic approaches employing unique NK cytotoxicity against human malignancies.